A tale of two bioconjugations: pH controlled divergent reactivity of protein a-oxo aldehydes in competing a-oxo-Mannich and catalyst-free aldol ligations

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Site-selective chemical methods for protein bioconjugation have revolutionised the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic or even toxic cargos. Herein we report two new methods for the bioconjugation of a-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The ‘a-oxo-Mannich’ and ‘catalyst-free aldol’ ligations both compete for the electrophilic a-oxo aldehyde which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalised bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalised bioconjugates. We explore the substrate scope and utility of both these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH.
Original languageEnglish
Pages (from-to)2387-2400
Number of pages14
JournalACS Chemical Biology
Issue number11
Early online date9 Nov 2021
Publication statusPublished - 19 Nov 2021

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© 2021 American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details


  • Aldehydes/chemistry
  • Aniline Compounds/chemistry
  • Catalysis
  • Hydrogen-Ion Concentration
  • Mannich Bases/chemistry
  • Peptides/chemistry
  • Proteins/chemistry

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