SK70898A3 - Process for the preparation of dipeptide of l-alanyl-l-glutamin - Google Patents

Abstract

The process is based on the fact that the L- -lanine ester together with L-glutamine or L-glutamine in the reaction medium on an organic basis solvents, miscible or immiscible with water, with the addition of alkali metal or alkaline salts soil and other necessary factors in contact with microbial protease, free or immobilized, at a temperature of 20 to 60 ° C and a pH of 8 to 10. The protected dipeptide is an important starting material for the preparation of L-alanyl-L-glutamine which has been found in the art infusion solutions used in the treatment of severe postoperative, traumatic, catabolic, septic and other stress conditions.

Description

A process for preparing a protected L-alanyl-L-glutamine dipeptide

Technical field

The invention relates to a process for the preparation of a protected L-alanyl-L-glutamine dipeptide of the general formula I

X-L-alanyl-L-glutamine-Y (I) wherein X is a protecting group N-alpha-formyl, N-alpha-furylacryloyl, N-alpha-benzoyl, N-alpha-benzoloxycarbonyl, N-alpha-phenylacetyl, N-alpha-tert-butyloxycarbonyl or N-alpha-fluorenylmethoxycarbonyl and Y is hydroxyl or amino. The protected dipeptide is the preferred starting material for the preparation of free L-alanyl-Lglutamine, which is used in the form of infusion solutions in the treatment of severe post-operative, traumatic, catabolic, septic and other stress conditions.

BACKGROUND OF THE INVENTION

As is known, synthetic dipeptides (or tripeptides) are used as components of different solutions for parenteral nutrition. These are peptides of those L-amino acids that are necessary for the body, but which are either unstable (glutamine) or only partially soluble (cysteine, tyrosine) in solution. Compared to free amino acids, dipeptides are more stable and soluble. Because the bioavailability of peptides and thus their usefulness in the body is very good, the application of dipeptides can increase the supply of amino acids without undesirable strain on the organism (A. Veselkova et al., Ces. And Slov. Farm. 45, No. 1, p. 14) -16,1996).

Glutamine is an important nitrogen donor in the body. Especially in stress states, its consumption increases, but in many cases its natural production is not enough. However, the additional supply is hampered by the instability of glutamine in the aqueous environment and its decomposition when sterilizing other solutions. (P. Fiirst et al., Proc. Nutr. Soc. 49, 343359, 1990). Glutamine, however, can be supplied to the body in the form of dipeptides, of which L-alanyl-L-glutamine has proven to be the best, which is rapidly degraded by plasma protease to alanine and glutamine in the plasma of healthy probands with rapid utilization of components (Fiirst et al. H. Lochs et al., Metabolism 39, 833-836 (1990). conventional

However, the nutritional therapy of these stress states has failed and the only possible route is to supply glutamine to the body in the form of infusion solutions supplemented with alanylglutamine.

Glutamine applied in the form of the dipeptide is very stable, does not decompose into undesirable products and is much more soluble than in the free state as experimentally demonstrated (P. Stehle, Klinische Emährung 35, 1991).

Alanylglutamine can be prepared by chemical synthesis (P. Stehle et al., J. Appl. Biochem, 4, 280-285, 1982), which is, however, disadvantageous for production for a number of technical, economic and environmental reasons. Furthermore, the first model enzyme synthesis of alanylglutamine for research purposes was accomplished using cysteine proteases of papain (from Carica papaya) and ficin (from Ficus carica). However, with these plant proteases, alanylglutamine cannot be produced in large quantities because they are expensive and hardly available proteases, not usable for economic production.

Animal protease production is also disadvantageous because it is expensive and cumbersome and only certain proteases for special purposes can be obtained, such as pepsin from bovine stomachs or alpha-chymotrypsin from porcine pancreas. In addition, no protease capable of synthesizing alanylglutamine was found among the proteases of animal origin in the available literature.

Attention has therefore been paid to the research of microbial proteases which are capable of synthesizing the desired dipeptide and which are affordable, inexpensive and allow economical production in bulk. To date, no microbial protease capable of synthesizing the desired dipeptide is known in the scientific and patent literature, i. alanyl. However, based on systematic research, in particular available commercial microbial proteases and microorganisms, suitable proteases capable of synthesizing protected alanylglutamine have been found.

SUMMARY OF THE INVENTION

The invention therefore relates to a process for the preparation of the protected L-alanyl-L-glutamine dipeptide of formula I

X - L - alanyl - L - glutamine - Y (I),

-3where X is N-alpha-formyl, N-alpha-furylacryloyl, N-alpha-benzoyl, N-alpha-benzoloxycarbonyl, N-alpha-phenylacetyl, N-alpha-tert-butyloxycarbonyl or N-alpha-fluorenylmethoxycarbonyl and Y is hydroxyl or amino. It is an object of the present invention to provide a protected L-alanine ester of formula II

XL-Ala-R (Π), where X is the same as in formula I and R is alkyl of 1 to 3 carbon atoms or benzyl, together with Lglutamine or L-glutaminamide are contacted with free or immobilized microbial protease or mixtures of these proteases, in a water-miscible or water-immiscible organic solvent medium, comprising C1-C5 alkanols, acetonitrile, dimethylformamide, dioxane, dimethylsulfoxide, ethyl acetate, carbon tetrachloride or mixtures thereof, at a concentration of 0,5 to 99,5% by volume , with an addition of alkali metal or alkaline earth metal salts with inorganic acids and disodium ethylenediaminetetraacetic acid (EDTA), at room temperature up to 60 ° C and at a pH of 8 to 10 ° C.

Suitably and preferably, the free immobilized microbial proteases used are proteases selected from the group consisting of protease from Aspergillus oryzae, Streptomyces griseus, Vibrio proteolyticus ATCC 15338, carboxypeptidase Y from Saccharomyces cerevisiae or Saccharomyces coreanus, proteinase K from peptidase-Tritirachase from Pseudomonas fluorescens RK-9 (Collection of the Antibiotic and Biotransformation Research Institute), alone or in a mixture.

As additives to the reaction, respectively. in the culture medium alkali metal or alkaline earth metal salts with inorganic acids, in particular sulphates, nitrates, chlorides or phosphates, are required individually or in a mixture; in some proteases, the addition of zinc chloride or the addition of L-cysteine is expedient.

The advantages of the method according to the invention are that the production of microbial proteases, starting from the available raw materials, is inexpensive, allows easy isolation of the enzyme in one place by a sequence of conventional operations and conventional equipment. For the production of dipeptides, it is sufficient, for example, to isolate the protease in question after cultivation of the microorganism only in the form of a technical preparation (no isolation and purification up to the acid state is necessary), because it is immobilized (for example by crosslinking agents, polymerisation into a polymer network or by binding to organic porous inorganic materials), whereby the enzyme becomes small solid particles (it is also possible to use directly soluble

(By entrapment into ultrafiltration membranes) and used repeatedly in a batch device (for example, 100-1000 times depending on enzyme activity stability and mechanical resistance of the immobilized preparation) or in a continuously operating reactor for several hours when the enzyme material has to be replenished for handling losses. After completion of the reaction, the supernatant (reaction mixture) is separated from the immobilized protease and collected for further processing.

The chemical production of L-alanyl-L-glutamine works solely in batches, limited by the carrying capacity of the reaction mixture, using toxic substances (eg phosgene), high temperatures and pressures, and polluting the environment. Groups that do not react together need to be protected, but unwanted reactions may occur. In chemical production, there is a particular risk of epimerization on the chiral carbon of the prepared peptide (formation of a racemic mixture). Also, the isolation of the protected peptide to the pure form is complicated.

On the other hand, the process according to the invention is easier to implement in bioreactors, working at acceptable temperatures and pressures, with conventional substances, while maintaining environmental conditions. There are no adverse reactions on the secondary groups and, in particular, there is no danger of the epimerization mentioned, since the enzymes are strictly stereospecific, thus giving (homogeneous) a dipeptide. Systematic investigation of microbial proteases has found several microorganisms producing proteases capable of synthesizing protected alanylglutamine. They are protease from Aspergillus oryzae (fungus) and Streptomyces griseus (actinomycetes), carboxypeptidase Y from Saccharomyces cerevisiae or Saccharomyces coreanus (yeast), proteinase K from Tritirachium album (fungus), protease from Vibrio proteolyticue-peptidase (bacteria), from Pseudomonas fragi and metalloendopeptidase from Pseudomonas fluorescens (pseudomonas). All of the proteases mentioned are endopeptidases or carboxypeptidases because only they are capable of producing the desired dipeptide. Most preferred are serine proteases, as they use a dipeptide in a relatively short time and in a high yield. Also in the enzyme preparation it is necessary to use protecting groups, but only for the alpha-amino group of alanine. Suitable protecting groups are listed above. For the synthesis, alanine is used in the form of an ester, which is also mentioned above.

In contacting the method of the invention, the starting compounds are contacted with one of the aforementioned microbial proteases in a suitable reaction medium. The reaction medium contains a miscible or immiscible organic solvent

With water and, depending on the nature of the protease used, other necessary ingredients such as salts, EDTA, buffers, cysteine and others. While stirring, the temperature and pH are kept within the necessary range.

When working with the immobilized microbial protease, the reaction mixture is gradually collected and then removed in a one-off process from the resulting alanylglutamine derivative either chemically (e.g., by contact hydrogenolysis, etc.) or preferably enzymatically, utilizing the ability of some amidases to separate the protecting groups ( penicilidase removes the phenylacetyl group, peptidamidase cleaves the amide group of glutaminamide and the like). The enzymatic cleavage method of the protecting groups allows, for example, a series of two flow reactors to be operated in the process of choosing a suitable protecting group, in which the protected alanylglutamine is synthesized in the first and the protecting group is removed in a second, so that the desired product is obtained directly. for example, ion exchange chromatography, gel filtration, optionally hydrophobic or affinity chromatography.

The details of the process according to the invention are illustrated in more detail by the following examples, which are illustrative but not limiting.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

In a 50 ml thermostatic flask, 30 ml of a mixture containing 0.1 M potassium chloride, 5 mM EDTA, 10 mM calcium chloride, 10 mM L cysteine, 0.663 g N-phenylacetyl-L-alanine methyl ester (0.1 M) was prepared, 3.5 g of L-glutamine (0.8 M), 5 vol% ethanol and water to a total volume of 30 ml. The pH was adjusted to 9.5 with 2M sodium hydroxide with stirring, and after the mixture was allowed to warm to 35 ° C, 10 mg of carboxypeptidase Z from Saccharomyces cerevisiae was added with stirring to initiate the reaction. The reaction mixture was stirred vigorously and the temperature was maintained at 35 ° C with an ultratermostat during the reaction and the pH was maintained at 9.5 with a pH stat. During the reaction, 0.5 ml samples were taken in which the reaction was stopped by adding 0.5 ml of 2 M hydrochloric acid. Samples were evaluated by capillary electrophoresis coupled to an evaluation computer. After a reaction time of 180 min, the end of which was indicated by almost complete consumption of N-phenylacetyl-L-alanine methyl ester, the concentration of N-phenylacetyl-L-alanyl-6L-glutamine was found to be 26.7 g / l, representing a conversion of 79.7% to the starting acyldonorester.

Example 2

Analogously to Example 1, 30 ml of a mixture containing 0.1 M potassium chloride, 30 mM sodium nitrate, 1 mM EDTA, 0.621 g of N-benzoyl-lalanine methyl ester (0.1 M), 2.61 g of L-glutamine, were prepared. (0.6 M), 50% by volume of dimethylformamide and water to a total volume of 30 ml. While stirring, the pH of the mixture was adjusted to 9.0 with 2M sodium hydroxide, and after the mixture was allowed to warm to 40 ° C, 1.26 g of Aspergillus orazae protease was added, leading to the start of the reaction. With vigorous stirring of the reaction mixture, the temperature was maintained at 40 ° C with an ultratermostat during the reaction and the pH was kept at a pH of 9.0 using a pH-stat. Analogously to Example 1, samples were taken from the reaction mixture and measured by capillary electrophoresis. After a reaction of 5 hours, when the N-benzoyl-L-alanine methyl ester concentration was maintained at practically the same level, the production of N-benzoyl-L-alanyl-L-glutamine amide was found to be 15.6 g / l, representing the starting acyldonorester. conversion of 48.8%.

Example 3

Analogously to Example 1, 30 ml of a mixture containing 0.1 M potassium chloride, 5 mM EDTA, 50 mM sodium sulfate, 4.38 g L-glutamine (IM), 0.747 g Nbenzyloxycarbonyl-L-alanine methyl ester (0.1 ml) were prepared. M), 63 vol% methanol and water to a total volume of 30 ml. With stirring, the pH of the mixture was adjusted to 8.0 with 2M sodium hydroxide, and after the mixture was allowed to warm to 30 ° C, 15 mg of Streptomyces griseus protease was added with stirring to initiate the reaction. The reaction mixture was stirred vigorously, the temperature was maintained at 30 ° C with an ultrathermostet, and the pH was maintained at 8.0 using a Ph-stat. Samples were taken and measured analogously to Example 1. After 120 min of reaction, 12.7 g / L of N-benzyloxycarbonyl-L-alanyl-L-glutamine was measured, which represents a conversion of 39.3% relative to the starting acyldonorester.

Example 4

(a) preparation of a partially isolated technical protease from Vibrio proteolyticus ATCC 15338

The culture of said strain was maintained on sloping agar with the composition:

Nutrient Agar Difco 3.1%

7Difco agar sodium chloride pH 7.0

0.5% 2.2%

The cultivation was carried out at 28 DEG C. on a rotary shaking machine at speeds 240 rpm in ¹ 500 ml round bottom flask filled with 100 ml of culture medium having the composition:

sucrose 4% com-steep liquor (CLS) 4% disodium hydrogen phosphate dodecahydrate 0.5% ammonium sulfate 0.5% potassium sulfate 0.1% calcium chloride 0.1% magnesium sulfate heptahydrate 0.05% sodium chloride 2.0% pH 7.0

Generation 1 flasks were inoculated with sterile slant agar and the first generation was cultivated for 24 h. Inoculation of 1% inoculum from first generation flasks was followed by cultivation Π. generation also 24 h. O.D. at the end of the cultivation it was 5.4 and the pH was 7.9. The contents of the flasks were centrifuged at 5000 g for 30 min to give a clear brownish supernatant with a protease activity of 0.28 mg azocasin / min / ml. The supernatant was concentrated on a rotary vacuum evaporator equipped with a powerful rotary oil pump at 25 ° C approximately

5.5x, whereby a protease activity of the concentrated supernatant of 0.65 mg azocazein / min / ml was achieved.

b) preparation of the dipeptide

Analogously to Example 1, 10 ml of a mixture containing 30 ml of 0.1 M potassium chloride, 10 mM calcium chloride, 1 mM zinc chloride, 1 mM EDTA, 0.2385 g N-formyl-L- alanine propyl ester (50 mM), 5.256 g of L-glutamine (1.2 M), 20 vol% propanol and water. After warming the mixture to the appropriate temperature and adjusting the pH with 2M sodium hydroxide to the appropriate value, 20 ml of concentrated Vibrio proteolyticus ATCC 15338 culture supernatant was added with stirring to initiate a reaction where the temperature of the vigorously stirred mixture was maintained at 40 ° C using an ultratermostat. The pH was maintained at a pH of 9.5.

In analogous sampling and analysis of the samples as in Example 1, after 3 hours of reaction, 7.2 g / l of N-formyl-L-alanyl-L-glutamine was found at a conversion of 58.7% based on the starting acyldonorester.

Example 5

(a) preparation of immobilized protease

In a 50 ml vial, the spherical organic copolymer particles of methacrylaldehyddivinylbenzene containing 11.8% by weight of methacrylaldehyde groups with a diameter of 150 to 300 µm in a stoichiometric ratio were activated with hexamethylenediamine and mixed with 2 g of 2 g proteinase K protein album in the presence of 1.6 mL of 50 vol% glutaraldehyde at room temperature for 6 h. After the reaction, the immobilized particles were separated from the supernatant by decantation and thoroughly washed with water. 5.4 g of wet mass of immobilized proteinase K with a specific activity of 4.3 pmol L-leucine / min / mg wet mass were obtained (hereinafter referred to as j / min / mg - protease activity was 0.11 mg azocazein / min / mg wet mass). ). At the original proteinase K activity of 13.7 µ / min / mg preparation (0.37 mg azocazein / min / mg), this represents an immobilization yield of about 85%.

b) preparation of the dipeptide

A 50 ml reactor equipped with a 150 min * ¹ anchor stirrer, which kept the biocatalysts in perfect buoyancy, minimally mechanically damaging them, and which ensured perfect mixing of the reaction mixture, was charged with 4.5 g of wet catalyst (approximately 19000 µl). 30 ml of a mixture containing 0.1 M potassium chloride, 5 mM EDTA, 10 mM calcium chloride, 50 mM tripotassium phosphate monohydrate, 0.663 g N-phenylacetyl-L / min. -alanine methyl ester (0.1 M), 5.256 g of L-glutamine (1.2 M), 30 vol% ethanol and water to a total volume of 30 ml. The pH of the mixture was always adjusted to 9.7 with 2M sodium hydroxide. In the stirred reactor, the batch reaction was always in contact with the immobilized proteinase K while maintaining the temperature with an ultratermostat at 28 ° C and pH with a pH-value of 9.7 for 60 to 80 minutes. After the reaction, the reaction mixture was always drawn off with a siphon equipped with a sintered frit and collected for further processing.

-9 In this way, 120 repeated batch conversions were performed; For example, for an analogous sampling and measurement as in Example 1, the average production of N-phenylacetyl-L-alanyl-L-glutamine was 29.8 g / L, which represents an average conversion of the starting acyldonorester of about 90%. The specific activity of the immobilized proteinase decreased after 120 repeated batch conversions, i. after about 140 h of operation, at 3.9 j / min / mg wet matter, which represents the operating half-life of the biocatalyst of about 950 h.

Industrial use

Protected L-alanyl-L-glutamine derivatives prepared by microbial proteases according to the invention are preferred starting materials for the preparation of the L-alanyl-L-glutamine dipeptide, which is used in the form of infusion solutions for the treatment of severe postoperative, traumatic, catabolic, septic and other stress states.

Claims (4)

PATENT CLAIMS A process for the preparation of a protected L-alanyl-L-glutamine dipeptide of general formula I X-L-alanyl-L-glutamine-Y (I) wherein X is a N-alpha-formyl, N-alpha-furylacryloyl, N-alpha-benzoyl, N-alpha-benzyloxycarbonyl, N-alpha-phenylacetyl, N-alpha-tert-butyloxycarbonyl or N-alpha-fluorenylmethoxycarbonyl and Y is a hydroxyl or amino group, characterized in that the protected L-alanine ester of the general formula Π XL-Ala-R (H) wherein X is the same as in Formula I and R is C 1 -C 3 alkyl or benzyl, together with Lglutamine or L-glutaminamide, to contact with a free or immobilized microbial protease or mixtures of these proteases in a water-miscible or water-immiscible organic solvent selected from the group consisting of C 1 -C 5 alkanols, acetonitrile, dimethylformamide, dioxane, dimethylsulfoxide, ethyl acetate, carbon tetrachloride or mixtures thereof, at a concentration of 0.5 to 99 5% by volume, with the addition of alkali metal or alkaline earth metal salts with inorganic acids and disodium ethylenediaminetetraacetic acid, at room temperature up to 60 ° C and at a pH of 8 to 10. Method according to claim 1, characterized in that proteases selected from the group consisting of protease from Aspergillus oryzae, Streptomyces griseus, Vibrio proteolyticus ATCC 15338, carboxypeptidase Y from Saccharomyces cerevisiae or Saccharomyces corean are used as free or immobilized microbial proteases. proteinase K from Tritirachium album, peptidyl-Asp-metalloendopeptidase from Pseudomonas fragi or metalloendopeptidase from Pseudomonas fluorescens. -113. Process according to claim 1 or 2, characterized in that the alkali metal or alkaline earth metal salts with inorganic acids are used as sulphates, nitrates, chlorides or phosphates, individually or in a mixture. The process according to claims 1 to 3, wherein the reaction medium comprises a zinc chloride additive. The process according to claims 1 to 3, wherein the reaction medium comprises a cysteine additive.