CZ283098B6 - Process for preparing peptides by making use of proteolytic enzymes - Google Patents

Abstract

A method of preparing peptides wherein a mixture of amino acid derivatives or peptides in the presence of sodium carbonate decahydrate is spread with proteolytic enzymes, thereby forming peptide bonds. Preferably proteolytic enzymes are -chymotrypsin, trypsin, proteinase K, subtilisin, elastase, and papain. This method avoids the use of toxic chemicals and solvents

Description

The invention relates to a process for the preparation of peptides by means of proteolytic enzymes.

BACKGROUND OF THE INVENTION

It has previously been found that proteolytic enzymes whose natural function is the hydrolysis of peptide bonds can also be used in the opposite reaction under certain conditions, i. for the synthesis of peptide bonds (review: Čeřovský V .: Universe 69.76 (1990). Jakubke H.-D. Kuhl K., Konecke A .: Angew. Chem. 24.85 (1985), Morihara K. Trends. Biotechnol. 5, 164 1987). The synthesis of peptides by proteolytic enzymes is carried out in aqueous solutions, in aqueous solutions with the addition of a water-miscible organic solvent, in two-phase or multiphase systems, or in low-water organic solvents.

It has now been found that this synthesis can also be carried out on a dry route by simply triturating amino acid derivatives or peptides with sodium carbonate decahydrate in the presence of the appropriate proteolytic enzyme. The process is advantageous both economically and ecologically, since its use eliminates toxic chemicals and solvents.

SUMMARY OF THE INVENTION

The process for the preparation of peptides by proteolytic enzymes according to the invention is characterized in that the mixture, acylating components consisting of an amino acid ester or peptide in free or protected form, and amino components consisting of amino acid or peptide in free or protected form, is spread with proteolytic enzymes. chymotrypsin, trypsin, proteinase K, subtilisin, elastase or papain in the presence of sodium carbonate decahydrate.

This procedure produces peptide bonds without the addition of any liquid.

Examples

Mixture of ester N of α-protected amino acid or peptide (0,1 mmol), C α-protected amino acid or peptide in hydrochloride, hydrobromide or free base form (0,1 mmol 0,2 mmol) and sodium carbonate decahydrate (0, 05 - 0.2 mmol) was spread in a glass homogenizer with an internal diameter of 9 mm with the appropriate proteolytic enzyme (2 mg), such as α-chymotrypsin, trypsin, proteinase K, subtilisin, elastase or papain, at room temperature for 1 - 24 hours.

The resulting products were isolated to confirm their identity by preparative HPCL. All peptides thus synthesized had the corresponding amino acid composition, mass spectrum and were homogeneous in thin layer analysis and HPCL.

Examples of individual syntheses with the yield of peptides in percent after the optimum reaction time are shown in the following tables, together with the yields of enzymatic hydrolysis of the ester of the acylating component, which always accompanies the desired synthesis.

Table 1. Synthesis of peptides in a solvent-free system, catalysed by α-chymotrypsin

Acylating component (0.1 mmol) Amino component (0.2 mmol) Synthesis of peptides (%) Hydrolysis of ester (%) Ac-Tyr-OEt Leu-NH ₂ .HCl 96 3 Ac-Tyr-OEt Gly-NH ₂ .HBr 82 16 Ac-Tyr-OEt Phe-NH ₂ .HCl 81 12 Ac-Tyr-OEt Phe-NH ₂ 61 4 Ac-Tyr-OEt Phe-NH? 40 3 Boc-Trp-OMe Leu-NH ₂ .HCl 59 3 Boc-Trp-OMe Met-NH ₂ .HCl 57 (77) ^c 2 (3) ^c Boc-Trp-OMe Gly-NH ₂ .HBr 5 1 Z-Gly-Phe-OMe Met-NH ₂ .HCl 93 6

Na ₂ CO ₃ .10H ₂ O 0.1 mmol, 4h. reaction time, α-chymotrypsin 2 mg, ^and monohydrate, ^b without use

Na ₂ CO ₃ .10H ₂ O, ^c 20 h after addition of the second portion of enzyme

Table 2. Papain-catalyzed peptide synthesis in a solvent-free system

Acylating component (0.1 mmol) Amino component (0.2 mmol) Synthesis of peptides (%) Hydrolysis of ester (%) Z-Glu-Oh ^and Leu-OBu'.HCl 61 34 Bz-Arg-OEt.HCl ^a Leu-OBu‘.HCl 47 24 Z-Ala-Ome ^b Phe-NH ₂ .HCl 97 2 Z-Gly-Gly-OMe ^b Phe-NH ₂ .HCl 39 4

papain 2 mg, Cys.HCl 0.02 mmol, 4 h reaction time, ^a 0.2 mmol, ^b 0.15 mmol Na ₂ CO ₃ .10H ₂ O

Table 3. Synthesis of peptides in a solvent-free system, catalyzed by trypsin protei15 name K, subtilisin and elastase

Acylating component (0.1 mmol) Amino component (0.2 mmol) Peptide synthesis I (%) ester ester hydrolysis (%) Bz-Arg-OEt.HCl Ala-Ala-OH trypsin 97 2 Boc-T y r-Ly s-Ome Met-NH ₂ .HCl 97 2 Z-Leu-Ala-Om Gly-Gly-OH proteinase K 72 26 Ac-Tyr-Oet _Ser-NH2 90 9 From-For-Leu-Ome GLy-NH ₂ .HBr 62 5 Ac-Tyr-Oet Gly-NH ₂ .HBr subtilisin 92 7 Z-Gly-Ala-Om _Gly-Gly-NH2 79 21 Z-Ala-Ala-Om Phe-NH ₂ elastase 65 18 Z-Ala-Ala-Om Trp-NH ₂ .HCl 81 19 Dec Z-Val-Ala-Om Trp-NH ₂ .HCl 53 3 Z-Ala-Ala-Om Leu-NH ₂ .HCl 28 20 May Na ₂ CO ₃ .10H ₂ O 0.1 mmol, enzyme 2 mg, 2 h. reaction time (extension there was no reaction time

no effect on yields)

-2GB 283098 B6

Industrial applicability

The method of the invention is useful for preparing peptides with biological activity, or fragments thereof.

Claims (1)

Process for the preparation of peptides by proteolytic enzymes, characterized in that a mixture of an acylating component consisting of an amino acid ester or peptide in free or protected form and amino component consisting of an amino acid or peptide in free or protected form is spread with proteolytic enzymes α-chymotrypsin, trypsin, proteinase K, subtilisin, elastase or papain in the presence of sodium carbonate decahydrate.