From the journal RSC Chemical Biology Peer review history
Design and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteins
Round 1
Manuscript submitted on 13 jan 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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06-Feb-2021
Dear Prof Pratt:
Manuscript ID: CB-REV-01-2021-000010
TITLE: Design and Synthesis of Metabolic Chemical Reporters for the Visualization and Identification of Glycoproteins
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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Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology
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Reviewer 1
The manuscript by Pedowitz and Pratt is exceptionally well-written, covering the large field of metabolic reporters of glycosylation. The review comprehensively covers both the history of MCRs as well more recent discoveries and will be extremely useful for colleagues in the field.
While this is an outstanding account already, there are a few points the authors may wish to add:
- The review rightfully focuses of the most widely used functional groups of reporters. Maybe the authors could briefly mention other groups such as cyclopropenones (Row et al., JACS 2017) and sydnones (e.g. Chinoy et al., ANIE 2019).
- In section 5.3, it would be good to add the reference Schart et al., Chembiochem, 2018.
- In the Fucose MCR section, it would be good to add the new paper by Ma et al., Int. J. Mol. Sci. 2020, covering the comparison between different Fucose reporter molecules, as well as Schneider et al., Nat Chem Biol. 2018, on inhibition of Notch signalling by Fucose analogs.
- In the figure on page 17, it might be good to add the probe developed by Debets et al., PNAS 2020 that is not epimerized.
- Shajahan et al. have recently profiled in detail the metabolic destination of azido-sugars (ACS Chem. Biol. 2020). It would be great to include this work as it adds important information to the field.
- The authors mention BTTES as a ligand for Cu-click chemistry. Could they maybe also add the other common ligands TBTA, THPTA and BTTAA, mentioning the kinetic superiority of BTTES and BTTAA? Further on p. 8, I am not sure if CuAAC will ever be used in living animals as stated in the text. Maybe better to modify this statement to “…in living systems”, which includes complex models such as organoids?
- Figure 4 states that IEDDA functional groups are too large for sugar salvage pathways. This is certainly true for many TCO reagents, but probably not for cyclopropenes?
Minor typos etc.:
- P. 4, “can also be <b>synthesized</b>”
- P. 6, “an expansive library of biorthogonal reactions” may be better called “a large number” or “a plethora”?
- P. 7 “are otherwise <b>chemically</b> inert”
- P. 8, “This bond deformation generates enough free energy” should be “generates strain that releases enough free energy upon 1,3-dipolar cycloaddition to proceed without…”
- P. 10, “consisting <b>of</b> the addition of four mannose…”
- P. 10, “N-linked glycans can <b>be</b> transferred…”
- P. 12, “generate double mutant <b>cell</b> lines…”
- P. 13, “incorporation <b>into</b> the cell surface…”
- P. 13, “Compared to healthy tissue indicating.” – sentence ends here.
- P. 14 “Okeley et al <b>were</b> able to…”
- In the figure on p. 17, the compound in the mucin section has 6 carbons and should thus be “Ac4GalN-hexynoic acid”
Reviewer 2
The manuscript by Pedowitz and Pratt reviews the development and application of chemical reporters for protein glycosylation. Glycosylation, which involves the attachment of different sugar moieties to proteins, is a common type of posttranslational modification that regulates a wide range of cellular processes. The heterogeneous and dynamic nature of protein glycosylation makes it challenging to detect this modification by traditional methods (e.g., antibodies). The development of metabolic chemical reporters has provided valuable tools to visualize and identify the glycoproteins, and largely advanced our understanding of the regulatory mechanism and biological functions of protein glycosylation. In this manuscript, the authors provided a historic overview of how the chemical reporters were designed and discussed examples of how the reporters were applied in different model systems. The current limitations and possible future directions in this field have also been discussed. Overall, this manuscript is informative and carefully prepared. I support its publication in RSC Chemical Biology.
A minor issue is that the layout of the manuscript should be adjusted. The legends of Figs. 2, 6, and 7 are not fully displayed. No legend has been found for Fig. 5.
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This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.
Thank you for the opportunity to submit a revised manuscript. Please see our "Response to Referees" PDF for our point by point response. We have also included a Manuscript file with the changes to the text highlighted in track changes.
Responses to referee comments:
Referee 1 General Comment - The manuscript by Pedowitz and Pratt is exceptionally well-written, covering the large field of metabolic reporters of glycosylation. The review comprehensively covers both the history of MCRs as well more recent discoveries and will be extremely useful for colleagues in the field.
Response - We thank Referee 1 for their kind words! We are proud of this work and excited to introduce it to the field.
While this is an outstanding account already, there are a few points the authors may wish to add: Comment #1 - The review rightfully focuses of the most widely used functional groups of reporters. Maybe the authors could briefly mention other groups such as cyclopropenones (Row et al., JACS 2017) and sydnones (e.g. Chinoy et al., ANIE 2019).
Response - We agree that other functional groups offer a more complete view of reporters. We have added a paragraph describing cyclopropenones section 2. After reviewing Chinoy et al., we believe it also serves as an important addition to the review. However, in terms of placement we feel it is better suited for the sialic acid section (4.3) as the authors detail experiments specifically with sialic acid MCRs and instead of broadly defining the functional group.
Comment #2 - In section 5.3, it would be good to add the reference Schart et al., Chembiochem, 2018.
Response - We agree with this suggestion and have added a few sentences and citation for this example of multi-labeling.
Comment #3 - In the Fucose MCR section, it would be good to add the new paper by Ma et al., Int. J. Mol. Sci. 2020, covering the comparison between different Fucose reporter molecules, as well as Schneider et al., Nat Chem Biol. 2018, on inhibition of Notch signalling by Fucose analogs.
Response - Both of these examples have been added to the fucose section.
Comment #4 - In the figure on page 17, it might be good to add the probe developed by Debets et al., PNAS 2020 that is not epimerized.
Response - While we agree that the probe described in this publication is noteworthy, due to space constraints in the figure’s design we find it difficult to add. We sincerely hope that the referee is ok with that decision. However, this paper is cited in the text and an example of a “bump-and-hole” probe designed and tested by the same group of researchers is included in the figure.
Comment #5 - Shajahan et al. have recently profiled in detail the metabolic destination of azido-sugars (ACS Chem. Biol. 2020). It would be great to include this work as it adds important information to the field. Page 3 of 29 RSC Chemical Biology
Response - We agree that this report adds important information to the field. We have included a citation in section 6.2 for the discussion of the interconversion of various MCRs and the following sentence in section 6.3: “This was exemplified in recent work from the Kohler lab108. Here, efforts to generate a probe-free technique for the identification of glycoproteins illuminated the vast metabolic cross talk that interconverts GalNaz, GlcNAz and ManNAz.”
Comment #6 - The authors mention BTTES as a ligand for Cu-click chemistry. Could they maybe also add the other common ligands TBTA, THPTA and BTTAA, mentioning the kinetic superiority of BTTES and BTTAA? Further on p. 8, I am not sure if CuAAC will ever be used in living animals as stated in the text. Maybe better to modify this statement to “…in living systems”, which includes complex models such as organoids?
Response - We have reviewed this section and agree that those changes would be an improvement. A sentence listing all chelating ligands has been added followed by a statement about the superiority of BTTES and BTTAA.
Comment #7 - Figure 4 states that IEDDA functional groups are too large for sugar salvage pathways. This is certainly true for many TCO reagents, but probably not for cyclopropenes?
Response - Figure 4 has been edited to say “Most functional groups…” to clarify.
Minor typos etc.: - P. 4, “can also be synthesized” - P. 6, “an expansive library of biorthogonal reactions” may be better called “a large number” or “a plethora”? - P. 7 “are otherwise chemically inert” - P. 8, “This bond deformation generates enough free energy” should be “generates strain that releases enough free energy upon 1,3-dipolar cycloaddition to proceed without…” - P. 10, “consisting of the addition of four mannose…” - P. 10, “N-linked glycans can be transferred…” - P. 12, “generate double mutant cell lines…” - P. 13, “incorporation into the cell surface…” - P. 13, “Compared to healthy tissue indicating.” – sentence ends here. - P. 14 “Okeley et al were able to…”
Response - These typos have been corrected in the text.
In the figure on p. 17, the compound in the mucin section has 6 carbons and should thus be “Ac4GalN-hexynoic acid”
This typo has been corrected in Figure 5.
Referee: 2 Comments to the Author
The manuscript by Pedowitz and Pratt reviews the development and application of chemical reporters for protein glycosylation. Glycosylation, which involves the attachment of different sugar moieties to proteins, is a common type of posttranslational modification that regulates a wide range of cellular processes. The heterogeneous and dynamic nature of protein glycosylation makes it challenging to detect this modification by traditional methods (e.g., antibodies). The development of metabolic chemical reporters has provided valuable tools to RSC Chemical Biology Page 4 of 29 visualize and identify the glycoproteins, and largely advanced our understanding of the regulatory mechanism and biological functions of protein glycosylation. In this manuscript, the authors provided a historic overview of how the chemical reporters were designed and discussed examples of how the reporters were applied in different model systems. The current limitations and possible future directions in this field have also been discussed. Overall, this manuscript is informative and carefully prepared. I support its publication in RSC Chemical Biology.
Response - We thank Referee 2 for their kind words and support for the publication of this manuscript.
Comment #1 - A minor issue is that the layout of the manuscript should be adjusted. The legends of Figs. 2, 6, and 7 are not fully displayed. No legend has been found for Fig. 5. Response - These issues have been fixed to allow for the full figure legends to be visible. We have also included separate figure files in the resubmission
Round 2
Revised manuscript submitted on 10 feb 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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11-Feb-2021
Dear Dr Pratt:
Manuscript ID: CB-REV-01-2021-000010.R1
TITLE: Design and Synthesis of Metabolic Chemical Reporters for the Visualization and Identification of Glycoproteins
Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below. Please correct the typo mentioned by the reviewer.
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With best wishes,
Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology
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Reviewer 1
The authors have incorporated all suggestions. There is only one typo in "cyclopropenones" on p. 10: "Cyclorpropenones are small in size and react readily with triaryl phosphines"
Congratulations to a great review!
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