Photo-Tunable Azobenzene-Anthraquinone Schiff Base Copper Complexes as Mediators for Laccase in Biofuel Cell Cathode
Round 1
Reviewer 1 Report
There are many sections in the manuscript that are poorly written, due to both language and scientific presentation.
Here are some issues which should be corrected before a proper review:
1. Reference 3 is missing! Actually it is the template style. For example Gaussian is cited in text as 30, however it is actually reference 29. Reference 44 and 45 are actually one citation split in two.
2. Line 46: how can molecular docking predict redox activity?
3. Line 60: label of Figure 1 - that is the structure of the enzyme.
4. Line 120: DFT spectra should be compared with experimental ones and inserted in ESI.
5. Line 135: "geometries ... were similar to DFT structures" - this should be supported by a table where the corresponding bond angles and lengths are compared.
6. Line 239: it should be "hydrogens (probably polar) were added", not "hydrogen bonds"
7. Line 240: the grid box definition is unclear - does 30 represents the number of points (in which case it is not Angstroms, since the spacing is 0.375) or the size of the cell?
8. The docking results are unclear: first, it is mentioned that the coordination geometry is square planar. However figure 9 doesn't suggest this. On the other hand it is difficult to observe, if the docked ligands incorporate Cu atoms?
Line 253: the listed amino acids (his395, etc.) are not visible in figure 9.
Figure 9 should focus on the ligand-receptor interactions, only (b,d and f) should be presented in a clear manner. Some suggestions: labels are huge, and should be positioned, backbone atoms should be hidden.
Table 2: interactions should be discussed in text.
Figure 10 is not cited in text. Mediator is not colored in grey! How are the Cu atoms involved in the mediator orientation? If so, then why are they not present in figure 9?
Author Response
April 23, 2020
Dear The editor of Symmetry:
Please find an attached the revised manuscript of article symmetry-788035.
The answers for query and revised points of the text (which have been written in the revised manuscript as red letters) are as follows:
<Editors>
Q1. Please revise the manuscript carefully to ensure the consecutive words plagiaristic are less than 12 within your revision.
A1. According to your comment, we fixed the sentences. However, references and acknowledgement could not be fixed.
Q2. Abstract section of papers so that readers could know clearly the paper's relevance to our journal Symmetry when they start to read the papers.
A2. According to your comment, we added this explanation associating with chirality (induced CD namely chiroptical spectroscopy) of biomolecules in abstract. “Induced chirality (achiral target in chiral matrix such as proteins) sometimes play a useful role in evaluation of supramolecular systems involving biomolecules.”
<Reviewer1>
Q1. Reference 3 is missing! Actually it is the template style. For example Gaussian is cited in text as 30, however it is actually reference 29. Reference 44 and 45 are actually one citation split in two.
A1. Thank you. According to your comment, we fixed references.
Q2. Line 46: how can molecular docking predict redox activity?
A2. According to your opinion, we changed “redox activity” into “the distance or path of electron transfer”.
Q3. Line 60: label of Figure 1 - that is the structure of the enzyme.
A3. According to your comment we added “(the structure of) the enzyme” in Figure 1’s legend.
Q4. Line 120: DFT spectra should be compared with experimental ones and inserted in ESI.
A4. Because DFT results were used mainly for obtaining optimized structures, we omitted the description about UV-vis spectra around Line 120.
Q5. Line 135: "geometries ... were similar to DFT structures" - this should be supported by a table where the corresponding bond angles and lengths are compared.
A5. As your comments, strictly speaking, crystal structures, structures in solutions, and DFT optimized structures are different from each other. Therefore, the similarity of “overall structure” was stated in the text, and we fixed the sentences.
Q6. Line 239: it should be "hydrogens (probably polar) were added", not "hydrogen bonds"
A6. According to your comment, we fixed as such.
Q7. Line 240: the grid box definition is unclear - does 30 represents the number of points (in which case it is not Angstroms, since the spacing is 0.375) or the size of the cell?
A7. According to your comments, we fixed to “points”.
Q8. The docking results are unclear: first, it is mentioned that the coordination geometry is square planar. However figure 9 doesn't suggest this. On the other hand it is difficult to observe, if the docked ligands incorporate Cu atoms?
A8. The docking results are about the best-fit ligand conformations which were selected based on their minimum binding energies. Complex 1 (ligand1) showed the highest docking score, i.e. -10.5 kcal/mol (-8.6 and -9.9 kcal/mol for 2 and 3, respectively).Since ligand1 has the most negative energy -10.5 kcal it is found to be best-docked molecule.
According to your comments, we added Figure 9(g). Because of optimized structure, Cu complex afford a slightly distorted planar coordination environment.
Q9. Line 253: the listed amino acids (his395, etc.) are not visible in figure 9.
A9. According to your comments, we re-drew Figure 9(g) with clear marked amino acids. Amino acids (his395) is not bonded with the three copper ligands so it was not shown on figure 9. May be reviewer wanted to pointed out figure number 10 where his395 is shown ,but there also it was attached to the laccase’s copper not to the copper ligand.
Q10. Figure 9 should focus on the ligand-receptor interactions, only (b,d and f) should be presented in a clear manner. Some suggestions: labels are huge, and should be positioned, backbone atoms should be hidden.
A10. According to your comments, we added re-drawn Figure 9(g) using bold models.
Q11. Table 2: interactions should be discussed in text.
A11. According to your comments, we added “Interaction were not only predominanotly hydrogen bonds but also hydrophobic or electrostatic ones, as mentioned in Table 2.”.
Q12. Figure 10 is not cited in text. Mediator is not colored in grey! How are the Cu atoms involved in the mediator orientation? If so, then why are they not present in figure 9?
A12. According to your comment, we omitted unimportant color in Figure 10’s legend, and sited in text with a sentence of “Electron transfer from mediator to copper sites are shown in Figure 10, in which redox properties of each metal sites are important.”.
Figure 10 is now cited in text. Mediator is colored in black, change in inserted in manuscript. Cu atoms are present in figure 9(a,c,e) in the form of sky blue balls.
<Reviewer 2>
Q1. In line 200-201 the authors claim: “Moreover, the reversible cis-trans photoisomerization, which converts the cis- to the trans-form by visible light irradiation, was observed for all complexes, although the photoisomerization for 3 did not occur as smoothly compared to that of 1 or 2” The phenomenon strictly related to the molecular structure should be deepened, the results explained. Also in the Introduction, the photochromic (azobenzene) moiety was cited without delving into these important features. In this regard it is suggested to add recent specific literature such as: A symmetrical azo-based fluorophore and the derived salen multipurpose framework for emissive Layers (2019) Inorganic Chemistry Communications, 104, pp. 186-189. Spectroscopic behaviour of two novel azobenzene fluorescent dyes and their polymeric blends (2020) Molecules, 25 (6), art. no. 1368.
A1. Thank you. We added suggested reference papers and explanation concerning azobezene just after the part of Line 200-201 (not introduction) as follows: “Azobenzene dyes ending with electron-withdrawing or donor groups were reported to shift absorption or emission wavelengths and even affect on photoisomerization.”.
Q2. Again, in the Introduction, some words should be spent to present the relevant group of such salen Cu(II) complexes adding recent related literature.
A2. According to your comment, we referred a recent review about salen complexes and added the sentence, “Generally, (chiral) salen metal complexes have been employed as (asymmetric) catalysis because of their advantage of stereochemical tuning (with respect to chirality) and redox properties with proper metal ions”.
Q3. In line 93 apparatus for register fluorescence spectra was mentioned but no fluorescence data have been presented. Have the ligands and complexes emission properties to be underlined?
A3. Thank you. According to your comment, fluorescence spectra was omitted.
Q4. The theoretical studies, central tools of the work, suggesting that the electron transfer was related not only to electronic states but to the molecular orientation provide interesting but not definitive conclusions.
A4. According to your comments, we rephrased the sentence before this part as “Even the theoretical studies can investigate electronic structures of photoisomers at molecular level, experimental results herein also suggested that…”.
Q5. All in all, the work is well presented, the conclusions are rather clear and consequential, even if some aspects are not completely resolved. English needs to be improved.
A5. Thank you for suggestion for future works. Prior to first submission, English was corrected by a native professional editor.
Q6. In addition, there are some errors in the references. In particular: ref 3 is empty; the name of the first author of ref 40 it’s wrong; ref 42 it’s strange; ref 45 does not exist, it is the continuation of ref 44.
A6. Thank you. We fixed these references.
<Reviewer 3>
Q1. My major concern with the article is the following. Despite the fact that the Authors made several experiments to synthetize, investigate and evaluate the current Cu-salen-laccase systems, the last part of the article cannot summarize sufficiently the findings, and cannot put this research in context. There are several sentences in this part that were copied from p1. without modification (p. 16. l. 341-347.). The conclusions are soft or even pointless in a few instances.
A1. According to your opinion, we rewrote the conclusion part.
Q2. Which one from the three complexes is the best mediator for laccase? Why?
A2. In conclusion, we added the comparison as “Among three complexes, 1 without disturbing substituent groups of electronic effects was comprehensively suitable for this purpose.”
Q3. How does these results compare to other salen-type mediators having related structure?
A3. Typical other metal complexes (ferrocene and [Fe(CN)6]3-/4-) for mediators and merits of these complexes were added in conclusion. To our knowledge, however, there were few study of other salen-type mediators.
Q4. How does these results compare to the best-in-class mediators?
A4. In conclusion, we added the comparision as “Besides these types of potential mediators or common organic mediators, the present azo-anthraquinone salen Cu complexes was confirmed to have advantage of photo-tuning their electronic states and spatial orientation.”.
Q5. Based on the current findings, what are the next steps to improve the performance of these mediators? (There are several sites on the mediators that can be tweaked by functional groups)
A5. We have also submitted this special issue of “Symmetry” a modified electrode of laccase-mediator systems connected to enantiomers of oligopeptides for spin filters as a next step, though it is under review now, too.
Q6. All data of Table 1 and Fig 6 is necessary, or some of this data can be put to the Supporting Information?
A6. According to your comment, we moved Table 1 and Fig. 6 (DOS) to Supporting Information.
Q7. Table 2 having a length of 2 pages in unnecessary in its current form. It can be shortened, and the rest of the data could be moved to the Supporting Information. Or the whole table can be moved to the supporting information.
A7. According to your comment, we moved a part of Table 2 to Supporting Information.
Q8. Maybe more emphasis should be put on the part of the ms which are in strong correlation with the aims and scope of the journal.
A8. In abstract, we wrote a sentence to emphasis symmetry (chirality and induced CD for supramolecular docking) aspect of this study to meet the aims and scope of the journal. And experimental section was also modified slightly.
Q9. 1. ‘tested for use as mediators’ ‘In the following ways will be explored to solve the disadvantage of low power.’ ‘Generally, a redox substance called a mediator – commas should be added’‘assists to donate and receive – assists the donation and reception’ etc.
A9. According to your comments, we fixed the representation.
Q10. 2. l. 50. References 16, 17 are in wrong order.
A10. Thank you. We fixed the order.
Q11. p.3. l. 82. The amount of hexane used for recrystallization is missing.
A11. Thank you. We added this information (starting amount for evaporating and cooling).
Q12.3. l. 83-85. There is no point to show the first decimals of the yields.
A12. Thank you. We removed them.
Q13. 10. The reference for Fig. 10 is missing in the corresponding text.
A13. Thank you. We added text for Fig. 10.
Q14. Fig 2. The N=N double bond is not visible.
A14. Thank you. We re-drew Fig.2.
Q15. Fig 7. The blue line is barely visible.
A15. The explanation of “Slow photoisomeriztion resulted in overlap of red and bule lines in Figure 7.” was added in text for this situation.
Q16. Fig 9. The pink colored molecules are barely visible in the pocket. Another color and better contrast is needed.
A16. According to your comment, Fig.9 (g) was re-drawn.
Q17. References: Ref. 3 is missing.
A17. Thank you. We added ref.3.
Q18. Ref. 16. There is an unnecessary underline.
A18. Thank you. We removed it.
That’s all.
We would like to say thanks in advance for your prompt treatment of our manuscript.
Best regards,
Prof. Dr. Takashiro Akitsu
Department of Chemistry, Faculty of Science, Tokyo University of Science
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Tel. +81-3-5228-8271, Fax. +81-3-5261-4631
E-mail: [email protected]
Reviewer 2 Report
Three new azo-anthraquinone salen Cu(II) complexes for cathodic compartment of enzymatic biofuel cells were designed and tested as a mediator. The Schiff base ligands consisted of an anthraquinone redox unit and a photochromic azobenzene moiety. The interaction between laccase and mediator was examined and the photo-induced tuning of cis-trans photoisomerization and orientation were studied. Theoretical simulations were employed to examine electrochemical and photochromic properties.
In line 200-201 the authors claim: “Moreover, the reversible cis-trans photoisomerization, which converts the cis- to the trans-form by visible light irradiation, was observed for all complexes, although the photoisomerization for 3 did not occur as smoothly compared to that of 1 or 2” The phenomenon strictly related to the molecular structure should be deepened, the results explained. Also in the Introduction, the photochromic (azobenzene) moiety was cited without delving into these important features. In this regard it is suggested to add recent specific literature such as: A symmetrical azo-based fluorophore and the derived salen multipurpose framework for emissive Layers (2019) Inorganic Chemistry Communications, 104, pp. 186-189. Spectroscopic behaviour of two novel azobenzene fluorescent dyes and their polymeric blends (2020) Molecules, 25 (6), art. no. 1368.
Again, in the Introduction, some words should be spent to present the relevant group of such salen Cu(II) complexes adding recent related literature.
In line 93 apparatus for register fluorescence spectra was mentioned but no fluorescence data have been presented. Have the ligands and complexes emission properties to be underlined?
The theoretical studies, central tools of the work, suggesting that the electron transfer was related not only to electronic states but to the molecular orientation provide interesting but not definitive conclusions.
All in all, the work is well presented, the conclusions are rather clear and consequential, even if some aspects are not completely resolved. English needs to be improved.
In addition, there are some errors in the references. In particular: ref 3 is empty; the name of the first author of ref 40 it’s wrong; ref 42 it’s strange; ref 45 does not exist, it is the continuation of ref 44.
Author Response
April 23, 2020
Dear The editor of Symmetry:
Please find an attached the revised manuscript of article symmetry-788035.
The answers for query and revised points of the text (which have been written in the revised manuscript as red letters) are as follows:
<Editors>
Q1. Please revise the manuscript carefully to ensure the consecutive words plagiaristic are less than 12 within your revision.
A1. According to your comment, we fixed the sentences. However, references and acknowledgement could not be fixed.
Q2. Abstract section of papers so that readers could know clearly the paper's relevance to our journal Symmetry when they start to read the papers.
A2. According to your comment, we added this explanation associating with chirality (induced CD namely chiroptical spectroscopy) of biomolecules in abstract. “Induced chirality (achiral target in chiral matrix such as proteins) sometimes play a useful role in evaluation of supramolecular systems involving biomolecules.”
<Reviewer1>
Q1. Reference 3 is missing! Actually it is the template style. For example Gaussian is cited in text as 30, however it is actually reference 29. Reference 44 and 45 are actually one citation split in two.
A1. Thank you. According to your comment, we fixed references.
Q2. Line 46: how can molecular docking predict redox activity?
A2. According to your opinion, we changed “redox activity” into “the distance or path of electron transfer”.
Q3. Line 60: label of Figure 1 - that is the structure of the enzyme.
A3. According to your comment we added “(the structure of) the enzyme” in Figure 1’s legend.
Q4. Line 120: DFT spectra should be compared with experimental ones and inserted in ESI.
A4. Because DFT results were used mainly for obtaining optimized structures, we omitted the description about UV-vis spectra around Line 120.
Q5. Line 135: "geometries ... were similar to DFT structures" - this should be supported by a table where the corresponding bond angles and lengths are compared.
A5. As your comments, strictly speaking, crystal structures, structures in solutions, and DFT optimized structures are different from each other. Therefore, the similarity of “overall structure” was stated in the text, and we fixed the sentences.
Q6. Line 239: it should be "hydrogens (probably polar) were added", not "hydrogen bonds"
A6. According to your comment, we fixed as such.
Q7. Line 240: the grid box definition is unclear - does 30 represents the number of points (in which case it is not Angstroms, since the spacing is 0.375) or the size of the cell?
A7. According to your comments, we fixed to “points”.
Q8. The docking results are unclear: first, it is mentioned that the coordination geometry is square planar. However figure 9 doesn't suggest this. On the other hand it is difficult to observe, if the docked ligands incorporate Cu atoms?
A8. The docking results are about the best-fit ligand conformations which were selected based on their minimum binding energies. Complex 1 (ligand1) showed the highest docking score, i.e. -10.5 kcal/mol (-8.6 and -9.9 kcal/mol for 2 and 3, respectively).Since ligand1 has the most negative energy -10.5 kcal it is found to be best-docked molecule.
According to your comments, we added Figure 9(g). Because of optimized structure, Cu complex afford a slightly distorted planar coordination environment.
Q9. Line 253: the listed amino acids (his395, etc.) are not visible in figure 9.
A9. According to your comments, we re-drew Figure 9(g) with clear marked amino acids. Amino acids (his395) is not bonded with the three copper ligands so it was not shown on figure 9. May be reviewer wanted to pointed out figure number 10 where his395 is shown ,but there also it was attached to the laccase’s copper not to the copper ligand.
Q10. Figure 9 should focus on the ligand-receptor interactions, only (b,d and f) should be presented in a clear manner. Some suggestions: labels are huge, and should be positioned, backbone atoms should be hidden.
A10. According to your comments, we added re-drawn Figure 9(g) using bold models.
Q11. Table 2: interactions should be discussed in text.
A11. According to your comments, we added “Interaction were not only predominanotly hydrogen bonds but also hydrophobic or electrostatic ones, as mentioned in Table 2.”.
Q12. Figure 10 is not cited in text. Mediator is not colored in grey! How are the Cu atoms involved in the mediator orientation? If so, then why are they not present in figure 9?
A12. According to your comment, we omitted unimportant color in Figure 10’s legend, and sited in text with a sentence of “Electron transfer from mediator to copper sites are shown in Figure 10, in which redox properties of each metal sites are important.”.
Figure 10 is now cited in text. Mediator is colored in black, change in inserted in manuscript. Cu atoms are present in figure 9(a,c,e) in the form of sky blue balls.
<Reviewer 2>
Q1. In line 200-201 the authors claim: “Moreover, the reversible cis-trans photoisomerization, which converts the cis- to the trans-form by visible light irradiation, was observed for all complexes, although the photoisomerization for 3 did not occur as smoothly compared to that of 1 or 2” The phenomenon strictly related to the molecular structure should be deepened, the results explained. Also in the Introduction, the photochromic (azobenzene) moiety was cited without delving into these important features. In this regard it is suggested to add recent specific literature such as: A symmetrical azo-based fluorophore and the derived salen multipurpose framework for emissive Layers (2019) Inorganic Chemistry Communications, 104, pp. 186-189. Spectroscopic behaviour of two novel azobenzene fluorescent dyes and their polymeric blends (2020) Molecules, 25 (6), art. no. 1368.
A1. Thank you. We added suggested reference papers and explanation concerning azobezene just after the part of Line 200-201 (not introduction) as follows: “Azobenzene dyes ending with electron-withdrawing or donor groups were reported to shift absorption or emission wavelengths and even affect on photoisomerization.”.
Q2. Again, in the Introduction, some words should be spent to present the relevant group of such salen Cu(II) complexes adding recent related literature.
A2. According to your comment, we referred a recent review about salen complexes and added the sentence, “Generally, (chiral) salen metal complexes have been employed as (asymmetric) catalysis because of their advantage of stereochemical tuning (with respect to chirality) and redox properties with proper metal ions”.
Q3. In line 93 apparatus for register fluorescence spectra was mentioned but no fluorescence data have been presented. Have the ligands and complexes emission properties to be underlined?
A3. Thank you. According to your comment, fluorescence spectra was omitted.
Q4. The theoretical studies, central tools of the work, suggesting that the electron transfer was related not only to electronic states but to the molecular orientation provide interesting but not definitive conclusions.
A4. According to your comments, we rephrased the sentence before this part as “Even the theoretical studies can investigate electronic structures of photoisomers at molecular level, experimental results herein also suggested that…”.
Q5. All in all, the work is well presented, the conclusions are rather clear and consequential, even if some aspects are not completely resolved. English needs to be improved.
A5. Thank you for suggestion for future works. Prior to first submission, English was corrected by a native professional editor.
Q6. In addition, there are some errors in the references. In particular: ref 3 is empty; the name of the first author of ref 40 it’s wrong; ref 42 it’s strange; ref 45 does not exist, it is the continuation of ref 44.
A6. Thank you. We fixed these references.
<Reviewer 3>
Q1. My major concern with the article is the following. Despite the fact that the Authors made several experiments to synthetize, investigate and evaluate the current Cu-salen-laccase systems, the last part of the article cannot summarize sufficiently the findings, and cannot put this research in context. There are several sentences in this part that were copied from p1. without modification (p. 16. l. 341-347.). The conclusions are soft or even pointless in a few instances.
A1. According to your opinion, we rewrote the conclusion part.
Q2. Which one from the three complexes is the best mediator for laccase? Why?
A2. In conclusion, we added the comparison as “Among three complexes, 1 without disturbing substituent groups of electronic effects was comprehensively suitable for this purpose.”
Q3. How does these results compare to other salen-type mediators having related structure?
A3. Typical other metal complexes (ferrocene and [Fe(CN)6]3-/4-) for mediators and merits of these complexes were added in conclusion. To our knowledge, however, there were few study of other salen-type mediators.
Q4. How does these results compare to the best-in-class mediators?
A4. In conclusion, we added the comparision as “Besides these types of potential mediators or common organic mediators, the present azo-anthraquinone salen Cu complexes was confirmed to have advantage of photo-tuning their electronic states and spatial orientation.”.
Q5. Based on the current findings, what are the next steps to improve the performance of these mediators? (There are several sites on the mediators that can be tweaked by functional groups)
A5. We have also submitted this special issue of “Symmetry” a modified electrode of laccase-mediator systems connected to enantiomers of oligopeptides for spin filters as a next step, though it is under review now, too.
Q6. All data of Table 1 and Fig 6 is necessary, or some of this data can be put to the Supporting Information?
A6. According to your comment, we moved Table 1 and Fig. 6 (DOS) to Supporting Information.
Q7. Table 2 having a length of 2 pages in unnecessary in its current form. It can be shortened, and the rest of the data could be moved to the Supporting Information. Or the whole table can be moved to the supporting information.
A7. According to your comment, we moved a part of Table 2 to Supporting Information.
Q8. Maybe more emphasis should be put on the part of the ms which are in strong correlation with the aims and scope of the journal.
A8. In abstract, we wrote a sentence to emphasis symmetry (chirality and induced CD for supramolecular docking) aspect of this study to meet the aims and scope of the journal. And experimental section was also modified slightly.
Q9. 1. ‘tested for use as mediators’ ‘In the following ways will be explored to solve the disadvantage of low power.’ ‘Generally, a redox substance called a mediator – commas should be added’‘assists to donate and receive – assists the donation and reception’ etc.
A9. According to your comments, we fixed the representation.
Q10. 2. l. 50. References 16, 17 are in wrong order.
A10. Thank you. We fixed the order.
Q11. p.3. l. 82. The amount of hexane used for recrystallization is missing.
A11. Thank you. We added this information (starting amount for evaporating and cooling).
Q12.3. l. 83-85. There is no point to show the first decimals of the yields.
A12. Thank you. We removed them.
Q13. 10. The reference for Fig. 10 is missing in the corresponding text.
A13. Thank you. We added text for Fig. 10.
Q14. Fig 2. The N=N double bond is not visible.
A14. Thank you. We re-drew Fig.2.
Q15. Fig 7. The blue line is barely visible.
A15. The explanation of “Slow photoisomeriztion resulted in overlap of red and bule lines in Figure 7.” was added in text for this situation.
Q16. Fig 9. The pink colored molecules are barely visible in the pocket. Another color and better contrast is needed.
A16. According to your comment, Fig.9 (g) was re-drawn.
Q17. References: Ref. 3 is missing.
A17. Thank you. We added ref.3.
Q18. Ref. 16. There is an unnecessary underline.
A18. Thank you. We removed it.
That’s all.
We would like to say thanks in advance for your prompt treatment of our manuscript.
Best regards,
Prof. Dr. Takashiro Akitsu
Department of Chemistry, Faculty of Science, Tokyo University of Science
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Tel. +81-3-5228-8271, Fax. +81-3-5261-4631
E-mail: [email protected]
Reviewer 3 Report
In their article, the Authors describe three Cu-salen complexes, their crystallographic and spectroscopic characterization. The Authors also made computations to model the docking of these complexes to laccase, and they also conducted thorough electrochemical measurements to evaluate the improved electron transfer properties.
The paper contains a considerable amount of valuable data. However, the overall representation is inadequate in its current form, and the article needs a major revision.
My major concern with the article is the following. Despite the fact that the Authors made several experiments to synthetize, investigate and evaluate the current Cu-salen-laccase systems, the last part of the article cannot summarize sufficiently the findings, and cannot put this research in context. There are several sentences in this part that were copied from p1. without modification (p. 16. l. 341-347.). The conclusions are soft or even pointless in a few instances.
It was really hard for the reviewer to find answers to the following questions:
Which one from the three complexes is the best mediator for laccase? Why?
How does these results compare to other salen-type mediators having related structure?
How does these results compare to the best-in-class mediators?
Based on the current findings, what are the next steps to improve the performance of these mediators? (There are several sites on the mediators that can be tweaked by functional groups)
Thus, this part of the article must be improved! Please rewrite the conclusion part!
Overall, I find this Article a bit lengthy. There are a few ways to solve this issue.
- 6-7. All data of Table 1 and Fig 6 is necessary, or some of this data can be put to the Supporting Information?
- 11. Table 2 having a length of 2 pages in unnecessary in its current form. It can be shortened, and the rest of the data could be moved to the Supporting Information. Or the whole table can be moved to the supporting information.
I am not fully convinced that the choice of Journal is suitable for such a paper, however, some similar ones had been published here. Maybe more emphasis should be put on the part of the ms which are in strong correlation with the aims and scope of the journal.
The article needs a thorough proofreading and the removal of typos, grammatical mistakes and other elements of imperfect style:
Grammatical mistakes:
- 1. ‘tested for use as mediators’ ‘In the following ways will be explored to solve the disadvantage of low power.’ ‘Generally, a redox substance called a mediator – commas should be added’‘assists to donate and receive – assists the donation and reception’ etc.
Minor remarks:
- 2. l. 50. References 16, 17 are in wrong order.
p.3. l. 82. The amount of hexane used for recrystallization is missing.
- 3. l. 83-85. There is no point to show the first decimals of the yields.
- 10. The reference for Fig. 10 is missing in the corresponding text.
Fig 2. The N=N double bond is not visible.
Fig 7. The blue line is barely visible.
Fig 9. The pink colored molecules are barely visible in the pocket. Another color and better contrast is needed.
References:
Ref. 3 is missing.
Ref. 16. There is an unnecessary underline.
Author Response
April 23, 2020
Dear The editor of Symmetry:
Please find an attached the revised manuscript of article symmetry-788035.
The answers for query and revised points of the text (which have been written in the revised manuscript as red letters) are as follows:
<Editors>
Q1. Please revise the manuscript carefully to ensure the consecutive words plagiaristic are less than 12 within your revision.
A1. According to your comment, we fixed the sentences. However, references and acknowledgement could not be fixed.
Q2. Abstract section of papers so that readers could know clearly the paper's relevance to our journal Symmetry when they start to read the papers.
A2. According to your comment, we added this explanation associating with chirality (induced CD namely chiroptical spectroscopy) of biomolecules in abstract. “Induced chirality (achiral target in chiral matrix such as proteins) sometimes play a useful role in evaluation of supramolecular systems involving biomolecules.”
<Reviewer1>
Q1. Reference 3 is missing! Actually it is the template style. For example Gaussian is cited in text as 30, however it is actually reference 29. Reference 44 and 45 are actually one citation split in two.
A1. Thank you. According to your comment, we fixed references.
Q2. Line 46: how can molecular docking predict redox activity?
A2. According to your opinion, we changed “redox activity” into “the distance or path of electron transfer”.
Q3. Line 60: label of Figure 1 - that is the structure of the enzyme.
A3. According to your comment we added “(the structure of) the enzyme” in Figure 1’s legend.
Q4. Line 120: DFT spectra should be compared with experimental ones and inserted in ESI.
A4. Because DFT results were used mainly for obtaining optimized structures, we omitted the description about UV-vis spectra around Line 120.
Q5. Line 135: "geometries ... were similar to DFT structures" - this should be supported by a table where the corresponding bond angles and lengths are compared.
A5. As your comments, strictly speaking, crystal structures, structures in solutions, and DFT optimized structures are different from each other. Therefore, the similarity of “overall structure” was stated in the text, and we fixed the sentences.
Q6. Line 239: it should be "hydrogens (probably polar) were added", not "hydrogen bonds"
A6. According to your comment, we fixed as such.
Q7. Line 240: the grid box definition is unclear - does 30 represents the number of points (in which case it is not Angstroms, since the spacing is 0.375) or the size of the cell?
A7. According to your comments, we fixed to “points”.
Q8. The docking results are unclear: first, it is mentioned that the coordination geometry is square planar. However figure 9 doesn't suggest this. On the other hand it is difficult to observe, if the docked ligands incorporate Cu atoms?
A8. The docking results are about the best-fit ligand conformations which were selected based on their minimum binding energies. Complex 1 (ligand1) showed the highest docking score, i.e. -10.5 kcal/mol (-8.6 and -9.9 kcal/mol for 2 and 3, respectively).Since ligand1 has the most negative energy -10.5 kcal it is found to be best-docked molecule.
According to your comments, we added Figure 9(g). Because of optimized structure, Cu complex afford a slightly distorted planar coordination environment.
Q9. Line 253: the listed amino acids (his395, etc.) are not visible in figure 9.
A9. According to your comments, we re-drew Figure 9(g) with clear marked amino acids. Amino acids (his395) is not bonded with the three copper ligands so it was not shown on figure 9. May be reviewer wanted to pointed out figure number 10 where his395 is shown ,but there also it was attached to the laccase’s copper not to the copper ligand.
Q10. Figure 9 should focus on the ligand-receptor interactions, only (b,d and f) should be presented in a clear manner. Some suggestions: labels are huge, and should be positioned, backbone atoms should be hidden.
A10. According to your comments, we added re-drawn Figure 9(g) using bold models.
Q11. Table 2: interactions should be discussed in text.
A11. According to your comments, we added “Interaction were not only predominanotly hydrogen bonds but also hydrophobic or electrostatic ones, as mentioned in Table 2.”.
Q12. Figure 10 is not cited in text. Mediator is not colored in grey! How are the Cu atoms involved in the mediator orientation? If so, then why are they not present in figure 9?
A12. According to your comment, we omitted unimportant color in Figure 10’s legend, and sited in text with a sentence of “Electron transfer from mediator to copper sites are shown in Figure 10, in which redox properties of each metal sites are important.”.
Figure 10 is now cited in text. Mediator is colored in black, change in inserted in manuscript. Cu atoms are present in figure 9(a,c,e) in the form of sky blue balls.
<Reviewer 2>
Q1. In line 200-201 the authors claim: “Moreover, the reversible cis-trans photoisomerization, which converts the cis- to the trans-form by visible light irradiation, was observed for all complexes, although the photoisomerization for 3 did not occur as smoothly compared to that of 1 or 2” The phenomenon strictly related to the molecular structure should be deepened, the results explained. Also in the Introduction, the photochromic (azobenzene) moiety was cited without delving into these important features. In this regard it is suggested to add recent specific literature such as: A symmetrical azo-based fluorophore and the derived salen multipurpose framework for emissive Layers (2019) Inorganic Chemistry Communications, 104, pp. 186-189. Spectroscopic behaviour of two novel azobenzene fluorescent dyes and their polymeric blends (2020) Molecules, 25 (6), art. no. 1368.
A1. Thank you. We added suggested reference papers and explanation concerning azobezene just after the part of Line 200-201 (not introduction) as follows: “Azobenzene dyes ending with electron-withdrawing or donor groups were reported to shift absorption or emission wavelengths and even affect on photoisomerization.”.
Q2. Again, in the Introduction, some words should be spent to present the relevant group of such salen Cu(II) complexes adding recent related literature.
A2. According to your comment, we referred a recent review about salen complexes and added the sentence, “Generally, (chiral) salen metal complexes have been employed as (asymmetric) catalysis because of their advantage of stereochemical tuning (with respect to chirality) and redox properties with proper metal ions”.
Q3. In line 93 apparatus for register fluorescence spectra was mentioned but no fluorescence data have been presented. Have the ligands and complexes emission properties to be underlined?
A3. Thank you. According to your comment, fluorescence spectra was omitted.
Q4. The theoretical studies, central tools of the work, suggesting that the electron transfer was related not only to electronic states but to the molecular orientation provide interesting but not definitive conclusions.
A4. According to your comments, we rephrased the sentence before this part as “Even the theoretical studies can investigate electronic structures of photoisomers at molecular level, experimental results herein also suggested that…”.
Q5. All in all, the work is well presented, the conclusions are rather clear and consequential, even if some aspects are not completely resolved. English needs to be improved.
A5. Thank you for suggestion for future works. Prior to first submission, English was corrected by a native professional editor.
Q6. In addition, there are some errors in the references. In particular: ref 3 is empty; the name of the first author of ref 40 it’s wrong; ref 42 it’s strange; ref 45 does not exist, it is the continuation of ref 44.
A6. Thank you. We fixed these references.
<Reviewer 3>
Q1. My major concern with the article is the following. Despite the fact that the Authors made several experiments to synthetize, investigate and evaluate the current Cu-salen-laccase systems, the last part of the article cannot summarize sufficiently the findings, and cannot put this research in context. There are several sentences in this part that were copied from p1. without modification (p. 16. l. 341-347.). The conclusions are soft or even pointless in a few instances.
A1. According to your opinion, we rewrote the conclusion part.
Q2. Which one from the three complexes is the best mediator for laccase? Why?
A2. In conclusion, we added the comparison as “Among three complexes, 1 without disturbing substituent groups of electronic effects was comprehensively suitable for this purpose.”
Q3. How does these results compare to other salen-type mediators having related structure?
A3. Typical other metal complexes (ferrocene and [Fe(CN)6]3-/4-) for mediators and merits of these complexes were added in conclusion. To our knowledge, however, there were few study of other salen-type mediators.
Q4. How does these results compare to the best-in-class mediators?
A4. In conclusion, we added the comparision as “Besides these types of potential mediators or common organic mediators, the present azo-anthraquinone salen Cu complexes was confirmed to have advantage of photo-tuning their electronic states and spatial orientation.”.
Q5. Based on the current findings, what are the next steps to improve the performance of these mediators? (There are several sites on the mediators that can be tweaked by functional groups)
A5. We have also submitted this special issue of “Symmetry” a modified electrode of laccase-mediator systems connected to enantiomers of oligopeptides for spin filters as a next step, though it is under review now, too.
Q6. All data of Table 1 and Fig 6 is necessary, or some of this data can be put to the Supporting Information?
A6. According to your comment, we moved Table 1 and Fig. 6 (DOS) to Supporting Information.
Q7. Table 2 having a length of 2 pages in unnecessary in its current form. It can be shortened, and the rest of the data could be moved to the Supporting Information. Or the whole table can be moved to the supporting information.
A7. According to your comment, we moved a part of Table 2 to Supporting Information.
Q8. Maybe more emphasis should be put on the part of the ms which are in strong correlation with the aims and scope of the journal.
A8. In abstract, we wrote a sentence to emphasis symmetry (chirality and induced CD for supramolecular docking) aspect of this study to meet the aims and scope of the journal. And experimental section was also modified slightly.
Q9. 1. ‘tested for use as mediators’ ‘In the following ways will be explored to solve the disadvantage of low power.’ ‘Generally, a redox substance called a mediator – commas should be added’‘assists to donate and receive – assists the donation and reception’ etc.
A9. According to your comments, we fixed the representation.
Q10. 2. l. 50. References 16, 17 are in wrong order.
A10. Thank you. We fixed the order.
Q11. p.3. l. 82. The amount of hexane used for recrystallization is missing.
A11. Thank you. We added this information (starting amount for evaporating and cooling).
Q12.3. l. 83-85. There is no point to show the first decimals of the yields.
A12. Thank you. We removed them.
Q13. 10. The reference for Fig. 10 is missing in the corresponding text.
A13. Thank you. We added text for Fig. 10.
Q14. Fig 2. The N=N double bond is not visible.
A14. Thank you. We re-drew Fig.2.
Q15. Fig 7. The blue line is barely visible.
A15. The explanation of “Slow photoisomeriztion resulted in overlap of red and bule lines in Figure 7.” was added in text for this situation.
Q16. Fig 9. The pink colored molecules are barely visible in the pocket. Another color and better contrast is needed.
A16. According to your comment, Fig.9 (g) was re-drawn.
Q17. References: Ref. 3 is missing.
A17. Thank you. We added ref.3.
Q18. Ref. 16. There is an unnecessary underline.
A18. Thank you. We removed it.
That’s all.
We would like to say thanks in advance for your prompt treatment of our manuscript.
Best regards,
Prof. Dr. Takashiro Akitsu
Department of Chemistry, Faculty of Science, Tokyo University of Science
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Tel. +81-3-5228-8271, Fax. +81-3-5261-4631
E-mail: [email protected]
Reviewer 4 Report
Title: Photo-tunable Azo-anthraquinone Schiff Base Copper Complexes as Mediators for Laccase in Biofuel Cell Cathode
Article Type: full article
Manuscript Number: Symmetry-787035
My recommendation is that the authors carefully consider the below points, revise appropriately.
- Page 1, line 76~78; Schiff base as a four dentate over one decade. The authors prepared a special functional ligand in this article that the original report by Hugo should be cited as well as reference 14.
- Page 3~4, line 108~116; crystal structure of 1~3 were determined by X-ray diffraction method. But the Rwp value are quiet not acceptable. Especially, the compound 3 have the higher Rwp value than the others. Does the bond distance and bond angle as well as geometry is acceptable?
- Page 3~4, line 157~158; my suggestion is that the bond distance and bond angles for Figure3~5 should be checked carefully for literal error such as “O1-Cu1-N1 = 157 176.590(2), O1-Cu1-N2 = 94.63(3), O2-Cu1-N1 = 92.35(3), O2-Cu1-N2 = 171.250(4)”.
- Page 9, line 230; the authors may consider overlap the line of Laccase in Figure 8 as the others between 200~500 nm.
- Page 10, line 250~251; The authors should clarify that three salen derivatives of Cu(II) complexes are bound to the domain three of laccase binding pockets as previous article reported by using azo-anthraquinone Schiff base Mn complexes as mediators.
- Page 15, line 327~332; the authors claim that the path and mechanism of the electron transfer is not clear and could not be reliably explained with the present systems. Can the authors show the contribution in this article or the segments with Mn complexes that have been cited in reference 14.
Author Response
Please see attached file.
Author Response File: 
Author Response.doc
Round 2
Reviewer 1 Report
After the revision of the paper, there are many serious flaws regarding the molecular docking study:
1. I have verified the gridbox in autodocktools as described in line 239, and it does not include the T1Cu atom. since this atom plays a major role in the mechanism, it should be included in the search space.
2. The size of the grid box is too small compared to the substrate. A rough estimate of the diameter of the ligand is ~25 Angstrom, however, the length of the Grid Box is just 11.25 Angstrom.
This is completely wrong, an optimal box should be used, which could easily accommodate the ligand molecule.
3. The parameter files of AutoDock (see http://autodock.scripps.edu/resources/parameters) do not contain the parametrization for copper atoms. Therefore, I find the results of the docking computations unreliable.
It can be observed in Figure 9, that the geometry around the Cu atoms deviates from the square planar coordination of the DFT geometries. This indicates that AutoDock cannot handle such ligands, and a QM/MM study should be performed to find the correct binding pose.
I have asked the authors to replace the figures 9b, 9d, 9f with a more detailed version, and remove 9a, 9b and 9c, as they don't contain any information. They have included a new figure 9g, however it is not mentioned in the caption which one of the ligands it represents. Once again I suggest, that the figures should also include the T1Cu atom.
Three decimals are enough to describe the interaction lengths which appear in Table 2 and Table S2, and the corresponding discussion starting at Line 251.
Line 258: This sentence is nonsense: "The amino acids around the ligands also contribute that ligands are bound to the domain three of laccase."
Gaussian is cited as [31] in text, and appears at [30] in the reference list.
AutoDock should be cited!
Overall, I don't find any connection with the topic of the journal.
Author Response
Please see attached file.
Author Response File: Author Response.doc
Reviewer 2 Report
The authors accepted the suggestions and corrections the referee made. After a detailed screening of the revised copy of the work, I find the manuscript improved enough for publication.
Author Response
Please see attached file.
Author Response File: Author Response.doc
Reviewer 3 Report
In the revision, the Authors made several corrections and answered the questions of the reviews. The Authors increased the quality of the paper significantly. Therefore, I recommend it for publication in its current form.
Author Response
Please see attached file.
Author Response File: Author Response.doc
Round 3
Reviewer 1 Report
It is a good thing that the authors have provided a screenshot of the grid box in AutoDockTools (ADT), because it proves that the molecular docking is done wrong:
- the size of the used grid box (more precisely the edge length) as described in the manuscript is just 11.25 Angstroms, since the number of points are 30, and the grid point spacing is 0.375 Angstrom only. Size of the box = number of points * spacing.
This is more than half smaller than the size of the studied ligands!
Also, as I have stated in my previous review, yet the authors prefer to omit, it is completely wrong to use a method which is not parametrized in this case for Copper atom type.
My suggestion is, that the authors should repeat the molecular docking part, provide molecular coordinates in the supplementary file of both the optimized DFT geometries, and the docking pose which is considered optimal.
However, considering, that the crystal structure of 1GYC doesn't have a co-crystallized ligand, the docking results should be validated by QM/MM studies.
