Structure determination of oleanolic and ursolic acids: a combined density functional theory/vibrational spectroscopy methodology

Raw samples of oleanolic and ursolic acids, a class of terpenoid acids mainly found in the leaf and fruit cuticles of some plant species, can be defined as a blend of clusters of different conformers aggregated in dimers and tetramers by means of hydrogen bonds and stabilized by non-electrostatic interactions.


1.
The authors interpret that the raw samples consist of dimers and tetramers that are stabilized by way of hydrogen bonds and non-electrostatic interactions. Can the authors describe these non-electrostatic interactions better? The authors may perhaps find it useful to compare the energetics with and without the empirical dispersion corrections. Additionally, energy decomposition analysis may throw some light on the nature of these non-electrostatic interactions. 2.
The authors have reported relative binding energies. But, I do not see it defined in the methodology section. It is important to define it for the benefit of the readers. 3.
The authors have characterized the hydrogen bond features by way of Mulliken charge analysis. The reported charge differences in Fig. S2 do not contain any units. Can the authors describe the data more comprehensively? 4.
From the geometries of the dimers and tetramers, it is clear that the entropic contribution to the energetics might be playing a crucial role. Can the authors assess the relative importance of enthalpic and entropic features in governing the binding of the various aggregates? 5.
In the second paragraph of the results section, the authors mention about a red-shifted C-O bond. However, it is not clear that the red-shift is with respect to which position. Can the authors address this better? 6.
In the second paragraph of the section on "From doubly hydrogen-bonded dimers", I see that the authors mention about high tendency of the molecules to assemble by non-electronic interactions. What do the authors mean by this? 7.
The authors report Maxwell-Boltzmann weighted theoretical spectra towards the end of the manuscript. The equation provided by the authors has Boltzmann constant missing in it! Can the authors carefully revise this? 8.
The presentation of the content in the manuscript needs significant improvement. Punctuations are not carefully followed (for example, no space is introduced between numerical value of a parameter and its units) and some sentences need to be rewritten (for example, "…will have an impact in molecule interaction"). In the caption of Fig. 7 and in SI, "tetramers" is misspelt.

Decision letter (RSOS-210162.R0)
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Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Heredia and coworkers determine the possible structures of oleanolic acid and ursolic acid using a combination of DFT calculations and IR experiments. The IR spectra calculated from monomeric acid units do not agree well with the experimental spectra. The authors have used different hydrogen-bonded aggregates to match the FTIR spectra. My comments are as follows: 1. The introduction should be more focused on this work. 2. The authors constructed the theoretical IR spectra using a FWHM of 10 cm-1. Why a uniform FWHM of 10 cm-1 is applicable to all the different conformers/aggregates/ functional groups? The authors need to explain. 3. The authors fitted the experiments FTIR spectra with 4 peaks. The FWHM of the peaks (same color) varies drastically from oleanolic acid and ursolic acid. Given the similarity in the structures of the two molecules, the authors need to explain this huge variation in the FWHM. Are the fits unique? Can we fit the same peak of more/less number of peaks with different FWHM? The authors should show the goodness of the fits by plotting the experimental spectra the fitted spectra in the same plot. The explanation heavily relies on deconvolution.
Reviewer: 2 Comments to the Author(s) The manuscript by Heredia and co-workers reports computational investigations in conjunction with IR spectroscopic measurements on Oleanolic and Ursolic acids. The authors have investigated a series of dimeric and tetrameric configurations using DFT. The computed IR spectra of various aggregates are compared with the experimental IR spectra of raw samples to interpret the presence of aggregates in the raw samples. The computations are carried out using the B3LYP functional with an empirical dispersion correction. The reported findings are interesting. However, the manuscript does need some improvement before it could be considered ready for publication. My comments are: 1. The authors interpret that the raw samples consist of dimers and tetramers that are stabilized by way of hydrogen bonds and non-electrostatic interactions. Can the authors describe these nonelectrostatic interactions better? The authors may perhaps find it useful to compare the energetics with and without the empirical dispersion corrections. Additionally, energy decomposition analysis may throw some light on the nature of these non-electrostatic interactions. 2. The authors have reported relative binding energies. But, I do not see it defined in the methodology section. It is important to define it for the benefit of the readers. 3. The authors have characterized the hydrogen bond features by way of Mulliken charge analysis. The reported charge differences in Fig. S2 do not contain any units. Can the authors describe the data more comprehensively? 4. From the geometries of the dimers and tetramers, it is clear that the entropic contribution to the energetics might be playing a crucial role. Can the authors assess the relative importance of enthalpic and entropic features in governing the binding of the various aggregates? 5. In the second paragraph of the results section, the authors mention about a red-shifted C-O bond. However, it is not clear that the red-shift is with respect to which position. Can the authors address this better? 6. In the second paragraph of the section on "From doubly hydrogen-bonded dimers", I see that the authors mention about high tendency of the molecules to assemble by non-electronic interactions. What do the authors mean by this? 7. The authors report Maxwell-Boltzmann weighted theoretical spectra towards the end of the manuscript. The equation provided by the authors has Boltzmann constant missing in it! Can the authors carefully revise this? 8. The presentation of the content in the manuscript needs significant improvement. Punctuations are not carefully followed (for example, no space is introduced between numerical value of a parameter and its units) and some sentences need to be rewritten (for example, "…will have an impact in molecule interaction"). In the caption of Fig. 7 and in SI, "tetramers" is misspelt.

See Appendix A.
Decision letter (RSOS-210162.R1) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr González-Cano: Title: Structure Determination of Oleanolic and Ursolic acids: a Combined DFT/Vibrational Spectroscopy Methodology Manuscript ID: RSOS-210162.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal. Reviewer: 1 Comments to the Author(s) Heredia and coworkers determine the possible structures of oleanolic acid and ursolic acid using a combination of DFT calculations and IR experiments. The IR spectra calculated from monomeric acid units do not agree well with the experimental spectra. The authors have used different hydrogen-bonded aggregates to match the FTIR spectra. My comments are as follows: 1. The introduction should be more focused on this work. The ms focuses in the arrangement of oleanolic acid and ursolic acid within a raw sample in order to explain their behavior within the plant cuticle and also their physicochemical properties. In this sense, the introduction is focused on a description of the structure and composition of the plant cuticle, the location of these acids within the cuticle, as well as some aspects of the structure and chemistry of triterpenoid acids and their uses in different fields. Nevertheless, we have simplified the text in the introduction.
2. The authors constructed the theoretical IR spectra using a FWHM of 10 cm-1. Why a uniform FWHM of 10 cm-1 is applicable to all the different conformers/aggregates/functional groups? The authors need to explain. Theoretical spectra were built with a standard FWHM of 10 cm -1 . This parameter didn't affect the conclusions of the study, based on the wavenumber and relative intensity of the IR band spectra.
3. The authors fitted the experiments FTIR spectra with 4 peaks. The FWHM of the peaks (same color) varies drastically from oleanolic acid and ursolic acid. Given the similarity in the structures of the two molecules, the authors need to explain this huge variation in the FWHM. Are the fits unique? Can we fit the same peak of more/less number of peaks with different FWHM? The authors should show the goodness of the fits by plotting the experimental spectra the fitted spectra in the same plot. The explanation heavily relies on deconvolution. The aim of the study is to investigate the ability of these molecules to form different aggregates based on the relative position of the monomers looking for the formation of hydrogen bonds. The first step is to point out that in the experimental FTIR spectra the νC=O band is splitted in different contributions. The aim of the deconvolution represented in this work is to demonstrate the existence and the shifting of these contributions. The reviewer is right mentioning this point. In this sense the ms has been updated in order to clarify it. The Figure 3 has been modified including the corresponding experimental spectra as a black line.