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Defined Conjugation of Glycans to the Lysines of CRM 197 Guided by their Reactivity Mapping
Author(s) -
Crotti Stefano,
Zhai Huili,
Zhou Jing,
Allan Martin,
Proietti Daniela,
Pansegrau Werner,
Hu QiYing,
Berti Francesco,
Adamo Roberto
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300785
Subject(s) - lysine , glycoconjugate , chemistry , reactivity (psychology) , glycan , click chemistry , residue (chemistry) , combinatorial chemistry , conjugate , biochemistry , stereochemistry , glycoprotein , amino acid , medicine , alternative medicine , pathology , mathematical analysis , mathematics
Systematic characterisation of the reactivity of the lysine moieties in CRM 197 towards N ‐hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two‐step conjugation of various glycans to CRM 197 by click chemistry in a well‐defined manner. By semiquantitative LC‐MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM 197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine‐mediated glycoconjugates with improved batch‐to‐batch reproducibility, thereby producing neo‐glycoconjugates with more‐consistent biological activity.