The role of acyl donor structure on the course of peptide bond formation catalyzed by SDS-subtilisin in ethanol was investigated. In the reaction ZAlaAlaLeuOR+HPhepNA→ZAlaAlaLeuPhepNA, nearly quantitative product yields were observed after 2h, regardless of whether an activated (R=CH3, p-C6H5Cl) or non-activated (R=H) acyl donor was used. It was found that the enzyme can accept as acyl donors N-protected tri-peptides containing basic or acidic amino acid residues in the P1-position. Tetra-peptides of general formula ZAlaAlaP1P1′pNA, where P1=Glu, Asp, Lys, Arg or His and P1′=Phe, Arg or Glu have been obtained in good yield.

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom ( A ) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly- A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly- A were obtained. It was shown that the condensation products can be split off from the support using Met residue cleavage by BrCN.

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using the Met residue cleavage by BrCN.

The solubility, stability, and activity of native subtilisin 72 and of its complex with SDS were comparatively studied in a number of polar organic solvents. Subtilisin was found to catalyze peptide bond formation when suspended in acetonitrile or solubilized as a complex with SDS in ethanol and isopropanol. Tripeptide Z-Ala-Ala-Leu-pNA, tetrapeptides A-Ala-Ala-P1-P1'-B, where A = Z or Abz; P1 = Leu, Phe, Met, Trp, Ile, Tyr, Phe(NO2), or Glu(OMe), P1' = Leu, Phe, Glu, Ala, Ile, Val, or Arg; B = NH2, pNA, or 2-(2,4-dinitrophenyl)aminoethylamine residue (Ded); pentapeptides Z-Ala-Ala-Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA; and hexapeptide Abz-Val-Ala-Phe-Phe-Ala-Ala-Ded were synthesized using the SDS-subtilisin complex. The complex also efficiently catalyzed the oligomerization of tripeptide H-Phe-Ala-Leu-OCH3 in ethanol, which resulted in a 63:37 mixture of trioligomer and tetraoligomer. It was demonstrated that SDS-subtilisin is a much more efficient catalyst than the suspension of native enzyme.

The behavior of subtilisin 72 in some aprotic solvents (acetonitrile, dioxane, and tetrahydrofurane) was studied. The enzyme was shown to be partially soluble in tetrahydrofurane, but it is rendered profoundly inactive in this solution. In acetonitrile and dioxane, subtilisin formed dilute suspensions whose activities were measured after dilution with water. Under these conditions, subtilisin suspended in acetonitrile manifested an activity that was an order of magnitude higher than that of its dioxane suspension, and this activity continued for a long time. Z-Ala-Ala-Leu-pNA was synthesized from Z-Ala-Ala-OCH3 and Leu-pNA under the catalysis by dilute suspension of subtilisin in acetonitrile. p-Nitroanilides of tetrapeptides, Z-Ala-Ala-P1-P'1-pNA, where P1 and P'1 were either Leu or Phe, were similarly synthesized in acetonitrile under catalysis by dilute subtilisin suspension at [S]:[E] = 10(5):1. p-Nitroanilides of tripeptides, Z-Ala-Ala-Leu-pNA, Z-Ala-Ala-Phe-pNA, and Z-Ala-Ala-Phe-NH2, were also synthesized in the presence of a concentrated subtilisin suspension at [S]:[E] = 10(3):1. It was shown that the increase in enzyme concentration resulted in the double coupling of nucleophile, and Z-Ala-Ala-Leu-Leu-pNA, Z-Ala-Ala-Phe-Phe-pNA, and Z-Ala-Ala-Phe-Phe-NH2 were obtained with 13, 33, and 40% yields, respectively. Therefore, such reaction systems can be used for creating long hydrophobic peptides whose synthesis in water-organic mixtures is difficult due to the poor solubility of starting components in aqueous buffer solutions.

An enzymatic synthesis of tripeptide Z-Ala-Ala-Leu-pNA, tetrapeptides Z-Ala-Ala-P1-P1′-Xaa, where P1 = Leu, Trp, Met, Ala, Ile, Phe; P1′ =Phe, Ala, Leu; Xaa = pNA, NH2, pentapeptides Z-Ala-Ala-Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA is described. The reactions were performed in organic solvents using SDS-subtilisin complex as a catalyst of the peptide bond synthesis.