DD260084A1 - PROCESS FOR THE PRODUCTION OF PEPTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of arginine-containing peptides. The aim of the invention is the preparation of chirally uniform arginine peptides with unblocked ganido function by means of proteolytic enzymes. The object is achieved in accordance with the invention by reacting an N-acylamino acid ester with an arginine ester whose amino group and guanidofunction are unblocked in an aqueous medium with catalysis of a cysteine proteinase, with one to three arginine units being controlled in a reaction step as a function of the reaction can be inserted into the peptide.

Description

Field of application of the invention

The invention relates to a process for the preparation of arginine-containing peptides, which can serve as active ingredients or as intermediates of active compounds after complete or partial removal of the protective groups.

Characteristic of the known technical solutions

For the preparation of peptides, various organic-chemical methods (see Overview: WÜNSCH, E. [1974] Synthesis of peptides, in HOUBEN-WEYL, Vol 15.1 / 2, Methods of Organic Chemistry, MÜLLER, E. [Hrsg .] Georg Thieme Verlag, Stuttgart) are used. In the course of chemical peptide syntheses, undesirable side reactions are frequently observed which reduce the yield and necessitate difficult and lengthy purification processes. A particularly serious disadvantage of the conventional processes is the unresolved problem of racemization, which occurs in particular in segmental condensation by means of chemical linking methods. Since stereoisomers can hardly be completely separated and the optical purity of the synthesis products is a necessary prerequisite for the biological activity, the industrial synthesis of peptides by means of organic-chemical processes has considerable disadvantages. Furthermore, in all chemical peptide synthetic operations, the third functions of amino acid building blocks must be reversibly masked due to the risk of side reactions, although there is still no ideal protecting group for the strongly basic guanidino function of arginine (see H.-D. Jakubke and H. Jeschkeit , Amino acids, peptides, proteins, 3rd ed., Akademie-Verlag Berlin, 1982, pp. 143-146). The use of biocatalysts for the catalysis of the peptide-linking step can be used to circumvent the difficulties described (for review, see: Jakubke, H.-D., and Kuhl, P. [1982] Pharmazy 37, 89; Fruton, JS [1982], in A. Meister: Adv. Enzymol., Relat. Areas Mol. Biol. 53, 239; Jakubke, H.-D. Kühl, P., and Könnecke, A. [1985] Angew Chem.97, 79). The stereospecificity of the proteases used as biocatalysts preserves the chiral integrity and the high degree of reaction control makes it possible to largely dispense with the protection of third-party functions.

Object of the invention

The aim of the invention is the preparation of chirally uniform arginine-containing peptides using biocatalysts.

Explanation of the essence of the invention

The invention has for its object to implement N-Acylaminosäurealkylester in the presence of proteases with side chain protected Argininalkylestern as a nucleophile.

According to the invention arginine-containing peptides are prepared from a selectively protected amino acid or a selectively protected peptide whose reacting carboxyl group is present as an alkyl ester, and a Argininalkylester in which the reacting amino group and the guanido are unblocked, in the presence of soluble or immobilized proteases as the reaction medium, water or a suitable buffer to which, if appropriate, proportions of organic solvents may be added. Depending on the reaction conditions, arginine-like peptides are formed in a controlled manner, which can be separated preparatively by suitable chromatographic techniques. After completion of the work-up, if necessary, the protective groups can be partially or completely removed by known methods. In contrast to known syntheses of arginine-containing peptides, the use according to the invention of arginine alkyl esters as amino component, depending on the reaction conditions and the type of alkyl ester grouping, makes it possible to build up peptides in which one, two or three arginine units can be sequentially added, which are separated on a preparative scale can be. In addition, arginine-containing peptides of the general sequence Y-Xxx- (Arg) _n -OR are interesting starting materials for the construction of longer peptide sequences, whereby kinetically controlled trypsin-catalyzed segment linkages can be made.

embodiments

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

Z benzyloxycarbonyl OMe Methylester OCAM Carboxamidomethylester OiPr isopropyl OiBu isobutyl

example 1 Synthesis of Z-Ala-Arg-OiPr

4 ml of water / methanol (4/1, v / v) containing 0.03M Z-Ala-OCAM and 0.1M Arg-OiPr and 1.3mM EDTA were adjusted to pH8.0 on pH-Stat and thereafter the reaction started by adding 1.5mg papain. After 2 minutes, the pH was adjusted to 3 by the addition of 6N HCl and the reaction was stopped. The yield was determined analytically by ion exchange HPLC on a TSK CM-3SW, 7.5 x 150 mm, and found to be 87% of theory.

Example 2

As Example 1, but with 195mg wet Enzacryl-AA-Papain. After 7 min 84% of theory Yield.

Example 3 Synthesis of Z-Ala-Arg-Arg-OMe

As in Example 1, 0.03 M Z-Ala-OCAM were reacted with 0.3 M Arg-OMe, enzyme being added again after 30 min. After 45 min was stopped, and it had 53% of theory. Tripeptide ester formed.

Example 4 Synthesis of Z-Ala-Arg-Arg-Arg-OMe

As Example 3, but with 0.2 M Arg-OMe. After 45 minutes 21% of theory Tetrapeptidester.

Example 5 Synthesis of Z-Gly-Arg-OMe

0.03 M Z-Gly-OCAM and 0.2 M Arg-OMe were reacted as in Example 1 with 7.5 mg papain. After 1 min 83% of theory Product.

Claims (6)

1. A process for the preparation of arginine-containing peptides from amino acids or corresponding peptides which are blocked with an alpha-amino protecting group and whose carboxyl function in reaction carries an ester grouping, characterized in that the reactants used is an arginine ester, the amino group thereof which reacts and guanido function are unblocked, being used in aqueous solution, which optionally contains organic solvent fractions and / or buffer substances, in the presence of an enzyme, with increasing protease addition and a longer period of time increasingly more Argininbausteine be cited, and after completion of the coupling and workup protective groups are partially or completely removed , 2. The method according to claim 1, characterized in that the enzyme is a plant cysteine protease such as papain, Brommelain o. The like. Used. 3. The method according to claim 1 and 2, characterized in that Z-Ala-OCAM is adjusted with Ar-OiPr in aqueous methanolic solution in the presence of EDTA to pH = 8.0, the reaction is started by the addition of papain, after Stopped for 2 min by acidification to pH = 3 and the Z-Ala-Arg-OiPr is separated. 4. The method according to claim 1 and 2, characterized in that Z-Ala-OCAM is adjusted to pH = 8.0 with Arg-OMe in aqueous methanolic solution in the presence of EDTA, the reaction is started by the addition of papain, after 30 min further enzyme is added, after 45 min by acidification to pH = 3, the reaction is stopped and the tripeptide ester is separated. 5. The method according to claim 1 and 2, characterized in that Z-Ala-OCAM is adjusted with Arg-OMe in aqueous methanolic solution in the presence of EDTA to pH = 8.0, the reaction is started by the addition of papain, after 45min by acidification to pH = 3 is stopped and the tetrapeptide ester is separated. 6. The method according to claim 1 to 5, characterized in that the protease is covalently bound to an insoluble carrier.