DD274235A1 - PROCESS FOR THE PRODUCTION OF PEPTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of peptides which, after complete or partial deprotection, can serve as enzyme substrates or peptide active agents or as intermediates of active compounds. The object of the invention is the preparation of sterically uniform peptides in the homogeneous phase under conditions of the backward penetration of undesired protease-catalyzed bond cleavages both in the educts and in the product. The object is achieved according to the invention that N-Acylaminosaeureester be used with high Kcat / KM values in excess, which decisively back Sekundaerhydrolyse and beyond allow the incorporation of Acyldonoraminosaeuren that do not correspond to the literature known Proteasepezifitaet, creating a significant broadening of the use of serine proteases such as alpha-chymotrypsin results as biocatalysts for enzymatic peptide syntheses.

Description

Scope of the invention

The invention relates to a process for the preparation of peptides which, after optionally complete or partial removal of the protective groups, can serve as enzyme substrates or active substances or as intermediates of active substances.

Characteristic of the known technical solutions

For the production of peptides, more than 140 different organic-chemical processes can be used (for review, see: Wünsch, E. [1974] Synthesis of Peptides, Houben-Weyl, Vol. 15,1 / 2, Methods of Organic Chemistry, Müller, E. (Ed.) Georg Thieme Verlag, Stuttgart; Jakubke, H.-D., and Jeschkeit, H., Amino Acids, Peptides, Proteins, 3rd ed., Akademie-Verlag, Berl'n [1982]). In chemical peptide syntheses, undesirable side reactions are often observed, which have the effect of reducing the yield and necessitating lengthy purification processes. A particularly serious disadvantage of the conventional chemical Ver Ahren ^f is the unsolved problem of racemization, which makes its appearance in particular in the chemical linkage of peptide segments (see., Inter alia, Meienhofer, J., [1981] Biopolymers 20.1761 to 1784). Since it is difficult to completely separate diastereomers and the chiral integrity is a necessary prerequisite for the biological action, the industrial processes for the synthesis of peptides by means of chemical coupling methods have distinct disadvantages. Furthermore, in all thematic peptide synthetic operations, the third functions of A.nino acid building blocks must be reversibly blocked because of the risk of side reactions. To circumvent the difficulties indicated, the use of biocatalysts for the catalysis of peptide bond formation is useful (for review, see: Jakubke, H.-D., and Kühl, P., [1982] Pharm. 37, 89-106; Fruton, JS, [1982] Adv. Enzymol., Rel. Areas Mol. Biol., 53, 23, 39-306, Jakubke, H.-D., Kühl, P., and Könnecke, A., (1985) Angew. Chem 97, 79-87). The stereochemistry and regioselectivity of the proteases used as biocatalysts ensures, on the one hand, the optic purity of the synthesis products and, on the other hand, the high degree of reaction control makes it possible to largely dispense with the protection of third-order functions.

In general, a distinction is made between equilibrium-controlled and kinetically controlled enzyme-catalyzed peptide syntheses. While the first variant involves the direct reversal of proteolysis, in kinetically controlled syntheses, alkyl esters are used as the carboxy components in the reg, which favor the necessary temporary acylation of the serine or cysteine proteases which can only be used as biocatalysts. The kinetically controlled enzymatic peptide synthesis has clear advantages over equilibrium-controlled enzymatic peptide synthesis because of the short duration of the reaction and the lower enzyme requirement (see Overview: Jakubke, H.-D., [1987] in: The Peptides [S. Udenfriend and J. Meienhofer , Ed.) Vol. 9, pp. 103-165, Academic Press, New York). However, in syntheses of inhomogeneous phase there is a permanent risk that the proteases used as biocatalysts catalyze unwanted bond cleavage.

Object of the invention

The aim of the invention is the production of chirally uniform peptides in a cost effective manner, in good yield, with simple reaction.

Explanation of the nature of an invention

The object of the invention is to provide a process for the preparation of chirally uniform peptides in a homogeneous phase using biocatalysts under conditions of repulsion of undesired prctease-catalyzed bond cleavages. According to the invention, the object is achieved by preparing peptides from a selectively protected amino acid or a selectively protected peptide whose esterifying carboxyl group is present as an ester, and an amino component in which the amino group entering the reaction is unblocked, more soluble in the presence or immobilized series or cysteine proteases, wherein the reaction medium used is water or a suitable Puffor, to which may optionally be added proportions: organic solvents. For this purpose, N-acyl amino acid esters are reacted in the presence of proteases with amino components which themselves contain proteolysis-sensitive cleavage sites. After stopping the reaction after erruichone of the product maximum or before complete. If the ester is consumed and after customary workup, the protective groups can be removed, if necessary, partially or completely by known methods. In contrast to known kinetically controlled protease-catalyzed syntheses using serion and cysteine proteases and simple alkyl esters, including methyl and ethyl esters, the use of N-acylamino acid residues ^r n according to the invention gives high K _cs , / K _M value 3n as a function of the nature of the ester grouping and the conditions of realization of the construction of peptides in which, in addition to the newly knotted bond, further protease-sensitive cleavage sites are located. In addition, the C-terminal acyl donor amino acid does not have to correspond to the protease specificity known from the literature, which additionally reduces the risk of secondary hydrolysis. The method according to the invention for the production of peptides has the advantage over previously known methods of protease-catalyzed kinotically controlled peptide synthesis that a soluble peptide product can be obtained in high yield despite protease-sensitive peptide bonds in the sequence under the new synthesis conditions. It is a common and prevalent opinion that a homogeneous-phase peptide with protease-sensitive cleavage sites is subject to proteolysis that is in sync formation. It is shown for the first time that it is possible to synthesize enzyme substrates for the protease which catalyzes the peptide-linking reaction in homogeneous solution. Unexpectedly, the protease-catalyzed coupling reaction leads to good yields of peptide in very short reaction times at room temperature. The invention will be explained in more detail below by exemplary embodiments.

embodiments

In the examples, the amino acids are abbreviated according to the internationally valid rules. In addition, the following abbreviations are used:

pNA 4-nitroanilide Git Glutalryl Times Maleyl (3-carboxyacryloyl) OMe Methylester OBzl benzyl ester ON b 4-nitrobenzyl DMSO dimethyl sulfoxide

example 1 Synthesis of glutaryl-L-leucyl-L-phenylalanine-4-nitroanilide

a) starting from Glt-Leu-0Bz1

500μl water / DMSO (9: 1, v / v) containing 20mM Glt-Lou-OBz1 and 5mM Phe-pNA were adjusted to pH 9 on pH-stat, and thereafter the reaction was stopped by adding 0.15mg chymotrypsin started at 25 ° C. After 5 minutes, the pH was adjusted to 3 by the addition of 6N HCl and the Rec k'.ion stopped. The yield was determined analytically by HPLC and was 34% of theory.

b) starting from Glt-Leu-ONb

As Example 1 a, but with 2OmM Glt-Leu-ONb instead of Glt-Leu-OBz1. After 5 min 63% of theory

Example 2

Synthesis of maleyl-L-leucyl-L-phenylalanine-4-nitroanilide a) starting from Mal-Leu-OMe

As Example 1a, but with 2OmM Mal-l.eu-OMe instead of Glt-i äu-OBz1. After 10min 3% of theory i from Mal-Leu-OBz1 As Example 1 a but with 2OmM Mal-Leu-OBz 1 instead of Glt-Leu-OBz1. After 5 min 65% of theory

c) starting from Mal-Leu-ONb

Like Beir; -iei 1 a, but with 2OmM Mal-Leu-ONb instead of GIt-Leu-OBz1. After 5 minutes 80.5% of theory

Claims (5)

1. A process for the preparation of peptides from amino acids or corresponding peptides which are blocked with an alpha-amino protecting group and their esterifying carboxyl group is esterified, characterized in that an acylamino acid ester is used whose Kcat / KM value is greater than that of and an amino component is used as the reaction partner whose amino function is unblocked in the sequence and protease-sensitive peptide bonds are contained, wherein in aqueous solution, optionally containing organic solvent and / or buffer substances, is operated in the presence of an enzyme, wherein after reaching the product maximum or in the event of incomplete ester consumption, the coupling reaction is stopped by addition of acid or filtration of the immobilized enzyme and after usual workup protective groups are partially or completely removed if necessary. 2. The method according to claim 1, characterized in that the enzyme is a serine protease such as chymotrypsin, elastase o. Like., Or a cysteine protease such as papain o. The like. Is used. 3. The method according to claim 1, characterized in that the acyldione is used in excess compared to the amino component. 4. The method according to claim 1 to 2, characterized in that the protease is bound to an insoluble carrier. 5. The method according to claim 1-3, characterized in that the C-terminal Acyldonoi amino acid does not correspond to the literature-known protease specificity.