title: Side reactions in enzymatic peptide synthesis in organic media: effects of enzyme, solvent, and substrate concentrations.
abstract: The progress of enzymatic peptide synthesis catalyzed by alpha-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH2 (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the L-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group donor, quantitative formation of MalAlaAlaPheXaaNH2 or ZAlaAlaPheXaaNH2 occurred. As a result, a method for the synthesis of polypeptide amides was developed. At low concentration of the acyl-group donor and excess of the nucleophile, the condensation by-products with two and three nucleophile molecules were found in the reaction mixtures. The data obtained provided evidence that organic solvents affected the S'1-specificity of alpha-chymotrypsin and the S1-specificity of subtilisin 72, while the S1-specificity of alpha-chymotrypsin and the S'1-specificity of subtilisin 72 were not affected. When the DMF content was increased, the rate of the alpha-chymotrypsin-catalyzed reactions decreased. In contrast to this, an increase in DMF content accelerated the subtilisin 72-catalyzed reactions. Hydrolysis of the acyl-group donor did not occur in the alpha-chymotrypsin-catalyzed reactions. Significant (up to 50%) formation of MalAlaAlaPheOH was observed at the early stage of the subtilisin 72-catalyzed reactions. Later MalAlaAlaPheOH underwent synthesis.
authors.affiliation: Department of Biotechnology, Chemical Center, Lund University, Sweden.
authors.lastname: Gololobov MYu
authors.forename: V M
authors.initials: VM
authors.lastname: Stepanov
authors.forename: T L
authors.initials: TL
authors.lastname: Voyushina
authors.forename: I P
authors.initials: IP
authors.lastname: Morozova
authors.forename: P
authors.initials: P
authors.lastname: Adlercreutz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis / Q000138:chemical synthesis*; D010179:Pancreas / Q000201:enzymology; D012997:Solvents; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D009842:Oligopeptides; D012997:Solvents; D002918:Chymotrypsin