A new technique for conversion of carboxylic acids to carbinols under mild conditions has been developed. This technique requires first formation of enol esters by coupling N-ethyl-5-phenylisoxazolium3' -sulfonate with an appropriate carboxylic acid in acetonitrile, followed by reduction with a 10-fold molar excess of NaBH₄. In addition five carboxyl group "specific" reagents were surveyed for their ability to inactivate the sulfhydryl proteinase papain (EC 3.4.4.10). No inactivation of the enzyme was observed with three of the reagents, diazoacetamide, triethyloxonium tetrafluoroborate, and N-ethyl-5-phenylisoxazolium-3'-sulfonate. The remaining two reagents, N-ethoxycarbony1-2-ethoxy-1,2-dihydroquinoline and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide did indeed inactivate papain. Inactivation by N-ethoxycarbony1-2-ethoxy-1,2-dihydroquinoline appears to be the result of reaction of this reagent with the free thiol group of the enzyme. Treatment of papain with l-ethyl-J-(3-dimethylaminopropyl) carbodiimide resulted in modification of the free thiol group, in 6 to 10 of the nineteen tyrosyl groups, and in six of the fifteen free carboxyl groups. The free thiol group could be essentially completely protected from reaction by converting it to its mercuric derivative with HgCl₂, and the tyrosyl modification was shown to have no effect on enzymatic activity. Reaction of active or mercuripapain with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of glycine ethyl ester resulted in incorporation of 6 ethyl glycinate residues per molecule of enzyme, with essentially complete inactivation. In the presence of benzamidoacetonitrile, a competitive inhibitor of papain, only four ethyl glycinate residues were incorporated into active papain with retention of ~80% of enzymatic activity, thus establishing that at least one and perhaps two of the six modified carboxyl groups were located in the active site of the enzyme. The identity of one of these carboxyl groups was postulated to be Asp-158, and thus some evidence was provided for the tentative assignment of a direct mechanistic role in catalysis for this amino acid residue. / Ph. D.