NL8620072A - N-Formyl peptide prodn. - from N-formyl cpd. and protected amino acid in presence of protease enzyme - Google Patents

Abstract

Prodn. of N-formyl-L-peptides of formula OHC-A-B-X (I) is effected by reacting an N-formyl amino acid or peptide of formula OHC-A-OH (II) with a carboxy-protected amino acid of formula H.B-X (III) at 0-60 deg.C and pH 6-9 in an aq. or aq.-organic medium in the presence of a metalloprotease (IV) derived from a Bacillus sp.: In the formulae, A = Asp, Met, Gly, Ala, Gly-Gly, Gly-Ala, Ala-Gly, Ala-Ala, Tyr or Phe; B = Phe, Leu, Ile or Val; X = OMe, OEt, OCH2Ph, NH2, NHPh, p-nitroanilino, NHNH2 or NHNHPh.

Description

-1-25881 / Vk / mvl 8620072

Short designation: Process for the preparation of N-formyl-L-peptides.

The present invention relates to the field of peptide chemistry and more particularly to a process for the preparation of formyl peptides, in particular to a process for the preparation of N-formy L-L peptides.

Formyl peptides are applicable in biology and in medicine to investigate phenomena such as phagocytosis and chemio-10 taxis. Phagocytosis, which is the absorption of large solid particles by a cell, relates to the nutrition and self-defense of cells. Chemiotaxis refers to the movement of cells under the influence of various chemical compounds. Both of these phenomena play a major role in the protective mechanism of the organisms, in particular in processes that take place under the influence of fever. As phagocyte stimulating and chemiotaxic agents, use is made of peptides containing in the N-terminal part a methionine unit acylated by formic acid. The presence of formylmethionine in a molecule is necessary because it is responsible for the interaction of peptides of the chemiotaxis and phagocytosis stimulating peptides with specific cell surface receptors. The same peptides also stimulate some other functions of the cell such as aggregation, secretion of enzymes, formation of toxic oxygen metabolites.

As examples of formyl peptides may be mentioned a precursor of aspartame-formylaspartame, namely methyl ester of N-formyl-L-aspartyl-L-phenylalanine.

Known state of the art.

According to the prior art, there are several approaches to the synthesis of formyl peptides. Classical methods for peptide synthesis are the carbodiimide method, the method of the mixed anhydrides or activated esters. For example, a synthesis of formyl-methionyl-leucyl-phenylalanine and formyl-methionyl-lysyl-prolyl-arginine is known by the successive condensation of tert-butyloxycarbonyl-L-amino acids with C-protected amino acids. After termination of a stepwise extension of the peptide chain, the N-terminal amino acid is unblocked and the partially protected peptide is formylated by activated esters of formic acid (see J. Martinez, J. Laur,

Synthesis, 1982, No. 11, pp. 979-981).

8620072

-2-25881 / Vk / mvL

This approach to the synthesis of phenyl peptides makes it possible to prepare compounds of high optical purity. The longer time of synthesis by introducing additional steps may result in a significant reduction in the total yield of desired product. Furthermore, the use of polyfunctional amino acids in the synthesis is hindered in this case. To prevent side reactions at this stage of the formylation, initial protection of all additional functional groups is required, which also complicates product synthesis, making it expensive and not feasible.

Well known in the art is an azide method for the preparation of peptides, which makes it possible to obtain products which exhibit a high degree of optical purity (see FE King, JW Clark-Lewis, DAA Kidd, 6. R. Smith, J. Chem. Soc. 1954, pp. 1039-1043). However, in the synthesis of formyl-containing peptides by the azide method, the authors exemplify the preparation of formyl-glycine anilide and p- (formyl-glycyl) -amino benzoic acid.

Formyl glycine hydrazide is obtained in 74% yield by treating a solution of formyl glycine ethylate in ethanol. The solution of formylglycine in 1N HCl is then stirred at a temperature of 0 ° C with an aqueous solution of sodium nitrite, mixed with aniline after neutralization with sodium carbonate and after 2 hours the desired reaction product, i.e. formylglycine anilide, is recovered. However, this method gives the desired product only with a low yield.

After the reaction of formyl-glycineazide prepared as indicated above with p-aminobenzoic acid, the yield of the reaction product is 13%. As already mentioned above, the azide method makes it possible to obtain optically pure peptides. However, as given examples, the synthesis of formyl-glycine anilide and ethyl ester of p- (formyl-glycyl) -30 amino benzoic acid has been reported, which are compounds without optical activity.

Therefore, it is difficult to discuss the advantages of the azide method in the preparation of the formyl containing peptides. Furthermore, the azide method can be carried out in two steps, whereby the protection of all additional functional groups with subsequent removal is necessary, which makes the process considerably more difficult and more expensive for the preparation of the desired compounds.

It is quite natural that classical methods for the 8620072

-3- 25881 / Vk / tnvL

peptide synthesis are suitable for the preparation of a sweet peptide aspartame, its analogs and precursors.

Thus, there is known in the prior art a method for the synthesis of aspartame, wherein α-2,4,5-trichlorophenyl ester-5 (β-benzyl ester Hl-α-benzyloxycarbonylaspargic acid is condensed with methylalanine methyl acrylate according to the method of activated esters to obtain of a fully protected aspartame-methyl ester precursor of Na-benzyloxycarbonyl- (B-benzyl ester) -aspartylphenylalanine Subsequent removal of the protecting groups, 10 β-benzyl ester and N-orbenzyloxycarbonyl group methyl ester of aspartyphenylalanine, is prepared by catalytic hydrogenation , namely aspartame (see JM Davey, AH Laird, JS Morley, J. Chem. Sod, (C), 1966, pp. 555-566) This method is sufficiently labor intensive and consists of many steps, in addition, the optical purity of the condensation product 15. The product yield in the condensation step is 96%.

A method is also known for the preparation of aspartame using metal proteinases in the formation of a peptide bond. In this case, benzyloxycarbonyl or p-methoxybenzyloxy-20-carbonylaspargic acid is condensed with two equivalents of phenylalanine methyl acrylate in the presence of a metal proteinase produced by microorganisms of genus Bacillus thermoproteolyticus, thermolysin, the reaction taking place at a pH of 6-8.40 ° C for 3-5 hours, the desired product being formed as a salt of phenylalanine methylate at the β-carbonyl group of aspartic acid with the methyl ester of N-benzyloxycarbonyl or Np-methoxybenzyloxycarbonyl-aspartylphenylalanine, respectively knocked down. After decomposition of this product in an acidic medium, a precursor of asparatame is obtained, namely the methyl ester of N-benzyloxycarbonyl, or N-p-methoxybenzyloxycarbonyl-aspartyl-phenylalanine, respectively.

This reaction takes place in a high yield which can become as high as 90%, while retaining an optical purity of the starting amino acids and this ensures the progress of the process only in the α-carboxy group of aspartic acid (see Y. Isowa, M Ohmori, T. Ichikawa, 35 K. Mori, Y. Nonaka, K. Kihara, K. Oyama, H. Saton, S. Nishimura, Tetrahedron Lett., 1979, No. 28, pp. 2611-2612).

A disadvantage of this method for the synthesis of a precursor 8620072

-4- 25881 / Vk / mvL

of aspartame refers to use as a protecting group for the amine function of aspartic acid of benzyloxycarbonyl or p-methoxybenzyloxycarbonyl protecting groups. As the starting acylating agent, the introduction of such protecting groups in such a case uses, for example, carbobenzoxychloride benzyl ester of chlorocarboxylic acid, but its preparation involves the use of a large amount of phosgene.

In the large-scale preparation of aspartame, it is recommended to use a cheaper and easier to obtain protecting group for the α-amino function of aspartic acid.

In the prior art, the synthesis of a precursor of aspartame is known using, as a protecting group for the amine function of aspartic acid, the formyl protecting group. This group contains a formic acid unit and is the cheapest and easiest amino protecting group available. In such a synthesis of the aspartame precursor, the internal anhydride of formylasparic acid is condensed with phenylalanine methyl hydrochloride. The reaction product in this case consists of a mixture of isomers at the α and β-carboxyl group of aspartic acid in the amount of 4: 1, i.e. the methyl ester of crN-formylaspartylphenylalaine and the methyl ester of β-form-formylaspartylphenylalanine ( PCT / FR Application 82/00181, filing date: November 5, 1982).

It has previously been shown that only the asisomer of aspartame has a sweet taste, while the β-isomer of aspartame has a bitter taste, thus special techniques have been developed for separating the mixture of isomers and recovering the required orisomer, which complicates the process as a whole and optionally reduces the yield of desired product.

The present invention is directed to the provision of a method for the preparation of N-formyl-L-peptides using enzymes which can make it possible to simplify the preparation of the desired optically active product and to increase the yield of this.

This object is achieved with the method of preparing N 'formyl-L-peptides of general formula:

For-A-B-X (1) where For = formyl; A = Asp, Met, Gly, Ala, Gly-Gly, Gly-Ala, Ala-Gly, 8620072 -5- 25881 / Vk / mvl

Ala-Ala, Tyr, Phe, B is Phe, Leu, lie, Val, X is -0CH3 / -OC2H5, -CH2COH5, -NH2, -NH-C ^, -p-NH-C6H4-NO2, -NH- NH 2, -NH-NH-C 1 H H, wherein according to the present invention N-formyl-5 protected carboxy compound of general formula:

For-A-OH (2) where For = formyl; A = Asp, Met, Gly, Ala, Gly-Gly, Gly-Ala, Ala-Gly, Ala-Ala, Tyr, Phe, is condensed with an amino acid protected at the carboxy group, of general formula: HBX (3) where : B = Phe, Leu, Ile, Val, x = -och3, -oc2h5, -och2h6h5, -nh2, -nh-c6h5, 15 -p-NH-C6H4 ~ NO2, -NH-NH2, -NH-NH- CóH5, in the presence of metalloproteinases, prepared by microorganisms of the genus Bacillus in an aqueous or aqueous-organic medium at a pH of 6.0-9.0 and at a temperature in the range of 0 to 60 ° C.

For example, the present invention has made it possible to prepare both substrates of serine proteinases from subtilysin or formyl-methionine-aminopeptidase as a precursor or aspartame-methyl ester of N-formyl-L-aspartyl-L-phenylalanine. These compounds corresponding to formula 1 are optically active products. The preparation thereof is simpler than compared to the known methods in which comparable products are prepared. The yield of the desired products in the process of the present invention is high, i.e. about 95.5%.

In order to broaden the scope of the starting compounds, i.e., to use synthetic amino acids for the preparation of the desired product of the present invention, it is recommended that N-formyl protected carboxy compound and amino acids are protected in the carboxy group, using DL derivatives of these compounds or optically active L-amino acids.

According to the present invention, it is preferable to use as microorganisms of the genus Bacillus the following species of microorganisms: Bacillus thermoproteolyticus, Bacillus 8620072

-6- 25881 / Vk / mvL

stearothermophylus, Baci LLus ami loliquefaciens, Baci ILus subti Lis,

Baci LLus polymyxa.

In order to ensure a high yield of desired products, it is recommended according to the present invention to carry out the condensation at a molar ratio of the N-formyl-protected carboxy compound (2) to the m-amino acid protected at the carboxy group (3) equal to 1: 1-1: 2.

In order to ensure a high yield of the desired product and to carry out the synthesis process in the shortest possible time, it is recommended according to the present invention to use the metalloproteinases prepared by the microorganisms of genus Bacillus in an amount of 1-10 x 10 3 mmol per mmol N-formyl protected carboxy compound (2).

An embodiment of the present invention relates to the use of an aqueous-organic medium such as an aqueous dimethylformamide medium.

Further objects and advantages of the present invention will become more apparent from the following detailed description of the process for the preparation of N-formyl-L peptides and examples further illustrating this process.

An advantage of the enzymatic synthesis of the peptide bond lies in the high stereo and region selectivity of the method. The use of an enzyme as a biocatalyst in peptide formation ensures the maintenance of the optical purity of the product and the occurrence of the reaction with the formation of a peptide bond of a natural L configuration. No prior protection of amino acids with additional functional groups in the side radicals is necessary. For example, in the case of aspartic acid, condensation only takes place at the orcarboxy group, while the 8-carboxy group remains free.

In the enzymatic synthesis of peptides, one of the possibilities for shifting the reaction equilibrium to the synthesis side is to remove the desired product from the reaction zone in the form of a precipitate. In addition, for the protection of the amino group, it is of great advantage to use amino acids or peptides that participate in the formation of a peptide bond by the carboxy group, hydrophobic protecting groups such as benzyloxycarbonyl or p-methoxybenzyl oxycarbonyl group (see Y. Isowa, M, Ohmori, T. Ichikawa, K. Mori, Y. Nonaka, 8620072-725881 / Vk / mvl K. Kihara, K. Oyama, H. Satoh, S. Nishimura, Tetrahedron Lett., 1979, No. 28, biz 2611-2612).

These protecting groups, while making a significant contribution to the hydrophobic character of the reaction product, shift the equilibrium towards the formation of the desired product.

A similar degree of the hydrophilic character of the formyl protecting group does not result in an appreciable reduction in the solubility of the reaction product and therefore in a shift of the reaction equilibrium to the formation of a peptide bond.

Special investigations and experiments have been conducted and it has been found that the conditions ensure a successful condensation of the selected starting materials in the presence of the selected enzymes.

The use of metalloproteinases such as thermolysin as biocatalyst at the peptide formation stage influences the selection of the amino acid units placed in positions A and B of the prepared peptides of general formula:

For-A-B-X.

It is known that metalloproteinases give a cleavage and thus give the peptide bonds at a highest rate where donors of amino groups are residues of hydrophobic amino acids or amino acids with branched side chains such as phenylalanine, leucine, isoleucine, valine. It has been found experimentally that the most advantageous group in the position B of a fragment H-B-X are the units leucine and phenylalanine.

The carboxylic acid group of the amino acid residue, placed in position B and participating in the formation of a peptide bond by its amino group, may be protected by virtually any protecting group of both the ester type and the amide type. As the ester type protecting group 30, methyl, ethyl, benzyl esters can be used. As amide-type protecting group, use can be made of an amide, anilide, substituted anilides, in particular p-nitroanilides, hydrazide and substituted hydrazides.

The specificity of metalloproteinases does not overly restrict the character of the amino acid units placed in position A of the prepared peptide of general formula

For-A-B-X.

8620072 -8- 25881 / Vk / mvl

In position A may be placed residues of aliphatic amino acids-glycine and alanine, aromatic or hydrophobic amino acids-phenyl-alanine, tyrosine, methionine, dicarboxylic amino acids-aspartic acid or dipeptide fragments containing these amino acid residues such as alanyl-alanyl, 5-alanyl-glycyl, glycyl -alanyl, glycyl-glycyl.

Various metalloproteinases, also referred to as neutral proteinases, can be used as a biocatalyst in peptide bond formation. A metal ion is present in the active center of the enzymes of this kind, all of which are sensitive to such metal chelating agents such as ethylenediamine tetraacetic acid, O-phenanthroline and the like. An optimal value of the enzymatic activity of these enzymes is usually a pH of about 7.0, although for individual enzymes the location of the pH optimum may differ significantly from each other. The most representative and probably most investigated enzymes of this kind is thermolysin metalloproteinase produced by Bacillus thermoproteolyticus. Also other metalloproteinases are known produced by various species of the bacilli such as enzymes produced by Bacillus stearo-thermophylus. Bacillus amyloliquefaciens. Bacillus subtilis, Bacillus 20 polymyxa, which are similar in terms of the properties and specificity of the substrate to thermolysin and can be used in the reaction of the peptide formation such as thermolysin.

The choice of a particular enzyme firstly depends on the availability as a preparation.

It has been found experimentally that for carrying out the process it is most advisable to use formyl-containing derivatives of methionine, phenyl-alanine, alanyl-alanine, aspartic acid as starting material for the carboxyl compound, while the amino component is preferred p-nitroanilides of phenylalanine and leucine or the methyl ester of phenylalanine.

The use of a strong stereoselective enzyme used in the condensation of a formylamino acid or a formyl peptide with a C-protected amino acid in the enzymatic method of forming a peptide bond ensures an optical purity of the desired product. Even the use of racemic DL amino acids in the composition of both carboxylic and amino compounds results in a product containing amino acid units of a natural L-L configuration. For example, in the preparation of formyl-aspartyl-phenylalanine methylate based on formyl-DL ** aspartic acid, only formoyl-L-aspartic acid participates in the condensation reaction, while the D-862-72 -9- 25881 / Vk / mvl isomer remains in the reaction mixture. Similarly, in the formation of a peptide bond of the L-L configuration, the synthesis takes place based on the use of DL racemic amino acids in the composition of an amine compound. In the preparation of the methyl ester of formyl-aspartyl-phenylalanine from the methyl ester of DL-phenylalanine, only the L-isomer of the methyl ester of phenylalanine has participated in the peptide formation reaction. Simultaneously, the unnatural D-isomer of phenylalanine methylate forms a salt at the β-carboxylic acid group of the aspartic acid residue to completely simulate the behavior of the methyl ester of L-phenylalanine in this reaction. The yield and optical purity of the methyl ester of formyl-aspartyl-phenyl-15-alanine formed in this case is substantially coincident with the results obtained using the starting methyl compound of L-phenylalanine as the amino compound. This also eliminates other side reactions associated with the commonly used condensing agents. In the enzymatic method for the synthesis of peptides it is possible to use 20 amino acids with laterally functional groups without any prior protection thereof. Thus, after the occurrence of formylaspartic acid in the reaction, the condensation occurs only at the α-carboxyl group of the amino acid residue, completely eliminating the formation of the β-isomer, so that the purification is considerably facilitated and thus the yield the desired product is increased.

The method of preparing N-formyl-L-peptides of the present invention makes it possible to prepare protected di- and tripeptides, which are suitable as substrates for eg proteolytic enzymes, rather efficiently.

The method of the present invention makes it possible to use amino acids more intensively in the peptide preparation with a formyl protection of the amino function. Today, the use of this type of protection is limited, although the synthesis of formylamino acids has been described long ago and its preparation requires fairly readily available reactants such as formic acid and acetic anhydride. Run the reaction at a temperature in 8620072

-10-25881 / Vk / mvL

range from 5 to 10 ° C significantly reduces the risk of racemization of the optically active amino acids in the acylation step. Both the process and the preparation of an acylating agent, such as an intermediate formic anhydride and the method of formylation as such, are carried out in single equipment. This makes it possible to avoid the preparation in advance of an acylating agent whose synthesis, in the case of urethane-type protection, is associated with the use of highly toxic compounds such as phosgene.

Despite the availability of formylamino acids, this protecting group has found no intensive use in peptide chemistry, because in most cases the reaction of the formation of a peptide bond proceeds insufficiently. When using the syntheses involving the formation of an anhydride, the racemization reaction occurs rather easily through the formation of azolactone compounds.

In the process for the preparation of N-formyl-L-peptides of the present invention, a high stereo selectivity of the enzyme used in the formation of a peptide bond guarantees the formation of an optically pure product, in which the main side reaction limiting use of formylamino acids in peptide synthesis, namely the racemization reaction, is excluded.

One of the most remarkable features of the method of the present invention is that the condensation reaction is carried out at a pH of the medium within the range of 6.0 to 9.0. The pH range used in the peptide bond synthesis reactions is usually ensured by the equimolar amounts of reactants, the required pH value being assured, if necessary, by the addition of a concentrated solution of sodium hydroxide. At a pH below 6, the yield of the product is sharply reduced (to 2% and below); at a pH above 9.0, some side reactions occur, which are possible and are related to the lability of the protecting groups such as methyl esters under these conditions.

The reaction usually takes place at a temperature of from 20 to 25 ° C. For example, in the preparation of formyl-alanyl-alanyl-phenyl-alanine p-nitroanilide, the product is obtained at a temperature of 22 ° C in an amount of 93Z. The temperature drop below 6 ° C

results in a delay of the enzymatic reaction and thus in a reduced yield of the peptide; at 6 ° C, the product yield is 8623072

-11- 25881 / Vk / mvL

62%. The use of a temperature above 60 ° C results in an increased solubility of the reaction product and thus in a reduced yield of the desired product. For example, in the preparation of the methyl ester of formyl-aspartyl-phenylalanine in the presence of metallo-5 proteinase Bacillus subtilis, the yield of the reaction product at the temperature of 15 ° C is 85%, at 2 ° C 18%, at a significant elongation of the working time, while at 60 ° C the yield is only 11%.

The reaction time is varied within the range of 6 to 48 hours and in each case depends on the rate of formation of a residue of the reaction product. For example, in the synthesis of formyl "alanyl-alanyl-phenyl-alanine-p-nitroanilide, the yield of the product after 3 hours is 54%, after 24 and 48 hours, 85 and 87%, respectively. Therefore, the reduction in reaction time has a significant influence on the yield of product At the same time, the extension of the working time over the optimum period does not give an appreciable increase in the yield (90% over 72 hours) and is therefore unsuitable The completeness of the process is strictly dependent on the individual reaction and under the chosen conditions depending on the structure of the starting compounds.

The amount of enzyme used in the reaction can be varied over a wide range from 1.10 * to 10.10 ^ mmol per mmol carboxy compound. For example, in the preparation of formyl-alanyl-alanyl-phenylalanine-p-nitroanilide, a decrease in the amount of enzyme to 1.10 ^ mmol results in a process slowdown and a decrease in yield to 40%, while after addition of 6x10 ^ or 10x10 4 mmole enzyme the reaction takes place to 89 and 95%, respectively; the use of excessive amounts of enzyme is not advisable.

The reaction is carried out in an aqueous medium or an aqueous-organic medium. Dimethylformamide 30 is usually used as the solvent. The content thereof in the reaction mixture becomes up to 50% and is entirely determined by the solubility of the starting amino and carboxy compounds.

The best way to carry out the method according to the invention.

It is proposed to conduct the condensation of an N-formyl protect the carboxyl compound of general formula (2), namely For-A-OH, where For is formyl, A is Asp, Met, Gly, Ala, Gly- Gly, Gly-Ala,? i; l. . Γ ^ W> W. A ^. ^ *. L '-12- 25881 / Vk / mvl

Ala-Gly, Ala-Ala, Tyr, Phe, with an amino acid protected at the carboxy group of general formula (3): H-B-X, where B is Phe, Leu, lie, Val; X is -0CH3 / -0C2H5 / -NH2, -NH-C0H5 / -p-NHC6H ^ -NO2 / -NH "NH2, -NH-NH-C1, in the presence of metalloproteinases prepared by microorganisms of the following species: Bacillus thermoproteolyticus, Bacillus stearothermophylus, Bacillus amyloliquefaciens, Bacillus subtillis, Bacillus polymyxa.

The reaction is carried out in an aqueous or an aqueous dimethylformamide medium under mild conditions, i.e. at a pH near neutral values and at room temperature. A catalytic amount of enzyme is required for the reaction to take place, i.e. a molar ratio of a carboxylic acid compound and an enzyme of 1-10x10 wherein the reaction time under these conditions is defined by the degree of precipitation of the reaction product, which in each case depends on the structure of the starting amino acids. The yield of reaction products obtained is varied within the range of from 25 to 95%.

For a better understanding of the present invention, some specific examples illustrate the particular embodiments set forth below. All amino acids, unless otherwise indicated, belong to the L-series. The homogeneity of the final compounds was determined by thin layer chromatography (TLC) using "Silufol" plates available in Czechoslovakia in the following systems: (1): n-butanol-pyridine-water-acetic acid 15:10: 12: 3, (2): methanol-chloroform 3: 7, 25 where the angles of agitation were measured on a spectro-polarimeter "Hilger" (Great Britain).

Thermolysin was obtained from the company "Serva" (B.R.D.). Other metalloproteinases were secreted in the laboratory. The amino acids used were obtained from "Reanal" (a company in Hungary), p-nitroanilides of phenylalanine and leucine were products available in Russia, and the resin SP-Sephadex C-25 was obtained from the Swedish company "Pharmacia".

The amino acids analysis was carried out in an amino acid analyzer "Durrum" D-500 (11.SA) after hydrolysis of the peptide for 24 hours in a closed ampoule with 5.7N HCl at a temperature of 110 ° C.

8 § 2 0 0 7 2

-13- 25881 / Vk / mvL

Example I

The preparation of formyl-methyonyl-leucine p-nitroanilide.

A mixture of 0.354 g (2 mmol) formylmethionine and 0.502 g (2 mmol) leucine p-nitroanilide was dissolved in 1.5 ml dimethylformamide, 2.5 ml 5x10 M solution of calcium chloride at a pH of 7.1 were carefully was added with stirring and then 5 mg (133 mmol) of thermolysin was added. Stirring was continued at a temperature of 20 ° C for 6 hours after which the reaction mixture was stored in a refrigerator overnight. The precipitate was filtered off and washed on the filter with 0.5N HCl (2 x 0.5ml), twice with 0.5ml water, twice with 0.5ml 0.5N NaHCO-2, twice with 0.5 ml of water and dried in a drying oven under reduced pressure. The yield was 0.784 g (95.5%). The Rf ^ value was 0.77 and Rfg is 0.54; [α] D = + 5.5 ° (c = 0.5, DMFA). The melting point was 164 ° C.

The amino acid composition was mMet 0.89, Leu = 1.11.

Examples II to IX

These examples are summarized in Table A.

TABLE A

Relationship between the yield of formyl-methionyl-leucine p-nitro-2 anilide to the pH of the reaction mixture. *) Example No. pH of the medium yield (%) II 5.6 1.97 III 6.0 8.8 IV 6.5 17.2 25 V 7.0 77.0 VI 7.3 79.8 VII 7.5 84.4 VIII 8.0 70.3 IX 8.4 60.4 30 --- *) The reaction mixture contained 0.63 mmol formylmethionine and 0.63 mmol leucine p-nitroanilide in 0.6 ml dimethylformamide in 2 ml 0.1M citrate bufer, Examples II to IV; 2 ml of a 0.05M borate buffer, examples V to IX; 2.5 mg (63 mmol) thermolysin; the mixture was kept at 20 ° C for 2 hours and in a refrigerator overnight, the residue was filtered off and washed as in Example I.

862 0 072 -14- 25881 / Vk / mvl

Example X.

Preparation of formyl-alanyl-alanyl-phenylalanine p-nitroanitide.

A mixture of 0.188 g (1 mmol) formyl-alanyl-alanine and 0.290 g (1 mmol) phenylalanine p-nitroanilide was dissolved in 0.4 ml dimethylformamide; 0.6 ml of a 1x10 ^ M solution of CaClg (pH = 6.9) was carefully added to this with stirring, after which 40 μl solution of thermolysin from a standard solution of the enzyme of 37.5 mg / ml (1 mM solution, molecular weight thermolysin is 37,500 Daltons) in a 1x10 2 solution of CaCl 2, pH 6.9, was added; the reaction mixture was kept at room temperature for 24 hours after which the resulting precipitate was filtered off, washed on the filter in the same manner as described in Example I above and dried in a drying oven under reduced pressure. The yield was 0.350 g (85%). Rf ^ = 0.94, Rf ^ = 0.88; [aj ^ = 7.5 ° (c = 0.5; DMFA). The melting point is 201 ° C.

The amino acid composition is: 1.99 Ala; 1.01 Phe.

Examples XI to XIV

These are summarized in Table B.

TABLE B

Relationship between the yield of formyl-alanyl-alanyl-phenylalanine 20 p-nitroanilide with the amount of enzyme. * 5.

amount of thermolysin example no. - yield in 1 μΐ of. ". (%). "^,. in nmol 1 mM solution 25 XI 10 10 40 XII 20 20 64 X 40 40 85 XIII 60 60 89 *** XIV __100__100__95 **} 30 *) The reaction mixture contains 1 mmol formyl-alanyl-alanine and 1 mmol phenyl alanine p- nitroanilide in 0.4 ml dimethylformamide and 0.6 ml CaCl 2 solution, pH 6.9 thermolysin was added from a standard solution of 37.5 mg / ml (1 mM solution) while the further operations were carried out as described in the previous example X.

**) The reaction mixture contained 0.5 ml of a 1x10 ^ M solution of CaCl2, pH = 6.9.

862 0 072 -15-

Examples XV to XIX

These examples are summarized in Table C.

TABLE C

Relationship between the yield of formyl-alanyl-alanyl-phenylaniline p-nitroanilide with respect to temperature. 1). ,, temperature of the,.

example no ... , 0- ,. yield CA) reaction mixture (C) 3 XV 6 62 1Q XVI 15 85 XVII 22 93 XVIII 37 80 XIX 45 51 *) The reaction mixture contains 0.5 mmole formyl-alanyl-alanine and 0.5 mmole 15 phenylalanine p-nitroanilide, 0 2 ml of dimethylformamide and 0.3 ml of water (pH = 6.7); 20 µl thermolysin was added from a standard solution of 37.5 mg / ml, viz. 20 nmol enzyme; stored for 24 hours at the above temperature. The further treatments are similar to those described in Example I.

Examples XX to XXIII

These examples are summarized in Table D.

TABLE D

Relationship of formyl-alanyl-alanyl-phenylalanine p-nitroanilide yield to reaction time.1) 25; example no. reaction time (hours) yield (%) XX 3 54 XXI 6 64 XVI 24 85 30 XXII 48 87 XXIII 72 90 862 0 072

The reaction was carried out in a manner similar to that described in Example XVI, the reaction mixture being kept at room temperature for the above time while the further steps are similar to those described in Example I.

-16- 25881 / Vk / mvl

Examples XXIV-XXVI

These examples are summarized in Table E.

TABLE E

Relationship between the yield of formyl-alanyl-alanyl-phenyl-alanine 5 p-nitroanilide to the amounts of the amino and carboxy compounds. *),. . formyl-alanyl-phenylalanine. .

Example No. <- „n + 4 a» yield C%) alanine immoU p-nitroanilide ___ (mmol) __ 10 XXIV 0.8 1.0 96 XXV 1.0 2.0 22 XXVI 1.0 0.8 70 *) The reaction mixture contained the above amount of the amino-15 and carboxy compounds, 0.2 ml of dimethylformamide, 0.3 ml of water, pH = 6.7 and 20 µl of a 1 mM solution of thermolysin; the mixture was kept at room temperature for 24 hours and then treated as described in example I.

Example XXVII

The preparation of formyl-alanyl-alanyl leucine p-nitroanilide.

A mixture of 0.188 g (1 mmol) formyl-alanyl-alanine and 0.251 g (1 mmol) leucine p-nitroanilide was dissolved in 1 ml dimethylformamide to which 5 ml of a solution of -2 5x10 M CaCl 2, pH was added with stirring = 6.8; 5 mg (133 mmol) of thermolysin was added to this and kept at a temperature of 22 ° C for 6 hours and then stored in a refrigerator overnight. The precipitate formed was filtered, washed on the filter, followed by an operation similar to that in Example 1, and dried in a drying oven under reduced pressure to obtain 0.305 g (72.5%) of the product. Rf ^ = 0.70; Rf ^ = 0.71; [α] D = 21 ° (c = 0.5, DMFA), melting point is 176 ° C.

The amino acid composition was: Ala 1.93, Leu 1.08. Example XXVIII

The preparation of formyl-phenylalanyl-leucine p-nitroanilide.

A mixture of 19 mg (100 ymol) formyl-phenylalanyl and 25 mg 35 (100 ymol) leucine p-nitroanilide was dissolved in 0.15 ml dimethylformamide and carefully added with stirring 0.2 ml water (pH = 6, 8) then 0.7 mg (20 nmol) of thermolysin and S020 072 -17-25881 / Vk / mvl stored at room temperature for 24 hours. The resulting precipitate was dissolved in 5 ml of ethyl acetate and washed with Q, 5N citric acid (1x1 ml), water (1x1 ml), 0.5N NaHCO2 (1x1 ml), dried over sodium sulfate and evaporated under reduced pressure. The yield was 27 mg (66%). Rf ^ = 0.81, Ri ^ = 0.91; 5 La] J8 = Ί60 (c = 0.5; DMFA). Melting point is 181 ° C.

The composition of the amino acid: Leu 1.0, Phe = 1.0.

Example XXXIX

The preparation of the methyl ester of formyl-aspartylphenylalanine.

To 0. 6 g (10 mmol) phenylalanine methyl acrylate hydrochloride was added 0.66 g (3.1 mmol) formylaspartic acid in 2.0 ml water. After dissolving the starting compounds, the pH of the reaction mixture was adjusted to 6.6-6.8 by addition of 6N NaOH solution. Then the reaction mixture was treated with 20 mg (530 nmol) thermolysin and stirred on a magnetic stirrer at 25 ° C for 24 hours. The residue was dissolved in 25 ml 1M acetic acid and treated with 30 ml SP-Sephadex C-25, after which the resin was filtered off and washed with 1M acetic acid. The peptide was extracted from the acetic acid solution using a mixture of ethyl acetate-water (9: 1) (8x8 ml). The combined extracts were evaporated under reduced pressure. The oil crystallized out with ether at a reduced temperature. The yield was 0.420 g (35%). [α] J8 = -24.5 ° (c = 0.5; HgO; [α] J8 = -15 ° (c = 1; AcOH).

The composition of the amino acid: Asp = 1.03; Phe = 0.97.

Example XXX

The preparation of the methyl ester of formyl-aspartyl-phenylalanine.

The preparation was carried out in a similar manner as described in Example XXIX from 2 mmol phenylalanine methyl acrylate and 1 mmol formyl aspartic acid.

To recover the reaction product, the precipitate was dissolved in 4.5 ml of acetic acid, the solution diluted by the addition of 40 ml of ethyl acetate; the resulting precipitate was separated, the organic layer washed with water (7x2 ml), dried over sodium sulfate and evaporated under reduced pressure. The yield was 0.140 mg (43%).

Example XXXI

The preparation of the methyl ester of N-formyl-alanyl-alanyl-phenylalanine.

A mixture of 0.376 g (2 mmol) N-formyl-alanyl-alanine and 662 0 072

-18- 25881 / Vk / mvL

0.430 g (2 mmol) phenylalanine methyl hydrochloride was dissolved in 0.5 ml 4N solution of sodium hydroxide (2 mmol), bringing the pH of the reaction mixture to 6.7, adding 3 mg metalloproteinase Bacillus polymyxa and stirring for 24 hours at a temperature 5 of 25 ° C. The precipitate was dissolved in ethyl acetate and washed with water. The organic layer was dried over sodium sulfate, then evaporated under reduced pressure. The yield of product was 0.25 g (32%);

Rf1 = 0.71.

The amino acid composition was: Ala 1.93; Phe = 1.06.

Example XXXII

The preparation of the methyl ester of N-formyl-alanyl-alanyl-leucine.

A mixture of 0.376g (2mmol) N-formyl-alanyl-alanine and 0.362g (2mmol) leucine methylate hydrochloride was dissolved in 0.33ml 15N solution of 2mmol sodium hydroxide, adjusting the pH of the reaction mixture to 6.7, after which 3 mg metalloproteinase Bacillus stearothermophylus were added, stirred at a temperature of 25 ° C for 24 hours. The precipitate was dissolved in ethyl acetate, washed as described in Example XXXI, dried over sodium sulfate and evaporated under reduced pressure. The yield was 0.240 g (36%); Rf ^ = 0.69.

The amino acid composition was: Ala = 1.98; Leu = 1.02. Example XXXIII

The preparation of N-formyl-alanyl-leucine p-nitroanilide.

A mixture of 10.3 mg (100 ymol) N-formyl-alanine and 25 mg of leucine p-nitroanilide was dissolved in 0.15 ml of dimethylformamide, to which was added 0.2 ml of water adjusted to a pH of 8, 7, after which 0.5 mg of metalloproteinase Bacillus amyloliquefaciens was added and kept at a temperature of 25 ° C for 24 hours. The precipitate was dissolved in ethyl acetate and then washed with water, 0.5N HCl, water, 0.5N 30 HCl, water, 0.5N sodium bicarbonate solution and water, dried over sodium sulfate and evaporated under reduced pressure. The yield was 12 mg (30%). The melting point was 143 ° C.

Rf 1 = 0.79; Rf2 = 0/81; [ajJ8 = -0.5 ° (c = 0.5; DMFA).

The amino acid composition was: Ala = 0.98; Leu = 1.06.

Example XXXIV

The preparation of N-formyl-alanyl-phenylalanine-p-nitroanilide.

A mixture of 10.3 mg (100 pmol) N-formylalanine and 29 mg 882 0 072 -19- 25881 / Vk / mvl (100 pmol) phenylalanine p-nitroanilide was dissolved in 0.15 ml dimethyl formamide to which 0, 2 ml of water was added, adjusted to a pH of 8.7 and then 0.5 mg of metalloproteinase Bacillus amyloliquefaciens was added and kept at a temperature of 25 ° C for 24 hours.

The precipitate was treated in a manner similar to that described in Example XXXIII. The yield of product was 8.5 mg (25%); the melting point was 159 ° C. Rf1 = 0.80; Rf2 = 0.84; [α] J8 = + 30.0 ° (c = 0.5; DMFA).

The amino acid composition was Ala = 0.96; Phe = 1.03.

Example XXXV

The preparation of N-formyl-phenylalanyl-phenylalanine p-nitroanilide.

A mixture of 19 mg (100 ymol) formyl-phenylalanine and 29 mg (100 ymol) phenylalanine p-nitroanilide was dissolved in 0.2 ml of dimethylformamide and 0.2 ml of water was added to this at pH 8, 7, after which 0.5 mg of metalloproteinase Bacillus amyloliquefaciens was added and kept at a temperature of 25 ° C for 24 hours. The precipitate was treated in a manner similar to that in Example XXXIII. The product was obtained in an amount of 35.5 mg (76%).

The melting point was 188 ° C; Rf ^ = 0.84; Rf2 = 0.91; 20 | ajJ8 = + 4 ° (c = 0.5; DMFA).

Example XXXVI

The preparation of the methyl ester of N "formyl-aspartyl" phenylalanine.

0.64 g (4 mmol) N-formyl aspartic acid in 2 ml water was added to 1.72 g (8 mmol) phenylalanine methyl hydrochloride. The reaction mixture was brought to a pH of 6.6 by the addition of a 6N solution of sodium hydroxide, after which 20 mg of metalloproteinase Bacillus subtilis was added and the mixture was stirred at a temperature of 15 ° C for 24 hours. The precipitate was dissolved in acetic acid, diluted with a tenfold volume by volume of ethyl acetate. The resulting precipitate was separated, the organic layer washed with water to a neutral pH, dried over sodium sulfate and evaporated under reduced pressure. The product was obtained in an amount of 1.1 g (85%), [α] J 8 = -15.2 ° (c = 1; AcOH).

The amino acid composition was: Asp = 0.98; Phe = 1.03.

8620072 -20-25881 / Vk / mvl

Example XXXVII

The preparation of the methyl ester of N-formyl-aspartyl-phenyl-alanine.

The procedure of Example XXXVI was repeated, except that the reaction mixture was stored at 10 ° C for 24 hours. The yield of the product was 0.51 g (40¾).

Example XXXVIII

The preparation of N-formyl-aspartyl-phenylalanine methyl ester.

The procedure of Example XXXVI was repeated, except that the reaction mixture was stored for 72 hours at a temperature of 2 ° C. The product was obtained in an amount of 0.230 g (18%).

Example XXXIX

The preparation of N-formyl-aspartyl-phenylalanine methyl ester.

The procedure of Example XXXVI was repeated, except that the reaction mixture was stored at a temperature of 60 ° C for 24 hours. The product was obtained in an amount of 0.14 g (11%).

Example XL

The preparation of N-formyl-aspartyl-phenylalanine methyl ester.

The procedure of Example XXXVI was repeated, except that DL-phenylalanine methyl hydrochloride was used instead of L-phenylalanine methyl hydrochloride. The product was obtained in an amount of 0.75 g (58%); [ctj ^ 8 = -15.1 ° (c = 1; AcOH).

Example XLI

The preparation of the methyl ester of N-formyl-aspartyl-phenylalanine.

1.28g (8mmol) N-formyl-DL-aspartic acid in 4ml water was added to 1.72g (8mmol) phenylalanine methyl hydrochloride and the reaction mixture was adjusted to a pH of 6.6 by adding 6N solution of sodium hydroxide, after which 20 mg of metalloproteinase Bacillus 30 subtilis was added and stirred at a temperature of 20 ° C for 48 hours. The resulting precipitate was treated in a manner similar to that in Example XXXVI. The yield of product was 18 o 0.35 g (27%) based on N-formyl-L-aspartic acid [α] Β = 14.8 ° (c = 1; AcOH).

35 Industrial applicability.

N-formyl L-peptides of the present invention are useful in experimental biology and in medicine; in particular formyl-862 0 372 -21- 25881 / Vk / mvl peptides containing chromogenic groups can be used as substrate for some enzymes such as formylmethionine aminopeptidase because such substrates are considerably hydrophilic and thus easily soluble in an aqueous medium making them subject to are affected by the action of the enzyme.

As mentioned previously, one of the examples of the formyl peptides is a precursor of aspartam-formylaspartam, namely formyl-L-aspartyl-L-phenylalane with hyylate.

As is known, aspartame is about 200 times sweeter than sugar 10 and may be suitable as a sugar substituent. Particularly promising is the use of aspartame as a sugar substituent in patients suffering from diabetes mellitis. Aspartame is also suitable in diets that have a low calorific value in the preparation of non-alcoholic drinks and in cosmetics.

Because aspartame is indifferent to microorganisms, it is particularly suitable for pharmaceutical applications such as sugary pharmaceuticals, which is important in the treatment of children, especially since aspartame is just as safe for children as it is for adults. From a medical point of view, aspartame is even more important than sucrose because it does not stimulate the development of dental caries.

In summary, the invention therefore also relates to N-formyl-L-peptides of general formula 1, prepared by the condensation of N-formyl-protected carboxy compound of general formula 2 with an amino acid protected at the carboxyl group and of general formula 3 in the presence of a metalloproteinase prepared by microorganisms of the genus Bacillus. The condensation is carried out in an aqueous or an aqueous-organic medium at a pH from 6.0 to 9.0 and at a temperature from 0 to 60 ° C.

For-A-B-X (1) 30 For-A-0H (2) H-B-X (3), where For = formyl; A = Asp, Met, Gly, Ala, Gly-Gly, Gly-Ala, Ala-Gly, Ala-Ala, Tyr, Phe; B = Phe, Leu, He, Val; X = -OCH ^; -0C2H5; -CH2C6H5; -NH2, -NH-C6H5; -p-NH-C6 H4-NO2; -NH-NH2; -NH-NH-C1.

35 862 0 072

Claims (6)

1. Process for the preparation of N-formyl-L-peptides of general formula: Method according to claim 1, characterized in that as N 'formyl-protected carboxyl compound and amino acids protected in the carboxyl group are used racemic DL derivatives of the compounds or optically active L-amino acids. Process for the preparation of N-formyl-L-peptides according to claims 1 and 2, characterized in that as microorganisms of the genus Bacillus the following species of the microorganisms are used: Bacillus thermoproteolyticus, Bacillus stearothermophylus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus polymyxa. Process according to any one of claims 1 to 3, characterized in that the condensation is carried out at a molar ratio of the N-formyl-protected carboxyl compound to the amino acid protected at the carboxyl group equal to 1: 1 to 1: 2. 862Q 072 -23- 25881 / Vk / mvl A method according to any one of claims 1 to 4, characterized in that the metalloproteinase prepared by the microorganisms of genus Bacillus is used in an amount of 1-10x10 mmol per mmol N "formyl" protected carboxyl compound. For-A-B-X (1) where For = formyl, A = Asp, Met, Gly, Ala, Gly-Gly, Gly-Ala, Ala-Gly, Ala-Ala, Tyr, Phe; B = Phe, Leu, lie, Val; 10 x = -och3, -oc2h5, -och2c6h5, -nh2, -nh-c6h5, -p-NH-C ^ H ^ -N02, -NH-NH2, -NH-NH-C ^ H ^, characterized that an N-formyl-protected carboxyl compound of general formula: For-A-OH (2) wherein For = formyl; A = Asp, Met, Gly, Ala, Gly-Gly, Gly-Ala, Ala-Gly, Ala-Ala, Tyr, Phe; is condensed with an amino acid protected at the carboxyl group and of general formula: H-B-X (3) 20 where B = Phe, Leu, Ile; Fall; X = -0CH3, -0C2H5, -0CH2C0H5, -NH2, -NH-C0H5, p-NH-C6H4 ~ NO2, -NH-NH2, -NH-NH-C1 in the presence of a metalloproteinase prepared by microorganisms by the genus Bacillus in an aqueous or aqueous-organic medium at a pH from 6.0 to 9.0 and at a temperature from 0 to 60 ° C. Process according to any one of claims 1 to 5, characterized in that an aqueous dimethylformamide medium is used as the aqueous organic acid. Eindhoven, September 1986 8 61 0 ^ 7 2