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Updated: Dec 20, 2025

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
Site-selective modification of proteins using cucurbit[7]uril as supramolecular protection for N-terminal aromatic
Anton A A Smith1, Caitlin L Maikawa2, Gillie A Roth2
1Department of Materials Science & Engineering, Stanford University, Stanford, CA 94305, USA. eappel@stanford.edu and Department of Chemistry, Aarhus University, 8000, Denmark.
Abstract:
Cucurbit[7,8]urils are known to form inclusion complexes with aromatic amino acids, hosting the hydrohobic side chains within the cavity and adjacent cations within the portal of the macrocyclic host. Here we show that cucurbit[7]uril binding with N-terminal phenylalanine significantly reduces the nucleophilicity of the amine, likely due to an increase in stability of the ammonium ion, rendering it unreactive at neutral pH. Using insulin as a model protein, we show that this supramolecular protection strategy can drive selectivity of N-terminal amine conjugation away from the preferred B chain N-terminal phenylalanine towards the A chain N-terminal glycine. Cucurbit[7]uril can therefore be used as a supramolecular protecting group for site-selective protein modification.
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