D,L-Citrullinato-bipyridine Copper Complex: Experimental and Theoretical Characterization

Round 1

Reviewer 1 Report

The work presents a new experimentally determined crystal structure and its solid theoretical investigation on the basis of DFT calculations and Hirshfeld surface analysis. Concerning the experimental data, the title compound is not well characterzied at the moment, despite authors' claim in the abstract. IR and Raman typically show only molecular consitiuents and some additional information on coordination modes, modes of hydrogen bonding etc. No compound individuality can be proven by both IR and Raman. Similarly, UV-vis spectroscopy can not confirm individuality of the compounds in a general case, due to typically very high width of the absorption bands. 

From all the row of presented analyses, the only MS could be suitable for the verification of the purity and individuality of the Cu-citrullinate complex. However, one can see (Fig. 8) additional band with m/z = 518 and other weaker bands with m/z > 456, which cannot be attribited to the title complex. Please provide experimental CHN content data and powder X-ray diffractogram compared to the theoretical one for the careful evaluation of the compound purity and individuality. 

Moreover, the staiblity of complex in water must be shown, at least, to verify the provided UV-vis spectra. Additionally, its stability in the vital media or at the biological pH values may be checked. 

Author Response

Reviewer 1

 

Answer are provided as (*)

 

The work presents a new experimentally determined crystal structure and its solid theoretical investigation on the basis of DFT calculations and Hirshfeld surface analysis. Concerning the experimental data, the title compound is not well characterized at the moment, despite authors' claim in the abstract. IR and Raman typically show only molecular constituents and some additional information on coordination modes, modes of hydrogen bonding etc. No compound individuality can be proven by both IR and Raman. Similarly, UV-vis spectroscopy cannot confirm individuality of the compounds in a general case, due to typically very high width of the absorption bands.

 

*The central part of article is the crystal structure of the compound, the spectroscopic characterization only confirm the presence of the components and the way they surround the copper (II) ion. The word “fully” has been removed since it does not reflect a complete characterization.

 

From all the row of presented analyses, the only MS could be suitable for the verification of the purity and individuality of the Cu-citrullinate complex. However, one can see (Fig. 8) additional band with m/z = 518 and other weaker bands with m/z > 456, which cannot be attributed to the title complex.

 

*The small peak at 518 could correspond to a small impurity of the dinitrate complex.

 

Please provide experimental CHN content data and powder X-ray diffractogram compared to the theoretical one for the careful evaluation of the compound purity and individuality.

 

*Unfortunately we cannot perform elemental analysis, since our equipment has been down since the pandemia and has not been recovered. As for the powder X-ray difractogram, we feel it can not surpass a single crystal X-ray structure determination.

 

Moreover, the stability of complex in water must be shown, at least, to verify the provided UV-vis spectra. Additionally, its stability in the vital media or at the biological pH values may be checked.

 

*Actually the stability has been checked and currently the compound have been tested for cytotoxicity in several cancer cell lines with promising results (manuscript in preparation).

 

Thank you so much for your kind observations.

Reviewer 2 Report

The discussion section contains repetition of introductory items. A discussion, focused on results only is desirable.

Author Response

Reviewer 2

Thank you so much for your important observation. A whole paragraph at the beginning of the discussion section has been removed.

     The coordination chemistry of Citrulline started in 1939 when Kurtz proposed the structure of its copper complex. However, as mentioned in the introduction, only a few examples of coordination compounds have been reported [10]. In this regard, the Cambridge Crystallographic Database contains only two reports on crystal structure: the palladium complex's structure was described by Mascaliovas et al. [12], and the recent report of our team [13]. Although the Citrulline complexes exhibit a similar composition to those formed by other conventional amino acids, in physiological conditions, Citrulline also exists predominantly as a zwitterion. In alkaline conditions, amine groups can function as Lewis donors; thus, the deprotonated carboxyl and amino groups allow the Citrulline molecule to act as a bidentate chelating ligand. The research conducted by Ganadu et al. [11] regarding the structure of [Pd(Citr)₂] was elucidated using spectroscopic techniques and corresponds to the first effort to characterize Citrulline complexes. The coordination of this compound involves the participation of two Citrulline molecules, which function as bidentate ligands within a square planar geometry. Mascaliovas et al. used powder diffraction to determine that the nitrogen and oxygen atoms in [Pd(Citr)₂] are arranged in trans orientations. The chemical crystallized in the P-1 space group, thus possessing an inversion center, which necessitates the coexistence of L and D-Citrulline. However, the authors do not refer to this fact [12].

 

Reviewer 3 Report

Line 19, what do you mean “under normal conditions”? Do you mean in solution (aqueous) or in the solid state?

Why is “Citrulline” written with capital “C”?

Line 28, please avoid using “fully”, the material can never be “fully” characterized, i.e. the authors haven’t performed ssNMR

In the introduction the figure presenting the structure of citrulline must be introduced

Line 30, why “structures” and not “structure”?

Line 49, exactly what medicinal properties?

Line 52, it should be “environment”

Lines 63-65, what is the CCDC refcode of this structure? The unit cell of it should be introduced in the form of a figure?

Lines 71-73, was this observation explained?

Line 98, please state the name of this supplier

Lines 103-105, more details are needed. Have you used KBr or ATR? How many scans? What was the resolution? Etc.

Line 157, why have you calculated the NMR shifts (and why only H?) while the experimental NMR spectrum was not recorded.

Please explain, despite having access to the crystal structure, the authors have decided to perform the DFT calculations on isolated molecule (in gas state) instead on the periodic crystal using i.e. CRYSTAL or CASTEP software. This would really increase the quality and accuracy of the obtained results.

Line 187-188, this must be described in the more detailed way. Since Z=2, therefore it should be 50% and 50%. Do you suggest that in some unit cells there are two D isomers?

Line 216, it’s not a methodology but method, or functional

Have you tried to deconvolute the spectra present in figures 4 and 5?

Mass spectrometry results should be deeper discussed, i.e. by introducing a table comparing the theoretical and experimentally observed values

Lines 353-399, My another major question is why the authors expect the optimized complexes to differ in their structural properties as they are simply the enantiomers? Therefore the optimized geometry should be the same.

The number of references is enormous, over 70 positions. This list should be shortened.

 

Author Response

Reviewer 3

 

Line 19, what do you mean “under normal conditions”? Do you mean in solution (aqueous) or in the solid state?

 

Thank you for all your valuable observations. The answers will be proceeded with(*)

 

*In aqueous solutions. It has been changed

 

Why is “Citrulline” written with capital “C”?

 

*Normally, the amino acids are capitalized in the first letter; also as, one of the ligands, Bipyridine, was capitalized in the whole text.

 

Line 28, please avoid using “fully”, the material can never be “fully” characterized, i.e. the authors haven’t performed NMR

 

*Thank you for the observation, the word “fully” has been removed

 

In the introduction, the figure presenting the structure of citrulline must be introduced

 

*Thank you. A figure presenting the structure of the Citrulline molecule has been included.

 

Line 30, why “structures” and not “structure”?

 

*Thank you. The letter “s” has been removed.

 

Line 49, exactly what medicinal properties?

 

*It is used for athletic performance, sickle cell disease, erectile dysfunction (ED), high blood pressure in the lungs, and heart disease since it improves blood flow and reduces blood pressure.

 

Line 52, it should be “environment”

 

*The letter “s” has been removed

 

Lines 63-65, what is the CCDC refcode of this structure? The unit cell of it should be introduced in the form of a figure?

 

*In these lines, we are in the introduction. The refcode, which is 2287583, is introduced in the crystallographic refinement section (2.3), and Figure 2 introduces the crystal structure of the compound.

 

Lines 71-73, was this observation explained?

 

* Although the starting material has an L-configuration, the surprising result was that the compound crystallized with two D- Citrulline molecules coordinating the copper(II) ion. In reference 13, an explanation was presented.

 

Line 98, please state the name of this supplier

* Beyond Raw Chemistry Labs

 

Lines 103-105, more details are needed. Have you used KBr or ATR? How many scans? What was the resolution? Etc.

 

*The Fourier-transform infrared (FT-IR) spectrum was recorded from 4000 to 400 cm-1 using an ALPHA II Platinum FT-IR Spectrophotometer (Bruker), with attenuated Total Reflectance (ATR).

 

Line 157, why have you calculated the NMR shifts (and why only H?) while the experimental NMR spectrum was not recorded?

 

*Indeed, the experimental ¹H NMR is not presented since the presence of paramagnetic copper ions makes the signal assignments in the spectrum difficult. Therefore, we have removed that phrase.

 

Please explain, despite having access to the crystal structure, the authors have decided to perform the DFT calculations on isolated molecule (in gas state) instead on the periodic crystal using i.e. CRYSTAL or CASTEP software. This would really increase the quality and accuracy of the obtained results.

 

*Theoretical calculations were carried out in the solution phase, using water as the solvent, as in experimental conditions the complex is spectroscopically characterized. Additionally, previous works in our group with other functionals using the solvent effect have reproduced and successfully predicted the experimental measurements.

 

Lines 187-188, this must be described in the more detailed way. Since Z=2, therefore it should be 50% and 50%. Do you suggest that in some unit cells there are two D isomers?

 

Indeed, we can find the D - D, D - L, or L - L isomers in the cell. We have no way of telling that we will only find the D - L configurations in each cell; we only see the average across the packing so that we can observe the presence of the enantiomers with the same absolute configuration in the cell and in this case the probability is 50 -50 for each one.

 

Line 216, it’s not a methodology but a method, or functional

 

*You are correct; it has been changed to functional.

 

Have you tried to deconvolute the spectra present in Figures 4 and 5?

 

*The spectra of copper(II) complexes in distorted octahedral environments are well known to have an unsymmetrical broad band containing three electronic transitions, as calculated and presented in Table 3. We believe it was not necessary to deconvolute the broadband.

 

 

Mass spectrometry results should be deeper discussed, i.e., by introducing a table comparing the theoretical and experimentally observed values.

 

*The paper describes mainly a new crystal structure, the mass spectrum is only presented as a confirmation of some important fragments and the molecular ion.

 

Lines 353-399, My another major question is why the authors expect the optimized complexes to differ in their structural properties as they are simply the enantiomers. Therefore, the optimized geometry should

be the same.

 

*You are correct. Although we have found it is important to calculate both isomers for our work in docking with relevant target proteins, it is unnecessary in this case. Therefore, we removed the D-isomer calculations. Figures 9, 10, 11 have been corrected.

 

The number of references is enormous, over 70 positions. This list should be shortened.

 

*Some references have been removed. However, we consider the paper to be well-referenced.

 

Thank you so much for all your kind observations.

 

 

Round 2

Reviewer 1 Report

Accept

Author Response

Thank you very much for your kind suggestions

Reviewer 3 Report

The Authors have answered most of my previous questions and queries. However, there is one more correction that is mandatory. Since, as stated by the Authors “Indeed, we can find the D - D, D - L, or L - L isomers in the cell. We have no way of telling that we will only find the D - L configurations in each cell; we only see the average across the packing so that we can observe the presence of the enantiomers with the same absolute configuration in the cell and in this case the probability is 50 -50 for each one.” I strongly recommend creating such unit cells (D-D, L-L, D-L, L-D) and add them as supplementary materials (cif files) to this study.

Author Response

Thank you for your kind suggestions. The supplementary material will be modified accordingly.