Labeling of the active site of α-chymotrypsin with N-nitroso-N-benzylacetamide and related-compounds

α-Chymotrypsin (CT) is slowly inhibited by N-nitroso-N-benzylacetamide (1) at 25^ΰC. The ¹³C-NMR spectrum of the hydrolysate of ¹³C-enriched 1-inhibited CT shows five new signals at 52.33, 44.55, 43.72, 36.79, and 32.90 ppm resulting from alkylation of the side chains and also of amide linkages of the enzyme backbone by benzyl carbocations produced in the active site. The alkylation pattern is different from those observed in the inhibitions of CT with D-N-nitroso-N-[α-¹³C]-benzyl-N′-isobutyrylalaninamide (2a) and D-N-nitroso-N-[α-¹³C]-benzyl-N′-isobutyrylphenylalaninamide (2b) (White et al. JACS1990, 112, 1956 -1961). The chemical shift data suggest that the 52.33 ppm signal stems from N-benzylglycine, the 36.79 ppm signal from S-benzylcysteine, and the 32.90 ppm signal from 2-benzyltryptophan. A synthesis of the latter compound was developed.