Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1993-02-12
1994-09-27
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435213, 435219, C12P 2100, C12N 976, C12N 950
Patent
active
053506820
DESCRIPTION:
BRIEF SUMMARY
AREA OF APPLICATION OF THE INVENTION
The invention relates to a process for preparing peptides which, after the splitting off of protective groups, can serve as active substances or intermediates of active substances.
DESCRIPTION OF THE KNOWN STATE OF THE ART
Various organic chemical processes can be employed for the preparation of peptides (cf. Review: Wunsch, E. (1974) Synthesis of Peptides, in Houben-Weyl, Vol. 15, 1/2, Methoden der organischen Chemie (Methods of organic chemistry), Muller, E. (Ed.) Georg Thieme Verlag, Stuttgart). During the course of chemical peptide syntheses, undesirable side reactions are frequently observed which decrease the yield and render necessary difficult and lengthy purification procedures. A particularly serious disadvantage of the conventional processes is the unsolved problem of racemization which appears in particular in relation to segment condensation using chemical coupling methods. Since it is very difficult to separate stereoisomers from each other completely, and the optical purity of the products of synthesis is a necessary prerequisite for biological activity, the industrial synthesis of peptides by means of organic chemical processes has substantial disadvantages. Furthermore, because of the danger of side reactions, the third functionalities of amino acid building blocks must be reversibly blocked in all chemical operations for synthesizing peptides. The use of biocatalysts for the catalysis of the peptide coupling step offers a means of circumventing the difficulties described (cf. Reviews: Jakubke, H.-D., and Kuhl, P. (1982) Pharmazie 37 89; Fruton, J. S. (1982) in A. Meister: Adv. Enzymmol. Relat. Areas Mol. Biol. 53, 239; Jakubke, H.-D., Kuhl, P. and Konnecke, A. (1985) Angew. Chem. 97, 79). As a result of the stereospecificity of the proteases employed as biocatalyst, chiral integrity is preserved and the high degree of reaction control makes it possible, to a large extent, to dispense with protection of third functionalities. Kinetic control of the reaction plays a key role within the framework of enzyme-catalyzed peptide synthesis. The hydrolysis of the acyl-enzyme intermediate that is involved with peptide product formation in this method still represents a problem for many synthesis reactions, since the yield of peptide product remains limited.
AIM OF THE INVENTION
The aim of the invention is to prepare chirally uniform peptides with greatly reduced hydrolysis of the acyl-enzyme intermediate as compared with previously employed methods.
EXPLANATION OF THE NATURE OF THE INVENTION
The basic object of the invention is to react alkyl esters of N-acylamino acids in the presence of proteases with amino acids, amino acid derivatives or peptides with an unprotected N-terminal alpha-amino group as the amino component.
According to the invention, peptides are prepared from an amino acid with a protected alpha-amino group or a peptide with a protected alpha-amino group, whose carboxyl group entering into the reaction is present as an ester, and from an amino acid, an amino acid derivative or a peptide, in which the amino group entering into the reaction is unblocked, in the presence of a protease in frozen aqueous solution, which optionally contains organic solvent constituents and/or buffer substances. Peptides are formed in high yields and can be preparatively separated by suitable chromatographic or extractive techniques. In contrast to known syntheses of peptides using proteases, a far higher yield of peptide is obtained by the freezing of the reaction mixture according to the invention. In one embodiment the enzyme-catalyzed reactions are carried out in a temperature range from -1.degree. C. to -40.degree. C.
This is the first time that the enzyme-catalyzed synthesis of the peptide class of compounds has been described in frozen aqueous systems. The effect of the invention is surprising inasmuchas a decrease in yield, which is essentially caused by the formation of byproducts, is generally associated with a lowering of temperature and the consequ
REFERENCES:
patent: 4256836 (1981-03-01), Isowa et al.
patent: 4935355 (1990-06-01), Ulmer
"Grundprinzipien der proteasekatalysierten Knupfung der Peptidbindung", Jakubke, Kuhl und Konnecke, Angewandte Chemie, 97 (1985) 79-87.
"Basic Principles of Protease-Catalyzed Peptide Bond Formation", Jakubke et al., Angew. Chem. Int. Ed. Engl. 24 (1985), No. 2, pp. 85-93.
"Proteinase-Catalyzed Synthesis of Peptide Bonds", Fruton, A. Meister Adv. Enzymmol. Relat. Areas Mol. Biol., 53, (1982), pp. 239-306.
"Proteasen als Biokatalysatoren fur die Peptidsynthese", Jakubke et al., Die Pharmazie 37, (Feb. 1982), pp. 89-106.
"Protease-catalyzed peptide synthesis: effect of temperature on the kinetics of acyl transfer catalyzed by papain", Gololobov et al., Chemical Abstracts, abstract No. 232227p, 112(25):314 (1990).
"Enzyme-catalyzed Peptide Synthesis In Ice", Schuster et al., Tetrahedron, 46(24): 8093-8102 (1990).
Aaviksaar Aavo
Jakubke Hans-Dieter
Schuster Matthias
Hoechst Aktiengesellschaft
Lilling Herbert J.
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