EPR Spectroscopy of Triplet Aryl Nitrenes Covalently Bound to α-Chymotrypsin. Application of Low-Temperature Methods to Photoaffinity Labeling

Article abstract of DOI:10.1021/ja00227a039

Reaction of α-chymotrypsin with various aryl azido acyl imidazoles leads to covalent attachment of the aryl azide to the enzyme via an ester linkage.Photolysis of frozen solutions of the modified enzymes at 77 K leads to intense EPR signals of the related triplet nitrenes.The position of the EPR resonance fields and the decay kinetics of the nitrenes can be interpreted in terms of the label being within or outside the binding pocket of the enzyme.Photolysis of methyl m-azidobenzoate in toluene at 298 K gives very little nitrene-derived product.At this temperature, ring expansion to a ketenimine that ultimately polymerizes is the major process.Photolysis of methyl m-azidobenzoate in frozen toluene at 77 K gives the triplet nitrene, which undergoes secondary photolysis leading to formal CH insertion into the solvent matrix.It is demonstrated that photolysis of α-chymotrypsin covalently modified with a m-azidobenzoyl group at serine-195 leads to more efficient labeling at 77 K than at 298 K.