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A series of N alpha-1-(dimethylamino)-5-naphthalenesfulfonyl (dansyl) derivatives of peptidyl chloromethanes (chloromethyl ketones) were synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active sites of proteinases via affinity labeling. Dansylalanyllysychloromethane (DALCM) was utilized to inactivate and fluorescently label trypsin and the trypsin-like enzyme thrombin. Dansylleucylphenylalanylchloromethane (DLPCM) was synthesized and selectively employed as an inhibitor of chymotrypsin. The di-, tri-, and tetrapeptides--dansylprolylalanylchloromethane (DPACM), dansylalanylprolylalanylchloromethane (DAPACM), and dansylprolylalanylprolylalanylchloromethane (DPAPACM)--were synthesizedand their interaction with elastase was evaluated. The compounds DALCM, DLPCM, and DAPACM all proved to be effective, fast-acting proteinase inhibitors. Studies of energy transfer in the enzyme-inhibitor conjugates led to results entirely consistent with the proposed conformational bomology of thrombin with the other serine proteinases studied. The fluorescent affinity labels are believed to posses enormous potential for the localization, isolation, and characterization of enzymes.

The 1-dimethylamino-5-naphthalene sulfonyl (dansyl) derivative of alanyllsyl chloromethane was synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active site of trypsin via affinity labelling. The potential of dansylalanyllysyl chloromethane lies in its high degree of selectivity and markedly faster rate of enzyme inactivation when compared to previously synthesized, single residue affinity label chromophores. This permits the practical utilization of stoichiometric amounts of the inhibitor to achieve 100% inactivation of trypsin, even at high dilutions. The transfer of energy between the four tryptophan residues of trypsin and the bound dansyl group has been investigated in the fluorescent inhibitor-enzyme conjugate. From transfer efficiency measurements mean distances of 19.0 A and 19.3 A between the point of attachment of the dansyl group and the four tryptophan residues of trypsin have been calculated. These compare well with the mean value of 18.8 A derived from calculations based on crystallographic model studies.