Exo-Mechanism Proximity-Accelerated Alkylations: Investigations of Linkers, Electrophiles and Surface Mutations in Engineered Cyclophilin-Cyclosporin Systems

• Published in: Chembiochem : a European journal of chemical biology Vol. 6; no. 5; pp. 890 - 899
• Main Authors: Levitsky, Konstantin, Boersma, Melissa D., Ciolli, Christopher J., Belshaw, Peter J.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.05.2005; WILEY‐VCH Verlag
• Subjects: Alkylation; Cross-Linking Reagents; Cyclophilins - chemistry; Cyclophilins - genetics; Cyclophilins - metabolism; cyclosporin; Cyclosporins - chemical synthesis; Cyclosporins - chemistry; Cyclosporins - metabolism; Cysteine - genetics; Cysteine - metabolism; Hydrogen-Ion Concentration; Michael addition; Models, Molecular; Mutation - genetics; Protein Engineering; protein modifications; Protein Structure, Tertiary; Static Electricity; Substrate Specificity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200400383

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. Reaching further. Exo‐mechanism proximity‐accelerated alkylations in engineered cyclophilin–cyclosporin systems were systematically investigated. We found that acrylamides and epoxides were the optimal electrophiles, remote cysteine residues could be alkylated efficiently, and that the local environment dominates the reactivity of surface cysteines.