Experimental and Theoretical Studies on DNA Binding and Anticancer Activity of Nickel(II) and Zinc(II) Complexes with N– (8–Quinolyl) Salicylaldimine Schiff Base Ligands
, Ratanon Chotima 2, Malinee Promkatkaew 1, Sunan Kitjaruwankul 1, Kittipong Chainok 3 and Teerawat Khudkham 2Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsTwo new organic compounds 5-bromo-N-(8-quinolyl)salicylaldimine and 3,5-dibromo-N-(8-quinolyl)salicylaldimine were synthesized and characterized by different spectroscopic techniques. The newly synthesized organic compounds were used as ligands to prepare three new Ni(II) and Zn(II) complexes. The structures of the metal complexes were proved by IR, UV-Vis, TGA, NMR and mass spectral analyses. The molecular structures of ligand HL2 and complex 3 were investigated by X-ray crystallography. The structures of ligands and complexes 1-3 were also optimized by DFT analysis.
I have some notes and remarks
1. I recommend that the authors compare the X-ray analysis data for bond lengths, angles, etc. of HL2 and Ni-HL2. Instead of Table 5, a new table could be constructed in which the data for bond lengths, angles, etc. of HL2 could be compared with those of complex 3.
2. It would be useful to comment on the structures of the other two complexes based on the molecular structure of complex 3.
3. In section 3. Results, 3.3. Thermogravimetric analysis it is commented that the new complexes 1-3 are decomposed in three steps. But the products obtained are not given correctly. How does this relate to the other analyses?
4. The cytotoxic activity of the newly synthesized compounds was evaluated on only one human tumor cell line. I recommend that more cell lines be investigated. My question is how exactly does one determine which cell line to use?
Comments on the Quality of English Language
Moderate editing of English language required.
Author Response
Comment 1: I recommend that the authors compare the X-ray analysis data for bond lengths, angles, etc. of HL2and Ni-HL2. Instead of Table 5, a new table could be constructed in which the data for bond lengths, angles, etc. of HL2 could be compared with those of complex 3.
Response 1: I have already constructed a new table that includes the comparative data for HL2 and complex 3, replacing the current Table 5.
Comment 2: It would be useful to comment on the structures of the other two complexes based on the molecular structure of complex 3.
Response 2: Thank you for your feedback. Based on your suggestion, in addition to the comments on the structures, I have also expanded the discussion on how the structures of these two complexes lead to discrepancies between computational calculations and experimental results. More detailed analyses have been already added to the manuscript (line 481), and some selected bond lengths [Å], angles [°] and torsion angles [°] for complex 1 from the crystallographic data have been presented in the supporting information.
Comment 3: In section 3. Results, 3.3. Thermogravimetric analysis it is commented that the new complexes 1-3are decomposed in three steps. But the products obtained are not given correctly. How does this relate to the other analyses?
Response 3: Initially, when the crystallization of these complexes presented difficulties, we employed TGA as one of the techniques to verify formation of the synthesized complexes. From the results, we observed that complexes 1 and 2 yield residues consisting of metal oxide and carbon atoms. For complex 1 with the formula C32H20Br2N4NiO2, the decomposition products include Br2, C9H2NO, C20H18N3, and NiO. I would like to clarify that the notation of "C atoms" refers to residual carbon ash that could not be further identified or assigned a specific molecular formula.
Thereafter, we eventually succeeded in crystallizing complex 1, however; this structure does not meet the publication standards according to the CheckCIF reports, so it is not included in this manuscript. For complex 2 with Zn(II) metal center, we provided NMR spectrum result, which correspond to the formation of this complex. Therefore, I would like to confirm that the TGA results are in agreement with other analytical techniques, supporting the successful synthesis and characterization of the complexes.
If there are any specific points you would like me to focus on, please let me know.
Comment 4: The cytotoxic activity of the newly synthesized compounds was evaluated on only one human tumor cell line. I recommend that more cell lines be investigated. My question is how exactly does one determine which cell line to use?
Response 4: The reasons for determining this cell line are added in the sentences before the last paragraph of the introduction section as highlighted.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsReviewer:
Recommendation: This paper may be publishable after major revision; I would like to be invited to further review.
Comments:
Pinchaipat et al. conducted synthesis, characterization and studies on the interaction with calf thymus DNA, CT-DNA and application in anticancer activity. They have successfully synthesized two different nickel compounds along with one zinc compound, which were then characterized by various techniques including IR, mass, NMR, TGA, DFT, and X-ray crystallography. Their steady-state spectroscopy (UV-Vis and fluorescence) results and molecular docking studies suggests that intercalation mode of binding occurs between newly synthesized metal (nickel and zinc) compounds with the CT-DNA. Based on the binding energy and association binding constant (Kb) values, it appears that nickel compound 3, [Ni(qsalBr2)2] has higher tendency to bind with CT-DNA, whereas zinc compound 2, [Zn(qsalBr)2], has greater potential to bind with human lung cancer cells.
These remarkable findings could significantly advance new drug design, synthesis, and cancer therapy, in addition to contributing to oncology studies.
Overall, the methodology and findings are clearly articulated, and the presentation is coherent and well-organized.
This research represents a potentially significant contribution to anticancer therapy. The paper merits publication after major revisions.
Regarding questions, I would like the authors to address the following queries prior to the acceptance of this manuscript:
1. I am curious about the stability of the duplex DNA upon intercalating binding with the newly synthesized compounds 1, 2 and 3. I strongly suggest that the authors assess the thermal stability of duplex DNA by measuring the melting temperature in the absence and presence of the newly synthesized compounds 1, 2 and 3.
2. In section 3.8.1, the statement reads, “In addition, a red shift also indicates the stability of DNA base pairs [27].” It is unclear what stability the author is referring to. Is it the stability of the newly synthesized compounds 1, 2 and 3 or the stability of the double-stranded DNA? Please clarify this statement.
3. From Figure 8 and Table 7, the authors have estimated the quenching constant (Kq) between the Ethidium Bromide-CT DNA (EB-DNA) complex and newly synthesized compounds 1, 2 and 3. For estimating Kq, knowledge of the excited state average lifetime of the EB-DNA complex is required. However, the average lifetime of EB-DNA complex was not measured throughout the manuscript. I suggest that the authors measure and provide the lifetime of these EB-DNA complex. If they have estimated these lifetime values somewhere, they should provide these parameters.
4. Throughout the manuscript, there is inconsistency concerning the terms “Luminescence” and “Fluorescence,” which are quite different. I recommend using one consistent term throughout.
5. There are several spelling mistakes. Please go through the manuscript thoroughly.
6. It is important to understand the stoichiometric relationship between a ligand and DNA in ligand-DNA interactions. What is the stoichiometry between the newly synthesized compounds 1, 2, and 3 and CT-DNA? You could determine this by constructing a Job's plot for the interaction of DNA with each of the newly synthesized compounds 1, 2, and 3. Please provide in the manuscript.
Comments for author File: 
Comments.pdf
Author Response
Comment 1: I am curious about the stability of the duplex DNA upon intercalating binding with the newly synthesized compounds 1, 2 and 3. I strongly suggest that the authors assess the thermal stability of duplex DNA by measuring the melting temperature in the absence and presence of the newly synthesized compounds 1, 2 and 3.
Response 1: We appreciate the reviewer’s suggestion to assess the thermal stability of duplex DNA by measuring the melting temperature in the absence and presence of the newly synthesized complexes 1-3. While we recognize the importance of these measurements, due to time constraints, we were unable to conduct the full set of melting temperature experiments within the current study. Therefore, we have referenced relevant studies.
The attempt to relate existing research to this point, we have found a study on the thermal denaturation of calf thymus DNA by H.J. Li, B. Brand, and A. Rotter (1974)[1]. This research revealed a major melting band at approximately 81°C with a shoulder around 55°C. In the presence of polylysine, polylysine-DNA complexes exhibited significant melting at 106.5°C and a smaller fraction at 50°C. These results suggested that the compounds can selectively stabilize different regions of DNA, leading to distinct melting behaviors.
In our study, preliminary binding studies and spectroscopic analyses have shown strong interactions between our synthesized compounds and DNA, suggesting similar stabilization effects. Based on this evidence, it is reasonable to predict that the melting temperature of DNA upon intercalation with the newly synthesized compounds 1, 2, and 3 might be similar to previous results, where the complexes caused an increase in the DNA melting point. Additionally, due to selective stabilization, we may observe a change with a shoulder around 55°C.
We agree that including detailed thermal stability data in future studies will provide valuable insights into the binding interactions and enhance the strength of our findings. We plan to conduct these experiments to further elucidate the effects of our complexes on DNA stability.
[1] Li, H.J.; Brand, B.; Rotter, A. Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction. Nucleic Acids Res. 1974, 1(2), 257-65. https://doi.org/10.1093/nar/1.2.257
Comment 2:In section 3.8.1, the statement reads, “In addition, a red shift also indicates the stability of DNA base pairs [27].” It is unclear what stability the author is referring to. Is it the stability of the newly synthesized compounds 1, 2 and 3 or the stability of the double-stranded DNA? Please clarify this statement.
Response 2: The stability referred here involves the double-stranded DNA. A red shift in spectroscopic studies indicates a change in the electronic environment of the DNA base pairs, suggesting interactions that stabilize the DNA structure. The complexes contribute to this stabilization of the DNA structure by intercalating or binding to the DNA, causing a red shift in the spectra.
Therefore, I have already changed the sentence to: “In addition, a red shift in spectroscopic studies indicates a change in the electronic environment of the DNA base pairs, suggesting that the synthesized complexes contribute to the stabilization of the DNA structure.” as highlighted.
Comment 3: From Figure 8 and Table 7, the authors have estimated the quenching constant (Kq) between the Ethidium Bromide-CT DNA (EB-DNA) complex and newly synthesized compounds 1, 2 and 3. For estimating Kq, knowledge of the excited state average lifetime of the EB-DNA complex is required. However, the average lifetime of EB-DNA complex was not measured throughout the manuscript. I suggest that the authors measure and provide the lifetime of these EB-DNA complex. If they have estimated these lifetime values somewhere, they should provide these parameters.
Response 3: The parameter has been successfully added in line 187 as highlighted.
Comment 4:Throughout the manuscript, there is inconsistency concerning the terms “Luminescence” and “Fluorescence,” which are quite different. I recommend using one consistent term throughout.
Response 4: All ‘Luminescence’ terms have been changed to ‘Fluorescence’, which is directly related to the experimental conditions.
Comment 5:There are several spelling mistakes. Please go through the manuscript thoroughly.
Response 5: I thoroughly reviewed the manuscript for any spelling mistakes and correct them.
Comment 6:It is important to understand the stoichiometric relationship between a ligand and DNA in ligand-DNA interactions. What is the stoichiometry between the newly synthesized compounds 1, 2, and 3 and CT-DNA? You could determine this by constructing a Job's plot for the interaction of DNA with each of the newly synthesized compounds 1, 2, and 3. Please provide in the manuscript.
Response 6: To determine the stoichiometry between the synthesized complexes and CT-DNA, fluorescence measurements and a modified Scatchard analysis were employed, as shown in equation 10. The results have already been added to the manuscript (line 445) as highlighted. The plots for the determination of the number of binding sites are also presented in the supporting information.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThis research article will published in its current form.
