Interaction of dansylated peptidyl chloromethanes with trypsin, chymotrypsin, elastase, and thrombin.

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.