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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 mechanism of inactivation of serine proteinases by peptide halomethyl ketone inhibitors was studied through the inhibition of trypsin with a series of model peptide ketones (Lys-Ala-LysCH2X). In this series, X is a poor leaving group with increasing electron-withdrawing capacity (X = H, CH2CO2CH3, COCH3, OCOCH3, and F), and as expected, the peptide ketones are reversible, competitive inhibitors of trypsin. The strength of binding of these inhibitors to trypsin increases with the electron-withdrawing ability of X, indicating that the inhibition constant Ki obtained is a measure of reversible hemiketal formation between the inhibitor ketone carbonyl group and the hydroxyl group of the active site serine. A Hammett plot of -log Ki vs. sigma I, the inductive substituent constant of X, reveals a linear relationship between the free energy of binding and the electron-withdrawing power of X. The reversible binding constant obtained for the corresponding chloromethyl ketone Lys-Ala-LysCH2Cl falls on this line, indicating that the reversible binding involves hemiketal formation, which is followed by alkylation of the enzyme.