Alkaline protease catalysis of a secondary amine to form a peptide bond

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Abstract
Procedures for peptide bond formation using esters of L-amino acids as the acyl donor and secondary amines such as derivatives of D- or L-proline, or pyroglutamic acid as the nucleophile in anhydrous 2-methyl-2-propanol catalyzed by alcalase or subtilisin Carlsberg have been developed. Kinetic studies have shown that all secondary amines tested had catalytic efficiencies (kcat/km) in the range 84-423 min-1 M-1. Both enzymes have identical catalytic properties. The selectivity of the enzyme-catalyzed reaction in 2-methyl-2-propanol shows that at the s-1 subsite of the enzyme only L-amino acids are substrates as acyl donors, and at the s-1' subsite both D- and L-amino acids are substrates. Optimal conditions for preparing precursors of the renin inhibitors Z-Ala-Pro-OBzl, Z-Lys(Z)-Pro-OBzl and Z-Lys(TFA)-Pro-OBzl were studied.