A Pathway for Aldol Additions Catalyzed by l-Hydroxyproline-Peptides via a β-Hydroxyketone Hemiaminal Intermediate
Round 1
Reviewer 1 Report
Please, check the attached file
Comments for author File: 
Comments.pdf
Author Response
Major revisions
“Please check carefully the assignment 1H-NMR (for compound 4 and 5 in consideration of the corresponding Supporting information, there are some mistakes).”
We apologize for minor inconsistencies of chemical shifts reported in the Materials and Methods section with the spectra given in the SI. We have revised the corresponding shifts accordingly (highlighted in the revised manuscript).
“In the 1H-NMR of trans-4 there are some impurities, add asterisks to indicate them or replace the NMR experiment.“
We have added asterisks to signals we attribute to solvent impurities in Figure S6. The caption now reads: “Figure S6. 1H-NMR of trans-4 in CDCl3 (signals due to solvent impurities marked with asterisks *).”
“Move all the title of 13C-NMR, because some chemical shifts are not readable.”
For better readability, we have removed all titles in the upper left corner of NMR figures (1H and 13C NMR).
“The style of 1H-NMR of cis-6 is completely different from the previous spectra, add the same information on it.”
All 1H-NMR have been modified to contain the same information in the same format.
“Check carefully the assignment of 1H-NMR of trans-6 and trans-7 and label the impurities with asterisks.”
Assignments have been adjusted in the materials and Methods section (highlighted in yellow). Impurities have been marked with asterisks in Figures S21 as in Figure S6 (see above).
“Check the retention time (there are differences between the experimental and assignment at page 5)”
The retention times on Page 5 have been corrected: “HPLC retention time (tR): (R)-9: 12.2 min; (S)-9: 15.4 min (Figures S30–S39).”
Justify all the peaks in the figures of the following chromatograms: FIGURE S32, S33, S34, S36.
In the captions of Figures S32 to S37, we added (where applicable) “The peak at … min is from starting material.” or “Peaks at … min and … min are from injection and starting material, respectively.”
“There are so many peaks in the chromatograms S38 and S39 that are not acceptable, please provide a new HPLC traces.“
All the experimental data in this paper has been acquired during a short-term scholarship project. Therefore, re-running single experiments cannot be easily accomplished. Anyway, as the chromatograms in both experiments are very similar in terms of additional peaks, we are convinced that this is rather an effect of the reaction with catalyst 7 itself than a problem with data acquisition. As high substrate conversion does not necessarily mean high yield of product, the additional peaks may have their origin in by-products formed through an alternative mechanism as proposed in the main text. To highlight this possibility, we added:
“Furthermore, despite high substrate conversion, considerable formation of by-products was observed both in presence of cis-7 and trans-7 (Figures S38 and S39). Possibly, in pure acetone, where higher amounts of 10 or even trimer 11 may be present, alternative pathways may exist…”
And in the captions of Figures S38 and S39:
“Additional peaks such as the broad peak at 27.7 min presumably have their origin in by-products formed in the course of an alternative pathway with cis-7 / trans-7 (see main article).”
Minor revisions:
“Generally, is better to report all the structure like 1 without brackets, check in all the text.”
We intended to use compound numbers in parentheses at first mention after the full compound names only. However, we found some instances where full names plus compound numbers in parentheses appeared at second or third mention as well. These instances have been replaced by the compound numbers without parentheses.
“Page 4 line 130, which HPLC instrument is used for all the enantiomeric ratio? Report in details all the information”
We used an HP 1100 chromatography system by Agilent. This information has been added to “General Experimental Conditions”.
“Page 4, line 133, write how many mL of DCM are used in this reaction.”
This information (“10 mL”) has been added to the section “Dipeptide Coupling”.
“Page 4, line 167 check carefully the notation about the volume of TEA” (sic, presumable refers to line 137)
The volume notation has been corrected.
“Page 4, Choose only one way t write the information at lines 145-146 in comparison with line 160-161.”
We changed the wording in the second instance to match the first instance.
“Page 4, My suggestion is to report (several rotamers) after the 1H-NMR not at the end of 13C-NMR.”
We have moved “several rotamers” to the beginning of the 13C NMR signal assignments: “13C NMR (CDCl3, several rotamers)…”
“Page 5, line 184 write the formula of acetone like previously.”
In the context of an NMR solvent it now reads “acetone-d6”.
“Page 5, it is better to write room temperature, check in all the text”
We changed “ambient temperature” to “room temperature” throughout the text.
“Page 7, write the same notation for time in all the text, I suggest h and not hrs.”
We changed “hrs” to “h” throughout the text and reaction schemes, if applicable.
“Check carefully Table 1, all the catalysts are not in bold style.”
Table 1 has been formatted with compound numbers in boldface.
“Page 8, check the sentence at line 302. Page 13, check the sentence at line 476”
The empty parentheses should refer to Table 1. This has been added.
“Check carefully the Table 2, all the notations cis and trans are not in italic style.”
Table 2 has been formatted accordingly.
Reviewer 2 Report
Comments
The manuscript entitled “A pathway for aldol additions catalyzed by ÊŸ-Hydroxyproline peptides via a β-hydroxyketone hemiaminal intermediate” describes the synthesis of hydroxyproline-based dipeptides through amide coupling of Boc-protected cis- or trans-4-ÊŸ-hydroxyproline (cis- or trans-4-Hyp) to benzylated glycine (Gly-OBn) and ÊŸ-valine (ÊŸ-Val-OBn) and their application as catalysts for a model aldol reaction and further computational studies.
1. However, there are certain areas in this manuscript in which improvement is needed. The author should mention the exact temperature on page 4, line 140.
2. Line 133, Page 4: Please mention whether two different diastereomer of amino acid 3 were reacted separately?
3. In Scheme 2: After TFA treatment only cis product formation is represented, please correct the scheme by mentioning the formation of trans product with yield.
Decision: Accept after minor revision.
Comments for author File: Comments.pdf
Author Response
“The author should mention the exact temperature on page 4, line 140.”
The reaction mixture was stirred at room temperature. This has been added to the section “Dipeptide Coupling” as well as to Scheme 2.
“Line 133, Page 4: Please mention whether two different diastereomer of amino acid 3 were reacted separately?”
To clarify this, the section now reads: “To a solution of 1.0 eq. of enantiomerically pure amino acid cis-3 or trans-3 (200 mg, 0.87 mmol) in dichloromethane (10 mL)…”
“In Scheme 2: After TFA treatment only cis product formation is represented, please correct the scheme by mentioning the formation of trans product with yield.”
We corrected the scheme by adding trans-6 and trans-7. Removal of the Boc group was achieved in quantitative yield, which is given in the foot note.
Reviewer 3 Report
Line 258: I would not use the term “free amines” for Gly-OBn.
Scheme 2: the conditions need to be added.
Compounds 2, 3: it is better to provide the absolute configuration then cis/trans descriptors.
Table 1: it is not clear why the ee was determined by NMR, but er by HPLC. Since the ee if function of er there is no need for different measurements.
Line 302, 476: please check.
The detailed stepwise protocol how to calculated er (based on DFT data) should be presented in manuscript and ESI.
Table 2. could Authors predict er based on assumption that 10 is involved it to the synthesis?
Table 1, 2: please confirm that R-product was major in all the cases. If not the explanation of reverse of stereoselectivity should be proposed.
Could Authors explain how the absolute configuration of valine effects on the reaction outcome?
Could Authors explain the role of -CO2Bn group in the reaction studied?
Author Response
“Line 258: I would not use the term ‘free amines’ for Gly-OBn.”
We have removed the term in this context. The corresponding section in Materials and methods now reads “Preparation of N-Deprotected Dipeptides”
“Scheme 2: the conditions need to be added.”
The conditions are now given on the arrows.
“Compounds 2, 3: it is better to provide the absolute configuration then cis/trans descriptors.”
We chose cis/trans descriptors because this is the way hydroxylprolines are usually named in the literature.
“Table 1: it is not clear why the ee was determined by NMR, but er by HPLC. Since the ee if function of er there is no need for different measurements.”
The ee and er were determined by HPLC, while the conversions were determined by H-NMR. This is now clarified by addition of a footnote to ee in both table 1 and Table 2. We apologize for any confusion about this issue.
“Line 302, 476: please check.”
In both instances, empty parentheses should reference to Table 1. This has been added.
“The detailed stepwise protocol how to calculated er (based on DFT data) should be presented in manuscript and ESI.”
A brief protocol and the formula used to calculate er from the calculated energy barriers have been added to Materials and Methods, Quantum chemical calculations. The equation is, for example, given in reference 39 (now added to the cited literature).
“Table 2. could Authors predict er based on assumption that 10 is involved it to the synthesis?”
If the catalytic cycle was entered by 10 instead of acetone, it would still arrive at the very same enamine intermediate as in the Houk–List pathway (see Scheme 4). Therefore, the stereoselective step would be the same as above and the theoretical er values would be the same as in Table 1.
“Table 1, 2: please confirm that R-product was major in all the cases. If not the explanation of reverse of stereoselectivity should be proposed.”
To clarify that the major product was R-configured in all cases, we added to Materials and Methods, Aldol Reactions: “The absolute configuration of the major product was determined as R in accordance with the elution order in previously published data on chiral separations of 9.”
“Could Authors explain how the absolute configuration of valine effects on the reaction outcome?”
The er reported in Table 1 shows that there is no impact in DMSO/acetone. In pure acetone, i. e. large excess of acetone, we hypothesize that acetone self-adduct(s) are also involved in later reaction steps (see last paragraph of Results section, “Possibly, in pure acetone, where higher amounts of 10 or even trimer 11 may be present, alternative pathways are possible involving acetone self-adduct intermediates even at the stereoselective C,C-coupling step”). The higher steric demand of such a transition state would render steric clashes with the ÊŸ-Val-sidechain more likely, thereby leading to decreased stereoselectivity, and, despite of high substrate conversion, the occurrence of by-products (Figures S38 and S39). However, regarding this work, this is a side aspect and proving this hypothesis is beyond the scope of this study. This also applies to studying the effect a á´…-Val sidechain may have in comparison to the ÊŸ-Val-sidechain.
“Could Authors explain the role of -CO2Bn group in the reaction studied?”
While this group served as a protective group to carry out peptide coupling, it also blocks the proton donor properties of the dipeptide C-terminus in the aldol reactions. Furthermore, it increases conformational space of the catalysts.
Round 2
Reviewer 1 Report
The paper could be published in this current form because authors made all the required corrections
Reviewer 3 Report
Authors clarified the issues had mentioned previously.
