From the journal RSC Chemical Biology Peer review history

Berlin, 24 February 2023

Dear Dr Büll:

Manuscript ID: CB-ART-01-2023-000006
TITLE: UV light-induced spatial loss of sialic acid capping using a photoactivatable sialyltransferase inhibitor

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

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Reviewer 1

This is a very nice piece of work, building on previous designs to create in situ sialic acid transferase inhibitors, now masked with a photolabile group to allow inhibition of cell-surface sialic acid glycan formation within tissue culture. The actual formation, in situ, of the CMP-sialic acid inhibitor, but indirect proof is of course evident and as well this part builds on previous art from the same group. In all, and also because of the pleasant read and the care with which the experiments are executed and documented, I recommend acceptance as is.

Reviewer 2

In this manuscript, the synthesis and application of the photocaged sialyltransferase inhibitor 4 is reported. Compound 4 is a variant of the known inhibitor 5 being equipped with a photolabile o-nitrobenzyl group and was used in several cell experiments. It is shown that irradiation of 4 with 365 nm light results in an active inhibitor of cell surface sialylation. Irradiation was either carried out before addition of the compound to HEK293 cells or after. Once photo deprotection has occurred, the effect of the inhibitor is observed after additional 3 days of cell culture. This is a proof-of-principle study with potential application for the treatment of superficial tumors penetrable by UV light. However, it should be kept in mind that irradiation of cells with UV light is very problematic even though the authors have shown that the used UV light dose does not impact cell viability under the conditions tested. I think that this manuscript might be suitable for publication in RSC Chem. Biol. after consideration of the following issues.

Major aspects
1.) The efficiency of photo deprotection has been demonstrated only qualitatively by observation of the formation of a brown color (Fig 2A, not further characterized) or indirectly via the effect of inhibition of sialylation after varying irradiation times (Fig 3). These experiments are scientifically not sound. Photo deprotection should be investigated and quantified by following the reaction outcome, for example by HPLC. In this way, it can be easily demonstrated that deprotection is complete after 30 sec of radiation. By the way, why is the brown color developing only after 900 sec (Fig 3A) if the deprotection is complete after 30 sec?
2.) On page 3 of the manuscript (right column, first paragraph), it is described that deprotection occurs specifically at 365 nm. However, only 3 wavelengths have been investigated. It is surprising that deprotection does not occur at 312 nm. Is there any explanation? The UV/VIS absorption spectrum of compound 4 should be shown.
3.) Experiments shown in Fig 4: In the manuscript (p. 4, last paragraph before Conclusion) it is mentioned that no signs of cell death were observed and the cell nuclei were regularly shaped. This is not visible in Fig 4B. An additional magnification should be shown in which individual cells and the shape of their nuclei is visible. Also, the legend to Fig 4 is not detailed enough and the scale bars in the figure are too small.

Minor aspects
4.) Page 2, right column, last line before Fig 2: ‘ortho-nitrobenzyl’ instead of ‘orthophenyl’
5.) Legend to Fig 3A: What is shown in the image below the diagram? (TLC plate? photograph of the reaction vessel?) The legend to the Fig 3B is not complete (‘After ...?’)
6.) Page S4, UV light exposure: Used solvent?
7.) Page S4, flow cytometry: Number of experiments/cells?
8.) Page S4, Immunocytochemistry: Check concentration: 100 or 150 microM?
9.) Page S9, Figure S4: Error bars from a single experiment?
10.) Page S10. Figure S5: Treatment with compound 5? (or 4?)
11.) Page S11-S13: Figures of NMR spectra should be properly labeled (frequency, solvent, ...)

Nijmegen, April 8th 2023

Subject: Revised article submission to RSC Chemical Biology CB-ART-01-2023-000006

Dear Prof. Dr. Roderich Süssmuth

Thank you for considering our work and the opportunity to revise our manuscript entitled ‘UV light-induced spatial loss of sialic acid capping using a photoactivatable sialyltransferase inhibitor’ by Moons et al.

We have addressed the constructive comments of both reviewers. Please find enclosed our revised marked and unmarked manuscript and a point-by-point reply.

We hope that you find the revised manuscript suitable for publication in RSC Chemical Biology.

Sincerely,

Christian Büll
On behalf of all authors


Reviewer #1
This is a very nice piece of work, building on previous designs to create in situ sialic acid transferase inhibitors, now masked with a photolabile group to allow inhibition of cell-surface sialic acid glycan formation within tissue culture. The actual formation, in situ, of the CMP-sialic acid inhibitor, but indirect proof is of course evident and as well this part builds on previous art from the same group. In all, and also because of the pleasant read and the care with which the experiments are executed and documented, I recommend acceptance as is.

Response: We thank the reviewer for the positive feedback. Previously, we have shown formation of CMP-SiaFNAc in situ (Heise et 2019) of compound 5 and agree that inhibition of sialylation with UV-SiaFNEtoc indirectly proofs formation of active inhibitor.

Action: We have added a reference to our previous work showing formation CMP-SiaFNAc to the introduction: ‘Upon entering the cell, P-SiaFNAc is deacetylated by esterases and converted into the active nucleotide sugar CMP-SiaFNAc by the CMP-sialic acid synthetase (CMAS).’17


Reviewer #2
In this manuscript, the synthesis and application of the photocaged sialyltransferase inhibitor 4 is reported. Compound 4 is a variant of the known inhibitor 5 being equipped with a photolabile o-nitrobenzyl group and was used in several cell experiments. It is shown that irradiation of 4 with 365 nm light results in an active inhibitor of cell surface sialylation. Irradiation was either carried out before addition of the compound to HEK293 cells or after. Once photo deprotection has occurred, the effect of the inhibitor is observed after additional 3 days of cell culture. This is a proof-of-principle study with potential application for the treatment of superficial tumors penetrable by UV light. However, it should be kept in mind that irradiation of cells with UV light is very problematic even though the authors have shown that the used UV light dose does not impact cell viability under the conditions tested. I think that this manuscript might be suitable for publication in RSC Chem. Biol. after consideration of the following issues.

Major aspects:
1.) The efficiency of photo deprotection has been demonstrated only qualitatively by observation of the formation of a brown color (Fig 2A, not further characterized) or indirectly via the effect of inhibition of sialylation after varying irradiation times (Fig 3). These experiments are scientifically not sound. Photo deprotection should be investigated and quantified by following the reaction outcome, for example by HPLC. In this way, it can be easily demonstrated that deprotection is complete after 30 sec of radiation. By the way, why is the brown color developing only after 900 sec (Fig 3A) if the deprotection is complete after 30 sec?

Response: We agree and have analyzed 4 using LED-NMR which showed efficient photolysis at 365 nm and formation of the unprotected inhibitor 2. We speculate that the decolouration derives from reactions between the solvent (DMSO) and released side products after photocleavage with UV light.

Action: We have added a new Fig. S3 showing LED-NMR analysis of 4 and the text on page 3: ‘LED-NMR analysis of 4 confirmed photolysis at 365 nm and formation of the active inhibitor (Fig. S3).’

2.) On page 3 of the manuscript (right column, first paragraph), it is described that deprotection occurs specifically at 365 nm. However, only 3 wavelengths have been investigated. It is surprising that deprotection does not occur at 312 nm. Is there any explanation? The UV/VIS absorption spectrum of compound 4 should be shown.

Response: Indeed, interestingly the absorbance maximum of 4 is between 250-300 nm, but 312 nm light treatment did yield sialylation inhibition. Literature reports that the substituents of the nitrobenzyl group influences absorbance and that the wavelength with maximum absorbance does not necessarily correspond to the wavelength at which photolysis occurs (Hansen et al 2015). This is in line with the wide use of 365 nm for photocleaving of such derivatives.

Action: We have included the UV-vis spectrum of 4 and 5 as new Fig. S4 and added the text on page 3. ‘Notably, 4 showed an absorbance peak between 250-300 nm, but remained inactive when treated with 312 nm light (Fig. S4). Presumably, the substituent may influence the absorption which has been reported for other nitrobenzyl derivatives while photolysis occurs most efficiently at 365 nm.40’

3.) Experiments shown in Fig 4: In the manuscript (p. 4, last paragraph before Conclusion) it is mentioned that no signs of cell death were observed and the cell nuclei were regularly shaped. This is not visible in Fig 4B. An additional magnification should be shown in which individual cells and the shape of their nuclei is visible. Also, the legend to Fig 4 is not detailed enough and the scale bars in the figure are too small.

Response: We agree.

Action: We have added 60x magnifications of control and UV-treated areas of HEK293 monolayers that show that nuclei are regularly shaped after treatment (new Fig. S8). We provide more readable scale bars and an extended legend to Figure 4 in the revised manuscript.

Minor aspects:
4.) Page 2, right column, last line before Fig 2: ‘ortho-nitrobenzyl’ instead of ‘orthophenyl’

Response: Thank you.

Action: We have changed the text accordingly.

5.) Legend to Fig 3A: What is shown in the image below the diagram? (TLC plate? photograph of the reaction vessel?) The legend to the Fig 3B is not complete (‘After ...?’)

Response: The image shows samples of compound 4 in a multi-well plate that were UV-treated in separate tubes. Thank you, the legend to Fig. 3B was accidentally cut out.

Action: We have revised the legend to Figure 3.

6.) Page S4, UV light exposure: Used solvent?

Response: Dimethyl sulfoxide (DMSO) was used as a solvent

Action: We have added this to the revised supplementary information S4.


7.) Page S4, flow cytometry: Number of experiments/cells?

Response: All flow cytometry experiments were performed 2-3 times and 10.000 cells were measured/sample.

Action: We have added this information to page S4 and specified the number of replicated in the supplementary figure legend.

8.) Page S4, Immunocytochemistry: Check concentration: 100 or 150 microM?

Response: Thank you, pulsing with 150 uM was used for immunocytochemistry.

Action: We have corrected this in the revised manuscript and supplementary information.

9.) Page S9, Figure S4: Error bars from a single experiment?

Response: Error bars show SD from three independent experiments.

Action: We have added this information to the revised legend of Figure S4.

10.) Page S10. Figure S5: Treatment with compound 5? (or 4?)

Response: Treatment was with DMSO (A) or 5 (B).

Action: We have labelled the revised images to clarify that compound 5 was used.

11.) Page S11-S13: Figures of NMR spectra should be properly labeled (frequency, solvent, ...)

Response: The information has been added.

Action: Labeling of Fig. S11-13 have been revised.

Berlin, 6 May 2023

Dear Dr Büll:

Manuscript ID: CB-ART-01-2023-000006.R1
TITLE: UV light-induced spatial loss of sialic acid capping using a photoactivatable sialyltransferase inhibitor

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

Reviewer 1

The authors did a good job in addressing the concerns of the second reviewer and so I remain of the position the manuscript can be accepted for publication in RSC Chem Biol, as is.

Reviewer 2

The authors did a great job in addressing all reviewer issues. The manuscript can now be published as is.