From the journal RSC Chemical Biology Peer review history

Berlin, 4. February 2022

Dear Dr Unsworth:

Manuscript ID: CB-ART-12-2021-000245
TITLE: Synthesis of medium-ring lactams and macrocyclic peptide mimetics via conjugate addition/ring expansion cascade reactions

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

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Yours sincerely,
Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

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Reviewer 1

The authors report on a novel synthetic strategy that gives access to lactams of medium ring size and macrocyclic peptide mimetics using a conjugate addition/ring expansion cascade reaction named CARE. In their very carefully performed study, scope and limitation of this approach has been elucidate in detail concerning ring size of the lactam, structural variations within the lactam moiety, variation of the Michael acceptor, as well as the chemical nature of the amine. In Addition, the concept of iterative CARE reactions is very interesting, because it provides beta-peptoid-based macrocycles in a straightforward manner. To my opinion, this CARE approach supplements in an elegant way the established set of reactions yielding such medium-ring lactams and macrocyclic peptide mimetics. Furthermore, the flexibility of the CARE approach is also of great interest to provide molecules in a systematic manner that possibly show biological activity, because the accessible structural space can be diversified simultaneously at four positions within the resulting product molecule. In conclusion, the manuscript, presenting the synthetic data in a very clear and easy to read manner, is recommended for publication in RSC Chemical Biology.

Reviewer 2

In this manuscript, Unsworth and coworkers report a new cascade reaction consisting of conjugate addition of an amine to an acrylamide followed by ring expansion through nucleophilic attack of an imide carbonyl group (CARE). This work is an extension of previous methodology developed by the group named SuRE. In SuRE, an acid chloride containing a protected amine (or another nucleophile) is reacted with a lactam followed by a deprotection and subsequently the ring expansion step. The first step has now been improved by using acryloyl chloride that modifies the lactam and can then be attacked by an amine that subsequently induces the ring expansion by the same mechanism. The advantages of CARE over SuRE are fewer synthetic steps for the preparation of the starting materials and the avoidance of unstable carbamate-protected amino acid chlorides. A limitation compared to SuRE, however, seems to be that only ring expansion by 4 atoms are possible whereas SuRE allows 3- and 4-atom expansions.

The authors nicely show the scope and limitations of the method by synthesizing a variety of compound with varying lactams, Michael acceptors, and amines. In general, yields are quite high although some substrates do not react or lead to side reactions. In addition, SuRE and CARE or two times CARE can be carried out iteratively. The manuscript is well-written, the experiments are well-documented, and new compounds are well-characterized. Although this manuscript is purely synthetic (as pointed out in the cover letter) it would fit to a themed collection on ‘Synthesis and Chemical Biology of Macrocycles’. I recommend publication after having considered the following minor issues.

1. Table 1, entry 14: How can it be that the isolated yield is higher than the one determined by 19F NMR spectroscopy?
2. Scheme 2A: Numbering of lactams 11a-e: I would have expected that ‘a’ corresponds to n=1, ‘b’ corresponds to n=2 and so on. The chosen assignment (a: n=3, b: n=1, c: n=2) is confusing.
3. Scheme 2E: The reactant on the first arrow should be 15 and not 15a.
4. Page 7 of 188 (last paragraph before the Conclusions): I would not call the 14-membered compounds 31a-c ‘tri-peptide mimetics’ because they are not built from ‘normal’ amino acids but from beta-amino acids. For the same reason I also do not see how this approach can be used for the preparation of RGD peptide analogs. The synthesized compounds are beta-peptoids and the stereoselective introduction of substituent in alpha or beta position is not possible with this approach. This part should be rephrased.

Dear Professors Yudin, Raj and Chen, and the rest of the editorial team at RSC Chemical Biology,

We were delighted to learn of your decision to accept the manuscript for publication after revisions, and also that both referees were so positive about the work. Referee 1 did provide any criticism, or request any revisions be made. Referee 2 recommended publication after considering four minor issues, listed below, along with our response. All changes made to the manuscript can be seen in a version of the manuscript with yellow highlighting.

1. Table 1, entry 14: How can it be that the isolated yield is higher than the one determined by 19F NMR spectroscopy?
Response: The synthetic reaction was done on a much larger scale (5 mmol vs 0.5 mmol). The Table 1 captions have been altered to more clearly reflect this difference.

2. Scheme 2A: Numbering of lactams 11a-e: I would have expected that ‘a’ corresponds to n=1, ‘b’ corresponds to n=2 and so on. The chosen assignment (a: n=3, b: n=1, c: n=2) is confusing.
Response: we agree with the referee that in Scheme 2A, the numbering looks a little odd. However, changing the numbering to the system the referee suggests would not be ideal either; the problem is that the 6-membered lactam (11a as we numbered it) appears earlier in the manuscript than the others, and we think that calling it 11c before introducing 11a and 11b could be confusing. Therefore, on balance, we would prefer to leave the numbering as is, if the editors agree.

3. Scheme 2E: The reactant on the first arrow should be 15 and not 15a.
Response: corrected

4. Page 7 of 188 (last paragraph before the Conclusions): I would not call the 14-membered compounds 31a-c ‘tri-peptide mimetics’ because they are not built from ‘normal’ amino acids but from beta-amino acids. For the same reason I also do not see how this approach can be used for the preparation of RGD peptide analogs. The synthesized compounds are beta-peptoids and the stereoselective introduction of substituent in alpha or beta position is not possible with this approach. This part should be rephrased.
Response: we agree with the referee here and have toned down/clarified this section accordingly. The phrase ‘tri-peptide mimetics’ has been replaced simply with ‘compounds’. Also, an extra sentence has been added after mention of RGD peptides, to emphasize that CARE can only able be used to insert beta-amino acid fragments and hence cannot be used to target proteinogenic amino acid sequences. The RGD peptide comparison was made to highlight the importance of sequence specific cyclic peptides, and we hope the extra sentence added helps to clarify this point.

Thus, having addressed all the reviewer comments, and with both referees having judged the manuscript favourably, we hope that you will now be in a position to formally accept the manuscript for publication. If there is anything else that we can do to help, please don’t hesitate to ask.

With best wishes, Will

Berlin 8. February 2022

Dear Dr Unsworth:

Manuscript ID: CB-ART-12-2021-000245.R1
TITLE: Synthesis of medium-ring lactams and macrocyclic peptide mimetics via conjugate addition/ring expansion cascade reactions

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor