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Abstract

Reaction of Ac-Tyr-OEt with HBr.Gly-NH₂, catalyzed by free proteinase K in various water-miscible organic solvents in the presence of triethylamine and 5 vol.% of water, was studied. Some aliphatic alcohols and acetonitrile proved to be suitable solvents. The effect of water content (2%-20%) on the synthesis of Ac-Tyr-Gly-NH₂ was studied using acetonitrile as solvent. Lowering of the water content to 5% or 2% led to almost 100% yield of the desired dipeptide; higher water content accelerated the reaction, reducing at the same time the yield of Ac-Tyr-Gly-NH₂ due to the concurrent hydrolysis of the ester Ac-Tyr-OEt. No reaction was observed in the absence of base (triethylamine), whereas an excess of base only retarded the reaction. The enzyme is capable of catalyzing the peptide bond synthesis with N-acylamino acids or N-acyl peptides as acylating components, which may contain all types of L-amino acid residues (except Pro) in the P₁ position. However, the peptide bond synthesis depends strongly on the amino component composition, particularly on the amino acid residue in the P'₁ position. Only amides of glycine and of hydrophilic amino acids were acylated with Ac-Tyr-OEt; amides of hydrophobic amino acids enter the reaction only reluctantly or not at all. The presence of Leu or Phe in position P'₂ and Leu in position P'₃ has not so negative effect on acylation of the amino component as has its presence in the P'₁ position. The choice of protecting groups for the α-carboxyl of the amino component is restricted only to amide and in some cases its undesired enzymatic removal was observed. Unprotected peptides seem to be suitable amino components.