Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript titled: "Comprehensive Structural, Electronic, and Biological Characterization of fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br]: X-ray, Aromaticity, Electrochemistry, and HeLa Cell Viability" by authors Alexander Carreño et. al. is well designed, organized, the applied methods were well executed and results confirm the experimental assumptions. 

In my opinion the manuscript should be accepted after some minor corrections.

I am aware of the fact that synthesis follow literature procedure but it would be helpful if authors add reaction scheme and label C atoms at complex structure. In the NMR spectra authors should add integrals values, and in the Experimental and Theoretical Details (Synthesis of fac-[Re(CO)3(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br] (ReL)) authors should add numbers of atoms for ppm values (for example: δ = 9.10 [d, J=4.4Hz, 1H, C1] which is in accordance with labeled chemical structure. 

 

Author Response

 

Dear Referees,

We appreciate your thorough review of the manuscript and your valuable suggestions. Your comments have been very helpful in improving the quality and clarity of our work. We have provided appropriate responses and implemented the corresponding changes for each of your questions and recommendations.

Best Regards

The authors

 

Referee 1

COMMENT 1:

Manuscript titled: "Comprehensive Structural, Electronic, and Biological Characterization of fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br]: X-ray, Aromaticity, Electrochemistry, and HeLa Cell Viability" by authors Alexander Carreño et. al. is well designed, organized, the applied methods were well executed and results confirm the experimental assumptions.

In my opinion the manuscript should be accepted after some minor corrections.

I am aware of the fact that synthesis follow literature procedure but it would be helpful if authors add reaction scheme and label C atoms at complex structure. In the NMR spectra authors should add integrals values, and in the Experimental and Theoretical Details (Synthesis of fac-[Re(CO)3(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br] (ReL)) authors should add numbers of atoms for ppm values (for example: δ = 9.10 [d, J=4.4Hz, 1H, C1] which is in accordance with labeled chemical structure.

RESPONSE TO COMMENT 1:

Response: We sincerely appreciate your positive feedback regarding the design, organization, and execution of the methods, as well as your observation that the results confirm the experimental assumptions. Thank you for recognizing these aspects of our work.

In line with your suggestion, we have made the following improvements in the revised manuscript: the complex structure with labeled carbon atoms has been added to Figure 1, and the reaction scheme for the synthesis of ReL is now included in the Experimental Section as Figure 11. We believe these additions enhance the clarity and completeness of the manuscript.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Review on:

 Comprehensive Structural, Electronic, and Biological Characterization of fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br]: X-ray, Aromaticity, Electrochemistry, and HeLa Cell Viability

The article discusses the synthesis, characterization, electrochemical analysis, computational and cytotoxic study of one compound, fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br].

The article is well written and contains a significant amount of work, albeit only focused on one compound, which is somewhat concerning. Usually, a series of similar compounds is discussed. But I will refer this point to the editor to decide whether the study of one compound is adequate.

Figure 1 shows the electrochemical reaction scheme of the electrochemical part of the study. This should be shown with the electrochemical study and not at the beginning of the paper. Showing the structure of the compound is relevant here, but the reaction scheme should be moved to later in the article.

The NMR’s shown in the supplementary material does not show the detail discussed in the paper. Particularly comparison made between the cis and trans forms in figures S3 – S9. The scale of the spectra is not consistent and you cannot see what the authors are trying to convey. Please display all spectra on a similar size level and show inserts of zoomed in peaks to display the detail discussed.

Figure S19 shows the CV of ferrocene, which is used as internal standard. Ferrocene is advised by IUPAC to be used as internal standard, because it conforms to certain requirements, which includes electrochemical reversibility. This is described by a ∆E value of less than 90 mV, however the CV shown displays a Epa of 0.752 V and an Epc of 0.396. This gives a ∆E of 356 mV, which is too large. Please clarify the discrepancy here. Something is troublesome with this CV.

Additionally, it should be used as an internal standard, yet no CV is shown where ferrocene is used as internal standard. This must be shown, even if just in the supplementary material.

P11 discusses the electrochemical study. It states that various oxidation and reduction processes were observed … as a result of byproducts. However, this is not completely true and can be clarified a bit more, since the computational work is included anyway. Also, it will be much more relevant to show and explain the structures shown in figure 1 here.

Two additional reduction waves are seen at aprox.  0.9 and 0.5 V. These should be explained. In the working window study, it can be clarified that reduction wave at 0.9 V is related to the second oxidation at 2.1 V, while the reduction at 0.5 V is related to the oxidation at 1.6 V. This is still not considered the reverse of the oxidation waves, but only reduction waves associated with the newly formed species as discussed in the text.

On p13 reference is made to figure 21, this should be figure S21.

On page 15, references are listed incorrectly. Please check reference manager.

It is reported that the ReL complex has a concentration dependent cytotoxic effect. Please calculate the IC50 value of this and report it.

Author Response

 

Dear Referees,

We appreciate your thorough review of the manuscript and your valuable suggestions. Your comments have been very helpful in improving the quality and clarity of our work. We have provided appropriate responses and implemented the corresponding changes for each of your questions and recommendations.

Best Regards

The authors

 

Referee 2

COMMENT 1:

Comprehensive Structural, Electronic, and Biological Characterization of fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br]: X-ray, Aromaticity, Electrochemistry, and HeLa Cell Viability

The article discusses the synthesis, characterization, electrochemical analysis, computational and cytotoxic study of one compound, fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br].

The article is well written and contains a significant amount of work, albeit only focused on one compound, which is somewhat concerning. Usually, a series of similar compounds is discussed. But I will refer this point to the editor to decide whether the study of one compound is adequate.

RESPONSE TO COMMENT 1:

We appreciate your thorough review of the manuscript and your valuable observations. This work represents a new and significantly expanded study, continuing our previous research on the rhenium(I) tricarbonyl complex fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br] (ReL), which had shown promising luminescent properties for staining walled cells. In this study, we provide a comprehensive characterization of ReL using advanced techniques not previously reported. Single-crystal X-ray diffraction confirmed its pseudo-octahedral geometry, while DFT calculations and aromaticity analysis revealed minimal electronic differences between cis and trans isomers. Electrochemical studies through cyclic voltammetry identified two oxidation and two reduction events, including a Re(I) → Re(0) transition, with theoretical redox potentials in agreement with experimental data. Finally, biological assays demonstrated a dose-dependent cytotoxic effect on HeLa cells, contrasting with its previously reported low toxicity in microbial systems. These findings highlight ReL’s multifunctional nature and its potential as a bioactive and imaging agent. Although focused on one neutral rhenium tricarbonyl complex, this work is, to the best of our knowledge, the first report of efficient staining of walled cells using such complexes, underscoring the novelty and relevance of our study.

 

COMMENT 2:

-Figure 1 shows the electrochemical reaction scheme of the electrochemical part of the study. This should be shown with the electrochemical study and not at the beginning of the paper. Showing the structure of the compound is relevant here, but the reaction scheme should be moved to later in the article.

RESPONSE TO COMMENT 2:

Response: The structure of the compound has been included as the new Figure 1, and the electrochemical reaction scheme has been relocated to the end of the electrochemical section of the manuscript.

 

COMMENT 3:

-The NMR’s shown in the supplementary material does not show the detail discussed in the paper. Particularly comparison made between the cis and trans forms in figures S3 – S9. The scale of the spectra is not consistent and you cannot see what the authors are trying to convey. Please display all spectra on a similar size level and show inserts of zoomed in peaks to display the detail discussed.

RESPONSE TO COMMENT 3:

Response: Done in the manuscript

 

 

 

 

 

COMMENT 4:

-Figure S19 shows the CV of ferrocene, which is used as internal standard. Ferrocene is advised by IUPAC to be used as internal standard, because it conforms to certain requirements, which includes electrochemical reversibility. This is described by a ∆E value of less than 90 mV, however the CV shown displays a Epa of 0.752 V and an Epc of 0.396. This gives a ∆E of 356 mV, which is too large. Please clarify the discrepancy here. Something is troublesome with this CV.

-Additionally, it should be used as an internal standard, yet no CV is shown where ferrocene is used as internal standard. This must be shown, even if just in the supplementary material.

RESPONSE TO COMMENT 4:

Response: We appreciate your valuable comment and suggestion. The reviewer is correct: ferrocene was not used as an internal reference in solution as it should have been. The potentials reported in the manuscript are referenced to an Ag/AgCl indicator electrode, as described in East & del Valle (J. Chem. Educ., 2000, 77, 97), now explicitly cited in the Experimental Section. Ferrocene measurements were included only to confirm the proper behavior of this reference electrode; however, they are not required for the analysis or discussion of the results and have therefore been removed in the revised version.

 

COMMENT 5:

-P11 discusses the electrochemical study. It states that various oxidation and reduction processes were observed … as a result of byproducts. However, this is not completely true and can be clarified a bit more, since the computational work is included anyway. Also, it will be much more relevant to show and explain the structures shown in figure 1 here.

RESPONSE TO COMMENT 5:

Response: Figure 1 has been moved to this section as recommended by the reviewer and nor is used to summarize the electrochemical behavior description made.

 

 

 

COMMENT 6:

-Two additional reduction waves are seen at aprox.  0.9 and 0.5 V. These should be explained. In the working window study, it can be clarified that reduction wave at 0.9 V is related to the second oxidation at 2.1 V, while the reduction at 0.5 V is related to the oxidation at 1.6 V. This is still not considered the reverse of the oxidation waves, but only reduction waves associated with the newly formed species as discussed in the text.

RESPONSE TO COMMENT 6:

The reviewer suggestion is right. Further discussion of these signals has been included and not only regarded to byproducts as recommended. Please check discussion at page 11, lines 326 to 341.

 

COMMENT 7:

-On p13 reference is made to figure 21, this should be figure S21.

-On page 15, references are listed incorrectly. Please check reference manager.

RESPONSE TO COMMENT 7:

Response: Done in the manuscript

 

COMMENT 8:

-It is reported that the ReL complex has a concentration dependent cytotoxic effect. Please calculate the IC50 value of this and report it.

RESPONSE TO COMMENT 8:

Response: We appreciate your comment regarding the cytotoxicity results. In this study, ReL exhibited a clear dose-dependent effect on HeLa cells, with an IC₅₀ of 182.31 μM, significantly higher than cis-platin and much higher than the IC₅₀ values typically reported for cationic rhenium(I) tricarbonyl complexes. This relatively low cytotoxicity indicates that neutral ReL meets key requirements for an effective luminescent probe, including minimal toxicity and efficient cellular uptake without permeabilizing agents, making it suitable for future bioimaging experiments in epithelial cell models. Previous studies have also shown that ReL interacts effectively with walled cells such as Gram-negative bacteria and yeasts, displaying minimal toxicity and enabling direct microbial imaging. These findings highlight the multifunctional nature of ReL and, in our view, contribute to advancing the study of neutral rhenium tricarbonyl species as novel fluorophores for potential biotechnological applications. Furthermore, a detailed discussion reinforced with specific literature on rhenium tricarbonyl complexes has been added to the revised manuscript to provide broader context and support for these observations.

Author Response File: Author Response.docx