Karalé and co-workers report on the development of two phosphoramidite-containing BCN derivatives suitable for solid-phase ON synthesis. They evaluated two types of BCN precursors: the conventional BNC carbinol and a more recent carboxylate homologue, allowing the formation of carbamate and amide bonds, respectively. While these two chemical groups had no influence on the stability of the resulting probes, the alkyne moiety was found to be acid-sensitive – as expected –, thus limiting their use to 5’-modifications of ON. While this work could be of interest to the community of chemical biologists, I find the scope of the present study to be quite narrow: only a single T5 model ON has been used by the authors, and a single example of a SPAAC using a free model amino acid is reported. This prevents the reader from assessing the wider application of this approach and its value compared with others that are already available (e.g., chemo-/regio-selective modification of a 5’-amine-modified ON with activated ester/carbonate of BCN). In addition, the SI needs profound revision, e.g.:
- there is insufficient characterisation at the moment: HRMS data is absent; 1H signals are not assigned; coupling constant values are sometimes missing (see compound 7 for example); some information is missing (have the authors used 19F-decoupled NMR analyses? If so, it needs to be stated; if not, multiplicity and 13C-19F coupling constant need to be provided); etc.
- the data provided are sometimes incorrect or lacunar: 13C chemical shifts are given with two decimals; yields and reaction conditions are missing on all the schemes; the number of 13C signals is slightly off in a number of compounds; there is a huge unlabelled peak in the 13C spectrum of compound 3 that could be consistent with CH2Cl2; etc.
- all RP-HPLC-MS data could benefit from better explanations: labelling the peaks; clarifying the reaction conditions and chemical structures to which the chromatograms and spectra correspond; explicit the differences between S21 and S22; clarify the meaning of “isomers”; etc.
- the procedure for stability studies is not sufficiently detailed: have calibration curves been made? How many replicates did the authors perform?
Besides these critical points, here are some additional comments that could help to improve the quality of the manuscript:
- why exactly did the authors add two BCN groups per compounds and not just one?
- some typos are present throughout the manuscript: SPACC instead of SPAAC; issues with references (“Error! Reference source not found”); the definitions of the abbreviations are given in parentheses while it should be the opposite (e.g., dibenzoazooctyne (DBCO)).
- what do the authors mean by “isomers of both heptamers”? It is unclear at the present.
- why was the stability study conducted with 1a (the exo isomer) and not 2 (the endo isomer)? And why was the exo isomer employed for the formation of linker B?
- when the authors mention that BCN carbamates are more stable than O-alkylated derivatives, to what type of stability do they refer to?
- I am not sure to understand how DBCO can form “diastereomeric adducts”: can the authors clarify this statement?
- the authors claim in their conclusion that sequential conjugation of azide is possible but this statement seems not to be supported by experimental evidences; can the authors bring evidence for this sequential conjugation?
In conclusion, this work could be published in the journal RSC Advances but only after substantial modifications.

This revised version of the manuscript submitted by Karalé and co-authors allows a better understanding of their work and its utility. While several of my comments have been addressed, I still find the scope of the work to be quite narrow (comment #1 from my previous review, which has not been really addressed in my opinion) and that the SI could be clearer and benefit from more information (e.g., how do the authors manage to determine a concentration in mg/mL in Fig 2 without resorting to calibration curves; better description of what the arrows correspond to on the chromatograms; analytical data missing for linkers A and B). Finally, the CH-CH2-O bond connecting the BCN ring to the carbamate's alcohol needs to be redrawn -- it is currently a straight line, it needs to be bent.