SE194137C1 - - Google Patents

Description

Inventors: G Arniard, R. Heymes and L. Velluz Priority given from December 30, 1955 (France) The present invention relates to a novel method of preparing γ-glutamyl peptides by direct reaction of amino acid esters or peptides with free N-tritylglutamic acid in the presence of a disubstituted carbodiimide. It has been found that blocking by tritylation of the nitrogen of glutamic acid is beneficial to the selective condensation of the γ-carboxyl of glutamic acid and of the aminated function of α-amino acids in peptidic condensation in the presence of a disubstituted carbodiide. This new method of arriving at γ-glutamyl peptides constitutes a considerable improvement over older procedures for producing said γ-glutamyl peptides, which will be described briefly in the following (see also: G. Amiard, R. Heymes and L. Velluz. Bull Soc. Chim. 1956, p. 97). Part French Patent 1,129,627 discloses a method of preparing α- and γ-glutamyl peptides which are derived from glutamic acid according to the following formula.

HOOC-CH-CH, -CH, -COOH aI NH, Formula I According to the procedure described in this patent, the α-glutamyl peptides are prepared by reacting an N-tritylated α- or γ-glutamic acid monoester with the ester of an amino acid or peptide in the presence of dicyclohexylcarbodiimide, as well as alkaline confluence and detritylation by attic acid diluted with water. The γ-glutamyl peptides are obtained by reacting the N-trityl-α-benzyl-glutamate with the benzyl ester of an amino acid or peptide in the presence of dicyclohexylcarbodiimide and selective hydrogenolysis of the obtained Ntritylated derivative of the benzyl ester of the benzyl folate amylglylamide. In this procedure, use the ronet, Dupl. at 12 o: 16; 12 d: 16 that the dibenzyl esters of glutamic acid are characterized by an extremely high stability of the ester group in the α-position in relation to alkali and alcoholic agents, so that one can completely prepare γ-alkylated α-benzyl esters, which give the α-benzyl monoester by mono-decomposition, or the yalkyl monoester by hydrogenolysis.

In the practice of this process, it has been found that only the α-glutamic acid benzyl ester is suitable for the preparation of pure γ-glutamyl peptides, since it is possible to avoid an alkaline treatment during the release of the α-carboxyl group after the γ-peptide is formed. It has thus been found that alkaline treatment of a diester of N-trityl-γ-glutamyl peptide (Formula II) above gives the α-glutamyl peptide (Formula IV) likely to give the cyclic derivative (Formula III).

R'0 0C— CH — CH, —CH, —CO — NH — CH — COOR "NH — C (CRI Formula II CH —CO.

CH / \ 2 N — CH — COOR "CH CO / I R1 NH — C (C, 11,), Formula III HOOC — CH — NH — CO — CH — CH2 — C112 — COOH R, NH, Formula IV R, denotes an alkyl or aryl radical or a heterocyclic radical R 'denotes an alkyl 2 - - R "denotes an alkyl which is optionally identical to R'.

A method has been proposed to prepare Ly-glutamyl-L-cysteinylglycine, the last step in tripeptide synthesis being that the S-tritylL-cysteinylethylglyeinate is condensed with Ntritylglutamic acid so that the desired ester of S, N-ditritylylt-γ-gleptide. By blocking by tritylation at the nitrogen of the glutamic acid, in this case the selective condensation of the γ-carboxyl and the aminated function of the S-trityl-Lcysteinylethylglycinate is favored. It has now been found that the selectivity in the γ-condensation described above also occurs with α-amino acids, which, however, have considerably lower monocylar barriers to the S-trityl-L-eysteinylethylglycinate. This ratio of a surprising salt has made it possible, according to the present invention, to generalize the use of the N-tritylglutamic acid to obtain γ-glutamyl peptides while avoiding the need to prepare the N-tritylated α-benzylglutamic acid ester as an intermediate by N-trutylate trituration. -benzyl and γ-methyl, prepared according to patent 177 462.

The N-trityl-L-glutamic acid is easily obtained by hydrogenolysis of the N-trityl-L-dibenzyl glutamate, which is prepared according to the above patent. Although this method does not form part of the present invention, it will also be described herein.

The process of the present invention consists in dissolving in the soluble solvent the N-trityl-Lglutamic acid (see formula V in the attached scheme), a disubstituted carbodiimide and an amino ester of the general formula (formula VI), in which n is 0 or a content of between 1 and 8. R 1 represents hydrogen, an alkyl radical, an aralkyl radical, an aryl radical or a heterocyclic radical, which may have a hydroxylated function or a sulfur radical, and R represents a lower alkyl, such as methyl or ethyl, after which the reaction mixture is left to stand at a temperature between 0 ° C and the boiling point of the solvent used, The excess carbodiimide is removed by treatment with acid, the reaction mixture is pre-cooled by alkaline treatment, the N-trityl peptide (formula VID is isolated during application, the N-trityl peptide (formula VII) is isolated during application of i and wash procedures, extractions and purifications, after which the latter product is detritylated by heating in water or alcohol haltig, sur miljo.

A preferred embodiment of this process is described as follows: The N-trityl-L-glutamic acid in the form of its acidic salt of triethylamine is dissolved in methylene chloride and the ester of the aminated acid which is now condensed, as well as an equimolar amount of discybdododihexyl. The reaction mixture is allowed to stand at room temperature, treated with acetic acid and suction filtered of the dicyclohexylurea which has formed. After washing in the usual way, the solution is collected to dryness, and the product is pre-cooled with the aid of alcoholic sodium hydroxide solution. The N-trityl-γ-glutamyl peptide (Formula VII) is isolated and then detritylated by heating in aqueous or alcoholic acetic acid.

The following examples illustrate the invention without limiting it. It is thus possible to vary the solvents, temperatures and reaction times, as well as to order them in the introduction of the chemical substances without exceeding the scope of the invention.

The specified melting points are the Instantaneous melting points and have been determined on Maquenne blocks.

Example 1. Preparation of γ-L-glutamylL-tyrosine (Formula VIII n 1111 - C61-14-0H.) A) Condensation of L-ethyltyrosinate with Ntrityl L-glutamic acid and preservation of ester function. 3.85 g of L-ethyl tyrosinate and 3.8 g of dicyclohexylcarbodiimide are dissolved in 50 ml of methyl chloride, 8.8 g of triethylamine salt of L-tritylglutamic acid are added and the reaction mixture is allowed to stand overnight at room temperature. The product is cooled to 0 ° C, the dicyclohexylurea is suction filtered and washed with methylene chloride, after which the filtrate and the solution are combined and washed with 45 ml of 0.5 N hydrochloric acid, then with water, dried over magnesium sulphate and evaporated to dryness in vacuo The resulting residue is dissolved in 20 ml of ethanol, 55 ml of N sodium hydroxide solution are added, the product is heated for 10 minutes to 60 ° C, 70 ml of ice water and then 60 ml of N hydrochloric acid are added, the resulting product is suction filtered, washed with water and evaporated to dryness. under vacuum.

This gives a product which, after recrystallization from 25 ml of absolute alcohol, gives pure N-trityl-αL-glutamyl-L-tyrosine, m.p. = 145 ° C, [at ° = + 340 ± 2 (c = 1% 0.06% sodium hydroxide solution in 75% methanol). This product has the form of small uncoloured prisms, such as Oro insoluble in water, ether, acetone, benzene and chloroform, as well as highly insoluble in cold alcohol. It contains 2 moles of crystallization ethanol, which emit successively on heating to 80 ° C and 110 ° C. This product has been previously described.

Analysis of the product dried at 80 ° C: C331-1,206N2 C2110H = 598.67 Calculated: C% 70.2 H% 6.4 0% 18.7 N% 4.7 Found: 70.26.319.14.9 ( Detritylation of N-trityl-γL-glutamyl-L-tyrosine.

Man info '. 3.5 g of N-trityl-γ-L-glutarnyl-L-tyrosine in 20 ml of 50% acetic acid (diluted with water) and heat on a water bath for 10 minutes. The triphenylcarbinol is filtered off with suction and washed with water, the washings are added to the filtrate and evaporated to dryness under vacuum at a temperature of at most 30 ° C. The residue is dissolved in 6 ml of water, -3,200 ml of acetone are added successively and the solution is allowed to stand overnight at room temperature. The product is filtered and washed with acetone and ether to give 1.7 g (97% γL-glutamyl-Ltyrosine, m.p. 265-270 ° C, [at ° = 260 + 2 (c = 2% water). This product is identical to an authentic sample of γL-glutamyl-L-tyrosine prepared according to G. Amiard, R. Heymes and L. Velluz, Bull. Soc. Chim. 1956, p. 97.

Example 2. Preparation of γ-L-glutamyl-Lleucine.

CH (Formula VIII, nR1 = -CH / ') \ CH, a) Condensation of L-ethyl leucinate with N-tritylL-glutamic acid and preselection of the ester function. 1.95 g of chlorohydrate of ethyl leucinate are dissolved in 3 ml of chloroform, the solution is cooled to 0 ° C, diethylamine is added dropwise until pH 9 is obtained, after which ml of cold sulfuric acid ether is added to the mixture thus obtained. The diethylamine chlorohydrate is suction filtered and the filtrate is evaporated to dryness without heating. The residue, consisting of the free amino ester, is taken up in 10 ml of methylene chloride, 4.9 g of triethylamine salt of N-trityl-L-glutamic acid dissolved in 10 ml of methylene chloride were added first and then 2.06 g of dicyclohexylcarbodiimide, dissolved in 5 ml of the methylene chloride, the reaction mixture for 1 hour at room temperature, treated with 0.5 ml of acetic acid, and the dicyclohexylurea formed is separated by filtration. The filtrate is washed with water, dilute hydrochloric acid, water, dried over magnesium sulphate and evaporated to dryness in vacuo without heating. For preselection, the residue obtained is dissolved in 5 ml of ethanol, after which 15.4 ml of N sodium hydroxide solution are added and heated for 110 minutes at 70 ° C, and 50 ml of ice water and then 15.4 ml of N hydrochloric acid are added. The N-trityl dipeptide is extracted with chloroform, the chloroform solution is washed with water, dried over magnesium sulfate and evaporated to dryness under vacuum. A residue is thus obtained which, after recrystallization from 2.5 ml of absolute alcohol, gives pure N-trityl-γ-L-glutamyl-L-leucine, mp 141-144 ° C, [.alpha.] D @ 20 - 0 ± 2 (c = 2%). , chloroform). This product dr fly.

Analysis: C1-1340N2 = 502.59 Berdknat: C% 71.69 H% 6.33 N% 5.57 Found: 71.47,05.8 b) Detritylation of N-trityl-γ-L-glutamyl-Lleucine. 2.8 g of N-trityl-γ-L-glutamyl-L-leucine are introduced into 10 ml of 50% acetic acid (diluted with water) and heated on a boiling water bath for 7 minutes. The triphenylcarbinol is suction filtered and washed with water, the wash water is added to the filtrate and concentrated in vacuo at a temperature of not more than 40 ° C, until the solution has reached a volume of about 2 nil. 100 ml of acetone were added, the mixture was suction filtered, washed with acetone and ether and then dried under vacuum. There is thus obtained 1.19 g (83%) of γ-L-glutamyl-L-leucine, mp 221-222 ° C, [α] D = -170 ± 2 (c = 2%, water). This product is identical to a sample of α-L-glutamyl-L-leucine prepared according to G. Amiard R. Heymes and L. Velluz, Bull. Soc. Chim., 1956, p. 97.

Example 3. Preparation of γ-L-glutamyl-L-methionine.

Formula VIII, n. = 1 R, = -CH 2, S CH,) Condensation of the methyl ester of L-methionine with N-trityl L-glutamic acid and preservation of the ester function. 0.9 g of hydrochloride of the methyl ester of L-methionine is dissolved in 3 ml of chloroform, cooled to 0 ° C, diethylamine was added dropwise until the pH rises to 9, after which 30 ml of cold sulfuric acid ether were added. The diethylamine chlorohydrate is suction filtered and the filtrate is evaporated to dryness under vacuum without fermentation. The residue is taken up in 3 ml of methylene chloride, 2.2 g of the triethylamine salt of the N-tritylglutamic acid dissolved in 5 ml of methylene chloride were added first and then 0.98 g of dicyclohexylcarbodiimide lbst in 4 ml of methylene chloride. The reaction mixture is left for 3 hours at room temperature. ml of acetic acid and filtered. The filtrate is washed with water, dilute hydrochloric acid, water, dried over magnesium sulphate and evaporated to dryness under vacuum without heating. For reconstitution, dissolve the residue obtained in 3 ml of absolute alcohol, then add 6.9 ml of sodium hydroxide solution, heat to 65 ° C for 10 minutes, add 30 ml of ice water and then 6.9 ml of N hydrochloric acid, extract with chloroform, wash the chloroform solution with water. , it dries over magnesium sulfate and cools to dryness under vacuum. A residue is thus obtained which, after recrystallization from 5 ml of absolute alcohol, gives pure N-trityl-γ-L-glutamyl-L-methionine, m.p. about 140 DEG C.

This product is fly.

Detritylation of N-trityl-α-L-glutamyl-Lmethionine. 520 mg of N-trityl-γ-L-glutamyl-L-methionine are suspended in 2.5 ml of 50% acetic acid (diluted with water), heated for 7 minutes in a boiling water bath and cooled, after which 2 ml of water are added and the triphenylcarbinol is separated by filtration. The filtrate is evaporated in vacuo at a temperature of not more than 30 ° C, the residue 40 ml are added with acetone, filtered off with suction, washed with acetone and then with ether and dried under vacuum.

175 mg (65%) of γ-L-glutamyl-L-methionine, mp 228-231 ° C, are obtained, [at ° = -9 ° in 1 (c = 1.4%, water). This product, which was not previously described, took the form of uncolored needles, such as Arc 10 soluble in water and insoluble in alcohol, ether, acetone, benzene and chloroform.

Analysis: C 10 H 11 O, N, S = 278.32 Calculated: C% 43.2 H% 6.5 N% 10.1 Found: 43.26.9.8 4 - Example 4. Preparation of γL-glutamyl-glycine .

(Formula VIII, n = 0.111 = H) Condensation of N-trityl-L-glutamic acid with ethyl glycinate and preselection of the ester. 1.4 g of hydrochloride of ethyl glycinate are suspended in 25 ml of methylene chloride, cooled to 0 ° C and then 1 ml of diethylamine is added with stirring. 4.9 g of triethylamine salt of N-trityl-L-glutamic acid and 2.1 g of dicyclohexylcarbodiimide are then introduced into the solution. The mixture is allowed to stand overnight at room temperature, 0.5 ml of acetic acid are added and the dicyclohexylurea is filtered off with suction and washed with 10 ml of methylene chloride. The filtrate and washings are combined, washed with 0.5 N hydrochloric acid, ice water, dried over sodium sulfate and evaporated to dryness in vacuo. The residue is taken up in 125 ml of ethyl acetate, 1 ml of diethylamine is added and the solution is allowed to stand for 10 hours under cooling. The product is suction filtered and 3.65 g (67%) of the diethylamine salt of the N-trityl-γ-L-glutamylethylglycinate are obtained. 2.45 g of this salt Rises in 20 ml of water, 6 ml of N hydrochloric acid are added, the product is extracted with 50 ml of ether, the ether solution is washed with ice water, dried over magnesium sulphate and evaporated to dryness under vacuum. The product obtained (γ-N-trityl-L-glutamylethylglycinate) is dissolved in 10 ml of alcohol. 10 ml of N sodium hydroxide solution were added and the product was allowed to stand for 1.5 hours at room temperature. Cool to 0 ° C, add 11 ml of N hydrochloric acid and extract with 25 ml of chloroform. The chloroform solution is washed three times with ice water and evaporated to 5 ml under vacuum. The product is suction filtered, washed with chloroform and dried. N-trityl-γ-L-glutamylglycine is thus obtained, m.p. about 145 ° C. This product, which has not been previously described, is in the form of undyed needles, which are insoluble in water and soluble in chloroform and ether, and soluble in alcohol and acetone.

Detritylation of N-trityl-γ-L-glutamyl-glydine.

The detritylation can be performed with aqueous acetic acid in the manner described in lb, 2b and 3b. However, it is also possible to work in an alcoholic environment, as described below: 1 g of N-trityl-γL-glutamylglycine is dissolved in 9 ml of 95% alcohol, 0.9 ml of acetic acid was added and the mixture was heated to 65 ° C for 1 hour. cooled to room temperature, suction filtered, washed with alcohol and then with acetone and dried at 90 ° C to give 0.3 g (66%) of γL-glutamylglycine, which is identical to an authentic sample prepared according to G. Amiard, R. Heymes and L. Velluz, Bull. Soc. Chim., 1956, p. 97.

Preparation of N-trityl glutamic acid. a) Preparation of N-trityl-L-dibenzylglutamate. 18.2 g of chlorohydrate of L (+) - dibenzyl glutamate prepared according to the instructions of H. Sachs and E. Brand (J. Am. Chem. Soc., 1953, 75, 4610) dissolve 1100 ml of chloroform, the solution is cooled to 0 ° C and continued with 15 ml of triethylamine. While the temperature was measured at 0 ° C, 14 g of trityl chloride were then added and the solution was allowed to stand for 48 hours at room temperature. The solution, in which the triethylamine chlorohydrate has partially crystallized, is washed with water, then with dilute hydrochloric acid and again with water, until the aqueous solutions are neutral, the chloroform solution is dried over magnesium sulphate and evaporated to dryness in a water bath at 40 ° C. , hot alcohol, of which about 15 ml is evaporated to completely remove the chloroform. To carry out crystallization, the rockers of the flask are scraped with a stirrer, then cooled, suction filtered, washed with methanol to obtain 23.1 g of the product directly usable for hydrogenolysis (yield 81 cy ()). b) Hydrogenolysis of N-trityl-L-dibenzylglutamate. 57 g of the N-trityl-L-dibenzyl glutamate are dissolved in 500 ml of ethyl acetate, added with palladium black, prepared from 15 g of charcoal and 5 ml of a 20% aqueous solution of palladium chloride, and then with 27 ml of triethylamine. The product is hydrogenated until the theoretical amount of water has been bandaged, which takes about 45 minutes, the catalyst is suction filtered, washed with ethyl acetate, the wash liquor is combined with the filtrate and concentrated to about 100 ml. After cooling, 100 ml of ether were added, the product was filtered off with suction, washed with ether and dried at 80 ° C to obtain 40 g (81%) of the acidic triethylamine salt of the N-trityl-L-glutamic acid, which is used in carrying out the procedure of the present invention. and whose representation Mr has only been described for the sake of completeness. The N-trityl-L-glutamic acid can also be prepared by direct tritylation of the L-glutamic acid or by pre-digestion of an N-trityl-L-glutamic acid ester.

CO, H / C61- CH — NH — C — C, H, CH, \ CH, (CH,) R 1 CO, HH, N — CH-O 0, R Formula VFormula VI CO, HC, H, CH — NH —C — C6H Formula VII CH, C61-1, CH, (CH2) 11111 CO — NH — CH — CO, H - - CO, H CH — NH, Foimel VIII CH, CH, (CH2) .R1 CO — NH —CH — CO, H

Claims (8)

Patent claim: Set to prepare γ-L-glutamyl peptides of the general formula: CO, H CH-NH, CH, R 1 CH, (CH 2). CO-HN-CH-CO, HI which n represents 0 or an integer between 1 and 8, R1 represents vate or an alkyl radical, aralkyl radical or aryl radical or heterocyclic radical, which may have a hydroxylated function or a sulfur radical, in a suitable solvent, N-trityl-L-glutamic acid, a disubstituted carbodiimide and an amino ester of the general formula are dissolved: (CH2) n H2N-CH - CO, R in which n and R have the same meaning as above , and R represents a lower alkyl, such as ethyl or methyl, after which the reaction mixture is solid. at a temperature between 0 ° C and the boiling point of the solvent used, the excess carbodiimide is removed by acid treatment, and the product is condensed by alkaline treatment, the N-trityl peptide is isolated by application of kanda procedures for washing, extraction and purification, after which the product is detritylated . 2. A kit according to claim 1, characterized in that the N-trityl-L-glutamic acid was used in the form of sieve acidic triethylamine salt 3. A kit according to claims 1 and 2, characterized in that the solvent is prepared from methylene chloride. 4. Set according to claims 1-3, characterized in that the reaction temperature is measured at the laboratory temperature (room temperature). 5. Mt according to claims 1-4, characterized in that the disubstituted carbodiimide is formed from dicyclohexylcarbodiimide. 6. A claim according to the preceding patent claim, characterized in that the excess dicyclohexylcarbodiimide is eliminated with acetic acid at room temperature, and the carboxylene is released by treatment with alcoholic sodium hydroxide solution and the product is detritylated by heating in attic acid, diluted with water or alcohol. 7. A claim according to any one of the preceding claims, characterized in that N-trityl-L-glutamic acid is condensed with esters of glycine, L-methionine, L-leucine and L-tyrosine to obtain the corresponding γ-L-glutamyl peptides. Request publications: American chemical society. Easton. Journal 77 (1955), pp. 1067. Applied Chemistry 66 (1954), pp. 50 8. Journal of Natural Research 8 B (1953), p. 44 5.