DD236346A1 - METHOD FOR THE ENZYMATIC ABOLITION OF THIOL PROTECTION GROUPS - Google Patents

Abstract

The invention relates to the enzymatic cleavage of thiol protecting groups in amino acid and peptide derivatives in the context of peptide synthesis. The method is for the recovery of peptide derivatives containing thiol components, with high purity of the products, high yield and gentle reaction conditions. The invention involves the enzymatic cleavage of S-phenacetamidomethyl or S-phenoxyacetamidomethyl groups in a two-step process using enzymes of the penicillinamidohydrolase type. The enzyme preparations can also be used in immobilized form.

Description

Field of application of the invention

The invention relates to a method of peptide synthesis and its peripheral regions, as far as the amino acid cysteine or its homologs (such as, for example, homocysteine), the corresponding deamin derivatives or decarboxy compounds are used.

Characteristic of the known technical solutions

To use thiol group-containing compounds in peptide synthesis, it is necessary to temporarily protect the SH function. About 100 protective groups have been described in the literature (Amino Acids, Peptides and Proteins, Volumes 1-15, The Royal Society of Chemistry, London 1968-1984, E. Wünsch [Ed.]: Synthesis of Peptides, Houben-Weyl Vol 15/1 and 15/2,

G. Thieme Verlag, Stuttgart 1974). Generally, it is known that cleavage of thiol protecting groups requires drastic conditions (e.g., sodium in liquid ammonia, liquid hydrogen fluoride, iodine in methanol, mercury salts, trifluoromethanesulfonic acid), often causing side reactions which affect the yield and purity of the products.

The advantageous properties of S-acylamidomethyl protecting groups as S-protecting groups in the synthesis of thiol-containing peptides or their disulfide derivatives (D.F. Veber, J.D. Milkowski, R.G. Denkewalter, R. Hirschmann: Tetrahedron Lett., 1968, 3057;

D.F. Veber, J.D. Milkowski, S.L. Varga, R.G. Denkewalter, R. Hirschmann: J. Amer. Chem. Soc. 94, 9456 [1972]; H. Arold, M. Eule in: Peptides 1972 [Eds. H. Hanson, H.-D. Jakubke] p. 78, North Holland. Publ. Co, Amsterdam 1973; P. Hermann, E. Schreier: ibid.

p. 123; R.G. Denkewalter, D.F. Veber, F.W. Holly, R-. Hirschmann: J. Amer. Chem. Soc. 91, 502 [1969]; P. Hermann, E. Schreier: J.

prakt. Chem. 316, 719 [1974]) are limited by the previously known cleavage methods (eg Hg ²⁺ ions, I ₂ in methanol).

Object of the invention

The aim of the invention, while maintaining the S-Acylamidorriethyl protective groups for the thiol function, in the synthesis of thiol-containing amino acid and peptide derivatives to introduce a gentle, selective, economically satisfactory cleavage method for Thiolschutzgruppen that provides high purity products.

Explanation of the essence of the invention

The invention has for its object to use enzymes for the removal of S-Acylamidomethyl groups and thus to overcome the shortcoming of the previous procedure

Features of the invention are therefore according to Scheme 1

Sn z 37m "acyl-ΠΗ-CrL.-SR + H ₂ O". H ₂ Ii-CH ₂ -SR + acyl-OH

Scheme 1 H ₂ O

spontaneous

^2V , CH ₂ O, HS-R

- 2 - 7b3 8 /

1. To run the cleavage of the S-protecting group in a two-step process, wherein only the first step is enzyme-catalyzed, the second step takes place spontaneously and provides the desired thiol compound.

2. In the first phase to use such enzymes that are of the amidohydrolase type and whose specificity is particularly aligned with the acyl residue of the bond to be cleaved.

3. To use those acylamidomethyl protective groups whose acyl radical corresponds to the requirements of the specificity of the amidohydrolase-type enzymes to be used.

According to the invention enzymes of the type of penicillin amidohydrolases (EC 3.5.1.11) are used from microorganisms. Peptides containing thiol-containing building blocks are constructed using S-phenylacetamidomethyl or S-phenoxyacetamidomethyl protecting groups or other S-acylamidomethyl protecting groups which have a substrate function as described above, and at the desired time of synthesis in aqueous solution or in water the mixture of water with organic solvents in the pH range of the optimal effect exposed to the catalytic influence of Penicillinamidohydrolasen. This can be done in a homogeneous or heterogeneous system, whereby the penicillin amidohydrolases can also be bound or otherwise immobilized on soluble or insoluble macromolecular carriers. Reaction temperature and enzyme / substrate ratio are determined and optimized by preliminary experiments. The thiol compounds obtained after this cleavage process can be further reacted in the desired manner, for. B. to disulfides.

embodiments

1. In an inert gas atmosphere at pH 7 and 310 Kin 10% dimethylformamide 0.2 mmol

tert-butyloxycarbonyl-S-phenoxyacetamidomethyl-L-cysteine with 1 mg of partially purified penicillinamidohydrolase from bacteria (TA Savidge, M. Cole: Methods Enzymol., 43, 705 [1975]) or fungi (H. Vanderhaeghe: Methods Enzymol., 43, 721 [1975]). After reaching the quantitative conversion, the resulting tert-butyloxycarbonyl-L-cysteine is oxidized by known methods to the disulfide, isolated and after recrystallization from ethyl acetate / hexane are crystals of di-tert-butyloxycarbonyl-L-cystine in 90% yield Received fp 418-419 K. The preparation is chromatographically uniform in several solvent mixtures.

2. Analogously to Example 1 0.2 mmol S ^ henacetamidomethyl glutathione, which by known methods from tert-butyloxycarbonyl-L-glutamyl (a-tert-butyl ester) -YS-phenacetamidomethyl-L-cysteinyl-glycine were incubated with 1 mg penicillinamidohydrolase from bacteria. The resulting glutathione was oxidized to disulfide with hydrogen peroxide and purified by column chromatography on a molecular sieve. In 77% yield glutathione disulfide with a mp. 448-453 K is obtained. [cc] ² _D ° = -105 °.

3. Tert-butyloxycarbonyl-S-phenacetamidomethyl-L-cysteine was immobilized at pH 7.6 and 318 K in the presence of 1% dimethylformamide by an immobilized penicillin amidohydrolase (covalently bonded to a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate which had been modified by epoxy groups). reacted analogously to Example 1 to tert-butyloxycarbonyl-L-cysteine.

4. S-phenacetamidomethyl-.beta.-mercaptopropionic acid (10 ~ ¹³ mol / l) was reacted at pH 8 and 298 K by penicillin amidohydrolase from bacteria to give β-mercaptopropionic acid.

Claims (7)

- I - / UvI U / Claim: 1. A process for the enzymatic cleavage of thiol protecting groups, characterized in that are used as thiol protecting groups those which are first cleaved as S-Acylamidomethyl groups enzymatically on the acid amide bond of the ^ protecting group and then the remaining S-Aminomethylthioverbindung spontaneously to release the Thiol function is hydrolyzed using amidohydrolase-type enzymes, which are particularly adjusted to the acyl radical involved in the S-acylamidomethyl protective group. 2. The method according to item 1, characterized in that are used as thiol protecting groups, the S-Phenacetamidomethyl- and the S-Phenoxyacetamidomethyl groups and other S-Acylamidomethyl groups which can also be cleaved enzymatically. 3. The method according to item 1, characterized in that enzymes of the type of penicillinamidohydrolases from microorganisms are used as enzymes. 4. The method according to items 1-3, characterized in that this method is preferably used for the synthesis of peptides, peptide derivatives and amino acid derivatives whose thiol-containing blocks must be temporarily blocked by protecting groups. 5. The method according to items 1-4, characterized in that the cleavage of the acyl group and the subsequent release of the thiol function in water or in mixtures with water and organic solvents in the pH range 6-10. 6. The method according to items 1-5, characterized in that the enzymes can also be used in immobilized form. 7. The method according to items 1 -6, characterized in that the enzyme action in the temperature range 283-323 K is used.