*Article* **Enantioseparation, Stereochemical Assignment and Chiral Recognition Mechanism of Sulfoxide-Containing Drugs**

#### **Fei Xiong, Bei-Bei Yang, Jie Zhang and Li Li \***

Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; xxf@imm.ac.cn (F.X.); yangbetty@imm.ac.cn (B.-B.Y.); zhd@imm.ac.cn (J.Z.)

**\*** Correspondence: annaleelin@imm.ac.cn; Tel.: +86-10-6316-5247

Received: 15 September 2018; Accepted: 15 October 2018; Published: 18 October 2018

**Abstract:** The distinct pharmacodynamic and pharmacokinetic properties of enantiopure sulfoxide drugs have stimulated us to systematically investigate their chiral separation, stereochemical assignment, and chiral recognition mechanism. Herein, four clinically widely-used sulfoxide drugs were chosen and optically resolved on various chiral stationary phases (CSPs). Theoretical simulations including electronic circular dichroism (ECD) calculation and molecular docking were adopted to assign the stereochemistry and reveal the underlying chiral recognition mechanism. Our results showed that the sequence of calculated mean binding energies between each pair of enantiomers and CSP matched exactly with experimentally observed enantiomeric elution order (EEO). It was also found that the length of hydrogen bond might contribute dominantly the interaction between two enantiomers and CSP. We hope our study could provide a fresh perspective to explore the stereochemistry and chiral recognition mechanism of chiral drugs.

**Keywords:** chiral sulfoxide drugs; enantiomer elution order; electronic circular dichroism; quantum chemical calculation; molecular docking

#### **1. Introduction**

Chirality has a profound impact on the chemical processes of biology. Sulfur is an important bio-element, whose certain compounds can exhibit chirality such as sulfoxides, or sulfoximines. Although chiral sulfur compounds have received much less attention than chiral carbon compounds, chiral sulfoxides possessing a pyramidal structure comprise a unique class of drugs containing a chiral heteroatom [1]. The high enantioselectivity of biological events has prompted researchers to develop enantiopure sulfoxide drugs due to their distinct pharmacodynamic and pharmacokinetic properties [2,3]. In addition to its application in medicinal chemistry, sulfoxide is also significant functional group for its use as a ligand and chiral auxiliary in asymmetric synthesis [4,5].

Compounds **1**–**4** (Figure 1) are clinically widely-used sulfoxide-containing drugs, and represent two typical structures of sulfoxides, i.e., aromatic alkyl sulfoxides (**1**, **2**, **4**) and alkyl sulfoxide (**3**). Both **1** and **2** earned enough attention because of marvelous market success, and **3** is a wake-promoting drug used in the war [6–8]. Recently, the non-steroidal anti-inflammatory drug **4** and its derivatives aroused the interest of researchers for its potent anticancer effect [9,10].

In the last two decades, hundreds of papers reported the chiral separation methods of sulfoxides including capillary electrophoresis [11,12], chemical resolution [13,14], HPLC [15–18], and supercritical fluid chromatography [19,20]. Amongst all, we focused on chiral HPLC, which might be a favorable and effective way for both analytical and preparative separations of chiral compounds [21]. There are several HPLC methods using different types of CSPs for **1**, **2**, and 3 [7,16,22–30]. Relatively few studies have been reported on the synthesis and chiral enantioseparation of **4** [31–36]. Although the piecemeal chiral separation of these compounds was previously reported, a systematic and comprehensive study is beneficial to seek for general clues to the separation of chiral sulfoxide drugs.

**Figure 1.** Chemical structures of chiral sulfoxides.

Absolute configuration (AC) assignment is crucial for the stereochemical characterization of chiral compounds. For these four drugs, this was previously fulfilled by single-crystal X-ray diffraction, chemical synthesis, or comparison of circular dichroism spectra with their strongly correlatives [7,24,29,34,37–39]. However, it might be interesting and useful to establish directly their stereochemistry without reference samples and spectra. Presently, ECD spectra together with quantum chemical calculations using time-dependent density functional theory (TDDFT) have provided a feasible and reliable way to facilitate the AC assignments of chiral drugs and natural products [15,18,40–44]. Hence, TDDFT calculations will be adopted to simulate ECD spectra of sulfoxides in this study.

Recently, rapid development of binding energy computation software has revolutionized the study and elucidation of chiral separation mechanism, and binding energy computation could be applied to reproduce the elution orders of enantiomers [44,45]. In this work, we selected these four sulfoxide drugs to take a systematic experimental and theoretical investigation, for the aim of disclosing some potential regulation during the process of chiral separation and characterization of sulfoxides. Specially, chiral resolution of **1**–**4** will be tested on commercially available and cost-effective Chiralpak AD-H, AS-H, Chiralcel OD-H, and OJ-H chiral columns, followed by AC assignment and chiral separation mechanism analysis.

#### **2. Results and Discussion**

#### *2.1. Chiral Resolution of* **1***–***4**

#### 2.1.1. Factors Affecting Optical Resolution

The influence of the flow rate, mobile phase composition, column temperature, and acidic modifier on resolution was taken into consideration. With the increasing proportion of n-hexane (n-Hex) in the mobile phase, Rs increased in a non-linear manner (Figure S1A). It was manifested that Rs changed slightly with increasing temperature (Figure S1B) and steadily decreased when the flow rate increased from 0.5 to 1.0 mL/min (Figure S1C). Additionally, the column temperature can also affect the protonation of analytes and the CSP, which can then alter the chiral interactions between the solute and the stationary phase.

The great concern about the tailing phenomenon of acid compound **4** can be overcome using an acidic additive like formic acid (FA). Preliminary study showed that resolution of racemic **4** could only be attained on the Chiralpak AD-H column herein, and FA was then added to make a flagrant contrast. Fortunately, FA could achieve better separation of **4** through decreasing tailing, sharpening the peaks, and improving the selectivity (Table S1 and Figure S2). FA in the mobile phase might protonate both **4** and residual -NH- groups on CSP arising from the weak protonation capability of FA. Hence, non-enantioselective association between **4** and the CSP was partly inhibited. Subsequently, it improved the mass transfer, which greatly enhanced the chiral recognition ability of the stationary phase.

#### 2.1.2. The Optimal Resolution and Enantiomeric Elution Order

The separation conditions for compounds **1**–**4** on four chiral columns are listed in Table 1, and the optimal UV and ECD chromatograms are shown in Figure 2 and Figures S3–S6. The more satisfactory results of optimal resolution of racemic **1** on the Chiralpak AD-H column, **2** on the Chiralcel OD-H column, **3** on the Chiralpak AS-H column, and **4** on the Chiralpak AD-H column were 6.63, 3.48, 7.46, and 8.39, respectively. Most of the separations were obtained by using *n*-Hex/EtOH as the mobile phase. The different elution abilities between EtOH and IPA may be due to the polarity and capability to form hydrogen bonds with CSPs.


**Table 1.** Comparison of sulfoxide drugs on chiral columns.

Retention factor k = (t1 − t0)/t0, Resolution factor Rs = 2(t2 − t1)/(w1 + w2). t1, t2 is retention time of enantiomer. t0 is dead time. w1, w2 is peak width of enantiomer. Selectivity factor α = k2/k1 = (t2 − t0)/(t1 − t0)]. Detection wavelengths of **1**–**4** are 275, 275, 240, and 285 nm, respectively. <sup>a</sup> Flow rate: 1 mL/min. <sup>b</sup> Flow rate: 0.8 mL/min. <sup>c</sup> Column temperature: 30 ◦C. <sup>d</sup> Column temperature: 25 ◦C. <sup>e</sup> Sign of the absolute configuration of the first eluted enantiomer.

**Figure 2.** The UV (**a**) and ECD chromatograms (**b**) of racemic **1**–**4** on various columns. **1** on the ChiralPak AD-H column, **2** on the Chiralcel OD-H column, **3** on the ChiralPak AS-H column, and **4** on the ChiralPak AD-H column. Detection wavelengths of **1**–**4** are 275, 275, 240, and 285 nm, respectively.

It is an important issue to determine the EEO in chiral HPLC separations [46]. As demonstrated in Table 1 and Figure S7, the EEO on polysaccharide-based CSP was reversed (the details shown in Section 2.2) when the type of chiral columns or mobile phase composition were changed, which was consistent with the literature [24,47]. For compound **1**, the EEO changed when EtOH was replaced with IPA as the polar modifier on Chiralpak AD-H column (Figure S7). Similar situation was reported earlier for several chiral sulfoxides, showing that the change in the structure of chiral selector and composition of mobile phase might cause an opposite affinity pattern of enantiomers [16,23,24,39].

#### *2.2. AC Assignments of* **1***–***4**

#### 2.2.1. Experimental UV and ECD Spectra

It is significant to directly assign the AC of each peak in the HPLC chromatograms. Without available ECD spectra of enantiopure standard samples, it is essential to establish their AC using a reliable method. In this work, we fell back on ECD spectroscopy together with quantum chemical calculations, which has been widely applied in the AC assignments of chiral organic molecules.

Experimental ECD and UV spectra of all enantiomers of **1**–**4** in MeOH were obtained (Figure 3). The experimental UV spectra of aromatic alkyl sulfoxides (**1**, **2**) seem to be similar. The UV spectrum of **1** manifests two absorption bands at 205 nm and 300 nm, and that of **2** is slightly blue shifted with an absorption peak at 285 nm, but a shoulder peak at 222 nm. Only one shoulder peak at 225 nm appeared in the UV spectrum of alkyl sulfoxide **3**. For aromatic methyl sulfoxide **4**, there are two absorption bands at 290 nm and 330 nm in its UV spectrum.

It is well known that a pair of enantiomers would share the same UV spectra and have mirrored ECD spectra. Though aromatic alkyl sulfoxides (**1**, **2**, **4**) show the broad peak in the ECD spectra, all these ECD spectra possess an obvious couplet-like feature, which is common in sulfoxide-containing compounds [48]. In the ECD spectra of enantiomeric **1**, peak **1**a presented two negative Cotton effects (CEs) at 300 nm and 268 nm, followed by an intense positive CE at 232 nm, which could be regarded as two branches of a negative, non-degenerate couplet-like feature. Peak **2**a shows a strong positive CE at 270 nm and a negative CE at 222 nm in the ECD spectrum. The ECD spectrum of peak **3**a shows a positive couplet-like feature consisting of two bands, with the first positive CE being at 238 nm and the second negative peak at 216 nm. Nevertheless, peak **4**a gives a wide negative CE signal over a broad range of 235–375 nm, followed by an obvious positive CE peak at 217 nm.

**Figure 3.** Comparison of experimental and calculated ECD (top) and UV (bottom) spectra of the stereoisomers (**a**–**d**). Solid: experimental in methanol, Dashed: theoretical values, and UV-corrected, bandwidth of **1**–**4** is 0.20 eV, 0.30 eV, 0.30 eV, and 0.40 eV, respectively. The enantiopure isomers of **1**–**4** were prepared under the optimal separation condition of our study, on ChiralPak AD-H, Chiralcel OD-H, ChiralPak AS-H and AD-H columns, respectively. Calculated spectra are Boltzmann averages from calculated spectra of each single conformer.

#### 2.2.2. TDDFT Calculation of UV/ECD Spectra

To simulate UV and ECD spectra of **1**–**4**, it is vital to choose suitable combinations of hybrid functional and basis sets [49]. These calculation parameters might greatly affect TDDFT calculated spectra, thus leading to ambiguous AC judgment. Hence, four different hybrid functional/basis set combinations were adopted to verify the consistency of AC assignments. Geometry optimizations and frequency calculations were run at the B3LYP/6-31G(d) and B3LYP/6-31+G(d,p) levels. ECD spectra were predicted employing various combinations of B3LYP and Cam-B3LYP hybrid functionals and 6-31G(d) and 6-311+G(d,p) basis sets. Fortunately, the calculation did give an unambiguous answer to the AC assignment after UV correction if necessary (Figure S8).

Typically, both UV corrections and intensity scaling are applied when the calculated spectra is compared with an experimentally collected one. The best performing calculation method of sulfoxides **1**–**3** was the B3LYP/6-31G(d)//B3LYP/6-31G(d) basis set level, and that of **4** was B3LYP/6-311+G(d,p)//CAM- B3LYP/6-311+G(d,p).

The optimal match with the experimental spectra is shown in Figure 3**.** The first eluted enantiomer of **1** on AD-H, **3** on AS-H and **4** on AD-H were thus assigned as *S*, and **2** on OD-H was assigned as *R*. The agreement between the calculated and experimental ECD spectra of compounds **1**–**3** is almost perfect. The maximum absorption peak of the calculated UV spectra of **4** is at 370 nm, and the experimental UV spectra of **4** shows two absorption bands at 290 nm and 330 nm. In the ECD spectra of enantiomer of **4**, two separate CEs over the range of 240–350 nm are merged into one broad CE.

Moreover, the AC of each peak of **1**–**4** on different chiral columns was also assigned (). The AC judgments of **1**, **2**, and **4** obtained by the TDDFT calculation are consistent with the previously reported results [24,34].

#### 2.2.3. Electron Transitions

Chemical structures of both alkyl sulfoxides **1** and **2** include a substituted pyridyl ring, a benzimidazole ring, and a methyl sulfoxide group. In their ECD spectra, the CEs at 270 nm are associated with two electronic transitions from the sulfoxide chiral center to the benzimidazole ring and the pyridine ring itself. The CE at 230 nm is assignable to π→π\* transitions from π-type S=O orbital to the benzene ring and the charge transfer transition in the pyridine ring itself. Compared with **1**, the ECD spectrum of **2** was slightly blue-shifted, which was due to the electron-withdrawing effect of CF3 group.

The first CE of alkyl sulfoxide **3** at 238 nm resulted from benzene 1La transition, and the second band at 216 nm might be attributed to the sulfinyl group n-π\* transition. The oxygen lone pair and π\*-type S=O orbitals are heavily mixed with σ and σ\*-type S-C orbitals, respectively [50]. The large conjugated aromatic ring leads to a UV spectrum of aromatic methyl sulfoxide **4**, which is distinct from the obtained spectra of the other sulfoxide drugs. The wide absorption band may be assigned to the phenyl 1La and 1Lb transitions. The experimental ECD spectra of **4** appeared a broad peak in the range of 235–375 nm. The second CE of **4** at 220 nm may correspond to the sulfoxide-centered (O=S<) n→π\* transition.

#### *2.3. Chiral Separation Mechanism and Molecular Docking*

The chiral recognition mechanism of polymer-based CSPs is much more complex, because their chiral recognition usually depends on their higher-order structure and the steric fit of the analyte inside the chiral cavity [51,52]. The sulfinyl (>S=O) group, the carbonyl (C=O) group, -OH and -NH groups, the phenyl moiety, benzimidazole, and the pyridyl group in the structure of chiral sulfoxide drugs may form a hydrogen bond, a dipole–dipole bond, π-π interactions and hydrophobic interaction with C=O, -NH- group and the aromatic ring of CSPs. The π-electron density, steric hindrance, various conformations and spatial structures of the polysaccharide glucose unit on CSPs will affect the interaction between the CSPs and chiral molecules [53]. The different types and intensity of the interactions between the sulfoxide compounds and CSPs bring about different chiral separation phenomena. However, recent studies and real understanding of the recognition mechanisms of polysaccharide-based CSPs are far behind their practical applications.

#### 2.3.1. Common Structural Features

During the molecular docking process of **1**–**4**, 100 conformers of each enantiomer were generated and divided into different clusters (Figure S9). The common features of the most populated conformational cluster of **1**–**4** (Figure 4) were created to describe the structural characteristics and better explain the chiral separation mechanisms. There are three types of groups in **1** and **2** which may interact with the CSP, namely, hydrogen bond acceptors (HBA) O=S<, hydrogen bond donors (HBD) -NH-, and hydrophobic moieties (HY) -phenyl/-CF3. The features of **3** include HBA O=S<, HBD NH2 and aromatic ring (AR) phenyl. The features of **4** are HBA (O=S<), HY (-CH3), AR (phenyl) and negative ionizable (NI, -COOH). The apparent difference between the *R* and *S* enantiomer of **1**–**4** is the distance of pharmacophores shown in Figure 4. The differences of distance may influence the stability of the complexes during separation, resulting in different elution of enantiomers.

**Figure 4.** Pharmacophores of **1**–**4** conformers over the most populated cluster. HBA: green, HBD: purple, HY: light blue, AR: orange, NI: mazarine.

#### 2.3.2. Molecular Interactions between Analytes and CSPs

The interactions between the enantiomers of **1**–**4** and CSP of the Chiralpak AD-H column are shown in Figure S10. The ligand is depicted as sticks, surrounded by a molecular surface, which is colored according to the interaction with the CSP. Meanwhile, hydrogen bonds are shown as a string of small green spheres. Both enantiomers of **1**–**4** can form one hydrogen bond with the CSP of the Chiralpak AD-H column.

For the enantiomers of **1**, the methoxy group forms a hydrogen bond with an -NH- group of CSP. As for the enantiomers of **2**, one hydrogen bond exists between the O=S< and the amino group of CSP. The carbonyl of **3** interacts with amino moiety of the CSP through a hydrogen bond. There is a hydrogen bond existing between -COOH of **4** and -NH- of the CSP. For **1**, **2**, and **4**, the length of the hydrogen bond of the (*R*)-stereoisomer is shorter than that of the (*S*)-stereoisomer and this is in accordance with the elution sequence on the Chiralpak AD-H column. On the contrary, the length of the hydrogen bond of (*R*)-**3** (2.195 Å) is longer than that of (*S*)-**3** (2.09 Å), and the elution time of (*S*)-**3** is longer on the Chiralpak AD-H column. These results suggest that a shorter hydrogen bond may ensure the stability of the enantiomer in the stationary phase.

#### 2.3.3. Mean Binding Energy

As we know, the enantiomer with lower binding energy could bind more closely to the CSPs and will subsequently be eluted after the other enantiomer. Mean binding energy between the two enantiomers of **1**–**4** and the CSP of the Chiralpak AD-H column are listed in Table 2. The results are concurrent with the elution orders observed in the experiment.

It is obvious that the enantiomer with a shorter hydrogen bond is more stable than the other enantiomer during the process of elution. Consequently, hydrogen bonding plays a highly important role in the chromatographic separation of sulfoxides on Chiralpak AD-H column. Our result is consistent with high-resolution magic angle spinning nuclear magnetic resonance spectroscopy experiments that the molecular bases for the chiral recognition were the proton interactions between **1** and amylose-based CSP [54]. This also suggests that docking simulations could be adopted to reproduce the elution orders of the enantiomers by the mean binding energy calculations and explain the chiral separation mechanisms visually through the image. Since the mechanisms involved in chiral recognition are complex, a perfect molecular docking method is still a challenging task. We believe our results will aid the development of molecular docking in the application of chiral sulfoxide drugs separation on polysaccharide-based columns.


**Table 2.** The calculated average binding energies for compounds **1**–**4** on the ChiralPak AD-H column.

#### **3. Materials and Methods**

#### *3.1. Materials and Reagents*

Sulfoxides **1**–**4** were purchased from National Institutes for Food and Drug Control of China (Beijing, China). All solvents including n-hexane (n-Hex), iso-propyl alcohol (IPA), methanol (MeOH), and ethanol (EtOH) were of HPLC grade. Formic acid (FA) is used as an acidic additive.

#### *3.2. Chromatographic Conditions*

Compounds **1**–**4** were separated on a Jasco HPLC system consisting of a PU-2089 pump, an AS-2055 sampler, a CO-2060 column thermostat, a MD-2010 detector and a CD-2095 detector. All the chiral columns including Chiralpak AD-H, AS-H, Chiralcel OD-H, and OJ-H (Daicel Chemical Industries, Tokyo, Japan) were 250 × 4.6 mm i.d. with a 5 μm particle size. The detection wavelength of **1**–**4** was set at 275, 275, 240, and 285 nm, respectively. The chromatographic parameters for the enantioselectivity evaluation of the CSPs including retention factor (k), separation factor (α), and resolution factor (Rs) for the enantiomers were calculated.

The samples dissolved in EtOH: n-Hex (80:20, *v*/*v*) were formulated as the solution of 2 mg/mL for analysis and 5 mg/mL for preparation. The enantiopure isomers of **1**–**4** were prepared under the optimal separation condition of our study, on Chiralpak AD-H, Chiralcel OD-H, Chiralpak AS-H and AD-H columns, respectively. Also, the high enantiomeric purities (enantiomeric excesses >99%) of the isolated sulfoxide enantiomers were verified by the enantioselective HPLC system.

#### *3.3. ECD Experiments*

ECD analysis of the sulfoxide enantiomers was carried out in MeOH on a Jasco J-815 spectrometer. A quartz cuvette with a 1 mm path length was used. The detection wavelength was at 200–400 nm. The spectra were baseline-corrected against MeOH.

#### *3.4. Computational Methods*

#### 3.4.1. TDDFT Computations

All calculations have been performed on S configuration of **1**–**4**. Preliminary conformational analysis was carried out with the use of the MMFF94 molecular mechanics force field via the MOE software package (Chemical Computing Group, Montreal, QC, Canada) [55]. Geometry optimization and frequency calculation of the MMFF94 conformers were then performed at the B3LYP/6-31G(d) and B3LYP/6-311+G(d,p) levels by using Gaussian 09 (Gaussian, Wallingford, CT, USA) [56]. ECD spectra were predicted employing various combinations of B3LYP or Cam-B3LYP hybrid functionals and 6-31G(d), 6-311+G(d,p) basis sets. All calculations were conducted with the PCM solvation model for MeOH. Calculated UV and ECD spectra of each conformer were simulated at the bandwidth of 0.20–0.40 eV, and the overall spectra were obtained according to the Boltzmann weighting of all conformers. Theoretical ECD and UV spectra were blue-shifted to facilitate the comparison with experimental data if necessary.

#### 3.4.2. Molecular Docking

The 3D-polymer structure of Chiralpak AD-H amylose derivative CSP (AD-12mer.pdb) was downloaded from http://pubs.acs.org [57]. The structure of chiral selector is composed of two AD-12mer.pdb molecules to form "tube-mode" [44]. The structure of AD-H was minimized by means of R2 Dreiding force field using Discovery Studio 2017 software. The structures of the enantiomers of **1**–**4** were minimized in CHARMM force field. AutoDock 4.2.6 (Scripps Research Institute, La Jolla, CA, USA) [58] was adopted to simulate molecular docking. During the docking process, the grid box was set to 30 × 30 × 30 (Å) with 0.375 Å spacing for **1**, **3**, and **4**, 40 × 40 × 40 (Å) with 0.375 Å spacing for **2**.

In consideration of the solvent effect of the mobile phase, the dielectric constant of **1**–**4** was respectively set as 10.668, 5.248, 6.124, and 6.124 based on the weighted average of the mixed mobile phase on the AD-H chiral column. Lamarckian genetic algorithm was used to generate 100 conformations.

#### **4. Conclusions**

Herein, a systematic study including chromatographic resolution, stereochemical assignment and chiral recognition mechanism of four chiral sulfoxide drugs (**1**–**4**) is conducted. All four compounds have been completely separated on a Chiralpak AD-H column (Figure S11). Among the tested conditions, n-Hex/EtOH as mobile phase is shown to be favorable for the separation of sulfoxides. EEO inversions were observed when the types of chiral columns or mobile phase composition changed. In this work, comparison of the ECD spectra with the TDDFT calculated data provided a robust way to assign the AC of their stereoisomers. Moreover, the observed EEOs were found to be in accord with mean binding energy calculations. The docking simulation could also explain visually the underlying chiral separation mechanisms. This work has the potential to build up an experimental and theoretical methodology to facilitate the stereochemistry and chiral recognition mechanism of chiral drugs.

**Supplementary Materials:** The following materials are available online. (1) Figure S1. Plots showing resolution factors of the enantiomers of **1**–**3** as a function of the n-Hex content in the mobile phase (A), temperature (B) and flow rate (C). (2) Table S1. Effect of the acidic additive on the resolution of **4** on the AD-H column. (3) Figure S2. Comparison of the UV (upper) and ECD (lower) chromatograms of **4** on ChiralPak AD-H column with acidic additive. (4) Figure S3. The UV (upper) and ECD (lower) chromatograms of **1** on ChiralPak AD-H column under the optimal condition. (5) Figure S4. The UV (upper) and ECD (lower) chromatograms of **2** on Chiralcel OD-H column under the optimal condition. (6) Figure S5. The UV (upper) and ECD (lower) chromatograms of **3** on ChiralPak AS-H column under the optimal condition. (7) Figure S6. The UV (upper) and ECD (lower) chromatograms of **4** on ChiralPak AD-H column under the optimal condition. (8) Figure S7. Comparison of the UV (upper) and ECD (lower) chromatograms of **1** on ChiralPak AD-H column with EtOH and IPA. (9) Figure S8. Conformational distribution of enantiomers **1**–**4** during the docking process. (10) Figure S9. Comparison of TDDFT-calculated ECD and UV spectra. (11) Figure S10. Interactions between two enantiomers of **1**–**4** and the CSP of the AD-H column. (12) Figure S11. Graphic illustrating the resolution of chiral sulfoxides on the chiral columns.

**Author Contributions:** Conceptualization, L.L.; Methodology, B.-B.Y.; Investigation, F.X., J.Z., and B.-B.Y.; Resources, L.L.; Funding acquisition, L.L.; Writing-Original Draft Preparation, F.X.; Writing-Review & Editing, L.L.

**Funding:** This study was financially supported by the CAMS Innovation Fund for Medical Sciences (CIFMS, No. 2016-I2M-3-009).

**Conflicts of Interest:** The authors have declared no conflict of interest.

#### **References**


**Sample Availability:** Samples of the enantiopure isomers of compounds **1**–**4** are available from the authors.

© 2018 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 (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Enantioselective Drug Recognition by Drug Transporters**

#### **Yuichi Uwai**

School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650, Japan; yuuwai@dpc.agu.ac.jp; Tel.: +81-52-757-6785

Academic Editor: Maria Elizabeth Tiritan Received: 25 October 2018; Accepted: 22 November 2018; Published: 22 November 2018

**Abstract:** Drug transporters mediate the absorption, tissue distribution, and excretion of drugs. The cDNAs of P-glycoprotein, multidrug resistance proteins (MRPs/ABCC), breast cancer resistance protein (BCRP/ABCG2), peptide transporters (PEPTs/SLC15), proton-coupled folate transporters (PCFT/SLC46A1), organic anion transporting polypeptides (OATPs/SLCO), organic anion transporters (OATs/SLC22), organic cation transporters (OCTs/SLC22), and multidrug and toxin extrusions (MATEs/SLC47) have been isolated, and their functions have been elucidated. Enantioselectivity has been demonstrated in the pharmacokinetics and efficacy of drugs, and is important for elucidating the relationship with recognition of drugs by drug transporters from a chiral aspect. Enantioselectivity in the transport of drugs by drug transporters and the inhibitory effects of drugs on drug transporters has been summarized in this review.

**Keywords:** drug transporter; enantioselectivity; transport; inhibition; pharmacokinetics

#### **1. Introduction**

Many drugs are chiral, and each enantiomer may exhibit specific therapeutic efficacy. For example, several nonsteroidal antiinflammatory drugs (NSAIDs) have an asymmetric carbon in their chemical structures, and the (*S*)-enantiomers exhibit stronger inhibitory potencies against cyclooxygenases [1]. The (*S*)-enantiomer of naproxen is commercially available. We examined the enantioselective effects of flurbiprofen on the disposition of lithium in rats, and demonstrated that the (*S*)-enantiomer, but not the (*R*)-enantiomer, decreased the renal clearance of lithium with the impairment of renal function [2]. The anticoagulant drug warfarin is also chiral. Warfarin is orally administered as the racemate and exhibits enantioselectivity not only in its pharmacological effects, but also in its pharmacokinetics. (*S*)-Warfarin has greater anticoagulant potency than the (*R*)-enantiomer [3]. (*S*)-Warfarin is metabolized by cytochrome P450 (CYP) 2C9, and the metabolism of the (*R*)-enantiomer is mediated by CYP1A2 and CYP3A4 [4]. The development of drugs with enantioselective pharmacokinetic properties, such as esomeprazole, has recently been accomplished. This proton pump inhibitor is the (*S*)-enantiomer of omeprazole, which is the racemate. Both enantiomers exert identical effects on H+, K+-ATPase [5], but exhibit different pharmacokinetic characteristics. The clearance of esomeprazole is lower than that of the (*R*)-enantiomer [6]. Furthermore, the metabolism of the (*R*)-enantiomer is mainly mediated by CYP2C19, which exhibits a genetic polymorphism, and the contribution of CYP2C19 to the metabolism of esomeprazole is negligible [6].

In 1988, CYP2D6 was the first cytochrome P450 enzyme whose cDNA was identified [7]. The cDNAs of organic ion transporters have been identified since the 1990s. These findings promoted research on the function and expression of drug transporters. Drug transporters are expressed in the plasma membranes of a number of cells including enterocytes, hepatocytes, brain microvascular endothelial cells, and renal epithelial cells, and facilitate the transport of drugs across the plasma membrane. Drug transporters are now known to play important roles in the absorption, tissue distribution, and excretion of drugs. Previous studies reported enantioselective drug transport by drug transporters. Furthermore, the enantioselective inhibitory effects of drugs on drug transporters has been demonstrated.

Representative drug transporters functioning in the intestinal absorption, biliary excretion, and renal tubular secretion of drugs have been summarized in this review. The interactions between chiral drugs and drug transporters have also been discussed. Figure 1 shows the chemical structures of the chiral drugs described herein.

**Figure 1.** Chemical structures of chiral drugs described in this review.

#### **2. Role of Drug Transporters in the Intestinal Absorption, Biliary Excretion, and Renal Tubular Secretion of Drugs**

#### *2.1. Intestinal Drug Absorption by Drug Transporters*

Drugs are absorbed by the small intestine after their oral administration. Figure 2 shows drug transporters that are involved in the intestinal absorption of drugs. Peptide transporter PEPT1 (SLC15A1) is one of the most extensively studied drug transporters in the small intestine. It is expressed in the brush-border membrane of intestinal epithelial cells and is responsible for the uptake of dipeptides and tripeptides from the lumen using an inward H+-electrochemical gradient [8]. PEPT1 recognizes peptide-like β-lactam antibiotics that are orally administered [8]. PEPT1 is used as a target molecule for improving the absorption of poorly absorbed drugs. Valganciclovir, the valine ester prodrug of ganciclovir, was developed to enhance the low bioavailability of ganciclovir, and the mechanism responsible for improved absorption was identified as drug recognition by PEPT1 [9].

The proton-coupled folate transporter PCFT (SLC46A1) is localized to the brush-border membrane of enterocytes, and mediates the absorption of folate [10]. PCFT recognizes the antifolates, methotrexate and aminopterin, as its substrates [11,12]. Aminopterin has not yet been approved as a medicine, and methotrexate is used in the treatment of neoplasia and rheumatoid arthritis. PCFT is considered to play a role in the intestinal absorption of orally administered methotrexate [13].

The organic anion transporting polypeptide OATP2B1 (SLCO2B1), which is expressed in the brush-border membrane of intestinal epithelial cells, is crucially involved in the uptake of fexofenadine, celiprolol, and montelukast from the lumen [14,15].

P-glycoprotein (P-gp; ABCB1) and the breast cancer resistance protein BCRP (ABCG2) are ATP-binding cassette (ABC) transporter proteins that are expressed in the brush-border membrane of enterocytes. These transporters are efflux pumps. P-gp transports various types of drugs, including anticancer agents, antihypertensive agents, antiarrhythmics, antidepressants, antimicrobial agents, anti-human immunodeficiency virus agents, anticonvulsants, antiemetics, immunosuppressants, neuroleptics, and opioids, and there is a broad range of overlapping substrate specificities for CYP3A4 and P-gp [16]. BCRP has also been shown to transport anticancer agents [17]. The tyrosine kinase inhibitors, imatinib, gefitinib, and nilotinib, have also been identified as substrates of BCRP [18–20]. P-gp and BCRP prevent drug absorption in the intestine.

**Figure 2.** Drug transporters in the small intestine.

In the basolateral membrane, the organic cation transporter OCT1 (SLC22A1) and the multidrug resistance protein MRP3 (ABCC3) mediate drug transport [21]. In OCT1 knockout mice, the intestinal excretion of the typical substrate, tetraethylammonium, was reduced [22]. The serosal efflux of the glucuronide conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38: the active metabolite of irinotecan) and acetaminophen in the jejunal everted sacs was decreased in Mrp3 knockout mice [23].

#### *2.2. Hepatic Transport of Drugs by Drug Transporters*

Drug transporters in the plasma membrane of hepatocytes are shown in Figure 3. The OATP family members OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) mediate the hepatic uptake of various drugs, such as HMG-CoA reductase inhibitors, angiotensin II receptor antagonists, nateglinide, asunaprevir, pemafibrate, and bosentan, from the circulation [14,24]. OCT1 functions in the sinusoidal uptake of organic cations, including metformin, tropisetron, sumatriptan and fenoterol, from the circulation [25–28]. P-gp, MRP2 (ABCC2), BCRP, and multidrug and toxin extrusion MATE1 (SLC47A1), which are expressed in the canalicular membrane, mediate the efflux of drugs into bile [24]. MRP2, a member of the ABC family of transporters, excretes monoglucuronosyl bilirubin and monoglucuronosyl bilirubin into bile, and genetic mutations in MRP2 cause Dubin-Johnson syndrome, an autosomal recessive disease characterized by conjugated hyperbilirubinemia [29]. MRP3 is localized to the sinusoidal membrane of hepatocytes [30]. Kitamura et al. reported decreased plasma levels and increased clearance for the biliary excretion of methotrexate in MRP3 knockout mice, suggesting that MRP3 transports methotrexate from hepatocytes into plasma [31].

**Figure 3.** Drug transporters in the liver.

#### *2.3. Renal Tubular Secretion of Drugs*

The renal tubular secretion of drugs is mediated by drug transporters, as shown in Figure 4. Organic anion and cation transport systems are present in renal proximal epithelial cells. The organic anion transporter system is involved in the tubular secretion of anionic drugs, including anticancer agents, β-lactam antibiotics, antivirals, and diuretics [24,32]. The organic anion transporters OAT1 (SLC22A6) and OAT3 (SLC22A8) are responsible for the basolateral uptake of anionic drugs from the circulation via an exchange with intracellular α-ketoglutarate, an intermediate in the Krebs cycle. Information on the drug transporters responsible for mediating the efflux of drugs via the brush-border membrane of proximal epithelial cells is more limited than that on basolateral transporters. Studies using rat renal brush-border membrane vesicles indicated that a potential-sensitive organic anion transporter and an anion/organic anion exchange transporter function [33–35]. However, their genes have not yet been identified. Previous studies reported reductions in the urinary excretion of adefovir, tenofovir, hydrochlorothiazide, furosemide, ceftizoxime, and cefazolin in MRP4 knockout mice [36–38]. Based on these findings, MRP4 has been suggested to play an important role in drug transport in the brush-border membrane. MRP2 and OAT4 are known to be expressed in the brush-border membrane of proximal epithelial cells [39,40]. Although their interactions with drugs have been investigated in in vitro experiments, the roles of MRP2 and OAT4 in the tubular transport of drugs currently remain unclear.

The organic cation transport system consists of the uptake type of OCT2 (SLC22A2) and the efflux modes of MATE1 and MATE2-K (SLC47A2) [24,32,41]. Drug transport by OCT2 is electrogenic, and is driven by an internal negative membrane potential. The efflux of drugs by MATE1 and MATE2-K is dominated by a H+/organic cation antiport, which involves electroneutral transport [41]. Cationic drugs, including cimetidine, metformin, procainamide, memantine, and amantadine, are secreted into urine by the organic cation transport system [32].

P-gp functions in the renal tubular secretion of the cardiac glycoside, digoxin [42]. Although a wide variety of drugs are transported by P-gp [16], most are not recovered into urine. The substrate drugs of P-gp may be reabsorbed by the distal tubules via simple diffusion after tubular secretion.

**Figure 4.** Drug transporters mediating renal tubular secretion drugs.

#### **3. Enantioselective Drug Transport by Drug Transporters**

#### *3.1. Enantioselective Transport of Antifolates by PCFT*

Previous studies reported enantioselectivity in drug transport by drug transporters, with PCFT as a representative. The antifolates methotrexate and aminopterin have an asymmetric carbon in their structures (Figure 1), and their enantioselective transport was examined. Narawa et al. constructed stably transfected human embryonic kidney cells 293 (HEK293 cells) expressing PCFT, and conducted a methotrexate uptake experiment using these cells [43]. Menter et al. examined aminopterin transport using Chinese hamster ovary (CHO) cells expressing PCFT [12]. The transport of both antifolates showed enantioselectivity, and kinetic parameters were summarized in Table 1. The (*S*)-enantiomers were found to have greater affinity to PCFT than each (*R*)-enantiomer. Menter et al. performed a pharmacokinetic study on aminopterin in dogs and patients with psoriasis, and the findings obtained revealed a correlation with enantioselective absorption and in vitro findings [12].

**Table 1.** Kinetic parameters of the PCFT-mediated transport of methotrexate and aminopterin.


#### *3.2. Enantioselectivity in the Pharmacokinetics of Fexofenadine and Its Transport by OATP2B1*

Fexofenadine, a histamine H1-receptor antagonist, has non-sedative properties that have been attributed to the restriction of its brain penetration by P-gp [44]. Enantioselectivity has been reported in its pharmacokinetics. Miura et al. demonstrated that the maximum plasma concentration and area under the plasma concentration-time curve of (*R*)-fexofenadine were higher than those of the (*S*)-enantiomer after the single oral administration of racemic fexofenadine to healthy volunteers [45]. Sakugawa et al. examined effect of verapamil, an inhibitor of P-gp, on the disposition of each enantiomer of fexofenadine in healthy volunteers, and suggested that the other mechanisms in addition to P-gp contribute to the stereoselective pharmacokinetics of fexofenadine [46]. To my knowledge, there is no reports representative of enantioselective transport of fexofenadine by P-gp from in vitro experiments. Most of the dosage of fexofenadine administered is excreted into urine in its unmetabolized form, and various drug transporters have been shown to contribute to its pharmacokinetics. The OATP family members, OATP1A2, OATP1B1, OATP1B3, and OATP2B1, were proposed to be responsible for the intestinal uptake of fexofenadine or its distribution into the liver [47–52]. MRP2 and the bile salt export pump BSEP (ABCB11) mediate the efflux of fexofenadine from hepatocytes into bile, while MRP3 transports it into plasma [52,53]. OAT3 plays a role in the renal tubular uptake of fexofenadine, and MATE1 contributes to its efflux into urine [54,55]. At least one of the drug transporters described above may be responsible for the enantioselective pharmacokinetics of fexofenadine. The enantioselective transport of fexofenadine has only been reported to occur by OATP2B1. Akamine et al. found greater uptake amounts of (*R*)-fexofenadine in *Xenopus* oocytes injected with OATP2B1 cRNA than that of the (*S*)-enantiomer [56]. They also demonstrated that apple juice decreased the absorption of fexofenadine orally administered to healthy volunteers, and that the juice inhibited its transport by OATP2B1 [56]. Accordingly, OATP2B1 appears to mediate the enantioselective absorption of fexofenadine by the small intestine. Akamine et al. did not describe the kinetic parameters of the transport of enantiomers by OATP2B1. The renal clearance of (*S*)-fexofenadine was higher than that of the (*R*)-enantiomer [45,56], whereas Kusuhara et al. reported no enantioselective transport of fexofenadine by OAT3 and MATE1 [57]. In addition, they showed the similar transport of both enantiomers by OATP1B3 [57]. Unidentified drug transporter(s) may be responsible for the enantioselective disposition of fexofenadine.

#### *3.3. Enantioselective Secretion of Pantoprazole into Milk by BCRP*

Enantioselective drug transport was demonstrated with the combination of pantoprazole and BCRP. BCRP affects the absorption, distribution, and excretion of drugs, and BCRP actively secretes xenobiotics, including drugs and carcinogens, and riboflavin into milk [58–60]. Wang et al. showed the greater accumulation of (−)-pantoprazole in the milk of lactating rats infused with racemic pantoprazole than that of (+)-pantoprazole [61], and the higher affinity of the (−)-enantiomer with BCRP [62].

#### **4. Enantioselective Inhibitory Effects of Drugs on Drug Transporters**

#### *4.1. Enantioselectivity in Inhibitory Effects of Drugs on OCT1, and Binding Affinities*

Enantioselectivity was shown in the inhibitory effect of disopyramide and propranolol on OCT1. The (*R*)-disopyramide inhibited the uptake of tetraethylammonium by HeLa cells expressing OCT1 more strongly than the (*S*)-enantiomer [63], and also for propanol the inhibitory effect of (*S*)-enantiomer is stronger than for the (*R*)-enantiomer [64]. The IC50 values of each enantiomer were described in Table 2.

**Table 2.** IC50 values of each enantiomer of disopyramide and propranolol for OCT1.


Moaddel et al. studied the binding of drugs to OCT1 with a liquid chromatography stationary phase containing immobilized membranes obtained from a cell line that expresses OCT1, and estimated

their binding affinities using frontal displacement chromatography with [3H]1-methyl 4-phenyl pyridinium as the marker ligand. The significant enantioselectivity on the binding to OCT1 was recognized in verapamil, atenolol, and propranolol [64,65]. (*R*)-Verapamil, (*S*)-atenolol, and (*S*)-propranolol showed the higher affinities than each enantiomer. In Table 3, their Kd values are summarized.


**Table 3.** Kd values of each enantiomer of verapamil, atenolol, and propranolol for OCT1.

#### *4.2. Enantioselective Inhibitory Effects of NSAIDs and Lansoprazole on OAT1 and OAT3*

The inhibition of renal organic anion transporters leads to the delayed elimination of their substrates from the circulation. NSAIDs interfere with the renal excretion of methotrexate, and this combination is a representative among drug interactions via OAT1 and OAT3. The interaction is fatal when high-dose methotrexate therapy is given to a patient [66,67]. Previous studies demonstrated the inhibitory effects of NSAIDs, including cyclooxygenase-2 inhibitors, on methotrexate uptake by OAT1 and OAT3 [68–72]. Some NSAIDs are chiral (Figure 1). We conducted a drug transport experiment using the *Xenopus* oocyte expression system in order to examine enantioselectivity in the inhibition of OAT1 and OAT3 by NSAIDs [73]. The findings obtained showed the stronger inhibitory effects of the (*S*)-enantiomers of flurbiprofen, ibuprofen, and naproxen on the transport of *p*-aminohippurate and methotrexate by OAT1 than that of each (*R*)-enantiomer. The inhibitory mechanisms of flurbiprofen were investigated, and both enantiomers were found to competitively inhibit OAT1. Enantioselective differences were not observed in the inhibition of OAT3.

Proton pump inhibitors also interact with methotrexate [74,75]; OAT1 and OAT3 were found to be inhibited by omeprazole, lansoprazole, pantoprazole, rabeprazole, and esomeprazole [76,77]. Chirality exists in the structures of proton pump inhibitors (Figure 1), and enantioselectivity was examined in the inhibition of OAT1 and OAT3 by lansoprazole [78]. The inhibitory effects of (*S*)-lansoprazole on the transport of estrone sulfate, methotrexate, and pemetrexed by OAT3 were stronger than those of the (*R*)-enantiomer. Furthermore, enantioselectivity was not recognized against OAT1. Enantioselectivity has been demonstrated in the pharmacokinetics of lansoprazole. The slower elimination of the (*R*)-enantiomer from plasma has been reported in healthy subjects [79]. The faster metabolism of (*S*)-lansoprazole by human liver microsomes was also shown [80]. This information is useful when considering drug interactions between lansoprazole and the substrate drugs of OAT3.

#### *4.3. Enantioselective Inhibitory Effects of NSAIDs on MRP2 and MRP4*

The enantioselective inhibitory effects of NSAIDs on MRP2 and MRP4 have been examined. Kawase et al. performed uptake experiments on methotrexate using MRP2- and MRP4-expressing inside-out vesicles, and showed the stronger inhibition of MRP2 by the (*S*)-enantiomers of flurbiprofen, ibuprofen, and naproxen than by each (*R*)-enantiomer [81]. In the case of MRP4, the (*R*)-enantiomers exerted strong inhibitory effects.

Table 4 summarizes the IC50 values of each enantiomer of flurbiprofen, ibuprofen, naproxen, and lansoprazole described above. The most prominent enantioselective difference was noted in the inhibition of MRP2 by naproxen.



#### **5. Conclusions**

Enantioselectivity has been demonstrated in the transport of methotrexate and aminopterin by PCFT, pantoprazole by BCRP, and fexofenadine by OATP2B1. Enantioselective differences were reported in the inhibitory effects of flurbiprofen, ibuprofen, and naproxen on OAT1, MRP2, and MRP4, and of lansoprazole on OAT3. The number of studies on enantioselective drug recognition by drug transporters is markedly smaller than those on drug metabolism enzymes. In research on enantioselectivity, data on drug metabolism accumulate, and Niwa et al. performed meta-analysis based on the reported values regarding the Michaelis-Menten constant, maximal velocity, intrinsic clearance, and inhibition constants [82]. They considered that there is a limited number of reports regarding stereoselective inhibition and induction in vitro [82]. Because drug transporters are also involved in drug interactions, it is desired to pay attention to in enantioselectivity in inhibition as well as in substrate recognition of drug transporters.

Computational methods such as quantitative structure-activity relationship (QSAR) and pharmacophore approaches have become more widely applied to assess interactions between drugs and drug transporters and predictions for substrates and inhibitors for several transporters were described [83]. Because ignoring stereoselectivity reduces the accuracy of the QSAR and modelling analysis, stereoselectivity should become a key aspect of the modelling of interactions between drugs and drug transporters [84].

In the future, research on drug transporters from the chiral aspect of drugs will provide important insights into pharmacokinetics, pharmacodynamics, and drug toxicity.

**Author Contributions:** Y.U. conceived the study, reviewed the literature, and wrote the manuscript.

**Funding:** This work was supported by a grant from the Japan Society for the Promotion of Science through a Grant-in-Aid for Scientific Research (KAKENHI, 16K08419 to Y.U.).

**Conflicts of Interest:** The author states that there are no conflicts of interest.

#### **References**


© 2018 by the author. 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 (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Enantiomeric Recognition and Separation by Chiral Nanoparticles**

**Ankur Gogoi 1,† , Nirmal Mazumder 2,†, Surajit Konwer 3, Harsh Ranawat <sup>2</sup> , Nai-Tzu Chen <sup>4</sup> and Guan-Yu Zhuo 4,5,\***


Academic Editors: Maria Elizabeth Tiritan, Madalena Pinto and Carla Sofia Garcia Fernandes Received: 3 February 2019; Accepted: 10 March 2019; Published: 13 March 2019

**Abstract:** Chiral molecules are stereoselective with regard to specific biological functions. Enantiomers differ considerably in their physiological reactions with the human body. Safeguarding the quality and safety of drugs requires an efficient analytical platform by which to selectively probe chiral compounds to ensure the extraction of single enantiomers. Asymmetric synthesis is a mature approach to the production of single enantiomers; however, it is poorly suited to mass production and allows for only specific enantioselective reactions. Furthermore, it is too expensive and time-consuming for the evaluation of therapeutic drugs in the early stages of development. These limitations have prompted the development of surface-modified nanoparticles using amino acids, chiral organic ligands, or functional groups as chiral selectors applicable to a racemic mixture of chiral molecules. The fact that these combinations can be optimized in terms of sensitivity, specificity, and enantioselectivity makes them ideal for enantiomeric recognition and separation. In chiral resolution, molecules bond selectively to particle surfaces according to homochiral interactions, whereupon an enantiopure compound is extracted from the solution through a simple filtration process. In this review article, we discuss the fabrication of chiral nanoparticles and look at the ways their distinctive surface properties have been adopted in enantiomeric recognition and separation.

**Keywords:** chirality; racemic mixture; enantiomer; enantiomeric recognition; enantiomeric separation; surface-modified nanoparticle; chiral ligand

#### **1. Introduction**

Chirality is one of those fundamental properties of molecular systems which are ubiquitous in nature [1,2]. Notably, a number of important biological compounds including proteins, amino acids, nucleosides, sugars, and a number of hormones, which are the basic building blocks of life, are chiral and thus the chemistry of many fundamental biological (metabolic and regulatory) processes are directly controlled by such molecular systems [3,4]. Interestingly, enantiomers are mirror images of a chiral compound, e.g., chiral drugs, pesticides, herbicides, etc., which often exhibit profoundly different metabolic, pharmacological, therapeutic and/or toxicological properties [5]. Consequently, the two enantiomers of a chiral compound will react differently with the complementary receptor or

enzyme molecule in a biological environment which is highly stereoselective or enantioselective [6]. This issue is particularly important for the pharmaceutical industry where many drugs currently in use are known to be chiral (about 56%) [4] out of which only about 25% are pure enantiomers. Besides, chirality is also important for other industries like agrochemical, food, petroleum, etc. [7–9]. For example, approximately 30–40% of the currently registered pesticides, insecticides and herbicides are chiral [6,8,10]. Most of these currently used chiral compounds are racemates or mixtures [11]. Importantly, while one enantiomer of a chiral drug or agrochemical product has desired effect (eutomer), the other may be inactive and/or cause adverse side effects in many cases (distomer) [3,6].

Given the tremendous importance of chirality in many biochemical processes, recent years witnessed enormous efforts among the researchers and manufacturing industries to prepare enantiopure compounds so that an administered chiral compound (drug or other chemical compounds) fits properly to the target binding site or receptor molecule. In this regard, the ideal solution would be the enantioselective synthesis of just one of the enantiomers (synthetic or chiral approach [9,12,13]) by using methods like isolation of natural compounds [14,15], fermentation [16], asymmetric synthesis [17], etc. Notably, the Nobel Prize in Chemistry for 2001 was awarded for the development of catalytic asymmetric synthesis with one half jointly to Knowles and Noyori "for their work on chirally catalyzed hydrogenation reactions" and the other half to Sharpless "for his work on chirally catalyzed oxidation reactions" [18]. Unfortunately very few enantiopure compounds can be obtained from natural sources. On the other hand, the use of asymmetric synthesis is limited due to the high catalyst cost and time-consuming synthesis procedure, albeit it is one of the most powerful methods to produce 100% enantiopure compounds [12,19,20].

As compared to chiral approach, the racemic approach involves recognition of racemates or mixtures and subsequent separation of the enantiomers, which is relatively cost-effective and presents lower level of difficulty [20]. This approach is based on a three-point interaction between the analyte and a chiral selector [12,21–23]. Briefly, a matched pair undergoes a three-point interaction whereas only one or two point interaction takes place in case of a mismatched pair, as shown in Figure 1. A number of chiral selectors, including proteins, cyclodextrin (CD), crown ethers, oligo- and polysaccharides, etc., have been developed till date. Unfortunately there is no universal chiral selector that can be efficiently applied for the recognition and/or separation of all types of chiral compounds [24].

**Figure 1.** Three-point interaction model for describing the homochiral (true) and heterochiral (false) interactions.

A number of techniques have been developed during the recent past for recognition and/or separation of enantiomers obtained by using the racemic approach [11,25]. Most commonly used methods include chromatographic methods such as high-performance liquid chromatography (HPLC), liquid chromatography (LC), gas chromatography (GC), capillary electrochromatography (CEC), thin-layer chromatography (TLC), micellar chromatography (MC), supercritical fluid chromatography (SFC), and high-speed countercurrent chromatography (HSCCC) [26–36]. Other methods are capillary electrophoresis (CE) [29,30,37], crystallization resolution [38,39], liquid-liquid extraction (LLE) [12,40], membrane separation [41–43], kinetic resolution [13], etc. On the other hand, self-disproportionation of enantiomers (SDE) introduced by Soloshonok is another process which efficiently transforms an enantiomerically enriched compound (scalemic mixture, i.e., a mixture of enantiomers at a ratio other than 50:50 or 100:0) into completely racemic (enantio-depleted) and enantiomerically pure (enantioenriched) fractions [44–47]. Importantly, SDE occurs spontaneously whenever nonracemic compounds are subjected to any physiochemical processes, such as, precipitation, centrifugation, recrystallization, sublimation, force field, achiral chromatography, etc., under totally achiral conditions [48].

Each of these methods has their unique abilities for enantiomeric recognition, separation and quantification. Nevertheless, despite the availability of such advanced chiral resolution methods, it is still very difficult to separate the enantiomers with high efficiency because of the intrinsic difficulties associated with these methods, mainly due to the exactly same physiochemical properties of the enantiomer pairs [24,49,50]. In addition, most of them require using chiral columns in place of stationary phase and such processes are often expensive and cumbersome, and do not allow real time analysis. The advantages and limitations of the aforementioned methods that are widely used for chiral resolution are tabulated in Table 1.

**Table 1.** Comparison of currently existing chiral resolution methods. The table and the caption have been adapted with permission from [50], Copyright © 2008 The Royal Society of Chemistry. Note that the original Table is slightly modified and the method "Self-disproportionation of enantiomers" has been added.


*<sup>a</sup>* Note: Advantages of (c1) and (c2) were obtained by comparing with high performance liquid chromatography.

Nanoparticles (NPs) represent an entirely new approach to chiral resolution. At present, this generally involves surface modification using chiral ligands; however, recent advances are making the recognition and separation of enantiomers far simpler. One particular achievement in enantiomeric recognition is colorimetric detection, which uses surface-modified NPs to convert recognition events into color changes observable to the naked eye or a UV-Vis spectrometer [52–62]. This makes it ideal for on-site chiral analysis and provides results instantaneously. The working principle is that interactions with specific enantiomers occur on the surface of metal NPs, which means that interactions can be monitored according to changes in surface plasmon resonance (SPR). Furthermore, chiral ligand-capped quantum dots (QDs) have also received considerable attention in this field, due to the size-dependent optical properties, bright chemiluminescence, and excellent chemical stability [63–65]. Beside the applications of magnetic nanoparticles (MNPs) such as catalysts, targeted administration and magnetic resonance imaging (MRI) [66,67], researchers have even capped the surfaces of MNPs with chiral ligands to promote specific interactions with the target enantiomers. It may find potential use in the design of new magneto-chiroptical devices [68]. A wide variety of chiral NPs have been developed for enantiomeric recognition, many of which are examined in Section 2. In enantiomeric separation, surface-modified NPs, as chiral selectors, are exposed to a racemic mixture of chiral molecules to perform the selective adsorption of one enantiomer, leaving an excess of the other enantiomer in solution to be removed through multiple rounds of centrifugation. Following centrifugation, the target enantiomers co-precipitate with the chiral NPs, whereas their enantiomeric counterparts remain in the supernatant [52,53,69]. Note that the chiral ligands chosen for enantiomeric separation are susceptible to denaturation and renaturation manipulated by external perturbations such as temperature and magnetic field, making them switchable and reusable. The used nanomaterials and chiral ligands are discussed in Section 3.

#### **2. Enantiomeric Recognition by Chiral Nanoparticles**

The ability to recognize the molecular chirality of enantiomers is significantly important owing to their critical role in drug development and biochemistry. Current discrimination of enantiomers has remained a challenge due to lack of efficient methods. NP-based enantiomeric recognition and separation have been widely discussed and studied in the past decade. Studies reported chiral -modified nanomaterials for catalysis [70,71], chiral drug separation [71] and sensing [54–60,63,68,72–74]. In this section, we will focus on the enantiomeric recognition and the fabrication of most commonly used gold and silver materials for chiral NPs.

#### *2.1. Gold-Based Nanomaterials*

Gold-based nanomaterials are the most widely used material for chiral NPs due to its unique optical properties with different shape and size, easy for surface medication and highly biocompatibility [75–77]. In addition, gold nanomaterials can be finely tuned by varying the morphological and reaction characteristic to yield desired size and shape of gold nanoparticles (AuNPs) including nanosphere, nanorod, nanocage and nanoflower. The majority of fabrication of gold materials is through gold-sulphur (Au-S) interaction formed between gold surface and either thiol or dithiol group. The surface-assembly monolayer (SAM) of gold-sulphur bond composes a strong and stable linkage for gold materials and the surface-modified moieties such as proteins, antibodies or polymers. Thiol-gold bond fabrication strategy for AuNP was used in several studies [54,58,70,71,73]. Kang et al. [54] developed an electrochemical sensor for enantioselective recognition of 3,4-dihydroxyphenylalanine (DOPA) based on penicillamine-modified AuNPs (Pen-AuNPs). Chiroptical activity was observed in small AuNPs (~2 nm) which are protected by thiolated Pen. This study was based on the chiral interaction between electroactive DOPA as a target molecule and Pen-AuNPs as chiral NPs. Keshvari's group applied L-cysteine (Cys)-capped AuNPs as a colorimetric sensor for enantioselective detection of naproxen racemic mixtures [73]. The enantioselective and rapid aggregation of L-Cys-modified AuNPs in the presence of *R*-naproxen

makes this design capable of visual chiral sensing. The aggregation/agglomeration morphology of AuNPs were successfully demonstrated though transmission electron microscope (TEM), UV-visible spectroscopy, zeta potential, and Fourier-transform infrared spectroscopy (FTIR) measurements.

AuNP bound with chiral molecules on their surface have been used in many studies for enantiomeric recognition. While most commonly employed surface molecules are L-Cys and its derivatives, owing to the ability of the sulfur group easily to bind with NP surface. Other chiral moieties like ionic liquids are now being investigated [78,79]. Ionic liquids are non-molecular ionic solvents where the ions are loosely coordinated, hence their low melting point. They are also called molten or fused salts, with distinct properties such as low vapour pressure, good thermal and chemical stability and fast diffusion and migration of ions. Ionic liquids have been employed successfully in asymmetric synthesis, LC, CE and GC studies. Huang et al. [60] synthesized AuNPs adsorbed with 1-ethyl-3-methylimidazole L-tartrate (EMIML-Tar) and 1-ethyl-3-methylimidazole Lactate (EMIML-Lac) as chiral molecules. EMIML-Tar-AuNPs recognized chiral samples more potently between L-tyrosine (Tyr) and D-Tyr samples, and were considered for the rest of the study. An exclusive red-to-purple color change was observed when L-Tyr was added to the EMIML-Tar-AuNP solution, unlike the D enantiomer. The absorption peaks had also broadened, and spectral differences were quantified using a plot of the extinction ratio (A650/A520) against logarithmic concentration of the Tyr sample solution, as shown in Figure 2. Extinction coefficient values were significantly higher for L-Tyr exposed samples, demonstrating the chiral potency of the EMIML-Tar-AuNP solution. CE analysis was used to characterize the chiral interactions with different amino acids, where it was observed that EMIML-Tar-AuNPs interacted with L-Tyr, tryptophan (Trp) and phenylalanine (Phe) in decreasing order of strength. Visual color changes and absorption spectra changes were confirmed by TEM analysis which showed exclusive aggregation of EMIM-Tar-AuNPs caused by L-Tyr. The proposed mechanism of selective binding was based on shorter distance of the amino group of L-Tyr to carboxyl group of EMIM-Tar-AuNP when comparing the enantiomers of Tyr, and hydroxyl group of Tyr allowing for three-point contact with the NPs when comparing Tyr with Trp and Phe.

**Figure 2.** Photographs and UV-vis spectra of EMIML-Tar-AuNPs in the presence of D-Tyr or L-Tyr. Experimental conditions: 1.5 mL EMIML-Tar-AuNPs added with 0.5 mL 1.25 mmol/L D-Tyr or L-Tyr. The figure and the caption have been adapted with permission from [60], Copyright © 2016 OSA.

When AuNPs are used to discriminate between enantiomer they mostly require surface modification with chiral ligands that enable chiral recognition. Unlike nanospheres, gold nanorods (AuNRs) can take part in enantiomeric recognition independent of surface modifications as they exhibit chirality of their own. They differ from conventional NPs by being slightly elongated in structure and possessing two SPR bands, one for transverse and the other for longitudinal direction of NR. They are also more sensitive to their micro-environment. Therefore, another simple, sensitive, cheap, and easy to operate enantiorecognition method was developed by Wang's group [58]. Naked AuNR was employed as colorimetric probes for visual recognition of glutamine (Gln) enantiomers. The inherent chirality of AuNRs only aggregated in the presence of D-Gln, thereby resulting in appreciable blue-gray color changes of AuNR solution. On the other hand, no color changes of AuNR solution in the presence of L-Gln. D-Gln also caused a significant decrease in absorbance at 620 nm (longitudinal SPR band). The experiment was also modified and tried to detect enantiomeric excess and was successful in doing so. The experiment was also performed to test the enantiomeric recognition of other amino acids. The results did show some difference for the different enantiomers of these α-amino acids. However, this may be improved by changing the experimental conditions that cater to the specific amino acid being used. Cysteine however showed aggregation of the AuNRs when either of the enantiomers was present. The aggregations could be observed by visualizing the solution using a TEM.

Amino acids play a key role in cellular metabolism and protein composition. All amino acids except glycine exhibit chirality. Contemporary techniques of chirality-dependent separation of these amino acids, such as HPLC, CE and GC are expensive and require complicated upstream treatments. For this purpose, Song et al. [56] devised an inexpensive and easy colorimetric probe for visual enantiomeric recognition of right-handed and left-handed amino acid. The synthesis strategy for AuNP was NaBH4 reduction method. L-Tartaric acid (TA) was found to be structure-similar to citric acid, which was the commonly used as both a reducing and capping agent for AuNP synthesis. Instead of three carboxyl group (-COOH) as citric acid, L-TA consists of two carboxyl groups, and is adsorbed onto the gold surface through the carboxyl group and provides steric barriers for repulsion. L-TA was also regarded as a chiral selector to separate various enantiomers, including many amino acids. The principle of the proposed technique (Figure 3) is that the change of dispersed AuNP solution to an aggregated state, caused by selective binding of enantiomers, is demonstrated by a red to blue shift. Enantiomer induced calorimetric changes for all 19 α-amino acids at 0.1 mM were observed, whose both L and D forms induced AuNP aggregation. Unlike the other 19 α-amino acids, Cys is the only molecule with a thiol-group, the sulfur atom of which can bind to the surface of AuNP causing the blue shift. On assessing optical rotation of L-TA-capped AuNPs and the amino acids, it was found that all caused a positive rotation, in accordance to the general observation of stronger homochiral bindings relative to heterochiral interactions. Hence, left-handed amino acids, which do not cause aggregation, can be discriminated from right-handed forms. For this, histidine (His) was analyzed further as a sample to prove this model. Solutions of L-His and D-His were analyzed against blank L-TA-capped AuNP solution. A visual change in the color of the solution was observed exclusively in L-His sample, measured as a reduction in the UV-Vis absorption spectra at 520 nm and emergence of a new 700 nm peak. TEM and dynamic light scattering (DLS) measurements established that the aggregation was exclusive to the L-His sample and the D-His sample did not affect the dispersity of L-TA-capped AuNPs in solution. The ratio of absorbance at 700 nm and 520 nm (A700/A520) was studied against different concentrations and enantiomer compositions to enable quantitative analysis. Using this technique for L-His, the limit of detection was 0.015 mM, the binding constant of His enantiomers with L-TA-capped AuNPs being 40.82 and 0.23 of L and D types, respectively. Mechanism of interaction between the right-handed amino acids and L-TA-capped AuNPs was proposed to be through carboxylic, hydroxyl and amino groups by hydrogen bonds. This was supported with FTIR spectral data. Owing to high stability of the synthesized NPs for a considerable amount of time and in a pH range of 3.0–9.0, along with the ability to discriminate enantiomers of 18 amino acids with cheap spectrophotometers as the chief measurement device, this technique can potentially be used for high-throughput applications.

**Figure 3.** Schematic illustration of visual chiral recognition of right-handed and left-handed amino acid using L-tartaric acid-capped AuNPs as colorimetric probes. The figure and the caption have been adapted with permission from [56], Copyright © 2016 The Royal Society of Chemistry.

Additionally, Zhou et al. [57] established a carbon dots-gold nanoparticle (C-dots@AuNP) complex for chiral discrimination of glucose enantiomers according to colorimetric and fluorescence dual-mode signals. Cysteine was selected as a precursor to generate sulfhydryl decorated-C-dots, which is responsible for the formation of the C-dots@AuNP complex based on the strong tendency of the sulphur element to conjugate onto the surface of AuNPs. H2O2 is produced as a result of the enzymatic action of glucose oxidase (Gox) and this by-product helps formation of AuNPs by facilitating the reduction chloroauric acid. As shown in Figure 4, in the presence of carbon dots decorated with sulfhydryl groups (derived from Cys), the AuNPs form a complex—C-dots@AuNP. The carbon dots used show an absorption peak at around 350 nm and at an excitation wavelength of 340 nm, they fluoresce with maximum emission at 424 nm. The intensity of this emission is quenched due to the AuNPs when the C-dots@AuNPs complexes are formed. Thus fluorescence spectra in this case can be used to tell D- and L-glucose apart. Such a method is much simpler and faster when compared to using chiral ligands to modify the surface of NPs. The difference can also be seen visually since in the presence of D-glucose the solution turns reddish while it remains colorless when L-glucose is used. TEM images of glucose oxidase catalytic reaction solution show that C-dots cluster around AuNPs when D-glucose is used whereas no AuNPs are formed in the first place when L-glucose is used and C-dots are seen in a monodisperse state.

**Figure 4.** Schematic representation for the generation of C-dots@AuNP complex trigged by the stereoselective enzymatic reaction for the discrimination of glucose enantiomers. The figure and the caption have been adapted with permission from [57], Copyright © 2018 The Royal Society of Chemistry.

#### *2.2. Silver-Based Nanomaterials*

Silver-based nanomaterials are other widely used materials for enantiomeric recognition, separation and sensing. Silver nanomaterials possess unique chemical and physical properties like

higher extinction coefficient, excellent biocompatibility and strong SPR absorption. This phenomenon depends on the size, shape and inter-particle distances of metal NPs and the surrounding environment. In addition, similar to AuNPs, AgNPs were found to have the inherent chirality. Those properties make AgNPs suitable for the fabrication of chiral sensors. Sun's group designed a sulfonated-substituted zinc tetraphenylporphyrin (ZnTPPS)-modified AgNP as colorimetric sensor for chiral detection of L-arginine (Arg) and His [55]. ZnTPPS was selected to fabricate the AgNPs for not only providing stability of NPs but also introducing enantiomeric recognition for L-Arg and L-His via Zn binding. ZnTPPS-AgNPs were found to be coordinately bound to N-H of L-Arg leading to formation of AgNP clusters (L-Arg-ZnTPPS-AgNPs) which were then characterized by TEM and UV-vis spectroscopy. In the presence of L-Arg, a color change from light yellow to yellow can be observed accompanied by a decrease in absorbance at 400 nm that arises from the SPR absorption of dispersed AgNPs. L-Arg induces aggregation of the ZnTPPS-AgNPs to form AgNP clusters, L-Arg-ZnTPPS-AgNPs. This was confirmed by TEM. Methionine (Met), His, Phe, Tyr and glutamic acid were tested using these modified AgNPs but only Met and His showed an obvious color change and His was chosen for further investigation. It was found that only L-His induced a color change from yellow brown to red accompanied by a significant decrease in the absorbance at 400 nm (Figure 5). A new band at 550 nm was observed and the absorbance ratio (R) at the two wavelengths were determined and was found to be enhanced only in the presence of L-His. D-His on the other hand had none of these effects, thereby proving the good chiral selectivity of these AgNPs. L-His induced further aggregation of the modified AgNPs and caused them to form larger agglomerates and this was clearly seen using TEM. In circular dichroism spectra, these NPs showed a slight chiral signal in the presence of D-His whereas L-His had a much stronger one. To satisfy the three-point contact model for enantiomeric recognition, they proposed a possible interaction mode between L-His and the AgNPs clusters via the carboxylic, amino and imidazole groups through electrostatic and hydrogen bond interactions. In a quantitative analysis study, they found that at very low concentrations of His, the NPs showed low chiral selectivity. Hence it showed in the end that this method was effective for enantiomeric recognition of His and Met.

**Figure 5.** Schematic representation The UV-vis spectra (**a**), photographic images (**b**) and the value of R (A550/A400) (**c**) of silver nanoparticle cluster solution in the presence of D- or L-His. It is operated as follows: 0.5 mL of 1 mM solution of D- or L-His was added to 1.5 mL L-Arg-ZnTPPS-AgNPs solution and mixed for 10 min before measuring, respectively. The figure and the caption have been adapted with permission from [55], Copyright © 2012 The Royal Society of Chemistry.

Marzieh et al. [61] used chitosan-capped silver nanoparticles (CS-AgNPs) for enantiomeric recognition of the essential amino acid, Trp. They scanned optical cells containing the NPs and D- or L-Trp and the color values of each optical cell were then analyzed. The NPs were characterized using FTIR spectra, TEM, X-ray diffractometer (XRD) and UV-vis spectroscopy after their synthesis. When given concentrations of the Trp enantiomers were added separately to a solution of CS-AgNPs, UV-vis spectra were recorded after 30 min. A control without Trp was also tested in the same manner. While scanning for the color of the solution at selected areas of the scanned image for RGB

analysis, the values were averaged. MATLAB software was used for assessing different types of colors and obtaining the CMYK values. Differences between CMYK values of the blank control were compared to those of solutions containing either of the Trp enantiomers. The maximum absorbance of the AgNPs was observed at 404 nm and represented the characteristic SPR band of the AgNPs. TEM images of the CS-AgNPs showed that they were uniformly distributed in aqueous solution with an average diameter of about 15 nm and their concentration was calculated using Beer's law. In the presence of L-Trp at sufficiently detectable concentrations, a color change from a yellowish to brown was observed and the CS-AgNPs were found to be aggregated when observed using TEM. No such observations were detected when D-Trp was used. Experiments were also performed by varying various experimental conditions to find the optimal settings of the experiment. They also found that enantiomeric composition of Trp could be determined from corresponding scanometry and spectrophotometric linear calibration curves. The possible interaction mechanism between the CS-AgNPs and L-Trp was proposed to be the hydrogen bond formation between -OH groups of surface of CS-AgNPs and the amine and carboxyl groups of L-Trp.

AgNPs exhibit a distance-relevant color and higher extinction coefficient than that of AuNPs of the same size, thereby making them more reliable as color reporting agents for colorimetric sensor design. Zhang et al. [52] used uridine 5 -trisphosphate (UTP)-capped AgNPs for chiral detection of Cys enantiomers. It was found that in the presence of D-Cys an appreciable color change from yellow to red took place but L-Cys showed no such change, as shown in Figure 6. Moreover, when these UTP-capped AgNPs were added to a racemic solution of Cys, they interacted specifically with one enantiomer leaving an excess of the other after centrifugation thus enabling enantiomeric separation. When UV-vis spectroscopy was performed, the peak at 400 nm was seen to undergo a red shift to 520 nm in the presence of D-Cys due to the SPR of AgNPs. Aggregation of the NPs also took place resulting in the decrease of the SPR band at 400 nm. This band was consequently broadened around 450-600 nm. No such effects were reported when L-Cys was used. The absorbance ratio at 520 nm and 400 nm relates to the quantities of dispersed and aggregated AgNPs and thus this was chosen to evaluate the performance of the AgNPs.

**Figure 6.** Colorimetric Discrimination of L- and D-Cys Using UTP-Capped AgNPs. The figure and the caption have been adapted with permission from [52], Copyright © 2011 American Chemical Society.

This ratio resulted in significant differences when D- and L-Cys were used. The ratio was found to increase with increasing concentration of D-Cys from 0.1 to 100 micromolars. No such event took place in case of L-Cys except when its concentration was above 100 micromolars where it may cause the AgNPs to lose stability and aggregate, but data suggested that aggregation caused by D-Cys was much more sensitive by at least two times that of L-Cys. UTP analogs were also tested in their role as stabilizing agents on the surface of the AgNPs. When adenosine 5 -triphosphate (ATP) was used, there was negligible discrimination between the two enantiomeric forms of Cys but gradual decrease in the 400 nm and increase in the 520 nm was observed along with the same color change as before. This proved that ATP coated AgNPs could be used to quantitatively detect Cys but not discriminate between the enantiomeric forms. Furthermore, Tashkhourian et al. [62] established AgNP-based colorimetric sensor to discriminate *R*-citalopram (Cit). Citalopram is a selective serotonin reuptake inhibitor used as an antidepressant drug. *S*-Cit was found 150 more potent than racemic Cit or *R*-Cit. Therefore, a simple and reliable detection system for chiral Cit is ponderable in pharmaceutical field.

Gold and silver nanoparticle-based enantiomeric recognition have been widely discussed and studied in the past decade. However, Bruckner et al. [80] demonstrated the use of polysaccharide-ester based amino acid enantiomers using gas chromatography-mass spectrometry (GCMS) in analysis of 24 h urine and blood sera. The experimental procedure involved GC of *N(O)*-pentafluoropropionyl (PFP)-amino acid-(2)-propyl esters on *N*-propionyl-L-valine-*tert*-butylamide polysiloxane, and subsequent filtration by selected ion monitoring mass spectrometry (SIM-MS). The PFP-(2)-propyl esters were synthesized using a mixture of 14 D,L-amino acids and γ-aminobutyric acid (Gaba). The highest amounts of chiral amino acids were determined as D-Serine and D-Alanine, both in urine samples and blood sera, with blood sera in relatively much lower amounts. Samples from fasting patients revealed decreased levels of D-amino acids, while time dependent analysis showed a continuous excretion in the urine.

#### **3. Enantiomeric Separation by Chiral Nanoparticles**

Applications of NPs in enantiomeric separation have shown promising results by enhancing the resolution, processing time and efficiency of the conventional separation methods [81]. The use of NPs in enantiomeric separation dates back to 1989 when Wallingford and Ewing reported the first application of polymer NPs (mentioned as monomolecular particles) in CE [82–84]. Since then a varieties of NPs have been used for efficient separation of enantiomers. Table 2 summarizes some of the recent applications of nanostructures in enantioseparation science.

**2.** Some representative examples in which nanomaterials have been used for the separation of chiral compounds [24,85,86].

**Table**

Metal Oxide

Iron Oxide

(Fe3O4)

HPLC

HPLC

HPLC

HPLC

Titanium

dioxide (TiO2) CE

Tris-H3PO4 solution containing TiO2 NPs as

background electrolytes (BGEs)

Carboxymethyl-β-CD

 BSA Cellulose

tris-(3,5-dimethyl-phenylcarbamate)-coated

TiO2/SiO2 chiral stationary phase (CSP)

 Bovine serum albumin (BSA)

thickness of silica layer in

Fe3O4@SiO2:

 60 nm

> -

 Average size: 13.3 nm

Size of TiO2/SiO2 spheres: ~6

nm; pore diameter: ~7 nm


Trp, Phe and His

D,L-Trp, Phe and Tyr

 Ibuprofen and ofloxacin

Eight basic indole ring derivative

enantiomers β-adrenergic drugs (atenolol,

eliprolol, clorprenaline, metoprolol, propranolol, and

terbutaline) and clenbuterol

 fenoterol,

[95]

[94]

 [92]

 [93]

 [66]

nanoparticles


**Table 2.** *Cont.*

TLC

D-TA-graphene

Thickness of graphene nanosheet:

Racemic drugs (propranolol and

ofloxacin)

[106]

2–3 nm


Notably, NPs represent a state of matter in the transition region between solids and molecular structures in the size range of 1~100 nm [103], where charge carriers are confined in one, two or all of the three dimensions resulting in quantum well structures (nanosheets), quantum wire structures (nanowires) or quantum dot structures (nanoparticles), respectively. Due to quantum confinement and large surface-to-volume ratio (*s*/*v* > 1), such structures exhibit unique size-dependent optical, electronic, magnetic, and catalytic properties [120]. In addition, nanostructures possess better chemical stability, low cytotoxicity, significant mechanical strength, and are easily modifiable in terms of their size, shape and surface properties [91,103]. Importantly, NPs enhance the performance of enantioseparation process due to their extremely large *s*/*v* ratio (e.g., nanospheres of 10 nm diameter can have a surface area as large as 600 m2/cm3 [121]) that maximizes the amount of binding sites [69,81]. In the following sections we summarize some of the recent applications of different types of nanostructures for the separation of enantiomers.

#### *3.1. Metal Nanoparticles*

Recent years have witnessed tremendous applications of metal NPs in diverse fields of research including material science, biotechnology, biomedical engineering, targeted drug delivery, environmental, etc. [122–124]. In addition, the potential of metal NPs in separation science has been already well recognized due to their easy synthesis procedure, large *s*/*v* ratio, controllable particle size, narrow size distribution, extraordinary biocompatibility, molecular detection properties, etc. [24,89,125].

In a pioneering work in 2005, Choi et al. [87] employed sulfonated β-CD as a chiral selector and Ag colloids as an additive in CE for the enantiomeric separation of arylalcohols (1-phenyl-1-propanol, 1-phenyl-2-propanol, and 2-phenyl-1-propanol). The group observed that the addition of Ag colloid to the running buffer improves the resolution significantly. Another simple, reliable and highly efficient colorimetric platform for enantiomeric separation and detection was proposed by Zhang et al. [52] by using UTP nucleotide-capped AgNPs. The enantioselective aggregation of AgNPs allowed the group achieving rapid colorimetric separation of D- and L-Cys.

AuNPs have also been applied extensively for enantiomeric separation. In 2005, Shao et al. [126] synthesized human serum albumin (HSA) immobilized gold nanotube membranes for the separation of warfarin enantiomers. Li et al. [127] used bovine serum albumin-gold nanoparticles (BSA-AuNPs) conjugate as chiral stationary phases (CSPs) in open-tubular capillary electrochromatography (OTCEC) for the enantiomeric separation of ephedrine and norephedrine isomers. Good resolutions of 1.18 and 2.15 were obtained for norephedrine and ephedrine isomers, respectively, within 250 s at an effective separation channel length of 36 mm. In another work, Yang et al. [90] employed thiolated β-CD-modified AuNPs as chiral selector in pseudostationary phase-CEC to separate four pairs of dinitrophenyl-labeled amino acid enantiomers and three pairs of drug enantiomers with enantioseparation resolution up to 4.7. The repeatability of this method has been improvised in a later work by immobilizing the thiolated β-CD-modified AuNPs onto the inner wall of a capillary to serve as stationary phase for enantioselective OTCEC separation [128]. Interestingly, Shukla et al. [88] reported a unique strategy for enantioselective adsorption of propylene oxide (PO) by using either D- or L-Cys-modified AuNPs. In particular, the group demonstrated the ability L-Cys (D-Cys)-modified AuNPs to selectively adsorb the (*R*)-propylene oxide ((*S*)-propylene oxide). Notably, this work was extended by using D- or L-Cys to functionalize tetrahexahedral (THH, 24-sided) AuNPs to separate a real chiral pharmaceutical-propranolol [89]. Recently, nucleic acid aptamer-functionalized AuNPs were used as chiral selector for the separation of racemic D,L-Trp. The method used centrifugation to separate the precipitate formed by the aptamer-specific enantiomer (L-Trp) bounded AuNPs (Figure 7) [69]. In another work, Su et al. [53] demonstrated the potential of laboratory synthesized chiral *N*-acetyl-L-Cys-capped AuNPs for high throughput enantiomeric separation of amino acid enantiomers via centrifugation. Recently, monolayer and multilayer AuNPs film capillary columns were fabricated through layer-by-layer self-assembly of AuNPs and their

subsequent functionalization through self-assembly of thiolated β-CD in OTCEC [129]. It was observed that, as compared to monolayer AuNPs film capillary column, the three layer AuNPs film capillary column possess superior enantioseparation performance. Further, in 2018, Liu et al. [86] successfully added streptomycin-modified AuNPs in background electrolyte (BGE) solution in CE for the first time to separate a number of drug racemates, such as, adrenaline hydrochloride, noradrenaline bitartrate, and isoprenaline hydrochloride.

**Figure 7.** The schematic of the enantioseparation of D,L-Trp based on functional nucleic acids modified Au nanoparticles. The figure and the caption have been adapted with permission from [69], Copyright © 2013 Wiley.

#### *3.2. Metal Oxide Nanoparticles*

In recent years, metal oxide nanoparticles such as magnetite (Fe3O4), titania (TiO2), Zirconia (ZrO2), etc., have become crucial in enantiomeric separations, catalysis, sensing devices, cell labeling, drug delivery, and biomedical applications [101,130–132]. Notably, nanoscale Fe3O4 is one of the important phases of iron oxide and has been extensively used in the field of enantiomeric separation due to the possibility of their easy manipulation even at a lower magnetic field [85,133]. In a seminal work, Choi et al. [91] prepared magnetic silica nanoparticles, MSNPs (spherical SiO2 NPs containing Fe3O4) of average size 300 nm in order to separate enantiomers of *N*-(3,5-dinitrobenzoyl)-α-amino acid *N*-propylamides. Interestingly, the group named the enantioseparation process as "enantioselective fishing" since the MSNPs tagged with an appropriate chiral selector readily formed complexes with one of the enantiomers which was subsequently separated simply by using a magnet. Enantiomeric separation of amino acids were also performed by employing water-soluble β-CD-modified Fe3O4 NPs fabricated by using a simple and convenient chemical route [134]. The group cleverly utilized a strategy to chirally select amino acids by β-CD and subsequently use Fe3O4 NPs as magnetic separators. In another work, enantiomeric separation of aromatic amino acids (D,L-Trp, Phe and Tyr) has also been achieved by using carboxymethyl-β-cyclodextrin (CM-β-CD)-functionalized MSNPs [92]. In the meantime, Arslan et al. [132] developed a novel enantioselective sorption approach based on Fe3O4 NPs functionalized with three different β-CDs. The group established that the novel CD-grafted Fe3O4 NPs significantly enhance their enantioseparation capabilities towards a set of chiral carboxylic acid molecules. Notably, Sayin et al. [135] grafted *p-tert*-butylcalix[8]arene derivative (C[8]-C4-COOH) onto Fe3O4 NPs and used both C[8]-C4-COOH and Fe3O4 NPs for the encapsulation of lipase in order to achieve enhanced catalysis and enantioselective resolution of racemic naproxen methyl ester. Chiral MNPs based on BSA and Fe3O4 NPs have been used recently to separate chiral drugs (ibuprofen and ofloxacin) [93] and amino acids (Figure 8) [66]. Most recently, chiral core/shell structured

MNPs were fabricated by using poly(*N*-isopropylacrylamide-co-glycidyl methacrylate)-β-cyclodextrin (PNG-CD) to form smart polymer brushes (shell) onto polydopamine coated Fe3O4 MNPs (core) [136]. The smart NPs (Fe3O4@PDA@PNG-CD), fabricated in this work, showed excellent magnetic separability where the β-CD units acted efficiently as the chiral selector to selectively recognize and bind the desired enantiomer forming host–guest inclusion complexes.

**Figure 8.** Preparation of BSA–PMNPs and the direct separation of the racemates. The figure and the caption have been adapted with permission from [66], Copyright © 2013 The Royal Society of Chemistry.

On the other hand, TiO2 and ZrO2, have also been well recognized as a stationary phase due to their favorable physiochemical properties [137–140]. The applicability of TiO2 NPs in enantiomeric separation has been demonstrated by first coating it onto the micron sized silica spheres [94]. The TiO2/SiO2 particles were further coated with cellulose tris-(3,5-dimethylphenylcarbamate) (CDMPC) to prepare the CSP for the separation of eight indole ring derivative enantiomers. Notably, the use of TiO2 NPs as additive in CE for the simultaneous separation of eight β-adrenergic drugs has also been reported [96]. In another work, a ZrO2 based HPLC packing material ZrO2/SiO2 was prepared by multilayer coating of ZrO2 NPs on the surfaces of silica spheres using layer-by-layer self-assembly technique [141]. The group analyzed the potential of ZrO2/SiO2 material for enantioseparation applications. Moreover, in another work, Kumar et al. [96] reported the fabrication of Fe3O4@ZrO2 microspheres (Fe3O4 magnetic core covered by a ZrO2 shell) of average size 340 nm, functionalized with CDMPC, for the separation of racemic chiral drugs. Interestingly, the excellent recyclability of the synthesized chiral ZrO2 magnetic microspheres is quite promising for their use in the multiple enantiomeric separations.

#### *3.3. Carbon-Based Nanomaterials*

Importantly, among different varieties nano-engineered materials used for enantiomeric separation, carbon nanostructures have also attracted much attention due to their high mechanical strength, good chemical stability, high elasticity, and high thermal conductivity [142,143]. So far, a number of allotropic carbonaceous nanomaterials, such as, carbon nanotubes (CNTs), graphene and graphene oxides (GO) have been successfully applied in this area.

#### 3.3.1. Carbon Nanotubes

Notably, CNTs can be considered as graphite sheets (sp2 hybridised carbon atoms) wrapped up in the form of a cylinder that are usually capped by a fullerene-like structure with diameters ranging from few Å [144–146] to tens of a nanometer [102,147] and length up to a few micrometer (may extend to centimetres in certain occasions) [103,148,149]. CNTs were first discovered by Iijima [150] and have been one of the most researched materials of 21st century [151]. CNTs can be broadly classified into two types: multi-walled carbon nanotubes (MWCNTs) comprised of more than one coaxially rolled up graphite sheets and single-walled carbon nanotubes (SWCNTs). Due to their unique physicochemical properties and large chemically active surface area, which have been thoroughly discussed in several review articles [152–155], both MWCNTS and SWCNTs are emerging as one of the most promising candidates for numerous potential applications [156–158]. In the context of enantiomeric separation, CNTs are capable of improving the speed, selectivity, stability, and efficiency in chiral chromatographic separation [24,159]. In an exciting work, Ahmed et al. [100] demonstrated the creation of a CSP with good enantioselectivity by encapsulating small amount of SWCNTs into polymer monolithic backbones. Using enantioselective nano-HPLC separation, the group successfully separated twelve classes of pharmaceutical racemates. Under optimum concentration conditions, the group achieved satisfactory repeatability and observed that (6,5) SWCNTs displayed higher enantioselectivity as compared to the (7,6) SWCNTs. Notwithstanding the enormous potentials of CNTs to be used as sorptive material for enantiomeric separation, there are very few reports where such non-functionalized CNTs have been used for the separation of chiral compounds through affinity chromatography. This might be due to the reason that CNTs, especially chiral CNTs, alone may not be an effective adsorbent for enantiomeric separation [160,161].

Nevertheless under strong and specific chemical conditions the surfaces CNTs can be modified/functionalized to enhance their solubility and other intrinsic properties which render them CSPs or pseudostationary phases [24,97]. There are a number of efforts in the recent past where enantiomeric separation was successfully performed by the modification of CNTs with chiral selectors such as β-CD. For example, Na et al. [101] demonstrated a new strategy for enantiomeric separation of clenbuterol by CE using modified CNT as chiral selector in 2004. The group reported the formation of a large surface area platform by the NPs modified with β-CD that served as a pseudostationary chiral phase for the enhanced separation of enantiomers. In a similar work to separate the same kind of enantiomers, Yu et al. [102] reported a method to functionalize MWCNTs with hydroxypropyl-β-cyclodextrin (HP-β-CD) in order to use as a stationary phase additive in TLC, as shown in Figure 9. Recently, carbogenic NPs, carboxylated SWCNTs and carboxylated MWCNTs were used to modify enantioseparation systems to form pseudostationary phases with dextrin as chiral selectors in electrokinetic chromatography (EKC) [104]. Particularly the group established that the introduction of NPs into the buffer significantly could enhance the resolution of several drug enantiomers namely sulconazole, ketoconazole, citalopram hydrobromide, and nefopam hydrochloride. Under optimized dextrin concentration, buffer pH and buffer concentration resolution time of 15 min with resolution values in the range 1.41–4.52 were achieved in this work. In another work, a novel chiral selector was developed by conjugating BSA which is a type of protein with carboxylic SWCNTs [98]. The efficient enantioseparation ability of the BSA-SWCNT conjugate was illustrated by the successful separation of Trp enantiomers within 70 s with a resolution factor of 1.35 and separation length of 32 mm obtained by using poly(methyl methacrylate) based microchip electrophoresis. The potential of concentrated surfactant-coated MWCNTs as an alternative to other chiral selectors like CDs were also successfully demonstrated by using partial filling of the capillary in EKC at different experimental conditions such as pH, addition of organic modifiers, potential and injection time [97].

Recently, Zhao et al. [162] allowed SWCNTs to bind with the inner wall of a capillary column in GC in order to enhance the enantiomeric separation by achieving larger surface for the chiral ionic liquid stationary phase, which in turn increased the interaction between the stationary phases and the analytes. They prepared two capillary columns for the chromatography process: column A containing only the chiral ionic liquid and column B containing a mixture of the chiral ionic liquid and SWCNTs. Interestingly out of 12 model racemates, column B successfully separated eight chiral compounds as compared to the column A which separated only four of such compounds. In another work, Guillaume et al. [99] successfully developed a simple approach by using HPLC for the enantiomeric separation of amino acids by functionalizing SWCNTs with pyrenyl derivative of

a chiral aminoglycoside called neomycin A (PNA). Magnetization of CNTs also showed promising results in the separation of enantiomers. Tarigh et al. [163] used the chiral selector L-threonine (Thr), anchored to the surface of magnetic MWCNTs, to successfully separate D,L-mandelic acid within a time period of 10 min.

**Figure 9.** Schematic presentation of preparation of HP-β-CD modified MWCNTs. The figure and the caption have been adapted with permission from [102], Copyright © 2011 Wiley.

#### 3.3.2. Graphene and Graphene Oxide Nanomaterials

Graphene is, on the other hand, a two-dimensional (2D) sheet of sp<sup>2</sup> hybridized carbon atoms with single atom thickness. In principle, graphene can be regarded as the basic building block of fullerenes (0D structure), carbon nanotubes (1D structure) and graphite (3D structure) [164,165]. It is the thinnest yet the strongest known material which exhibit exceptional optical, electronic, thermal, and adsorptive properties [166,167]. The profound impact of graphene in the field of material science has been well recognized by the Nobel Prize in Physics for 2010 awarded jointly to Geim and Novoselov of The University of Manchester, UK for their groundbreaking experiments related to graphene [168]. On the other hand, oxidization of graphite leads to the formation of graphene oxide (GO) decorated with various oxygen containing functionalities such as epoxide, carbonyl, carboxyl, and hydroxyl groups [169]. GO is attracting enormous attention due to its easy synthesis procedure, high yield and satisfactory dispersibility in organic solvents [170].

Notably, graphene and GO exhibit considerable potential in separation science [165] and has been used by several research groups for the enantiomeric separation of chiral molecules. For instance, Tu et al. [106] reported the graphene assisted resolution of two racemic drugs propranolol and ofloxacin using pure D-(–)-TA as the chiral selector in TLC. Interestingly, computational simulations using density functional theory also showed the applicability of nanoporous graphene, when functionalized by a chiral bouncer molecule [171]. In another exciting work, Candelaria et al. [105] demonstrated the fabrication of new type of chemically stable, versatile and cost-effective graphene-based CSPs for enantiomeric separation using LC. The group employed functionalized mesoporous 3D graphene nanosheets for the enantiomeric separation of pharmaceutical grade racemic mixtures of model ibuprofen and thalidomide.

In case of GO, Liang and his coworkers, in a series of publications, employed OTCEC for the separation of different types of enantiomers by using β-CD conjugated GO-magnetic nanocomposites (GO/Fe3O4 NCs) [108], BSA-conjugated GO-magnetic nanocomposites (GO/Fe3O4) [172] and BSA conjugated polydopamine-GO nanocomposites (PDA/GO/BSA) [109] as the stationary phases. Exploiting the large surface area, excellent biocompatibility of graphene and rough surface morphology of GO, the group used the OTCEC microdevices to successfully separate Trp, Thr and propranolol enantiomers with reasonably good resolution factors. Notably, as compared to other similar techniques, the group reported better resolution factor for the enantiomeric separation of D,L-Trp by using the

novel PDA/GO/BSA stationary phase. Li et al. [170] reported a facile and efficient strategy to chirally functionalize GO with optically active helical polyacetylene chains. The group successfully used the GO hybrid as a chiral inducer for the enantioselective crystallization of alanine enantiomers where L-alanine was induced to crystallize in the form of rodlike crystals. There are also examples where GO coated fused silica capillaries were applied for the enantiomeric separation of the ephedrine–pseudoephedrine (E-PE) and -methylphenethylamine (-Me-PEA) isomers by using CE method [173]. On the other hand, GO-polymer coated fused-silica capillary columns were used to improve the enantiomeric separation of three anionic racemic drugs (naproxen, warfarin and pranoprofen) by using CEC with methyl-β-cyclodextrin (Me-β-CD) as chiral selector [107]. Using molecular modeling with AutoDock, the group studied the mechanism of GO-modification effect on the enantioseparation efficiency of the CEC system. In another work, Hong et al. [174] reported for the first time the development of GO-based affinity capillary silica monolith with HSA or pepsin as chiral selector for enantiomeric separation and proteolysis by using CEC. It was observed that as compared to affinity monoliths without GO, HSA-modified affinity capillary silica monoliths (HSA-GO-EDA@CSM) possess better enantiorecognition ability. Recently, Li et al. [110] reported better HPLC enantiomeric separation of benzene-enriched enantiomers by using a novel CSP based on GO. In this method, GO was first covalently coupled to silica gel microspheres which was subsequently reduced with hydrazine to form reduced graphene oxide@silica gel (rGO@SiO2). Finally, the surfaces of rGO@SiO2 were physically coated with cellulose tris-(3,5-dimethylphenylcarbamate) to prepare the CSP. Most recently, a smart multifunctional graphene oxide nanocomposite was prepared which showed exceptionally good selectivity, thermosensitivity and magnetic separability for the identification and enantiomeric separation of Trp enantiomers [111]. The synthesized nanocomposite was composed of GO nanosheets, immobilized superparamagnetic Fe3O4 nanoparticles, poly(*N*-isopropylacrylamide-co-glycidyl methacrylate) (PNG) chains and β-CD which was fabricated through a combination of surface-initiated atom transfer radical polymerization (SI-ATRP) and a ring-opening reaction, as shown in Figure 10.

**Figure 10.** Schematic illustration of the thermosensitive chiral recognition and enantioseparation of AAs enantiomers by MGO@PNG-CD. (A) As the temperature is below the LCST of PNIPAM chains (T1), the β-CD units in PNG-CD can selectively recognize and accommodate L-enantiomers to form stable host-guest inclusion complexes, while as the temperature is above the LCST of PNIPAM chains (T2), the loaded L-enantiomers can desorb from the β-CD cavities automatically because of the reduction in the inclusion constants of β-CD/L-enantiomers complexes; (B) The MGO@PNG-CD is added in the AAs enantiomeric solution at T1 and the PNIPAM chains are swollen, and then L-enantiomers are recognized by β-CD; (C) Through an external magnetic field L-enantiomers are loaded on the MGO@PNG-CD, and D-enantiomers are remained in the enantiomeric solution for subsequent separation; (D) while the operating temperature is above the LCST of PNIPAM (T2), the PNIPAM chains occur to collapse, and the loaded L-enantiomers are released, and the MGO@PNG-CD is recycled; (E and F) the regenerated MGO@PNG-CD is recovered using a magnet and reused. The figure and the caption have been adapted with permission from [111], Copyright © 2018 American Chemical Society.

#### **4. Conclusions**

Enantiomeric recognition and separation is one of the most challenging tasks, particularly in the fields of contemporary pharmaceutical science, agrochemical science, material science, and many other rapidly expanding areas of research. Notably, the advent of modern high-throughput experimentation (HTE) technology has enabled the research and industrial laboratories to produce a large number of samples within a very short period of time. In comparison, the analytical methods (e.g., HPLC, GC, CE, etc.) which are generally employed for screening the reaction yield, enantiomeric purity, stability to racemization, enantiomeric excess (ee), and concentration of chiral compounds are relatively expensive, time consuming, laborious and produces solvent waste [175–177]. In this context, chiral sensing methods, capable of performing real time analysis of mixtures of enantiomers by utilizing inexpensive instrumentation with almost zero waste of expensive reagents, have significant scientific and industrial relevance and, therefore, are gaining increasing attention [177]. To date, a number of methods including electrochemical sensors, gravimetric-mass sensors-resonators, electrical sensors and chiroptical/spectroscopic sensors have been developed for fast and accurate differentiation of enantiomer [178]. Table 3 summarizes some of the recently used chiral sensing methods. Importantly, there are also examples where NPs have been used for the purpose of chiral sensing. For example, Tsourkas et al. [179] reported a enantioselective immunosensor by utilizing dextran-coated MNPs as magnetic relaxation switch that decreased the T2 relaxation time of water by 100 ms. The group successfully detected 0.1 μM of D-Phe in the presence of 10 mM of L-Phe (99.998% ee) by using NMR measurement of the T2 parameter. In another work, Wang et al. [58] reported a visual sensing platform based on AuNPs for visual recognition of Gln enantiomers. Silver nanoparticles (~400 nm) have been used to enhance the circular dichroism by two orders of magnitude [180]. Moreover, enantioselective sensors based on microcantilevers (MCs) with nanostructured (roughened) gold surfaces on one side showed promising results for the label-free stereoselective detection of α-amino acids [181]. Further, use of molecular imprinted polymer (MIP) nanowires/nanofibers showed immense potential to enhance the sensitivity of chiral sensors by allowing more binding sites (or cavities) due to the high surface area of such nanostructures [182,183]. Thus, integration of nanoscience and nanotechnology has a lot of potential to enhance the molecular sensing and signal transduction, which are the basic processes of chiral sensing [184]. A more detailed analysis of the chiral sensing methods can be found in several recent reviews [177,184,185] and are not discussed here.

In this review, we mainly focus on the application of nanostructured materials to amplify chiral recognition and separation. Notably, a wide array of nanostructures get benefited from the rapid development of nanotechnology are synthesized for the characterization and purification of racemates. The surfaces of NPs coated with chiral ligands (chiral NPs) have proved the potentialities for enantiomeric recognition based on the specific interactions between analytes and target enantiomers. Chiral NPs have paved a new route for enantiomeric recognition and surpassed the current cumbersome methods in this field, offering a relatively simple examination platform as discussed in Section 2. Notably, chiral NPs accommodate various nanocomposites to manipulate the molecular adsorption and aggregation, colloidal assembly, and to characterize chemical dynamics at particle surfaces as a useful modality for the understanding of enantioselective mechanism and future pharmaceutical applications. These surface effects also raise questions concerning the means by which biologically active compounds interact with chiral molecules, particularly with regard to enantioselective mechanisms at the nanoscale.


**Table 3.** Representative list of chiral sensing methods. The table derives from Manoli et al. [177].

On the other hand, separation of racemic compounds is accomplished by selecting the desired enantiomer by introducing chiral selectors of compatible size and structure to the separation system as a part of the CSP or as chiral additive in the mobile phases (CAMP) [9,204]. Notably, there still remain three major issues on the use of chiral selectors for the detection and/or separation of enantiomers. These are: (a) the broad spectrum of applications, (b) the cost of the selector and (c) the productivity of the separation (gram of pure enantiomers/kg of CSP/day). In this regard, it is noteworthy to mention that α-, β- and γ-CDs (oligosaccharides) and their derivatives seem to represent a kind of universal chiral selector not only due to their broad-spectrum of chiral selectivity and relatively low cost but also their applicability in most of the separation techniques, such as, TLC, CEC, HPLC, CE, crystallization, etc. In addition, modified polysaccharides, proline derivatives, Pirkle type chiral selectors are also finding widespread applications in both analytical and preparative scale separations [9,205,206]. Recently, the most important and widely used CSPs for enantio-recognition/separation mechanisms were discussed in several reviews [9,21,207–211]. Importantly, economics of the separation process including the selection of the CSP/CAMP and the separation technique depends mainly on the stage of production. For example, in early stage small scale laboratory productions, finding an optimum separation method which is capable of producing large amount of desired enantiomers within a suitable time period becomes more important than the cost of the process. For preparative scale productions, however, both cost of the process as well as its productivity plays an important role [212,213]. Importantly, during the transition from analytical to preparative scale productions, conventional separation techniques (e.g., batch chromatography) are often replaced by high throughput large scale production techniques like stimulated moving bed (SMB) technology [214]. Further, in preparative scale separations the cost of production may significantly vary depending on the type of CSP/CAMP used and the technique in which the separation process is conducted. Thus the primary challenge is to find an optimum (cost effective and most productive) separation process, especially by

using all possible combinations of more efficient CSPs/CAMPs [213,215]. In this regard, the use of nanoparticles and nanocomposites in separation processes has the potential to circumvent such challenges. In Section 3, we described the state of the art in the application of nanostructured materials in the field of enantiomeric separation. Arguably the nanostructured materials will continue to play a vital role in the separation of chiral molecules, especially due to their extraordinary capacity to enhance the enantioseparation ability of conventional techniques.

To summarize, we note that enantioseparation science has completed a phenomenal journey of around 170 years since its first demonstration in 1848 by Louis Pasteur [216]. At the present time, combination of conventional enantioseparation techniques with the recent advances in nanoscience and nanotechnology is proving to be quite synergetic by producing exciting results. Despite all these developments, the major challenge that still remains is the development of a more flexible, efficient and cost-effective chiral resolution technique and/or chiral selector to efficiently separate the numerous newer and newer chiral compounds, especially chiral drugs that are continuously being introduced. Nevertheless, without forgetting the enormous research efforts being devoted in this area during the last few years, we anticipate that the near future shall witness smarter techniques with more universal abilities for enantiomeric sensing, recognition, and separation.

**Author Contributions:** A.G., N.M., S.K., H.R., N.-T.C., and G.-Y.Z. wrote and editing the manuscript, G.-Y.Z. conceived the work and organized the manuscript.

**Funding:** This work was funded by Ministry of Science and Technology, Taiwan, 107-2112-M-039-001.

**Acknowledgments:** We thank Arbaaz Sait and Shih-Ting Lin for the help on editing the manuscript.

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

#### **References**


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