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Exo‐Mechanism Proximity‐Accelerated Alkylations: Investigations of Linkers, Electrophiles and Surface Mutations in Engineered Cyclophilin–Cyclosporin Systems
Author(s) -
Levitsky Konstantin,
Boersma Melissa D.,
Ciolli Christopher J.,
Belshaw Peter J.
Publication year - 2005
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.200400383
Subject(s) - electrophile , chemistry , cyclophilin a , reactivity (psychology) , alkylation , ligand (biochemistry) , cyclophilin , linker , cysteine , stereochemistry , combinatorial chemistry , receptor , biochemistry , biology , medicine , enzyme , alternative medicine , microbiology and biotechnology , pathology , gene , computer science , operating system , catalysis
Investigations on the scope and utility of exo‐mechanism proximity‐accelerated reactions in engineered receptor–ligand systems are reported. We synthesized a series of electrophilic cyclosporin (CsA) derivatives by varying electrophiles and linker lengths, prepared a series of nucleophilic cysteine mutations on the surface of cyclophilin A (Cyp), and examined their reactivity and specificity in proximity‐accelerated reactions. Acrylamide and epoxide electrophiles afforded useful reactivity and high specificity for alkylation of engineered receptors in Jurkat cell extracts. We found that remote cysteines (>17 Å from the ligand) could be alkylated with useful rates under physiological conditions. The results from mutations of the receptor surface suggest that the dominant factors governing the rates of proximity‐accelerated reactions are related to the local environment of the reactive group on the protein surface. This study defines several parameters affecting reactivity in exo‐mechanism proximity‐accelerated reactions and provides guidance for the design of experiments for biological investigations involving proximity‐accelerated reactions.