DE19834308A1 - Use of proteases as biocatalysts for production of organic acid amides from carboxylic acid esters - Google Patents

Abstract

Proteases are used as biocatalysts for the production of organic acid amides from carboxylic acid esters that are protease substrate mimetics. Process for producing an organic acid amide comprises using a biocatalyst comprising a protease coupled (sic) with a substrate mimetic.

Description

The invention relates to a method for the biocatalytic production of organic chemical compounds containing an acid amide bond and the Use of biocatalysts.

For the production of acid amides as precursors for various types of commercial Products are currently mainly different organic chemical Process (see overview: Houben-Weyl, Methods of Organic Chemistry, Volume E5, carboxylic acids and carboxylic acid derivatives (Ed .: J. Falbe) 1985, pp. 934-1312) used. In the course of chemical amide syntheses, especially those with additional functional groupings are very often undesirable Side reactions observed when not looking at the knotting reactions the amide bond uses a selective protecting group tactic. A special one the serious disadvantage of conventional chemical processes is the unsolved one Problem of maintaining chiral integrity. Because stereoisomers are extremely difficult Allow to separate completely, but the optical purity of the synthesis product is a necessary prerequisite for a specific effect Industrial synthesis of acid amides using organic chemical processes significant disadvantages. In contrast, there is an increasing need for chiral Amides and environmental aspects also require emphatic, mild ones to develop biocatalytic production processes.

There are currently two biocatalytic methods for the production of simple amides known. In the first method, under the catalysis of nitrile hydratase (EC 4.2.1.84) organic nitriles (hereinafter referred to only as nitriles) to the corresponding amides hydrolyzed, while in the second method suitable Enzymes that catalyze the ammonolysis of carboxylic acid esters.

The chemical conversation of nitriles has several disadvantages, such as the requirement of strongly acidic or basic reaction conditions, one  high energy consumption, the formation of unwanted by-products, low Yields and environmental problems in the generation of waste salts. The Bioconversation of nitriles (see reviews: T. Nagasawa and H. Yamada (1989) TIBTECH 7, 153-158; J.M. Wyatt et al. E.A. Linton, The industrial potential of microbial nitrile biochemistry, in D. Evered and S. Harnett (eds.) Cyanide compounds in biology, Ciba Foundation Symposium 1988, 140, 32-48) is supported by those in nature widely catalyzed nitrile-hydrolyzing enzymes. The disadvantage here is that z. B. Nitrile hydratase-producing microorganisms also enzymes synthesize, which hydrolyze the amide to acid in a subsequent step and thus form undesirable by-products (A. de Raadt, N. Klempier, K. Faber, H. Griengl, J. Chem. Soc. Perkin Trans. 1992, 1, (1) 137-140). They are also selective Nitrile-hydrolyzing enzymes are not currently commercial products available.

The second, little used method of enzymatic amide formation Ammonolysis of carboxylic acid esters is restricted in that only on Amide nitrogen unsubstituted amides can be produced.

The object of the invention is the biocatalytic production of organic Acid amides with the complete exclusion of side reactions.

This object is achieved with a method according to claim 1.

According to the present invention, the biocatalytic production of a organic acid amides not with amidases as usual, but contrary to prevailing opinion of the professional world with proteases, whereby by a programmed substrate manipulation the usual specificity of the protease used switched off and thereby the catalysis of the formation of acid amide bonds also enables nonpeptide educts. Because proteases are common Peptide bonds, i. H. cleave substituted amides, and these proteolytic Activities with substrate specificity for specific side chain functions of the Amino acid building blocks are coupled, the results according to the invention are very good surprised.  

Preferred are for the biocatalytic amide syntheses according to the invention the educts quite common in amide syntheses in the form of a carboxylic acid and a primary amine derivative used. As carboxylic acid derivatives are esters suitable, whose leaving groups determine the specificity of the serine used or contain cysteine protease and hereinafter referred to as substrate mimetics (Tab. 1). This excludes the use of charge-bearing ester leaving groups for proteases with a native specificity for hydrophobic amino acid side chains a, which can also mediate highly specific enzyme-substrate contacts. The overall procedure, d. H. the choice of the starting materials, the buffer, the medium, the Response time, etc., is relatively uncritical and can be done by a person skilled in the art biocatalytic transformations can be easily determined.

Table 1

Selection of specificity determinants for substrate mimetic-mediated protease catalysis

Chiral substrate mimetics can also be used as carboxylic acid derivatives. As a rule, functional groups not involved in the implementation must be involved  not temporarily blocked during the enzymatic amide linkage reaction become.

Depending on the protease used, suitable amine components are Educts with a primarily primary but also secondary amino function, where other acylatable third party functions can be freely available.

The choice of possible derivatizations and starting substrates depends on this basically according to the desired end product and the possible requirement, To prevent side reactions. This choice is therefore in the usual professional Act.

The method according to the invention discloses due to the stereo and Regioselectivity compared to the previous use of chemical processes special advantage that educts with chiral centers and other acylatable Functions without experimentally complex and additional side reactions leading temporary selective blocking measures can.

According to the invention, organic acid amides are preferably produced from a Carboxylic acid derivative, the carboxyl group which reacts as an ester with a Specificity determinant is present in the leaving group, the enzyme used corresponds, and an organic amine derivative in which the reacting Amino function is unblocked, in the presence of a protease, e.g. B. a thiol protease such as clostripain in solution or suspension at room temperature, the proportions, if any contains organic solvents and / or buffer substances, or even with deeper ones Temperatures.

The reaction is preferably carried out in an aqueous medium, u. U. low temperatures are advantageous.  

The amides formed can be used with suitable chromatographic or extractive Techniques are preparatively separated. After processing has been completed, existing protective groups are removed by known methods.

In contrast to the methods described for the synthesis of amides using other enzymes is in the inventive use of proteases Combination with the substrate mimetics the risk of unwanted amide cleavages prevented. It will be the first time for the organic acid amide class biocatalytic synthesis carried out with proteases. The effect of the invention is to the extent that it is surprising that with proteolytic enzymes a non-peptidic one Amide linkage could not be expected.

The amides which can be prepared with the present invention are suitable, if appropriate, after Removal of essential protective groups, e.g. B. as active ingredients or Intermediate products of active ingredients.

The present invention will be explained in more detail below with the aid of examples executed.

example 1 Synthesis of phenylbutterylargininamide with chymotrypsin

1 ml 0.05 M HEPES buffer, pH 8, containing 0.1 M NaCl, 0.01 M CaCl ₂ , 2 mM phenylbutyric acid 4-guanidinophenyl ester, 34 mM argininamide (effective proportion: 1 / 1.72) and 30 µM chymotrypsin are stirred at 25 ° C under pH control. After 10 min, the reaction solution with 0.5 percent. Trifluoroacetic acid in methanol / water (1: 1; v / v) brought to pH 2. The yield is determined analytically by HPLC and is 90% of theory. Th. Parallel runs after 1, 2 and 5 h led to the same yields.

Example 2 Synthesis of phenylbutterylalanylalaninamide with chymotrypsin

1 ml 0.05 M HEPES buffer, pH 8, containing 0.1 M NaCl, 0.01 M CaCl ₂ , 2 mM phenylbutyric acid 4-guanidinophenyl ester, 39 mM alanylalaninamine (effective proportion: 1 / 1.96) and 30 µM chymotrypsin are stirred at 25 ° C under pH control. After 10 min, the reaction solution with 0.5 percent. Trifluoroacetic acid in methanol / water (1: 1; v / v) brought to pH 2. The yield is determined analytically by HPLC and is 80% of theory. Th. Parallel runs after 1, 2 and 5 h led to the same yields.

Example 3 Synthesis of phenylbutterylpentylamide with clostripain

1 ml 0.05 M HEPES buffer, pH 8, containing 0.1 M NaCl, 0.01 M CaCl ₂ , 2 mM phenylbutyric acid 4-guanidinophenyl ester, 109 mM pentylamine (effective fraction: 1/448) and 7 µM clostripain are stirred at 25 ° C under pH control. After 1h the reaction solution with 0.5 percent. Trifluoroacetic acid in methanol / water (1: 1; v / v) brought to pH 2. The yield is determined analytically by HPLC and is 81.5% of theory. Th. Parallel runs after 2, 5 and 24h led to the same yields.

Example 4 Synthesis of phenylbutteryl-3-propanol-1-amide with clostripain

1 ml 0.05 M HEPES buffer, pH 8, containing 0.1 M NaCl, 0.01 M CaCl ₂ , 2 mM phenylbutyric acid 4-guanidinophenyl ester, 38 mM 1-amino-3-propanol (effective proportion: 1 / 92) and 7 μM clostripain are stirred at 25 ° C. under pH control. After 1 h, the reaction solution with 0.5 percent. Trifluoroacetic acid in methanol / water (1: 1; v / v) brought to pH 2. The yield is determined analytically by HPLC and is 39.0% of theory. Th. An O-acylation was not found. Parallel runs after 2, 5 and 24 h led to the same yields.

Example 5 Synthesis of phenylbutteryl-1,2-propanediol-3-amide with clostripain

1 ml 0.05 M HEPES buffer, pH 8, containing 0.1 M NaCl, 0.01 M CaCl ₂ , 2 mM phenylbutyric acid 4-guanidinophenyl ester, 25 mM 3-amino-1,2-propanediol (effective proportion: 1/27) and 7 µM clostripain are stirred at 25 ° C under pH control. After 1h the reaction solution with 0.5 percent. Trifluoroacetic acid in methanol / water (1: 1; v / v) brought to pH 2. The yield is determined analytically by HPLC and is 81.1% of theory. Th. An O-acylation was not found. Parallel runs after 2, 5 and 24 h led to the same yields.

Example 6 Synthesis of phenylbutteryl-1,3-diaminopentan-1-amide with clostripain

1 ml 0.05 M HEPES buffer, pH 8, containing 0.1 M NaCl, 0.01 M CaCl ₂ , 2 mM phenylbutyric acid 4-guanidinophenyl ester, 51.7 mM 1,3-diaminopentane (effective proportion: 1 / 160) and 7 μM clostripain are stirred at 25 ° C. under pH control. After 1h the reaction solution with 0.5 percent. Trifluoroacetic acid in methanol / water (1: 1; v / v) brought to pH 2. The yield is determined analytically by HPLC and is 35% of theory. Th. N-acylation of the primary amino function in position 3 was not found. Parallel runs after 2, 5 and 24 h led to the same yields.

Claims (5)

1. A process for the biocatalytic preparation of an organic acid amide, characterized in that a suitable protease coupled with a substrate mimetic is used as the biocatalyst. 2. The method according to claim 1, characterized, that the acid amide from a carboxylic acid ester with the Specificity determinant of the protease used in the ester leaving group and builds up an amine derivative. 3. The method according to claim 1 or 2, characterized, that one carries out the reaction in an aqueous medium. 4. The method according to any one of the preceding claims, characterized, that as a protease clostripain, trypsin, chymotrypsin, V8 protease, subtilisin or mutants of these enzymes and carboxylic acid esters as substrate mimetics the enzyme specific specificity determinants in the ester leaving group starts. 5. Use of proteases as biocatalysts characterized, that the production of. by proteases in conjunction with carboxylic acid esters organic acid amides is catalyzed, as far as the carboxylic acid ester in turn represent substrate mimetics.