[The chemoselective fluorescence labeling of alpha-chymotrypsin]

The covalent fixation of the phosphinoyl residues in the active site of alpha-chymotrypsin is proved by the application of the fluorescent phosphinoyl fluorides 1 [( 5-(dimethylamino)-1-naphthyl]phenylphosphinoyl-fluoride) or 4 [(5-methoxy-1-naphthyl)phenyl-phosphinoylfluoride]. The differences in the rates of the phosphinoylation of alpha-chymotrypsin and "methyl-alpha-chymotrypsin" as compared to 1 agree with model reactions. In both enzymes the serine-OH in the active site is phosphinoylated. The non-fluorescent 4-nitrophenyl [5-(dimethylamino)-1-naphthyl]phenylphospinate (3) and the corresponding non-fluorescent 5-methoxynaphthyl derivative 5 inhibit alpha-chymotrypsin far more slowly than the corresponding fluorides 1 and 4. The phosphinoyl residues of the nitrophenyl esters 3 and 5 are covalently linked in a yield of 80% to the active site of the enzyme with evolution of fluorescence. 20% of the nitrophenyl ester inhibits the enzyme by adsorption.