From the journal RSC Chemical Biology Peer review history

03-Jan-2023

Dear Dr Bode:

Manuscript ID: CB-ART-11-2022-000234
TITLE: Synthesis of Multi-Module Low Density Lipoprotein Receptor Class A Domains with Acid Labile Cyanopyridiniumylides (CyPY) as Aspartic Acid Masking Groups

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I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

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Reviewer 1

In the current manuscript, the authors identified cyanopyridiniumylide (CyPy) based carboxylic acid protecting groups, which suppress the aspartimide formation during solid phase peptide synthesis (SPPS). The authors showed that CyPy’s can be easily prepared from commercial sources, exemplified with Fmoc-Asp(CyPy)-OH from Fmoc-Asp(OtBu)-OH, and are completely compatible with all standard amino acid residues and protecting group strategies. The authors separately employed Fmoc-Asp(CyPy)-OH and Fmoc-Asp(OtBu)-OH in the synthesis of the peptide NN92, and compared the aspartimide formation by treating peptide, on resin, with bases such as piperidine or DBU, and revealed that aspartimide formation was very low in the case of CyPy protection. The authors showed that these ylide based protecting groups can be removed under standard resin cleavage and global deprotection conditions. The authors exemplified the utility of CyPy’s in the synthesis of Cys- and Asp-rich containing monomeric and dimeric low-density lipoprotein receptor class A (LA module). The authors performed the KAHA ligation between two LA modules, one with an alpha-ketoacid at C-terminal and another with a (S)-5-oxaproline at the N-terminal in presence of CyPy’s followed by its selective removal and multiple disulfide bond formation.
In this current manuscript, the authors identified a new class of acid protecting group to repress the aspartimide formation in peptide synthesis was good. Also, the authors nicely illustrate the difference between the standard Fmoc-Asp(OtBu)-OH and the current identified Fmoc-Asp(CyPy)-OH in aspartimide formation. So, the current work is very appropriate to publish in RSC Chemical Biology as is.

Reviewer 2

Low-density lipoprotein receptor class A domains (LA modules) mediate many cellular processes including endosytosis of extracellular targets, cell migration and others. Owing to the important functions, LA modules have been considered as therapeutic targets or as therapeutics. Chemically synthetic LA modules can facilitate their therapeutic applications. However, the chemical synthesis of LA modules is challenging due to the high density of aspartic acid residues which cause the undesired aspartimides. In this manuscript, the authors reported a robust chemical synthesis strategy for LA modules. The authors developed cyanopyridiumylides (CyPY) as a new type of protecting group for Asp to prevent the aspartimide formation during Fmoc SPPS. Unlike their previously reported cyanosulfurylides (CSY) using the oxidative deprotection conditions, the new CyPY protecting groups can be removed under aqueous acid conditions (e.g. 350 mM HCl I H2O/DMSO) which are compatible with all common residues. Using the new CyPY groups, the authors prepared and folded the LA3 and LA4 modules, as well as a heterodimeric LA3-LA4. It is expected that other difficult-to-obtain Asp-rich peptides/proteins can be chemically synthesized by using the new CyPY groups.
Overall, the manuscript should be published in RSC Chem Biol after addressing the comments below.

Comments:
1. Did the authors test the removal of the Acm and cyanosulfurylides (CSY) in one-pot fashion?
2. I wonder if the authors used the recent reported disulfide-bond formation strategies (Nat Commun 2021, 12 (1), 870; Angew Chem Int Ed 2019, 58 (17), 5729) to fold the LA modules. Maybe these methods can achieve the “one-pot” de-Acm and S-S formation.
3. the legends of Fig 1, 2 and 4 shouldn’t be a part of pictures.

Thank you for the positive reviewers of our manuscript. Please see the response to reviewers letter for specific changes made to the manuscript. In addition, a version of the manuscript with highlights of the specific changes made in included in the submitted files.

With my best regards,

Jeffrey

This text has been copied from the Microsoft Word response to reviewers and does not include any figures, images or special characters:

Point-To-Point Response

Referee: 1

The current work is very appropriate to publish in RSC Chemical Biology as is.

Response: We thank referee 1 for evaluating our manuscript and recommending publication in RSC Chemical Biology.

Referee: 2

1) Did the authors test the removal of the Acm and cyanosulfurylides (CSY) in one-pot fashion?

Response: We thank referee 2 for this suggestion. Indeed, the Acm-residue can be removed under acidic condition in the presence of Ag(OAc). We did not evaluate if these conditions can be implemented in the process of CyPY-removal. Given the complexity and high abundance of Acm-residues on LA modules, we preferred to purify and characterize the modules prior Acm-removal. In our experience, best results are obtained for Acm-removal when the peptide is purified by HPLC.

2) I wonder if the authors used the recent reported disulfide-bond formation strategies (Nat Commun 2021, 12 (1), 870; Angew Chem Int Ed 2019, 58 (17), 5729) to fold the LA modules. Maybe these methods can achieve the “one-pot” de-Acm and S-S formation.

Response: We thank referee 2 for suggesting one-pot Acm removal and disulfide bond formation. We identified that calcium ions are necessary for correct folding and that the folding is sensitive to small changes in temperature, ion concentration and redox equilibrium. For that reason, we preferred to carry out the removal of Acm and folding separately. However, since the combination of Acm-removal and folding would reduce the synthesis by one step, we added a sentence that describes this possibility with the corresponding references.

“While we carried out the Acm-removal and folding separately, one can consider combining these steps in the future.24,25”

3) the legends of Fig 1, 2 and 4 shouldn’t be a part of pictures.

Response: We apologize for the inconvenience and ungrouped captions and figures.

20-Jan-2023

Dear Dr Bode:

Manuscript ID: CB-ART-11-2022-000234.R1
TITLE: Synthesis of Multi-Module Low Density Lipoprotein Receptor Class A Domains with Acid Labile Cyanopyridiniumylides (CyPY) as Aspartic Acid Masking Groups

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With best wishes,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 2

The authors answered the concerns properly. I agree on the publication of the revised manuscript.