*Review* **Trends in the Hydrogen**−**Deuterium Exchange at the Carbon Centers. Preparation of Internal Standards for Quantitative Analysis by LC-MS**

**Paulina Grocholska and Remigiusz B ˛achor \***

Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland; paulina.grocholska@chem.uni.wroc.pl

**\*** Correspondence: remigiusz.bachor@chem.uni.wroc.pl; Tel.: +48-71-375-7218; Fax: +48-71-328-2348

**Abstract:** The application of internal standards in quantitative and qualitative bioanalysis is a commonly used procedure. They are usually isotopically labeled analogs of the analyte, used in quantitative LC-MS analysis. Usually, <sup>2</sup>H, <sup>13</sup>C, <sup>15</sup>N and <sup>18</sup>O isotopes are used. The synthesis of deuterated isotopologues is relatively inexpensive, however, due to the isotopic effect of deuterium and the lack of isotopologue co-elution, usually they are not considered as good internal standards for LC-MS quantification. On the other hand, the preparation of <sup>13</sup>C, <sup>15</sup>N and <sup>18</sup>O containing standards of drugs and their metabolites requires a complicated multistep de novo synthesis, starting from the isotopically labeled substrates, which are usually expensive. Therefore, there is a strong need for the development of low-cost methods for isotope-labeled standard preparations for quantitative analysis by LC-MS. The presented review concentrates on the preparation of deuterium-labeled standards by hydrogen−deuterium exchange reactions at the carbon centers. Recent advances in the development of the methods of isotopologues preparation and their application in quantitative analysis by LC-MS are evaluated.

**Keywords:** hydrogen−deuterium exchange; liquid chromatography-mass spectrometry; *N*-methylated amino acids; quantitative LC-MS analysis

#### **1. Introduction**

Hydrogen−deuterium exchange (HDX) is a process involving the substitution of a hydrogen atom by a deuterium atom in a molecule of a chemical compound, in the presence of a deuterating agent, e.g., deuterium oxide (D2O), or another source of dissociating deuterons [1,2]. Labile hydrogens in the backbone and side-chain functional groups of chemical compounds undergo exchange with protons of the solvent within a few minutes [1]. Due to the basic mechanism of the H/D exchange reaction, including acid-base catalysis, the degree of the reaction depends strongly on the pH of the solution [2]. The decimal logarithm of the reaction rate constant versus pH curve takes a V-shape with a characteristic minimum between pH 2 and 3, where the average half-time of the exchange at 0◦ C is tens of minutes. The rate of H/D exchange increases significantly with increasing pH. The rate of the H/D exchange reaction depends also on the acidity of the hydrogens bounded with heteroatoms, which is a consequence of the electronegativity differences between these atoms. For this reason, hydrogens from carboxyl or hydroxyl groups undergo the H/D exchange reaction much more easily than amide ones. The role of the inductive effects of the individual functional groups present in the vicinity of the hydrogen undergoing H/D exchange, and of steric hindrance, hindering the access of the acid or basic catalyst to the exchangeable proton is also described [1,2].

The influence of the molecular structure of the compound on the isotope exchange has also been observed, especially in the case of peptides and proteins. It was found that amide hydrogens participating in intramolecular hydrogen bonds were much less susceptible

**Citation:** Grocholska, P.; B ˛achor, R. Trends in the Hydrogen−Deuterium Exchange at the Carbon Centers. Preparation of Internal Standards for Quantitative Analysis by LC-MS. *Molecules* **2021**, *26*, 2989. https:// doi.org/10.3390/molecules26102989

Academic Editors: Gavino Sanna and Stefan Leonidov Tsakovski

Received: 6 April 2021 Accepted: 14 May 2021 Published: 18 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to isotopic exchange reactions. Therefore, the H/D exchange was found as a tool in the conformation analysis of biologically active compounds. Additionally, the isotope exchange reactions enabled the analysis of the mechanisms of chemical processes [3]. The compelling advantage of mass spectrometric analysis of the H/D exchange of peptides and proteins is the high sensitivity of the method, the low concentrations of the analyte which can be used and the ability to simultaneously analyze individual components of the complex mixtures [4]. the conformation analysis of biologically active compounds. Additionally, the isotope exchange reactions enabled the analysis of the mechanisms of chemical processes [3]. The compelling advantage of mass spectrometric analysis of the H/D exchange of peptides and proteins is the high sensitivity of the method, the low concentrations of the analyte which can be used and the ability to simultaneously analyze individual components of the complex mixtures [4]. In contrast to the hydrogens attached to heteroatoms, the hydrogen atoms bound to

amide hydrogens participating in intramolecular hydrogen bonds were much less susceptible to isotopic exchange reactions. Therefore, the H/D exchange was found as a tool in

In contrast to the hydrogens attached to heteroatoms, the hydrogen atoms bound to the carbon atoms are usually not exchangeable, however, the specific reaction conditions, including pH-dependent and metal-dependent catalysis, may promote the replacement of carbon-attached protons by deuterons (Figure 1) [5–8]. Such compounds may serve as internal standards in quantitative LC-MS analysis. Therefore, the exchange of hydrogen attached to the carbon by its heavier isotopes are of interest for mechanistic, productorientated research and quantitative analysis [3]. The existing methods of isotope exchange on the α-carbon atoms of amino acids are expensive and time-consuming, because they require multistep de novo synthesis with the application of isotopically labeled substrates. Usually preparation of such deuterated derivatives by hydrogen−deuterium exchange is easier and more cost effective than by classical de novo synthesis [7]. Therefore, the development of new, 'gentle' methods of isotope exchange on the α-C carbon atoms of amino acid residues in peptides seems to be an important issue. At present, isotopically labeled compounds at the carbon atoms are of interest, especially due to their application as internal standards in quantitative mass spectrometry in the analysis of fragmentation mechanisms. the carbon atoms are usually not exchangeable, however, the specific reaction conditions, including pH-dependent and metal-dependent catalysis, may promote the replacement of carbon-attached protons by deuterons (Figure 1) [5–8]. Such compounds may serve as internal standards in quantitative LC-MS analysis. Therefore, the exchange of hydrogen attached to the carbon by its heavier isotopes are of interest for mechanistic, product-orientated research and quantitative analysis [3]. The existing methods of isotope exchange on the α-carbon atoms of amino acids are expensive and time-consuming, because they require multistep de novo synthesis with the application of isotopically labeled substrates. Usually preparation of such deuterated derivatives by hydrogen−deuterium exchange is easier and more cost effective than by classical de novo synthesis [7]. Therefore, the development of new, 'gentle' methods of isotope exchange on the α-C carbon atoms of amino acid residues in peptides seems to be an important issue. At present, isotopically labeled compounds at the carbon atoms are of interest, especially due to their application as internal standards in quantitative mass spectrometry in the analysis of fragmentation mechanisms.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 2 of 26

**Figure 1.** Hydrogen−deuterium exchange catalyzed by base, acid or metal catalyst. **Figure 1.** Hydrogen−deuterium exchange catalyzed by base, acid or metal catalyst.

Liquid chromatography-mass spectrometry (LC-MS) quantification frequently is performed in the presence of isotopically labeled standards which in most cases have to be synthesized de novo [9,10]. There are certain requirements for these standards: isotopologues should be characterized by identical chromatographic behavior, the mass difference between isotopologues should be at least 2 Daltons and the introduced isotopes cannot undergo back exchange during LC-MS separation conditions [11]. Quantification is Liquid chromatography-mass spectrometry (LC-MS) quantification frequently is performed in the presence of isotopically labeled standards which in most cases have to be synthesized de novo [9,10]. There are certain requirements for these standards: isotopologues should be characterized by identical chromatographic behavior, the mass difference between isotopologues should be at least 2 Daltons and the introduced isotopes cannot undergo back exchange during LC-MS separation conditions [11]. Quantification is performed by MS analysis, by comparing the extracted ion chromatograms (peak area) of the isotope-labeled and nonlabeled compounds. Although various stable isotope-labeled quantification reagents have been described containing <sup>2</sup>H, <sup>13</sup>C, <sup>15</sup>N and <sup>18</sup>O isotopes, due to their complicated and expensive chemical synthesis, there is still a strong need to develop

a method of preparation of new isotopically-labeled standards. Usually, the preparation of deuterated standards is relatively inexpensive, however, the possibility of isotope effect affecting their co-elution during LC-MS would limit their possible application in LC-MS quantification [12,13]. It was found that the isotope effect on the chromatographic behavior of deuterated and nondeuterated isotopologues depends on the number and place of the introduced deuterons [14,15]. Additionally, deuterium-labeled compounds cannot be used for in vivo studies due to the possible loss of deuterium or different metabolism pathways. Therefore, there is a strong need for low-cost methods for isotope-labeled standard preparation for quantitative analysis by LC-MS. Although, the incorporation of deuterium into the target molecules may present some drawbacks, nevertheless the most important advantage of such a procedure is its low cost and simplicity of preparation.

In this review, we present the methods of deuterium incorporation into the molecules of compounds by exchange reactions and the possible application of deuterated standards in quantitative analysis by LC-MS.

### **2. Hydrogen**−**Deuterium Exchange at Carbon Centers**

#### *2.1. Acid- and Base-Catalyzed HDX*

The pH-dependent hydrogen−deuterium exchange reactions are the first reported methods in the presented field [16]. The acid and base-catalyzed HDX process involves enolization which makes the H/D exchange at activated carbon centers possible in the presence of a source of deuterium, including deuterated Brønsted acids or bases. The back exchange of introduced deuterons is of course possible, therefore further deactivation of the analyzed compound is required.

In most cases, acid-catalyzed H/D-exchange reactions are used to incorporate the deuterium into the aromatic molecules. In these cases, strong deuterated Brønsted or Lewis acids, in the presence of a deuterium source are commonly used. The application of Lewis acids, including AlBr<sup>3</sup> or MoCl<sup>5</sup> is mostly restricted to the nonpolar arenes. In acidcatalyzed HDX, the incorporation of the <sup>2</sup>H isotope to the aromatic compounds exhibits limited regioselectivity. The effect of the aromatic ring substituents on the deuteration regioselectivity was analyzed in the acid-catalyzed HDX on ferrocenes. It was found that the electrophilic aromatic deuteration of the cyclopentadienyl rings was favored by alkyl groups whereas enolization of the carbonyl group in ketones led to the selective and complete H−D exchange of all three hydrogens of the acyl residue [17].

Base-catalyzed HDX is also a facile method for deuterium incorporation by means of keto–enol equilibria. Due to the higher acidity of carbon-bound hydrogen atoms in carbonyl compounds, including ketones [18], aldehydes [19], esters [20] and carboxylic acids [21], they undergo H/D exchange with high selectivity (>90% D) and yield. The γ hydrogens in α,β-unsaturated carbonyl compounds are also able to exchange through conjugation, as presented on the steroid framework of androstenedione, testosterone, and cortisone [22].

The deuteration of the methyl group in aryl methyl ketones and aryl methyl sulfones under basic conditions was presented by Berthelette and Scheigetz [23]. It was found that the reaction efficiency and rate depended on the base, the substrate and the solvent nature. Whereas 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was found as a base allowing high deuteration efficiency, *N,N,N*-triethylamine (TEA) was less effective in the corresponding HDX processes (Figure 2). TEA was found as a base allowing methyl group deuteration in the base-sensitive ketones without decomposition. *Molecules* **2021**, *26*, x FOR PEER REVIEW 4 of 26

> carbonyl compounds, including *N*-substituted acetamides or diketopiperazines are usually highly exchanged [5]. The presented H/D exchange involved the application of acetone-d6, *N*,*N*,*N*-triethylamine (TEA) or a stronger base in the form of diazabicycloundec-7-ene (DBU) and incubation at higher temperatures (35 °C to 50 °C). The acidity of αcarbon hydrogens of amino acids and peptides is of interest because the corresponding enolates play an important role in nonenzymatic racemization during peptide synthesis and enzyme-catalyzed racemization in different biochemical transformations [5,24]. Till now only a few quantitative investigations on their stability in water have been published. Their formation rate constants have been analyzed and determined in studies of α-hydrogen exchange or racemization reactions of amino acids and peptides at high temperatures [25,26] for the development of base-catalyzed methods for the preparation of α-C-deuterated amino acids is an important research task [8,27]. Generally, synthetic methods are based on the glycine derivatives application, which are subjected to a basic HDX and the

> The acidity of α-C hydrogens of various amino acids and their derivatives have been extensively investigated. Ho et al. informed that α-C hydrogens in *N*-methylated analogs of cyclic dipeptides are more acidic than those in nonmethylated compounds [5,28]. It was also confirmed that the substitution of the amino group with electron-withdrawing substituents, such as the acetyl group, facilitated the HDX of α-C hydrogens. It was found by Rios and co-workers that the acidity of the α-C hydrogens depended on the ionization state of the amino acid, additionally the exhaustive methylation of α amino group also affected the pKa of the presented hydrogens [29,30]. Up to now, several different compounds containing *N*-methylated amino acids in their chemical structure have been described. These compounds belongs to the group of important drugs and natural tissue metabolites or substances commonly used in industry and households. In many cases

> Moozeh and co-workers presented the stereoinversion of L-alanine to α-C deuterated D-alanine by base catalysis [31]. In the presence of salicylaldehyde and a chiral base, 87% deuterium incorporation at α-C of L-alanine and an inversion to D-alanine, was obtained. The enantiomeric excess (ee) was 67% (Figure 3). The developed method was also successfully applied for the deuteration of another 11 natural amino acids (threonine, tryptophan, phenylalanine, methionine, glutamic acid, glycine, glutamine, asparagine, serine, lysine

and leucine). Stereoinversion for the presented examples was not reported.

**Figure 2.** Deuteration of diketone presented by Berthelette and co-workers [23]. **Figure 2.** Deuteration of diketone presented by Berthelette and co-workers [23].

stereoselective insertion of the desired side chain.

there is a need to quantify them.

The base-catalyzed HDX reaction is a simple method for the acidic hydrogen exchange for deuterium by keto-enol equilibria [16]. The carbon-bound acidic hydrogens in carbonyl compounds, including *N*-substituted acetamides or diketopiperazines are usually highly exchanged [5]. The presented H/D exchange involved the application of acetone-d6, *N*,*N*,*N*-triethylamine (TEA) or a stronger base in the form of diazabicycloundec-7-ene (DBU) and incubation at higher temperatures (35 ◦C to 50 ◦C). The acidity of α-carbon hydrogens of amino acids and peptides is of interest because the corresponding enolates play an important role in nonenzymatic racemization during peptide synthesis and enzymecatalyzed racemization in different biochemical transformations [5,24]. Till now only a few quantitative investigations on their stability in water have been published. Their formation rate constants have been analyzed and determined in studies of α-hydrogen exchange or racemization reactions of amino acids and peptides at high temperatures [25,26] for the development of base-catalyzed methods for the preparation of α-C-deuterated amino acids is an important research task [8,27]. Generally, synthetic methods are based on the glycine derivatives application, which are subjected to a basic HDX and the stereoselective insertion of the desired side chain.

The acidity of α-C hydrogens of various amino acids and their derivatives have been extensively investigated. Ho et al. informed that α-C hydrogens in *N*-methylated analogs of cyclic dipeptides are more acidic than those in nonmethylated compounds [5,28]. It was also confirmed that the substitution of the amino group with electron-withdrawing substituents, such as the acetyl group, facilitated the HDX of α-C hydrogens. It was found by Rios and co-workers that the acidity of the α-C hydrogens depended on the ionization state of the amino acid, additionally the exhaustive methylation of α amino group also affected the pKa of the presented hydrogens [29,30]. Up to now, several different compounds containing *N*-methylated amino acids in their chemical structure have been described. These compounds belongs to the group of important drugs and natural tissue metabolites or substances commonly used in industry and households. In many cases there is a need to quantify them.

Moozeh and co-workers presented the stereoinversion of L-alanine to α-C deuterated D-alanine by base catalysis [31]. In the presence of salicylaldehyde and a chiral base, 87% deuterium incorporation at α-C of L-alanine and an inversion to D-alanine, was obtained. The enantiomeric excess (ee) was 67% (Figure 3). The developed method was also successfully applied for the deuteration of another 11 natural amino acids (threonine, tryptophan, phenylalanine, methionine, glutamic acid, glycine, glutamine, asparagine, serine, lysine and leucine). Stereoinversion for the presented examples was not reported. *Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 26

**Figure 3.** Catalytic stereoselective deuteration of L-alanine to deuterated D-alanine in the presence of 3,5-dichloro-2-hydroxybenzaldehyde and a chiral base [31]. **Figure 3.** Catalytic stereoselective deuteration of L-alanine to deuterated D-alanine in the presence of 3,5-dichloro-2-hydroxybenzaldehyde and a chiral base [31].

Mitulovi and co-workers [32] presented the method of acid-catalyzed deuteration of Mitulovi and co-workers [32] presented the method of acid-catalyzed deuteration of αamino acids (i.e., alanine, leucine, phenylalanine). In the presence of [D1]acetic acid (excess)

α-amino acids (i.e., alanine, leucine, phenylalanine). In the presence of [D1]acetic acid (ex-

mation (Figure 4). The obtained compound was converted into the tertbutoxycarbonyl (Boc) protected derivative and the resulting enantiomeric mixture was separated by pre-

The methods for the preparation of enantiomerically pure α-C-deuterated amino acids in the presence of a base are based on the application of glycine or its derivatives, which are subjected to a basic HDX. Finally, the side chain is inserted stereoselectively

Lankiewicz and co-workers [7], described the method of the preparation of deuterated glycine derivative in the mixture of MeOD/D2O and the presence of catalytic amounts of Na2CO3 as a base. After three reaction steps of reaction the obtained derivative was characterized by a deuterium content greater than 98%. Then the reaction with the Oppolzer sultam provide an intermediate for the subsequent stereoselective alkylation [33]. After removal of the auxiliary, the chiral Boc-protected amino acid (glycine, alanine, leucine, phenylalanine, O-benzyltyrosine) was isolated almost enantiomerically pure (>99%

parative high-performance liquid chromatography on a chiral stationary phase.

**Figure 4.** Deuteration of amino acids under acidic conditions [16].

**Figure 5.** Stereoselective synthesis of deuterated amino acids [16].

with the aid of chiral auxiliaries [6–8].

ee) in high yield (Figure 5).

and catalytic amounts of aldehyde, the reaction is characterized by good yield and the deuterium incorporation at the level of 95% via the corresponding Schiff's base formation (Figure 4). The obtained compound was converted into the tertbutoxycarbonyl (Boc) protected derivative and the resulting enantiomeric mixture was separated by preparative high-performance liquid chromatography on a chiral stationary phase. the deuterium incorporation at the level of 95% via the corresponding Schiff's base formation (Figure 4). The obtained compound was converted into the tertbutoxycarbonyl (Boc) protected derivative and the resulting enantiomeric mixture was separated by preparative high-performance liquid chromatography on a chiral stationary phase. mation (Figure 4). The obtained compound was converted into the tertbutoxycarbonyl (Boc) protected derivative and the resulting enantiomeric mixture was separated by preparative high-performance liquid chromatography on a chiral stationary phase.

**Figure 3.** Catalytic stereoselective deuteration of L-alanine to deuterated D-alanine in the presence

**Figure 3.** Catalytic stereoselective deuteration of L-alanine to deuterated D-alanine in the presence

α-amino acids (i.e., alanine, leucine, phenylalanine). In the presence of [D1]acetic acid (excess) and catalytic amounts of aldehyde, the reaction is characterized by good yield and

Mitulovi and co-workers [32] presented the method of acid-catalyzed deuteration of

Mitulovi and co-workers [32] presented the method of acid-catalyzed deuteration of α-amino acids (i.e., alanine, leucine, phenylalanine). In the presence of [D1]acetic acid (excess) and catalytic amounts of aldehyde, the reaction is characterized by good yield and the deuterium incorporation at the level of 95% via the corresponding Schiff's base for-

of 3,5-dichloro-2-hydroxybenzaldehyde and a chiral base [31].

of 3,5-dichloro-2-hydroxybenzaldehyde and a chiral base [31].

*Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 26

*Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 26

The methods for the preparation of enantiomerically pure α-C-deuterated amino acids in the presence of a base are based on the application of glycine or its derivatives, which are subjected to a basic HDX. Finally, the side chain is inserted stereoselectively The methods for the preparation of enantiomerically pure α-C-deuterated amino acids in the presence of a base are based on the application of glycine or its derivatives, which are subjected to a basic HDX. Finally, the side chain is inserted stereoselectively with the aid of chiral auxiliaries [6–8]. ids in the presence of a base are based on the application of glycine or its derivatives, which are subjected to a basic HDX. Finally, the side chain is inserted stereoselectively with the aid of chiral auxiliaries [6–8]. Lankiewicz and co-workers [7], described the method of the preparation of deuter-

with the aid of chiral auxiliaries [6–8]. Lankiewicz and co-workers [7], described the method of the preparation of deuterated glycine derivative in the mixture of MeOD/D2O and the presence of catalytic amounts of Na2CO3 as a base. After three reaction steps of reaction the obtained derivative was characterized by a deuterium content greater than 98%. Then the reaction with the Oppolzer sultam provide an intermediate for the subsequent stereoselective alkylation [33]. After removal of the auxiliary, the chiral Boc-protected amino acid (glycine, alanine, leu-Lankiewicz and co-workers [7], described the method of the preparation of deuterated glycine derivative in the mixture of MeOD/D2O and the presence of catalytic amounts of Na2CO<sup>3</sup> as a base. After three reaction steps of reaction the obtained derivative was characterized by a deuterium content greater than 98%. Then the reaction with the Oppolzer sultam provide an intermediate for the subsequent stereoselective alkylation [33]. After removal of the auxiliary, the chiral Boc-protected amino acid (glycine, alanine, leucine, phenylalanine, O-benzyltyrosine) was isolated almost enantiomerically pure (>99% ee) in high yield (Figure 5). ated glycine derivative in the mixture of MeOD/D2O and the presence of catalytic amounts of Na2CO3 as a base. After three reaction steps of reaction the obtained derivative was characterized by a deuterium content greater than 98%. Then the reaction with the Oppolzer sultam provide an intermediate for the subsequent stereoselective alkylation [33]. After removal of the auxiliary, the chiral Boc-protected amino acid (glycine, alanine, leucine, phenylalanine, O-benzyltyrosine) was isolated almost enantiomerically pure (>99% ee) in high yield (Figure 5).

**Figure 5. Figure 5.** Stereoselective synthesis of deuterated amino acids [16]. Stereoselective synthesis of deuterated amino acids [16].

**Figure 5.** Stereoselective synthesis of deuterated amino acids [16]. Rose and et al. [8], inspired by the bislactim ether method developed by Schöllkopf and co-workers [34], described a base-catalyzed method of C6-position deuteration of the dihydropyrazine in boiling mixture of MeOD/D2O (Figure 6). No hydrogen−deuterium exchange was observed at the C3-position due to the steric hindrance of the isopropyl group in the transition state. The obtained [6-D2]isotopologue was stereoselectively alkylated at the C6-position, thereby giving access to a series of α-C-deuterated amino acids (serine, phenylalanine, allylglycine, aspartic acid) in good yields, high degrees of deuteration and enantiomeric excesses (>95%) [8].

lyst [16].

(serine, phenylalanine, allylglycine, aspartic acid) in good yields, high degrees of deuter-

Rose and et al. [8], inspired by the bislactim ether method developed by Schöllkopf and co-workers [34], described a base-catalyzed method of C6-position deuteration of the dihydropyrazine in boiling mixture of MeOD/D2O (Figure 6). No hydrogen−deuterium exchange was observed at the C3-position due to the steric hindrance of the isopropyl group in the transition state. The obtained [6-D2]isotopologue was stereoselectively alkyl-

ation and enantiomeric excesses (>95%) [8].

ation and enantiomeric excesses (>95%) [8].

*Molecules* **2021**, *26*, x FOR PEER REVIEW 6 of 26

**Figure 6.** Schӧllkopf bislactim ether application in the preparation of deuterated amino acids [16]. **Figure 6.** Schöllkopf bislactim ether application in the preparation of deuterated amino acids [16]. **Figure 6.** Schӧllkopf bislactim ether application in the preparation of deuterated amino acids [16].

Rose and et al. [8], inspired by the bislactim ether method developed by Schöllkopf and co-workers [34], described a base-catalyzed method of C6-position deuteration of the dihydropyrazine in boiling mixture of MeOD/D2O (Figure 6). No hydrogen−deuterium exchange was observed at the C3-position due to the steric hindrance of the isopropyl group in the transition state. The obtained [6-D2]isotopologue was stereoselectively alkylated at the C6-position, thereby giving access to a series of α-C-deuterated amino acids (serine, phenylalanine, allylglycine, aspartic acid) in good yields, high degrees of deuter-

A further enantioselective synthesis of α-C-deuterated (Figure 7) proceeds by asymmetric alkylation of the activated glycine in the presence of the chiral phase-transfer catalyst. The HDX in the presence of KOD in D2O and the introduction of the side chain were performed in a single reaction step. After imine hydrolysis the amino acid *tert*-butyl esters were isolated in good yields and with high deuterium incorporation of more than 90% [6]. A further enantioselective synthesis of α-C-deuterated (Figure 7) proceeds by asymmetric alkylation of the activated glycine in the presence of the chiral phase-transfer catalyst. The HDX in the presence of KOD in D2O and the introduction of the side chain were performed in a single reaction step. After imine hydrolysis the amino acid *tert*-butyl esters were isolated in good yields and with high deuterium incorporation of more than 90% [6]. A further enantioselective synthesis of α-C-deuterated (Figure 7) proceeds by asymmetric alkylation of the activated glycine in the presence of the chiral phase-transfer catalyst. The HDX in the presence of KOD in D2O and the introduction of the side chain were performed in a single reaction step. After imine hydrolysis the amino acid *tert*-butyl esters were isolated in good yields and with high deuterium incorporation of more than 90% [6].

**Figure 7.** Enantioselective deuteration of amino acids in the presence of chiral phase transfer cata-**Figure 7.** Enantioselective deuteration of amino acids in the presence of chiral phase transfer cata-**Figure 7.** Enantioselective deuteration of amino acids in the presence of chiral phase transfer catalyst [16].

lyst [16]. In addition to hydrogen−deuterium exchange based on the keto-enol tautomerism, the deuterolysis of an organometallic compound is also the chemical tool used for the synthesis of deuterated derivatives. In this reaction, the intermediate organometallic compound is formed by deprotonation in the presence of strong bases (i.e., Grignard reagent or alkyl–lithium compound), and subsequently deuterated with electrophiles in the form of D2O, MeOD, or AcOD; which formally correspond to the H/D exchange [35]. Using this approach complete ortho-deuteration of aromatic amides and aromatic carbamates was achieved. Moreover, due to the large kinetic isotope effect (KIE), incorporated deuterium In addition to hydrogen−deuterium exchange based on the keto-enol tautomerism, the deuterolysis of an organometallic compound is also the chemical tool used for the synthesis of deuterated derivatives. In this reaction, the intermediate organometallic compound is formed by deprotonation in the presence of strong bases (i.e., Grignard reagent or alkyl–lithium compound), and subsequently deuterated with electrophiles in the form of D2O, MeOD, or AcOD; which formally correspond to the H/D exchange [35]. Using this approach complete ortho-deuteration of aromatic amides and aromatic carbamates was achieved. Moreover, due to the large kinetic isotope effect (KIE), incorporated deuterium In addition to hydrogen−deuterium exchange based on the keto-enol tautomerism, the deuterolysis of an organometallic compound is also the chemical tool used for the synthesis of deuterated derivatives. In this reaction, the intermediate organometallic compound is formed by deprotonation in the presence of strong bases (i.e., Grignard reagent or alkyl–lithium compound), and subsequently deuterated with electrophiles in the form of D2O, MeOD, or AcOD; which formally correspond to the H/D exchange [35]. Using this approach complete ortho-deuteration of aromatic amides and aromatic carbamates was achieved. Moreover, due to the large kinetic isotope effect (KIE), incorporated deuterium served as a protecting group for the carbon center, allowing the control of the regioselectivity of the subsequent lithiation.

served as a protecting group for the carbon center, allowing the control of the regioselectivity of the subsequent lithiation. served as a protecting group for the carbon center, allowing the control of the regioselectivity of the subsequent lithiation. Hydrogen−deuterium exchange reactions can also be performed without the addition of acids and bases. Such transformations are characteristic for the acidic CH centers which may be deuterated simply by the incubation of compound in deuterium oxide. The autoprotolysis equilibrium of D2O, makes it possible to act as either an acid or a base. For example, the synthesis of [1,1,3,3-D4]2-indanone was achieved by the heating of the compound of interest in the D2O [36]. Other reactions, depending on the compound, which were suspected to be HDX, required sometimes drastic reaction conditions which cannot be applied for most organic molecules.

> A simple strategy was presented by Pacchioni et al. [37]. It was found that the formation of *N,N,N*-trinitroso derivative of the 1,4,7-triazacyclononane made the α-methylene hydrogen atoms more acidic allowing HDX in the presence of base.

A variant of acid-catalyzed HDX, which uses only D2O during the deuteration process and is accelerated by microwave irradiation, was presented by Barthez et al. [38]. The developed strategy was also successfully applied to aminopyridine derivative preparation [39]. In order to avoid any proton sources, the labile hydrogen atoms bound to the nitrogen were exchanged to deuterons in the presence of D2O. The applied strategy allowed complete deuteration within a few minutes and a high deuterium content at the *ortho* and *para* positions in the amino group.

Very recently, the application of microwaves in HDX processes significantly increased, especially due to the higher degree of deuteration, shorter reaction times as compared to the classical heating conditions. Based on this technique several MS standards of bleomycin A2 for quantitative MS analysis were successfully prepared in D2O after a two-minute heating at 165 ◦C [40]. Additionally, some physicochemical reports have been published in which the kinetics of noncatalyzed HDX and energetic investigations were described [41].

It was previously reported that the base-catalyzed hydrogen−deuterium exchange at the carbon centers of aldehydes and ketones, thioesters and oxygen esters or amides occurred via a stepwise mechanism involving the enolate intermediate formation when the enolate was sufficiently stable to exist for the time of a bond vibration [30].

*N*-methylglycine, also called sarcosine, represents a natural, achiral compound with a methylated amino group which plays an important role in biological systems [42]. This amino acid residue is present in cyclosporine A, a cyclic nonribosomal peptide, commonly used as immunosuppressant [43]. Methods for the quantitative analysis of cyclosporine A and its metabolites have been developed [44] however, due to the necessity of preparation of its isotopically labeled standards for MS quantification, the costs of such analysis were very high. Joining the mainstream of the base-catalyzed HDX, previously, we developed several methods of preparation of deuterated standards of compounds containing *N*-substituted glycine derivative in their chemical structure, including denatonium benzoate, peptomers, cyclosporin A and creatinine. Additionally, the applicability of the obtained deuterated standards were tested in the quantitative analysis of these compounds by LC-MS.

In our previous work, the base-catalyzed HDX of α-C hydrogens in sarcosine residue and specific hydrogen scrambling in such peptides were investigated [45]. We found the unusual hydrogen−deuterium exchange at the α-carbon in *N*-methy- and *N*-benzylglycine residues in the presence of 1% solution of *N,N,N*-triethylamine in D2O (Figure 8). We found that the observed HDX proceeded at a much slower rate as compared to the hydrogen−deuterium exchange of hydrogens present in amines or amides. Moreover, we observed the hydrogen scrambling during the collision-induced dissociation experiment which suggested the lability of such hydrogens. The presented work opened a wider possibility of application of the presented HDX reaction in peptide chemistry and mass spectrometry. *Molecules* **2021**, *26*, x FOR PEER REVIEW 8 of 26

**Figure 8.** Schematic presentation of H/D exchange reaction at the α carbon in *N*-methylglycine moiety [45]. TEA-*N,N,N*-triethylamine. **Figure 8.** Schematic presentation of H/D exchange reaction at the α carbon in *N*-methylglycine moiety [45]. TEA-*N,N,N*-triethylamine.

Our investigation on compounds containing sarcosine residue revealed its presence in cyclosporin A molecule. The analysis of the possibility of the α-C deuteration of cyclosporine A (CsA) in *N*-methylated amino acid residues was performed [46]. The proposed reaction is based on the method previously reported by us [45], proceeds under basic conditions in the presence of TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene) or MTBD (7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene) at pH 13.4 (Figure 9). The obtained results revealed that there is a possibility of three deuteron incorporation, two at the α-C of *N*-methylglycine and one at the α-C of 2-*N*-methyl-(R)-((E)-2-butenyl)-4-methyl-L-threonine (MeBmt) Our investigation on compounds containing sarcosine residue revealed its presence in cyclosporin A molecule. The analysis of the possibility of the α-C deuteration of cyclosporine A (CsA) in *N*-methylated amino acid residues was performed [46]. The proposed reaction is based on the method previously reported by us [45], proceeds under basic conditions in the presence of TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene) or MTBD (7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene) at pH 13.4 (Figure 9). The obtained results revealed that there is a possibility of three deuteron incorporation, two at the α-C of *N*-methylglycine and one at the α-C of 2-*N*-methyl-(R)-((E)-2-butenyl)-4-methyl-L-threonine (MeBmt) residue. The prepared isotopologues were stable (did not undergo back exchange) under neutral

residue. The prepared isotopologues were stable (did not undergo back exchange) under

revealed co-elution, which make their application for the quantitative analysis by using isotope dilution strategy possible. The developed strategy of the CsA deuteration is rapid, cost-efficient and does not require special reaction conditions, other reagents or further

**Figure 9.** Preparation of deuterated analogs of cyclosporin A as described by Bąchor et al. [46].

The synthesis of the α-C deuterium-labeled *N*-substituted glycine residues in peptomers—oligomers composed of both α-amino acids and *N*-substituted glycine monomers—at basic conditions at room temperature was also analyzed by Bąchor et al. [47]. The developed method covered the deuterium labeling of peptomers at the α-C atom of *N*-substituted glycine residues by using simple HDX. The proposed labeling procedure is easy, inexpensive, and does not require any derivatization reagents or further purification. The introduced deuterons do not undergo a back-exchange under neutral and acidic conditions during LC-MS separation. The LC-MS analysis of an isotopologue mixture showed their co-elution. Therefore the developed strategy may be applied in the quantitative isotope dilution analysis of peptoids and other derivatives of *N*-substituted gly-

Very recently we developed a method of deuterium-labeled standard preparation of creatinine, a breakdown product of creatine phosphate in muscle and a molecular biomarker of renal function [48]. The *N*-methylated glycine moiety was also presented within the creatinine molecule. The performed investigation allowed the doubly deuterated Cre analogue to be obtained, even after 60 min incubation in 1% TEA/D2O solution at room temperature (Figure 10). We found that the introduced deuterons were stable under acidic and neutral conditions and any back exchange was observed. The obtained

purification [46].

cines.

cines.

and acidic conditions. Additionally, the deuterated and nondeuterated derivatives revealed co-elution, which make their application for the quantitative analysis by using isotope dilution strategy possible. The developed strategy of the CsA deuteration is rapid, cost-efficient and does not require special reaction conditions, other reagents or further purification [46]. revealed co-elution, which make their application for the quantitative analysis by using isotope dilution strategy possible. The developed strategy of the CsA deuteration is rapid, cost-efficient and does not require special reaction conditions, other reagents or further purification [46].

**Figure 8.** Schematic presentation of H/D exchange reaction at the α carbon in *N*-methylglycine

Our investigation on compounds containing sarcosine residue revealed its presence in cyclosporin A molecule. The analysis of the possibility of the α-C deuteration of cyclosporine A (CsA) in *N*-methylated amino acid residues was performed [46]. The proposed reaction is based on the method previously reported by us [45], proceeds under basic conditions in the presence of TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene) or MTBD (7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene) at pH 13.4 (Figure 9). The obtained results revealed that there is a possibility of three deuteron incorporation, two at the α-C of *N*-methylgly-

residue. The prepared isotopologues were stable (did not undergo back exchange) under neutral and acidic conditions. Additionally, the deuterated and nondeuterated derivatives

*Molecules* **2021**, *26*, x FOR PEER REVIEW 8 of 26

moiety [45]. TEA-*N,N,N*-triethylamine.

**Figure 9.** Preparation of deuterated analogs of cyclosporin A as described by Bąchor et al. [46]. **Figure 9.** Preparation of deuterated analogs of cyclosporin A as described by B ˛achor et al. [46].

The synthesis of the α-C deuterium-labeled *N*-substituted glycine residues in peptomers—oligomers composed of both α-amino acids and *N*-substituted glycine monomers—at basic conditions at room temperature was also analyzed by Bąchor et al. [47]. The developed method covered the deuterium labeling of peptomers at the α-C atom of *N*-substituted glycine residues by using simple HDX. The proposed labeling procedure is easy, inexpensive, and does not require any derivatization reagents or further purification. The introduced deuterons do not undergo a back-exchange under neutral and acidic conditions during LC-MS separation. The LC-MS analysis of an isotopologue mixture The synthesis of the α-C deuterium-labeled *N*-substituted glycine residues in peptomers—oligomers composed of both α-amino acids and *N*-substituted glycine monomers—at basic conditions at room temperature was also analyzed by B ˛achor et al. [47]. The developed method covered the deuterium labeling of peptomers at the α-C atom of *N*substituted glycine residues by using simple HDX. The proposed labeling procedure is easy, inexpensive, and does not require any derivatization reagents or further purification. The introduced deuterons do not undergo a back-exchange under neutral and acidic conditions during LC-MS separation. The LC-MS analysis of an isotopologue mixture showed their co-elution. Therefore the developed strategy may be applied in the quantitative isotope dilution analysis of peptoids and other derivatives of *N*-substituted glycines.

showed their co-elution. Therefore the developed strategy may be applied in the quantitative isotope dilution analysis of peptoids and other derivatives of *N*-substituted gly-Very recently we developed a method of deuterium-labeled standard preparation of creatinine, a breakdown product of creatine phosphate in muscle and a molecular biomarker of renal function [48]. The *N*-methylated glycine moiety was also presented within the creatinine molecule. The performed investigation allowed the doubly deuterated Cre analogue to be obtained, even after 60 min incubation in 1% TEA/D2O solution at room temperature (Figure 10). We found that the introduced deuterons were stable under acidic and neutral conditions and any back exchange was observed. The obtained Very recently we developed a method of deuterium-labeled standard preparation of creatinine, a breakdown product of creatine phosphate in muscle and a molecular biomarker of renal function [48]. The *N*-methylated glycine moiety was also presented within the creatinine molecule. The performed investigation allowed the doubly deuterated Cre analogue to be obtained, even after 60 min incubation in 1% TEA/D2O solution at room temperature (Figure 10). We found that the introduced deuterons were stable under acidic and neutral conditions and any back exchange was observed. The obtained results suggest that the obtained deuterated Cre analogue may serve as a good internal standard for quantitative analysis by ESI-MS by using the isotope dilution method. The proposed methodology is a new, inexpensive and simple way for creatinine quantification. Additionally the performed quantification in the presence of the obtained deuterated Cre standard correlates with the Jaffe test method. *Molecules* **2021**, *26*, x FOR PEER REVIEW 9 of 26 results suggest that the obtained deuterated Cre analogue may serve as a good internal standard for quantitative analysis by ESI-MS by using the isotope dilution method. The proposed methodology is a new, inexpensive and simple way for creatinine quantification. Additionally the performed quantification in the presence of the obtained deuterated Cre standard correlates with the Jaffe test method.

**Figure 10.** H/D exchange at the carbon center of creatinine molecule [48]. **Figure 10.** H/D exchange at the carbon center of creatinine molecule [48].

In 2015, the method of denatonium benzoate (Bitrex) deuteration via HDX of α-carbon hydrogens located in the CH2 group, situated between carbonyl carbon and quaternary nitrogen atom (Figure 11) [49]. The reaction proceeded at room temperature and the In 2015, the method of denatonium benzoate (Bitrex) deuteration via HDX of α-carbon hydrogens located in the CH<sup>2</sup> group, situated between carbonyl carbon and quaternary nitrogen atom (Figure 11) [49]. The reaction proceeded at room temperature and the deuteration

deuteration was completed after 1 h of sample incubation in 1% TEA/D2O mixture. We

neutral conditions. We also found that the isotopologues—deuterated and nondeuterated—denatonium cation co-elute during the chromatographic separation. The applicability of the obtained deuterated denatonium cation as the internal standard for quantitative analysis of Bitrex was confirmed by the LC-MS analysis of various Bitrex-containing household products. The proposed strategy is a new and simple solution for sensitive Bitrex quantification by LC-MS method. We found that the presence of a quaternary nitrogen atom connected with the α-C atom facilitated the H/D exchange. Based on this observation, we focused our attention on the compounds containing quaternary ammonium groups in the form of *N*-substituted glycine derivatives. We reported the influence of the quaternary ammonium group on HDX at the α-C of sarcosine and *N*-methylalanine in peptides [50–52]. The significant acceleration of the HDX in sarcosine residue caused by the presence of a fixed charge tag was found. The effect depended on the distance between the sarcosine residue and the quaternary nitrogen atom. The deuterium atoms introduced at the α-C, did not undergo back-exchange under acidic aqueous solution. The tandem mass spectrometry analysis of the deuterated analogs of quaternary ammonium-tagged oligosarcosine peptides without mobile hydrogen showed the mobilization of the hydro-

**Figure 11.** Preparation of deuterated Bitrex standard for quantitative analysis by mass spectrome-

It was presented previously that the racemization and hydrogen−deuterium exchange at the α-amino carbon atoms in dipeptides may proceed via the reversible diketopiperazine intermediate formation [53,54]. It was also assumed by Rios and co-workers that the exchange of hydrogens into deuterons at the α carbon atom in amino acid residues

is formed [30]. The most important advantage of the acid or base catalyzed HDX is their simplicity of preparation, relatively low costs to perform, and mostly high efficiency. It should be also be pointed out that sometimes the hard reaction conditions, including high

anion where the enolate intermediate

gens localized at α-C atom of sarcosine residue.

occurs via a stepwise mechanism catalyzed by DO-

or low pH values, may lead to the compound decomposition.

try [49].

was completed after 1 h of sample incubation in 1% TEA/D2O mixture. We found that the introduced deuterons did not undergo back exchange under acidic and neutral conditions. We also found that the isotopologues—deuterated and nondeuterated—denatonium cation co-elute during the chromatographic separation. The applicability of the obtained deuterated denatonium cation as the internal standard for quantitative analysis of Bitrex was confirmed by the LC-MS analysis of various Bitrex-containing household products. The proposed strategy is a new and simple solution for sensitive Bitrex quantification by LC-MS method. We found that the presence of a quaternary nitrogen atom connected with the α-C atom facilitated the H/D exchange. Based on this observation, we focused our attention on the compounds containing quaternary ammonium groups in the form of *N*-substituted glycine derivatives. We reported the influence of the quaternary ammonium group on HDX at the α-C of sarcosine and *N*-methylalanine in peptides [50–52]. The significant acceleration of the HDX in sarcosine residue caused by the presence of a fixed charge tag was found. The effect depended on the distance between the sarcosine residue and the quaternary nitrogen atom. The deuterium atoms introduced at the α-C, did not undergo back-exchange under acidic aqueous solution. The tandem mass spectrometry analysis of the deuterated analogs of quaternary ammonium-tagged oligosarcosine peptides without mobile hydrogen showed the mobilization of the hydrogens localized at α-C atom of sarcosine residue. neutral conditions. We also found that the isotopologues—deuterated and nondeuterated—denatonium cation co-elute during the chromatographic separation. The applicability of the obtained deuterated denatonium cation as the internal standard for quantitative analysis of Bitrex was confirmed by the LC-MS analysis of various Bitrex-containing household products. The proposed strategy is a new and simple solution for sensitive Bitrex quantification by LC-MS method. We found that the presence of a quaternary nitrogen atom connected with the α-C atom facilitated the H/D exchange. Based on this observation, we focused our attention on the compounds containing quaternary ammonium groups in the form of *N*-substituted glycine derivatives. We reported the influence of the quaternary ammonium group on HDX at the α-C of sarcosine and *N*-methylalanine in peptides [50–52]. The significant acceleration of the HDX in sarcosine residue caused by the presence of a fixed charge tag was found. The effect depended on the distance between the sarcosine residue and the quaternary nitrogen atom. The deuterium atoms introduced at the α-C, did not undergo back-exchange under acidic aqueous solution. The tandem mass spectrometry analysis of the deuterated analogs of quaternary ammonium-tagged oligosarcosine peptides without mobile hydrogen showed the mobilization of the hydrogens localized at α-C atom of sarcosine residue.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 9 of 26

**Figure 10.** H/D exchange at the carbon center of creatinine molecule [48].

Cre standard correlates with the Jaffe test method.

results suggest that the obtained deuterated Cre analogue may serve as a good internal standard for quantitative analysis by ESI-MS by using the isotope dilution method. The proposed methodology is a new, inexpensive and simple way for creatinine quantification. Additionally the performed quantification in the presence of the obtained deuterated

In 2015, the method of denatonium benzoate (Bitrex) deuteration via HDX of α-carbon hydrogens located in the CH2 group, situated between carbonyl carbon and quaternary nitrogen atom (Figure 11) [49]. The reaction proceeded at room temperature and the deuteration was completed after 1 h of sample incubation in 1% TEA/D2O mixture. We found that the introduced deuterons did not undergo back exchange under acidic and

**Figure 11.** Preparation of deuterated Bitrex standard for quantitative analysis by mass spectrome-**Figure 11.** Preparation of deuterated Bitrex standard for quantitative analysis by mass spectrometry [49].

try [49]. It was presented previously that the racemization and hydrogen−deuterium exchange at the α-amino carbon atoms in dipeptides may proceed via the reversible diketopiperazine intermediate formation [53,54]. It was also assumed by Rios and co-workers that the exchange of hydrogens into deuterons at the α carbon atom in amino acid residues occurs via a stepwise mechanism catalyzed by DO anion where the enolate intermediate is formed [30]. The most important advantage of the acid or base catalyzed HDX is their simplicity of preparation, relatively low costs to perform, and mostly high efficiency. It It was presented previously that the racemization and hydrogen−deuterium exchange at the α-amino carbon atoms in dipeptides may proceed via the reversible diketopiperazine intermediate formation [53,54]. It was also assumed by Rios and co-workers that the exchange of hydrogens into deuterons at the α carbon atom in amino acid residues occurs via a stepwise mechanism catalyzed by DO- anion where the enolate intermediate is formed [30]. The most important advantage of the acid or base catalyzed HDX is their simplicity of preparation, relatively low costs to perform, and mostly high efficiency. It should be also be pointed out that sometimes the hard reaction conditions, including high or low pH values, may lead to the compound decomposition.

#### should be also be pointed out that sometimes the hard reaction conditions, including high *2.2. Metal-Catalyzed HDX Adjacent to Oxygen Atom*

or low pH values, may lead to the compound decomposition. The first example of H/D exchange adjacent to oxygen was discovered in 1974 by Regen. In this study, the deuteration of 1-butanol at the α carbon atom was performed in the presence of a catalyst in the form of (tris(triphenylphosphine) ruthenium dichloride [RuCl2(PPh3)3] [55]. In the proposed method 1-butanol was incubated at 200 ◦C for one hour in the presence of a catalyst, which allowed the exchange of hydrogen at the α carbon position by deuterium bonded to the oxygen atom (Figure 12). The proposed reaction conditions were also applied to other deuterated alcohols at the α-C atom. The obtained results also revealed that the addition of D2O to the reaction mixture significantly increased the deuteration efficiency and that the degree of deuteration depended on the D2O/alcohol ratio.

ratio.

ratio.

**Figure 12.** H/D exchange adjacent to oxygen atom, presented by Regen [55]. Ishibashi et al. presented the efficient deuteration of compounds containing electron donors in the form of double bonds, hydroxyl groups, in the presence of ruthenium cata-

*2.2. Metal-Catalyzed HDX Adjacent to Oxygen Atom* 

*2.2. Metal-Catalyzed HDX Adjacent to Oxygen Atom* 

*Molecules* **2021**, *26*, x FOR PEER REVIEW 10 of 26

The first example of H/D exchange adjacent to oxygen was discovered in 1974 by

Regen. In this study, the deuteration of 1-butanol at the α carbon atom was performed in the presence of a catalyst in the form of (tris(triphenylphosphine) ruthenium dichloride [RuCl2(PPh3)3] [55]. In the proposed method 1-butanol was incubated at 200 °C for one hour in the presence of a catalyst, which allowed the exchange of hydrogen at the α carbon position by deuterium bonded to the oxygen atom (Figure 12). The proposed reaction conditions were also applied to other deuterated alcohols at the α-C atom. The obtained results also revealed that the addition of D2O to the reaction mixture significantly increased the deuteration efficiency and that the degree of deuteration depended on the D2O/alcohol

The first example of H/D exchange adjacent to oxygen was discovered in 1974 by Regen. In this study, the deuteration of 1-butanol at the α carbon atom was performed in the presence of a catalyst in the form of (tris(triphenylphosphine) ruthenium dichloride [RuCl2(PPh3)3] [55]. In the proposed method 1-butanol was incubated at 200 °C for one hour in the presence of a catalyst, which allowed the exchange of hydrogen at the α carbon position by deuterium bonded to the oxygen atom (Figure 12). The proposed reaction conditions were also applied to other deuterated alcohols at the α-C atom. The obtained results also revealed that the addition of D2O to the reaction mixture significantly increased the deuteration efficiency and that the degree of deuteration depended on the D2O/alcohol

**Figure 12.** H/D exchange adjacent to oxygen atom, presented by Regen [55]. Ishibashi et al. presented the efficient deuteration of compounds containing electron donors in the form of double bonds, hydroxyl groups, in the presence of ruthenium catalyst. It was found that alkenols were efficiently deuterated in D2O by the migration of the double bond and isomerization to ketones in the presence of ruthenium catalyst (Figure 13) [56]. It was found that primary alcohols were oxidated to aldehydes on the selective way in the presence of RuCl2(PPh3)3 catalyst which was hampered in the presence of small amounts of water. The reaction temperature around 150 °C in microwave synthesizer, al-Ishibashi et al. presented the efficient deuteration of compounds containing electron donors in the form of double bonds, hydroxyl groups, in the presence of ruthenium catalyst. It was found that alkenols were efficiently deuterated in D2O by the migration of the double bond and isomerization to ketones in the presence of ruthenium catalyst (Figure 13) [56]. It was found that primary alcohols were oxidated to aldehydes on the selective way in the presence of RuCl2(PPh3)<sup>3</sup> catalyst which was hampered in the presence of small amounts of water. The reaction temperature around 150 ◦C in microwave synthesizer, allowed an efficient HDX at the α carbon atom of the primary alcohols with small epimerization observed in the case of chiral compounds. Under lower temperatures around 100 ◦C the epimerization was sufficiently suppressed [57]. lyst. It was found that alkenols were efficiently deuterated in D2O by the migration of the double bond and isomerization to ketones in the presence of ruthenium catalyst (Figure 13) [56]. It was found that primary alcohols were oxidated to aldehydes on the selective way in the presence of RuCl2(PPh3)3 catalyst which was hampered in the presence of small amounts of water. The reaction temperature around 150 °C in microwave synthesizer, allowed an efficient HDX at the α carbon atom of the primary alcohols with small epimerization observed in the case of chiral compounds. Under lower temperatures around 100 °C the epimerization was sufficiently suppressed [57].

**Figure 13.** Ruthenium-mediated deuteration according to Ishibashi et al. [56].

**Figure 13.** Ruthenium-mediated deuteration according to Ishibashi et al. [56].

**Figure 13.** Ruthenium-mediated deuteration according to Ishibashi et al. [56]. In 2011, Bossi and co-authors presented a method for selective deuteration of alcohols In 2011, Bossi and co-authors presented a method for selective deuteration of alcohols at the α position in the presence of ruthenium and osmium pincer catalysts [58]. In the presence of isopropanol-d8 as a source of deuterium, Bossi was able to obtain high deuterium incorporation at the C1 carbon atom of primary and secondary alcohols, and within the case of secondary alcohols also the deuteration at the C2 position was observed. In 2013, Khaskin and Milstein proposed another ruthenium pincer catalyst allowing deu-In 2011, Bossi and co-authors presented a method for selective deuteration of alcohols at the α position in the presence of ruthenium and osmium pincer catalysts [58]. In the presence of isopropanol-d8 as a source of deuterium, Bossi was able to obtain high deuterium incorporation at the C1 carbon atom of primary and secondary alcohols, and within the case of secondary alcohols also the deuteration at the C2 position was observed. In 2013, Khaskin and Milstein proposed another ruthenium pincer catalyst allowing deuteration at the carbon centers in the presence of D2O and with a lower catalyst loading [59]. Other substrates, including secondary alcohols also presented the possibility for β deuteration, which was frequent, with deuterium incorporation up to 97%.

at the α position in the presence of ruthenium and osmium pincer catalysts [58]. In the presence of isopropanol-d8 as a source of deuterium, Bossi was able to obtain high deuterium incorporation at the C1 carbon atom of primary and secondary alcohols, and within the case of secondary alcohols also the deuteration at the C2 position was observed. In 2013, Khaskin and Milstein proposed another ruthenium pincer catalyst allowing deuteration at the carbon centers in the presence of D2O and with a lower catalyst loading [59]. Other substrates, including secondary alcohols also presented the possibility for β teration at the carbon centers in the presence of D2O and with a lower catalyst loading [59]. Other substrates, including secondary alcohols also presented the possibility for β deuteration, which was frequent, with deuterium incorporation up to 97%. In 2015, Bai et al. [60] proposed a selective α and β deuteration method of alcohols in the presence of D2O as a deuterating agent. The reaction optimization process revealed that octahedral ruthenium complexes with the amine ligand presented higher activity in HDX promotion (Figure 14). It was also found that the (η6-cymene) ruthenium complex In 2015, Bai et al. [60] proposed a selective α and β deuteration method of alcohols in the presence of D2O as a deuterating agent. The reaction optimization process revealed that octahedral ruthenium complexes with the amine ligand presented higher activity in HDX promotion (Figure 14). It was also found that the (η6-cymene) ruthenium complex allowed deuteration only at the β position. The mechanism of this process involves oxidation of the alcohol to an aldehyde followed by base-mediated β deuteration. The formed aldehyde is then reduced to an alcohol by means of the deuterated ruthenium complex which results in the formation of α,β-deuterated derivative.

deuteration, which was frequent, with deuterium incorporation up to 97%.

In 2015, Bai et al. [60] proposed a selective α and β deuteration method of alcohols in

the presence of D2O as a deuterating agent. The reaction optimization process revealed that octahedral ruthenium complexes with the amine ligand presented higher activity in HDX promotion (Figure 14). It was also found that the (η6-cymene) ruthenium complex

which results in the formation of α,β-deuterated derivative.

which results in the formation of α,β-deuterated derivative.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 11 of 26

**Figure 14.** Khaskin's work on alcohol deuteration catalyzed by ruthenium pincer catalyst [59]. **Figure 14.** Khaskin's work on alcohol deuteration catalyzed by ruthenium pincer catalyst [59].

In the same year, Chatterje et al. presented the method of selective α and α,β deuter-

allowed deuteration only at the β position. The mechanism of this process involves oxidation of the alcohol to an aldehyde followed by base-mediated β deuteration. The formed aldehyde is then reduced to an alcohol by means of the deuterated ruthenium complex

allowed deuteration only at the β position. The mechanism of this process involves oxidation of the alcohol to an aldehyde followed by base-mediated β deuteration. The formed aldehyde is then reduced to an alcohol by means of the deuterated ruthenium complex

In the same year, Chatterje et al. presented the method of selective α and α,β deuteration of alcohols in the presence of a low-loading and commercially available ruthenium pincer catalyst (Ru-MACHO), the base in the form of potassium *tert*-butoxide and D2O as the deuterating agent (Figure 15) [61]. The proposed reaction condition optimization on aromatic benzylic alcohols revealed that mild heating (60–100 °C) was sufficient to obtain 95% of deuterium incorporation. It was also presented, that in this method linear primary alcohols were also able to undergo β deuteration at 10–20%, while in the case of secondary alcohols, deuteration at the β carbon was higher. The mechanism of this reaction involves the oxidation and reduction of the alcohol. Based on this, the authors proposed that the intermediate, in the form of ketones, was a more long-living species than the aldehydes and therefore, β deuteration proceeded at a higher rate in the case of secondary alcohols. In the same year, Chatterje et al. presented the method of selective α and α,β deuteration of alcohols in the presence of a low-loading and commercially available ruthenium pincer catalyst (Ru-MACHO), the base in the form of potassium *tert*-butoxide and D2O as the deuterating agent (Figure 15) [61]. The proposed reaction condition optimization on aromatic benzylic alcohols revealed that mild heating (60–100 ◦C) was sufficient to obtain 95% of deuterium incorporation. It was also presented, that in this method linear primary alcohols were also able to undergo β deuteration at 10–20%, while in the case of secondary alcohols, deuteration at the β carbon was higher. The mechanism of this reaction involves the oxidation and reduction of the alcohol. Based on this, the authors proposed that the intermediate, in the form of ketones, was a more long-living species than the aldehydes and therefore, β deuteration proceeded at a higher rate in the case of secondary alcohols. What is interesting, in the case of diols, only the deuteration at the α position was observed. ation of alcohols in the presence of a low-loading and commercially available ruthenium pincer catalyst (Ru-MACHO), the base in the form of potassium *tert*-butoxide and D2O as the deuterating agent (Figure 15) [61]. The proposed reaction condition optimization on aromatic benzylic alcohols revealed that mild heating (60–100 °C) was sufficient to obtain 95% of deuterium incorporation. It was also presented, that in this method linear primary alcohols were also able to undergo β deuteration at 10–20%, while in the case of secondary alcohols, deuteration at the β carbon was higher. The mechanism of this reaction involves the oxidation and reduction of the alcohol. Based on this, the authors proposed that the intermediate, in the form of ketones, was a more long-living species than the aldehydes and therefore, β deuteration proceeded at a higher rate in the case of secondary alcohols. What is interesting, in the case of diols, only the deuteration at the α position was observed.

**Figure 15.** H/D exchange catalyzed by Ru-MACHO catalyst [61]. **Figure 15.** H/D exchange catalyzed by Ru-MACHO catalyst [61].

**Figure 15.** H/D exchange catalyzed by Ru-MACHO catalyst [61]. Similarly to Khaskin [59], in 2016 Gauvin and co-workers focused their attention on the application of ruthenium pincer complexes in the transformation of alcohols to carboxylic acids. They performed the reaction in a closed vessel to displace the reaction equilibrium toward the substrate (alcohol), thus allowing deuteration in the presence of D2O [62]. They also decided to optimize the catalyst by switching from the Ru-MACHO to its analog containing cyclohexyl substituents, resulting in the higher activity and selectivity in α carbon deuteration.

Using a similar strategy to Regen's method, Koch and Stuart found that there was a possibility for primary and secondary alcohol deuteration at the α carbon by refluxing the alcohol in the presence of D2O and Raney nickel as the catalyst [63]. The proposed strategy was successfully applied in the preparation of deuterated nonreducing carbohydrates. Additionally, it was proposed that the alcohol should undergo a redox process in the presence of Raney nickel as hydrogen-transfer catalyst. The observed retention of the configuration was explained by the polyhydric cyclic structure of the carbohydrate [64]. The isomerization of methyl α-D-mannopyranoside and methyl β-D-galactopyranoside to corresponding D-gluco isomers was also found after several days of reaction. Using a similar strategy to Regen's method, Koch and Stuart found that there was a possibility for primary and secondary alcohol deuteration at the α carbon by refluxing the alcohol in the presence of D2O and Raney nickel as the catalyst [63]. The proposed strategy was successfully applied in the preparation of deuterated nonreducing carbohydrates. Additionally, it was proposed that the alcohol should undergo a redox process in the presence of Raney nickel as hydrogen-transfer catalyst. The observed retention of the configuration was explained by the polyhydric cyclic structure of the carbohydrate [64]. The isomerization of methyl α-D-mannopyranoside and methyl β-D-galactopyranoside to corresponding D-gluco isomers was also found after several days of reaction. possibility for primary and secondary alcohol deuteration at the α carbon by refluxing the alcohol in the presence of D2O and Raney nickel as the catalyst [63]. The proposed strategy was successfully applied in the preparation of deuterated nonreducing carbohydrates. Additionally, it was proposed that the alcohol should undergo a redox process in the presence of Raney nickel as hydrogen-transfer catalyst. The observed retention of the configuration was explained by the polyhydric cyclic structure of the carbohydrate [64]. The isomerization of methyl α-D-mannopyranoside and methyl β-D-galactopyranoside to corresponding D-gluco isomers was also found after several days of reaction. As described by Cioffi and co-workers, the activation of Raney nickel by sonication

Using a similar strategy to Regen's method, Koch and Stuart found that there was a

Similarly to Khaskin [59], in 2016 Gauvin and co-workers focused their attention on the application of ruthenium pincer complexes in the transformation of alcohols to carboxylic acids. They performed the reaction in a closed vessel to displace the reaction equilibrium toward the substrate (alcohol), thus allowing deuteration in the presence of D2O [62]. They also decided to optimize the catalyst by switching from the Ru-MACHO to its analog containing cyclohexyl substituents, resulting in the higher activity and selectivity

Similarly to Khaskin [59], in 2016 Gauvin and co-workers focused their attention on the application of ruthenium pincer complexes in the transformation of alcohols to carboxylic acids. They performed the reaction in a closed vessel to displace the reaction equilibrium toward the substrate (alcohol), thus allowing deuteration in the presence of D2O [62]. They also decided to optimize the catalyst by switching from the Ru-MACHO to its analog containing cyclohexyl substituents, resulting in the higher activity and selectivity

As described by Cioffi and co-workers, the activation of Raney nickel by sonication allowed a microwave-assisted deuteration of nonreducing carbohydrates without racemization [65]. The 1-O-methyl-β-d-galactopyranoside, used as a model compound, was heated in 15-s intervals up to 36 times, using a microwave oven. It was found that the deuterium was incorporated without epimerization or compound decomposition. Further optimization of the Stuart's method may be achieved by application of sonication which makes a higher level of deuterium incorporation possible (Figure 16) [66]. Microwave irradiation may also improve the deuteration however in this case the epimerization and substrate degradation was observed [65]. In the proposed solution deuteration was regioselective and occurred at the C2, C3 and C4 positions at the carbon atoms connected with hydroxyl group which was necessary for the HDX [67]. As described by Cioffi and co-workers, the activation of Raney nickel by sonication allowed a microwave-assisted deuteration of nonreducing carbohydrates without racemization [65]. The 1-O-methyl-β-d-galactopyranoside, used as a model compound, was heated in 15-second intervals up to 36 times, using a microwave oven. It was found that the deuterium was incorporated without epimerization or compound decomposition. Further optimization of the Stuart's method may be achieved by application of sonication which makes a higher level of deuterium incorporation possible (Figure 16) [66]. Microwave irradiation may also improve the deuteration however in this case the epimerization and substrate degradation was observed [65]. In the proposed solution deuteration was regioselective and occurred at the C2, C3 and C4 positions at the carbon atoms connected with hydroxyl group which was necessary for the HDX [67]. allowed a microwave-assisted deuteration of nonreducing carbohydrates without racemization [65]. The 1-O-methyl-β-d-galactopyranoside, used as a model compound, was heated in 15-second intervals up to 36 times, using a microwave oven. It was found that the deuterium was incorporated without epimerization or compound decomposition. Further optimization of the Stuart's method may be achieved by application of sonication which makes a higher level of deuterium incorporation possible (Figure 16) [66]. Microwave irradiation may also improve the deuteration however in this case the epimerization and substrate degradation was observed [65]. In the proposed solution deuteration was regioselective and occurred at the C2, C3 and C4 positions at the carbon atoms connected with hydroxyl group which was necessary for the HDX [67].

*Molecules* **2021**, *26*, x FOR PEER REVIEW 12 of 26

*Molecules* **2021**, *26*, x FOR PEER REVIEW 12 of 26

in α carbon deuteration.

in α carbon deuteration.

**Figure 16.** Methyl α-D-glucopyranoside deuteration catalyzed by Raney nickel under sonication as presented by Cioffi and co-workers [66]. **Figure 16.** Methyl α-D-glucopyranoside deuteration catalyzed by Raney nickel under sonication as presented by Cioffi and co-workers [66]. **Figure 16.** Methyl α-D-glucopyranoside deuteration catalyzed by Raney nickel under sonication as presented by Cioffi and co-workers [66].

Vert and co-workers applied the HSCIE (high-temperature solid-state catalytic isotope exchange) technique to obtain the selectively deuterated lactide [68] and glycolide derivatives [69], which were applied as substrates for the synthesis of isotopically labeled biocompatible absorbable poly-α-hydroxy acids. It was found that the optimal reaction temperature was close to the melting point of the substrate. The hydrogen−deuterium exchange of L-lactide at 120 °C in the presence of the Pd/CaCO3 catalyst resulted in incomplete deuterium incorporation (Figure 17), but occurred without epimerization. Additionally, the reaction conditions were suitable for the tritium incorporation [70]. Vert and co-workers applied the HSCIE (high-temperature solid-state catalytic isotope exchange) technique to obtain the selectively deuterated lactide [68] and glycolide derivatives [69], which were applied as substrates for the synthesis of isotopically labeled biocompatible absorbable poly-α-hydroxy acids. It was found that the optimal reaction temperature was close to the melting point of the substrate. The hydrogen−deuterium exchange of L-lactide at 120 ◦C in the presence of the Pd/CaCO<sup>3</sup> catalyst resulted in incomplete deuterium incorporation (Figure 17), but occurred without epimerization. Additionally, the reaction conditions were suitable for the tritium incorporation [70]. Vert and co-workers applied the HSCIE (high-temperature solid-state catalytic isotope exchange) technique to obtain the selectively deuterated lactide [68] and glycolide derivatives [69], which were applied as substrates for the synthesis of isotopically labeled biocompatible absorbable poly-α-hydroxy acids. It was found that the optimal reaction temperature was close to the melting point of the substrate. The hydrogen−deuterium exchange of L-lactide at 120 °C in the presence of the Pd/CaCO3 catalyst resulted in incomplete deuterium incorporation (Figure 17), but occurred without epimerization. Additionally, the reaction conditions were suitable for the tritium incorporation [70].

**Figure 17.** HSCIE method of selective H/D exchange at the carbon centers [68].

**Figure 17.** HSCIE method of selective H/D exchange at the carbon centers [68]. **Figure 17.** HSCIE method of selective H/D exchange at the carbon centers [68]. In 1990, Möbius and Schaaf [71] developed a method for the preparation of deuterated aliphatic hydrocarbons by metal-catalyzed HDX at higher temperatures (up to 290 ◦C). The reaction was performed in an autoclave, where a wire basket with the catalyst was placed above the substrate to be deuterated. The D2/D2O atmosphere was used to obtained deuterated derivatives under the pressure of around 25 Mpa. Under these conditions, water dissociates much more rapidly than at room temperature [72] and therefore Pd<sup>0</sup> is able to insert oxidatively into the H-OH bond with the formation of a Pd(II) derivative [73].

Based on the method developed by Matsubara and co-workers [74], complete deuteration of aromatic or aliphatic hydrocarbons was achieved by decarboxylation of carboxylic

ative [73].

ative [73].

acids under hydrothermal reaction conditions. In this method, the model lactone molecule in D2O allowed the formation of the phenol derivative with a high yield in the presence of 10% Pd/C (5 mol %) at 250 ◦C and a pressure of 4–5 Mpa (Figure 18). boxylic acids under hydrothermal reaction conditions. In this method, the model lactone molecule in D2O allowed the formation of the phenol derivative with a high yield in the presence of 10% Pd/C (5 mol%) at 250 °C and a pressure of 4–5 Mpa (Figure 18). presence of 10% Pd/C (5 mol%) at 250 °C and a pressure of 4–5 Mpa (Figure 18).

In 1990, Möbius and Schaaf [71] developed a method for the preparation of deuterated aliphatic hydrocarbons by metal-catalyzed HDX at higher temperatures (up to 290 °C). The reaction was performed in an autoclave, where a wire basket with the catalyst was placed above the substrate to be deuterated. The D2/D2O atmosphere was used to obtained deuterated derivatives under the pressure of around 25 Mpa. Under these conditions, water dissociates much more rapidly than at room temperature [72] and therefore Pd0 is able to insert oxidatively into the H-OH bond with the formation of a Pd(II) deriv-

In 1990, Möbius and Schaaf [71] developed a method for the preparation of deuterated aliphatic hydrocarbons by metal-catalyzed HDX at higher temperatures (up to 290 °C). The reaction was performed in an autoclave, where a wire basket with the catalyst was placed above the substrate to be deuterated. The D2/D2O atmosphere was used to obtained deuterated derivatives under the pressure of around 25 Mpa. Under these conditions, water dissociates much more rapidly than at room temperature [72] and therefore Pd0 is able to insert oxidatively into the H-OH bond with the formation of a Pd(II) deriv-

Based on the method developed by Matsubara and co-workers [74], complete deuteration of aromatic or aliphatic hydrocarbons was achieved by decarboxylation of car-

Based on the method developed by Matsubara and co-workers [74], complete deuteration of aromatic or aliphatic hydrocarbons was achieved by decarboxylation of carboxylic acids under hydrothermal reaction conditions. In this method, the model lactone molecule in D2O allowed the formation of the phenol derivative with a high yield in the

**Figure 18.** Lactone deuteration as presented by Matsubara et al. [74]. **Figure 18.** Lactone deuteration as presented by Matsubara et al. [74]. Previous studies by Sajiki and co-workers [75] on 5-phenylvaleric acid revealed the

*Molecules* **2021**, *26*, x FOR PEER REVIEW 13 of 26

*Molecules* **2021**, *26*, x FOR PEER REVIEW 13 of 26

Previous studies by Sajiki and co-workers [75] on 5-phenylvaleric acid revealed the influence of reaction temperatures on the regioselectivity and deuteration. It was found that the benzylic hydrogen atoms were selectively exchanged to deuterons at room temperature, whereas at the higher temperature (160 °C), the deuteration in less reactive positions was also found, resulting in the formation of multideuterated derivatives. The proposed reaction conditions were compatible with compounds containing different functional groups in the form of carboxy, keto or hydroxyl groups, but the described reaction was characteristic for those with aryl-linked side chains. The proposed Pd/C–H2/D2O system may be also applied for the preparative formation of the phenylalanine selectively deuterated at the β carbon atom which takes place at 110 °C (6 h, 96% D) without racemization [76]. It was also found that at 160 °C the α position is also able for HDX, but these Previous studies by Sajiki and co-workers [75] on 5-phenylvaleric acid revealed the influence of reaction temperatures on the regioselectivity and deuteration. It was found that the benzylic hydrogen atoms were selectively exchanged to deuterons at room temperature, whereas at the higher temperature (160 ◦C), the deuteration in less reactive positions was also found, resulting in the formation of multideuterated derivatives. The proposed reaction conditions were compatible with compounds containing different functional groups in the form of carboxy, keto or hydroxyl groups, but the described reaction was characteristic for those with aryl-linked side chains. The proposed Pd/C–H2/D2O system may be also applied for the preparative formation of the phenylalanine selectively deuterated at the β carbon atom which takes place at 110 ◦C (6 h, 96% D) without racemization [76]. It was also found that at 160 ◦C the α position is also able for HDX, but these reaction conditions promote racemization (17% ee). influence of reaction temperatures on the regioselectivity and deuteration. It was found that the benzylic hydrogen atoms were selectively exchanged to deuterons at room temperature, whereas at the higher temperature (160 °C), the deuteration in less reactive positions was also found, resulting in the formation of multideuterated derivatives. The proposed reaction conditions were compatible with compounds containing different functional groups in the form of carboxy, keto or hydroxyl groups, but the described reaction was characteristic for those with aryl-linked side chains. The proposed Pd/C–H2/D2O system may be also applied for the preparative formation of the phenylalanine selectively deuterated at the β carbon atom which takes place at 110 °C (6 h, 96% D) without racemization [76]. It was also found that at 160 °C the α position is also able for HDX, but these reaction conditions promote racemization (17% ee). In 2005, Proszenyák et al. [77] developed a method allowing higher efficiency of HDX

reaction conditions promote racemization (17% ee). In 2005, Proszenyák et al. [77] developed a method allowing higher efficiency of HDX for the benzylic hydrogen atoms of the piperidine derivative in the presence of Pd/C–H2– D2O, deuterated alcohols and DCl. Earlier, in 1986, Stock and Ofosu-Asante [78] presented a method for selective benzylic deuteration of the tetrahydronaphthalene carboxylic acid in the presence of a Pd/C catalyst under D2 atmosphere and deuterated acetic acid as the In 2005, Proszenyák et al. [77] developed a method allowing higher efficiency of HDX for the benzylic hydrogen atoms of the piperidine derivative in the presence of Pd/C–H2– D2O, deuterated alcohols and DCl. Earlier, in 1986, Stock and Ofosu-Asante [78] presented a method for selective benzylic deuteration of the tetrahydronaphthalene carboxylic acid in the presence of a Pd/C catalyst under D<sup>2</sup> atmosphere and deuterated acetic acid as the deuterating agent (Figure 19). for the benzylic hydrogen atoms of the piperidine derivative in the presence of Pd/C–H2– D2O, deuterated alcohols and DCl. Earlier, in 1986, Stock and Ofosu-Asante [78] presented a method for selective benzylic deuteration of the tetrahydronaphthalene carboxylic acid in the presence of a Pd/C catalyst under D2 atmosphere and deuterated acetic acid as the deuterating agent (Figure 19).

**Figure 19.** HDX reaction at the carbon center of piperidine derivative as presented by Proszenyák **Figure 19.** HDX reaction at the carbon center of piperidine derivative as presented by Proszenyák et al. [77]. In 2005, Sajiki and co-workers [79] revealed that platinum catalysts present a higher

**Figure 19.** HDX reaction at the carbon center of piperidine derivative as presented by Proszenyák et al. [77]. et al. [77]. In 2005, Sajiki and co-workers [79] revealed that platinum catalysts present a higher tendency towards the deuteration of aromatic positions, whereas palladium catalysts prefer mostly aliphatic ones (Figure 20). Using this method, the efficient deuteration of phenol was obtained in the presence of 5% Pt/C at room temperature, whereas the palladium-catalyzed reaction needed a higher temperature (180 ◦C) to obtain the same level of deuteration. tendency towards the deuteration of aromatic positions, whereas palladium catalysts prefer mostly aliphatic ones (Figure 20). Using this method, the efficient deuteration of phenol was obtained in the presence of 5% Pt/C at room temperature, whereas the palladiumcatalyzed reaction needed a higher temperature (180 °C) to obtain the same level of deuteration.

**Figure 20.** HDX at the carbon center presented by Sajiki et al. [79]. **Figure 20.** HDX at the carbon center presented by Sajiki et al. [79].

Palladium and platinum catalysts may also be applied in a mixed catalyst system for the preparation of deuteration derivatives on the sterically hindered aromatic positions. Palladium and platinum catalysts may also be applied in a mixed catalyst system for the preparation of deuteration derivatives on the sterically hindered aromatic positions. It was found that the deuterium incorporation at the *ortho* position in 5-phenylvaleric acid

It was found that the deuterium incorporation at the *ortho* position in 5-phenylvaleric acid in the presence of palladium (10% Pd/C) was only 14%, with platinum (5% Pt/C) 19%. In

(97% D) of the *ortho* position. Additionally, the synergistic effect of palladium and platinum complexes in stepwise deuteration was postulated as a useful tool in the case of a

HDX at the carbon center of alcohol molecules may also be catalyzed by molybdocenes. In this case, the reaction mechanism involves C–H bond activation by the metal catalyst. Deuteration may occur in different ranges, depending on the chemical structure of the alcohol used and can reach up to 99% in the case of benzylic hydrogens [81].

Most of the presented metal-catalyzed deuteration methods involve the application of second-row transition metals. In 2018, Prakash and co-workers developed a method of the regioselective deuteration of primary and secondary alcohols in the presence of firstrow transition-metal catalysts [82]. In 2007 Hamid et al. presented the 'borrowing hydrogen' method of deuteration [83] which allows deuteration at the α and β carbon atoms of alcohols and amines by using a hydrogen-transfer catalyst. It was reported that manganese and iron pincer catalysts increase the deuterium incorporation in primary and secondary alcohols especially in the presence of a base. The presented mechanism of HDX involves the amido-complex formation with the used base which is a key factor for catalytic cycle initiation. The formed aldehyde is subsequently reduced by the deuterated metal complex formed by HDX on the catalyst mediated by D2O. This method was successfully applied in the deuteration on the α and β carbons of primary and secondary

As presented by Bergman et al., the cationic iridium complexes may also activate C-H bonds [84], and therefore iridium-mediated HDX represents the largest number of published examples in the field of homogeneous metal catalysis. The most exploited area is the ortho-deuteration of aryl ketones and acetanilides. Starting with the investigations of the Heys [85] and Hesk [86] research groups, several different studies related to the effects of complex ligands [87], the deuterating agent [88], solvent [88–90], addition of bases [85] the amount of catalyst [36], the temperature, and the duration of the reaction [85] on the

degree of deuteration and the substitution pattern in the substrate (Figure 21).

low degree of deuterium incorporation. [80].

alcohols, as well as diols.

teration.

in the presence of palladium (10% Pd/C) was only 14%, with platinum (5% Pt/C) 19%. In a mixed catalyst, the same reaction was characterized by almost complete deuteration (97% D) of the *ortho* position. Additionally, the synergistic effect of palladium and platinum complexes in stepwise deuteration was postulated as a useful tool in the case of a low degree of deuterium incorporation. [80]. in the presence of palladium (10% Pd/C) was only 14%, with platinum (5% Pt/C) 19%. In a mixed catalyst, the same reaction was characterized by almost complete deuteration (97% D) of the *ortho* position. Additionally, the synergistic effect of palladium and platinum complexes in stepwise deuteration was postulated as a useful tool in the case of a low degree of deuterium incorporation. [80].

Palladium and platinum catalysts may also be applied in a mixed catalyst system for the preparation of deuteration derivatives on the sterically hindered aromatic positions. It was found that the deuterium incorporation at the *ortho* position in 5-phenylvaleric acid

In 2005, Sajiki and co-workers [79] revealed that platinum catalysts present a higher tendency towards the deuteration of aromatic positions, whereas palladium catalysts prefer mostly aliphatic ones (Figure 20). Using this method, the efficient deuteration of phenol was obtained in the presence of 5% Pt/C at room temperature, whereas the palladiumcatalyzed reaction needed a higher temperature (180 °C) to obtain the same level of deu-

*Molecules* **2021**, *26*, x FOR PEER REVIEW 14 of 26

**Figure 20.** HDX at the carbon center presented by Sajiki et al. [79].

HDX at the carbon center of alcohol molecules may also be catalyzed by molybdocenes. In this case, the reaction mechanism involves C–H bond activation by the metal catalyst. Deuteration may occur in different ranges, depending on the chemical structure of the alcohol used and can reach up to 99% in the case of benzylic hydrogens [81]. HDX at the carbon center of alcohol molecules may also be catalyzed by molybdocenes. In this case, the reaction mechanism involves C–H bond activation by the metal catalyst. Deuteration may occur in different ranges, depending on the chemical structure of the alcohol used and can reach up to 99% in the case of benzylic hydrogens [81].

Most of the presented metal-catalyzed deuteration methods involve the application of second-row transition metals. In 2018, Prakash and co-workers developed a method of the regioselective deuteration of primary and secondary alcohols in the presence of first-row transition-metal catalysts [82]. In 2007 Hamid et al. presented the 'borrowing hydrogen' method of deuteration [83] which allows deuteration at the α and β carbon atoms of alcohols and amines by using a hydrogen-transfer catalyst. It was reported that manganese and iron pincer catalysts increase the deuterium incorporation in primary and secondary alcohols especially in the presence of a base. The presented mechanism of HDX involves the amido-complex formation with the used base which is a key factor for catalytic cycle initiation. The formed aldehyde is subsequently reduced by the deuterated metal complex formed by HDX on the catalyst mediated by D2O. This method was successfully applied in the deuteration on the α and β carbons of primary and secondary alcohols, as well as diols. Most of the presented metal-catalyzed deuteration methods involve the application of second-row transition metals. In 2018, Prakash and co-workers developed a method of the regioselective deuteration of primary and secondary alcohols in the presence of firstrow transition-metal catalysts [82]. In 2007 Hamid et al. presented the 'borrowing hydrogen' method of deuteration [83] which allows deuteration at the α and β carbon atoms of alcohols and amines by using a hydrogen-transfer catalyst. It was reported that manganese and iron pincer catalysts increase the deuterium incorporation in primary and secondary alcohols especially in the presence of a base. The presented mechanism of HDX involves the amido-complex formation with the used base which is a key factor for catalytic cycle initiation. The formed aldehyde is subsequently reduced by the deuterated metal complex formed by HDX on the catalyst mediated by D2O. This method was successfully applied in the deuteration on the α and β carbons of primary and secondary alcohols, as well as diols.

As presented by Bergman et al., the cationic iridium complexes may also activate C-H bonds [84], and therefore iridium-mediated HDX represents the largest number of published examples in the field of homogeneous metal catalysis. The most exploited area is the ortho-deuteration of aryl ketones and acetanilides. Starting with the investigations of the Heys [85] and Hesk [86] research groups, several different studies related to the effects of complex ligands [87], the deuterating agent [88], solvent [88–90], addition of bases [85] the amount of catalyst [36], the temperature, and the duration of the reaction [85] on the degree of deuteration and the substitution pattern in the substrate (Figure 21). As presented by Bergman et al., the cationic iridium complexes may also activate C-H bonds [84], and therefore iridium-mediated HDX represents the largest number of published examples in the field of homogeneous metal catalysis. The most exploited area is the ortho-deuteration of aryl ketones and acetanilides. Starting with the investigations of the Heys [85] and Hesk [86] research groups, several different studies related to the effects of complex ligands [87], the deuterating agent [88], solvent [88–90], addition of bases [85] the amount of catalyst [36], the temperature, and the duration of the reaction [85] on the degree of deuteration and the substitution pattern in the substrate (Figure 21).

**Figure 21.** H/D exchange catalyzed by iridium catalyst as shown by Heys [85].

Further, Kröger et al. presented that the unsaturated carbonyl compounds were also suitable substrates for the above presented deuteration method reacting through a similar mechanism [88]. It was shown that β-hydrogen atoms underwent the H/D exchange with a good yield. It was also pointed out that the regioselectivity of the labeling depended on which deuterium source was used.

Buchanan et al., who developed catalysts for C-H bond activation [84] also presented the applicability of the soluble iridium complexes for the specific deuterium incorporation in aliphatic and nonfunctionalized aromatic substrates. A high degree of deuterium incorporation was obtained with hydrocarbons, alcohols, phenols, ethers, carboxylic acids, esters, and amides with D2O, [D6]acetone, or [D6]benzene [91].

In 2006, Peris et al. demonstrated efficient HDX with diethyl ether, ethyl methyl ketone, isopropanol, and styrene with the *N*-heterocyclic iridium–carbene complexes in the presence of [D4]methanol (Figure 22) [92].

ters, and amides with D2O, [D6]acetone, or [D6]benzene [91].

ters, and amides with D2O, [D6]acetone, or [D6]benzene [91].

**Figure 21.** H/D exchange catalyzed by iridium catalyst as shown by Heys [85].

**Figure 21.** H/D exchange catalyzed by iridium catalyst as shown by Heys [85].

*Molecules* **2021**, *26*, x FOR PEER REVIEW 15 of 26

Further, Kröger et al. presented that the unsaturated carbonyl compounds were also suitable substrates for the above presented deuteration method reacting through a similar mechanism [88]. It was shown that β-hydrogen atoms underwent the H/D exchange with a good yield. It was also pointed out that the regioselectivity of the labeling depended on

Further, Kröger et al. presented that the unsaturated carbonyl compounds were also suitable substrates for the above presented deuteration method reacting through a similar mechanism [88]. It was shown that β-hydrogen atoms underwent the H/D exchange with a good yield. It was also pointed out that the regioselectivity of the labeling depended on

Buchanan et al., who developed catalysts for C-H bond activation [84] also presented the applicability of the soluble iridium complexes for the specific deuterium incorporation in aliphatic and nonfunctionalized aromatic substrates. A high degree of deuterium incorporation was obtained with hydrocarbons, alcohols, phenols, ethers, carboxylic acids, es-

Buchanan et al., who developed catalysts for C-H bond activation [84] also presented the applicability of the soluble iridium complexes for the specific deuterium incorporation in aliphatic and nonfunctionalized aromatic substrates. A high degree of deuterium incorporation was obtained with hydrocarbons, alcohols, phenols, ethers, carboxylic acids, es-

In 2006, Peris et al. demonstrated efficient HDX with diethyl ether, ethyl methyl ketone, isopropanol, and styrene with the *N*-heterocyclic iridium–carbene complexes in the

In 2006, Peris et al. demonstrated efficient HDX with diethyl ether, ethyl methyl ketone, isopropanol, and styrene with the *N*-heterocyclic iridium–carbene complexes in the

**Figure 22.** Iridium-carbene complexes presented by Peris et al. [92]. **Figure 22.** Iridium-carbene complexes presented by Peris et al. [92].

#### *2.3. Metal-Catalyzed HDX Adjacent to Nitrogen Atom 2.3. Metal-Catalyzed HDX Adjacent to Nitrogen Atom*

which deuterium source was used.

which deuterium source was used.

presence of [D4]methanol (Figure 22) [92].

presence of [D4]methanol (Figure 22) [92].

*2.3. Metal-Catalyzed HDX Adjacent to Nitrogen Atom*  A first example of deuterium incorporation into the carbon centers in amines was reported in 1977, by Maeda et al. [93]. The hydrogen−deuterium exchange on the carbon atoms of primary and tertiary amines was performed in the presence of a deuterated form of Adam's catalyst (platinum oxide treated under reductive conditions by D2 in D2O) (Figure 23). Such a solution allowed the selective deuteration at the β carbon of primary amines and at the α-C of the tertiary. The applied catalyst was activated by UV light or γradiation irradiation. PtO2, after activation with D2, was applied for the selective deuterium incorporation in nucleosides [94]. A strong dependency of the exchange selectivity upon the number and steric demand of the substituents on the nitrogen atom has been observed for the exchange of α-hydrogen atoms of aliphatic amines and amino acids with Adam's catalyst (PtO2·H2O). It was found that the nitrogen atom bound to the surface of the applied catalyst. The efficiency of H/D exchange decreased in the following series tertiary > secondary > primary amines [93]. A first example of chiral carbon atom deuteration in amines with retention of configuration was presented by Jere et al. in 2003 [95]. In this case, the HDX of alanine or alaninol with complete retention was performed in the pres-A first example of deuterium incorporation into the carbon centers in amines was reported in 1977, by Maeda et al. [93]. The hydrogen−deuterium exchange on the carbon atoms of primary and tertiary amines was performed in the presence of a deuterated form of Adam's catalyst (platinum oxide treated under reductive conditions by D<sup>2</sup> in D2O) (Figure 23). Such a solution allowed the selective deuteration at the β carbon of primary amines and at the α-C of the tertiary. The applied catalyst was activated by UV light or γradiation irradiation. PtO2, after activation with D2, was applied for the selective deuterium incorporation in nucleosides [94]. A strong dependency of the exchange selectivity upon the number and steric demand of the substituents on the nitrogen atom has been observed for the exchange of α-hydrogen atoms of aliphatic amines and amino acids with Adam's catalyst (PtO2·H2O). It was found that the nitrogen atom bound to the surface of the applied catalyst. The efficiency of H/D exchange decreased in the following series tertiary > secondary > primary amines [93]. A first example of chiral carbon atom deuteration in amines with retention of configuration was presented by Jere et al. in 2003 [95]. In this case, the HDX of alanine or alaninol with complete retention was performed in the presence of ruthenium on carbon under D<sup>2</sup> in D2O. A first example of deuterium incorporation into the carbon centers in amines was reported in 1977, by Maeda et al. [93]. The hydrogen−deuterium exchange on the carbon atoms of primary and tertiary amines was performed in the presence of a deuterated form of Adam's catalyst (platinum oxide treated under reductive conditions by D2 in D2O) (Figure 23). Such a solution allowed the selective deuteration at the β carbon of primary amines and at the α-C of the tertiary. The applied catalyst was activated by UV light or γradiation irradiation. PtO2, after activation with D2, was applied for the selective deuterium incorporation in nucleosides [94]. A strong dependency of the exchange selectivity upon the number and steric demand of the substituents on the nitrogen atom has been observed for the exchange of α-hydrogen atoms of aliphatic amines and amino acids with Adam's catalyst (PtO2·H2O). It was found that the nitrogen atom bound to the surface of the applied catalyst. The efficiency of H/D exchange decreased in the following series tertiary > secondary > primary amines [93]. A first example of chiral carbon atom deuteration in amines with retention of configuration was presented by Jere et al. in 2003 [95]. In this case, the HDX of alanine or alaninol with complete retention was performed in the presence of ruthenium on carbon under D2 in D2O.

ence of ruthenium on carbon under D2 in D2O.

**Figure 23.** H/D exchange at carbon centers adjacent to nitrogen atom observed by Maeda et al. [93].

The possibility of secondary amine deuteration via H/D exchange in the presence of RuCl2(PPh3)<sup>3</sup> as the catalyst was investigated by Matsubara et al. [57]. It was found that the applied reaction conditions allowed selective deuteration at the α carbon atom with deuterium incorporations of up to 94% (Figure 24) The proposed reaction conditions were also applied in the deuteration of tertiary amines, resulting in 12% deuterium incorporation. As described, there was a possibility of selective amine deuteration at the α carbon atom under reaction conditions similar to those presented for alcohols. The configuration on the stereocenter in the β position depended on the temperature isotope exchange process and remained unaffected as long as the temperature did not exceed 100 ◦C [57].

[93].

[93].

**Figure 24.** HDX at the carbon centers adjacent to nitrogen proposed by Matsubara and co-workers with selected examples [57]. **Figure 24.** HDX at the carbon centers adjacent to nitrogen proposed by Matsubara and co-workers with selected examples [57]. with selected examples [57]. In 2015, Taglang and co-workers, reported that hydrogen−deuterium exchange at the

**Figure 23.** H/D exchange at carbon centers adjacent to nitrogen atom observed by Maeda et al.

**Figure 23.** H/D exchange at carbon centers adjacent to nitrogen atom observed by Maeda et al.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 16 of 26

The possibility of secondary amine deuteration via H/D exchange in the presence of RuCl2(PPh3)3 as the catalyst was investigated by Matsubara et al. [57]. It was found that the applied reaction conditions allowed selective deuteration at the α carbon atom with deuterium incorporations of up to 94% (Figure 24) The proposed reaction conditions were also applied in the deuteration of tertiary amines, resulting in 12% deuterium incorporation. As described, there was a possibility of selective amine deuteration at the α carbon atom under reaction conditions similar to those presented for alcohols. The configuration on the stereocenter in the β position depended on the temperature isotope exchange process and remained unaffected as long as the temperature did not exceed 100 °C [57].

The possibility of secondary amine deuteration via H/D exchange in the presence of RuCl2(PPh3)3 as the catalyst was investigated by Matsubara et al. [57]. It was found that the applied reaction conditions allowed selective deuteration at the α carbon atom with deuterium incorporations of up to 94% (Figure 24) The proposed reaction conditions were also applied in the deuteration of tertiary amines, resulting in 12% deuterium incorporation. As described, there was a possibility of selective amine deuteration at the α carbon atom under reaction conditions similar to those presented for alcohols. The configuration on the stereocenter in the β position depended on the temperature isotope exchange process and remained unaffected as long as the temperature did not exceed 100 °C [57].

In 2015, Taglang and co-workers, reported that hydrogen−deuterium exchange at the chiral carbon centers of amino acid derivatives turned out to be stereoretentive (Figure 25) [96]. The authors proposed a mechanism of reaction in which the key step is the coordination of the nitrogen atom of the amino group to the surface of the applied catalyst which makes a ruthenium site and enables the C–H activation at the α carbon atom. A molecule thus-activated undergoes HDX, resulting in a selective α carbon deuterated de-In 2015, Taglang and co-workers, reported that hydrogen−deuterium exchange at the chiral carbon centers of amino acid derivatives turned out to be stereoretentive (Figure 25) [96]. The authors proposed a mechanism of reaction in which the key step is the coordination of the nitrogen atom of the amino group to the surface of the applied catalyst which makes a ruthenium site and enables the C–H activation at the α carbon atom. A molecule thus-activated undergoes HDX, resulting in a selective α carbon deuterated derivative. chiral carbon centers of amino acid derivatives turned out to be stereoretentive (Figure 25) [96]. The authors proposed a mechanism of reaction in which the key step is the coordination of the nitrogen atom of the amino group to the surface of the applied catalyst which makes a ruthenium site and enables the C–H activation at the α carbon atom. A molecule thus-activated undergoes HDX, resulting in a selective α carbon deuterated derivative.

**Figure 25.** Schematic presentation of H/D exchange using ruthenium nanoparticles presented by **Figure 25.** Schematic presentation of H/D exchange using ruthenium nanoparticles presented by Taglang et al. [96] with selected examples. **Figure 25.** Schematic presentation of H/D exchange using ruthenium nanoparticles presented by Taglang et al. [96] with selected examples.

Taglang et al. [96] with selected examples. Alexakis et al. in 2005 analyzed different ruthenium(II) catalysts in the deuterium labeling reactions of piperidines, piperazines and several different dialkylamines in the presence of D2O as the deuterium source [97]. They found that while RuCl2(PPh3)3 was active for the deuteration of primary alcohols and amines, in the presence of RuCl4(CO)6 only secondary amine labeling was possible. The study on the deuteration of 4-benzylpi-Alexakis et al. in 2005 analyzed different ruthenium(II) catalysts in the deuterium labeling reactions of piperidines, piperazines and several different dialkylamines in the presence of D2O as the deuterium source [97]. They found that while RuCl2(PPh3)3 was active for the deuteration of primary alcohols and amines, in the presence of RuCl4(CO)6 only secondary amine labeling was possible. The study on the deuteration of 4-benzylpi-Alexakis et al. in 2005 analyzed different ruthenium(II) catalysts in the deuterium labeling reactions of piperidines, piperazines and several different dialkylamines in the presence of D2O as the deuterium source [97]. They found that while RuCl2(PPh3)<sup>3</sup> was active for the deuteration of primary alcohols and amines, in the presence of RuCl4(CO)<sup>6</sup> only secondary amine labeling was possible. The study on the deuteration of 4-benzylpiperidine in the presence of [Ru2Cl4(CO)6] as catalyst revealed the incorporation of an average of five deuterium atoms per molecule, however, the positions of the introduced deuterons were not precisely described [97].

The study of the increase of the deuterated yield led Roche's group [98] to demonstrate that similar deuteration as shown by Taglang and co-workers [96] could be achieved in the presence of a Ru/C catalyst instead of Ru nanoparticles, H<sup>2</sup> atmosphere in D2O and a base under mild temperature (70 ◦C) [99]. The presented methodology allowed the stereoretentive preparation of large quantities (up to 0.2 mols) of hydrosoluble amines with chiral centers, showing the applicability of this process for industrial application.

After successful deuteration of α, β carbons of alcohols in the presence of Ru-pincer complexes, Gunanathan and co-workers [100] presented the α deuteration of amines and amino acids by using D2O as deuterium source. The presented protocol is characterized by its selectivity for primary and secondary amines (no deuteration occurs at the α carbon of tertiary amines or alcohols). In the case of amino acids epimerization was observed.

In 2012, Neubert et al. applied 'borrowing hydrogen' catalysis to perform the deuteration of tertiary amines [101]. In this study the Shvo catalyst was used to optimize the reaction conditions (Figure 26) [102]. The mechanism involved the formation of an in-

termediate in the form of iminium ion (in equilibrium with the corresponding enamine) mediated by the monomer of the catalyst bearing an available position in the coordination sphere of the metal atom. The reduced form of the applied catalyst underwent HDX in the presence of deuterating agent (D2O, deuterated alcohols) and subsequently transferred the deuterium atoms to the unsaturated bond of the enamine, generating a doubly labeled tertiary amine. It was found in the case of *N*,*N*,*N*-trihexylamine as the substrate that the deuterium incorporation was as high as 93% at the α and β positions in the presence of isopropanol-d8 as the solvent. This protocol was also successfully applied in the formation of deuterated pharmaceutical compounds, such as sunitinib (a kinase inhibitor). diated by the monomer of the catalyst bearing an available position in the coordination sphere of the metal atom. The reduced form of the applied catalyst underwent HDX in the presence of deuterating agent (D2O, deuterated alcohols) and subsequently transferred the deuterium atoms to the unsaturated bond of the enamine, generating a doubly labeled tertiary amine. It was found in the case of *N*,*N*,*N*-trihexylamine as the substrate that the deuterium incorporation was as high as 93% at the α and β positions in the presence of isopropanol-d8 as the solvent. This protocol was also successfully applied in the formation of deuterated pharmaceutical compounds, such as sunitinib (a kinase inhibitor).

peridine in the presence of [Ru2Cl4(CO)6] as catalyst revealed the incorporation of an average of five deuterium atoms per molecule, however, the positions of the introduced

chiral centers, showing the applicability of this process for industrial application.

The study of the increase of the deuterated yield led Roche's group [98] to demonstrate that similar deuteration as shown by Taglang and co-workers [96] could be achieved in the presence of a Ru/C catalyst instead of Ru nanoparticles, H2 atmosphere in D2O and a base under mild temperature (70 °C) [99]. The presented methodology allowed the stereoretentive preparation of large quantities (up to 0.2 mols) of hydrosoluble amines with

After successful deuteration of α, β carbons of alcohols in the presence of Ru-pincer complexes, Gunanathan and co-workers [100] presented the α deuteration of amines and amino acids by using D2O as deuterium source. The presented protocol is characterized by its selectivity for primary and secondary amines (no deuteration occurs at the α carbon of tertiary amines or alcohols). In the case of amino acids epimerization was observed.

In 2012, Neubert et al. applied 'borrowing hydrogen' catalysis to perform the deuteration of tertiary amines [101]. In this study the Shvo catalyst was used to optimize the reaction conditions (Figure 26) [102]. The mechanism involved the formation of an intermediate in the form of iminium ion (in equilibrium with the corresponding enamine) me-

*Molecules* **2021**, *26*, x FOR PEER REVIEW 17 of 26

deuterons were not precisely described [97].

**Figure 26.** Schematic presentation of HDX in the presence of Shvo's catalyst [102]. **Figure 26.** Schematic presentation of HDX in the presence of Shvo's catalyst [102].

In 2014, Pieters et al. developed a method of regioselective α deuteration of nitrogencontaining bioactive compounds in the presence of ruthenium nanoparticles supported on polyvinylpyrrolidone under the D2 atmosphere [103] (Figure 27). Using this method, In 2014, Pieters et al. developed a method of regioselective α deuteration of nitrogencontaining bioactive compounds in the presence of ruthenium nanoparticles supported on polyvinylpyrrolidone under the D<sup>2</sup> atmosphere [103] (Figure 27). Using this method, the authors obtained high deuterium incorporation with complete regioselectivity.

the authors obtained high deuterium incorporation with complete regioselectivity. In 2016, Jackson et al. [105] developed a method of electrocatalytic deuteration of amines and alcohols which overcame the problem of the D<sup>2</sup> solubility in an aqueous environment, by in situ generation of D2. Using this method, different deuterated derivatives were prepared with high level of deuterium incorporation, but with low yield. *Molecules* **2021**, *26*, x FOR PEER REVIEW 18 of 26

**Figure 27.** Ruthenium nanoparticles-catalyzed HDX according to Beller [101] and Pieters [103].

alcohol deuteration, complete racemization occurred (Figure 28) [104].

**Figure 28.** Model metal-catalyzed HDX reactions presented by Szymczak et al. [104].

were prepared with high level of deuterium incorporation, but with low yield.

In 2016, Jackson et al. [105] developed a method of electrocatalytic deuteration of amines and alcohols which overcame the problem of the D2 solubility in an aqueous environment, by in situ generation of D2. Using this method, different deuterated derivatives

A main challenge in this field was the development a stereoretentive hydrogen−deuterium exchange at the chiral carbon centers. Although the Rousseau reaction can be considered as stereospecific, an example of deuteration at the chiral carbon atom was presented in his paper. Maeda et al. also investigated the stereospecificity of the HDX in amines and found that at temperatures above 100 °C, L-alanine started to racemize [94]. During the analysis of selective HDX at the β carbon of phenylalanine in the presence of Pd/C catalyst under H2, the additional deuteration at the α carbon was observed at 160°C,

Pyrimidine bases, including uracil or cytosine, may also be deuterated in the 5- and 6-positions in the presence of a Pd/C–H2/D2O mixture at 110 °C [106]. The deuterium was incorporated in the 5-methyl group of thymine in addition to the 6-position, and no side products have been reported. Purine nucleosides, including adenosine or inosine, were successfully and chemoselectively deuterated in the 2- and 8-positions [107]. The lower HDX levels were noted for the pyrimidine bases and analyzed nucleoside in the presence

In 2001, Hardacre and co-workers reported the application of a catalyst activated by hydrogen reduction in the preparation of deuterated imidazoles and imidazole salts. The substrate solution in D2O was treated by the reaction mixture, degassed by several cool-

**Figure 27.** Ruthenium nanoparticles-catalyzed HDX according to Beller [101] and Pieters [103]. In 2016, Hale and Szymczak proposed a method of stereoretentive deuteration in the presence of Ru-bMepi complex that avoided the use of D2. In this method the complete stereoretention in the case of a chiral amine was observed. However, in the case of chiral In 2016, Hale and Szymczak proposed a method of stereoretentive deuteration in the presence of Ru-bMepi complex that avoided the use of D2. In this method the complete stereoretention in the case of a chiral amine was observed. However, in the case of chiral alcohol deuteration, complete racemization occurred (Figure 28) [104].

with partial racemization of the chiral carbon atom.

ing/thawing cycles [108].

of CD3OD as a solvent and deuterating agent in the form of D2O.

**Figure 28.** Model metal-catalyzed HDX reactions presented by Szymczak et al. [104]. **Figure 28.** Model metal-catalyzed HDX reactions presented by Szymczak et al. [104].

In 2016, Jackson et al. [105] developed a method of electrocatalytic deuteration of amines and alcohols which overcame the problem of the D2 solubility in an aqueous environment, by in situ generation of D2. Using this method, different deuterated derivatives were prepared with high level of deuterium incorporation, but with low yield. A main challenge in this field was the development a stereoretentive hydrogen−deu-A main challenge in this field was the development a stereoretentive hydrogen−deuterium exchange at the chiral carbon centers. Although the Rousseau reaction can be considered as stereospecific, an example of deuteration at the chiral carbon atom was presented in his paper. Maeda et al. also investigated the stereospecificity of the HDX in amines and found that at temperatures above 100 ◦C, L-alanine started to racemize [94]. During the analysis of selective HDX at the β carbon of phenylalanine in the presence of Pd/C catalyst under H2, the additional deuteration at the α carbon was observed at 160◦C, with partial racemization of the chiral carbon atom.

alcohol deuteration, complete racemization occurred (Figure 28) [104].

**Figure 27.** Ruthenium nanoparticles-catalyzed HDX according to Beller [101] and Pieters [103].

In 2016, Hale and Szymczak proposed a method of stereoretentive deuteration in the

presence of Ru-bMepi complex that avoided the use of D2. In this method the complete stereoretention in the case of a chiral amine was observed. However, in the case of chiral

terium exchange at the chiral carbon centers. Although the Rousseau reaction can be considered as stereospecific, an example of deuteration at the chiral carbon atom was presented in his paper. Maeda et al. also investigated the stereospecificity of the HDX in amines and found that at temperatures above 100 °C, L-alanine started to racemize [94]. During the analysis of selective HDX at the β carbon of phenylalanine in the presence of Pyrimidine bases, including uracil or cytosine, may also be deuterated in the 5- and 6-positions in the presence of a Pd/C–H2/D2O mixture at 110 ◦C [106]. The deuterium was incorporated in the 5-methyl group of thymine in addition to the 6-position, and no side products have been reported. Purine nucleosides, including adenosine or inosine, were successfully and chemoselectively deuterated in the 2- and 8-positions [107]. The lower HDX levels were noted for the pyrimidine bases and analyzed nucleoside in the presence of CD3OD as a solvent and deuterating agent in the form of D2O.

Pd/C catalyst under H2, the additional deuteration at the α carbon was observed at 160°C, with partial racemization of the chiral carbon atom. Pyrimidine bases, including uracil or cytosine, may also be deuterated in the 5- and In 2001, Hardacre and co-workers reported the application of a catalyst activated by hydrogen reduction in the preparation of deuterated imidazoles and imidazole salts. The substrate solution in D2O was treated by the reaction mixture, degassed by several cooling/thawing cycles [108].

6-positions in the presence of a Pd/C–H2/D2O mixture at 110 °C [106]. The deuterium was incorporated in the 5-methyl group of thymine in addition to the 6-position, and no side products have been reported. Purine nucleosides, including adenosine or inosine, were successfully and chemoselectively deuterated in the 2- and 8-positions [107]. The lower HDX levels were noted for the pyrimidine bases and analyzed nucleoside in the presence of CD3OD as a solvent and deuterating agent in the form of D2O. In 2001, Hardacre and co-workers reported the application of a catalyst activated by hydrogen reduction in the preparation of deuterated imidazoles and imidazole salts. The Raney nickel catalyst in the presence of [D6]acetone, [D3]acetonitrile, [D1]chloroform, D2O or [D8] 2-propanol allowed the selective deuteration of specific positions in tryptophan derivatives, based on their nucleophilicity [109]. The influence of the differing nucleophilic potential of these positions and the indole ring orientation on the catalyst surface influenced by the solvent were responsible for the selectivity of deuteration (Figure 29). In the case of deuteration catalyzed by Raney nickel, it was found that only the hydrogen atoms at the α-carbons underwent selective deuteration in quinuclidine at higher temperatures, a longer reaction time and in the presence of D2O (100 ◦C, 40 h, 2 reaction cycles; ≥99.7% D). It was also reported that less than 1% of deuterium was incorporated into the β and γ carbon centers [110].

substrate solution in D2O was treated by the reaction mixture, degassed by several cooling/thawing cycles [108]. Previously Hickey et al. reported the extended spectrum of the substrate to aniline and benzylamine derivatives [111]. The application of [Ir(cod)(acac-F6)] (acac-F6= hexafluoroacetylacetonate) and D<sup>2</sup> allowed *ortho* HDX, relative to the position of the amino or methylamino group.

porated into the β and γ carbon centers [110].

**Figure 29.** H/D exchange described by Gavrisch Yau and Gawrisch [109]. **Figure 29.** H/D exchange described by Gavrisch Yau and Gawrisch [109].

Previously Hickey et al. reported the extended spectrum of the substrate to aniline and benzylamine derivatives [111]. The application of [Ir(cod)(acac-F6)] (acac-F6=hexafluoroacetylacetonate) and D2 allowed *ortho* HDX, relative to the position of the amino or methylamino group. In 2017, Loh et al. [112] proposed a new deuteration strategy, based on the application of photoredox deuteration and tritiation. The method was successfully applied in the formation of isotopically labeled derivatives of pharmaceuticals containing alkyl amine moieties. The developed method involved the formation of an α-amino radical via a single-electron transfer occurred in the presence of an iridium(III) catalyst, previously excited by visible blue light. The application of a hydrogen atom transfer (HAT) catalyst allowed the abstraction of deuterium (or tritium) to form labeled derivatives. It was also found that application of thiols as HAT catalysts was crucial for the preparation of labeled compounds. This process was optimized and then applied in the preparation of gram scale products. All of the reported examples met all the requirement characteristic for internal standards which may be applied in quantitative analysis by mass spectrometry (more than 4 D and less than 0.1% of the unlabeled compound). Moreover, the proposed process may be performed in the presence of several functional groups, presenting a high selectivity towards exchange at the C(sp3)–H centers adjacent to the nitrogen atom and retention of the stereochemistry, even when HDX proceeds at the chiral center. In 2015, Hu and In 2017, Loh et al. [112] proposed a new deuteration strategy, based on the application of photoredox deuteration and tritiation. The method was successfully applied in the formation of isotopically labeled derivatives of pharmaceuticals containing alkyl amine moieties. The developed method involved the formation of an α-amino radical via a single-electron transfer occurred in the presence of an iridium(III) catalyst, previously excited by visible blue light. The application of a hydrogen atom transfer (HAT) catalyst allowed the abstraction of deuterium (or tritium) to form labeled derivatives. It was also found that application of thiols as HAT catalysts was crucial for the preparation of labeled compounds. This process was optimized and then applied in the preparation of gram scale products. All of the reported examples met all the requirement characteristic for internal standards which may be applied in quantitative analysis by mass spectrometry (more than 4 D and less than 0.1% of the unlabeled compound). Moreover, the proposed process may be performed in the presence of several functional groups, presenting a high selectivity towards exchange at the C(sp<sup>3</sup> )–H centers adjacent to the nitrogen atom and retention of the stereochemistry, even when HDX proceeds at the chiral center. In 2015, Hu and co-workers described an example of deuteration based on the single-electron transfer mechanism [113]. In the presented process, deuteration took place only at the α carbon atom of the amine when the radical was stabilized at a benzylic position. It was also found that the presence of the nitrogen was not required for the deuterium incorporation.

Raney nickel catalyst in the presence of [D6]acetone, [D3]acetonitrile, [D1]chloroform, D2O or [D8] 2-propanol allowed the selective deuteration of specific positions in tryptophan derivatives, based on their nucleophilicity [109]. The influence of the differing nucleophilic potential of these positions and the indole ring orientation on the catalyst surface influenced by the solvent were responsible for the selectivity of deuteration (Figure 29). In the case of deuteration catalyzed by Raney nickel, it was found that only the hydrogen atoms at the α-carbons underwent selective deuteration in quinuclidine at higher temperatures, a longer reaction time and in the presence of D2O (100 °C, 40 h, 2 reaction cycles; ≥99.7% D). It was also reported that less than 1% of deuterium was incor-

#### co-workers described an example of deuteration based on the single-electron transfer *2.4. pH Dependent and Metal-Catalyzed HDX Adjacent to Sulfur Atom*

mechanism [113]. In the presented process, deuteration took place only at the α carbon atom of the amine when the radical was stabilized at a benzylic position. It was also found that the presence of the nitrogen was not required for the deuterium incorporation. *2.4. pH Dependent and Metal-Catalyzed HDX Adjacent to Sulfur Atom*  Sulfur-directed deuteration is not so common in scientific literature. Only a few papers report deuteration at the α carbon position of sulfur center. In 2018, the application Sulfur-directed deuteration is not so common in scientific literature. Only a few papers report deuteration at the α carbon position of sulfur center. In 2018, the application of ruthenium on carbon under D<sup>2</sup> in the H/D exchange in thioesters was presented by Gao et al. [114] By using this method a number of deuterium-labeled drugs or amino acids and peptides have been successfully prepared with a good regioselectivity towards the α carbon to the sulfur center. Additionally, in the case of chiral molecules involved in this process, the retention of the configuration was found. It was also suggested that the mechanism of the described process was the same as that proposed for the ruthenium nanoparticle-mediated deuteration of amino acids [96].

A great review presenting the possibility of pH dependent and metal-catalyzed hydrogen−deuterium exchange adjacent to the sulfur atom was presented by Michelotti and co-workers [115]. Rauk et al. [116] demonstrated a deuteration process in the presence of NaOD in D2O or just in D2O for substances containing the methylsulfonyl or methylsulfinyl groups. They reported that sulfonyl (-SO2-) and sulfinyl (-SO-) groups should present similar conjugative ability with adjacent carbanionic centers (Figure 30). It was also presented that -SO- group was much less effective than a -SO2- group in promoting deuteration at the α carbon center. The previously reported HDX of dimethyl

sulfoxide in the presence of deuteroxide may have resulted from the enhanced reactivity of applied bases in dimethyl sulfoxide solution. Additionally, an unexpected difference in the deuteration level at the two methylene hydrogens was found [101]. presence of deuteroxide may have resulted from the enhanced reactivity of applied bases in dimethyl sulfoxide solution. Additionally, an unexpected difference in the deuteration level at the two methylene hydrogens was found [101].

of ruthenium on carbon under D2 in the H/D exchange in thioesters was presented by Gao et al. [114] By using this method a number of deuterium-labeled drugs or amino acids and peptides have been successfully prepared with a good regioselectivity towards the α carbon to the sulfur center. Additionally, in the case of chiral molecules involved in this process, the retention of the configuration was found. It was also suggested that the mechanism of the described process was the same as that proposed for the ruthenium nanopar-

A great review presenting the possibility of pH dependent and metal-catalyzed hydrogen−deuterium exchange adjacent to the sulfur atom was presented by Michelotti and co-workers [115]. Rauk et al. [116] demonstrated a deuteration process in the presence of NaOD in D2O or just in D2O for substances containing the methylsulfonyl or methylsulfinyl groups. They reported that sulfonyl (-SO2-) and sulfinyl (-SO-) groups should present similar conjugative ability with adjacent carbanionic centers (Figure 30). It was also presented that -SO- group was much less effective than a -SO2- group in promoting deuteration at the α carbon center. The previously reported HDX of dimethyl sulfoxide in the

*Molecules* **2021**, *26*, x FOR PEER REVIEW 20 of 26

ticle-mediated deuteration of amino acids [96].

**Figure 30.** Schematic presentation of H/D exchange at the carbon center connected with the sulfur atom presented by Gao et al. [114] (**A**) and Rauk et al. [116] (**B**).

**Figure 30.** Schematic presentation of H/D exchange at the carbon center connected with the sulfur atom presented by Gao

et al. [114] (**A**) and Rauk et al. [116] (**B**). The possibility of complete deuteration of methylene hydrogens adjacent to the sulfinyl group was presented by Redondo and co-workers [117]. The reaction proceeded at room temperature when the sodium salts of compounds containing [(pyridylmethyl)sulfinyl]benzimidazole structural core were dissolved in a solvent serving as a deuterium source, including D2O and CD3OD. The presented process resulted from the weak acidity of the methylene hydrogen atoms, and was also observed in a nondeuterating solvent like The possibility of complete deuteration of methylene hydrogens adjacent to the sulfinyl group was presented by Redondo and co-workers [117]. The reaction proceeded at room temperature when the sodium salts of compounds containing [(pyridylmethyl)sulfinyl] benzimidazole structural core were dissolved in a solvent serving as a deuterium source, including D2O and CD3OD. The presented process resulted from the weak acidity of the methylene hydrogen atoms, and was also observed in a nondeuterating solvent like DMSOd<sup>6</sup> in the presence of aa catalytic amount of NaOH. The described HDX was monitored by <sup>1</sup>H NMR which also revealed the stereoselectivity of deuterium incorporation [116,118].

> DMSO-d6 in the presence of aa catalytic amount of NaOH. The described HDX was monitored by 1H NMR which also revealed the stereoselectivity of deuterium incorporation [116,118]. Transition metal-catalyzed H/D exchange can be classified into three categories. The first one involves the C−H bond activation which generates an organometallic intermedi-Transition metal-catalyzed H/D exchange can be classified into three categories. The first one involves the C−H bond activation which generates an organometallic intermediate, bearing a metal−carbon bond as a result of the carbon−hydrogen bond activation. The second one is denoted as C−H insertion catalysis as a key working mode, whereas the third one is related to the photoredox catalysis [119].

> ate, bearing a metal−carbon bond as a result of the carbon−hydrogen bond activation. The second one is denoted as C−H insertion catalysis as a key working mode, whereas the third one is related to the photoredox catalysis [119]. The advantages of metal catalyzed hydrogen−deuterium exchange processes are that they are usually easy to perform and most of the presented catalysts are commercially available. The limitation of the presented methods is the high price of the catalyst, necessity of compound purification, long reaction times or the necessity for the application of high pressure.

#### **3. Conclusions**

The presented manuscript provides an overview of the methods of H/D exchange reactions at the carbon centers. From the first reports of HDX at the carbon center in alcohols and amines, the state-of-the-art in this field has expanded enormously, mostly due to the application of metal catalysis. proving its validity and utility. Nowadays, the deuterated compounds play a crucial role as the internal standards in the quantitative analysis by the LC-MS technique. Although the possibilities of deuterated standards in quantitative investigation of compounds are beyond doubt, their application in medical diagnosis may be limited, due to the high costs of their preparation. Therefore, despite significant progress there is still a strong need for the development of new, simple, rapid and cost-efficient methods of carbon deuteration. It may be speculated that the new techniques of hydrogen−deuterium exchange at the carbon centers will be developed in the near future. It may be expected that they will be characterized by a low price of preparation, the stability of the introduced deuterons, long-term storage of the deuterated derivatives at room temperature, an appropriate mass difference for MS quantification, stability of introduced deuterons during chromatographic separation and co-elution with nondeuterated isotopologues. It may be expected that further work in the presented area will be forthcoming.

**Author Contributions:** Conceptualization, R.B.; writing—original draft preparation, P.G., R.B. The manuscript was written through contributions of all authors. Both authors commented on the manuscript. Both authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by NATIONAL SCIENCE CENTRE POLAND, grant number UMO-2016/23/B/ST4/01036.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Andrzej Reszka (Shim-Pol, Poland) for providing access to LCMS-8050, LCMS-9030 and LCMS IT-TOF instruments.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Fluorescent Probes for Live Cell Thiol Detection**

**Shenggang Wang † , Yue Huang † and Xiangming Guan \***

> Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, South Dakota State University, Box 2202C, Brookings, SD 57007, USA; Shenggang.wang@fda.hhs.gov (S.W.); Yue.huang@sdstate.edu (Y.H.)

**\*** Correspondence: Xiangming.Guan@sdstate.edu; Tel.: +1-605-688-5314; Fax: +1-605-688-5993

† Shenggang Wang and Yue Huang contributed equally to this article.

**Abstract:** Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols' concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

**Keywords:** live cell thiol fluorescence imaging; cellular thiols; subcellular thiols; non-protein thiols; protein thiols; mitochondrial thiols; Lysosomal thiols; glutathione; cysteine; homocysteine; thiol specific agents; thiol-sulfide exchange reaction; thiol-disulfide exchange reaction; Michael addition reaction

#### **1. Introduction**

Thiols, compounds that contain a sulfhydryl group (-SH), in the biological system are referred as biological thiols or biothiols [1,2]. Biothiols can be categorized into two classes: protein thiols (PSH) and non-protein thiols (NPSH). NPSH are mainly small molecule thiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), while PSH are primarily the cysteine residues in proteins [3]. Thiols play vital and irreplaceable roles in various cellular functions in the biological system [4] thanks to their unique chemical properties of strong nucleophilicity, reductivity, and chelating ability for metals. These roles include detoxification to remove reactive electrophiles, as an antioxidant to remove reactive oxygen species (ROS), and the ability of metal chelation. For example, GSH is the key compound in glutathione conjugation reaction—a phase II drug metabolism in the body to remove reactive electrophiles which are toxic. GSH is also the most abundant intracellular NPSH and serves as the most important antioxidant to prevent damages caused by ROS via termination of ROS. In addition, thiols are also involved in many other aspects of cellular functions, such as being part of enzyme active sites, being involved in signal transduction, being involved in cell division, etc. Abnormality of thiol levels has been linked with many diseases and biological disorders, such as cardiovascular diseases, cancer, Alzheimer's disease and etc [5].

Thiols are distributed heterogeneously and their levels change dynamically in the biological system. Subcellular organelles such as nucleus, mitochondria, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, cell surfaces, etc., are important structures that play critical roles in the normal function of cells. Disruption of the homeostasis of

**Citation:** Wang, S.; Huang, Y.; Guan, X. Fluorescent Probes for Live Cell Thiol Detection. *Molecules* **2021**, *26*, 3575. https://doi.org/10.3390/ molecules26123575

Academic Editors: Gavino Sanna and Stefan Leonidov Tsakovski

Received: 31 March 2021 Accepted: 26 May 2021 Published: 11 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

these structures can interrupt the normal function of cells resulting in many disorders and diseases [6,7]. Thiols distribute unevenly in these subcellular organelles [8]. Their levels in different subcellular organelles can serve as an indicator to reflect the status of the subcellular organelles. Thus, tools or methods that can map thiol distribution and monitor their status changes will provide valuable information to understand the relationship between thiols and cells' functions/dysfunctions, and help guide the correlation/treatment of these dysfunctions and their related diseases [9].

Many analytical methods have been developed for detecting thiols in the biological system. These methods include high performance liquid chromatography (HPLC/UV) [10–12], mass spectrometry (MS), HPLC-MS [13], colorimetric assay [14], enzyme assays, capillary electrophoresis, and gel electrophoresis [15–18]. Most of these methods need a complicated and lengthy sample preparation step and involve a homogenization process to break cells or tissues before an analytical method can be applied. Due to chemical instability of thiols, the sample process step quite often can introduce potential artifacts and cause a potential loss of the analyte.

In the past two decades, one of the most popular approaches being developed to address analyte loss during cell/tissue homogenization is to analyze the analyte in live cells using the fluorescence imagining technique via the use of a fluorescent probe [19]. Fluorescence imaging outstands itself by its simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way [1]. The non-invasive fluorescence imaging provides the advantage of visualizing an analyte without breaking the cell [20–27], enabling the determination of intracellular distribution and dynamitic movement of an analyte in its intact native environments, and reveal information that cannot be revealed after cells/tissues are homogenized.

Significant efforts have been made for the detection of thiols in the biological system via fluorescence imaging by developing a large number of fluorescent probes for thiols. These probes include BODIPY [28,29], rhodamine [30,31], monochlorobimane [32], fluorescein [33,34], tetraphenylethylene [35], coumarin [36,37], curcumin [38], mercury orange [32], rosamine-based [39], naphthalimide-based [40], polymethine [41], benzofurazan [42], and pyrene [43] (Figure 1). The optical spectra of these fluorescence probes cover visible fluorescence, near infra-red (NIR) fluorescence, single-photon or two-photon excitation fluorescence, or aggregation-induced emission (AIE) fluorescence. Some of these probes are developed as an "off-on" agents in a single channel which themselves are nonfluorescent due to a variety of quenching mechanisms (e.g., FRET, PET, and ICT) but can be turned on to a fluorescent product by thiols. The others are "ratiometric" probes utilizing dual-channel emission which themselves exhibit fluorescence but can be switched to a totally different fluorescence upon reaction with thiols and the thiol's level can be reflected by calculating the ratio of the two fluorescence intensities. Compared with "off-on" probes which measure the absolute intensity at only one wavelength, ratiometric fluorescent probes are believed to be more accurate since the use of two different emission wavelength can provide a built-in correction [44].

Among the reported probes, majority of them were designed to detect thiols from the whole cell despite the fact that thiols are distributed unevenly inside cells. Only a few probes were for the detection of mitochondrial thiols while fewer for lysosomes and endoplasmic reticulum (ER). Finding a method to detect thiols in subcellular organelles remains to be a challenge.

For a fluorescence imaging agent to image thiols in live cells, the agent needs to be cell membrane permeable, capable of turning thiols selectively, ideally specifically, into fluorescence strong enough for detection, and noncytotoxic or at least noncytotoxic during the imaging experiment. For a subcellular thiol imaging agent, the agent needs to be cell membrane permeable, capable of turning thiols selectively, ideally specifically, in the targeted subcellular organelle into fluorescence strong enough for detection, and noncytotoxic or at least noncytotoxic during the imaging experiment. For a cellular or subcellular thiol quantifying fluorescence imaging agent, it requires the reagent to quantitatively turn thiols

into fluorescence strong enough for detection within a reasonable time in addition to the requirements for a cellular or subcellular thiol fluorescence imagining agent. on" probes which measure the absolute intensity at only one wavelength, ratiometric fluorescent probes are believed to be more accurate since the use of two different emission wavelength can provide a built-in correction [44].

*Molecules* **2021**, *26*, x FOR PEER REVIEW 2 of 59

of these dysfunctions and their related diseases [9].

cause a potential loss of the analyte.

vealed after cells/tissues are homogenized.

their status changes will provide valuable information to understand the relationship between thiols and cells' functions/dysfunctions, and help guide the correlation/treatment

Many analytical methods have been developed for detecting thiols in the biological system. These methods include high performance liquid chromatography (HPLC/UV) [10–12], mass spectrometry (MS), HPLC-MS [13], colorimetric assay [14], enzyme assays, capillary electrophoresis, and gel electrophoresis [15–18]. Most of these methods need a complicated and lengthy sample preparation step and involve a homogenization process to break cells or tissues before an analytical method can be applied. Due to chemical instability of thiols, the sample process step quite often can introduce potential artifacts and

In the past two decades, one of the most popular approaches being developed to address analyte loss during cell/tissue homogenization is to analyze the analyte in live cells using the fluorescence imagining technique via the use of a fluorescent probe [19]. Fluorescence imaging outstands itself by its simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way [1]. The non-invasive fluorescence imaging provides the advantage of visualizing an analyte without breaking the cell [20– 27], enabling the determination of intracellular distribution and dynamitic movement of an analyte in its intact native environments, and reveal information that cannot be re-

Significant efforts have been made for the detection of thiols in the biological system via fluorescence imaging by developing a large number of fluorescent probes for thiols. These probes include BODIPY [28,29], rhodamine [30,31], monochlorobimane [32], fluorescein [33,34], tetraphenylethylene [35], coumarin [36,37], curcumin [38], mercury orange [32], rosamine-based [39], naphthalimide-based [40], polymethine [41], benzofurazan [42], and pyrene [43] (Figure 1). The optical spectra of these fluorescence probes cover visible fluorescence, near infra-red (NIR) fluorescence, single-photon or two-photon excitation fluorescence, or aggregation-induced emission (AIE) fluorescence. Some of these probes are developed as an "off-on" agents in a single channel which themselves are non-fluorescent due to a variety of quenching mechanisms (e.g., FRET, PET, and ICT) but can be turned on to a fluorescent product by thiols. The others are "ratiometric" probes utilizing dual-channel emission which themselves exhibit fluorescence but can be switched to a totally different fluorescence upon reaction with thiols and the thiol's level can be reflected by calculating the ratio of the two fluorescence intensities. Compared with "off-

**Figure 1.** Different fluorophores used for construction of thiol detection probes.

This review will provide an overview of the fluorescent probes that are developed for detecting thiols in live cells. These probes will be mainly classified into two categories: i. probes used for detection of GSH or total thiols; ii. probes used to detect Cys/Hcy. Intracellularly, GSH is much more abundant (1–10 mM) than Cys (30–200 µM) and Hcy (5–12 µM) [45]. The detection of intracellular GSH is less likely to be interfered by Cys/Hcy. As a result, GSH is often used to reflect the total NPSH in cells. Therefore, the probes claimed for GSH or total NPSH are summarized as one category in this review although some of the probes were claimed to be selective for GSH. In contrast, the detection of Cys/Hcy can be interfered by GSH. For that reason, the probes designed for distinguishing Cys/Hcy over GSH are summarized into a different category.

The purpose of this review is not trying to be inclusive in terms of thiol fluorescence probes since they have been covered by other comprehensive reviews [2,46–49]. Rather, this review focuses on summarizing some of the major strategies or mechanisms that are used for detecting GSH, Cys/Hcy, and how they are applied for the detection of cellular or subcellular thiols. The fluorophores being used, sensing mechanisms (for GSH or Cys/Hcy) and bio-applications of the probes will be illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

#### **2. Detection of Thiols in Cells**

#### *2.1. NPSH, GSH, Cys, and Hcy*

NPSH are thiols other than PSH. They include GSH, Cys, Hcy, and other small molecule thiols. Among them, GSH is the most abundant thiol with a concentration of millimolar in the biological system. GSH serves as the most important antioxidant to prevent damages caused by ROS [50,51] via terminating ROS with itself being converted to its oxidized form glutathione disulfide (GSSG). GSSG is then converted back to GSH by glutathione reductase. Cells maintain a normal ratio (>100) of GSH/GSSG through the GSH/GSSG cycle [52,53]. The abnormal level of GSH has been associated with various diseases such as cancer, AIDS, growth delay, neurodegenerative disease, and cardiovascular disease [54–57]. Thus, determination of GSH level in live cells is of particular interest.

Numerous fluorescent probes have been developed for the detection of intracellular GSH or thiols in live cells based on different mechanisms. Most of these methods were designed based on the high nucleophilicity of thiols [58,59].

Distinguishing GSH from Cys and Hcy remains a challenge due to the structure and reactivity similarities GSH shared with Cys/Hcy [60–62]. The majority of the thiol probes being developed are not able to distinguish GSH from other thiols including Cys/Hcy, since most of them undergo similar reactions and similar emission changes upon reaction with a thiol. However, as mentioned earlier Cys or Hcy is less likely to interfere with the detection of GSH since intracellular GSH level (1–10 mM) is much higher than Cys (30–200 µM), and Hcy (5–15 µM). Although some of the probes have been reported to selectively detect GSH and some reported for detecting NPSH, all these methods are included in the category of GSH and total thiols. This part will focus on the main strategies/mechanisms that have been developed for the detection of GSH and total thiols. The strategies/mechanisms included in these methods are: A. Michael addition reaction with α, β -unsaturated carbonyl derivatives [63,64]; B. Nucleophilic substitution with substrates bearing a leaving group (e.g., halogen, ether, or thioether) [29,65,66]; C. Cleavage of sulfonamide or sulfonate ester [67]; D. Disulfide–thiol exchange reaction [68]; E. others [30,46] (Figure 2). It is noted that although reaction with a thiol is preferred in all these reactions, other nucleophiles such as -OH, or -NH can also be involved in these reactions. The involvement of other nucleophiles in the reaction will affect thiol imaging especially quantitative thiol imaging. Nevertheless, thiol–disulfide exchange reaction (D) and thiol–sulfide exchange reaction (B) have been demonstrated to be thiol specific. Thiol specific fluorescence imaging agents based on the thiol-sulfide reaction have been developed and used successfully to image and quantify cellular thiols and subcellular thiols in live cells (refer to Sections 2.1.2, 3.1.2 and 3.3.2) *Molecules* **2021**, *26*, x FOR PEER REVIEW 4 of 59 with a thiol. However, as mentioned earlier Cys or Hcy is less likely to interfere with the detection of GSH since intracellular GSH level (1–10 mM) is much higher than Cys (30– 200 μM), and Hcy (5–15 μM). Although some of the probes have been reported to selectively detect GSH and some reported for detecting NPSH, all these methods are included in the category of GSH and total thiols. This part will focus on the main strategies/mechanisms that have been developed for the detection of GSH and total thiols. The strategies/mechanisms included in these methods are: A. Michael addition reaction with α, β unsaturated carbonyl derivatives [63,64]; B. Nucleophilic substitution with substrates bearing a leaving group (e.g., halogen, ether, or thioether) [29,65,66]; C. Cleavage of sulfonamide or sulfonate ester [67]; D. Disulfide–thiol exchange reaction [68]; E. others [30,46] (Figure 2). It is noted that although reaction with a thiol is preferred in all these reactions, other nucleophiles such as -OH, or -NH can also be involved in these reactions. The involvement of other nucleophiles in the reaction will affect thiol imaging especially quantitative thiol imaging. Nevertheless, thiol–disulfide exchange reaction (D) and thiol– sulfide exchange reaction (B) have been demonstrated to be thiol specific. Thiol specific fluorescence imaging agents based on the thiol-sulfide reaction have been developed and used successfully to image and quantify cellular thiols and subcellular thiols in live cells (refer to Sections 2.1.2., 3.1.2., and 3.3.2.)

**Figure 2.** Representative sensing mechanisms used for thiol detection. **Figure 2.** Representative sensing mechanisms used for thiol detection.

2.1.1. Detection of NPSH via a Michael Addition Reaction 2.1.1. Detection of NPSH via a Michael Addition Reaction

Michael addition is one of the most commonly used strategies for detection of thiols. Michael addition reaction has a high selectivity for thiols though it can also occur with other nucleophiles such as -OH, NH, or -COOH. Xiaoqiang Chen et al. [34] published their work on the development of a thiol imaging tool bearing fluorescein as the fluorophore (**Probe 1**). As shown in Figure 3, the process of turning on the fluorescence starts from a 1,4-addition of thiols to the α, β-unsaturated ketone in the probe. Subsequent spiro ring opening of fluorescein lights up the fluorescence (Ex. 485 nm, Em. 520 nm) which provides fluorescence enhancement by 61-fold compared with probe itself. The probe exhibits a similar fluorescence enhancement towards Cys, Hcy, and GSH. With fluorescein as a standard, the quantum yields of the thiols adducts with probe were determined to be 0.65, 0.91, and 0.47 for GSH, Cys, and Hcy, respectively. The probe is featured with a rapid Michael addition is one of the most commonly used strategies for detection of thiols. Michael addition reaction has a high selectivity for thiols though it can also occur with other nucleophiles such as -OH, NH, or -COOH. Xiaoqiang Chen et al. [34] published their work on the development of a thiol imaging tool bearing fluorescein as the fluorophore (**Probe 1**). As shown in Figure 3, the process of turning on the fluorescence starts from a 1,4-addition of thiols to the α, β-unsaturated ketone in the probe. Subsequent spiro ring opening of fluorescein lights up the fluorescence (Ex. 485 nm, Em. 520 nm) which provides fluorescence enhancement by 61-fold compared with probe itself. The probe exhibits a similar fluorescence enhancement towards Cys, Hcy, and GSH. With fluorescein as a standard, the quantum yields of the thiols adducts with probe were determined to be 0.65, 0.91, and 0.47 for GSH, Cys, and Hcy, respectively. The probe is featured with a rapid

response and high sensitivity with a detection limit of 10−7–10−8 M. Further, the probe was proved to be capable of detecting the thiol status change in live P19 cells. More im-

response and high sensitivity with a detection limit of 10−7–10−<sup>8</sup> M. Further, the probe was proved to be capable of detecting the thiol status change in live P19 cells. More importantly, the probe is also the first reagent being used to monitor the thiols status in zebrafish. A significant fluorescence intensity decrease was observed when the cells or zebrafish were treated with a thiol trapping reagent, N-methylmaleimide (NMM) (40–50 µM). zebrafish. A significant fluorescence intensity decrease was observed when the cells or zebrafish were treated with a thiol trapping reagent, N-methylmaleimide (NMM) (40–50 μM). μM).

**Figure 3.** Structure of **Probe 1** and its reaction mechanism with RSH. **Figure 3.** Structure of **Probe 1** and its reaction mechanism with RSH. 

*Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 59

*Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 59

In 2011, Gun-Joong Kim et al. [36] reported a Michael addition reaction based fluorescent probe (**Probe 2**) for ratiometric imaging of cellular GSH. As illustrated in Figure 4, the probe has three parts, a coumarin moiety serving as the signaling fluorophore, a thiol reactive enone linker, and an *O*-hydroxyl group as an activation unit through a resonanceassisted hydrogen bond [69,70]. Once a reaction occurs between thiol (2-mercaptoethanol as a representative agent) and the Michael acceptor structure of the probe, the fluorescence emission band will shift from 553 to 466 nm (Ex. 420 nm). The ratio of the fluorescence intensity under these wavelength (F466 nm/F553 nm) changes ratiometrically upon reaction with thiol such as Hcy, Cys, and GSH. The probe was successfully applied for monitoring cellular thiol status in live cells. The fluorescence ratio (Fblue 410 nm–460 nm/Fgreen 490–540 nm) increased significantly when the cells were pretreated with R-lipoic acid, an enhancer of GSH. In contrast, a remarkable ratio decrease was observed when cells were pretreated with NEM, a scavenger of GSH. In 2011, Gun-Joong Kim et al. [36] reported a Michael addition reaction based fluorescent probe (**Probe 2**) for ratiometric imaging of cellular GSH. As illustrated in Figure 4, the probe has three parts, a coumarin moiety serving as the signaling fluorophore, a thiol reactive enone linker, and an *O*-hydroxyl group as an activation unit through a resonanceassisted hydrogen bond [69,70]. Once a reaction occurs between thiol (2-mercaptoethanol as a representative agent) and the Michael acceptor structure of the probe, the fluorescence emission band will shift from 553 to 466 nm (Ex. 420 nm). The ratio of the fluorescence intensity under these wavelength (F466 nm/F553 nm) changes ratiometrically upon reaction with thiol such as Hcy, Cys, and GSH. The probe was successfully applied for monitoring cellular thiol status in live cells. The fluorescence ratio (Fblue 410 nm–460 nm/Fgreen 490–540 nm) increased significantly when the cells were pretreated with R-lipoic acid, an enhancer of GSH. In contrast, a remarkable ratio decrease was observed when cells were pretreated with NEM, a scavenger of GSH. rescent probe (**Probe2**) for ratiometric imaging of cellular GSH. As illustrated in Figure 4, the probe has three parts, a coumarin moiety serving as the signaling fluorophore, a thiol reactive enone linker, and an *O*-hydroxyl group as an activation unit through a resonanceassisted hydrogen bond [69,70]. Once a reaction occurs between thiol (2-mercaptoethanol 466 nm553 nmwith thiol such as Hcy, Cys,and GSH. Theprobe was successfully applied formonitoring cellular thiol status in live cells. The fluorescence ratio (F/F) in-

**Figure 4.** Structure of **Probe 2** and its reaction with RSH. **Figure 4.** Structure of **Probe2**andits reaction with RSH.**Figure 4.** Structure of **Probe 2** and its reaction with RSH.

It is known that maleimide can react selectively toward thiols than other nucleophiles via a Michael addition reaction. In the meantime, maleimide can also serve as a fluorescence quencher in their conjugated form. Thus, maleimide has been widely utilized by many groups to build the thiols selective probes. For instance, in 2017, Hai Shu et al. [71] reported a rhodamine B-based probe (L1, **Probe 3**) with maleimide serving as a thiol recognition unit. Upon reaction with GSH, the probe can be turned on from non-fluorescent to a strong fluorescent thiol-rhodamine adduct (586 nm) due to a Michael addition reaction followed by a spirolactam ring opening (Figure 5). A more than 200-fold increase of fluorescence intensity was observed when excessive GSH was added. In addition, the fluorescence intensity was found to be linearly increased with an increase in GSH (2–26 μM) with a detection limit of 0.219 μM. The probe shows a high selectivity toward GSH than other thiols. Further, cellular experiments demonstrated that L1 can be used to monitor intracellular GSH in HepG-2 and HUVEC cells. Another similar approach was reported by Tao Liu et al. [72] in 2016 for selective detection of thiols in live cells with a high sensitivity (**Probe 4**). The detection limits were determined to be 0.085, 0.12, and 0.13 μM It is known that maleimide can react selectively toward thiols than other nucleophiles reported a rhodamine B-based probe (L1, **Probe 3**) with maleimide serving as a thiol recognition unit. Upon reaction with GSH, the probe can be turned on from non-fluoresfluorescence intensity was found to be linearly increased with an increase in GSH (2–26 μM) with a detection limit of 0.219 μM. The probe shows a high selectivity toward GSH than other thiols. Further, cellular experiments demonstrated that L1 can be used to monsensitivity (**Probe4**). The detection limits were determined to be 0.085, 0.12,and 0.13 μM for GSH, Hcy, and Cys, respectively. Similarly, the probe was also successfully applied in It is known that maleimide can react selectively toward thiols than other nucleophiles via a Michael addition reaction. In the meantime, maleimide can also serve as a fluorescence quencher in their conjugated form. Thus, maleimide has been widely utilized by many groups to build the thiols selective probes. For instance, in 2017, Hai Shu et al. [71] reported a rhodamine B-based probe (L1, **Probe 3**) with maleimide serving as a thiol recognition unit. Upon reaction with GSH, the probe can be turned on from non-fluorescent to a strong fluorescent thiol-rhodamine adduct (586 nm) due to a Michael addition reaction followed by a spirolactam ring opening (Figure 5). A more than 200-fold increase of fluorescence intensity was observed when excessive GSH was added. In addition, the fluorescence intensity was found to be linearly increased with an increase in GSH (2–26 µM) with a detection limit of 0.219 µM. The probe shows a high selectivity toward GSH than other thiols. Further, cellular experiments demonstrated that L1 can be used to monitor intracellular GSH in HepG-2 and HUVEC cells. Another similar approach was reported by Tao Liu et al. [72] in 2016 for selective detection of thiols in live cells with a high sensitivity (**Probe 4**). The detection limits were determined to be 0.085, 0.12, and 0.13 µM

for GSH, Hcy, and Cys, respectively. Similarly, the probe was also successfully applied in

for GSH, Hcy, and Cys, respectively. Similarly, the probe was also successfully applied in the determination of intracellular GSH in live HepG2 cells. *Molecules* **2021**, *26*, x FOR PEER REVIEW 6 of 59

**Figure 5.** Structures of **Probes 3** and **4** and their reactions with GSH or thiols, respectively. **Figure 5.** Structures of **Probes 3** and **4** and their reactions with GSH or thiols, respectively.

In contrast to traditional fluorescent methods, reagents which can detect a thiol in a reversible manner was developed. In 2015, Xiqian Jiang et al. [73] reported a reversible and ratiometric fluorescent probe-ThiolQuant Green (TQ Green, **Probe 5**) for quantitative imaging of GSH. The probe is composed of a 7-amino coumarin group as the fluorophore and an aromatic structure to extend the absorption wavelength of coumarin. These two parts are linked together by a thiol reactive group, Michael acceptor. The probe's reaction with GSH is reversible when GSH is depleted. TQ Green (Ex. 488 nm, Em. 590 nm) is turned into to TQ Green-GSH (Ex. 405 nm, Em. 463 nm) upon reaction with GSH (Figure 6). The ratio of signal intensities (absorbance or fluorescence) between TQ Green-GSH and TQ Green is proportional to the GSH concentration enabling the ratiometric detection of GSH with a detection limit of 20 nM. No reaction was observed when the probe was exposed to an excessive ratio of bovine serum albumin (BSA) indicating the probe does not react with PSH. Cellular experiments with multiple cell lines, including 3T3-L1, HepG2, PANC-1, and PANC-28 cells, proved that the GSH concentration obtained from TQ Green live imaging were well correlated with the values achieved from bulk lysate measure-In contrast to traditional fluorescent methods, reagents which can detect a thiol in a reversible manner was developed. In 2015, Xiqian Jiang et al. [73] reported a reversible and ratiometric fluorescent probe-ThiolQuant Green (TQ Green, **Probe 5**) for quantitative imaging of GSH. The probe is composed of a 7-amino coumarin group as the fluorophore and an aromatic structure to extend the absorption wavelength of coumarin. These two parts are linked together by a thiol reactive group, Michael acceptor. The probe's reaction with GSH is reversible when GSH is depleted. TQ Green (Ex. 488 nm, Em. 590 nm) is turned into to TQ Green-GSH (Ex. 405 nm, Em. 463 nm) upon reaction with GSH (Figure 6). The ratio of signal intensities (absorbance or fluorescence) between TQ Green-GSH and TQ Green is proportional to the GSH concentration enabling the ratiometric detection of GSH with a detection limit of 20 nM. No reaction was observed when the probe was exposed to an excessive ratio of bovine serum albumin (BSA) indicating the probe does not react with PSH. Cellular experiments with multiple cell lines, including 3T3-L1, HepG2, PANC-1, and PANC-28 cells, proved that the GSH concentration obtained from TQ Green live imaging were well correlated with the values achieved from bulk lysate measurements. The probe can well reflect the GSH level changes in live cells when the GSH level was reduced by diethylmaleate (DEM). One drawback of the method is that the reverse reaction between GSH and TQ Green is slow leading to the inability of the probe for a quick detection of a decrease of GSH in live cells.

ments. The probe can well reflect the GSH level changes in live cells when the GSH level was reduced by diethylmaleate (DEM). One drawback of the method is that the reverse reaction between GSH and TQ Green is slow leading to the inability of the probe for a

**Figure 6.** Structures of **Probes 5**, **6** and their reversible reactions with GSH.

quick detection of a decrease of GSH in live cells.

**Figure 5.** Structures of **Probes 3** and **4** and their reactions with GSH or thiols, respectively.

In contrast to traditional fluorescent methods, reagents which can detect a thiol in a

reversible manner was developed. In 2015, Xiqian Jiang et al. [73] reported a reversible and ratiometric fluorescent probe-ThiolQuant Green (TQ Green, **Probe 5**) for quantitative imaging of GSH. The probe is composed of a 7-amino coumarin group as the fluorophore and an aromatic structure to extend the absorption wavelength of coumarin. These two parts are linked together by a thiol reactive group, Michael acceptor. The probe's reaction with GSH is reversible when GSH is depleted. TQ Green (Ex. 488 nm, Em. 590 nm) is turned into to TQ Green-GSH (Ex. 405 nm, Em. 463 nm) upon reaction with GSH (Figure 6). The ratio of signal intensities (absorbance or fluorescence) between TQ Green-GSH and TQ Green is proportional to the GSH concentration enabling the ratiometric detection of GSH with a detection limit of 20 nM. No reaction was observed when the probe was exposed to an excessive ratio of bovine serum albumin (BSA) indicating the probe does not react with PSH. Cellular experiments with multiple cell lines, including 3T3-L1, HepG2, PANC-1, and PANC-28 cells, proved that the GSH concentration obtained from TQ Green live imaging were well correlated with the values achieved from bulk lysate measurements. The probe can well reflect the GSH level changes in live cells when the GSH level was reduced by diethylmaleate (DEM). One drawback of the method is that the reverse reaction between GSH and TQ Green is slow leading to the inability of the probe for a

**Figure 6. Figure 6.**  Structures of Structures of **Probes 5 Probes 5**, **6** and their reversible reactions with GSH. , **6** and their reversible reactions with GSH.

quick detection of a decrease of GSH in live cells.

Continuing with their work, Xiqian Jiang et al. [74] published another reversible fluorescent probe named as RealThiol (RT, **Probe 6**) in 2017 for quantitatively real-time monitor of intracellular GSH in live cells with a much higher reaction rate (50-fold faster than TQ Green). RT was developed based on a series of optimization of TQ Green to enable a quantitative real-time imaging of GSH in living cells with a much-enhanced reaction rate for both forward and reverse directions. Similar to TQ Green, RT shows ratiometric fluorescence response with a superb linear relationship to various concentrations of GSH in a range of 1–10 mM. In contrast to TQ Green which exhibits a second-order forward reaction rate of 0.15 M−<sup>1</sup> s −1 , the second-order reaction rate for RT is 7.5 M−<sup>1</sup> s <sup>−</sup><sup>1</sup> which is 50 times faster than TQ Green. No significant fluorescence change was observed when RT was mixed with cell lysates suggesting the inability of RT to react with PSH. Impressively, the probe could well reflect the GSH level change when HeLa cells were treated with different concentrations of H2O2. Later, the probe was demonstrated to quantitatively monitor GSH levels when cells were consecutively treated with H2O<sup>2</sup> (500 µM) and GSH ester (100 µM). Further, with the aid of RT, an enhanced antioxidant capability of activated neurons and dynamic GSH changes during ferroptosis were observed. RT was successfully applied for high-throughput quantification of GSH in single cells via flow cytometry.

It should be noted that a number of other reversible fluorescent probes have been developed for quantitatively monitoring GSH in real time in live cells with various fluorophores and sensing mechanisms. These probes include probe QG-1 developed by Zhixue Liu et al. [75], probe RP-1 and RP-2 by Ming Tian et al. [76,77], and a number of single-molecule localization microscopy (SMLM) applicable probes developed from Urano groups [78].

2.1.2. Detection of NPSH via a Nucleophilic Aromatic Substitution (SNAr) Reaction Using Halogen, Ether, Thioether as a Leaving Group

SNAr reaction is another widely used mechanism for imaging thiols in live cells since the thiol group can easily displace a leaving group that is attached to an aromatic ring in a fluorophore by a substitution reaction to form a thioether. These leaving groups include halogen, ether, thioether, etc. To be noted here, SNAr substitution reactions have also been commonly used to discriminatively detect Cys/Hcy due to a rearrangement reaction occurring to the thioether formed in the substitution reaction. The rearrangement reaction only occurs to the thioether derived from Cys and Hcy (Figure 7).

Cys and Hcy, while the rapid intramolecular displacement of S to N will shift the emission

maxima back to around 564 nm for Cys and Hcy, with GSH remaining at 588 nm.

**Figure 7. Probe 7** and its reaction with GSH or Cys (*n* = 1)/Hcy (*n* = 2). **Figure 7. Probe 7** and its reaction with GSH or Cys (*n* = 1)/Hcy (*n* = 2).

Detection of NPSH via a SNAr Reaction Using ether as a Leaving Group Detection of NPSH via a SNAr Reaction Using Halogen as a Leaving Group

In addition to halogen, other leaving groups, such as ether or thioether are also widely used as a thiol recognition unit for building a thiol selective probe. For example, Xinghui Gao et al. [80] developed a probe (**Probe 8**) in 2015 for detection of GSH and Cys in different emission channels by connecting 7-nitrobenzofurazan with resorufin through the ether bond in which the ether bond also served as a thiols recognition unit. Reaction with thiols (GSH, or Cys) yields a free strong fluorescent resorufin (Em. 585 nm) and a thiol conjugated nitrobenzofurazan. However, the thiol conjugated notrobenzofurazn derived from Cys will undergo an intramolecular rearrangement to generate a new fluorescent product with λem = 540 nm while the thiol conjugated notrobenzofurazn derived from GSH remained unchanged (Figure 8). Measuring the fluorescence intensity from resorufin (Em. 585 nm) enabled the determination of the total thiols (GSH, and Cys/Hcy) In 2012, Liya Niu and co-workers [29] reported their efforts on developing a fluorescent probe (**Probe 7**) for highly selective detection of GSH over Cys/Hcy based on monochlorinated BODIPY. As shown in Figure 7, a SNAr reaction occurred between the sulfur group (GSH, Cys, or Hcy) and the monochlorinated BODIPY resulting in a thioether. A fluorescence emission wavelength red shift (556 to ~588 nm) occurred after the reaction, and the ratio of fluorescence intensity (I588/I556) linearly increased as an increase in the concentration of GSH (0−<sup>60</sup> <sup>µ</sup>M) with the detection limit of 8.6 <sup>×</sup> <sup>10</sup>−<sup>8</sup> M, enabling the quantification of intracellular GSH in live cells. The probe was successfully applied for detecting the GSH levels' change when the cells were pretreated with diamide [79] in HeLa cells. The probe was claimed to be the first ratiometric fluorescent sensor developed for the selective detection of GSH over Cys/Hcy.

while measuring the fluorescence intensity of both resorufin and the thiol conjugated nitrobenzofurazan provided a method to distinguish GSH from Cys. The detection limits for GSH and Cys were reported to be 0.07 μM and 0.13 μM respectively with a linear relationship between the fluorescence intensity and the concentration of GSH (1–18 μM) and Cys (3.1–90 μM, 1–40 μM), respectively. The probe was employed to detect Cys and GSH in human plasma. A quick intramolecular displacement occurred with the thioether generated from Cys and Hcy to replace the thiolate with the amino group to form amino-substituted BODIPY, while the thioether generated from GSH was not able to undergo the intramolecular displacement due to the unavailability of an amino group. Since the photophysics of BODIPY derivatives is sensitive to substituents, the amino-substituted BODIPY exhibit a relative weaker and blue shifted fluorescence which enable the selective detection of GSH over Cys and Hcy, while the rapid intramolecular displacement of S to N will shift the emission maxima back to around 564 nm for Cys and Hcy, with GSH remaining at 588 nm.

#### Detection of NPSH via a SNAr Reaction Using ether as a Leaving Group

**Figure 8.** Structure of **Probe 8** and its reaction with GSH and Cys. In addition to halogen, other leaving groups, such as ether or thioether are also widely used as a thiol recognition unit for building a thiol selective probe. For example, Xinghui Gao et al. [80] developed a probe (**Probe 8**) in 2015 for detection of GSH and Cys in different emission channels by connecting 7-nitrobenzofurazan with resorufin through the ether bond in which the ether bond also served as a thiols recognition unit. Reaction with thiols (GSH, or Cys) yields a free strong fluorescent resorufin (Em. 585 nm) and a thiol conjugated nitrobenzofurazan. However, the thiol conjugated notrobenzofurazn derived from Cys will undergo an intramolecular rearrangement to generate a new fluorescent product with λem = 540 nm while the thiol conjugated notrobenzofurazn derived from GSH remained unchanged (Figure 8). Measuring the fluorescence intensity from resorufin (Em. 585 nm) enabled the determination of the total thiols (GSH, and Cys/Hcy) while measuring the fluorescence intensity of both resorufin and the thiol conjugated nitrobenzofurazan provided a method to distinguish GSH from Cys. The detection limits for GSH and Cys were reported to be 0.07 µM and 0.13 µM respectively with a linear relationship between

the fluorescence intensity and the concentration of GSH (1–18 µM) and Cys (3.1–90 µM, 1–40 µM), respectively. The probe was employed to detect Cys and GSH in human plasma. and Cys (3.1–90 μM, 1–40 μM), respectively. The probe was employed to detect Cys and GSH in human plasma.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 8 of 59

**Figure 7. Probe 7** and its reaction with GSH or Cys (*n* = 1)/Hcy (*n* = 2).

Detection of NPSH via a SNAr Reaction Using ether as a Leaving Group

In addition to halogen, other leaving groups, such as ether or thioether are also widely used as a thiol recognition unit for building a thiol selective probe. For example, Xinghui Gao et al. [80] developed a probe (**Probe 8**) in 2015 for detection of GSH and Cys in different emission channels by connecting 7-nitrobenzofurazan with resorufin through the ether bond in which the ether bond also served as a thiols recognition unit. Reaction with thiols (GSH, or Cys) yields a free strong fluorescent resorufin (Em. 585 nm) and a thiol conjugated nitrobenzofurazan. However, the thiol conjugated notrobenzofurazn derived from Cys will undergo an intramolecular rearrangement to generate a new fluorescent product with λem = 540 nm while the thiol conjugated notrobenzofurazn derived from GSH remained unchanged (Figure 8). Measuring the fluorescence intensity from resorufin (Em. 585 nm) enabled the determination of the total thiols (GSH, and Cys/Hcy) while measuring the fluorescence intensity of both resorufin and the thiol conjugated nitrobenzofurazan provided a method to distinguish GSH from Cys. The detection limits for GSH and Cys were reported to be 0.07 μM and 0.13 μM respectively with a linear relationship between the fluorescence intensity and the concentration of GSH (1–18 μM)

Cys and Hcy, while the rapid intramolecular displacement of S to N will shift the emission

maxima back to around 564 nm for Cys and Hcy, with GSH remaining at 588 nm.

**Figure 8.** Structure of **Probe 8** and its reaction with GSH and Cys. **Figure 8.** Structure of **Probe 8** and its reaction with GSH and Cys. Xilei Xie et al. [81] reported a probe (Res-Biot, **Probe 9**) in 2017 for selective detection

Xilei Xie et al. [81] reported a probe (Res-Biot, **Probe 9**) in 2017 for selective detection of GSH based on the ether bond. In this approach, pyrimidine moiety was for the first time investigated and optimized as a unique recognition unit for thiols. The pyrimidine moiety was connected with fluorophore resorufin via an ether linkage to generate probe Res-Biot. Upon reaction with a thiol, a high fluorescent resorufin is released with a strong fluorescence at 585 nm (Figure 9). Moreover, the fluorescence intensity at 585 nm was increased proportionally to the concentrations of GSH in a range of 0−20 µM with a detection limit of 0.29 µM. The probe was successfully used to detect a decrease in intracellular GSH concentration down-regulated by an oxidative stress inducer, 12-myristate 13-acetate (PMA), or by L-buthionine sulfoximine (BSO)—an inhibitor of γ-glutamylcysteine synthetase. of GSH based on the ether bond. In this approach, pyrimidine moiety was for the first time investigated and optimized as a unique recognition unit for thiols. The pyrimidine moiety was connected with fluorophore resorufin via an ether linkage to generate probe Res-Biot. Upon reaction with a thiol, a high fluorescent resorufin is released with a strong fluorescence at 585 nm (Figure 9). Moreover, the fluorescence intensity at 585 nm was increased proportionally to the concentrations of GSH in a range of 0−20 μM with a detection limit of 0.29 μM. The probe was successfully used to detect a decrease in intracellular GSH concentration down-regulated by an oxidative stress inducer, 12-myristate 13 acetate (PMA), or by L-buthionine sulfoximine (BSO)—an inhibitor of γ-glutamylcysteine synthetase.

**Figure 9. Probe 9** and its reaction with GSH. **Figure 9. Probe 9** and its reaction with GSH.

[42,82]

Detection of NPSH via a SNAr Reaction Using Thioether as a Leaving Group

Detection of NPSH via a SNAr Reaction Using Thioether as a Leaving Group In 2012, Yinghong Li et al. from our group reported a thiol specific probe [42] (GUALY's reagent, **Probe 10**) based on a symmetric benzofurazan sulfide for imaging and quantifying total thiols in live cells. The symmetric benzofurazan sulfide was constructed by combining two benzofurazan fluorophores with a thioether group (Figure 10). The probe itself is not fluorescent due to the self-quenching of the symmetric structure but turns into a fluorescent product (Ex. 430 nm, Em. 520 nm) rapidly upon reaction with thiols through a thiol specific thiol–sulfide exchange reaction. GUALY's reagent has been successfully applied for imaging and quantifying total thiols (PSH+NPSH) in live cells. In 2012, Yinghong Li et al. from our group reported a thiol specific probe [42] (GUALY's reagent, **Probe 10**) based on a symmetric benzofurazan sulfide for imaging and quantifying total thiols in live cells. The symmetric benzofurazan sulfide was constructed by combining two benzofurazan fluorophores with a thioether group (Figure 10). The probe itself is not fluorescent due to the self-quenching of the symmetric structure but turns into a fluorescent product (Ex. 430 nm, Em. 520 nm) rapidly upon reaction with thiols through a thiol specific thiol–sulfide exchange reaction. GUALY's reagent has been successfully applied for imaging and quantifying total thiols (PSH+NPSH) in live cells [42,82].

Jing Liu et al. [83] reported a GSH discriminating fluorescent probe (**Probe 11,** Figure 11) in 2014 based on pyronin B as the fluorophore and thioether as the thiol recognition group. In the approach, A methoxythiophenol group was connected to the fluorescent pyronin moiety to serve as a fluorescence quencher via a photoinduced electron transfer (PET) process. Reaction with thiols (GSH, Cys, Hcy) through SNAr reaction generates a highly fluorescent thiol conjugated pyronin with λem for GSH-pyronin and Cys/Hcy-pyronin at 622 nm and 546 nm respectively due to the additional intramolecular rearrangements of Cys/Hcy. The probe has been successfully applied for simultaneously detection

**Figure 10. Probe 10** and its reaction with thiols.

of Cys/Hcy and GSH in B16 cells.

Detection of NPSH via a SNAr Reaction Using Thioether as a Leaving Group

In 2012, Yinghong Li et al. from our group reported a thiol specific probe [42] (GUALY's reagent, **Probe 10**) based on a symmetric benzofurazan sulfide for imaging and quantifying total thiols in live cells. The symmetric benzofurazan sulfide was constructed by combining two benzofurazan fluorophores with a thioether group (Figure 10). The probe itself is not fluorescent due to the self-quenching of the symmetric structure but turns into a fluorescent product (Ex. 430 nm, Em. 520 nm) rapidly upon reaction with thiols through a thiol specific thiol–sulfide exchange reaction. GUALY's reagent has been successfully applied for imaging and quantifying total thiols (PSH+NPSH) in live cells.

Xilei Xie et al. [81] reported a probe (Res-Biot, **Probe 9**) in 2017 for selective detection

of GSH based on the ether bond. In this approach, pyrimidine moiety was for the first time investigated and optimized as a unique recognition unit for thiols. The pyrimidine moiety was connected with fluorophore resorufin via an ether linkage to generate probe Res-Biot. Upon reaction with a thiol, a high fluorescent resorufin is released with a strong fluorescence at 585 nm (Figure 9). Moreover, the fluorescence intensity at 585 nm was increased proportionally to the concentrations of GSH in a range of 0−20 μM with a detection limit of 0.29 μM. The probe was successfully used to detect a decrease in intracellular GSH concentration down-regulated by an oxidative stress inducer, 12-myristate 13 acetate (PMA), or by L-buthionine sulfoximine (BSO)—an inhibitor of γ-glutamylcysteine

**Figure 10. Probe 10** and its reaction with thiols. **Figure 10. Probe 10** and its reaction with thiols.

**Figure 9. Probe 9** and its reaction with GSH.

synthetase.

[42,82]

Jing Liu et al. [83] reported a GSH discriminating fluorescent probe (**Probe 11,** Figure 11) in 2014 based on pyronin B as the fluorophore and thioether as the thiol recognition group. In the approach, A methoxythiophenol group was connected to the fluorescent pyronin moiety to serve as a fluorescence quencher via a photoinduced electron transfer (PET) process. Reaction with thiols (GSH, Cys, Hcy) through SNAr reaction generates a highly fluorescent thiol conjugated pyronin with λem for GSH-pyronin and Cys/Hcy-pyronin at 622 nm and 546 nm respectively due to the additional intramolecular rearrange-Jing Liu et al. [83] reported a GSH discriminating fluorescent probe (**Probe 11**, Figure 11) in 2014 based on pyronin B as the fluorophore and thioether as the thiol recognition group. In the approach, A methoxythiophenol group was connected to the fluorescent pyronin moiety to serve as a fluorescence quencher via a photoinduced electron transfer (PET) process. Reaction with thiols (GSH, Cys, Hcy) through SNAr reaction generates a highly fluorescent thiol conjugated pyronin with λem for GSH-pyronin and Cys/Hcy-pyronin at 622 nm and 546 nm respectively due to the additional intramolecular rearrangements of Cys/Hcy. The probe has been successfully applied for simultaneously detection of Cys/Hcy and GSH in B16 cells. *Molecules* **2021**, *26*, x FOR PEER REVIEW 10 of 59

ments of Cys/Hcy. The probe has been successfully applied for simultaneously detection

**Figure 11. Probe 11** and its reaction with GSH or Cys/Hcy. **Figure 11. Probe 11** and its reaction with GSH or Cys/Hcy.

In 2015, Lun Song et al. [84] reported two water-soluble colorimetric and turn-on fluorescent probes (STP1, 2, **Probes 12, 13**) for selective detection of GSH using thioether as a thiol recognition group and naphthalimide as the fluorophore reporter. The probes are non-fluorescent due to a PET process from the naphthalimide electron donor to 4-nitrobenzene acceptor via the thioether bond (Figure 12). Upon reaction with GSH, a remarkable enhancement of fluorescence (~90-fold) at 487 nm was observed. A linear relationship was found between the fluorescence intensity (490 nm) and GSH concentration in the range of 0–100 μM with the detection limit of 84 nM. It should be noted that a linear relation was also found between the fluorescence intensity and thiol-containing proteins bo-In 2015, Lun Song et al. [84] reported two water-soluble colorimetric and turn-on fluorescent probes (STP1, 2, **Probes 12**, **13**) for selective detection of GSH using thioether as a thiol recognition group and naphthalimide as the fluorophore reporter. The probes are non-fluorescent due to a PET process from the naphthalimide electron donor to 4 nitrobenzene acceptor via the thioether bond (Figure 12). Upon reaction with GSH, a remarkable enhancement of fluorescence (~90-fold) at 487 nm was observed. A linear relationship was found between the fluorescence intensity (490 nm) and GSH concentration in the range of 0–100 µM with the detection limit of 84 nM. It should be noted that a linear relation was also found between the fluorescence intensity and thiol-containing proteins bovine serum albumin (BSA) and ovalbumin (OVA) suggesting that the probes can also imaging PSH. These probes have been demonstrated to be capable for fluorescence imaging of GSH in HeLa cells.

**Figure 12.** Structures of **Probes 12**, **13** and their reaction with GSH or Cys/Hcy.

2.1.3. Detection of NPSH via Cleavage of Sulfonamide or Sulfonate Ester

In 2014, Masafumi Yoshida et al. [85] reported a novel cell-membrane-permeable flu-

orescent probe (**Probe 14**) for detection of GSH based on the cleavage of sulfonamide as the thiol responsive mechanism. As illustrated in Figure 13, a fluorophore hydroxymethylrhodamine green (HMRG) and a 2,4-dinitrobenzenesulfonyl (DNBS) moiety were combined by a sulfonamide group in the approach,. The fluorescence of the probe was quenched by two fluorescence quenching mechanisms, intramolecular spirocyclization (close-ring form of HMRG) and intramolecular PET between HMRG and DNBS. Reaction with a thiol breaks the quenching by cleaving the sulfonamide bond to release a free open-ring high fluorescent HMRG with an up to 7000-fold increase in fluorescence intensity (520 nm) and the increase was proportional to the concentration of GSH around

vine serum albumin (BSA) and ovalbumin (OVA) suggesting that the probes can also imaging PSH. These probes have been demonstrated to be capable for fluorescence imaging

Detection of NPSH via Cleavage of Sulfonamide

of GSH in HeLa cells.

**Figure 11. Probe 11** and its reaction with GSH or Cys/Hcy.

In 2015, Lun Song et al. [84] reported two water-soluble colorimetric and turn-on fluorescent probes (STP1, 2, **Probes 12, 13**) for selective detection of GSH using thioether as a thiol recognition group and naphthalimide as the fluorophore reporter. The probes are non-fluorescent due to a PET process from the naphthalimide electron donor to 4-nitrobenzene acceptor via the thioether bond (Figure 12). Upon reaction with GSH, a remarkable enhancement of fluorescence (~90-fold) at 487 nm was observed. A linear relationship was found between the fluorescence intensity (490 nm) and GSH concentration in the range of 0–100 μM with the detection limit of 84 nM. It should be noted that a linear relation was also found between the fluorescence intensity and thiol-containing proteins bovine serum albumin (BSA) and ovalbumin (OVA) suggesting that the probes can also imaging PSH. These probes have been demonstrated to be capable for fluorescence imaging

of GSH in HeLa cells.

**Figure 12.** Structures of **Probes 12**, **13** and their reaction with GSH or Cys/Hcy. **Figure 12.** Structures of **Probes 12**, **13** and their reaction with GSH or Cys/Hcy.

2.1.3. Detection of NPSH via Cleavage of Sulfonamide or Sulfonate Ester Detection of NPSH via Cleavage of Sulfonamide 2.1.3. Detection of NPSH via Cleavage of Sulfonamide or Sulfonate Ester Detection of NPSH via Cleavage of Sulfonamide

In 2014, Masafumi Yoshida et al. [85] reported a novel cell-membrane-permeable fluorescent probe (**Probe 14**) for detection of GSH based on the cleavage of sulfonamide as the thiol responsive mechanism. As illustrated in Figure 13, a fluorophore hydroxymethylrhodamine green (HMRG) and a 2,4-dinitrobenzenesulfonyl (DNBS) moiety were combined by a sulfonamide group in the approach,. The fluorescence of the probe was quenched by two fluorescence quenching mechanisms, intramolecular spirocyclization (close-ring form of HMRG) and intramolecular PET between HMRG and DNBS. Reaction with a thiol breaks the quenching by cleaving the sulfonamide bond to release a free open-ring high fluorescent HMRG with an up to 7000-fold increase in fluorescence intensity (520 nm) and the increase was proportional to the concentration of GSH around In 2014, Masafumi Yoshida et al. [85] reported a novel cell-membrane-permeable fluorescent probe (**Probe 14**) for detection of GSH based on the cleavage of sulfonamide as the thiol responsive mechanism. As illustrated in Figure 13, a fluorophore hydroxymethylrhodamine green (HMRG) and a 2,4-dinitrobenzenesulfonyl (DNBS) moiety were combined by a sulfonamide group in the approach. The fluorescence of the probe was quenched by two fluorescence quenching mechanisms, intramolecular spirocyclization (close-ring form of HMRG) and intramolecular PET between HMRG and DNBS. Reaction with a thiol breaks the quenching by cleaving the sulfonamide bond to release a free open-ring high fluorescent HMRG with an up to 7000-fold increase in fluorescence intensity (520 nm) and the increase was proportional to the concentration of GSH around the physiological concentration. The probe was successfully applied for the detection of GSH levels' change in live cells. Further, the probe was used to detect tumor nodules in tumor bearing mice of SHIN-3 ovarian cancer by taking advantage of the fact that GSH level in tumor tissues is higher than normal tissues. *Molecules* **2021**, *26*, x FOR PEER REVIEW 11 of 59 the physiological concentration. The probe was successfully applied for the detection of GSH levels' change in live cells. Further, the probe was used to detect tumor nodules in tumor bearing mice of SHIN-3 ovarian cancer by taking advantage of the fact that GSH level in tumor tissues is higher than normal tissues.

**Figure 13.** Structure of **Probe 14** and its reaction with thiols. **Figure 13.** Structure of **Probe 14** and its reaction with thiols.

Fluorophores with emission in red or near NIR region have the advantages of low fluorescence background, deeper penetration, and less damages to live cells or tissues [86,87]. In 2014, a cyanine-based fluorescent probe (**Probe 15**, Figure 14) for highly selective detection of GSH in cells and live mouse tissues have been reported by Jun Yin et al. [88]. The probe utilizes a 5-(dimethylamino)naphthalenesulfonamide group for highly selective detection of GSH over Cys and Hcy. In vitro cellular experiments with HeLa cells proved that the probe was capable of monitoring GSH in live cells. When the cells were pretreated with *N*-methylmaleimide (NMM, a thiol-blocking agent) followed by treatment with the probe, it showed no red fluorescence while strong fluorescence was observed when no NMM was used confirming that the probe was detecting thiols. In addition, only a minor change of fluorescence intensity was observed when Cys (100 μM) or Hcy (100 μM) was used to the NMM-pretreated HeLa cells. In contrast, significant red Fluorophores with emission in red or near NIR region have the advantages of low fluorescence background, deeper penetration, and less damages to live cells or tissues [86,87]. In 2014, a cyanine-based fluorescent probe (**Probe 15**, Figure 14) for highly selective detection of GSH in cells and live mouse tissues have been reported by Jun Yin et al. [88]. The probe utilizes a 5-(dimethylamino)naphthalenesulfonamide group for highly selective detection of GSH over Cys and Hcy. In vitro cellular experiments with HeLa cells proved that the probe was capable of monitoring GSH in live cells. When the cells were pretreated with *N*-methylmaleimide (NMM, a thiol-blocking agent) followed by treatment with the probe, it showed no red fluorescence while strong fluorescence was observed when no NMM was used confirming that the probe was detecting thiols. In addition, only a minor change of fluorescence intensity was observed when Cys (100 µM) or Hcy (100 µM) was used to the NMM-pretreated HeLa cells. In contrast, significant red fluorescence emission was

fluorescence emission was observed when GSH (100 μM) was added suggesting that the probe can selectively detect GSH in the presence of Cys or Hcy. The probe was success-

In 2019, Xie Han et al. [89] reported an aggregation-induced emission (AIE) probe (TPE-Np, **Probe 16**, Figure 15) for selective detection of GSH. The probe is designed by modifying the widely used AIE fluorophore tetraphenylethene with a sulfonyl-based naphthalimide. Cleavage of sulfonamide by GSH induces a remarkable increase of fluorescence intensity at 496 nm which shows a good linear relationship with GSH concentrations in the range of 0–50 μM with a detection limit of 1.9 μM. The probe shows a high selectivity toward GSH than Cys and Hcy in the presence of cetyltrimethylammonium bromide. In addition, poly(ethyleneglycol)–polyethylenimine (PEG-PEI) nanogel was used as a carrier to cross-link TPE-Np to improve its solubility and biocompatibility. The probe was successfully applied for imaging and monitoring intracellular GSH level in

**Figure 14.** Structure of **Probe 15** and its reaction with GSH.

and spleen.

MCF-7 cells.

observed when GSH (100 µM) was added suggesting that the probe can selectively detect GSH in the presence of Cys or Hcy. The probe was successfully applied for monitoring the GSH levels in mouse tissues such as liver, kidney, lung, and spleen. fluorescence emission was observed when GSH (100 μM) was added suggesting that the probe can selectively detect GSH in the presence of Cys or Hcy. The probe was successfully applied for monitoring the GSH levels in mouse tissues such as liver, kidney, lung, and spleen.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 11 of 59

level in tumor tissues is higher than normal tissues.

**Figure 13.** Structure of **Probe 14** and its reaction with thiols.

the physiological concentration. The probe was successfully applied for the detection of GSH levels' change in live cells. Further, the probe was used to detect tumor nodules in tumor bearing mice of SHIN-3 ovarian cancer by taking advantage of the fact that GSH

Fluorophores with emission in red or near NIR region have the advantages of low fluorescence background, deeper penetration, and less damages to live cells or tissues [86,87]. In 2014, a cyanine-based fluorescent probe (**Probe 15**, Figure 14) for highly selective detection of GSH in cells and live mouse tissues have been reported by Jun Yin et al. [88]. The probe utilizes a 5-(dimethylamino)naphthalenesulfonamide group for highly selective detection of GSH over Cys and Hcy. In vitro cellular experiments with HeLa cells proved that the probe was capable of monitoring GSH in live cells. When the cells were pretreated with *N*-methylmaleimide (NMM, a thiol-blocking agent) followed by treatment with the probe, it showed no red fluorescence while strong fluorescence was observed when no NMM was used confirming that the probe was detecting thiols. In addition, only a minor change of fluorescence intensity was observed when Cys (100 μM) or Hcy (100 μM) was used to the NMM-pretreated HeLa cells. In contrast, significant red

**Figure 14.** Structure of **Probe 15** and its reaction with GSH. **Figure 14.** Structure of **Probe 15** and its reaction with GSH.

In 2019, Xie Han et al. [89] reported an aggregation-induced emission (AIE) probe (TPE-Np, **Probe 16**, Figure 15) for selective detection of GSH. The probe is designed by modifying the widely used AIE fluorophore tetraphenylethene with a sulfonyl-based naphthalimide. Cleavage of sulfonamide by GSH induces a remarkable increase of fluorescence intensity at 496 nm which shows a good linear relationship with GSH concentrations in the range of 0–50 μM with a detection limit of 1.9 μM. The probe shows a high selectivity toward GSH than Cys and Hcy in the presence of cetyltrimethylammonium bromide. In addition, poly(ethyleneglycol)–polyethylenimine (PEG-PEI) nanogel was used as a carrier to cross-link TPE-Np to improve its solubility and biocompatibility. The probe was successfully applied for imaging and monitoring intracellular GSH level in In 2019, Xie Han et al. [89] reported an aggregation-induced emission (AIE) probe (TPE-Np, **Probe 16**, Figure 15) for selective detection of GSH. The probe is designed by modifying the widely used AIE fluorophore tetraphenylethene with a sulfonyl-based naphthalimide. Cleavage of sulfonamide by GSH induces a remarkable increase of fluorescence intensity at 496 nm which shows a good linear relationship with GSH concentrations in the range of 0–50 µM with a detection limit of 1.9 µM. The probe shows a high selectivity toward GSH than Cys and Hcy in the presence of cetyltrimethylammonium bromide. In addition, poly(ethyleneglycol)–polyethylenimine (PEG-PEI) nanogel was used as a carrier to crosslink TPE-Np to improve its solubility and biocompatibility. The probe was successfully applied for imaging and monitoring intracellular GSH level in MCF-7 cells. *Molecules* **2021**, *26*, x FOR PEER REVIEW 12 of 59

**Figure 15.** Structure of **Probe 16**. **Figure 15.** Structure of **Probe 16**.

MCF-7 cells.

Detection of NPSH via Cleavage of Sulfonate Ester

Detection of NPSH via Cleavage of Sulfonate Ester Dicyanomethylene-4H-pyran, one of the fluorophores with emission in red or NIR region, is featured with its better stability than other NIR fluorophores such as cyanine and squaraine. In 2014, Meng Li et al. [61] reported a colorimetric and NIR fluorescence turn-on thiol probe (**Probe 17**, Figure 16) based on dicyanomethylene-4H-pyran as the fluorophore and 2,4-dinitrobenzenesulfonyl (DNBS) as the thiol recognition group and also as the fluorescence quencher. A benzene unit was introduced to the dicyanopyran to extend its conjugated system to make its emission wavelength into the desired NIR region [90]. The fluorescence of the compound switched from off to on when the DNBS was Dicyanomethylene-4H-pyran, one of the fluorophores with emission in red or NIR region, is featured with its better stability than other NIR fluorophores such as cyanine and squaraine. In 2014, Meng Li et al. [61] reported a colorimetric and NIR fluorescence turn-on thiol probe (**Probe 17**, Figure 16) based on dicyanomethylene-4H-pyran as the fluorophore and 2,4-dinitrobenzenesulfonyl (DNBS) as the thiol recognition group and also as the fluorescence quencher. A benzene unit was introduced to the dicyanopyran to extend its conjugated system to make its emission wavelength into the desired NIR region [90]. The fluorescence of the compound switched from off to on when the DNBS was cleaved by GSH (Ex. 560 nm, Em. 690 nm), and the fluorescence intensity increases linearly as the concentration of GSH increases (1 to 10 <sup>µ</sup>M) with a detection limit of 1.8 <sup>×</sup> <sup>10</sup>−<sup>8</sup> M. A similar response was observed for other sulfhydryl-containing compounds such as Cys, Hcy, and dithiothreitol. The probe was applied for imaging GSH in HeLa cells.

cleaved by GSH (Ex. 560 nm, Em. 690 nm), and the fluorescence intensity increases linearly as the concentration of GSH increases (1 to 10 μM) with a detection limit of 1.8 × 10−8 M. A similar response was observed for other sulfhydryl-containing compounds such as

In 2016, Ling Huang et al. [91] reported a soluble, distyryl boron dipyrromethene

In 2018, Xiang Xia et al. [67] reported a BODIPY disulfonate probe (BODIPY-diONs,

**Probe 19**, Figure 18) with a two-photon fluorescent turn-on effect for discriminately detecting GSH over Cys and Hcy. In the approach, the BODIPY dye was modified by extending the conjugated π-system to achieve a near-infrared emission. The DNBS moiety

(BODIPY)-based nanomicelles probe (**Probe 18**, Figure 17) with NIR properties. DNBS was attached to distyryl boron dipyrromethene (BODIPY) to serve as a fluorescence quencher through a PET mechanism and a thiols responsive unit. The probe will be lighted up by a thiol (Cys, GSH) with an NIR emission at 660 nm (Ex. 600 nm). A PLA-PEG unit is incorporated in the structure enabling micelle formation of the compound with an excellent water solubility. The probe can quantitatively detect the thiols' level

Cys, Hcy, and dithiothreitol. The probe was applied for imaging GSH in HeLa cells.

changes in HeLa cells.

**Figure 16.** Structure of **Probe 17** and its reaction with thiols.

**Figure 17.** Structure of **Probe 18** and its reaction with thiols.

**Figure 16.** Structure of **Probe 17** and its reaction with thiols. **Figure 16.** Structure of **Probe 17** and its reaction with thiols. **Figure 16.** Structure of **Probe 17** and its reaction with thiols.

changes in HeLa cells.

**Figure 15.** Structure of **Probe 16**.

**Figure 15.** Structure of **Probe 16**.

Detection of NPSH via Cleavage of Sulfonate Ester

Detection of NPSH via Cleavage of Sulfonate Ester

*Molecules* **2021**, *26*, x FOR PEER REVIEW 12 of 59

In 2016, Ling Huang et al. [91] reported a soluble, distyryl boron dipyrromethene (BODIPY)-based nanomicelles probe (**Probe 18**, Figure 17) with NIR properties. DNBS was attached to distyryl boron dipyrromethene (BODIPY) to serve as a fluorescence quencher through a PET mechanism and a thiols responsive unit. The probe will be lighted up by a thiol (Cys, GSH) with an NIR emission at 660 nm (Ex. 600 nm). A PLA-PEG unit is incorporated in the structure enabling micelle formation of the compound with an excellent water solubility. The probe can quantitatively detect the thiols' level In 2016, Ling Huang et al. [91] reported a soluble, distyryl boron dipyrromethene (BODIPY)-based nanomicelles probe (**Probe 18**, Figure 17) with NIR properties. DNBS was attached to distyryl boron dipyrromethene (BODIPY) to serve as a fluorescence quencher through a PET mechanism and a thiols responsive unit. The probe will be lighted up by a thiol (Cys, GSH) with an NIR emission at 660 nm (Ex. 600 nm). A PLA-PEG unit is incorporated in the structure enabling micelle formation of the compound with an excellent water solubility. The probe can quantitatively detect the thiols' level changes in HeLa cells. In 2016, Ling Huang et al. [91] reported a soluble, distyryl boron dipyrromethene (BODIPY)-based nanomicelles probe (**Probe 18**, Figure 17) with NIR properties. DNBS was attached to distyryl boron dipyrromethene (BODIPY) to serve as a fluorescence quencher through a PET mechanism and a thiols responsive unit. The probe will be lighted up by a thiol (Cys, GSH) with an NIR emission at 660 nm (Ex. 600 nm). A PLA-PEG unit is incorporated in the structure enabling micelle formation of the compound with an excellent water solubility. The probe can quantitatively detect the thiols' level changes in HeLa cells.

Dicyanomethylene-4H-pyran, one of the fluorophores with emission in red or NIR region, is featured with its better stability than other NIR fluorophores such as cyanine and squaraine. In 2014, Meng Li et al. [61] reported a colorimetric and NIR fluorescence turn-on thiol probe (**Probe 17**, Figure 16) based on dicyanomethylene-4H-pyran as the fluorophore and 2,4-dinitrobenzenesulfonyl (DNBS) as the thiol recognition group and also as the fluorescence quencher. A benzene unit was introduced to the dicyanopyran to extend its conjugated system to make its emission wavelength into the desired NIR region [90]. The fluorescence of the compound switched from off to on when the DNBS was cleaved by GSH (Ex. 560 nm, Em. 690 nm), and the fluorescence intensity increases linearly as the concentration of GSH increases (1 to 10 μM) with a detection limit of 1.8 × 10−8 M. A similar response was observed for other sulfhydryl-containing compounds such as Cys, Hcy, and dithiothreitol. The probe was applied for imaging GSH in HeLa cells.

Dicyanomethylene-4H-pyran, one of the fluorophores with emission in red or NIR region, is featured with its better stability than other NIR fluorophores such as cyanine and squaraine. In 2014, Meng Li et al. [61] reported a colorimetric and NIR fluorescence turn-on thiol probe (**Probe 17**, Figure 16) based on dicyanomethylene-4H-pyran as the fluorophore and 2,4-dinitrobenzenesulfonyl (DNBS) as the thiol recognition group and also as the fluorescence quencher. A benzene unit was introduced to the dicyanopyran to extend its conjugated system to make its emission wavelength into the desired NIR region [90]. The fluorescence of the compound switched from off to on when the DNBS was cleaved by GSH (Ex. 560 nm, Em. 690 nm), and the fluorescence intensity increases linearly as the concentration of GSH increases (1 to 10 μM) with a detection limit of 1.8 × 10−8 M. A similar response was observed for other sulfhydryl-containing compounds such as Cys, Hcy, and dithiothreitol. The probe was applied for imaging GSH in HeLa cells.

**Figure 17.** Structure of **Probe 18** and its reaction with thiols. **Figure 17.** Structure of **Probe 18** and its reaction with thiols. **Figure 17.** Structure of **Probe 18** and its reaction with thiols.

In 2018, Xiang Xia et al. [67] reported a BODIPY disulfonate probe (BODIPY-diONs, **Probe 19**, Figure 18) with a two-photon fluorescent turn-on effect for discriminately detecting GSH over Cys and Hcy. In the approach, the BODIPY dye was modified by extending the conjugated π-system to achieve a near-infrared emission. The DNBS moiety In 2018, Xiang Xia et al. [67] reported a BODIPY disulfonate probe (BODIPY-diONs, **Probe 19**, Figure 18) with a two-photon fluorescent turn-on effect for discriminately detecting GSH over Cys and Hcy. In the approach, the BODIPY dye was modified by extending the conjugated π-system to achieve a near-infrared emission. The DNBS moiety In 2018, Xiang Xia et al. [67] reported a BODIPY disulfonate probe (BODIPY-diONs, **Probe 19**, Figure 18) with a two-photon fluorescent turn-on effect for discriminately detecting GSH over Cys and Hcy. In the approach, the BODIPY dye was modified by extending the conjugated π-system to achieve a near-infrared emission. The DNBS moiety was utilized as the fluorescence quencher and also the thiol response group. A SNAr process will be initialized when the probe is exposed to GSH, resulting in the cleavage of DNBS and a significant increase of fluorescence (Em. 675 nm). Because of the double DNBSfunctionalization, a different reaction rate between the probe and Cys/Hcy vs. GSH was observed with GSH being the fastest one. Later, the probe BODIPY-diONs was demonstrated to be capable of detecting GSH in live cells. In comparison with other reported fluorescent probes, BODIPY-diONs has the advantages of longer emission wavelength and low detection limit of 0.17 µM. *Molecules* **2021**, *26*, x FOR PEER REVIEW 13 of 59 was utilized as the fluorescence quencher and also the thiol response group. A SNAr process will be initialized when the probe is exposed to GSH, resulting in the cleavage of DNBS and a significant increase of fluorescence (Em. 675 nm). Because of the double DNBS-functionalization, a different reaction rate between the probe and Cys/Hcy vs. GSH was observed with GSH being the fastest one. Later, the probe BODIPY-diONs was demonstrated to be capable of detecting GSH in live cells. In comparison with other reported fluorescent probes, BODIPY-diONs has the advantages of longer emission wavelength and low detection limit of 0.17 μM.

Disulfide bond (-S-S-) is another widely used thiol recognition unit for the designing

In 2016, Mingzhou Ye et al. [93] reported a probe (**Probe 21**, Figure 20) named as Cy-S-CPT (also a prodrug) in which a NIR cyanine dye and comptothecin (CPT) are connected

ostasis. Many thiol probes were designed based on the reaction of cleavage of a disulfide bond through a thiol-disulfide exchange reaction which has been demonstrated to be thiol specific. For example, a naphthalimide derivative probe (**Probe 20**, Figure 19) made by connecting two naphthalimide units through a disulfide bond for selective and ratiometric detection of thiols were reported by Baocun Zhu et al. in 2010 [92]. The probe was switched from colorless to green color after reacting with GSH through a cleavage of a disulfide bond followed by a cyclization to release 4-aminonaphthalimide as a strong green fluorescent compound with a remarkable shift of emissions from 485 to 533 nm. The fluorescence intensity ratio of (F533/F485) (Ex. 400 nm) increased linearly with the GSH concentration in a range of 0.5 to 10 mM. The detection limit of the probe for GSH was 28 μM, enabling the ratiometric detection of GSH. The probe can successfully detect the intracel-

**Figure 18. Probe 19** and its reaction with thiols. through a disulfide bond. In the approach, the cleavage of the disulfide bond in Cy-S-CPT **Figure 18. Probe 19** and its reaction with thiols.

lular GSH changes in live HeLa cells.

**Figure 19.** Structure of **Probe 20** and its reaction with GSH.

2.1.4. Detection of NPSH via Cleavage of a Disulfide Bond

#### 2.1.4. Detection of NPSH via Cleavage of a Disulfide Bond 2.1.4. Detection of NPSH via Cleavage of a Disulfide Bond

*Molecules* **2021**, *26*, x FOR PEER REVIEW 13 of 59

length and low detection limit of 0.17 μM.

**Figure 18. Probe 19** and its reaction with thiols.

was utilized as the fluorescence quencher and also the thiol response group. A SNAr process will be initialized when the probe is exposed to GSH, resulting in the cleavage of DNBS and a significant increase of fluorescence (Em. 675 nm). Because of the double DNBS-functionalization, a different reaction rate between the probe and Cys/Hcy vs. GSH was observed with GSH being the fastest one. Later, the probe BODIPY-diONs was demonstrated to be capable of detecting GSH in live cells. In comparison with other reported fluorescent probes, BODIPY-diONs has the advantages of longer emission wave-

Disulfide bond (-S-S-) is another widely used thiol recognition unit for the designing of a thiol detection probe due to its minimal perturbation to the intracellular redox homeostasis. Many thiol probes were designed based on the reaction of cleavage of a disulfide bond through a thiol-disulfide exchange reaction which has been demonstrated to be thiol specific. For example, a naphthalimide derivative probe (**Probe 20**, Figure 19) made by connecting two naphthalimide units through a disulfide bond for selective and ratiometric detection of thiols were reported by Baocun Zhu et al. in 2010 [92]. The probe was switched from colorless to green color after reacting with GSH through a cleavage of a disulfide bond followed by a cyclization to release 4-aminonaphthalimide as a strong green fluorescent compound with a remarkable shift of emissions from 485 to 533 nm. The fluorescence intensity ratio of (F533/F485) (Ex. 400 nm) increased linearly with the GSH concentration in a range of 0.5 to 10 mM. The detection limit of the probe for GSH was 28 µM, enabling the ratiometric detection of GSH. The probe can successfully detect the intracellular GSH changes in live HeLa cells. Disulfide bond (-S-S-) is another widely used thiol recognition unit for the designing of a thiol detection probe due to its minimal perturbation to the intracellular redox homeostasis. Many thiol probes were designed based on the reaction of cleavage of a disulfide bond through a thiol-disulfide exchange reaction which has been demonstrated to be thiol specific. For example, a naphthalimide derivative probe (**Probe 20**, Figure 19) made by connecting two naphthalimide units through a disulfide bond for selective and ratiometric detection of thiols were reported by Baocun Zhu et al. in 2010 [92]. The probe was switched from colorless to green color after reacting with GSH through a cleavage of a disulfide bond followed by a cyclization to release 4-aminonaphthalimide as a strong green fluorescent compound with a remarkable shift of emissions from 485 to 533 nm. The fluorescence intensity ratio of (F533/F485) (Ex. 400 nm) increased linearly with the GSH concentration in a range of 0.5 to 10 mM. The detection limit of the probe for GSH was 28 μM, enabling the ratiometric detection of GSH. The probe can successfully detect the intracellular GSH changes in live HeLa cells.

**Figure 19.** Structure of **Probe 20** and its reaction with GSH. **Figure 19.** Structure of **Probe 20** and its reaction with GSH.

In 2016, Mingzhou Ye et al. [93] reported a probe (**Probe 21**, Figure 20) named as Cy-S-CPT (also a prodrug) in which a NIR cyanine dye and comptothecin (CPT) are connected through a disulfide bond. In the approach, the cleavage of the disulfide bond in Cy-S-CPT In 2016, Mingzhou Ye et al. [93] reported a probe (**Probe 21**, Figure 20) named as Cy-S-CPT (also a prodrug) in which a NIR cyanine dye and comptothecin (CPT) are connected through a disulfide bond. In the approach, the cleavage of the disulfide bond in Cy-S-CPT by thiols will induce a dramatic NIR fluorescence shift from 825 to 650 nm. In the meantime, the cleavage activates the anti-cancer drug CPT enabling a real time tracking of the prodrug distribution at 825 nm and the activated drug at 650 nm. The whole process involved the cleavage of disulfide and a cyclization to release both the active CPT and a highly fluorescent compound CyA-K. More impressively, a PEG-PLA nanoparticle loaded with the probe was able to inhibit the tumor growth rate as high as 94.0% which is much higher than the clinical approved CPT-11 (55.8%). *Molecules* **2021**, *26*, x FOR PEER REVIEW 14 of 59 by thiols will induce a dramatic NIR fluorescence shift from 825 to 650 nm. In the meantime, the cleavage activates the anti-cancer drug CPT enabling a real time tracking of the prodrug distribution at 825 nm and the activated drug at 650 nm. The whole process involved the cleavage of disulfide and a cyclization to release both the active CPT and a highly fluorescent compound CyA-K. More impressively, a PEG-PLA nanoparticle loaded with the probe was able to inhibit the tumor growth rate as high as 94.0% which is much higher than the clinical approved CPT-11 (55.8%).

In 2018, Chao Yin et al. [94] developed a ratiometric photoacoustic imaging (PAI) fluorescence probe (**Probe 22**, Figure 21) for imaging GSH in vivo with a high resolution and deep penetration. The probe, IR806-pyridine dithioethylamine (PDA), employs a di-

unit. Upon reaction with GSH, the disulfide is cleaved followed by a subsequent intramolecular reaction to form a sulfydryl group leading to a ratiometrically signal changes of NIR-absorption peak from 658 to 820 nm. The detection limit for GSH is as low as 0.86 × 10−6 M. The probe was successfully applied for in vivo ratiometric PAI of upregulated

In addition to the thiol detection probes presented above, numerous other thiol detection probes have been developed based on other mechanisms or strategies [30,62,95]. Although not covered in this review, there are also a number of thiol detection probes which were developed based on nanoparticles and nanocomposites, and metal ion displacement and coordination [46]. These different strategies also emerged as promising

Cysteine (Cys) is involved in many biological processes, such as protein synthesis, detoxification, metabolism, and post-translational modification [61]. Abnormal Cys level

**Figure 20.** Structure of **Probe 21** and its reaction with GSH. **Figure 20.** Structure of **Probe 21** and its reaction with GSH.

GSH in the tumor of mice.

**Figure 21.** Structure of **Probe 22** and its reaction with GSH.

2.1.5. Detection of NPSH via Other Strategies

strategies for detection of thiols.

*2.2. Selective Detection of Cys and Hcy*

In 2018, Chao Yin et al. [94] developed a ratiometric photoacoustic imaging (PAI) fluorescence probe (**Probe 22**, Figure 21) for imaging GSH in vivo with a high resolution and deep penetration. The probe, IR806-pyridine dithioethylamine (PDA), employs a disulfide bond as the thiol recognition group and NIR cyanine as the fluorescence report unit. Upon reaction with GSH, the disulfide is cleaved followed by a subsequent intramolecular reaction to form a sulfydryl group leading to a ratiometrically signal changes of NIR-absorption peak from 658 to 820 nm. The detection limit for GSH is as low as 0.86 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M. The probe was successfully applied for in vivo ratiometric PAI of upregulated GSH in the tumor of mice. In 2018, Chao Yin et al. [94] developed a ratiometric photoacoustic imaging (PAI) fluorescence probe (**Probe 22**, Figure 21) for imaging GSH in vivo with a high resolution and deep penetration. The probe, IR806-pyridine dithioethylamine (PDA), employs a disulfide bond as the thiol recognition group and NIR cyanine as the fluorescence report unit. Upon reaction with GSH, the disulfide is cleaved followed by a subsequent intramolecular reaction to form a sulfydryl group leading to a ratiometrically signal changes of NIR-absorption peak from 658 to 820 nm. The detection limit for GSH is as low as 0.86 × 10−6 M. The probe was successfully applied for in vivo ratiometric PAI of upregulated GSH in the tumor of mice.

**Figure 20.** Structure of **Probe 21** and its reaction with GSH.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 14 of 59

higher than the clinical approved CPT-11 (55.8%).

by thiols will induce a dramatic NIR fluorescence shift from 825 to 650 nm. In the meantime, the cleavage activates the anti-cancer drug CPT enabling a real time tracking of the prodrug distribution at 825 nm and the activated drug at 650 nm. The whole process involved the cleavage of disulfide and a cyclization to release both the active CPT and a highly fluorescent compound CyA-K. More impressively, a PEG-PLA nanoparticle loaded with the probe was able to inhibit the tumor growth rate as high as 94.0% which is much

**Figure 21.** Structure of **Probe 22** and its reaction with GSH. **Figure 21.** Structure of **Probe 22** and its reaction with GSH.

#### 2.1.5. Detection of NPSH via Other Strategies 2.1.5. Detection of NPSH via Other Strategies

In addition to the thiol detection probes presented above, numerous other thiol detection probes have been developed based on other mechanisms or strategies [30,62,95]. Although not covered in this review, there are also a number of thiol detection probes which were developed based on nanoparticles and nanocomposites, and metal ion displacement and coordination [46]. These different strategies also emerged as promising strategies for detection of thiols. In addition to the thiol detection probes presented above, numerous other thiol detection probes have been developed based on other mechanisms or strategies [30,62,95]. Although not covered in this review, there are also a number of thiol detection probes which were developed based on nanoparticles and nanocomposites, and metal ion displacement and coordination [46]. These different strategies also emerged as promising strategies for detection of thiols.

#### *2.2. Selective Detection of Cys and Hcy 2.2. Selective Detection of Cys and Hcy*

Cysteine (Cys) is involved in many biological processes, such as protein synthesis, detoxification, metabolism, and post-translational modification [61]. Abnormal Cys level Cysteine (Cys) is involved in many biological processes, such as protein synthesis, detoxification, metabolism, and post-translational modification [61]. Abnormal Cys level has been linked with many diseases such as slow growth, Alzheimer's disease and cardiovascular diseases et al. Hcy is associated with activation of multiple signal pathways and is found to be associated with cardiovascular diseases [61]. Accurate and effective detection of Cys and Hcy are essential for further exploring the roles these thiols in the biological system, A tool that can measure Cys/Hcy is in need. Although many fluorescent probes have been developed for thiols, special probes capable of discriminating Cys, Hcy, and GSH are still limited. The high GSH concentration (1–10 mM) makes it even more challenge to detect Cys and Hcy since the concentrations of Cys (30−200 µM) and Hcy (5–15 µM) are in µM.

Nevertheless, many Cys selective detection probes have been reported based on various mechanisms. These mechanisms include native chemical ligation (NCL) [96,97], aromatic substitution–rearrangement [98], and cyclization with aldehydes or acrylates [43,99–101]. The fluorophores employed in these probes include curcumin [38], coumarin [102,103], BOD-IPY [98,104,105], naphthalimide [106], rhodamine [107], fluorescein [108], and pyrene [43]. In the following, some of the mechanisms used to distinguish Cys/Hcy are presented. Most of the probes take two steps to achieve the selective detection of Cys vs. Hcy. Step one usually is the same for all the thiols (GSH, Cys, or Hcy) that is a reaction between the highly nucleophilic thiolate and a probe to form a thiol adduct. Step two commonly involved an intramolecular rearrangement reaction or cyclization reaction which occurs for Cys/Hcy adducts or Cys adduct only. Many selective probes for Cys or Hcy were developed based on the extended version of these two steps although some other probes achieve the discrimination of Cys and Hcy by the kinetic difference of the reaction between the different thiols and probes.

2.2.1. Selective Detection of Cys and Hcy through Cyclization of Cys/Hcy with Acrylates or Aldehydes

Cyclization of Cys/Hcy with aldehydes or acrylates is one of the most extensively used mechanism for selectively detection of Cys/Hcy over GSH. As mentioned above, a two-step reaction takes place for the selective detection of Cys/Hcy. The method starts with a Michael addition reaction from the thiolate of a thiol (GSH, Cys, or Hcy) to form a thioether. The thioether derived from Cys or Hcy will go through a cyclization to produce a seven members-ring cyclic amide (Cys) or eight-member ring cyclic amide (Hcy) at a different reaction rate while the thioether derived from GSH is not able to undergo the cyclization reaction. Many probes have been designed based on this mechanism to selectively detect Cys/Hcy over GSH (Figure 22). For example, Xiaofeng Yang et al. [99] reported in 2011 a benzothiazole derivative which utilized an α, β-unsaturated carbonyl recognition as the thiol responsive unit to selectively detect Cys. *Molecules* **2021**, *26*, x FOR PEER REVIEW 16 of 59

**Figure 22. Probe 23** and its reaction mechanism for distinguishing Cys over GSH and Hcy. **Figure 22. Probe 23** and its reaction mechanism for distinguishing Cys over GSH and Hcy.

Colorimetric probes are another widely used reagents which enable the recognition of analytes by naked-eye and do not need an instrument [47,109]. Lanfang Pang et al. [38] published their work for development of a new curcumin-based fluorescent and colorimetric probe (CAC, **Probe 24**) for specific detection of Cys. Curcumin is used because of its excellent optical properties and great biocompatibility [110,111]. As depicted in Figure 23, CAC was made by incorporating two acrylate groups with curcumin to serve as thiol recognition sites. The CAC is easier to synthesize. The detection starts from a Michael addition reaction between the thiolate of Cys and the α, β-unsaturated ketone of CAC followed by a cyclization to produce a seven membered-ring cyclic amide and a free curcumin molecule [44,112,113]. Unlike other fluorescence probes, CAC itself carries a strong fluorescence (ΦF = 0.20) which will be quenched after being activated by Cys (ΦF = 0.02), Zhiqian Guo et al. [102] developed a ratiometric near-infrared fluorescent probe (CyAC, **Probe 23**) for selective detection of Cys over Hcy and GSH. As depicted in Figure22, CyAC is composed of a NIR cyanine fluorophore and an acrylate group to serve as the thiol response unit. A significant optical property change (from emission 780 nm to emission 570 nm) was observed upon reaction with Cys, while GSH and Hcy were not able to induce the change confirming the selectivity of the probe for Cys. Further, a linear relationship was found between the fluorescence intensity ratio I560 nm/I740 nm and Cys concentration in the range of 0–25 µM. The different kinetic of the cyclization reaction was the reason for inducing the difference between Cys and Hcy, since the seven-membered ring (Cys) should be formed easier and faster than eight-membered ring (Hcy). Under the same condition, GSH was not able to induce the cyclization reaction at all.

whereas the fluorescence quenching effect was not observed with other thiols like Hcy and GSH. The probe itself is colorless but can be turned into yellow color by Cys which could be easily visualized by naked eyes. In vitro cell experiments in PC12 cells showed that the probe was able to detect the change in Cys levels in live cell. The probe was further successfully applied for detecting Cys status on zebrafish. **Figure 23.** Structure of **Probe 24** and its detection mechanism with Cys. Colorimetric probes are another widely used reagents which enable the recognition of analytes by naked-eye and do not need an instrument [47,109]. Lanfang Pang et al. [38] published their work for development of a new curcumin-based fluorescent and colorimetric probe (CAC, **Probe 24**) for specific detection of Cys. Curcumin is used because of its excellent optical properties and great biocompatibility [110,111]. As depicted in Figure 23, CAC was made by incorporating two acrylate groups with curcumin to serve as thiol recognition sites. The CAC is easier to synthesize. The detection starts from a Michael addition reaction between the thiolate of Cys and the α, β-unsaturated ketone of CAC followed by a cyclization to produce a seven membered-ring cyclic amide and a free curcumin molecule [44,112,113]. Unlike other fluorescence probes, CAC itself carries a strong fluorescence (ΦF = 0.20) which will be quenched after being activated by Cys (ΦF = 0.02), whereas the fluorescence quenching effect was not observed with other thiols like Hcy and GSH. The probe itself is colorless but can be turned into yellow color by Cys which could be easily visualized by naked eyes. In vitro cell experiments in PC12 cells

Yao Liu et al. reported [44] a ratiometric fluorescent probe (**Probe 25**) for specific de-

fer (ESIPT) dye (4′-dimethylami- no-3-hydroxyflavone) as the fluorophore. As illustrated in Figure 24, an acrylate group was anchored to the fluorophore to block the ESIPT and consequently quench the fluorescence. Recognition and cleavage of acrylate moiety by Cys will restore the ESIPT process and induce a remarkable fluorescence enhancement and emission wavelength shift (471 to 550 nm). The "off to on" fluorescence switch starts from a Michael addition reaction between the thiolate of Cys and the α, β-unsaturated ketone of the acrylate group followed by an intramolecular cyclization to produce a seven membered-ring amide. A kinetic difference of the intramolecular cyclization resulting in a selective detection of Cys over Hcy and GSH. A steric effect could also possibly elevate the kinetic difference of the reaction from Cys over Hcy. Upon completion of the reaction,

showed that the probe was able to detect the change in Cys levels in live cell. The probe was further successfully applied for detecting Cys status on zebrafish. that the probe was able to detect the change in Cys levels in live cell. The probe was further successfully applied for detecting Cys status on zebrafish.

**Figure 22. Probe 23** and its reaction mechanism for distinguishing Cys over GSH and Hcy.

Colorimetric probes are another widely used reagents which enable the recognition

of analytes by naked-eye and do not need an instrument [47,109]. Lanfang Pang et al. [38] published their work for development of a new curcumin-based fluorescent and colorimetric probe (CAC, **Probe 24**) for specific detection of Cys. Curcumin is used because of its excellent optical properties and great biocompatibility [110,111]. As depicted in Figure 23, CAC was made by incorporating two acrylate groups with curcumin to serve as thiol recognition sites. The CAC is easier to synthesize. The detection starts from a Michael addition reaction between the thiolate of Cys and the α, β-unsaturated ketone of CAC followed by a cyclization to produce a seven membered-ring cyclic amide and a free curcumin molecule [44,112,113]. Unlike other fluorescence probes, CAC itself carries a strong fluorescence (ΦF = 0.20) which will be quenched after being activated by Cys (ΦF = 0.02), whereas the fluorescence quenching effect was not observed with other thiols like Hcy and GSH. The probe itself is colorless but can be turned into yellow color by Cys which could be easily visualized by naked eyes. In vitro cell experiments in PC12 cells showed

*Molecules* **2021**, *26*, x FOR PEER REVIEW 16 of 59

**Figure 23.** Structure of **Probe 24** and its detection mechanism with Cys. **Figure 23.** Structure of **Probe 24** and its detection mechanism with Cys.

Yao Liu et al. reported [44] a ratiometric fluorescent probe (**Probe 25**) for specific detection of Cys based on a visible-light excitable excited-state intramolecular proton transfer (ESIPT) dye (4′-dimethylami- no-3-hydroxyflavone) as the fluorophore. As illustrated in Figure 24, an acrylate group was anchored to the fluorophore to block the ESIPT and consequently quench the fluorescence. Recognition and cleavage of acrylate moiety by Cys will restore the ESIPT process and induce a remarkable fluorescence enhancement and emission wavelength shift (471 to 550 nm). The "off to on" fluorescence switch starts from a Michael addition reaction between the thiolate of Cys and the α, β-unsaturated ketone of the acrylate group followed by an intramolecular cyclization to produce a seven membered-ring amide. A kinetic difference of the intramolecular cyclization resulting in a selective detection of Cys over Hcy and GSH. A steric effect could also possibly elevate the kinetic difference of the reaction from Cys over Hcy. Upon completion of the reaction, Yao Liu et al. reported [44] a ratiometric fluorescent probe (**Probe 25**) for specific detection of Cys based on a visible-light excitable excited-state intramolecular proton transfer (ESIPT) dye (40 -dimethylami- no-3-hydroxyflavone) as the fluorophore. As illustrated in Figure 24, an acrylate group was anchored to the fluorophore to block the ESIPT and consequently quench the fluorescence. Recognition and cleavage of acrylate moiety by Cys will restore the ESIPT process and induce a remarkable fluorescence enhancement and emission wavelength shift (471 to 550 nm). The "off to on" fluorescence switch starts from a Michael addition reaction between the thiolate of Cys and the α, β-unsaturated ketone of the acrylate group followed by an intramolecular cyclization to produce a seven membered-ring amide. A kinetic difference of the intramolecular cyclization resulting in a selective detection of Cys over Hcy and GSH. A steric effect could also possibly elevate the kinetic difference of the reaction from Cys over Hcy. Upon completion of the reaction, a nearly 40-fold increase of ratiometric value of the emission intensities (I550/471) was induced. Additionally, the probe was able to detect Cys level changes in HeLa cells. Additional advantages of the probe include a large Stokes shift (>130 nm), fast response (within minutes) and high sensitivity (~0.2 µM) for Cys. *Molecules* **2021**, *26*, x FOR PEER REVIEW 17 of 59 a nearly 40-fold increase of ratiometric value of the emission intensities (I550/471) was induced. Additionally, the probe was able to detect Cys level changes in HeLa cells. Additional advantages of the probe include a large Stokes shift (>130 nm), fast response (within minutes) and high sensitivity (~0.2 μM) for Cys.

**Figure 24.** Structure of **Probe 25** and its reaction with Cys and Hcy. **Figure 24.** Structure of **Probe 25** and its reaction with Cys and Hcy.

Similar to the structure of acrylate, a bromoacetyl group was also used to develop probes for detection of Cys/Hcy employing the same sensing mechanism. In 2013, fluorescein chemodosimeter was developed by Keum-Hee Hong et al. [114] as a fluorescent probe (**Probe 26,** Figure 25) for detection of Cys over Hcy and GSH. The reagent was made by introducing a bromoacetyl group to fluorescein. The selectivity for Cys over Hcy and GSH in aqueous solution is due to the thermodynamically stable and kinetically rapid formation of six-member lactam ring in aqueous solution with Cys [115]. The reagent was capable of detecting Cys changes in HeLa cells. Similar to the structure of acrylate, a bromoacetyl group was also used to develop probes for detection of Cys/Hcy employing the same sensing mechanism. In 2013, fluorescein chemodosimeter was developed by Keum-Hee Hong et al. [114] as a fluorescent probe (**Probe 26**, Figure 25) for detection of Cys over Hcy and GSH. The reagent was made by introducing a bromoacetyl group to fluorescein. The selectivity for Cys over Hcy and GSH in aqueous solution is due to the thermodynamically stable and kinetically rapid formation of six-member lactam ring in aqueous solution with Cys [115]. The reagent was capable of detecting Cys changes in HeLa cells.

Hye Yeon Lee et al. [43] reported two new pyrene-based fluorescent probes (**Probes 27**, **28**, Figure 26) for selective detection of Hcy over Cys and other thiols. The two probes were converted rapidly (10 min) to a high fluorescence (350 nm and 450 nm) form when mixed with Hcy, while no fluorescence response was observed when mixed with Cys and GSH even over a long reaction time. The detection limit of Hcy was determined to be 1.44

−7 M with a linear range of 600–1000 nM in HEPES containing 10% DMSO (0.01 M and pH 7.4). The authors provided an explanation for the fluorescence response difference for Hcy over Cys and GSH. These probes have been applied for the detection of Hcy changes

in mammalian and showed no response to the change of GSH.

**Figure 26.** Structures of **Probes 27** and **28**.

**Figure 25.** Structure of **Probe 26** and its reaction with Cys.

× 10

**Figure 24.** Structure of **Probe 25** and its reaction with Cys and Hcy.

**Figure 24.** Structure of **Probe 25** and its reaction with Cys and Hcy.

minutes) and high sensitivity (~0.2 μM) for Cys.

minutes) and high sensitivity (~0.2 μM) for Cys.

**Figure 25.** Structure of **Probe 26** and its reaction with Cys. **Figure 25.** Structure of **Probe 26** and its reaction with Cys. **Figure 25.** Structure of **Probe 26** and its reaction with Cys.

Hye Yeon Lee et al. [43] reported two new pyrene-based fluorescent probes (**Probes 27**, **28**, Figure 26) for selective detection of Hcy over Cys and other thiols. The two probes were converted rapidly (10 min) to a high fluorescence (350 nm and 450 nm) form when mixed with Hcy, while no fluorescence response was observed when mixed with Cys and GSH even over a long reaction time. The detection limit of Hcy was determined to be 1.44 −7 M with a linear range of 600–1000 nM in HEPES containing 10% DMSO (0.01 M and pH 7.4). The authors provided an explanation for the fluorescence response difference for Hcy over Cys and GSH. These probes have been applied for the detection of Hcy changes in mammalian and showed no response to the change of GSH. Hye Yeon Lee et al. [43] reported two new pyrene-based fluorescent probes (**Probes 27**, **28**, Figure 26) for selective detection of Hcy over Cys and other thiols. The two probes were converted rapidly (10 min) to a high fluorescence (350 nm and 450 nm) form when mixed with Hcy, while no fluorescence response was observed when mixed with Cys and GSH even over a long reaction time. The detection limit of Hcy was determined to be 1.44 <sup>×</sup> <sup>10</sup>−<sup>7</sup> M with a linear range of 600–1000 nM in HEPES containing 10% DMSO (0.01 M and pH 7.4). The authors provided an explanation for the fluorescence response difference for Hcy over Cys and GSH. These probes have been applied for the detection of Hcy changes in mammalian and showed no response to the change of GSH. Hye Yeon Lee et al. [43] reported two new pyrene-based fluorescent probes (**Probes 27**, **28**, Figure 26) for selective detection of Hcy over Cys and other thiols. The two probes were converted rapidly (10 min) to a high fluorescence (350 nm and 450 nm) form when mixed with Hcy, while no fluorescence response was observed when mixed with Cys and GSH even over a long reaction time. The detection limit of Hcy was determined to be 1.44 × 10 −7 M with a linear range of 600–1000 nM in HEPES containing 10% DMSO (0.01 M and pH 7.4). The authors provided an explanation for the fluorescence response difference for Hcy over Cys and GSH. These probes have been applied for the detection of Hcy changes in mammalian and showed no response to the change of GSH.

a nearly 40-fold increase of ratiometric value of the emission intensities (I550/471) was induced. Additionally, the probe was able to detect Cys level changes in HeLa cells. Additional advantages of the probe include a large Stokes shift (>130 nm), fast response (within

a nearly 40-fold increase of ratiometric value of the emission intensities (I550/471) was induced. Additionally, the probe was able to detect Cys level changes in HeLa cells. Additional advantages of the probe include a large Stokes shift (>130 nm), fast response (within

*Molecules* **2021**, *26*, x FOR PEER REVIEW 17 of 59

Similar to the structure of acrylate, a bromoacetyl group was also used to develop probes for detection of Cys/Hcy employing the same sensing mechanism. In 2013, fluorescein chemodosimeter was developed by Keum-Hee Hong et al. [114] as a fluorescent probe (**Probe 26,** Figure 25) for detection of Cys over Hcy and GSH. The reagent was made by introducing a bromoacetyl group to fluorescein. The selectivity for Cys over Hcy and GSH in aqueous solution is due to the thermodynamically stable and kinetically rapid formation of six-member lactam ring in aqueous solution with Cys [115]. The reagent was

Similar to the structure of acrylate, a bromoacetyl group was also used to develop probes for detection of Cys/Hcy employing the same sensing mechanism. In 2013, fluorescein chemodosimeter was developed by Keum-Hee Hong et al. [114] as a fluorescent probe (**Probe 26,** Figure 25) for detection of Cys over Hcy and GSH. The reagent was made by introducing a bromoacetyl group to fluorescein. The selectivity for Cys over Hcy and GSH in aqueous solution is due to the thermodynamically stable and kinetically rapid formation of six-member lactam ring in aqueous solution with Cys [115]. The reagent was

× 10

**Figure 26.** Structures of **Probes 27** and **28**. **Figure 26.** Structures of **Probes 27** and **28**. **Figure 26.** Structures of **Probes 27** and **28**.

2.2.2. Selective Detection of Cys and Hcy through Cys-Induced SNAr Substitution−Rearrangement Reaction 2.2.2. Selective Detection of Cys and Hcy through Cys-Induced SNAr Substitution−Rearrangement Reaction The Cys-induced SNAr substitution−rearrangement reaction used for selective dis-

The Cys-induced SNAr substitution−rearrangement reaction used for selective discrimination of thiols has been widely explored. Many probes have been developed based on various fluorophores that possess the ability to undergo a *S* to *N* displacement rearrangement reaction presented early. The fluorophores employed include BODIPY, NBD, naphthalimide, and cyanine (Figure 27). The requirements for fluorophores to be applied for the purpose are that they should be able to form a thioether (GSH, or Cys/Hcy) first. Then the amino group of Cys/Hcy conjugated thioether will rearrange by displacing the sulfur of the thioether with the amino group to form amino-substituted product. As described early, the rearrangement does not occur with GSH thioether. An example of the mechanism is illustrated in Figure 28. The remarkable optical property differences between sulfur and amino substituted BODIPY enable the discrimination of GSH over Cys/Hcy. crimination of thiols has been widely explored. Many probes have been developed based on various fluorophores that possess the ability to undergo a *S* to *N* displacement rearrangement reaction presented early. The fluorophores employed include BODIPY, NBD, naphthalimide, and cyanine (Figure 27). The requirements for fluorophores to be applied for the purpose are that they should be able to form a thioether (GSH, or Cys/Hcy) first. Then the amino group of Cys/Hcy conjugated thioether will rearrange by displacing the sulfur of the thioether with the amino group to form amino-substituted product. As described early, the rearrangement does not occur with GSH thioether. An example of the mechanism is illustrated in Figure 28. The remarkable optical property differences between sulfur and amino substituted BODIPY enable the discrimination of GSH over Cys/Hcy.

**Figure 27.** Representative fluorophores used for selective discrimination of thiols via Cys-induced SNAr substitution−rearrangement reaction. **Figure 27.** Representative fluorophores used for selective discrimination of thiols via Cys-induced SNAr substitution−rearrangement reaction.

**Figure 28.** Illustration of the Cys-induced SNAr substitution−rearrangement reaction.

BODIPY fluorescent dyes featured with a high molar absorption coefficient, quantum

In 2012, Liya Niu et al. [29] developed monochlorinated boron dipyrromethene (BODIPY) derivatives to selectively detect GSH over Cys/Hcy. However, the emission band of amino-substituted BODIPY (Cys and Hcy) is close to the emission band of the original monochlorinated boron dipyrromethene (BODIPY) probe, Thus, the probe failed to detect Cys and Hcy. In 2013, a structure modification [117] by adding a nitrothiophenyl or nitrophenyl group to the same BODIPY fluorophore was employed to address the problem (**Probe 29**). As depicted in Figure 29, the probe itself displays no fluorescence (PET) but can be turned on to fluorescent products upon reaction with thiols followed by the occurrence of the rapid intramolecular displacement of sulfur with the amino group of

yield, and photo stability which enable them to be widely applied in many fields [116].

BODIPY

**Figure 27.** Representative fluorophores used for selective discrimination of thiols via Cys-induced

2.2.2. Selective Detection of Cys and Hcy through Cys-Induced SNAr Substitution−Rear-

The Cys-induced SNAr substitution−rearrangement reaction used for selective discrimination of thiols has been widely explored. Many probes have been developed based on various fluorophores that possess the ability to undergo a *S* to *N* displacement rearrangement reaction presented early. The fluorophores employed include BODIPY, NBD, naphthalimide, and cyanine (Figure 27). The requirements for fluorophores to be applied for the purpose are that they should be able to form a thioether (GSH, or Cys/Hcy) first. Then the amino group of Cys/Hcy conjugated thioether will rearrange by displacing the sulfur of the thioether with the amino group to form amino-substituted product. As described early, the rearrangement does not occur with GSH thioether. An example of the mechanism is illustrated in Figure 28. The remarkable optical property differences between sulfur and amino substituted BODIPY enable the discrimination of GSH over

**Figure 28.** Illustration of the Cys-induced SNAr substitution−rearrangement reaction. **Figure 28.** Illustration of the Cys-induced SNAr substitution−rearrangement reaction.

#### BODIPY BODIPY

rangement Reaction

Cys/Hcy.

SNAr substitution−rearrangement reaction.

BODIPY fluorescent dyes featured with a high molar absorption coefficient, quantum yield, and photo stability which enable them to be widely applied in many fields [116]. BODIPY fluorescent dyes featured with a high molar absorption coefficient, quantum yield, and photo stability which enable them to be widely applied in many fields [116].

In 2012, Liya Niu et al. [29] developed monochlorinated boron dipyrromethene (BODIPY) derivatives to selectively detect GSH over Cys/Hcy. However, the emission band of amino-substituted BODIPY (Cys and Hcy) is close to the emission band of the original monochlorinated boron dipyrromethene (BODIPY) probe, Thus, the probe failed to detect Cys and Hcy. In 2013, a structure modification [117] by adding a nitrothiophenyl or nitrophenyl group to the same BODIPY fluorophore was employed to address the problem (**Probe 29**). As depicted in Figure 29, the probe itself displays no fluorescence (PET) but can be turned on to fluorescent products upon reaction with thiols followed by the occurrence of the rapid intramolecular displacement of sulfur with the amino group of In 2012, Liya Niu et al. [29] developed monochlorinated boron dipyrromethene (BOD-IPY) derivatives to selectively detect GSH over Cys/Hcy. However, the emission band of amino-substituted BODIPY (Cys and Hcy) is close to the emission band of the original monochlorinated boron dipyrromethene (BODIPY) probe, Thus, the probe failed to detect Cys and Hcy. In 2013, a structure modification [117] by adding a nitrothiophenyl or nitrophenyl group to the same BODIPY fluorophore was employed to address the problem (**Probe 29**). As depicted in Figure 29, the probe itself displays no fluorescence (PET) but can be turned on to fluorescent products upon reaction with thiols followed by the occurrence of the rapid intramolecular displacement of sulfur with the amino group of Cys and Hcy. The reaction of Cys is much faster than Hcy while no such intramolecular displacement occurred for GSH achieving the ability of discrimination of Cys from Hcy and GSH. The probe itself shows barely any fluorescence but can be turned on to strong fluorescence by thiols with the product derived from Cys exhibiting emission wavelength at 564 nm while the products derived from GSH and Hcy emitting at 588 nm. The fluorescence intensity at 565 nm followed a nicely linear relationship with the Cys concentration (0–100 µM) with a detection limit of 2.12 <sup>×</sup> <sup>10</sup>−<sup>7</sup> M. The probe was successfully shown to be capable of detecting Cys status change in live cells. *Molecules* **2021**, *26*, x FOR PEER REVIEW 19 of 59 Cys and Hcy. The reaction of Cys is much faster than Hcy while no such intramolecular displacement occurred for GSH achieving the ability of discrimination of Cys from Hcy and GSH. The probe itself shows barely any fluorescence but can be turned on to strong fluorescence by thiols with the product derived from Cys exhibiting emission wavelength at 564 nm while the products derived from GSH and Hcy emitting at 588 nm. The fluorescence intensity at 565 nm followed a nicely linear relationship with the Cys concentration (0–100 μM) with a detection limit of 2.12 × 10−7 M. The probe was successfully shown to be capable of detecting Cys status change in live cells.

**Figure 29.** Structure of **Probe 29** and its sensing mechanisms for distinguish Cys/Hcy and GSH. **Figure 29.** Structure of **Probe 29** and its sensing mechanisms for distinguish Cys/Hcy and GSH.

7-Nitrobenzofurazan (NBD) is another widely used and extensively studied fluorophore that is able to go through the Cys-induced SNAr substitution−rearrangement reac-

through the intramolecular displacement mechanism to discriminatively detect different thiols. As shown in Figure 30, Cys/Hcy and GSH form different release fluorophores NBD, *N*-substitute products (Cys/Hcy) or *S*-substitute product (GSH). The *N*-substitute product (Cys/Hcy) exhibit significantly higher fluorescence than the *S*-substitute product (GSH). A simple probe NBD-Cl (**Probe 30**) was developed and successfully applied for detecting Cys in live cells. Pinaki Talukdar et al. [119] found the same phenomenon which further

push the development of Cys/Hcy selective probes based on NBD.

**Figure 30.** Structures of **Probes 30**, **31** and their reaction toward GSH and Cys/Hcy.

In 2019, Lihui Zhai et al. [120] developed a dual emission turn on fluorescent probe (CA-NBD, **Probe 31**) for discriminative detection of Cys/Hcy and GSH simultaneously based on NBD. The probe was designed by combing another fluorophore coumarin together with 7-nitrobenzofurazan (NBD). Two different NBD derivatives formed, one is *N*-

7-Nitrobenzofurazan (NBD)

#### 7-Nitrobenzofurazan (NBD) 7-Nitrobenzofurazan (NBD)

7-Nitrobenzofurazan (NBD) is another widely used and extensively studied fluorophore that is able to go through the Cys-induced SNAr substitution−rearrangement reaction for selective detection of thiols (Figure 28). 7-Nitrobenzofurazan (NBD) is another widely used and extensively studied fluorophore that is able to go through the Cys-induced SNAr substitution−rearrangement reaction for selective detection of thiols (Figure 28).

**Figure 29.** Structure of **Probe 29** and its sensing mechanisms for distinguish Cys/Hcy and GSH.

Cys and Hcy. The reaction of Cys is much faster than Hcy while no such intramolecular displacement occurred for GSH achieving the ability of discrimination of Cys from Hcy and GSH. The probe itself shows barely any fluorescence but can be turned on to strong fluorescence by thiols with the product derived from Cys exhibiting emission wavelength at 564 nm while the products derived from GSH and Hcy emitting at 588 nm. The fluorescence intensity at 565 nm followed a nicely linear relationship with the Cys concentration (0–100 μM) with a detection limit of 2.12 × 10−7 M. The probe was successfully shown to

In 2014, Liya Niu et al. [118] explored NBD as another fluorophore that can go through the intramolecular displacement mechanism to discriminatively detect different thiols. As shown in Figure 30, Cys/Hcy and GSH form different release fluorophores NBD, *N*substitute products (Cys/Hcy) or *S*-substitute product (GSH). The *N*-substitute product (Cys/Hcy) exhibit significantly higher fluorescence than the *S*-substitute product (GSH). A simple probe NBD-Cl (**Probe 30**) was developed and successfully applied for detecting Cys in live cells. Pinaki Talukdar et al. [119] found the same phenomenon which further push the development of Cys/Hcy selective probes based on NBD. In 2014, Liya Niu et al. [118] explored NBD as another fluorophore that can go through the intramolecular displacement mechanism to discriminatively detect different thiols. As shown in Figure 30, Cys/Hcy and GSH form different release fluorophores NBD, *N*-substitute products (Cys/Hcy) or *S*-substitute product (GSH). The *N*-substitute product (Cys/Hcy) exhibit significantly higher fluorescence than the *S*-substitute product (GSH). A simple probe NBD-Cl (**Probe 30**) was developed and successfully applied for detecting Cys in live cells. Pinaki Talukdar et al. [119] found the same phenomenon which further push the development of Cys/Hcy selective probes based on NBD.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 19 of 59

be capable of detecting Cys status change in live cells.

**Figure 30.** Structures of **Probes 30**, **31** and their reaction toward GSH and Cys/Hcy. **Figure 30.** Structures of **Probes 30**, **31** and their reaction toward GSH and Cys/Hcy.

In 2019, Lihui Zhai et al. [120] developed a dual emission turn on fluorescent probe (CA-NBD, **Probe 31**) for discriminative detection of Cys/Hcy and GSH simultaneously based on NBD. The probe was designed by combing another fluorophore coumarin together with 7-nitrobenzofurazan (NBD). Two different NBD derivatives formed, one is *N*-In 2019, Lihui Zhai et al. [120] developed a dual emission turn on fluorescent probe (CA-NBD, **Probe 31**) for discriminative detection of Cys/Hcy and GSH simultaneously based on NBD. The probe was designed by combing another fluorophore coumarin together with 7-nitrobenzofurazan (NBD). Two different NBD derivatives formed, one is *N*-substitute products (Cys/Hcy) and the other is S-substitute product (GSH). At the excitation wavelength of 460 nm, GSH-NBD (*S*-substitute) derivative was non-emissive. In contrast, the Cys/Hcy-NBD derivatives exhibited strong yellow fluorescence enabling the discrimination of Cys/Hcy over GSH. The fluorescence intensity was linearly increased when the thiol concentration with a detection limit of 2.00 <sup>×</sup> <sup>10</sup>−<sup>8</sup> M for Cys and 1.02 <sup>×</sup> <sup>10</sup>−<sup>8</sup> M for Hcy, respectively. The probe can successfully detect the Cys status change in live cells.

A dual channel responsive NIR fluorescent probe (**Probe 32**, Figure 31) for selective detection of Cys in live cells was reported by Zhuo Ye et al. [121] in 2017. The probe named as BDY-NBD is composed of two fluorophores, a NIR BODIPY fluorophore, BDY-OH, and a NBD fluorophore. In the structure, NBD is employed as the thiol responsive unit and the fluorescence of BDY-OH is quenched by NBD owing to PET. Once the probe reacts with a thiol, the nucleophilic thiolate will cleave the NBD from BDY-OH and attach to NBD to form a NBD thiol adduct. The NIR fluorescence of BDY-OH (λex = 650 nm, λem = 735 nm) will be recovered because of the termination of PET. Different from GSH, the thiol adduct (Cys-NBD) produced in the reaction between Cys and probe will subsequently undergo an S to N acyl shift reaction to form a much stronger fluorescent product with a much different emission wavelength (Ex. 466 nm, Em. 540 nm) than BDY-OH while the shift reaction does not occur for GSH. Therefore, a dual-emission mode can be utilized for the detection of Cys. The probe has a much low detection limitation (22 nM, 540 nm emission) that is lower than most of the fluorescent probes developed for the detection of Cys previously [100,122,123]. In vitro cell experiments with HeLa cells demonstrated that the probe can be applied for multicolor imaging of intracellular Cys, and a significant differences of fluorescence imaging were observed for GSH and Cys in live cells when their levels are manipulated.

their levels are manipulated.

Many other probes have been designed and applied based on different thiol recognition groups or other modifications. Shuangshuang Ding et al. [124] developed the probe (**Probe 33**, Figure 32) by combing a benzothiazole molecule and 7-nitrobenzofurazan (NBD) molecule through an ether bond. Longwei He et al. [125] designed and synthesized a probe (**Probe 34**, Figure 32) by attaching NBD to another fluorophore hydroxyphenyl benzothiazole merocyanine (HBTMC) through an ether bond. The probe was able to detect Cys/Hcy, GSH, and H2S at different emission wavelengths. Peng Wang et al. [126] developed a NIR probe (**Probe 35**, Figure 32) named as DCM-NBD which was made by combining a NIR fluorophore icyanomethylene-4*H*-pyran (DCM) derivatives and a NBD fluorophore via ether linkage. The probe was successfully being applied not only in vitro but also in animals. Many other probes have been designed and applied based on different thiol recognition groups or other modifications. Shuangshuang Ding et al. [124] developed the probe (**Probe 33**, Figure 32) by combing a benzothiazole molecule and 7-nitrobenzofurazan (NBD) molecule through an ether bond. Longwei He et al. [125] designed and synthesized a probe (**Probe 34**, Figure 32) by attaching NBD to another fluorophore hydroxyphenyl benzothiazole merocyanine (HBTMC) through an ether bond. The probe was able to detect Cys/Hcy, GSH, and H2S at different emission wavelengths. Peng Wang et al. [126] developed a NIR probe (**Probe 35**, Figure 32) named as DCM-NBD which was made by combining a NIR fluorophore icyanomethylene-4*H*-pyran (DCM) derivatives and a NBD fluorophore via ether linkage. The probe was successfully being applied not only in vitro but also in animals. *Molecules* **2021**, *26*, x FOR PEER REVIEW 21 of 59

substitute products (Cys/Hcy) and the other is S-substitute product (GSH). At the excitation wavelength of 460 nm, GSH-NBD (*S*-substitute) derivative was non-emissive. In contrast, the Cys/Hcy-NBD derivatives exhibited strong yellow fluorescence enabling the discrimination of Cys/Hcy over GSH. The fluorescence intensity was linearly increased when the thiol concentration with a detection limit of 2.00 × 10−8 M for Cys and 1.02 × 10−8 M for Hcy, respectively. The probe can successfully detect the Cys status change in live cells.

A dual channel responsive NIR fluorescent probe (**Probe 32**, Figure 31) for selective detection of Cys in live cells was reported by Zhuo Ye et al. [121] in 2017. The probe named as BDY-NBD is composed of two fluorophores, a NIR BODIPY fluorophore, BDY-OH, and a NBD fluorophore. In the structure, NBD is employed as the thiol responsive unit and the fluorescence of BDY-OH is quenched by NBD owing to PET. Once the probe reacts with a thiol, the nucleophilic thiolate will cleave the NBD from BDY-OH and attach to NBD to form a NBD thiol adduct. The NIR fluorescence of BDY-OH (λex = 650 nm, λem = 735 nm) will be recovered because of the termination of PET. Different from GSH, the thiol adduct (Cys-NBD) produced in the reaction between Cys and probe will subsequently undergo an S to N acyl shift reaction to form a much stronger fluorescent product with a much different emission wavelength (Ex. 466 nm, Em. 540 nm) than BDY-OH while the shift reaction does not occur for GSH. Therefore, a dual-emission mode can be utilized for the detection of Cys. The probe has a much low detection limitation (22 nM, 540 nm emission) that is lower than most of the fluorescent probes developed for the detection of Cys previously. [100,122,123]. In vitro cell experiments with HeLa cells demonstrated that the probe can be applied for multicolor imaging of intracellular Cys, and a significant differences of fluorescence imaging were observed for GSH and Cys in live cells when

**Figure 32.** Structures of **Probe 33**, **34**, **35**, and **36**. **Figure 32.** Structures of **Probe 33**, **34**, **35**, and **36**.

Similar with the structure of NBD, a nitrobenzothiadiazole probe (**Probe 36**, Figure 32) was also developed by Dayoung Lee et al. [127] to achieve the goal of selective detection of Cys and Hcy over GSH based on the same mechanism. The probe was found to increase fluorescence at pH 7.4 in the presence of Cys or Hcy, while at weakly acidic conditions (pH 6.0), only Cys can induce the fluorescence enhancement. Similar with the structure of NBD, a nitrobenzothiadiazole probe (**Probe 36**, Figure 32) was also developed by Dayoung Lee et al. [127] to achieve the goal of selective detection of Cys and Hcy over GSH based on the same mechanism. The probe was found to increase fluorescence at pH 7.4 in the presence of Cys or Hcy, while at weakly acidic conditions (pH 6.0), only Cys can induce the fluorescence enhancement.

#### Other Fluorophores Other Fluorophores

In addition to NBD, BODIPY, probes based on naphthalimide, and cyanine [118] have also been extensively developed, such as 4-nitro-1,8-naphthalic anhydride (NNA, **Probe 37**, Figure 33) which was reported by Limin Ma et al. [128] in 2012 based on nitronaphthalimides as the fluorophore and aromatic nitro part as the thiol recognition group. The probe can selectively detect Cys with a detection limit of 0.3 μM by following the same mechanism (Figure 28) exhibited for NBD and BODIPY. In addition to NBD, BODIPY, probes based on naphthalimide, and cyanine [118] have also been extensively developed, such as 4-nitro-1,8-naphthalic anhydride (NNA, **Probe 37**, Figure 33) which was reported by Limin Ma et al. [128] in 2012 based on nitronaphthalimides as the fluorophore and aromatic nitro part as the thiol recognition group. The probe can selectively detect Cys with a detection limit of 0.3 µM by following the same mechanism (Figure 28) exhibited for NBD and BODIPY.

Many other probes have been developed for discrimination of Cys/Hcy over GSH based on different sensing mechanism. For instance, Hyo Sung Jung et al. [129] developed a probe (**Probe 38**, Figure 34) in 2011 that can selectively detect intracellular Cys by the Michael addition reaction in combination with a steric hinderance factor. A bulky substituent was added to achieve the preferential response for Cys relative to GSH since the molecule of GSH or Hcy is larger than Cys. In addition, the lower pKa of Cys than Hcy and GSH is another factor leading to a higher nucleophilicity of Cys. The fluorescence intensity was found to be linearly increased with an increase in Cys concentration in a range of 0–0.9 mM. The detection limit was determined to be 10−7 M for Cys. Later, the probe was successfully applied for monitoring intracellular Cys in HepG2 cells. The fluorescence derived from the probe in live cells decreased after an increase in NEM concentration. Xin Zhou et al. [130] developed a Cys selective detection probe (**Probe 39**, Figure

2.2.3. Selective Detection of Cys and Hcy via Other Mechanisms

34) based on a Michael addition reaction assisted by an electrostatic attraction.

**Figure 33.** Structure of **Probe 37** and its reaction with Cys.

same mechanism (Figure 28) exhibited for NBD and BODIPY.

ditions (pH 6.0), only Cys can induce the fluorescence enhancement.

**Figure 33.** Structure of **Probe 37** and its reaction with Cys. **Figure 33.** Structure of **Probe 37** and its reaction with Cys.

**Figure 32.** Structures of **Probe 33**, **34**, **35**, and **36**.

Other Fluorophores

#### 2.2.3. Selective Detection of Cys and Hcy via Other Mechanisms 2.2.3. Selective Detection of Cys and Hcy via Other Mechanisms

Many other probes have been developed for discrimination of Cys/Hcy over GSH based on different sensing mechanism. For instance, Hyo Sung Jung et al. [129] developed a probe (**Probe 38**, Figure 34) in 2011 that can selectively detect intracellular Cys by the Michael addition reaction in combination with a steric hinderance factor. A bulky substituent was added to achieve the preferential response for Cys relative to GSH since the molecule of GSH or Hcy is larger than Cys. In addition, the lower pKa of Cys than Hcy and GSH is another factor leading to a higher nucleophilicity of Cys. The fluorescence intensity was found to be linearly increased with an increase in Cys concentration in a range of 0–0.9 mM. The detection limit was determined to be 10−7 M for Cys. Later, the probe was successfully applied for monitoring intracellular Cys in HepG2 cells. The fluorescence derived from the probe in live cells decreased after an increase in NEM concentration. Xin Zhou et al. [130] developed a Cys selective detection probe (**Probe 39**, Figure 34) based on a Michael addition reaction assisted by an electrostatic attraction. Many other probes have been developed for discrimination of Cys/Hcy over GSH based on different sensing mechanism. For instance, Hyo Sung Jung et al. [129] developed a probe (**Probe 38**, Figure 34) in 2011 that can selectively detect intracellular Cys by the Michael addition reaction in combination with a steric hinderance factor. A bulky substituent was added to achieve the preferential response for Cys relative to GSH since the molecule of GSH or Hcy is larger than Cys. In addition, the lower pKa of Cys than Hcy and GSH is another factor leading to a higher nucleophilicity of Cys. The fluorescence intensity was found to be linearly increased with an increase in Cys concentration in a range of 0–0.9 mM. The detection limit was determined to be 10−<sup>7</sup> M for Cys. Later, the probe was successfully applied for monitoring intracellular Cys in HepG2 cells. The fluorescence derived from the probe in live cells decreased after an increase in NEM concentration. Xin Zhou et al. [130] developed a Cys selective detection probe (**Probe 39**, Figure 34) based on a Michael addition reaction assisted by an electrostatic attraction. *Molecules* **2021**, *26*, x FOR PEER REVIEW 22 of 59

Similar with the structure of NBD, a nitrobenzothiadiazole probe (**Probe 36**, Figure 32) was also developed by Dayoung Lee et al. [127] to achieve the goal of selective detection of Cys and Hcy over GSH based on the same mechanism. The probe was found to increase fluorescence at pH 7.4 in the presence of Cys or Hcy, while at weakly acidic con-

In addition to NBD, BODIPY, probes based on naphthalimide, and cyanine [118] have also been extensively developed, such as 4-nitro-1,8-naphthalic anhydride (NNA, **Probe 37**, Figure 33) which was reported by Limin Ma et al. [128] in 2012 based on nitronaphthalimides as the fluorophore and aromatic nitro part as the thiol recognition group. The probe can selectively detect Cys with a detection limit of 0.3 μM by following the

**Figure 34.** Structures of **Probes 38**, **39**, and **40**. **Figure 34.** Structures of **Probes 38**, **39**, and **40**.

While most of the probes being developed are capable of sensing only one of the thiols at a time, a probe which can selectively detect two or three thiols simultaneously with different emissions is highly desirable. In order to address the need, Jing Liu et al. [102] developed a chlorinated coumarin-hemicyanine probe (**Probe 40**, Figure 34) which can simultaneously detects Cys and GSH separately from two different emission and excitation channels. Similar with the two steps strategy for discrimination of thiols, this new coumarin-hemicyanine dye utilize the same strategy starting from a SNAr nucleophilic substitution first to produce a thio-coumarin-hemicyanine, followed by a different intramolecular rearrangement reaction (GSH vs. Cys/Hcy) or different kinetic of the same intramolecular reaction (Cys vs. Hcy) to selectively detect GSH or Cys. The probe is nonemissive and can be turned on by Cys and GSH with emission maximum at 420 and 512 nm, respectively, indicting the high selectivity of the probe for Cys or GSH. For both Cys and GSH, a linear relationship was found between the fluorescence intensity at their emission wavelength and their concentrations (GSH, 0 to 0.9 equiv; Cys, 0.4 to 0.8 equiv). The probe was demonstrated to be able to simultaneously monitor Cys and GSH in COS-7 cells in multicolor imaging. While most of the probes being developed are capable of sensing only one of the thiols at a time, a probe which can selectively detect two or three thiols simultaneously with different emissions is highly desirable. In order to address the need, Jing Liu et al. [102] developed a chlorinated coumarin-hemicyanine probe (**Probe 40**, Figure 34) which can simultaneously detects Cys and GSH separately from two different emission and excitation channels. Similar with the two steps strategy for discrimination of thiols, this new coumarinhemicyanine dye utilize the same strategy starting from a SNAr nucleophilic substitution first to produce a thio-coumarin-hemicyanine, followed by a different intramolecular rearrangement reaction (GSH vs. Cys/Hcy) or different kinetic of the same intramolecular reaction (Cys vs. Hcy) to selectively detect GSH or Cys. The probe is non-emissive and can be turned on by Cys and GSH with emission maximum at 420 and 512 nm, respectively, indicting the high selectivity of the probe for Cys or GSH. For both Cys and GSH, a linear relationship was found between the fluorescence intensity at their emission wavelength and their concentrations (GSH, 0 to 0.9 equiv; Cys, 0.4 to 0.8 equiv). The probe was demonstrated to be able to simultaneously monitor Cys and GSH in COS-7 cells in multicolor imaging.

#### **3. Detection of Thiols in Subcellular Organelles 3. Detection of Thiols in Subcellular Organelles**

thiols probes will be discussed and summarized.

*3.1. Mitochondrial Total Thiols and GSH*

Thiols are known to distribute unevenly in subcellular organelles [8] such as mitochondria, lysosomes, endoplasmic reticulum (ER), golgi apparatus, nucleus, and cell sur-Thiols are known to distribute unevenly in subcellular organelles [8] such as mitochondria, lysosomes, endoplasmic reticulum (ER), golgi apparatus, nucleus, and cell surface.

face. Their levels in different organelles could be an indicator or biomarker to reflect the status of the organelles. Thus, tools or methods that can map thiol distribution and mon-

will provide valuable information to understand the relationship between thiols and cells' functions and dysfunctions, and reveal their correlation as well as find treatment for various thiol-associated diseases [9]. While extensive work has been made and plenty of probes have been developed for the detection of thiols for the whole cell, efforts have been made to develop tools for detecting thiols in subcellular organelles. A subcellular organelle targeting structure is usually needed for a subcellular thiol imaging probe to direct the probe into the targeted organelle. A few subcellular organelle targeting thiol probes have been developed based on the difference identified for subcellular organelles. Most of these probes are for mitochondria with a few for lysosome and ER. In the next part, the diverse targeting approaches and sensing mechanisms of subcellular organelle-targeted

As one of the most important subcellular organelles in cells, mitochondria are double-membrane constructed organelles that serve as the energy generator and also involved in many other biological process including calcium circulation, protein synthesis, apoptosis pathways, etc. A large amount of reactive oxygen species (ROS) are generated in mitochondria during the energy generation process [131–133]. With thiols (especially GSH) being the most important antioxidant to terminate ROS in cells, it is of importance to have a tool that can monitor mitochondrial thiols in live cells. Inspired by the knowledge that Their levels in different organelles could be an indicator or biomarker to reflect the status of the organelles. Thus, tools or methods that can map thiol distribution and monitoring their status changes not only for the whole cell but also for subcellular organelles will provide valuable information to understand the relationship between thiols and cells' functions and dysfunctions, and reveal their correlation as well as find treatment for various thiol-associated diseases [9]. While extensive work has been made and plenty of probes have been developed for the detection of thiols for the whole cell, efforts have been made to develop tools for detecting thiols in subcellular organelles. A subcellular organelle targeting structure is usually needed for a subcellular thiol imaging probe to direct the probe into the targeted organelle. A few subcellular organelle targeting thiol probes have been developed based on the difference identified for subcellular organelles. Most of these probes are for mitochondria with a few for lysosome and ER. In the next part, the diverse targeting approaches and sensing mechanisms of subcellular organelle-targeted thiols probes will be discussed and summarized.

#### *3.1. Mitochondrial Total Thiols and GSH*

As one of the most important subcellular organelles in cells, mitochondria are doublemembrane constructed organelles that serve as the energy generator and also involved in many other biological process including calcium circulation, protein synthesis, apoptosis pathways, etc. A large amount of reactive oxygen species (ROS) are generated in mitochondria during the energy generation process [131–133]. With thiols (especially GSH) being the most important antioxidant to terminate ROS in cells, it is of importance to have a tool that can monitor mitochondrial thiols in live cells. Inspired by the knowledge that mitochondrial membrane spans carry a negative charge [134], a lipophilic cationic structure are widely used for achieving the mitochondria targeting. These lipophilic cations include triphenylphosphonium (TPP), positive charged cyanine, and rhodamine (Figure 35). In addition to lipophilic cations, functional groups, such as peptides [135] have also been developed for mitochondria targeting [136]. In this section, probes developed for detection of mitochondria thiols will be illustrated based on their sensing mechanisms. *Molecules* **2021**, *26*, x FOR PEER REVIEW 23 of 59 mitochondrial membrane spans carry a negative charge [134], a lipophilic cationic structure are widely used for achieving the mitochondria targeting. These lipophilic cations include triphenylphosphonium (TPP), positive charged cyanine, and rhodamine (Figure 35). In addition to lipophilic cations, functional groups, such as peptides [135] have also been developed for mitochondria targeting [136]. In this section, probes developed for detection of mitochondria thiols will be illustrated based on their sensing mechanisms.

**Figure 35.** Representative mitochondrion-targeting. **Figure 35.** Representative mitochondrion-targeting.

3.1.1. Detection of Mitochondrial Thiols via a Michael Addition Reaction 3.1.1. Detection of Mitochondrial Thiols via a Michael Addition Reaction

In 2020, Yutao Yang et al. [137] developed a NIR fluorescent probe (NIR-HMPC, **Probe 41,** Figure 36) based on a Michael addition reaction followed by self-immolative reaction to selectively detect mitochondrial thiols. This NIR probe made by conjugating hemicyanine dye with a benzopylium was connected with a 7-hydroxymethyl-2,3- dihydro-1H-cyclopent-a[b]chromene-1-one with a carbonyl ester. The positive charged indoles iodized salt in the molecule attributes to the mitochondria selectivity. Upon exposure to thiols, nucleophilic addition of the sulfhydryl group will lead to the chromene ring-open to result in a released phenol anion. Subsequently, phenol anions will trigger a self-immolative reaction to generate carbon dioxide and release a strong fluorescent NIR product (λem 731 nm). The fluorescence intensity (731 nm) was found to be linearly increased as the concentration of GSH, Cys, and Hcy increased with the detection limit of 0.59 μM, 0.39 μM, and 0.54 μM, respectively. Colocalization studies with DAPI and Mito-Tracker Green in MCF-7 cells, HepG2 cells, and HeLa cells clearly confirmed that NIR-HMPC can effectively imaging mitochondrial thiols. When PMA (Phorbol 12-myristate 13-acetate, a cellu-In 2020, Yutao Yang et al. [137] developed a NIR fluorescent probe (NIR-HMPC, **Probe 41**, Figure 36) based on a Michael addition reaction followed by self-immolative reaction to selectively detect mitochondrial thiols. This NIR probe made by conjugating hemicyanine dye with a benzopylium was connected with a 7-hydroxymethyl-2,3 dihydro-1H-cyclopent-a[b]chromene-1-one with a carbonyl ester. The positive charged indoles iodized salt in the molecule attributes to the mitochondria selectivity. Upon exposure to thiols, nucleophilic addition of the sulfhydryl group will lead to the chromene ring-open to result in a released phenol anion. Subsequently, phenol anions will trigger a self-immolative reaction to generate carbon dioxide and release a strong fluorescent NIR product (λem 731 nm). The fluorescence intensity (731 nm) was found to be linearly increased as the concentration of GSH, Cys, and Hcy increased with the detection limit of 0.59 µM, 0.39 µM, and 0.54 µM, respectively. Colocalization studies with DAPI and Mito-Tracker Green in MCF-7 cells, HepG2 cells, and HeLa cells clearly confirmed that NIR-HMPC can effectively imaging mitochondrial thiols. When PMA (Phorbol 12-myristate

lar oxidative stress inducer) and BSO (a sulfoximine that can reduce GSH levels) were used to pretreat cells to induce an intracellular thiol decrease, a dramatic fluorescence de-

drial thiol change in live cells. More impressively, the probe can not only reflect thiol change in vitro but also reflect the change in vivo in a mouse model. The probe has also been successfully applied on sensing thiol change in a mouse mode of cerebral ischemia, warranting the application of the probe for monitoring the physiological and pathological

**Figure 36.** Structure of **Probe 41** and its reaction with thiols.

processes at the cellular and animal levels.

13-acetate, a cellular oxidative stress inducer) and BSO (a sulfoximine that can reduce GSH levels) were used to pretreat cells to induce an intracellular thiol decrease, a dramatic fluorescence decrease was observed revealing that the probe was able to visualize and reflect mitochondrial thiol change in live cells. More impressively, the probe can not only reflect thiol change in vitro but also reflect the change in vivo in a mouse model. The probe has also been successfully applied on sensing thiol change in a mouse mode of cerebral ischemia, warranting the application of the probe for monitoring the physiological and pathological processes at the cellular and animal levels. tively imaging mitochondrial thiols. When PMA (Phorbol 12-myristate 13-acetate, a cellular oxidative stress inducer) and BSO (a sulfoximine that can reduce GSH levels) were used to pretreat cells to induce an intracellular thiol decrease, a dramatic fluorescence decrease was observed revealing that the probe was able to visualize and reflect mitochondrial thiol change in live cells. More impressively, the probe can not only reflect thiol change in vitro but also reflect the change in vivo in a mouse model. The probe has also been successfully applied on sensing thiol change in a mouse mode of cerebral ischemia, warranting the application of the probe for monitoring the physiological and pathological processes at the cellular and animal levels.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 23 of 59

**Figure 35.** Representative mitochondrion-targeting.

3.1.1. Detection of Mitochondrial Thiols via a Michael Addition Reaction

In 2020, Yutao Yang et al. [137] developed a NIR fluorescent probe (NIR-HMPC, **Probe 41,** Figure 36) based on a Michael addition reaction followed by self-immolative reaction to selectively detect mitochondrial thiols. This NIR probe made by conjugating hemicyanine dye with a benzopylium was connected with a 7-hydroxymethyl-2,3- dihydro-1H-cyclopent-a[b]chromene-1-one with a carbonyl ester. The positive charged indoles iodized salt in the molecule attributes to the mitochondria selectivity. Upon exposure to thiols, nucleophilic addition of the sulfhydryl group will lead to the chromene ring-open to result in a released phenol anion. Subsequently, phenol anions will trigger a self-immolative reaction to generate carbon dioxide and release a strong fluorescent NIR product (λem 731 nm). The fluorescence intensity (731 nm) was found to be linearly increased as the concentration of GSH, Cys, and Hcy increased with the detection limit of 0.59 μM, 0.39 μM, and 0.54 μM, respectively. Colocalization studies with DAPI and Mito-Tracker Green in MCF-7 cells, HepG2 cells, and HeLa cells clearly confirmed that NIR-HMPC can effec-

mitochondrial membrane spans carry a negative charge [134], a lipophilic cationic structure are widely used for achieving the mitochondria targeting. These lipophilic cations include triphenylphosphonium (TPP), positive charged cyanine, and rhodamine (Figure 35). In addition to lipophilic cations, functional groups, such as peptides [135] have also been developed for mitochondria targeting [136]. In this section, probes developed for detection of mitochondria thiols will be illustrated based on their sensing mechanisms.

**Figure 36. Figure 36.**  Structure of Structure of **Probe 41 Probe 41** and its reaction with thiols. and its reaction with thiols.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 24 of 59

In 2017, Jianwei Chen et al. [138] developed a new mitochondrion-targeted probe (MitoRT, **Probe 42**, Figure 37) which could detected the mitochondrial GSH status reversibly. In the molecule, TPP, the positive charged mitochondria targeting group, was linked to a fluorophore via an optimized 4-carbon linker. The reagent is capable of reacting with GSH rapidly in both forward and reverse directions that enable the real time monitoring of mitochondrial GSH dynamically in live cells. In addition, MitoRT showed ratiometric fluorescence responses with a wide dynamic range when reacting with GSH. The probe has been demonstrated to be capable of monitoring mitochondrial GSH in live HeLa cells reversibly through fluorescence microscope. The probe can also be applied to monitor mitochondrial GSH levels in a high throughput manner (flow cytometry). In 2017, Jianwei Chen et al. [138] developed a new mitochondrion-targeted probe (MitoRT, **Probe 42**, Figure 37) which could detected the mitochondrial GSH status reversibly. In the molecule, TPP, the positive charged mitochondria targeting group, was linked to a fluorophore via an optimized 4-carbon linker. The reagent is capable of reacting with GSH rapidly in both forward and reverse directions that enable the real time monitoring of mitochondrial GSH dynamically in live cells. In addition, MitoRT showed ratiometric fluorescence responses with a wide dynamic range when reacting with GSH. The probe has been demonstrated to be capable of monitoring mitochondrial GSH in live HeLa cells reversibly through fluorescence microscope. The probe can also be applied to monitor

mitochondrial GSH levels in a high throughput manner (flow cytometry).

**Figure 37.** Structure of **Probe 42** and its reversible reaction with GSH. **Figure 37.** Structure of **Probe 42** and its reversible reaction with GSH.

In 2017, Keitaro Umezawa et al. [139] reported their work on developing reversible fluorescent probe (**Probe 43**) for live cell imaging and ratiometric quantification of fast GSH dynamics in mitochondria based on a cationic rhodamine fluorophore as an electrophile for Michael addition to reversibly react with thiols. The calculated rate constant *k* = 560 M–1 s –1 (t1/2 = 620 ms at GSH = 1 mM) which is a 3900-fold increase than TQ green [73]. Similar with GSH, other thiol-containing molecules such as Cys, Hcy, and H2S can also induce the fluorescence change if their concentration is in millimolar range. However, since their physiological concentration is much lower than GSH, their interference with GSH should be a minimum. As shown in Figure 38, a TMR (typical rhodamine structure) was introduced to the probe as a donor of the FRET. Based on that, two probes, QG0.6 and QG3.0 were designed and synthesized. Both probes exhibit ratiometric absorption/fluorescence changes in response to various concentrations of GSH. Co-staining experiments with MitoTracker Deep Red FM indicated that the compound could accumulate inside mitochondria. QG3.0 can quantitatively monitor GSH status change in real time In 2017, Keitaro Umezawa et al. [139] reported their work on developing reversible fluorescent probe (**Probe 43**) for live cell imaging and ratiometric quantification of fast GSH dynamics in mitochondria based on a cationic rhodamine fluorophore as an electrophile for Michael addition to reversibly react with thiols. The calculated rate constant *k* = 560 M−<sup>1</sup> s −1 (t1/2 = 620 ms at GSH = 1 mM) which is a 3900-fold increase than TQ green [73]. Similar with GSH, other thiol-containing molecules such as Cys, Hcy, and H2S can also induce the fluorescence change if their concentration is in millimolar range. However, since their physiological concentration is much lower than GSH, their interference with GSH should be a minimum. As shown in Figure 38, a TMR (typical rhodamine structure) was introduced to the probe as a donor of the FRET. Based on that, two probes, QG0.6 and QG3.0 were designed and synthesized. Both probes exhibit ratiometric absorption/fluorescence changes in response to various concentrations of GSH. Co-staining experiments with MitoTracker Deep Red FM indicated that the compound could accumulate inside mitochondria. QG3.0 can quantitatively monitor GSH status change in real time

when cells are consecutively treated with H2O<sup>2</sup> or GSH ester. The probe is also capable of

3.1.2. Detection of Mitochondrial NPSH via a SNAr Reaction Using Halogen, Ether, or

Detection of Mitochondrial NPSH via a SNAr Reaction Using Halogen as a Leaving

In 2017, Xueliang Liu et al. [140] reported a mitochondria-targeting fluorescent probe (BODIPY-PPh3,**Probe 44**) for selective and ratiometric detection of GSH over Cys and Hcy in line with the group's previous work [29]. As depicted in Figure 39, chlorinated BODIPY

monitoring the real time GSH level change after the cells being glucose deprived.

**Figure 38.** Structure of **Probe 43** and its reversible reaction with GSH.

Thioether as a Leaving Group

Group

560 M–1 s

Group

when cells are consecutively treated with H2O<sup>2</sup> or GSH ester. The probe is also capable of monitoring the real time GSH level change after the cells being glucose deprived. when cells are consecutively treated with H2O<sup>2</sup> or GSH ester. The probe is also capable of monitoring the real time GSH level change after the cells being glucose deprived.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 24 of 59

mitochondrial GSH levels in a high throughput manner (flow cytometry).

**Figure 37.** Structure of **Probe 42** and its reversible reaction with GSH.

In 2017, Jianwei Chen et al. [138] developed a new mitochondrion-targeted probe (MitoRT, **Probe 42**, Figure 37) which could detected the mitochondrial GSH status reversibly. In the molecule, TPP, the positive charged mitochondria targeting group, was linked to a fluorophore via an optimized 4-carbon linker. The reagent is capable of reacting with GSH rapidly in both forward and reverse directions that enable the real time monitoring of mitochondrial GSH dynamically in live cells. In addition, MitoRT showed ratiometric fluorescence responses with a wide dynamic range when reacting with GSH. The probe has been demonstrated to be capable of monitoring mitochondrial GSH in live HeLa cells reversibly through fluorescence microscope. The probe can also be applied to monitor

In 2017, Keitaro Umezawa et al. [139] reported their work on developing reversible fluorescent probe (**Probe 43**) for live cell imaging and ratiometric quantification of fast GSH dynamics in mitochondria based on a cationic rhodamine fluorophore as an electrophile for Michael addition to reversibly react with thiols. The calculated rate constant *k* =

–1 (t1/2 = 620 ms at GSH = 1 mM) which is a 3900-fold increase than TQ green [73].

Similar with GSH, other thiol-containing molecules such as Cys, Hcy, and H2S can also induce the fluorescence change if their concentration is in millimolar range. However, since their physiological concentration is much lower than GSH, their interference with GSH should be a minimum. As shown in Figure 38, a TMR (typical rhodamine structure) was introduced to the probe as a donor of the FRET. Based on that, two probes, QG0.6

periments with MitoTracker Deep Red FM indicated that the compound could accumulate inside mitochondria. QG3.0 can quantitatively monitor GSH status change in real time

**Figure 38.** Structure of **Probe 43** and its reversible reaction with GSH. **Figure 38.** Structure of **Probe 43** and its reversible reaction with GSH.

3.1.2. Detection of Mitochondrial NPSH via a SNAr Reaction Using Halogen, Ether, or Thioether as a Leaving Group 3.1.2. Detection of Mitochondrial NPSH via a SNAr Reaction Using Halogen, Ether, or Thioether as a Leaving Group

Detection of Mitochondrial NPSH via a SNAr Reaction Using Halogen as a Leaving Group

Detection of Mitochondrial NPSH via a SNAr Reaction Using Halogen as a Leaving In 2017, Xueliang Liu et al. [140] reported a mitochondria-targeting fluorescent probe (BODIPY-PPh3,**Probe 44**) for selective and ratiometric detection of GSH over Cys and Hcy in line with the group's previous work [29]. As depicted in Figure 39, chlorinated BODIPY In 2017, Xueliang Liu et al. [140] reported a mitochondria-targeting fluorescent probe (BODIPY-PPh3, **Probe 44**) for selective and ratiometric detection of GSH over Cys and Hcy in line with the group's previous work [29]. As depicted in Figure 39, chlorinated BODIPY was used as the fluorophore and the chlorinated site serves as thiol (GSH, Cys, or Hcy) recognition site through a SNAr reaction to afford the thioether. As discussed early, the thioether formed with Cys and Hcy underwent an intramolecular rearrangement to yield the amino derivatives resulting in a remarkable change of photophysical properties, while the thioether formed with GSH could not undergo such an intramolecular rearrangement. The difference in the intramolecular rearrangement reaction for GSH vs. Cys and Hcy attribute to the selective detection of GSH over Cys and Hcy. To achieve mitochondria targeting, a TPP structure was attached to the probe. The probe itself shows an intrinsic emission at 557 nm which decreases dramatically upon reaction with GSH and a new emission at 588 nm (Ex. 550 nm) increased significantly. Their ratio (*I*588/*I*557) was found to increase linearly with GSH concentration (0–80 µM) with a detection limit of 1.1 µM. The linearity enables the quantitative detection of GSH. The fluorescence behavior of the probe reacting with Cys and Hcy was totally different with a much lower emission at 588 nm and much smaller (*I*588/*I*557) ratio change. The colocalization study with rhodamine 123 found that BODIPY-PPh<sup>3</sup> could accurately locate in the mitochondria. Pretreatment of the cells with NEM to deplete GSH resulted in a lower red/green fluorescence ratio (~2). In contrast, pretreatment of cells with GSH before incubation with BODIPY-PPh<sup>3</sup> led to a higher red/green channels fluorescence ratio (~5). The results reveals that the BODIPY-PPh<sup>3</sup> can effectively and ratiometrically monitor the status of mitochondrial thiols. *Molecules* **2021**, *26*, x FOR PEER REVIEW 25 of 59 was used as the fluorophore and the chlorinated site serves as thiol (GSH, Cys, or Hcy) recognition site through a SNAr reaction to afford the thioether. As discussed early, the thioether formed with Cys and Hcy underwent an intramolecular rearrangement to yield the amino derivatives resulting in a remarkable change of photophysical properties, while the thioether formed with GSH could not undergo such an intramolecular rearrangement. The difference in the intramolecular rearrangement reaction for GSH vs. Cys and Hcy attribute to the selective detection of GSH over Cys and Hcy. To achieve mitochondria targeting, a TPP structure was attached to the probe. The probe itself shows an intrinsic emission at 557 nm which decreases dramatically upon reaction with GSH and a new emission at 588 nm (Ex. 550 nm) increased significantly. Their ratio (*I*588/*I*557) was found to increase linearly with GSH concentration (0–80 μM) with a detection limit of 1.1 μM. The linearity enables the quantitative detection of GSH. The fluorescence behavior of the probe reacting with Cys and Hcy was totally different with a much lower emission at 588 nm and much smaller (*I*588/*I*557) ratio change. The colocalization study with rhodamine 123 found that BODIPY-PPh<sup>3</sup> could accurately locate in the mitochondria. Pretreatment of the cells with NEM to deplete GSH resulted in a lower red/green fluorescence ratio (~2). In contrast, pretreatment of cells with GSH before incubation with BODIPY-PPh<sup>3</sup> led to a higher red/green channels fluorescence ratio (~5). The results reveals that the BODIPY-PPh<sup>3</sup> can effectively and ratiometrically monitor the status of mitochondrial thiols.

**Figure 39. Probe 44** and its reaction with GSH. **Figure 39. Probe 44** and its reaction with GSH.

In 2018, Sujie Qi et al. [141] presented a water-soluble ratiometric fluorescent probe (**Probe 45**) for selective detection of GSH in mitochondria of live cells. The probe was built based on colorimetric hemicyanine dye and was capable of discriminating GSH over Cys/Hcy in a quantitative manner in a range of 1.0–15.0 mM with a low detection limit in In 2018, Sujie Qi et al. [141] presented a water-soluble ratiometric fluorescent probe (**Probe 45**) for selective detection of GSH in mitochondria of live cells. The probe was built based on colorimetric hemicyanine dye and was capable of discriminating GSH over Cys/Hcy in a quantitative manner in a range of 1.0–15.0 mM with a low detection limit in

aqueous solution. In detail, the hemicyanine dye was modified by an aldehyde group and a chloro-substitution which equip the probe the ability to selectively react with GSH since

form a ring (Figure 40). The reaction was much faster than the reaction with Cys and Hcy since an intramolecular aldimine condensation to form a ring could not occur with Cys and Hcy [65,142]. The quaternary ammonium cation serves as the mitochondria targeting moiety and a hydrophilic sulfonate was added to enhance the water-solubility of the probe. Upon reaction with GSH, significant optical property (absorption and fluorescence) changes were induced which significantly dropped fluorescence intensity at 607 nm along with appearance of a new fluorescence peak at 648 nm. The ratio of emission intensities (I648/I607) displayed a linear relationship with GSH concentration (1.0 to 15.0 mM) with a detection limit of 24.16 μM, enabling the quantitative detection of GSH. The probe displays a great mitochondria targeting ability and can well reflect the status

changes of mitochondrial GSH in a ratiometrical manner.

aqueous solution. In detail, the hemicyanine dye was modified by an aldehyde group and a chloro-substitution which equip the probe the ability to selectively react with GSH since GSH reacts with the probe via a SNAr and an intramolecular aldimine condensation to form a ring (Figure 40). The reaction was much faster than the reaction with Cys and Hcy since an intramolecular aldimine condensation to form a ring could not occur with Cys and Hcy [65,142]. The quaternary ammonium cation serves as the mitochondria targeting moiety and a hydrophilic sulfonate was added to enhance the water-solubility of the probe. Upon reaction with GSH, significant optical property (absorption and fluorescence) changes were induced which significantly dropped fluorescence intensity at 607 nm along with appearance of a new fluorescence peak at 648 nm. The ratio of emission intensities (I648/I607) displayed a linear relationship with GSH concentration (1.0 to 15.0 mM) with a detection limit of 24.16 µM, enabling the quantitative detection of GSH. The probe displays a great mitochondria targeting ability and can well reflect the status changes of mitochondrial GSH in a ratiometrical manner. *Molecules* **2021**, *26*, x FOR PEER REVIEW 26 of 59 *Molecules* **2021**, *26*, x FOR PEER REVIEW 26 of 59

Group

**Figure 40.** Structure of **Probe 45** and its reaction with GSH. **Figure 40.** Structure of **Probe 45** and its reaction with GSH. **Figure 40.** Structure of **Probe 45** and its reaction with GSH.

Detection of Mitochondrial NPSH via a SNAr Reaction Using Thioether as a Leaving Group Detection of Mitochondrial NPSH via a SNAr Reaction Using Thioether as a Leaving Group Detection of Mitochondrial NPSH via a SNAr Reaction Using Thioether as a Leaving

successfully used to image and quantify NPSH in mitochondria in live cells.

In 2018, we developed a mitochondrial-targeting rhodamine based probe [22] (TBROS, **Probe 46**) for selective imaging and quantification of mitochondrial thiols in live cells. The probe was designed based on a thiol specific thiol–sulfide exchange reaction reported by us in 2012 [42]. Similar to the GUALY's reagent [42], two benzofurazan moieties were connected together by a thioether to maintain a symmetric structure (Figure 41). Two cationic rhodamine B units were introduced symmetrically to the benzofurazan structure to provide the mitochondria selectivity and also serve as a strong fluorophore to enhance the fluorescence intensity of benzofurazan. The probe itself is not fluorescent due to the self-quenching of the symmetric structure but turns into strong fluorescent products (Ex. 550 nm, Em. 580 nm) upon reaction with thiols through the thiol specific thiol–sulfide exchange reaction. TBROS has been confirmed nonreactive toward PSH and In 2018, we developed a mitochondrial-targeting rhodamine based probe [22] (TBROS, **Probe 46**) for selective imaging and quantification of mitochondrial thiols in live cells. The probe was designed based on a thiol specific thiol–sulfide exchange reaction reported by us in 2012 [42]. Similar to the GUALY's reagent [42], two benzofurazan moieties were connected together by a thioether to maintain a symmetric structure (Figure 41). Two cationic rhodamine B units were introduced symmetrically to the benzofurazan structure to provide the mitochondria selectivity and also serve as a strong fluorophore to enhance the fluorescence intensity of benzofurazan. The probe itself is not fluorescent due to the self-quenching of the symmetric structure but turns into strong fluorescent products (Ex. 550 nm, Em. 580 nm) upon reaction with thiols through the thiol specific thiol–sulfide exchange reaction. TBROS has been confirmed nonreactive toward PSH and successfully used to image and quantify NPSH in mitochondria in live cells. In 2018, we developed a mitochondrial-targeting rhodamine based probe [22] (TBROS, **Probe 46**) for selective imaging and quantification of mitochondrial thiols in live cells. The probe was designed based on a thiol specific thiol–sulfide exchange reaction reported by us in 2012 [42]. Similar to the GUALY's reagent [42], two benzofurazan moieties were connected together by a thioether to maintain a symmetric structure (Figure 41). Two cationic rhodamine B units were introduced symmetrically to the benzofurazan structure to provide the mitochondria selectivity and also serve as a strong fluorophore to enhance the fluorescence intensity of benzofurazan. The probe itself is not fluorescent due to the self-quenching of the symmetric structure but turns into strong fluorescent products (Ex. 550 nm, Em. 580 nm) upon reaction with thiols through the thiol specific thiol–sulfide exchange reaction. TBROS has been confirmed nonreactive toward PSH and successfully used to image and quantify NPSH in mitochondria in live cells.

**Figure 41.** Structure of **Probe 46** and its reaction with thiols. **Figure 41.** Structure of **Probe 46** and its reaction with thiols. **Figure 41.** Structure of **Probe 46** and its reaction with thiols.

In 2019, Zhiqiang Xu et al. [143] developed a NIR probe (Cy-S-Np, **Probe 47**, Figure 42) based on thioether as the thiol response unit to visualize mitochondrial and lysosomal GSH in live cells and also in a mouse model. The probe was constructed by connecting a In 2019, Zhiqiang Xu et al. [143] developed a NIR probe (Cy-S-Np, **Probe 47**, Figure 42) based on thioether as the thiol response unit to visualize mitochondrial and lysosomal GSH in live cells and also in a mouse model. The probe was constructed by connecting a In 2019, Zhiqiang Xu et al. [143] developed a NIR probe (Cy-S-Np, **Probe 47**, Figure 42) based on thioether as the thiol response unit to visualize mitochondrial and lysosomal GSH in live cells and also in a mouse model. The probe was constructed by connecting

> cationic cyanine IR-780 dye (mitochondria targeting) and a morpholine-coating naphthalimide (lysosome targeting) through a thioether (thiol response moiety). Cy-S-Np can

> its promising use for studying the interaction of cellular function between mitochondria and lysosome. After reaction with GSH, a remarkable fluorescence intensity enhancement at 812 nm (Ex. 710 nm) was observed. Impressively, the probe showed a detection limit of 11 nM. Cellular experiments showed that the probe was able to detect GSH changes both in mitochondria and in lysosome of live HeLa cells. Further, visualization of subcellular

cationic cyanine IR-780 dye (mitochondria targeting) and a morpholine-coating naphthalimide (lysosome targeting) through a thioether (thiol response moiety). Cy-S-Np can

its promising use for studying the interaction of cellular function between mitochondria and lysosome. After reaction with GSH, a remarkable fluorescence intensity enhancement at 812 nm (Ex. 710 nm) was observed. Impressively, the probe showed a detection limit of 11 nM. Cellular experiments showed that the probe was able to detect GSH changes both in mitochondria and in lysosome of live HeLa cells. Further, visualization of subcellular

GSH level at organism level in mice was achieved as well.

GSH level at organism level in mice was achieved as well.

a cationic cyanine IR-780 dye (mitochondria targeting) and a morpholine-coating naphthalimide (lysosome targeting) through a thioether (thiol response moiety). Cy-S-Np can effectively distinguish GSH over Cys and Hcy in mitochondria and lysosome, warranting its promising use for studying the interaction of cellular function between mitochondria and lysosome. After reaction with GSH, a remarkable fluorescence intensity enhancement at 812 nm (Ex. 710 nm) was observed. Impressively, the probe showed a detection limit of 11 nM. Cellular experiments showed that the probe was able to detect GSH changes both in mitochondria and in lysosome of live HeLa cells. Further, visualization of subcellular GSH level at organism level in mice was achieved as well. *Molecules* **2021**, *26*, x FOR PEER REVIEW 27 of 59

**Figure 42.** Structure of **Probe 47** and its reaction with GSH. **Figure 42.** Structure of **Probe 47** and its reaction with GSH.

Detection of Mitochondrial NPSH via a SNAr Reaction Using Ether as a Leaving Group

Detection of Mitochondrial NPSH via a SNAr Reaction Using Ether as a Leaving Group Yuan Gu et al. [35] developed a ratiometric reagent which can be used in two-photon fluorescence microscopy (TPFM). The two photon excitation fluorescent imaging has the advantage of less interference from autofluorescence background, deeper penetration to tissues, and low phototoxicity [144–147]. The regent (**Probe 48**, Figure 43) named as TPE-PBP showed a high sensitivity and selectivity toward thiols, including GSH, Cys, Hcy, etc., with a high selectivity for mitochondria. Cell experiments shows that TPE-PBP was able to measure the mitochondrial thiol status change in a ratiometric manner. The twophoton-absorption cross section enables the regent to apply in live cells, in living skeletal muscle tissues, and in two day old fish larva. The success of this reagent provides a new strategy for the construction of ratiometric two-photon active reagens on the application of in vivo biosensing and bioimaging application [35]. It needs to be noted that the application of traditional fluorophore probes was hampered by the aggregation-caused quenching (ACQ) effect that is that the probes' fluorescence will be quenched or weakened if the probes are concentrated or aggregated [148–150]. The ACQ effect makes the quantification of analytes in live cells more challenging. Tang and co-workers observed an unusual phenomenon on a class of molecules with propeller shape (e.g., tetraphenylethylene (TPE), siloles) which can turn on their fluorescence in the aggregate or solid states while keep non-emissive in diluted solutions. This phenomenon was termed Yuan Gu et al. [35] developed a ratiometric reagent which can be used in two-photon fluorescence microscopy (TPFM). The two photon excitation fluorescent imaging has the advantage of less interference from autofluorescence background, deeper penetration to tissues, and low phototoxicity [144–147]. The regent (**Probe 48**, Figure 43) named as TPE-PBP showed a high sensitivity and selectivity toward thiols, including GSH, Cys, Hcy, etc., with a high selectivity for mitochondria. Cell experiments shows that TPE-PBP was able to measure the mitochondrial thiol status change in a ratiometric manner. The two-photonabsorption cross section enables the regent to apply in live cells, in living skeletal muscle tissues, and in two day old fish larva. The success of this reagent provides a new strategy for the construction of ratiometric two-photon active reagens on the application of in vivo biosensing and bioimaging application [35]. It needs to be noted that the application of traditional fluorophore probes was hampered by the aggregation-caused quenching (ACQ) effect that is that the probes' fluorescence will be quenched or weakened if the probes are concentrated or aggregated [148–150]. The ACQ effect makes the quantification of analytes in live cells more challenging. Tang and co-workers observed an unusual phenomenon on a class of molecules with propeller shape (e.g., tetraphenylethylene (TPE), siloles) which can turn on their fluorescence in the aggregate or solid states while keep non-emissive in diluted solutions. This phenomenon was termed as "aggregation-induced emission (AIE)" and the mechanism can be explained by a restriction of intramolecular motions (RIMs), including restriction of intramolecular vibrations (RIVs) and restriction of intramolecular rotations (RIR) [151–159]. Herein, AIE probes can overcome the drawbacks of the traditional fluorophore probes.

probes can overcome the drawbacks of the traditional fluorophore probes.

as "aggregation-induced emission (AIE)" and the mechanism can be explained by a restriction of intramolecular motions (RIMs), including restriction of intramolecular vibrations (RIVs) and restriction of intramolecular rotations (RIR) [151–159]. Herein, AIE

In 2016, Jian Zhang et al. [160] developed a probe (**Probe 49**, Figure 44) for detecting

mitochondrial GSH with BODIPY serving as the fluorophore. In this approach, a self-immolative dinitrophenoxy benzyl pyridinium was connected with BODIPY to serve as the

**Figure 43.** Structure of **Probe 48** and its reaction with GSH.

probes can overcome the drawbacks of the traditional fluorophore probes.

**Figure 43.** Structure of **Probe 48** and its reaction with GSH. **Figure 43.** Structure of **Probe 48** and its reaction with GSH.

**Figure 42.** Structure of **Probe 47** and its reaction with GSH.

Detection of Mitochondrial NPSH via a SNAr Reaction Using Ether as a Leaving Group

Yuan Gu et al. [35] developed a ratiometric reagent which can be used in two-photon fluorescence microscopy (TPFM). The two photon excitation fluorescent imaging has the advantage of less interference from autofluorescence background, deeper penetration to tissues, and low phototoxicity [144–147]. The regent (**Probe 48**, Figure 43) named as TPE-PBP showed a high sensitivity and selectivity toward thiols, including GSH, Cys, Hcy, etc., with a high selectivity for mitochondria. Cell experiments shows that TPE-PBP was able to measure the mitochondrial thiol status change in a ratiometric manner. The twophoton-absorption cross section enables the regent to apply in live cells, in living skeletal muscle tissues, and in two day old fish larva. The success of this reagent provides a new strategy for the construction of ratiometric two-photon active reagens on the application of in vivo biosensing and bioimaging application [35]. It needs to be noted that the application of traditional fluorophore probes was hampered by the aggregation-caused quenching (ACQ) effect that is that the probes' fluorescence will be quenched or weakened if the probes are concentrated or aggregated [148–150]. The ACQ effect makes the quantification of analytes in live cells more challenging. Tang and co-workers observed an unusual phenomenon on a class of molecules with propeller shape (e.g., tetraphenylethylene (TPE), siloles) which can turn on their fluorescence in the aggregate or solid states while keep non-emissive in diluted solutions. This phenomenon was termed as "aggregation-induced emission (AIE)" and the mechanism can be explained by a restriction of intramolecular motions (RIMs), including restriction of intramolecular vibrations (RIVs) and restriction of intramolecular rotations (RIR) [151–159]. Herein, AIE

In 2016, Jian Zhang et al. [160] developed a probe (**Probe 49**, Figure 44) for detecting mitochondrial GSH with BODIPY serving as the fluorophore. In this approach, a self-immolative dinitrophenoxy benzyl pyridinium was connected with BODIPY to serve as the In 2016, Jian Zhang et al. [160] developed a probe (**Probe 49**, Figure 44) for detecting mitochondrial GSH with BODIPY serving as the fluorophore. In this approach, a selfimmolative dinitrophenoxy benzyl pyridinium was connected with BODIPY to serve as the targeting ligand and also the GSH recognition group. Once being exposed to GSH, the dinitrophenyl moiety will be cleaved through a SNAr reaction, followed by a selfimmolation reaction to release fluorophore BODIPY with strong fluorescence (599 nm). A linear relationship was observed between the fluorescence intensity and GSH concentration in a range of 1 to 15 µM with a detection limit of 109 nM. The selectivity of GSH over Cys and Hcy was possible due to an electrostatic interaction between the cationic pyridinium moiety and GSH: once the electrostatic interaction formed, GSH was believed to react much faster than Cys and Hcy. The probe has been successfully used to monitor the status of mitochondrial GSH in live HeLa cells. *Molecules* **2021**, *26*, x FOR PEER REVIEW 28 of 59 targeting ligand and also the GSH recognition group. Once being exposed to GSH, the dinitrophenyl moiety will be cleaved through a SNAr reaction, followed by a self-immolation reaction to release fluorophore BODIPY with strong fluorescence (599 nm). A linear relationship was observed between the fluorescence intensity and GSH concentration in a range of 1 to 15 μM with a detection limit of 109 nM. The selectivity of GSH over Cys and Hcy was possible due to an electrostatic interaction between the cationic pyridinium moiety and GSH: once the electrostatic interaction formed, GSH was believed to react much faster than Cys and Hcy. The probe has been successfully used to monitor the status of mitochondrial GSH in live HeLa cells.

sity was barely changed.

**Figure 44.** Structure of **Probe 49** and its reaction with GSH. **Figure 44.** Structure of **Probe 49** and its reaction with GSH.

In 2019, Yue Xu et al. [1] reported a visible and near-infrared, dual emission fluorescent probe (Cy-DC, **Probe 50**) for monitoring mitochondrial thiol in vitro. The probe also exhibits impressive ability to detect solid tumor by both naked eye and near-infrared fluorescence. As depicted in Figure 45, two fluorophores, dicyanomethylene-4H-pyran and cyanine, with distinct wavelength emission bands are linked together with a thiol responsive unit ether linker aryl ether group. The positive charged cyanine is the driving force for mitochondria targeting. The fluorescence activation by thiols was achieved with a nucleophilic aromatic substitution-rearrangement reaction between the thiolate of a thiol with the aryl ether to form a thioether which the thiol molecule is attached to the cyanine with strong NIR fluorescence (Em. 810 nm). In the meantime, the other fluorophore, dicyanomethylene-4H-pyran, was released and restored its visible fluorescence (520 nm). In contrast to GSH, the thioether formed from Cys and Hcy will undergo an intramolecular rearrangement vis a 5-(Cys) or 6-(Hcy) membered transition state resulting in the replace-In 2019, Yue Xu et al. [1] reported a visible and near-infrared, dual emission fluorescent probe (Cy-DC, **Probe 50**) for monitoring mitochondrial thiol in vitro. The probe also exhibits impressive ability to detect solid tumor by both naked eye and near-infrared fluorescence. As depicted in Figure 45, two fluorophores, dicyanomethylene-4H-pyran and cyanine, with distinct wavelength emission bands are linked together with a thiol responsive unit ether linker aryl ether group. The positive charged cyanine is the driving force for mitochondria targeting. The fluorescence activation by thiols was achieved with a nucleophilic aromatic substitution-rearrangement reaction between the thiolate of a thiol with the aryl ether to form a thioether which the thiol molecule is attached to the cyanine with strong NIR fluorescence (Em. 810 nm). In the meantime, the other fluorophore, dicyanomethylene-4H-pyran, was released and restored its visible fluorescence (520 nm). In contrast to GSH, the thioether formed from Cys and Hcy will undergo an intramolecular rearrangement vis a 5-(Cys) or 6-(Hcy) membered transition state resulting in the replace-

ment of *S* with *N* leading to a significant fluorescence elimination (810 nm). This provides the probe the ability for discrimination of GSH over Cys and Hcy. Briefly, GSH activated

Cys and Hcy only attribute to the fluorescent intensity of Cy-DC at 520 nm. The detection limits for GSH in two different channels were determined to be 24 nM (visible) and 32 nM (NIR). Additionally, an increase in the thiol blocking agent (NEM) led to a gradually decrease of fluorescence intensity in both channels. In line with the hypothesized mechanism, pretreatment of cells with NEM totally inhibited the fluorescence while addition of GSH to the cells induced a remarkable increase in fluorescence intensity in both channels (green visible channel and NIR red channel). In contrast, addition of Cys and Hcy only led to the fluorescence increase in green visible channel while NIR red fluorescence inten-

ment of *S* with *N* leading to a significant fluorescence elimination (810 nm). This provides the probe the ability for discrimination of GSH over Cys and Hcy. Briefly, GSH activated a remarkable increase of fluorescent intensity of Cy-DC both at 520 nm and 810 nm while Cys and Hcy only attribute to the fluorescent intensity of Cy-DC at 520 nm. The detection limits for GSH in two different channels were determined to be 24 nM (visible) and 32 nM (NIR). Additionally, an increase in the thiol blocking agent (NEM) led to a gradually decrease of fluorescence intensity in both channels. In line with the hypothesized mechanism, pretreatment of cells with NEM totally inhibited the fluorescence while addition of GSH to the cells induced a remarkable increase in fluorescence intensity in both channels (green visible channel and NIR red channel). In contrast, addition of Cys and Hcy only led to the fluorescence increase in green visible channel while NIR red fluorescence intensity was barely changed. *Molecules* **2021**, *26*, x FOR PEER REVIEW 29 of 59 *Molecules* **2021**, *26*, x FOR PEER REVIEW 29 of 59

**Figure 45.** Structure of **Probe 50** and its reaction with GSH and Cys/Hcy. **Figure 45.** Structure of **Probe 50** and its reaction with GSH and Cys/Hcy. **Figure 45.** Structure of **Probe 50** and its reaction with GSH and Cys/Hcy.

In 2019, Mingming Cui et al. [161] reported a turn on fluorescent probe (**Probe 51,**  Figure 46) for detection of mitochondrial GSH with twisted intramolecular charge transfer (TICT) and aggregation-enhanced emission (AEE) characteristics. Cleavage of the dinitrophenyl ether from QUPY-S by GSH followed by a self-immolation reaction results in a low water solute compound QUPY which aggregates to turn on the fluorescence at 516 nm. The detection limit was determined to 434 nM. The probe is featured with a large Stokes shift (131 nm) and capable of detecting GSH in HeLa cells. In 2019, Mingming Cui et al. [161] reported a turn on fluorescent probe (**Probe 51**, Figure 46) for detection of mitochondrial GSH with twisted intramolecular charge transfer (TICT) and aggregation-enhanced emission (AEE) characteristics. Cleavage of the dinitrophenyl ether from QUPY-S by GSH followed by a self-immolation reaction results in a low water solute compound QUPY which aggregates to turn on the fluorescence at 516 nm. The detection limit was determined to 434 nM. The probe is featured with a large Stokes shift (131 nm) and capable of detecting GSH in HeLa cells. In 2019, Mingming Cui et al. [161] reported a turn on fluorescent probe (**Probe 51,**  Figure 46) for detection of mitochondrial GSH with twisted intramolecular charge transfer (TICT) and aggregation-enhanced emission (AEE) characteristics. Cleavage of the dinitrophenyl ether from QUPY-S by GSH followed by a self-immolation reaction results in a low water solute compound QUPY which aggregates to turn on the fluorescence at 516 nm. The detection limit was determined to 434 nM. The probe is featured with a large Stokes shift (131 nm) and capable of detecting GSH in HeLa cells.

**Figure 46.** Structure of **Probe 51** and its reaction with GSH. **Figure 46.** Structure of **Probe 51** and its reaction with GSH. **Figure 46.** Structure of **Probe 51** and its reaction with GSH.

3.1.3. Detection of Mitochondrial NPSH via Cleavage of Sulfonamide or Sulfonate Ester Detection of Mitochondrial NPSH via Cleavage of Sulfonamide 3.1.3. Detection of Mitochondrial NPSH via Cleavage of Sulfonamide or Sulfonate Ester 3.1.3. Detection of Mitochondrial NPSH via Cleavage of Sulfonamide or Sulfonate Ester Detection of Mitochondrial NPSH via Cleavage of Sulfonamide

In 2018, Zhiqiang Xu et al. [162] developed a visible and near-infrared, dual-channel fluorescence-on robe (CyP-SNp, **Probe 52**, Figure 47) for monitoring mitochondrial GSH in a spatiotemporal and synchronous manner. In the approach, two widely used strong Detection of Mitochondrial NPSH via Cleavage of Sulfonamide In 2018, Zhiqiang Xu et al. [162] developed a visible and near-infrared, dual-channel fluorescence-on robe (CyP-SNp, **Probe 52**, Figure 47) for monitoring mitochondrial GSH In 2018, Zhiqiang Xu et al. [162] developed a visible and near-infrared, dual-channel fluorescence-on robe (CyP-SNp, **Probe 52**, Figure 47) for monitoring mitochondrial GSH in a spatiotemporal and synchronous manner. In the approach, two widely used strong

fluorophores, naphthalimide (a visible fluorophore) and cyanine (a NIR fluorophore), are

in a spatiotemporal and synchronous manner. In the approach, two widely used strong fluorophores, naphthalimide (a visible fluorophore) and cyanine (a NIR fluorophore), are

which displays a weak fluorescence will be turned on to two completely different fluorescence upon reaction with GSH, green fluorescence (Em. 495 nm, Ex. 370 nm) in the visible channel and red fluorescence (Em. 795 nm, Ex. 700 nm) in the near-infrared channel. The cationic cyanine dye accumulates in mitochondria. The detection mechanism involves a nucleophilic substitution reaction between the thiolate of GSH with sulfonamide to form an intermediate and subsequent cleavage of the sulfur–nitrogen bond of the intermediate will result in a stable NIR fluorescent product CyP and another reactive intermediate Np-GSH-SO<sup>2</sup> which will switch to a visible-light-emitting product Np-GSH after releasing SO2. Cys and Hcy follow the similar mechanistic pathway in a much slower manner. The kinetic differences enable the probe to discriminatively detect GSH over Cys and Hcy. The

chosen since they are emitting in two distinctly different wavelength regions. The probe which displays a weak fluorescence will be turned on to two completely different fluorescence upon reaction with GSH, green fluorescence (Em. 495 nm, Ex. 370 nm) in the visible channel and red fluorescence (Em. 795 nm, Ex. 700 nm) in the near-infrared channel. The cationic cyanine dye accumulates in mitochondria. The detection mechanism involves a nucleophilic substitution reaction between the thiolate of GSH with sulfonamide to form an intermediate and subsequent cleavage of the sulfur–nitrogen bond of the intermediate will result in a stable NIR fluorescent product CyP and another reactive intermediate Np-GSH-SO<sup>2</sup> which will switch to a visible-light-emitting product Np-GSH after releasing SO2. Cys and Hcy follow the similar mechanistic pathway in a much slower manner. The kinetic differences enable the probe to discriminatively detect GSH over Cys and Hcy. The

fluorophores, naphthalimide (a visible fluorophore) and cyanine (a NIR fluorophore), are bridged together by a thiol-reactive sulfonamide moiety. These two fluorophores were chosen since they are emitting in two distinctly different wavelength regions. The probe which displays a weak fluorescence will be turned on to two completely different fluorescence upon reaction with GSH, green fluorescence (Em. 495 nm, Ex. 370 nm) in the visible channel and red fluorescence (Em. 795 nm, Ex. 700 nm) in the near-infrared channel. The cationic cyanine dye accumulates in mitochondria. The detection mechanism involves a nucleophilic substitution reaction between the thiolate of GSH with sulfonamide to form an intermediate and subsequent cleavage of the sulfur–nitrogen bond of the intermediate will result in a stable NIR fluorescent product CyP and another reactive intermediate Np-GSH-SO<sup>2</sup> which will switch to a visible-light-emitting product Np-GSH after releasing SO2. Cys and Hcy follow the similar mechanistic pathway in a much slower manner. The kinetic differences enable the probe to discriminatively detect GSH over Cys and Hcy. The detection limits in the two different channels are 1.53 <sup>×</sup> <sup>10</sup>−<sup>7</sup> M (visible channel) and 1.71 <sup>×</sup> <sup>10</sup>−<sup>7</sup> <sup>M</sup> (near-infrared channel) respectively. Cellular experiments showed that the probe display good mitochondria-targeting capacity. *Molecules* **2021**, *26*, x FOR PEER REVIEW 30 of 59 detection limits in the two different channels are 1.53 × 10−7 M (visible channel) and 1.71 × 10−7 M (near-infrared channel) respectively. Cellular experiments showed that the probe display good mitochondria-targeting capacity. The probe was further demonstrated to be capable of monitoring GSH status in live cells with a minimum interference from Cys and Hcy. Results showed that addition of Cys and Hcy did not change the fluorescence intensity significantly while addition of GSH induced a remarkable intense fluorescence increase. Later the probe showed to be capable of tracking GSH levels in living tissues with imaging depths of up to 120 μm in the nearinfrared channel [163].

In 2020, Zhengkun Liu et al. [164] reported a two-photon probe MT-1 (**Probe 53**, Figure 48) based on a two-photon and fluorescence resonance energy transfer (FRET) strategy. In the molecule, naphthalimide was used as the two-photon receptor as well as the FRET donor while a rhodamine B group was used to serve as the FRET acceptor as well The probe was further demonstrated to be capable of monitoring GSH status in live cells with a minimum interference from Cys and Hcy. Results showed that addition of Cys and Hcy did not change the fluorescence intensity significantly while addition of GSH induced a remarkable intense fluorescence increase. Later the probe showed to be capable of tracking GSH levels in living tissues with imaging depths of up to 120 µm in the near-infrared channel [163].

as the mitochondria-targeting ligand. DNBS was attached to naphthalimide moiety via piperazine and utilized as thiol recognition group. In addition, DNBS is a high electron withdrawing group (EWG) which could withdraw the electron to induce intramolecular charge transfer (ICT) effect resulting in the quenching of the fluorescence [165,166]. More importantly, this is the first probe developed for mitochondrial thiols based on two-photon and Förster resonance energy transfer (TP-FRET) strategy, and the probe was successfully applied for detection of mitochondrial thiols in live cells and in mouse liver tissue slices. In 2020, Zhengkun Liu et al. [164] reported a two-photon probe MT-1 (**Probe 53**, Figure 48) based on a two-photon and fluorescence resonance energy transfer (FRET) strategy. In the molecule, naphthalimide was used as the two-photon receptor as well as the FRET donor while a rhodamine B group was used to serve as the FRET acceptor as well as the mitochondria-targeting ligand. DNBS was attached to naphthalimide moiety via piperazine and utilized as thiol recognition group. In addition, DNBS is a high electron withdrawing group (EWG) which could withdraw the electron to induce intramolecular charge transfer (ICT) effect resulting in the quenching of the fluorescence [165,166]. More importantly, this is the first probe developed for mitochondrial thiols based on two-photon and Förster resonance energy transfer (TP-FRET) strategy, and the probe was successfully applied for detection of mitochondrial thiols in live cells and in mouse liver tissue slices.

**Figure 48.** Structure of **Probe 53** and its reaction with thiols.

Detection of Mitochondrial NPSH via Cleavage of Sulfonate Ester

In 2018, Fangfang Wang et al. [167] presented a BODIPY-based fluorescent probe (**Probe 54**) for fast detection of intracellular mitochondrial thiols with high sensitivity and selectivity by introduction of a dual reactive group. Although BODIPY exhibits a number of advantages as a fluorophore, such as high fluorescence quantum yield, excellent photostability, and intense absorption of visible light [160,168–170], utilization of BODIPY for building a BODIPY-based turn-on fluorescent probes is hindered by the hardship of completely quenching the original fluorescence of BODIPY leading to a high background and

detection limits in the two different channels are 1.53 × 10−7 M (visible channel) and 1.71 × 10−7 M (near-infrared channel) respectively. Cellular experiments showed that the probe

The probe was further demonstrated to be capable of monitoring GSH status in live cells with a minimum interference from Cys and Hcy. Results showed that addition of Cys and Hcy did not change the fluorescence intensity significantly while addition of GSH induced a remarkable intense fluorescence increase. Later the probe showed to be capable of tracking GSH levels in living tissues with imaging depths of up to 120 μm in the near-

In 2020, Zhengkun Liu et al. [164] reported a two-photon probe MT-1 (**Probe 53**, Figure 48) based on a two-photon and fluorescence resonance energy transfer (FRET) strategy. In the molecule, naphthalimide was used as the two-photon receptor as well as the FRET donor while a rhodamine B group was used to serve as the FRET acceptor as well as the mitochondria-targeting ligand. DNBS was attached to naphthalimide moiety via piperazine and utilized as thiol recognition group. In addition, DNBS is a high electron withdrawing group (EWG) which could withdraw the electron to induce intramolecular charge transfer (ICT) effect resulting in the quenching of the fluorescence [165,166]. More importantly, this is the first probe developed for mitochondrial thiols based on two-photon and Förster resonance energy transfer (TP-FRET) strategy, and the probe was successfully applied for detection of mitochondrial thiols in live cells and in mouse liver tissue

display good mitochondria-targeting capacity.

**Figure 47.** Structure of **Probe 52** and its reactions with thiols.

infrared channel [163].

slices.

**Figure 48.** Structure of **Probe 53** and its reaction with thiols. **Figure 48.** Structure of **Probe 53** and its reaction with thiols.

Detection of Mitochondrial NPSH via Cleavage of Sulfonate Ester Detection of Mitochondrial NPSH via Cleavage of Sulfonate Ester

In 2018, Fangfang Wang et al. [167] presented a BODIPY-based fluorescent probe (**Probe 54**) for fast detection of intracellular mitochondrial thiols with high sensitivity and selectivity by introduction of a dual reactive group. Although BODIPY exhibits a number of advantages as a fluorophore, such as high fluorescence quantum yield, excellent photostability, and intense absorption of visible light [160,168–170], utilization of BODIPY for building a BODIPY-based turn-on fluorescent probes is hindered by the hardship of completely quenching the original fluorescence of BODIPY leading to a high background and In 2018, Fangfang Wang et al. [167] presented a BODIPY-based fluorescent probe (**Probe 54**) for fast detection of intracellular mitochondrial thiols with high sensitivity and selectivity by introduction of a dual reactive group. Although BODIPY exhibits a number of advantages as a fluorophore, such as high fluorescence quantum yield, excellent photostability, and intense absorption of visible light [160,168–170], utilization of BODIPY for building a BODIPY-based turn-on fluorescent probes is hindered by the hardship of completely quenching the original fluorescence of BODIPY leading to a high background and low sensitivity. To overcome the challenge, two quenching groups, a DNBS moiety and a nitroolefin moiety (-CH=CH-NO2) were introduced to the BODIPY fluorophore. These two groups also serve as the thiol recognition groups. In the molecule, DNBS serves as the acceptor of the PET and nitroolefin moiety (-CH=CH-NO2) serves as a Michael acceptor for the PET as well as a receptor for the intramolecular charge transfer (ICT) process. Thus, the fluorescence of the probe was heavily quenched by these dual quenching systems. The dual reaction of GSH with the probe (Figure 49) breaks the PET and ICT process and restores the strong fluorescence of BODIPY (Em. 517 nm). A linearly increase of fluorescence was observed with an increase in GSH concentration (0–350 µM), Hcy (0–300 µM), and Cys (0–100 µM), enabling the quantitatively detection of thiols. The detection limits of the probe for Hcy, Cys, and GSH were determined to be 87 nM, 147 nM, and 129 nM, respectively. Later, the probe was demonstrated to selectively accumulated inside mitochondria. The ability of the probe to reflect the mitochondrial thiols change was confirmed with live HeLa cells, a significant decrease of fluorescence was observed when the cells were pretreated with NEM. Medium wash was not needed in cell imaging since the probe exhibited a low fluorescence background. The probe was able to image mitochondrial thiols in ca. 10 min. *Molecules* **2021**, *26*, x FOR PEER REVIEW 31 of 59 low sensitivity. To overcome the challenge, two quenching groups, a DNBS moiety and a nitroolefin moiety (-CH=CH-NO2) were introduced to the BODIPY fluorophore. These two groups also serve as the thiol recognition groups. In the molecule, DNBS serves as the acceptor of the PET and nitroolefin moiety (-CH=CH-NO2) serves as a Michael acceptor for the PET as well as a receptor for the intramolecular charge transfer (ICT) process. Thus, the fluorescence of the probe was heavily quenched by these dual quenching systems. The dual reaction of GSH with the probe (Figure 49) breaks the PET and ICT process and restores the strong fluorescence of BODIPY (Em. 517 nm). A linearly increase of fluorescence was observed with an increase in GSH concentration (0–350 μM), Hcy (0–300 μM), and Cys (0–100 μM), enabling the quantitatively detection of thiols. The detection limits of the probe for Hcy, Cys, and GSH were determined to be 87 nM, 147 nM, and 129 nM, respectively. Later, the probe was demonstrated to selectively accumulated inside mitochondria. The ability of the probe to reflect the mitochondrial thiols change was confirmed with live HeLa cells, a significant decrease of fluorescence was observed when the cells were pretreated with NEM. Medium wash was not needed in cell imaging since the probe exhibited a low fluorescence background. The probe was able to image mitochondrial thiols in ca. 10 min.

**Figure 49.** Structure of **Probe 54** and its reaction with thiols. **Figure 49.** Structure of **Probe 54** and its reaction with thiols.

In 2017, three two-photon fluorescent mitochondrial thiol probes (**Probes 55–57**, Figure 50) were reported by Yi Li et al. [171] based on coumarin as the fluorophore. The probes employed an imidazolium cation to target mitochondria and a strong electronwithdrawing DNBS was introduced to serve as the thiol recognition site and to quench the fluorescence of the coumarin fluorophore via an intramolecular charge transfer (ICT) reaction. A thiol molecule will react with the probes and remove the DNBS structure to In 2017, three two-photon fluorescent mitochondrial thiol probes (**Probes 55**–**57**, Figure 50) were reported by Yi Li et al. [171] based on coumarin as the fluorophore. The probes employed an imidazolium cation to target mitochondria and a strong electronwithdrawing DNBS was introduced to serve as the thiol recognition site and to quench the fluorescence of the coumarin fluorophore via an intramolecular charge transfer (ICT) reaction. A thiol molecule will react with the probes and remove the DNBS structure to

turn on the fluorescence (Em. 482 nm). The probes showed dramatic optical responses to various thiols, such as GSH, Cys, and Hcy. Moreover, the probes showed response to BSA

mitochondria via a colocalization study with mito-tracker red while probes without the imidazolium cation distributed everywhere inside the cells. These probes were capable of monitoring thiol level change in A549 cells. The ability of detecting thiol level changes was further confirmed in the zebrafish. The penetration depth of these two-photon probes was determined to be 160 μm in the vasculature of an anaesthetized mouse using twophoton microscopy. Overall, these probes showed high mitochondrial selectivity, large two-photon absorption cross-section, and good biocompatibility. They are promising re-

agents for application for thiol imaging in vitro and in vivo.

turn on the fluorescence (Em. 482 nm). The probes showed dramatic optical responses to various thiols, such as GSH, Cys, and Hcy. Moreover, the probes showed response to BSA confirming they can also react with PSH. In contrast, negligible response was observed for non-thiol amino acids. The authors demonstrated that the probes were located inside mitochondria via a colocalization study with mito-tracker red while probes without the imidazolium cation distributed everywhere inside the cells. These probes were capable of monitoring thiol level change in A549 cells. The ability of detecting thiol level changes was further confirmed in the zebrafish. The penetration depth of these two-photon probes was determined to be 160 µm in the vasculature of an anaesthetized mouse using two-photon microscopy. Overall, these probes showed high mitochondrial selectivity, large two-photon absorption cross-section, and good biocompatibility. They are promising reagents for application for thiol imaging in vitro and in vivo. *Molecules* **2021**, *26*, x FOR PEER REVIEW 32 of 59 *Molecules* **2021**, *26*, x FOR PEER REVIEW 32 of 59

**Figure 50.** Structures of **Probes 55–57** and their reactions wih thiols. **Figure 50.** Structures of **Probes 55**–**57** and their reactions wih thiols. **Figure 50.** Structures of **Probes 55–57** and their reactions wih thiols.

In 2020, Xin Li et al. [172] developed a novel composite NIR dye (Hcyc-NO, **Probe 58**) for effective detection of thiols and oxidative stress inside mitochondria in live cells. In the approach (Figure 51), a novel NIR hemicyanine dye (Hcyc) composed by cyanine and coumarin were designed and synthesized. The probe itself exhibits weak fluorescence but can be turned on to a high fluorescence product (Ex. 723 nm, Em. 751 nm) upon reaction with thiols, such as GSH, Cys, Hcy H2S, and dithiothreitol (DTT). Removal of DNBS group from the probe induced the fluorescence enhancement. The probe displays an instantaneous response (*<*5 s) to thiols with high selectivity and sensitivity. A linear relationship was observed when the probe reacted with various concentrations of GSH (0–5.0 μM), Cys (0–2.0 μM), and Hcy (0–1.0 μM) with the detection limits of 0.11, 0.08, and 0.20 μM, respectively. A cellular colocalization study with Mito-Tracker demonstrated that the probe was localized inside mitochondria. The probe demonstrated the ability to detect a change in thiol levels in live cells. Furthermore, Hcyc-NO was confirmed to be capable of quickly detecting the content of thiols or oxidative stress in vivo in a mouse model. In 2020, Xin Li et al. [172] developed a novel composite NIR dye (Hcyc-NO, **Probe 58**) for effective detection of thiols and oxidative stress inside mitochondria in live cells. In the approach (Figure 51), a novel NIR hemicyanine dye (Hcyc) composed by cyanine and coumarin were designed and synthesized. The probe itself exhibits weak fluorescence but can be turned on to a high fluorescence product (Ex. 723 nm, Em. 751 nm) upon reaction with thiols, such as GSH, Cys, Hcy H2S, and dithiothreitol (DTT). Removal of DNBS group from the probe induced the fluorescence enhancement. The probe displays an instantaneous response (<5 s) to thiols with high selectivity and sensitivity. A linear relationship was observed when the probe reacted with various concentrations of GSH (0–5.0 µM), Cys (0–2.0 µM), and Hcy (0–1.0 µM) with the detection limits of 0.11, 0.08, and 0.20 µM, respectively. A cellular colocalization study with Mito-Tracker demonstrated that the probe was localized inside mitochondria. The probe demonstrated the ability to detect a change in thiol levels in live cells. Furthermore, Hcyc-NO was confirmed to be capable of quickly detecting the content of thiols or oxidative stress in vivo in a mouse model. In 2020, Xin Li et al. [172] developed a novel composite NIR dye (Hcyc-NO, **Probe 58**) for effective detection of thiols and oxidative stress inside mitochondria in live cells. In the approach (Figure 51), a novel NIR hemicyanine dye (Hcyc) composed by cyanine and coumarin were designed and synthesized. The probe itself exhibits weak fluorescence but can be turned on to a high fluorescence product (Ex. 723 nm, Em. 751 nm) upon reaction with thiols, such as GSH, Cys, Hcy H2S, and dithiothreitol (DTT). Removal of DNBS group from the probe induced the fluorescence enhancement. The probe displays an instantaneous response (*<*5 s) to thiols with high selectivity and sensitivity. A linear relationship was observed when the probe reacted with various concentrations of GSH (0–5.0 μM), Cys (0–2.0 μM), and Hcy (0–1.0 μM) with the detection limits of 0.11, 0.08, and 0.20 μM, respectively. A cellular colocalization study with Mito-Tracker demonstrated that the probe was localized inside mitochondria. The probe demonstrated the ability to detect a change in thiol levels in live cells. Furthermore, Hcyc-NO was confirmed to be capable of quickly detecting the content of thiols or oxidative stress in vivo in a mouse model.

3.1.4. Detection of Mitochondria NPSH via Cleavage of Disulfide Bond

3.1.4. Detection of Mitochondria NPSH via Cleavage of Disulfide Bond

In 2011, Chang Su Lim et al. [173] developed a two-photon probe (SSH-Mito, **Probe 59**) for ratiometrically detecting of mitochondrial thiols. In the approach, 6-(benzo[d]thiazol-20 -yl)-2-(*N,N*-dimethylamino)naphthalene (BTDAN) was used as the fluorophore,

In 2011, Chang Su Lim et al. [173] developed a two-photon probe (SSH-Mito, **Probe 59**) for ratiometrically detecting of mitochondrial thiols. In the approach, 6-(benzo[d]thiazol-20 -yl)-2-(*N,N*-dimethylamino)naphthalene (BTDAN) was used as the fluorophore,

bond followed by a cleavage of the C–N bond to afford the final product. The process will shift the fluorescence from blue color (462 nm (Φ = 0.82)) to yellow color (545 nm (Φ = 0.12)) and the ratio of the fluorescence intensity of Fyellow (525–575 nm) to Fblue (425–475 nm) elevated 42 to 77-fold in the presence of thiols (GSH, Cys, dithiothreitol (DTT), 2 mercaptoethanol (2-ME), and 2-AET). In contrast, no significant change was observed in the presence of non-thiol amino acids. In vitro cellular experiments demonstrated that SSH-Mito predominantly accumulated inside mitochondria. The probe was proved to be able to respond to thiols' changes in live cells. Moreover, the probe was successfully used

bond followed by a cleavage of the C–N bond to afford the final product. The process will shift the fluorescence from blue color (462 nm (Φ = 0.82)) to yellow color (545 nm (Φ = 0.12)) and the ratio of the fluorescence intensity of Fyellow (525–575 nm) to Fblue (425–475 nm) elevated 42 to 77-fold in the presence of thiols (GSH, Cys, dithiothreitol (DTT), 2 mercaptoethanol (2-ME), and 2-AET). In contrast, no significant change was observed in the presence of non-thiol amino acids. In vitro cellular experiments demonstrated that SSH-Mito predominantly accumulated inside mitochondria. The probe was proved to be able to respond to thiols' changes in live cells. Moreover, the probe was successfully used

**Figure 51.** Structure of **Probe 58** and its reaction with thiols. **Figure 51.** Structure of **Probe 58** and its reaction with thiols. **Figure 51.** Structure of **Probe 58** and its reaction with thiols.

for visualizing thiol levels in living tissue depths of 90–190 μm.

for visualizing thiol levels in living tissue depths of 90–190 μm.

#### 3.1.4. Detection of Mitochondria NPSH via Cleavage of Disulfide Bond

In 2011, Chang Su Lim et al. [173] developed a two-photon probe (SSH-Mito, **Probe 59**) for ratiometrically detecting of mitochondrial thiols. In the approach, 6-(benzo[d]thiazol-20-yl)-2-(*N*,*N*-dimethylamino)naphthalene (BTDAN) was used as the fluorophore, TPP as the mitochondrial-targeting site, and a disulfide group as the thiol recognition group. The reaction was illustrated in Figure 52. Thiol will first react with the disulfide bond followed by a cleavage of the C–N bond to afford the final product. The process will shift the fluorescence from blue color (462 nm (Φ = 0.82)) to yellow color (545 nm (Φ = 0.12)) and the ratio of the fluorescence intensity of Fyellow (525–575 nm) to Fblue (425–475 nm) elevated 42 to 77-fold in the presence of thiols (GSH, Cys, dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and 2-AET). In contrast, no significant change was observed in the presence of nonthiol amino acids. In vitro cellular experiments demonstrated that SSH-Mito predominantly accumulated inside mitochondria. The probe was proved to be able to respond to thiols' changes in live cells. Moreover, the probe was successfully used for visualizing thiol levels in living tissue depths of 90–190 µm. *Molecules* **2021**, *26*, x FOR PEER REVIEW 33 of 59

live HeLa cells.

In 2020, Lu Wang et al. [174] reported a ratiometric fluorescent probe (**Probe 60**, Figure 53) for detection of mitochondria thiols. The probe was built based on the FRET strategy. BODIPY and rhodamine were used in this approach to achieve the FRET-based fluorescent probes since there is a clear overlap between the emission of BODIPY and absorption of rhodamine [175,176]. Thus, BODIPY and a modified near-infrared rhodamine [26,177] were connected together with a disulfide to build an effective FRET structure with BODIPY serving as the donor and rhodamine as the acceptor. In the meantime, the disulfide is the thiol recognition group and also the switch for turning on-off the FRET. When the probe was excited at 488 nm (BODIPY's excitation wavelength), a strong emission with a peak at 656 nm of rhodamine acceptor was observed and a weak emission peak at 512 nm (BODIPY's emission) due to the high efficiency FRET process. Once the probe reacted with a thiol molecule (GSH, Cys, or Hcy), the disulfide was cleaved by thiols and the FRET was turned off resulting in an increase of the emission intensity at 512 nm (corresponding BODIPY fluorescence) while the fluorescence intensity at 656 nm decreased. The fluorescence ratio (F512 nm/F656 nm) increased linearly as GSH concentration increased from 10 to 100 In 2020, Lu Wang et al. [174] reported a ratiometric fluorescent probe (**Probe 60**, Figure 53) for detection of mitochondria thiols. The probe was built based on the FRET strategy. BODIPY and rhodamine were used in this approach to achieve the FRET-based fluorescent probes since there is a clear overlap between the emission of BODIPY and absorption of rhodamine [175,176]. Thus, BODIPY and a modified near-infrared rhodamine [26,177] were connected together with a disulfide to build an effective FRET structure with BODIPY serving as the donor and rhodamine as the acceptor. In the meantime, the disulfide is the thiol recognition group and also the switch for turning on-off the FRET. When the probe was excited at 488 nm (BODIPY's excitation wavelength), a strong emission with a peak at 656 nm of rhodamine acceptor was observed and a weak emission peak at 512 nm (BODIPY's emission) due to the high efficiency FRET process. Once the probe reacted with a thiol molecule (GSH, Cys, or Hcy), the disulfide was cleaved by thiols and the FRET was turned off resulting in an increase of the emission intensity at 512 nm (corresponding BODIPY fluorescence) while the fluorescence intensity at 656 nm decreased. The fluorescence ratio (F512 nm/F656 nm) increased linearly as GSH concentration increased from 10 to 100 µM with a detection limit of 0.26 µM suggesting that probe can be used as a ratiometric fluorescent probe for quantitative detection of thiols. The probe was demonstrated to be well localized inside mitochondria and able to detect the mitochondrial thiols' change in live HeLa cells.

μM with a detection limit of 0.26 μM suggesting that probe can be used as a ratiometric fluorescent probe for quantitative detection of thiols. The probe was demonstrated to be well localized inside mitochondria and able to detect the mitochondrial thiols' change in

In 2017, Qingbin Zeng et al. [178] developed a mitochondria targeting and intracellularthiol triggered hyperpolarized [128] Xe magnetofluorescent biosensor (**Probe 61**, Figure 54) based on a disulfide as the thiol recognition group. The probe composed of a TPP group as mitochondrial targeting ligand, a Xe encapsulated in a cryptophane-A cage as a [128] Xe NMR reporter, and a naphthalimide moiety as a fluorescent reporter. After the disulfide bond being cleavage by thiols, a cyclization reaction will occur to afford an amine product, resulting in a remarkable increase of fluorescence intensity at 560 nm and a significant change in the [128] Xe chemical shift. The probe exhibits an extremely low detection limit (10−10 M) by using Hyper-CEST (chemical exchange saturation transfer)

**Figure 53.** Structure of **Probe 60** and its reaction with thiols.

**Figure 52.** Structure of **Probe 59** and its reaction with GSH.

In 2020, Lu Wang et al. [174] reported a ratiometric fluorescent probe (**Probe 60**, Figure 53) for detection of mitochondria thiols. The probe was built based on the FRET strategy. BODIPY and rhodamine were used in this approach to achieve the FRET-based fluorescent probes since there is a clear overlap between the emission of BODIPY and absorption of rhodamine [175,176]. Thus, BODIPY and a modified near-infrared rhodamine [26,177] were connected together with a disulfide to build an effective FRET structure with BODIPY serving as the donor and rhodamine as the acceptor. In the meantime, the disulfide is the thiol recognition group and also the switch for turning on-off the FRET. When the probe was excited at 488 nm (BODIPY's excitation wavelength), a strong emission with a peak at 656 nm of rhodamine acceptor was observed and a weak emission peak at 512 nm (BODIPY's emission) due to the high efficiency FRET process. Once the probe reacted with a thiol molecule (GSH, Cys, or Hcy), the disulfide was cleaved by thiols and the FRET was turned off resulting in an increase of the emission intensity at 512 nm (corresponding BODIPY fluorescence) while the fluorescence intensity at 656 nm decreased. The fluorescence ratio (F512 nm/F656 nm) increased linearly as GSH concentration increased from 10 to 100 μM with a detection limit of 0.26 μM suggesting that probe can be used as a ratiometric fluorescent probe for quantitative detection of thiols. The probe was demonstrated to be well localized inside mitochondria and able to detect the mitochondrial thiols' change in

**Figure 53.** Structure of **Probe 60** and its reaction with thiols. **Figure 53.** Structure of **Probe 60** and its reaction with thiols.

live HeLa cells.

In 2017, Qingbin Zeng et al. [178] developed a mitochondria targeting and intracellularthiol triggered hyperpolarized [128] Xe magnetofluorescent biosensor (**Probe 61**, Figure 54) based on a disulfide as the thiol recognition group. The probe composed of a TPP group as mitochondrial targeting ligand, a Xe encapsulated in a cryptophane-A cage as a [128] Xe NMR reporter, and a naphthalimide moiety as a fluorescent reporter. After the disulfide bond being cleavage by thiols, a cyclization reaction will occur to afford an amine product, resulting in a remarkable increase of fluorescence intensity at 560 nm and a significant change in the [128] Xe chemical shift. The probe exhibits an extremely low detection limit (10−10 M) by using Hyper-CEST (chemical exchange saturation transfer) In 2017, Qingbin Zeng et al. [178] developed a mitochondria targeting and intracellular thiol triggered hyperpolarized [128] Xe magnetofluorescent biosensor (**Probe 61**, Figure 54) based on a disulfide as the thiol recognition group. The probe composed of a TPP group as mitochondrial targeting ligand, a Xe encapsulated in a cryptophane-A cage as a [128] Xe NMR reporter, and a naphthalimide moiety as a fluorescent reporter. After the disulfide bond being cleavage by thiols, a cyclization reaction will occur to afford an amine product, resulting in a remarkable increase of fluorescence intensity at 560 nm and a significant change in the [128] Xe chemical shift. The probe exhibits an extremely low detection limit (10−<sup>10</sup> M) by using Hyper-CEST (chemical exchange saturation transfer) NMR. The probe was successfully applied to effectively detect mitochondrial thiol changes in live cells. *Molecules* **2021**, *26*, x FOR PEER REVIEW 34 of 59 NMR. The probe was successfully applied to effectively detect mitochondrial thiol changes in live cells.

**Figure 54.** structure of **Probe 61**. **Figure 54.** Structure of **Probe 61**.

**Probe 63**.

#### 3.1.5. Detection of Mitochondrial NPSH through Other Methods 3.1.5. Detection of Mitochondrial NPSH through Other Methods

In 2014, a mitochondrion-targeting NIR probe (**Probe 62**, Figure 55) named MitoGP was developed by Soo-Yeon Lim et al. [179] for imaging and detecting mitochondrial thiols. Within the molecule, a heptamethine group was utilized as the mitochondrial targeting moiety and also served as the fluorophore, while a nitroazo group was used as the GSH recognition group as well as the fluorescence quencher. The probe showed a great selectivity for GSH over other amino acids including Cys and Hcy. The probe can be turned on to a strong fluorescence in the presences of GSH at 810 nm. The selectivity for GSH over Cys and Hcy was achieved by a specific 1, 6-conjugate addition and subsequent elimination reaction which triggered the release of the fluorophore [136]. In vitro cell experiments with HeLa cells demonstrated that the probe could effectively reflect the GSH level change in mitochondria. The authors believe that the MitoGP is much more superior to the commercially available thiol probe (mCB) or GSH-silent (rhodamine 123) Mito-Tracker and anticipated the possibility that the probe can be further used as a therapeutic reagent for mitochondrial GSH-related pathology. Moreover, the NIR emissive fluores-In 2014, a mitochondrion-targeting NIR probe (**Probe 62**, Figure 55) named MitoGP was developed by Soo-Yeon Lim et al. [179] for imaging and detecting mitochondrial thiols. Within the molecule, a heptamethine group was utilized as the mitochondrial targeting moiety and also served as the fluorophore, while a nitroazo group was used as the GSH recognition group as well as the fluorescence quencher. The probe showed a great selectivity for GSH over other amino acids including Cys and Hcy. The probe can be turned on to a strong fluorescence in the presences of GSH at 810 nm. The selectivity for GSH over Cys and Hcy was achieved by a specific 1, 6-conjugate addition and subsequent elimination reaction which triggered the release of the fluorophore [136]. In vitro cell experiments with HeLa cells demonstrated that the probe could effectively reflect the GSH level change in mitochondria. The authors believe that the MitoGP is much more superior to the commercially available thiol probe (mCB) or GSH-silent (rhodamine 123) MitoTracker and anticipated the possibility that the probe can be further used as a therapeutic reagent for mitochondrial GSH-related pathology. Moreover, the NIR emissive fluorescence property

cence property provides the advantage of avoiding the interference from cellular auto-

**Figure 55.** (**A**). Structure of **Probe 62** and its reaction with GSH and Cys/Hcy; (**B**). Structure of

In 2016, Fangfang Meng et al. [180] presented a two-photon fluorescent probe (**Probe 63**, Figure 55) for sensing mitochondrial thiols in live cells. Its two-photon properties enable the probe to be applied in mouse liver tissues. The probe named CA-TPP was developed by a combination of three parts, 9-ethyl-3-styryl-9*H*-carbazole (CA) as the TP fluorescent platform, aldehyde as the thiol recognition units, and a TPP as the mitochondrial targeting moiety. In vitro cellular experiments in A549 cells shows that pretreating the

fluorescence when used as noninvasive in vivo imaging tool.

**Probe 63**.

Tracker and anticipated the possibility that the probe can be further used as a therapeutic reagent for mitochondrial GSH-related pathology. Moreover, the NIR emissive fluores-

*Molecules* **2021**, *26*, x FOR PEER REVIEW 34 of 59

3.1.5. Detection of Mitochondrial NPSH through Other Methods

changes in live cells.

**Figure 54.** structure of **Probe 61**.

NMR. The probe was successfully applied to effectively detect mitochondrial thiol

In 2014, a mitochondrion-targeting NIR probe (**Probe 62**, Figure 55) named MitoGP

was developed by Soo-Yeon Lim et al. [179] for imaging and detecting mitochondrial thiols. Within the molecule, a heptamethine group was utilized as the mitochondrial targeting moiety and also served as the fluorophore, while a nitroazo group was used as the GSH recognition group as well as the fluorescence quencher. The probe showed a great selectivity for GSH over other amino acids including Cys and Hcy. The probe can be turned on to a strong fluorescence in the presences of GSH at 810 nm. The selectivity for GSH over Cys and Hcy was achieved by a specific 1, 6-conjugate addition and subsequent elimination reaction which triggered the release of the fluorophore [136]. In vitro cell experiments with HeLa cells demonstrated that the probe could effectively reflect the GSH level change in mitochondria. The authors believe that the MitoGP is much more superior

provides the advantage of avoiding the interference from cellular autofluorescence when used as noninvasive in vivo imaging tool. cence property provides the advantage of avoiding the interference from cellular autofluorescence when used as noninvasive in vivo imaging tool.

**Figure 55.** (**A**). Structure of **Probe 62** and its reaction with GSH and Cys/Hcy; (**B**). Structure of **Figure 55.** (**A**). Structure of **Probe 62** and its reaction with GSH and Cys/Hcy; (**B**). Structure of **Probe 63**.

In 2016, Fangfang Meng et al. [180] presented a two-photon fluorescent probe (**Probe 63**, Figure 55) for sensing mitochondrial thiols in live cells. Its two-photon properties enable the probe to be applied in mouse liver tissues. The probe named CA-TPP was developed by a combination of three parts, 9-ethyl-3-styryl-9*H*-carbazole (CA) as the TP fluorescent platform, aldehyde as the thiol recognition units, and a TPP as the mitochondrial targeting moiety. In vitro cellular experiments in A549 cells shows that pretreating the In 2016, Fangfang Meng et al. [180] presented a two-photon fluorescent probe (**Probe 63**, Figure 55) for sensing mitochondrial thiols in live cells. Its two-photon properties enable the probe to be applied in mouse liver tissues. The probe named CA-TPP was developed by a combination of three parts, 9-ethyl-3-styryl-9*H*-carbazole (CA) as the TP fluorescent platform, aldehyde as the thiol recognition units, and a TPP as the mitochondrial targeting moiety. In vitro cellular experiments in A549 cells shows that pretreating the cells with NEM to deplete the mitochondrial thiol could lead to a decrease in fluorescence intensity by ~64%, indicating CA-TPP is able to reflect the mitochondrial thiols in live cells. CA-TPP was successfully applied for monitoring mitochondrial thiols in living tissues as well.

> Hua Zhang et al. [181] developed an ultrasensitive ratiometric fluorescent probe (IQDC-M, **Probe 64**, Figure 56) for detection of ultratrace change of mitochondrial GSH in cancer cells with a detection limit to be as low as 2.02 nM. IQDC-M was designed by utilizing a modified sulfonamide as a thiol response moiety. Two naphthalene derivative fluorophores served as the fluorescence reporting groups are linked together by sulfonamide to maintain a fluorescence resonance energy transfer (FRET) process. In the presence of FRET, IQDC-M displays a maximum emission at 592 nm (Ex. 450 nm). Interestingly, IQDC-M show no fluorescence response when GSH changed at mM level due to the formation of N=N" group [182], while ultratrace change of GSH at nM could forbad the FRET and led to a significant fluorescence decrease at 592 nm and appearance of a new peak mainly at 520 nm from the product IQ-M which was a result of a thiol attack at the sulfonamide bond (addition reaction) followed by an elimination reaction to release IQ-M. The ratio of I520 nm/I592 nm increased linearly as the GSH concentration increases in nM scale. The probe showed a high selectivity for GSH than other thiols, such as Cys, Hcy, or DTT. The probe was proved to be well located inside mitochondria via a colocalization study with MitoTrackers Green FM. Interestingly, the probe was found to selectively enter cancer cells. The compound has been successfully applied to monitor the ultrachange of mitochondrial GSH in cancer cells during the apoptosis for the first time.

sues as well.

chondrial GSH in cancer cells during the apoptosis for the first time.

**Figure 56. Probe 64** and its reaction with different levels of GSH. **Figure 56. Probe 64** and its reaction with different levels of GSH.

In 2020, Pingru Su et al. [135] reported a TAT peptide-based ratiometric two-photon fluorescent probe (TAT-probe, **Probe 65**, Figure 57) which can detect thiols and simultaneously distinguish GSH in mitochondria. Cell penetrating peptide TAT (RRQRRKKRG) was widely used for delivering macromolecular substances into cells and mitochondria [183–185]. Thus, TAT was incorporated in the probe for achieving the mitochondria selectivity. In the meantime, a naphthalimide and a rhodamine B fluorophore were connected through a thioester bond to construct a Forster resonance energy transfer (FRET) system. A reaction with thiols breaks the thioester and releases a free green fluorescent naphthalimide derivative (Ex. 404/820 nm, Em. 520 nm) and a thiol-conjugated rhodamine. The GSH conjugated rhodamine displays a red fluorescence (Ex. 545 nm, Em. 585 nm) and Hcy/Cys conjugated rhodamine exhibit no fluorescence. TAT-probe was demonstrated to be able to detect GSH, Cys and Hcy with the detect limits of 5.15 μM, 0.865 μM, and 6.51 μM, respectively. Cellular experiments with HeLa cells showed that TAT-probe was able to detect thiols as well as discriminatively detect GSH over Cys/Hcy in mitochondria in live cells in different excitation channels. In 2020, Pingru Su et al. [135] reported a TAT peptide-based ratiometric two-photon fluorescent probe (TAT-probe, **Probe 65**, Figure 57) which can detect thiols and simultaneously distinguish GSH in mitochondria. Cell penetrating peptide TAT (RRQRRKKRG) was widely used for delivering macromolecular substances into cells and mitochondria [183–185]. Thus, TAT was incorporated in the probe for achieving the mitochondria selectivity. In the meantime, a naphthalimide and a rhodamine B fluorophore were connected through a thioester bond to construct a Forster resonance energy transfer (FRET) system. A reaction with thiols breaks the thioester and releases a free green fluorescent naphthalimide derivative (Ex. 404/820 nm, Em. 520 nm) and a thiol-conjugated rhodamine. The GSH conjugated rhodamine displays a red fluorescence (Ex. 545 nm, Em. 585 nm) and Hcy/Cys conjugated rhodamine exhibit no fluorescence. TAT-probe was demonstrated to be able to detect GSH, Cys and Hcy with the detect limits of 5.15 µM, 0.865 µM, and 6.51 µM, respectively. Cellular experiments with HeLa cells showed that TAT-probe was able to detect thiols as well as discriminatively detect GSH over Cys/Hcy in mitochondria in live cells in different excitation channels. *Molecules* **2021**, *26*, x FOR PEER REVIEW 36 of 59

cells with NEM to deplete the mitochondrial thiol could lead to a decrease in fluorescence intensity by ~64%, indicating CA-TPP is able to reflect the mitochondrial thiols in live cells. CA-TPP was successfully applied for monitoring mitochondrial thiols in living tis-

Hua Zhang et al. [181] developed an ultrasensitive ratiometric fluorescent probe (IQDC-M, **Probe 64**, Figure 56) for detection of ultratrace change of mitochondrial GSH in cancer cells with a detection limit to be as low as 2.02 nM. IQDC-M was designed by utilizing a modified sulfonamide as a thiol response moiety. Two naphthalene derivative fluorophores served as the fluorescence reporting groups are linked together by sulfonamide to maintain a fluorescence resonance energy transfer (FRET) process. In the presence of FRET, IQDC-M displays a maximum emission at 592 nm (Ex. 450 nm). Interestingly, IQDC-M show no fluorescence response when GSH changed at mM level due to the formation of N=N" group [182], while ultratrace change of GSH at nM could forbad the FRET and led to a significant fluorescence decrease at 592 nm and appearance of a new peak mainly at 520 nm from the product IQ-M which was a result of a thiol attack at the sulfonamide bond (addition reaction) followed by an elimination reaction to release IQ-M. The ratio of I520nm/I592nm increased linearly as the GSH concentration increases in nM scale. The probe showed a high selectivity for GSH than other thiols, such as Cys, Hcy, or DTT. The probe was proved to be well located inside mitochondria via a colocalization study with MitoTrackers Green FM. Interestingly, the probe was found to selectively enter cancer cells. The compound has been successfully applied to monitor the ultrachange of mito-

**Figure 57. Probe 65** and its reaction with GSH and Cys/Hcy. **Figure 57. Probe 65** and its reaction with GSH and Cys/Hcy.

In 2017, Chunlong Sun et al. [186] reported a mitochondria-targeted two-photon fluorescent probe (TP-Se, **Probe 66**, Figure 58) to monitor changes of ONOO- /GSH levels in cells based on a organoselenium moiety serving as the rection site for ONOO- /GSH and a methyl pyridinium moiety as a mitochondria-targeting functional group. The emission intensity at 565 nm (Ex. 430 nm) linearly increased upon reaction with ONOO- , while the fluorescence intensity increase was reversed when GSH was added. Cellular experiments In 2017, Chunlong Sun et al. [186] reported a mitochondria-targeted two-photon fluorescent probe (TP-Se, **Probe 66**, Figure 58) to monitor changes of ONOO−/GSH levels in cells based on a organoselenium moiety serving as the rection site for ONOO−/GSH and a methyl pyridinium moiety as a mitochondria-targeting functional group. The emission intensity at 565 nm (Ex. 430 nm) linearly increased upon reaction with ONOO−, while the fluorescence intensity increase was reversed when GSH was added. Cellular experiments revealed that TP-Se exhibited a good sensitivity and selectivity in monitoring ONOO−oxidation and GSH reduction events under physiological conditions in live cells.

idation and GSH reduction events under physiological conditions in live cells.

revealed that TP-Se exhibited a good sensitivity and selectivity in monitoring ONOO-ox-

In 2004, George T. Hanson et al. [187] reported redox-sensitive green fluorescence

protein indicators (roGFPs, **Probe 67**) for monitoring mitochondrial redox potential. RoG-FPs were constructed by introducing of redox-reactive groups (disulfides) to green fluorescence protein through gene modification. The leader sequence of the E1\_ subunit of pyruvate dehydrogenase was employed to direct the protein to mitochondria. The probe exhibits two maximum wavelength bands of around 400 and 490 nm while monitoring emission at 508 nm. The ratio of fluorescence intensity of two wavelengths responds to the changes in ambient redox potential rapidly, reversibly, and ratiometrically. RoGFPs were proved to be able to monitor the mitochondrial redox potential changes caused by cell treatment with a reductants (DTT) or an oxidants (H2O2) in these cells through fluo-

3.2.1. Selective Detection of Mitochondrial Cys and Hcy through Cyclization of Cys/Hcy

In 2015, Chunmiao Han et al. [188] presented a near-infrared mitochondria-targeting fluorescent probe (NFL1, **Probe 68**, Figure 59) for selective detection of Cys over Hcy and GSH with a low detection limit of 14.5 nM. NFL<sup>1</sup> is composed of a semiheptamethine derivate group to serve as the NIR fluorophore and an acryloyl group to serve as a thiol responsive moiety. The fluorescence of the semiheptamethine derivate group was quenched

/GSH.

**Figure 58.** Structure of **Probe 58** and its reaction with ONOO-

rescence microscopy or in cell suspension using a fluorometer.

*3.2. Selective Detection of Mitochondrial Cys/Hcy*

with Acrylates or Aldehydes

orescent probe (TP-Se, **Probe 66**, Figure 58) to monitor changes of ONOO-

cells based on a organoselenium moiety serving as the rection site for ONOO-

intensity at 565 nm (Ex. 430 nm) linearly increased upon reaction with ONOO-

idation and GSH reduction events under physiological conditions in live cells.

In 2017, Chunlong Sun et al. [186] reported a mitochondria-targeted two-photon flu-

methyl pyridinium moiety as a mitochondria-targeting functional group. The emission

fluorescence intensity increase was reversed when GSH was added. Cellular experiments revealed that TP-Se exhibited a good sensitivity and selectivity in monitoring ONOO-ox-

/GSH levels in

/GSH and a

, while the

**Figure 58.** Structure of **Probe 58** and its reaction with ONOO- /GSH. **Figure 58.** Structure of **Probe 66** and its reaction with ONOO−/GSH.

**Figure 57. Probe 65** and its reaction with GSH and Cys/Hcy.

In 2004, George T. Hanson et al. [187] reported redox-sensitive green fluorescence protein indicators (roGFPs, **Probe 67**) for monitoring mitochondrial redox potential. RoG-FPs were constructed by introducing of redox-reactive groups (disulfides) to green fluorescence protein through gene modification. The leader sequence of the E1\_ subunit of pyruvate dehydrogenase was employed to direct the protein to mitochondria. The probe exhibits two maximum wavelength bands of around 400 and 490 nm while monitoring emission at 508 nm. The ratio of fluorescence intensity of two wavelengths responds to the changes in ambient redox potential rapidly, reversibly, and ratiometrically. RoGFPs were proved to be able to monitor the mitochondrial redox potential changes caused by cell treatment with a reductants (DTT) or an oxidants (H2O2) in these cells through fluo-In 2004, George T. Hanson et al. [187] reported redox-sensitive green fluorescence protein indicators (roGFPs, **Probe 67**) for monitoring mitochondrial redox potential. RoGFPs were constructed by introducing of redox-reactive groups (disulfides) to green fluorescence protein through gene modification. The leader sequence of the E1\_ subunit of pyruvate dehydrogenase was employed to direct the protein to mitochondria. The probe exhibits two maximum wavelength bands of around 400 and 490 nm while monitoring emission at 508 nm. The ratio of fluorescence intensity of two wavelengths responds to the changes in ambient redox potential rapidly, reversibly, and ratiometrically. RoGFPs were proved to be able to monitor the mitochondrial redox potential changes caused by cell treatment with a reductants (DTT) or an oxidants (H2O2) in these cells through fluorescence microscopy or in cell suspension using a fluorometer.

#### rescence microscopy or in cell suspension using a fluorometer. *3.2. Selective Detection of Mitochondrial Cys/Hcy*

*3.2. Selective Detection of Mitochondrial Cys/Hcy* 3.2.1. Selective Detection of Mitochondrial Cys and Hcy through Cyclization of Cys/Hcy with Acrylates or Aldehydes

3.2.1. Selective Detection of Mitochondrial Cys and Hcy through Cyclization of Cys/Hcy with Acrylates or Aldehydes In 2015, Chunmiao Han et al. [188] presented a near-infrared mitochondria-targeting fluorescent probe (NFL1, **Probe 68**, Figure 59) for selective detection of Cys over Hcy and GSH with a low detection limit of 14.5 nM. NFL<sup>1</sup> is composed of a semiheptamethine derivate group to serve as the NIR fluorophore and an acryloyl group to serve as a thiol responsive moiety. The fluorescence of the semiheptamethine derivate group was quenched In 2015, Chunmiao Han et al. [188] presented a near-infrared mitochondria-targeting fluorescent probe (NFL1, **Probe 68**, Figure 59) for selective detection of Cys over Hcy and GSH with a low detection limit of 14.5 nM. NFL<sup>1</sup> is composed of a semiheptamethine derivate group to serve as the NIR fluorophore and an acryloyl group to serve as a thiol responsive moiety. The fluorescence of the semiheptamethine derivate group was quenched by acryloyl group due to a PET process. The semiheptamethine derivate group also provides the probe the ability to target mitochondria due to a positive charge. The off-on mechanism triggered by Cys can be explained in two steps. First, a Michael addition of the thiolate from Cys to the acryloyl group to yield a thioether. Second, the thioether will go through a rapid intramolecular cyclization to form a seven-membered ring and release a free semiheptamethine derivate. This destroys the PET process and restores the strong NIR fluorescence of the semiheptamethine derivate with the maximum emission at 735 nm. The high selectivity of NFL<sup>1</sup> toward Cys over other thiols is due to the fact that the kinetic rate of the intramolecular cyclization reaction with Cys is higher (formation of a sevenmembered ring) than Hcy (formation of an eight-membered ring). The intramolecular cyclization reaction does not occur for GSH products. The fluorescence intensity at 735 nm is linearly increased as the concentrations of Cys ascended from 5 to 15 µM with a detection limit of 14.5 nM, enabling the quantitative detection of Cys. The probe has been proved to successfully target mitochondria and monitor mitochondrial Cys change. The reagent is also capable of assessing mitochondrial oxidative stress that induced by LPS in cells, Moreover, NFL1 was successfully applied in vivo for the detection of mitochondrial Cys.

by acryloyl group due to a PET process. The semiheptamethine derivate group also provides the probe the ability to target mitochondria due to a positive charge. The off-on mechanism triggered by Cys can be explained in two steps. First, a Michael addition of the thiolate from Cys to the acryloyl group to yield a thioether. Second, the thioether will go through a rapid intramolecular cyclization to form a seven-membered ring and release a free semiheptamethine derivate. This destroys the PET process and restores the strong NIR fluorescence of the semiheptamethine derivate with the maximum emission at 735 nm. The high selectivity of NFL<sup>1</sup> toward Cys over other thiols is due to the fact that the kinetic rate of the intramolecular cyclization reaction with Cys is higher (formation of a seven-membered ring) than Hcy (formation of an eight-membered ring). The intramolecular cyclization reaction does not occur for GSH products. The fluorescence intensity at 735 nm is linearly increased as the concentrations of Cys ascended from 5 to 15 μM with a detection limit of 14.5 nM, enabling the quantitative detection of Cys. The probe has been proved to successfully target mitochondria and monitor mitochondrial Cys change. The reagent is also capable of assessing mitochondrial oxidative stress that induced by LPS in cells, Moreover, NFL1 was successfully applied in vivo for the detection of mito-

**Figure 59.** Structure of **Probe 68** and its reaction with Cys. **Figure 59.** Structure of **Probe 68** and its reaction with Cys.

chondrial Cys.

In 2016, Weifen Niu et al. [112] developed a two-photon fluorescent probe (**Probe 69)** for ratiometrically imaging and detection of Cys in mitochondria in live cells. As illustrated in Figure 60, the probe named ASMI is made by attaching an acrylate moiety to a highly two-photon active and biocompatible merocyanine fluorophore, in which the acrylate moiety is employed as a thiol responsive site and merocyanine as the mitochondria targeting group since it carries a positive charge. Same as other acrylate probes, the reaction between Cys and the probe started with a Michael addition of Cys to the acrylate followed by an intramolecular cyclization to release the merocyanine fluorophore and a seven membered-ring cyclic amide. The probe ASMI itself showed blue fluorescence with a maximum emission wavelength at 452 nm while the released free merocyanine exhibited green fluorescence with a maximum emission wavelength at 452 nm. In addition, both ASMI and merocyanine showed large two-photon action cross-section (Φσmax) of 65.2 GM (Ex. 740 nm) and 72.6 GM (Ex. = 760 nm), respectively. In addition, the ratio of fluorescence intensity of emission at 518 nm and 452 nm (F518/F452) is linearly proportional to Cys concentrations in the range of 0.5−40 μM, suggesting that the probe can detect Cys' level ratiometrically. All of these features raise the high potential to use the probe for high contrast and brightness ratiometric two-photon fluorescence imaging of live samples. Compared to other thiols such as Hcy or GSH, ASMI reacted much faster with Cys. Moreover, the probe was able to ratiometrically detect the mitochondrial Cys change in live cells when its level is elevated by addition of Cys or decreased by NEM. ASMI was also demonstrated to selectively detect mitochondrial Cys and monitor Cys status change in intact living tissues at the depth of 150 μm by the two-photon fluorescence microscopy in a mouse model. In 2016, Weifen Niu et al. [112] developed a two-photon fluorescent probe (**Probe 69**) for ratiometrically imaging and detection of Cys in mitochondria in live cells. As illustrated in Figure 60, the probe named ASMI is made by attaching an acrylate moiety to a highly two-photon active and biocompatible merocyanine fluorophore, in which the acrylate moiety is employed as a thiol responsive site and merocyanine as the mitochondria targeting group since it carries a positive charge. Same as other acrylate probes, the reaction between Cys and the probe started with a Michael addition of Cys to the acrylate followed by an intramolecular cyclization to release the merocyanine fluorophore and a seven membered-ring cyclic amide. The probe ASMI itself showed blue fluorescence with a maximum emission wavelength at 452 nm while the released free merocyanine exhibited green fluorescence with a maximum emission wavelength at 452 nm. In addition, both ASMI and merocyanine showed large two-photon action cross-section (Φσmax) of 65.2 GM (Ex. 740 nm) and 72.6 GM (Ex. = 760 nm), respectively. In addition, the ratio of fluorescence intensity of emission at 518 nm and 452 nm (F518/F452) is linearly proportional to Cys concentrations in the range of 0.5−40 µM, suggesting that the probe can detect Cys' level ratiometrically. All of these features raise the high potential to use the probe for high contrast and brightness ratiometric two-photon fluorescence imaging of live samples. Compared to other thiols such as Hcy or GSH, ASMI reacted much faster with Cys. Moreover, the probe was able to ratiometrically detect the mitochondrial Cys change in live cells when its level is elevated by addition of Cys or decreased by NEM. ASMI was also demonstrated to selectively detect mitochondrial Cys and monitor Cys status change in intact living tissues at the depth of 150 µm by the two-photon fluorescence microscopy in a mouse model. *Molecules* **2021**, *26*, x FOR PEER REVIEW 38 of 59 imaging probe by caged oxazolidinoindocyanine, Peng Zhang et al. [190] developed a NIR probe based on a difluoroboron curcuminoid scaffold, and LijunTang et al. [191] reported a far-red emissive fluorescence probe based on benzothiazole, etc.

**Figure 60.** Structure of **Probe 69** and its reaction with Cys. **Figure 60.** Structure of **Probe 69** and its reaction with Cys.

3.2.2. Selective Detection of Mitochondrial Cys/Hcy through Cys-induced SNAr Substitution−Rearrangement Reaction Mingwang Yang et al. [192] reported a colorimetric and ratiometric fluorescent chemosensor (**Probe 70**, Figure 61) for the detection of Cys/Hcy in mitochondria with a high selectivity and sensitivity (Cys, 22 nM; Hcy, 23 nM) in 2019. The probe is made by In addition, a number of other probes have been developed by using the same sensing mechanism. For instance, Chae Yeong Kim et al. [189] developed a mitochondrial Cys imaging probe by caged oxazolidinoindocyanine, Peng Zhang et al. [190] developed a NIR probe based on a difluoroboron curcuminoid scaffold, and LijunTang et al. [191] reported a far-red emissive fluorescence probe based on benzothiazole, etc.

connecting a 4-methlythiophenol to a positive charged pyronin fluorophore with a thioether bond which also serves as the thiol recognition unit. The selectivity of Cys/Hcy 3.2.2. Selective Detection of Mitochondrial Cys/Hcy through Cys-induced SNAr Substitution−Rearrangement Reaction

over GSH is achieved by a SNAr substitution reaction followed by an intramolecular rearrangement reaction while GSH is not able to undergo the intramolecular rearrangement reaction. Upon reaction with Cys/Hcy, fluorescence intensity ratio of F540/F<sup>620</sup> increases linearly with the concentration of Cys/Hcy, enabling the raitometric detection of Cys/Hcy. Mingwang Yang et al. [192] reported a colorimetric and ratiometric fluorescent chemosensor (**Probe 70**, Figure 61) for the detection of Cys/Hcy in mitochondria with a high selectivity and sensitivity (Cys, 22 nM; Hcy, 23 nM) in 2019. The probe is made by connecting

The probe was not only able to reflect mitochondrial thiol change in MCF-7 cells induced

In 2021, Xin Ji et al. [193] reported a pyridinium substituted BODIPY probe ((BDP-S-

, **Probe 71**, Figure 62) for selective and rapid detection of mitochondrial Cys in vitro and in vivo based on the SNAr substitution−rearrangement reaction. The pyridinium group was connected to a BODIPY fluorophore via a thioether bond to serve as a thiol recognition group and a mitochondrion targeting moiety. As a good leaving group, the pyridinium will be replaced by the thiolate of Cys upon reaction. Then an intramolecular rearrangement reaction occurred to produce an amine-substituted BODIPY with significantly different fluorescence properties. The detection limit of the compound was determined to be 72 nM with a linear relationship between the fluorescence intensity and Cys concentration in a range of 0 to 50 μM. The probe was successfully used to detect mitochondrial Cys change in HeLa cells with minimum interference from GSH and Hcy. The

**Figure 61.** Structure of **Probe 70** and its reaction with GSH and Cys/Hcy.

probe was capable of monitoring endogenous Cys in mice as well.

organisms.

*o*-Py<sup>+</sup>

a 4-methlythiophenol to a positive charged pyronin fluorophore with a thioether bond which also serves as the thiol recognition unit. The selectivity of Cys/Hcy over GSH is achieved by a SNAr substitution reaction followed by an intramolecular rearrangement reaction while GSH is not able to undergo the intramolecular rearrangement reaction. Upon reaction with Cys/Hcy, fluorescence intensity ratio of F540/F<sup>620</sup> increases linearly with the concentration of Cys/Hcy, enabling the raitometric detection of Cys/Hcy. The probe was not only able to reflect mitochondrial thiol change in MCF-7 cells induced by H2O2, but also able to ratiometrically image endogenous and exogenous thiols in living organisms. connecting a 4-methlythiophenol to a positive charged pyronin fluorophore with a thioether bond which also serves as the thiol recognition unit. The selectivity of Cys/Hcy over GSH is achieved by a SNAr substitution reaction followed by an intramolecular rearrangement reaction while GSH is not able to undergo the intramolecular rearrangement reaction. Upon reaction with Cys/Hcy, fluorescence intensity ratio of F540/F<sup>620</sup> increases linearly with the concentration of Cys/Hcy, enabling the raitometric detection of Cys/Hcy. The probe was not only able to reflect mitochondrial thiol change in MCF-7 cells induced by H2O2, but also able to ratiometrically image endogenous and exogenous thiols in living organisms.

3.2.2. Selective Detection of Mitochondrial Cys/Hcy through Cys-induced SNAr Substi-

Mingwang Yang et al. [192] reported a colorimetric and ratiometric fluorescent chemosensor (**Probe 70**, Figure 61) for the detection of Cys/Hcy in mitochondria with a high selectivity and sensitivity (Cys, 22 nM; Hcy, 23 nM) in 2019. The probe is made by

imaging probe by caged oxazolidinoindocyanine, Peng Zhang et al. [190] developed a NIR probe based on a difluoroboron curcuminoid scaffold, and LijunTang et al. [191] reported

*Molecules* **2021**, *26*, x FOR PEER REVIEW 38 of 59

**Figure 60.** Structure of **Probe 69** and its reaction with Cys.

tution−Rearrangement Reaction

a far-red emissive fluorescence probe based on benzothiazole, etc.

**Figure 61.** Structure of **Probe 70** and its reaction with GSH and Cys/Hcy. **Figure 61.** Structure of **Probe 70** and its reaction with GSH and Cys/Hcy.

In 2021, Xin Ji et al. [193] reported a pyridinium substituted BODIPY probe ((BDP-S*o*-Py<sup>+</sup> , **Probe 71**, Figure 62) for selective and rapid detection of mitochondrial Cys in vitro and in vivo based on the SNAr substitution−rearrangement reaction. The pyridinium group was connected to a BODIPY fluorophore via a thioether bond to serve as a thiol recognition group and a mitochondrion targeting moiety. As a good leaving group, the pyridinium will be replaced by the thiolate of Cys upon reaction. Then an intramolecular rearrangement reaction occurred to produce an amine-substituted BODIPY with significantly different fluorescence properties. The detection limit of the compound was determined to be 72 nM with a linear relationship between the fluorescence intensity and Cys concentration in a range of 0 to 50 μM. The probe was successfully used to detect mitochondrial Cys change in HeLa cells with minimum interference from GSH and Hcy. The probe was capable of monitoring endogenous Cys in mice as well. In 2021, Xin Ji et al. [193] reported a pyridinium substituted BODIPY probe ((BDP-S-*o*-Py<sup>+</sup> , **Probe 71**, Figure 62) for selective and rapid detection of mitochondrial Cys in vitro and in vivo based on the SNAr substitution−rearrangement reaction. The pyridinium group was connected to a BODIPY fluorophore via a thioether bond to serve as a thiol recognition group and a mitochondrion targeting moiety. As a good leaving group, the pyridinium will be replaced by the thiolate of Cys upon reaction. Then an intramolecular rearrangement reaction occurred to produce an amine-substituted BODIPY with significantly different fluorescence properties. The detection limit of the compound was determined to be 72 nM with a linear relationship between the fluorescence intensity and Cys concentration in a range of 0 to 50 µM. The probe was successfully used to detect mitochondrial Cys change in HeLa cells with minimum interference from GSH and Hcy. The probe was capable of monitoring endogenous Cys in mice as well. *Molecules* **2021**, *26*, x FOR PEER REVIEW 39 of 59

**Figure 62.** Structure of **Probe 71** and its reaction with Cys. **Figure 62.** Structure of **Probe 71** and its reaction with Cys.

3.2.3. Selective Detection of Mitochondrial Cys/Hcy through Other Mechanisms 3.2.3. Selective Detection of Mitochondrial Cys/Hcy through Other Mechanisms

In 2019, Li Fan et al. [194] reported the first mitochondria-targeted ratiometric twophoton (DNEPI) probe (**Probe 72**, Figure 63) based on the DNBS group. The probe is prepared by combining the two-photon fluorophore, merocyanine, and a thiol reaction unit DNBS, while a positive charge of the structure was accounted for mitochondria targeting. A cleavage of the sulfonic acid ester bond by Cys leads to a significant red shift of both fluorescence (485 to 583 nm) and absorption properties (352 to 392 nm). The ratio of fluo-In 2019, Li Fan et al. [194] reported the first mitochondria-targeted ratiometric twophoton (DNEPI) probe (**Probe 72**, Figure 63) based on the DNBS group. The probe is prepared by combining the two-photon fluorophore, merocyanine, and a thiol reaction unit DNBS, while a positive charge of the structure was accounted for mitochondria targeting. A cleavage of the sulfonic acid ester bond by Cys leads to a significant red shift of both fluorescence (485 to 583 nm) and absorption properties (352 to 392 nm). The

rescence intensity (F<sup>583</sup> nm/F485 nm) displays a linear relationship with Cys concentration

Kun Yin et al. [195] reported in 2015 a near-infrared ratiometric fluorescence probe Cy-NB (**Probe 73**) for detection of mitochondrial Cys. The probe is composed of heptamethine cyanine as fluorophore and *p*-nitrobenzoyl as the Cys recognition site. Heptamethine cyanine also serves as the mitochondria targeting moiety (Figure 64). The fluorescence properties of heptamethine cyanine are changed dramatically when *p*-nitrobenzoyl is attached to modulate the intramolecular polymethine π-electron system. Cleavage of *p*nitrobenzoyl from the probe by Cys will restore the polymethine π-electron system and induce a remarkable fluorescence emission shift simultaneously. The probe itself shows a maximum excitation wavelength and maximum emission wavelength of 720 nm and 785 nm, respectively. Upon reaction with thiols, the maximum excitation and emission wavelengths change to 580 nm and 640 nm due to the released heptamethine cyanine. The emission peak at 785 nm (Ex. 720 nm) of probe Cy-NB decreases gradually while the emission peak at 640 nm (Ex. 560 nm) increases gradually in response to an increase in Cys concentration. The ratio of two peaks (F<sup>640</sup> nm/F<sup>785</sup> nm) followed a linearly change when the concentration of cysteine is in the range of 0–35 μM and 35–100 μM, and the detection limit is 0.2 μM in 5 min. The lower pKa of Cys (8.53) than Hcy (10.00) and GSH (9.20) is believed to be one of the reasons attributing to the Cys selectivity because more nucleophilic ionized form will be formed for Cys. Moreover, the steric hindrance of tripeptide GSH is believed to be another reason for the selectivity. The pseudo-first-order reaction constant *k* for Cys is 330-fold higher than GSH and 78-fold higher than Hcy. The probe

capable of monitoring the level of intracellular Cys.

**Figure 63.** Structure of **Probe 72** and its reaction with Cys.

ratio of fluorescence intensity (F<sup>583</sup> nm/F<sup>485</sup> nm) displays a linear relationship with Cys concentration (2–10 µM), and the detection limit was determined to be 0.29 µM. A much higher reactivity of Cys than GSH and Hcy leads to the selectivity of the probe for Cys. The probe was capable of monitoring the level of intracellular Cys. fluorescence (485 to 583 nm) and absorption properties (352 to 392 nm). The ratio of fluorescence intensity (F<sup>583</sup> nm/F485 nm) displays a linear relationship with Cys concentration (2–10 μM), and the detection limit was determined to be 0.29 μM. A much higher reactivity of Cys than GSH and Hcy leads to the selectivity of the probe for Cys. The probe was

In 2019, Li Fan et al. [194] reported the first mitochondria-targeted ratiometric twophoton (DNEPI) probe (**Probe 72**, Figure 63) based on the DNBS group. The probe is prepared by combining the two-photon fluorophore, merocyanine, and a thiol reaction unit DNBS, while a positive charge of the structure was accounted for mitochondria targeting. A cleavage of the sulfonic acid ester bond by Cys leads to a significant red shift of both

3.2.3. Selective Detection of Mitochondrial Cys/Hcy through Other Mechanisms

**Figure 63.** Structure of **Probe 72** and its reaction with Cys. **Figure 63.** Structure of **Probe 72** and its reaction with Cys.

capable of monitoring the level of intracellular Cys.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 39 of 59

**Figure 62.** Structure of **Probe 71** and its reaction with Cys.

Kun Yin et al. [195] reported in 2015 a near-infrared ratiometric fluorescence probe Cy-NB (**Probe 73**) for detection of mitochondrial Cys. The probe is composed of heptamethine cyanine as fluorophore and *p*-nitrobenzoyl as the Cys recognition site. Heptamethine cyanine also serves as the mitochondria targeting moiety (Figure 64). The fluorescence properties of heptamethine cyanine are changed dramatically when *p*-nitrobenzoyl is attached to modulate the intramolecular polymethine π-electron system. Cleavage of *p*nitrobenzoyl from the probe by Cys will restore the polymethine π-electron system and induce a remarkable fluorescence emission shift simultaneously. The probe itself shows a maximum excitation wavelength and maximum emission wavelength of 720 nm and 785 nm, respectively. Upon reaction with thiols, the maximum excitation and emission wavelengths change to 580 nm and 640 nm due to the released heptamethine cyanine. The emission peak at 785 nm (Ex. 720 nm) of probe Cy-NB decreases gradually while the emission peak at 640 nm (Ex. 560 nm) increases gradually in response to an increase in Cys concentration. The ratio of two peaks (F<sup>640</sup> nm/F<sup>785</sup> nm) followed a linearly change when the concentration of cysteine is in the range of 0–35 μM and 35–100 μM, and the detection limit is 0.2 μM in 5 min. The lower pKa of Cys (8.53) than Hcy (10.00) and GSH (9.20) is believed to be one of the reasons attributing to the Cys selectivity because more nucleophilic ionized form will be formed for Cys. Moreover, the steric hindrance of tripeptide GSH is believed to be another reason for the selectivity. The pseudo-first-order reaction constant *k* for Cys is 330-fold higher than GSH and 78-fold higher than Hcy. The probe Kun Yin et al. [195] reported in 2015 a near-infrared ratiometric fluorescence probe Cy-NB (**Probe 73**) for detection of mitochondrial Cys. The probe is composed of heptamethine cyanine as fluorophore and *p*-nitrobenzoyl as the Cys recognition site. Heptamethine cyanine also serves as the mitochondria targeting moiety (Figure 64). The fluorescence properties of heptamethine cyanine are changed dramatically when *p*-nitrobenzoyl is attached to modulate the intramolecular polymethine π-electron system. Cleavage of *p*-nitrobenzoyl from the probe by Cys will restore the polymethine π-electron system and induce a remarkable fluorescence emission shift simultaneously. The probe itself shows a maximum excitation wavelength and maximum emission wavelength of 720 nm and 785 nm, respectively. Upon reaction with thiols, the maximum excitation and emission wavelengths change to 580 nm and 640 nm due to the released heptamethine cyanine. The emission peak at 785 nm (Ex. 720 nm) of probe Cy-NB decreases gradually while the emission peak at 640 nm (Ex. 560 nm) increases gradually in response to an increase in Cys concentration. The ratio of two peaks (F<sup>640</sup> nm/F<sup>785</sup> nm) followed a linearly change when the concentration of cysteine is in the range of 0–35 µM and 35–100 µM, and the detection limit is 0.2 µM in 5 min. The lower pKa of Cys (8.53) than Hcy (10.00) and GSH (9.20) is believed to be one of the reasons attributing to the Cys selectivity because more nucleophilic ionized form will be formed for Cys. Moreover, the steric hindrance of tripeptide GSH is believed to be another reason for the selectivity. The pseudo-first-order reaction constant *k* for Cys is 330-fold higher than GSH and 78-fold higher than Hcy. The probe was applied for detection of mitochondrial Cys' status in HepG2 cells successfully. The probe can reflect the Cys level change in mitochondria. The probe and is capable of detecting endogenous Cys level in mice. *Molecules* **2021**, *26*, x FOR PEER REVIEW 40 of 59 was applied for detection of mitochondrial Cys' status in HepG2 cells successfully. The probe can reflect the Cys level change in mitochondria. The probe and is capable of detecting endogenous Cys level in mice.

**Figure 64.** Structure of **Probe 73** and its reaction with Cys. **Figure 64.** Structure of **Probe 73** and its reaction with Cys.

In addition to the probes developed above, many other probes have been developed as well for selective detection of mitochondrial Cys or Hcy., Xiaopeng Yang et al. [196] developed a multi-signal fluorescent probe for selective detection of Cys and SO<sup>2</sup> based In addition to the probes developed above, many other probes have been developed as well for selective detection of mitochondrial Cys or Hcy., Xiaopeng Yang et al. [196]

drial Cys detection probe based on a coumarin-hemicyanine fluorophore.

*3.3. Lysosomal Total Thiols and GSH*

tecting thiols in lysosomes in live cells.

**Figure 65.** Representative lysosome-targeting.

the GSH level change in lysosomes in HeLa cells.

base molecules for lysosomes targeting [26,136] (Figure 65).

3.3.1. Detection of Lysosomal NPSH via a Michael Addition Reaction

on a coumarin fluorophore. Jing Liu et al. [197] developed a highly sensitive mitochon-

As another important subcellular organelle, lysosomes serve as the main digestive compartments with more than 50 hydrolases for digesting various exogenous and endogenous biomolecules. Thiols help with the processing of macromolecule degradation by the reduction of disulfide bonds [198–201]. Additionally, GSH has been reported to stabilize lysosome membranes while Cys is known for activating albumin degradation in liver lysosomes in mice [202]. Therefore, the importance of assessment of thiol levels in lysosomes has been well recognized. However, not many probes have been reported for de-

The design of most of lysosome targeting thiol probes involves the incorporation of a weak basic group which can be trapped in lysosomes which are acidic (pH ~5.0). 4-(2- Aminoethyl)morpholine and *N,N*-dimethylethylenediamine are the most widely used

In 2016, Rong Huang et al. [203] reported a lysosome targeting rhodamine B-based fluorescent probe (**Probe 74**, Figure 66) for rapid and sensitive detection of GSH utilizing a Michael acceptor as the thiol response group. The probe itself is non-fluorescent due to PET but can be turned on to fluorescent (Ex. 520 nm, Em. 582 nm) by GSH within in 10 s. The "turn on" can be explained by a Michael addition reaction followed by the formation of an H-bond between the rhodamine's carbonyl group and the *N*-H group of GSH to open the ring of rhodamine resulting in a remarkable fluorescence increase. The weak basicity of the probe attributes to the lysosome selectivity. The probe is featured with a high sensitivity with a detection limit of 190 nM. Cellular experiments and colocalization studies showed that the probe effectively accumulated inside lysosomes and could detect developed a multi-signal fluorescent probe for selective detection of Cys and SO<sup>2</sup> based on a coumarin fluorophore. Jing Liu et al. [197] developed a highly sensitive mitochondrial Cys detection probe based on a coumarin-hemicyanine fluorophore. As another important subcellular organelle, lysosomes serve as the main digestive compartments with more than 50 hydrolases for digesting various exogenous and endog-

drial Cys detection probe based on a coumarin-hemicyanine fluorophore.

**Figure 64.** Structure of **Probe 73** and its reaction with Cys.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 40 of 59

tecting endogenous Cys level in mice.

was applied for detection of mitochondrial Cys' status in HepG2 cells successfully. The probe can reflect the Cys level change in mitochondria. The probe and is capable of de-

In addition to the probes developed above, many other probes have been developed

as well for selective detection of mitochondrial Cys or Hcy., Xiaopeng Yang et al. [196] developed a multi-signal fluorescent probe for selective detection of Cys and SO<sup>2</sup> based on a coumarin fluorophore. Jing Liu et al. [197] developed a highly sensitive mitochon-

#### *3.3. Lysosomal Total Thiols and GSH* enous biomolecules. Thiols help with the processing of macromolecule degradation by

*3.3. Lysosomal Total Thiols and GSH*

As another important subcellular organelle, lysosomes serve as the main digestive compartments with more than 50 hydrolases for digesting various exogenous and endogenous biomolecules. Thiols help with the processing of macromolecule degradation by the reduction of disulfide bonds [198–201]. Additionally, GSH has been reported to stabilize lysosome membranes while Cys is known for activating albumin degradation in liver lysosomes in mice [202]. Therefore, the importance of assessment of thiol levels in lysosomes has been well recognized. However, not many probes have been reported for detecting thiols in lysosomes in live cells. the reduction of disulfide bonds [198–201]. Additionally, GSH has been reported to stabilize lysosome membranes while Cys is known for activating albumin degradation in liver lysosomes in mice [202]. Therefore, the importance of assessment of thiol levels in lysosomes has been well recognized. However, not many probes have been reported for detecting thiols in lysosomes in live cells. The design of most of lysosome targeting thiol probes involves the incorporation of

The design of most of lysosome targeting thiol probes involves the incorporation of a weak basic group which can be trapped in lysosomes which are acidic (pH ~5.0). 4-(2-Aminoethyl)morpholine and *N*,*N*-dimethylethylenediamine are the most widely used base molecules for lysosomes targeting [26,136] (Figure 65). a weak basic group which can be trapped in lysosomes which are acidic (pH ~5.0). 4-(2- Aminoethyl)morpholine and *N,N*-dimethylethylenediamine are the most widely used base molecules for lysosomes targeting [26,136] (Figure 65).

**Figure 65.** Representative lysosome-targeting. **Figure 65.** Representative lysosome-targeting.

3.3.1. Detection of Lysosomal NPSH via a Michael Addition Reaction

3.3.1. Detection of Lysosomal NPSH via a Michael Addition Reaction In 2016, Rong Huang et al. [203] reported a lysosome targeting rhodamine B-based fluorescent probe (**Probe 74**, Figure 66) for rapid and sensitive detection of GSH utilizing a Michael acceptor as the thiol response group. The probe itself is non-fluorescent due to PET but can be turned on to fluorescent (Ex. 520 nm, Em. 582 nm) by GSH within in 10 s. The "turn on" can be explained by a Michael addition reaction followed by the formation of an H-bond between the rhodamine's carbonyl group and the *N*-H group of GSH to open the ring of rhodamine resulting in a remarkable fluorescence increase. The weak In 2016, Rong Huang et al. [203] reported a lysosome targeting rhodamine B-based fluorescent probe (**Probe 74**, Figure 66) for rapid and sensitive detection of GSH utilizing a Michael acceptor as the thiol response group. The probe itself is non-fluorescent due to PET but can be turned on to fluorescent (Ex. 520 nm, Em. 582 nm) by GSH within in 10 s. The "turn on" can be explained by a Michael addition reaction followed by the formation of an H-bond between the rhodamine's carbonyl group and the *N*-H group of GSH to open the ring of rhodamine resulting in a remarkable fluorescence increase. The weak basicity of the probe attributes to the lysosome selectivity. The probe is featured with a high sensitivity with a detection limit of 190 nM. Cellular experiments and colocalization studies showed that the probe effectively accumulated inside lysosomes and could detect the GSH level change in lysosomes in HeLa cells. *Molecules* **2021**, *26*, x FOR PEER REVIEW 41 of 59

3.3.2. Detection of Lysosomal NPSH via SNAr Reactions Using Ether, Thioether as a

In 2018, Hui Zhang et al. [204] reported a lysosome targeting, coumarin and resorufin based fluorescent probe (**Probe 75**, Figure 67) for simultaneous detection of Cys/Hcy and GSH. A morpholine moiety was introduced to serve as the lysosome targeting group, and the coumarin derivative and resorufin were connected together by an ether bond which was the thiol response site. Reaction with different thiols by a nucleophilic substitution reaction will lead to the same free red fluorescent resorufin but different thio-coumarin. The thio-coumarin of Cys/Hcy undergoes an intramolecular rearrangement to switch from thio-coumarin to amino-coumarin (blue) with a significantly fluorescence change while the thio-coumarin (green) derived from GSH will remain unchanged. Thus, the probe can be used to detect the total thiols by measuring the red fluorescence of red fluorescent resorufin and detect Cys/Hcy, and GSH simultaneously from their corresponding coumarin adducts. A nearly linear relationship was found between the fluorescence intensity at 480 nm and Cys/Hcy concentration with a detection limit determining of 27 nM (or 33 nM for Hcy). A linear relationship and a detection limit of 16 nM were found for GSH with its corresponding fluorescence intensity at 542 nm. Cellular experiments with HeLa cells demonstrated that the probe was cable of discriminating intracellular Cys/Hcy,

**Figure 66.** Structure of **Probe 74** and its reaction with GSH. **Figure 66.** Structure of **Probe 74** and its reaction with GSH.

Leaving Group

GSH based on their different signal patterns.

**Figure 67.** Structure of **Probe 75** and its reaction with GSH and Cys/Hcy.

tensity at 490 nm with a detection limit of 3.3 × 10−8 M, 5.2 × 10

Xufen Song et al. [205] published their work in 2020 for the development of a lysosome-targeting fluorescent probe (**Probe 76**, Figure 68) for simultaneous detection and discrimination of Cys/Hcy and GSH by dual channels based on a similar mechanism. The probe named Lyso-O-NBD was constructed by combining a coumarin fluorophore and a NBD fluorophore via an ether bond, and a lysosome targeting moiety morpholine was attached to the Coumarin fluorophore. Similar with the probe discussed above, an intramolecular rearrangement after the nucleophilic substitution reaction attributed to the fluorescence difference of the GSH adduct and the Cys/Hcy adducts. A linear relationship was also found between the thiol (GSH, Cys, or Hcy) concentration and fluorescence in-

−8 M, and 3.9 × 10

−8 M for

Leaving Group

3.3.2. Detection of Lysosomal NPSH via SNAr Reactions Using Ether, Thioether as a Leaving Group based fluorescent probe (**Probe 75**, Figure 67) for simultaneous detection of Cys/Hcy and GSH. A morpholine moiety was introduced to serve as the lysosome targeting group, and

In 2018, Hui Zhang et al. [204] reported a lysosome targeting, coumarin and resorufin

3.3.2. Detection of Lysosomal NPSH via SNAr Reactions Using Ether, Thioether as a

*Molecules* **2021**, *26*, x FOR PEER REVIEW 41 of 59

**Figure 66.** Structure of **Probe 74** and its reaction with GSH.

Detection of Lysosomal NPSH via SNAr Reactions Using Ether as a Leaving Group the coumarin derivative and resorufin were connected together by an ether bond which

In 2018, Hui Zhang et al. [204] reported a lysosome targeting, coumarin and resorufin based fluorescent probe (**Probe 75**, Figure 67) for simultaneous detection of Cys/Hcy and GSH. A morpholine moiety was introduced to serve as the lysosome targeting group, and the coumarin derivative and resorufin were connected together by an ether bond which was the thiol response site. Reaction with different thiols by a nucleophilic substitution reaction will lead to the same free red fluorescent resorufin but different thio-coumarin. The thio-coumarin of Cys/Hcy undergoes an intramolecular rearrangement to switch from thio-coumarin to amino-coumarin (blue) with a significantly fluorescence change while the thio-coumarin (green) derived from GSH will remain unchanged. Thus, the probe can be used to detect the total thiols by measuring the red fluorescence of red fluorescent resorufin and detect Cys/Hcy, and GSH simultaneously from their corresponding coumarin adducts. A nearly linear relationship was found between the fluorescence intensity at 480 nm and Cys/Hcy concentration with a detection limit determining of 27 nM (or 33 nM for Hcy). A linear relationship and a detection limit of 16 nM were found for GSH with its corresponding fluorescence intensity at 542 nm. Cellular experiments with HeLa cells demonstrated that the probe was cable of discriminating intracellular Cys/Hcy, GSH based on their different signal patterns. was the thiol response site. Reaction with different thiols by a nucleophilic substitution reaction will lead to the same free red fluorescent resorufin but different thio-coumarin. The thio-coumarin of Cys/Hcy undergoes an intramolecular rearrangement to switch from thio-coumarin to amino-coumarin (blue) with a significantly fluorescence change while the thio-coumarin (green) derived from GSH will remain unchanged. Thus, the probe can be used to detect the total thiols by measuring the red fluorescence of red fluorescent resorufin and detect Cys/Hcy, and GSH simultaneously from their corresponding coumarin adducts. A nearly linear relationship was found between the fluorescence intensity at 480 nm and Cys/Hcy concentration with a detection limit determining of 27 nM (or 33 nM for Hcy). A linear relationship and a detection limit of 16 nM were found for GSH with its corresponding fluorescence intensity at 542 nm. Cellular experiments with HeLa cells demonstrated that the probe was cable of discriminating intracellular Cys/Hcy, GSH based on their different signal patterns.

**Figure 67.** Structure of **Probe 75** and its reaction with GSH and Cys/Hcy. **Figure 67.** Structure of **Probe 75** and its reaction with GSH and Cys/Hcy.

Xufen Song et al. [205] published their work in 2020 for the development of a lysosome-targeting fluorescent probe (**Probe 76**, Figure 68) for simultaneous detection and discrimination of Cys/Hcy and GSH by dual channels based on a similar mechanism. The probe named Lyso-O-NBD was constructed by combining a coumarin fluorophore and a NBD fluorophore via an ether bond, and a lysosome targeting moiety morpholine was attached to the Coumarin fluorophore. Similar with the probe discussed above, an intramolecular rearrangement after the nucleophilic substitution reaction attributed to the fluorescence difference of the GSH adduct and the Cys/Hcy adducts. A linear relationship was also found between the thiol (GSH, Cys, or Hcy) concentration and fluorescence intensity at 490 nm with a detection limit of 3.3 × 10−8 M, 5.2 × 10 −8 M, and 3.9 × 10 −8 M for Xufen Song et al. [205] published their work in 2020 for the development of a lysosome-targeting fluorescent probe (**Probe 76**, Figure 68) for simultaneous detection and discrimination of Cys/Hcy and GSH by dual channels based on a similar mechanism. The probe named Lyso-O-NBD was constructed by combining a coumarin fluorophore and a NBD fluorophore via an ether bond, and a lysosome targeting moiety morpholine was attached to the Coumarin fluorophore. Similar with the probe discussed above, an intramolecular rearrangement after the nucleophilic substitution reaction attributed to the fluorescence difference of the GSH adduct and the Cys/Hcy adducts. A linear relationship was also found between the thiol (GSH, Cys, or Hcy) concentration and fluorescence intensity at 490 nm with a detection limit of 3.3 <sup>×</sup> <sup>10</sup>−<sup>8</sup> M, 5.2 <sup>×</sup> <sup>10</sup>−<sup>8</sup> M, and 3.9 <sup>×</sup> <sup>10</sup>−<sup>8</sup> M for Cys, Hcy, and GSH, respectively. Later, Lyso-O-NBD was successfully applied to detect and discriminate Cys/Hcy and GSH in lysosomes in HeLa cells by confocal laser scanning microscopy.

microscopy.

microscopy.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 42 of 59

Cys, Hcy, and GSH, respectively. Later, Lyso-O-NBD was successfully applied to detect and discriminate Cys/Hcy and GSH in lysosomes in HeLa cells by confocal laser scanning

**Figure 68.** Structure of **Probe 76** and its reactions with GSH or Cys/Hcy. **Figure 68.** Structure of **Probe 76** and its reactions with GSH or Cys/Hcy. Detection of Lysosomal NPSH via SNAr Reactions Using Thioether as a Leaving Group

Detection of Lysosomal NPSH via SNAr Reactions Using Thioether as a Leaving Group Detection of Lysosomal NPSH via SNAr Reactions Using Thioether as a Leaving Group Based on the thiol specific thiol–sulfide exchange reaction reported from our group [42], we developed a thiol specific and lysosome-selective fluorogenic agent (BISMORX,

Cys, Hcy, and GSH, respectively. Later, Lyso-O-NBD was successfully applied to detect and discriminate Cys/Hcy and GSH in lysosomes in HeLa cells by confocal laser scanning

Based on the thiol specific thiol–sulfide exchange reaction reported from our group [42], we developed a thiol specific and lysosome-selective fluorogenic agent (BISMORX, **Probe 77**, Figure 69) [206] in 2019 by introducing a lysosome targeting structure morpholine symmetrically to the symmetric benzofurazan sulfide. BSMORX uses a thioether moiety to serve as the thiol recognition units. Similar to the early thiol specific fluorescence imaging probes developed from our lab, BISMORX itself is non-fluorescent due to the selfquenching of the symmetric structure but reacts readily with a thiol to form a strong fluorescence thiol adducts (Ex. 380 nm, Em. 540 nm). Cellular experiments showed that BIS-MORX was able to image, quantify, and detect the change of NPSH in lysosomes in live cells. We also developed another lysosomal NPSH imaging agent TBONES (**Probe 78**) [207]. Interestingly, the lysosome selectivity of this probe was likely due to its selective entry of lysosomes through an endocytosis pathway which is different than other lyso-Based on the thiol specific thiol–sulfide exchange reaction reported from our group [42], we developed a thiol specific and lysosome-selective fluorogenic agent (BISMORX, **Probe 77**, Figure 69) [206] in 2019 by introducing a lysosome targeting structure morpholine symmetrically to the symmetric benzofurazan sulfide. BSMORX uses a thioether moiety to serve as the thiol recognition units. Similar to the early thiol specific fluorescence imaging probes developed from our lab, BISMORX itself is non-fluorescent due to the self-quenching of the symmetric structure but reacts readily with a thiol to form a strong fluorescence thiol adducts (Ex. 380 nm, Em. 540 nm). Cellular experiments showed that BISMORX was able to image, quantify, and detect the change of NPSH in lysosomes in live cells. We also developed another lysosomal NPSH imaging agent TBONES (**Probe 78**) [207]. Interestingly, the lysosome selectivity of this probe was likely due to its selective entry of lysosomes through an endocytosis pathway which is different than other lysosome-targeting probes. **Probe 77**, Figure 69) [206] in 2019 by introducing a lysosome targeting structure morpholine symmetrically to the symmetric benzofurazan sulfide. BSMORX uses a thioether moiety to serve as the thiol recognition units. Similar to the early thiol specific fluorescence imaging probes developed from our lab, BISMORX itself is non-fluorescent due to the selfquenching of the symmetric structure but reacts readily with a thiol to form a strong fluorescence thiol adducts (Ex. 380 nm, Em. 540 nm). Cellular experiments showed that BIS-MORX was able to image, quantify, and detect the change of NPSH in lysosomes in live cells. We also developed another lysosomal NPSH imaging agent TBONES (**Probe 78**) [207]. Interestingly, the lysosome selectivity of this probe was likely due to its selective entry of lysosomes through an endocytosis pathway which is different than other lysosome-targeting probes.

some-targeting probes.

change in the levels of Cys and Hcy in HepG2 cells.

**Figure 69.** A. Structure of **Probe 77** and its reaction with thiols; B. Structure of **Probe 78**. **Figure 69.** A. Structure of **Probe 77** and its reaction with thiols; B. Structure of **Probe 78**. **Figure 69.** A. Structure of **Probe 77** and its reaction with thiols; B. Structure of **Probe 78**.

3.3.3. Detection of Lysosome NPSH via Cleavage of Sulfonamide and Sulfonate Detection of Lysosomal NPSH via Cleavage of Sulfonamide 3.3.3. Detection of Lysosome NPSH via Cleavage of Sulfonamide and Sulfonate 3.3.3. Detection of Lysosome NPSH via Cleavage of Sulfonamide and Sulfonate Detection of Lysosomal NPSH via Cleavage of Sulfonamide

In 2016, Meijiao Cao et al. [208] reported a lysosome targeting naphthalimide-based fluorescence probe (**Probe 79**, Figure 70) for selective detection of GSH in live cells based on cleavage of sulfonamide as the thiol recognition mechanism. A morpholine was attached to the probe to provide the lysosome selectivity. Cleavage of sulfonamide by GSH will produce a strong fluorescent GSH conjugated naphthalimide derivate (Ex. 370 nm, Em. 495 nm). Colocalization experiments with LysoTracker Red on HepG2 cells demonstrated that the probe effectively localized inside lysosomes. Further cellular experiments showed that the probe effectively reflected lysosome GSH changes but not affected by a change in the levels of Cys and Hcy in HepG2 cells. Detection of Lysosomal NPSH via Cleavage of Sulfonamide In 2016, Meijiao Cao et al. [208] reported a lysosome targeting naphthalimide-based fluorescence probe (**Probe 79**, Figure 70) for selective detection of GSH in live cells based on cleavage of sulfonamide as the thiol recognition mechanism. A morpholine was attached to the probe to provide the lysosome selectivity. Cleavage of sulfonamide by GSH will produce a strong fluorescent GSH conjugated naphthalimide derivate (Ex. 370 nm, Em. 495 nm). Colocalization experiments with LysoTracker Red on HepG2 cells demonstrated that the probe effectively localized inside lysosomes. Further cellular experiments showed that the probe effectively reflected lysosome GSH changes but not affected by a In 2016, Meijiao Cao et al. [208] reported a lysosome targeting naphthalimide-based fluorescence probe (**Probe 79**, Figure 70) for selective detection of GSH in live cells based on cleavage of sulfonamide as the thiol recognition mechanism. A morpholine was attached to the probe to provide the lysosome selectivity. Cleavage of sulfonamide by GSH will produce a strong fluorescent GSH conjugated naphthalimide derivate (Ex. 370 nm, Em. 495 nm). Colocalization experiments with LysoTracker Red on HepG2 cells demonstrated that the probe effectively localized inside lysosomes. Further cellular experiments showed that the probe effectively reflected lysosome GSH changes but not affected by a change in the levels of Cys and Hcy in HepG2 cells.

copy.

**Figure 70.** Structure of **Probe 79** and its reaction with GSH. **Figure 70.** Structure of **Probe 79** and its reaction with GSH. Jiangli Fan et al. [165] reported a similar work (**Probe 80**, Figure 71) in 2016 in which naphthalimide was used as the fluorophore. A DNBS group was attached to serve as a

*Molecules* **2021**, *26*, x FOR PEER REVIEW 43 of 59

Jiangli Fan et al. [165] reported a similar work (**Probe 80**, Figure 71) in 2016 in which naphthalimide was used as the fluorophore. A DNBS group was attached to serve as a thiol recognition group and a morpholine was incorporated as the lysosome targeting ligand. PET between DNBS and naphthalimide quenches the fluorescence of the probe. Upon reaction with thiols, DNBS will be detached and terminate the PET process, leading to the restoration of the fluorescence of naphthalimide (Ex. 400 nm, Em. 540 nm). A linear relationship was found between the fluorescence intensity at 540 nm and the concentra-Jiangli Fan et al. [165] reported a similar work (**Probe 80**, Figure 71) in 2016 in which naphthalimide was used as the fluorophore. A DNBS group was attached to serve as a thiol recognition group and a morpholine was incorporated as the lysosome targeting ligand. PET between DNBS and naphthalimide quenches the fluorescence of the probe. Upon reaction with thiols, DNBS will be detached and terminate the PET process, leading to the restoration of the fluorescence of naphthalimide (Ex. 400 nm, Em. 540 nm). A linear relationship was found between the fluorescence intensity at 540nm and the concentration of thiols with a detection limit of 2.6 <sup>×</sup> <sup>10</sup>−<sup>7</sup> M, 2.41 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M and 4.87 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M for Cys, GSH, and Hcy, respectively. The probe was successfully used to detect lysosomal thiol change in live HeLa cells and MCF-7 cells as well as in tissues by a two-photon microscopy. thiol recognition group and a morpholine was incorporated as the lysosome targeting ligand. PET between DNBS and naphthalimide quenches the fluorescence of the probe. Upon reaction with thiols, DNBS will be detached and terminate the PET process, leading to the restoration of the fluorescence of naphthalimide (Ex. 400 nm, Em. 540 nm). A linear relationship was found between the fluorescence intensity at 540 nm and the concentration of thiols with a detection limit of 2.6 × 10−7 M, 2.41 × 10−6 M and 4.87 × 10−6 M for Cys, GSH, and Hcy, respectively. The probe was successfully used to detect lysosomal thiol change in live HeLa cells and MCF-7 cells as well as in tissues by a two-photon micros-

tion of thiols with a detection limit of 2.6 × 10−7 M, 2.41 × 10−6 M and 4.87 × 10−6 M for Cys,

veloped by Quankun Gao et al. [209] in 2017 for selective detection of thiols based on the cleavage of sulfonate mechanism. A morpholine moiety was incorporated into the complex to achieve the lysosome selectivity, DNBS was attached to serve as an efficient PET quencher. Upon reaction with thiols, a phosphorescence enhancement was observed at 620 nm (Ex. 459 nm) due to the cleavage of sulfonate moiety. A linear relationship was observed between the phosphorescence intensity and the concentration of GSH in a range

**Figure 72.** (**A**). Structure of **Probe 81** and its reaction with thiols. (**B**). Structure of **Probe 82**.

**Figure 71.** Structure of **Probe 80** and its reaction with thiols. **Figure 71.** Structure of **Probe 80** and its reaction with thiols.

Detection of Lysosomal NPSH via Cleavage of Sulfonate

**Figure 71.** Structure of **Probe 80** and its reaction with thiols. Detection of Lysosomal NPSH via Cleavage of Sulfonate A lysosome targeting ruthenium(II) derivative probe (**Probe 81**, Figure 72) was de-Detection of Lysosomal NPSH via Cleavage of Sulfonate A lysosome targeting ruthenium(II) derivative probe (**Probe 81**, Figure 72) was developed by Quankun Gao et al. [209] in 2017 for selective detection of thiols based on the cleavage of sulfonate mechanism. A morpholine moiety was incorporated into the complex to achieve the lysosome selectivity, DNBS was attached to serve as an efficient PET quencher. Upon reaction with thiols, a phosphorescence enhancement was observed at 620 nm (Ex. 459 nm) due to the cleavage of sulfonate moiety. A linear relationship was observed between the phosphorescence intensity and the concentration of GSH in a range of 4.0 to 25 μM. The detection limit was determined to be 62 nM, 146 nM, and 115 nM for A lysosome targeting ruthenium(II) derivative probe (**Probe 81**, Figure 72) was developed by Quankun Gao et al. [209] in 2017 for selective detection of thiols based on the cleavage of sulfonate mechanism. A morpholine moiety was incorporated into the complex to achieve the lysosome selectivity, DNBS was attached to serve as an efficient PET quencher. Upon reaction with thiols, a phosphorescence enhancement was observed at 620 nm (Ex. 459 nm) due to the cleavage of sulfonate moiety. A linear relationship was observed between the phosphorescence intensity and the concentration of GSH in a range of 4.0 to 25 µM. The detection limit was determined to be 62 nM, 146 nM, and 115 nM for GSH, Cys, and Hcy, respectively. The probe was successfully applied for visualizations of thiols in lysosomes in live cells and in *Daphnia magna*.

**Figure 72.** (**A**). Structure of **Probe 81** and its reaction with thiols. (**B**). Structure of **Probe 82**.

GSH, Cys, and Hcy, respectively. The probe was successfully applied for visualizations of

thiols in lysosomes in live cells and in *Daphnia magna*.

thiols in lysosomes in live cells and in *Daphnia magna*.

**Figure 72.** (**A**). Structure of **Probe 81** and its reaction with thiols. (**B**). Structure of **Probe 82**. **Figure 72.** (**A**). Structure of **Probe 81** and its reaction with thiols. (**B**). Structure of **Probe 82**.

3.3.4. Detection of Lysosomal NPSH via Cleavage of Disulfide

**Figure 70.** Structure of **Probe 79** and its reaction with GSH.

**Figure 71.** Structure of **Probe 80** and its reaction with thiols.

thiols in lysosomes in live cells and in *Daphnia magna*.

Detection of Lysosomal NPSH via Cleavage of Sulfonate

copy.

Jiangli Fan et al. [165] reported a similar work (**Probe 80**, Figure 71) in 2016 in which naphthalimide was used as the fluorophore. A DNBS group was attached to serve as a thiol recognition group and a morpholine was incorporated as the lysosome targeting ligand. PET between DNBS and naphthalimide quenches the fluorescence of the probe. Upon reaction with thiols, DNBS will be detached and terminate the PET process, leading to the restoration of the fluorescence of naphthalimide (Ex. 400 nm, Em. 540 nm). A linear relationship was found between the fluorescence intensity at 540 nm and the concentration of thiols with a detection limit of 2.6 × 10−7 M, 2.41 × 10−6 M and 4.87 × 10−6 M for Cys, GSH, and Hcy, respectively. The probe was successfully used to detect lysosomal thiol change in live HeLa cells and MCF-7 cells as well as in tissues by a two-photon micros-

A lysosome targeting ruthenium(II) derivative probe (**Probe 81**, Figure 72) was developed by Quankun Gao et al. [209] in 2017 for selective detection of thiols based on the cleavage of sulfonate mechanism. A morpholine moiety was incorporated into the complex to achieve the lysosome selectivity, DNBS was attached to serve as an efficient PET quencher. Upon reaction with thiols, a phosphorescence enhancement was observed at 620 nm (Ex. 459 nm) due to the cleavage of sulfonate moiety. A linear relationship was observed between the phosphorescence intensity and the concentration of GSH in a range of 4.0 to 25 μM. The detection limit was determined to be 62 nM, 146 nM, and 115 nM for GSH, Cys, and Hcy, respectively. The probe was successfully applied for visualizations of

In 2019, Ziming Zheng et al. [210] reported a disulfide bond based probe (SQSS, **Probe 82**) for selective detection of lysosome thiols in cells with a high signal-to-noise ratio. Two squaraines (NiR fluorophore) were linked together via a disulfide bond and a high fluorescence resonance energy transfer (FRET) quenching effect was generated between the two squaraines. The two weakly alkaline groups in the structure might attribute to the lysosome selectivity. Upon reaction with GSH, the double bond will be broken leading to the restoration of the fluorescence (Em. 665 nm, Ex. 610 nm). A linear relationship was observed between the concentration of GSH and fluorescence intensity (665 nm) with a detection limit of 0.15 µM, and the probe showed a higher selectivity toward GSH than Cys and Hcy by a possible electrostatic interaction between the two carbonate anions of GSH and the thiazole cations of SQSS. Cell imaging experiments revealed that SQSS can monitor endogenous and exogenous GSH in tumor or normal cells.

#### 3.3.5. Detection of Lysosome NPSH via Other Methods

In 2020, Hong Wang et al. [211] built a dual targeting (cancer-specific and lysosometargeted) fluorescence nanoprobe (DTFN, **Probe 83**) for GSH imaging in live cells. The nanoprobe was constructed by combining folic acid-modified photostable aggregationinduced emission dots with manganese dioxide (MnO2) nanosheets (GSH responsive site) through electrostatic interactions. Folic acid was introduced to facilitate the probe to be taken by folate receptor (FR) over-expressed cancer cells, and the positively charged amino moiety helps the nanoparticles enter lysosomes. Fluorescence quenching is achieved vis a fluorescence resonance energy transfer (FRET) effect from AIE dots to the MnO<sup>2</sup> nanosheets. Reaction with GSH will reduce MnO<sup>2</sup> nanosheets to Mn2+ to break the FRET process resulting in the restoration of the fluorescence at 523 nm. The fluorescence intensity increased linearly as the concentration of GSH increased with a detection limit of 1.03 µM. Cellular experiments with live HeLa cells showed that the probe could successfully detect lysosomal GSH level in cancer cells.

#### *3.4. Selective Detection of Lysosomal Cys and Hcy*

Development of probes for detection of lysosomal Cys and Hcy are still in the early stage. In 2019, Jinhua Gao et al. [212] reported two lysosome targeting BODIPY fluorescence probes (**Probes 84–85**) for selective detection of Cys over GSH and Hcy based on a Cys induced SNAr substitution reaction followed by an intramolecular rearrangement. As illustrated in Figure 73, morpholinoethoxy group was introduced to achieve the lysosome selectivity, and a *p*-methoxyphenylmercapto moiety was attached to the meta position to serve as a Cys recognition unit. After reaction with Cys, the *p*-methoxyphenylmercapto moiety was replaced by Cys by a Cys-induced SNAr substitution followed by an intramolecular rearrangement to generate meso-amino-BODIPYs with strong fluorescence at 566 nm. The two probes displayed high selectivity for Cys over GSH and Hcy. A linear relationship was found between the fluorescence intensity (566 nm) and Cys concentration and a low detection limit was achieved for Cys (46 nM for Lyso-S and 76 nM for Lyso-D). Cellular

experiments in HeLa cells showed that the probes effective localized in lysosomes and imaged lysosome thiols. *Molecules* **2021**, *26*, x FOR PEER REVIEW 45 of 59

*Molecules* **2021**, *26*, x FOR PEER REVIEW 45 of 59

**Figure 73.** Structures of **Probes 84**, and **85**. **Figure 73.** Structures of **Probes 84**, and **85**. **Figure 73.** Structures of **Probes 84**, and **85**.

[115,213,214].

ER.

ER.

#### *3.5. Detection of NPSH in Endoplasmic Reticulum 3.5. Detection of NPSH in Endoplasmic Reticulum*

*3.5. Detection of NPSH in Endoplasmic Reticulum* Endoplasmic reticulum plays important roles [136] in protein synthesis, lipid and carbohydrate metabolism, calcium signaling, etc. Fluorescent probes for imaging thiols inside endoplasmic reticulum have been rarely reported. Among a very few reported probes, methyl benzene sulfonamide is the most commonly used as an ER-targeting moiety (Figure 74) Endoplasmic reticulum plays important roles [136] in protein synthesis, lipid and carbohydrate metabolism, calcium signaling, etc. Fluorescent probes for imaging thiols inside endoplasmic reticulum have been rarely reported. Among a very few reported probes, methyl benzene sulfonamide is the most commonly used as an ER-targeting moiety (Figure 74) [115,213,214]. Endoplasmic reticulum plays important roles [136] in protein synthesis, lipid and carbohydrate metabolism, calcium signaling, etc. Fluorescent probes for imaging thiols inside endoplasmic reticulum have been rarely reported. Among a very few reported probes, methyl benzene sulfonamide is the most commonly used as an ER-targeting moiety (Figure 74) [115,213,214].

**Figure 74.** ER-targeting thiols imaging probes. **Figure 74.** ER-targeting thiols imaging probes.

**Figure 74.** ER-targeting thiols imaging probes. In 2019, Chengshi Jiang et al. [215] developed an naphthalimide-based fluorescent probe (**Probe 86**, Figure 75) for imaging cellular GSH in the ER in live cells. The probe named ER-G was constructed by introducing a *p*-toluenesulfonamide unit to the top of naphthalimide as an ER-targeting unit and a DNBS was attached to the tail as the thiol recognition unit. The non-fluorescent ER-G can be turned on to a highly fluorescent naphthalimide derivate (Em. 558 nm, Ex. 470 nm) after reaction with GSH in which the DNBS was cleaved. The fluorescence intensity was found to be linearly associated with the concentration of GSH (75–300 μM). Cellular experiments with live HepG2 cells revealed that the probe detected the thiol level change by addition of NEM or addition of GSH. The In 2019, Chengshi Jiang et al. [215] developed an naphthalimide-based fluorescent probe (**Probe 86**, Figure 75) for imaging cellular GSH in the ER in live cells. The probe named ER-G was constructed by introducing a *p*-toluenesulfonamide unit to the top of naphthalimide as an ER-targeting unit and a DNBS was attached to the tail as the thiol recognition unit. The non-fluorescent ER-G can be turned on to a highly fluorescent naphthalimide derivate (Em. 558 nm, Ex. 470 nm) after reaction with GSH in which the DNBS was cleaved. The fluorescence intensity was found to be linearly associated with the concentration of GSH (75–300 μM). Cellular experiments with live HepG2 cells revealed that the probe detected the thiol level change by addition of NEM or addition of GSH. The probe was capable of monitoring H2O2-induced cellular GSH concentration change in the In 2019, Chengshi Jiang et al. [215] developed an naphthalimide-based fluorescent probe (**Probe 86**, Figure 75) for imaging cellular GSH in the ER in live cells. The probe named ER-G was constructed by introducing a *p*-toluenesulfonamide unit to the top of naphthalimide as an ER-targeting unit and a DNBS was attached to the tail as the thiol recognition unit. The non-fluorescent ER-G can be turned on to a highly fluorescent naphthalimide derivate (Em. 558 nm, Ex. 470 nm) after reaction with GSH in which the DNBS was cleaved. The fluorescence intensity was found to be linearly associated with the concentration of GSH (75–300 µM). Cellular experiments with live HepG2 cells revealed that the probe detected the thiol level change by addition of NEM or addition of GSH. The probe was capable of monitoring H2O2-induced cellular GSH concentration change in the ER.

probe was capable of monitoring H2O2-induced cellular GSH concentration change in the

**Figure 75.** (**A**). Structure of **Probe 86** and its reaction with GSH; (**B**). Structure of **Probe 87** and its reactions with thiols. **Figure 75.** (**A**). Structure of **Probe 86** and its reaction with GSH; (**B**). Structure of **Probe 87** and its reactions with thiols.

A ER-targeted TP fluorescent probe named as ER-SH (**Probe 87**, Figure 75) based the mechanism of cleavage of sulfonamide has been developed for imaging of thiols in live cells and in vivo by Ping Li et al. in 2019 [214]. The probe was constructed similarly with the probe mentioned above in which a naphthalimide was used as a two-photon fluorophore, a methyl benzene sulfonamide was used as an ER targeting moiety and DNBS was used as a thiol recognition group. ER-SH exhibits minimum fluorescence since the fluorescence of naphthalimide was quenched by DNBS due to a PET mechanism. Reaction with thiol will cleave the DNBS and restore the fluorescence (535 nm). Fluorescence intensity at 535 nm increased linearly with an increase in the concentrations of Cys, GSH, and Hcy with the detection limit of 1.67 × 10−7 M, 4.70 × 10−6 M, and 9.62 × 10−7 M, respectively. In addition to the ability to reflect thiol levels' change in live cells, the probe was demonstrated to measure thiols levels in vivo by revealing that the thiol levels were reduced in brains of mice with depression phenotypes. A ER-targeted TP fluorescent probe named as ER-SH (**Probe 87**, Figure 75) based the mechanism of cleavage of sulfonamide has been developed for imaging of thiols in live cells and in vivo by Ping Li et al. in 2019 [214]. The probe was constructed similarly with the probe mentioned above in which a naphthalimide was used as a two-photon fluorophore, a methyl benzene sulfonamide was used as an ER targeting moiety and DNBS was used as a thiol recognition group. ER-SH exhibits minimum fluorescence since the fluorescence of naphthalimide was quenched by DNBS due to a PET mechanism. Reaction with thiol will cleave the DNBS and restore the fluorescence (535 nm). Fluorescence intensity at 535 nm increased linearly with an increase in the concentrations of Cys, GSH, and Hcy with the detection limit of 1.67 <sup>×</sup> <sup>10</sup>−<sup>7</sup> M, 4.70 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M, and 9.62 <sup>×</sup> <sup>10</sup>−<sup>7</sup> M, respectively. In addition to the ability to reflect thiol levels' change in live cells, the probe was demonstrated to measure thiols levels in vivo by revealing that the thiol levels were reduced in brains of mice with depression phenotypes.

Xiuxiu Yue et al. [216] developed an ER selective fluorescent probe (ER-CP, **Probe 88**, Figure 76) to discriminatively detect Cys, Hcy, and GSH in live cells for the first time based on a SNAr substitution reaction followed by an intramolecular rearrangement reaction. ER-CP composed of three parts, a coumarin fluorophore, a methyl sulfonamide as an ERtargeting group, and 4-nitrobenzenethiol attached to the coumarin through a thioether bond as the thiol recognition group. PET presented between 4-nitrobenzenethiol and coumarin leads to a fluorescence quenching of ER-CP. A reaction with thiols will replace the nitrobenzenethiol moiety to form a green fluorescent thiol adducts, the same as other probes we described before. The thiol adducts with coumarin derived from Cys and Hcy will further undergo an intramolecular rearrangement reaction to afford a blue fluorescent amnio-substitution coumarin adducts (Ex. 370 nm, Em. 473 nm) although the reaction for Cys was much fast than Hcy while GSH adducts will remain unchanged as the green fluorescent thiol-substitution adducts (Ex. 427 nm, Em. 541 nm). A linear relationship was found between the concentration of thiols (Cys, Hcy, and GSH) and its corresponding fluorescence intensity with a detection limit of 14 nM, 16 nM, and 23 nM, respectively. The probe demonstrates an excellent ER-targeting property and is able to simultaneously Xiuxiu Yue et al. [216] developed an ER selective fluorescent probe (ER-CP, **Probe 88**, Figure 76) to discriminatively detect Cys, Hcy, and GSH in live cells for the first time based on a SNAr substitution reaction followed by an intramolecular rearrangement reaction. ER-CP composed of three parts, a coumarin fluorophore, a methyl sulfonamide as an ER-targeting group, and 4-nitrobenzenethiol attached to the coumarin through a thioether bond as the thiol recognition group. PET presented between 4-nitrobenzenethiol and coumarin leads to a fluorescence quenching of ER-CP. A reaction with thiols will replace the nitrobenzenethiol moiety to form a green fluorescent thiol adducts, the same as other probes we described before. The thiol adducts with coumarin derived from Cys and Hcy will further undergo an intramolecular rearrangement reaction to afford a blue fluorescent amnio-substitution coumarin adducts (Ex. 370 nm, Em. 473 nm) although the reaction for Cys was much fast than Hcy while GSH adducts will remain unchanged as the green fluorescent thiol-substitution adducts (Ex. 427 nm, Em. 541 nm). A linear relationship was found between the concentration of thiols (Cys, Hcy, and GSH) and its corresponding fluorescence intensity with a detection limit of 14 nM, 16 nM, and 23 nM, respectively. The probe demonstrates an excellent ER-targeting property and is able to simultaneously detect Cys, Hcy, and GSH in HeLa cells.

detect Cys, Hcy, and GSH in HeLa cells.

**Figure 76.** Structure of **Probe 88** and its different reactions mechanisms toward GSH or Cys/Hcy. **Figure 76.** Structure of **Probe 88** and its different reactions mechanisms toward GSH or Cys/Hcy.

#### **4. Conclusions 4. Conclusions**

In the past two decades, extensive efforts have been made in thiol detection in live cells with different approaches and mechanisms. Numerous fluorescent probes have been developed for detection of thiols in the whole cell as well as in subcellular organelles. The probes for imaging thiols in the whole cells are usually made up of two parts: a structure that can react with a thiol selectively, ideally specifically, and a structure that can be fluorescent for detection. The probes for imaging thiol in subcellular organelles will need to add a subcellular organelle targeting part on top of the probe for imaging thiols in the whole cells. The selectivity and reactivity of a probe for thiol is critical in ensuring only thiols are imaged. This is especially true for quantification. As discussed early, most of the employed reactions for thiol detection are based on a nucleophilic reaction. Although thiol is preferred in these reactions based on the fact that thiol is superior in terms of its nucleophilicity when compared with other common nucleophilic groups present in the biological system such as -OH, -NH, and COOH, these reactions are not thiol specific. A few reactions for thiol were reported to be thiol specific. These reactions include thiol–disulfide exchange reaction and thiol–sulfide exchange reaction (Sections 2.1.4, 3.1.4, 3.3.4, 2.1.2.3, 3.1.2.2, and 3.3.2.2). It is expected that significant progress will be made if more In the past two decades, extensive efforts have been made in thiol detection in live cells with different approaches and mechanisms. Numerous fluorescent probes have been developed for detection of thiols in the whole cell as well as in subcellular organelles. The probes for imaging thiols in the whole cells are usually made up of two parts: a structure that can react with a thiol selectively, ideally specifically, and a structure that can be fluorescent for detection. The probes for imaging thiol in subcellular organelles will need to add a subcellular organelle targeting part on top of the probe for imaging thiols in the whole cells. The selectivity and reactivity of a probe for thiol is critical in ensuring only thiols are imaged. This is especially true for quantification. As discussed early, most of the employed reactions for thiol detection are based on a nucleophilic reaction. Although thiol is preferred in these reactions based on the fact that thiol is superior in terms of its nucleophilicity when compared with other common nucleophilic groups present in the biological system such as -OH, -NH, and COOH, these reactions are not thiol specific. A few reactions for thiol were reported to be thiol specific. These reactions include thiol– disulfide exchange reaction and thiol–sulfide exchange reaction (Sections 2.1.4, 3.1.4 and 3.3.4 and Sections 2.1.2.3, 3.1.2.2 and 3.3.2.2). It is expected that significant progress will be made if more thiol specific reactions are identified.

thiol specific reactions are identified. This review summarizes various thiol imaging probes used for live cell thiol imaging. To help readers for the probes presented in this review, Table 1 lists the mechanisms of thiol detection, usage, detection limits, and references for most of the probes presented in this review. This review summarizes various thiol imaging probes used for live cell thiol imaging. To help readers for the probes presented in this review, Table 1 lists the mechanisms of thiol detection, usage, detection limits, and references for most of the probes presented in this review.


**Table 1.** Thiol imaging probes presented in the review.





Developing probes for imaging thiols in subcellular organelles obviously faces more challenges since it requires the availability of the subcellular targeting part. Thiol probes for many subcellular organelles including nucleus, ER, Golgi apparatus, and cell surface are still lacking due to a lack of an ideal targeting ligand for the subcellular organelle. Although a number of probes have been developed for imaging thiols in mitochondria and lysosomes, challenges still exist. For example, the majority of mitochondria targeting probes achieved their mitochondria targeting by attaching a lipophilic cationic structure which has the potential risk of decreasing negative mitochondrial transmembrane potential (MMP) [136]. The lysosome targeting group morpholine can induce certain toxicities by elevating the pH levels of lysosomes if a high amount of the probes are applied [136].

In addition, a low water solubility is another shortcoming for most of the probes. Organic solvents like DMSO are commonly used to improve water solubility to enable the probe to be applied in cells or tissues. Thus, a probe with adequate water solubility and sufficient permeability is desirable [136]. A membrane transporter can also be a problem. Although most of the probes can diffuse inside cells due to a good permeability, active transporters (e.g., P-gp, or MRP) on cell membrane can potentially pump the hydrophobic probes out of the cells rapidly, which can significantly affect the accuracy of thiol measurement in live cells. The potential interferences caused by active transporters should be studied.

Real-time thiol imaging in live cells is another challenge. Most of the probes developed are based on an irreversible reaction with thiols, which makes it difficult to monitor the dynamic change of thiols in real time. A few reagents that can detect the analysts of interest in a reversible manner have been developed in the past ~5 years to address this challenge [74–77,139,140,217]. These probes can monitor the dynamic status change of the analysts in real time. Some of the reversible probes have been well developed and characterized for quantifying and monitoring GSH changes at the cellular level and even subcellular levels with a number of probes showing the capability of quantifying and monitoring mitochondrial GSH changes [139,140].

More agents for different subcellular organelles are expected to be developed, and better fluorophores and sensing mechanisms are also anticipated to be discovered and applied in the future. Although majority of the developed probes can only be used for cells, quite a number of probes with NIR or two-photon fluorophores have been successfully applied at tissue levels or in in vivo models. More impressively, some of the probes were demonstrated to be capable of distinguishing tumor tissues from other tissues, warranting the possibility of employing these probes as clinical diagnostic tools in the future though much work is still needed.

In conclusion, we summarized the design strategies and sensing mechanisms of some of the most representative thiol imaging probes for live cell thiol imaging. We hope this review can provide the readers an overview and better understanding of the current status and challenges in the field.

**Author Contributions:** X.G. provided guidance and the outline of the review to co-authors and was responsible for the final editing and approval of the review. S.W. and Y.H. were responsible for the preparation of the manuscript draft and contributed equally to this article. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Article*
