Hemicyanine-Based Near-Infrared Fluorescence Off–On Probes for Imaging Intracellular and In Vivo Nitroreductase Activity

Round 1

Reviewer 1 Report

The authors developed three fluorescent probes to measure nitroreductase acvtivity. The probes are selective, sensitive and their emission falls in the near infrared range. The applications of the probes are demonstrated by microscopic images and by imaging in xenograft mice. These results are valuable and the manuscript is well-organized, however, it requires some corrections before publication.

- It is written in the abstract and also in the main text that the low fluorescence intensity of the probes is due to ‘interference of intramolecular charge transfer (ICT) by nitro group.’ It is a general experience that most of the aromatic nitro compounds show only a weak fluorescence. The quenching effect of the nitro substituents is explained most frequently by an interplay between the fluorescent ππ* transition and a forbidden nπ* transition (see e. g. ChemPlusChem 86, 11, 202 and refs therein).

- I recommend to complete the photophysical data with the fluorescence quantum yields and to summarize the spectral data (absorption wavelengths, absorption coefficients, fluorescence wavelengths, fluorescence quantum yields) in a Table in the main manuscript

- Some parts of the manuscript (e.g. section 4.1) require a thorough English revision

Author Response

Comments and Suggestions of Reviewer #1

Comment: The authors developed three fluorescent probes to measure nitroreductase activity. The probes are selective, sensitive and their emission falls in the near infrared range. The applications of the probes are demonstrated by microscopic images and by imaging in xenograft mice. These results are valuable and the manuscript is well-organized, however, it requires some corrections before publication.

>> Response: It is our pleasure that you notice the value for our study.

1. It is written in the abstract and also in the main text that the low fluorescence intensity of the probes is due to ‘interference of intramolecular charge transfer (ICT) by nitro group. It is a general experience that most of the aromatic nitro compounds show only a weak fluorescence. The quenching effect of the nitro substituents is explained most frequently by an interplay between the fluorescent ππ* transition and a forbidden nπ* transition (see e.g. ChemPlusChem 86, 11, 202 and refs therein).

>> Response: Thanks for your comment. Unfortunately, we could not find the reference you mentioned. In precedents, aromatic nitro group was explained as the interferent moiety for the ICT (Dyes Pigm. 2019, 171, 107779; New J. Chem. 2020, 44, 16265-16268; RSC Adv. 2021, 11, 8516-8520; ACS Appl. Bio Mater. 2021, 4, 2052–2057). The nitrogen cation is low electron density in NIR-HCy-NO₂ 1, however, the electron density become higher after reduction due to the donating effect of amine group. We consider that the electron delocalization in the amine structure is originated from the charge transfer between donor and acceptor. Thus, ICT concept could be sufficiently exploited to explain the fluorescence turn-off and on system for NIR-HCy-NO₂ 1 to 3.

Figure. Molecular orbital of NIR-HCy-NO₂ 1 and NIR-HCy-NH₂ 1. The calculated molecular models were from Chem3D (PerkinElmer Informatics, USA).

2. I recommend to complete the photophysical data with the fluorescence quantum yields and to summarize the spectral data (absorption wavelengths, absorption coefficients, fluorescence wavelengths, fluorescence quantum yields) in a Table in the main manuscript

>> Response: We mentioned the optical properties for NIR-HCy-NO₂ 1 to 3 in the Table S1.

3. Some parts of the manuscript (e.g. section 4.1) require a thorough English revision

>> Response: We completed the English proofreading for whole manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Hemicyanine-based near-infrared fluorescence off-on probes for imaging intracellular and in vivo nitroreductase activity

Presented by Sun Hyeok Lee et al.

The authors presented original research involving the design of three probes with a nitro group that is reduced by a process that includes NTR to the amino group on the aromatic ring. This manuscript has been previously reviewed by other colleagues, although the authors present the cover letter with the changes to improve their manuscript, it is important to clarify some points.

a) For figure 1 and figure 2, the authors must present the form of conjugation of the amine (EDG, compound red color) within the aromatic system, in this way the operation of the sensor would make more sense.

b) Figure 1 is presented in line 65 and is mentioned in the text up to lines 164 and 165. Could you mention it in lines close to 65?

c) lines 124 -127, authors must submit logP oct/water analyses, to demonstrate that the compounds are soluble in water.

d) In figure 2C, fluorophore 2 increases fluorescence 7-fold and in line 146, authors describe an increase 6-fold?.

 

e) the authors must perform the quantum yield of the three probes.

f) lines 240-241 here it is necessary to indicate references, “Thus, the NIR-HCy-NO2 1–3 had better affinity to NTR compared to the other probes reported in previous studies.”

g) in line 251, Please indicate what is the concentration? “All probes were non-toxic at high concentrations”

h) in line 254, Please indicate what is the concentration? “At a low concentration”

i) the materials and methods section (4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7) does not present references.

Please see supplementary section.

j) For the NMR studies, the authors clearly recorded the hydrogen spectra on a 400MHz equipment and the Carbon-13 (150MHz) spectra with a 600MHz equipment, so they must mention all the details of each equipment and in which university or institute it is located.

k) This reviewer does not understand why they performed the carbon-13 spectra in a different solvent than hydrogen, in some cases they used chloroform-d3 (1H-NMR) and DMSO-d6 (13C-NMR), in other cases DMSO-d6 (1H-NMR) and Methanol- d4? (13C-NMR), why is this due?

l) Authors must list the hydrogen and carbon of each compound and fully assign the 1H and 13C signals in each spectrum, especially putting special interest in the three probes since the compounds are more complex and present many signals in hydrogen and carbon-13. Two-dimensional experiments may be required (COSY, HSQC, HMBC, NOESY, etc.).

m) In the compound synthesis section, the authors only indicated the yield of compound 2 (82%), what is the yield of the rest of the compounds?

 

Author Response

Comments and Suggestions of Reviewer #2

Comment: The authors presented original research involving the design of three probes with a nitro group that is reduced by a process that includes NTR to the amino group on the aromatic ring. This manuscript has been previously reviewed by other colleagues, although the authors present the cover letter with the changes to improve their manuscript, it is important to clarify some points.

>> Response: We appreciate your feedback.

1. For figure 1 and figure 2, the authors must present the form of conjugation of the amine (EDG, compound red color) within the aromatic system, in this way the operation of the sensor would make more sense.

>> Response: We check the conjugation change from nitro group to amine group in Chem3D (PerkinElmer Informatics, USA). In NIR-HCy-NO₂ 1, the electrons accumulate on the nitro group, and after converting to amine group, the electrons delocalize on the pi conjugation. The conjugation change makes bathochromic shift and fluorescence intensity enhancement.

Figure. Molecular orbital of NIR-HCy-NO₂ 1 and NIR-HCy-NH₂ 1. The calculated molecular models were from Chem3D (PerkinElmer Informatics, USA).

2. Figure 1 is presented in line 65 and is mentioned in the text up to lines 164 and 165. Could you mention it in lines close to 65?

>> Response: We change the figure position close to figure 1-mentioned sentence.

3. lines 124 -127, authors must submit logP oct/water analyses, to demonstrate that the compounds are soluble in water.

>> Response: We tried to request logP analysis to Korean government research institute (KMEDI hub), however, minimum 100 mg is essential to perform the analysis. Unfortunately, we don’t have that much amount. We explain ‘ClogP’ which is possible to use the calculated value for ‘logP’. ClogP values for three probes (NIR-HCy-NO₂ 1 to 3) were obtained from ChemDraw Professional 15.1 (PerkinElmer Informatics, USA). The ClogP value of NIR-HCy-NO₂ 3 is the lowest (-0.578) among three of them and it means that NIR-HCy-NO₂ 3 is the most water-soluble. The calculated values are matched with our expectation. Sulfonate (-SO₃^-) and quaternary ammonium (-NMe₃⁺) were contributed to increase the water solubility of NIR-HCy-NO₂ 2 and NIR-HCy-NO₂ 3.

Figure. ClogP of NIR-HCy-NO₂

4. In figure 2C, fluorophore 2 increases fluorescence 7-fold and in line 146, authors describe an increase 6-fold?.

>> Response: The 7-fold is correct, and we change it in the sentence.

~ the fluorescence signals of all NIR-HCy-NO₂ 1–3 were enhanced 15-, 7-, and 9-fold, re-spectively, ~.

5. the authors must perform the quantum yield of the three probes.

>> Response: We calculated the quantum yields for NIR-HCy-NO₂ 1 to 3, which are mentioned in the Table S1.

6. lines 240-241 here it is necessary to indicate references, “Thus, the NIR-HCy-NO2 1–3 had better affinity to NTR compared to the other probes reported in previous studies.”

>> Response: The references were mentioned in Table S1. We change that sentence like below.

Thus, the NIR-HCy-NO₂ 1–3 had better affinity to NTR compared to the other probes re-ported in previous studies (Table S1).

7. in line 251, Please indicate what is the concentration? “All probes were non-toxic at high concentrations”

>> Response: We add the exact concentration information in the sentence.

All probes were non-toxic at high concentrations (≥ 20 μM) for cell imaging ~.

8. in line 254, Please indicate what is the concentration? “At a low concentration”

>> Response: We add the exact concentration information in the sentence.

At low concentrations (< 5 μM), NIR-HCy-NO2 1 induced ~.

9. the materials and methods section (4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7) does not present references.

>> Response: We do not have any reference for the method sections in the manuscript.

Please see supplementary section.

10. For the NMR studies, the authors clearly recorded the hydrogen spectra on a 400MHz equipment and the Carbon-13 (150MHz) spectra with a 600MHz equipment, so they must mention all the details of each equipment and in which university or institute it is located.

>> Response: We add the related information in the supporting information.

11. This reviewer does not understand why they performed the carbon-13 spectra in a different solvent than hydrogen, in some cases they used chloroform-d3 (1H-NMR) and DMSO-d6 (13C-NMR), in other cases DMSO-d6 (1H-NMR) and Methanol- d4? (13C-NMR), why is this due?

>> Response: We changed the NMR solvent in the altered NMR spectra for NIR-HCy-NO₂ probes. In the combination of CDCl₃ and DMSO-d6, we would like to make higher concentration samples for ¹³C NMR because DMSO is well known as the well-dissolved solvent for most of organic molecules.

12. Authors must list the hydrogen and carbon of each compound and fully assign the 1H and 13C signals in each spectrum, especially putting special interest in the three probes since the compounds are more complex and present many signals in hydrogen and carbon-13. Two-dimensional experiments may be required (COSY, HSQC, HMBC, NOESY, etc.).

>> Response: We add related 2D NMR spectra in the supporting information in figure S18-S26.

13. In the compound synthesis section, the authors only indicated the yield of compound 2 (82%), what is the yield of the rest of the compounds?

>> Response: We add the yield-related information for synthesized compounds.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,

In my opinion, the results given in your article are presented at a high theoretical and experimental level.

I have several non-fundamental recommendations and comments:

1. I suggest to transfer the scheme 1 from the Supplementary to the main text.

2. Figure 2 "C" indicates fluorescence enhancement of NIR-HCy-NO2 of 7 folds, while in the text on the page 5 line 146 - 6 folds.

3. Page 5, line 155 - not aminE group, but aminO group

Sincerely, Reviewer

Author Response

Comments and Suggestions of Reviewer #3

Comment: In my opinion, the results given in your article are presented at a high theoretical and experimental level. I have several non-fundamental recommendations and comments:

>> Response: Thanks for your comments.

1. I suggest to transfer the scheme 1 from the Supplementary to the main text.

>> Response: Thanks for your detailed feedback. However, we would like to more focus on the developed probes performance, not the synthesis. Additionally, it is less novel to highlight our synthesis approach in the main manuscript and we consider it as the minor claim point. It is the reason we put it in the supporting information, and we hope you respect our intention about it.

2. Figure 2 "C" indicates fluorescence enhancement of NIR-HCy-NO2 of 7 folds, while in the text on the page 5 line 146 - 6 folds.

>> Response: The 7-fold is correct, and we change it in the sentence.

~ the fluorescence signals of all NIR-HCy-NO2 1–3 were enhanced 15-, 7-, and 9-fold, re-spectively, ~.

3. Page 5, line 155 - not aminE group, but aminO group

>> Response: Thanks to your comment. However, amino group is the subcategory concept for the amine group. In the actual, amine and amino can be used as the similar meaning. For example, aniline can be expressed as ‘aminobenzene’ or ‘phenyl amine’. It seems no problem using ‘amine group’ in our manuscript. We consider that it does not make the readers confused.

 

Author Response File: Author Response.docx

Reviewer 4 Report

The manuscript described novel NIR probes (NIR-HCy-NO₂ 1-3) that introduced a nitro group to hemicyanine skeleton for fluorescence image of NTR activity including cellular and in vivo, and revealed promising prospects. NIR-HCy-NO2 1 showed best performance among three derivatives. NIR- 325 HCy-NO2 has potential to be applied to various fields related to NTR. The work is interesting and the data is solid, however, there are some issues to be clarified before it can be accepted. Specifically:

1.       Type I mitochondrial NTR is based on the bacterial origin of mitochondria, however, the viability of these probes for the detection of bacterial enzymatic activities was not assessed.

2.       Why NIR-Hcy-NH₂ 1 and NIR-Hcy-NH₂ 2 reacted with NTR in PBS and ACN solvent systems?

3.       For the cytotoxicity assay, co-incubation time with different concentrations of NIR-HCy-NO₂ 1-3 was only 1 hour, which should be extended, at least 24 or 48 h.

4.       The NIR-Hcy-NH₂ 1-3 obtained by reduction were not characterized by NMR.

5.       There are some typos.

Author Response

Comments and Suggestions of Reviewer #4

Comment: The manuscript described novel NIR probes (NIR-HCy-NO2 1-3) that introduced a nitro group to hemicyanine skeleton for fluorescence image of NTR activity including cellular and in vivo, and revealed promising prospects. NIR-HCy-NO2 1 showed best performance among three derivatives. NIR- 325 HCy-NO2 has potential to be applied to various fields related to NTR. The work is interesting and the data is solid, however, there are some issues to be clarified before it can be accepted. Specifically:

>> Response: I appreciate your interesting and the positive feedback for our study.

1. Type I mitochondrial NTR is based on the bacterial origin of mitochondria, however, the viability of these probes for the detection of bacterial enzymatic activities was not assessed.

>> Response: Thanks for your comment. For type I nitroreductase, it is considered as the bacteria origin due to the similar properties to the bacterial NTR. However, it does not mean that type I NTR and bacterial NTR, both are fully same. Despite, we consider that it has the potential to also detect the bacterial NTR because of the similarity.

In the manuscript, we already checked the possibility for the bacterial NTR detection. For in vitro experiments, we used NTR supplied from Sigma Aldrich and the company provides the specific gene information for nitroreductase (cat. N9284) on the website. The gene name is ‘NfsB’ which is bacterial NTR. It means that we confirmed the reactivity with bacterial NTR in the in vitro section.

 

2. Why NIR-Hcy-NH2 1 and NIR-Hcy-NH2 2 reacted with NTR in PBS and ACN solvent systems?

>> Response: NIR-HCy-NO₂ 1 and NIR-HCy-NO₂ 2 have relatively non-polar structure compared to NIR-HCy-NO₂ 3. It was considered to select the solvent system for in vitro experiments, and we decided the cosolvent system using ACN as the organic solvent. Although we used the cosolvent system for in vitro section, all DMSO stocks for the probes can be available to dilute it with PBS at the low concentration for cell and in vivo experiments.

3. For the cytotoxicity assay, co-incubation time with different concentrations of NIR-HCy-NO2 1-3 was only 1 hour, which should be extended, at least 24 or 48 h.

>> Response: As per reviewer’s valuable suggestion, we have analyzed the effects of NIR probes on cell viability after 24 hours treatment. At the concentration range of 0.5 to 20 µM, NIR-HCy-NO₂ 1 reduced the cell viability in a concentration-dependent manner; however, NIR-HCy-NO₂ 2 and 3 did not affect. Even though the cell viability was not changed by 1 hour treatment of NIR-HCy-NO₂ 1 for detection of fluorescence images (Fig. S29), the extended treatment of NIR-HCy-NO₂ 1 resulted in inhibition of cell viability indicating that the reduced NIR-HCy-NO₂ 1 by NTR may have induced cell toxicity. Further research is warranted to understand the exact mechanism of action.

Figure. Cell cytotoxicity test of NIR-HCy-NO₂ 1-3. Cell viability of A549 treated with various concentrations of NIR-HCy-NO₂ 1-3 for 24 hours. (A) NIR-HCy-NO₂ 1 in DMEM (10% FBS, 1% P/S), (B) NIR-HCy-NO₂ 2 in DMEM (10% FBS, 1% P/S), (C) NIR-HCy-NO₂ 3 in DMEM (10% FBS, 1% P/S).

4. The NIR-Hcy-NH2 1-3 obtained by reduction were not characterized by NMR.

>> Response: We add new NMR spectra for the mentioned compounds in figure S27-S29.

5. There are some typos.

>> Response: We completed the English proofreading for the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

the authors improved both the manuscript and the supplementary material

Reviewer 4 Report

With all the improvements and revisions the authors have made, this work could now be accepted for the publication.