The method of obtaining l-glutamyl-l-tryptophan and its salts

 

(57) Abstract:

The present invention relates to the chemistry of peptides. The method of obtaining L-glutamyl-L-tryptophan and its salts includes the successive stages of protection of the amino group of glutamic acid activation-carboxyl group of glutamic acid by formation of a complex of activated ester, interaction with tryptophan complex activated ester, removal of protective groups hydrogenated product of interaction with tryptophan in the presence of a palladium catalyst and a hydrogen donor and the selection of the finished product. The technical result consists in the simplification and cheapening of the process and increase the yield of the target product, is achieved by protection of the amino group of glutamic acid is performed by the interaction of glutamic acid with an excess of carbobenzoxy in an alkaline environment. Activation-carboxyl group of glutamic acid is performed by the interaction of benzyloxycarbonyl-L-glutamic acid with an excess of polyformaldehyde with 3-(N-benzyloxycarbonyl-oxazolidin-5 - one-4-yl)propionic acid, and the product of hydrogenation in spirtovodnogo the solution is treated with an equivalent amount of acid or near knogo metal use bicarbonates of sodium and potassium, and as the alkaline earth metal hydroxides of magnesium and calcium. 4 C.p. f-crystals.

The present invention relates to organic chemistry, and in particular to methods for producing peptides, in particular L-glutamyl-L-tryptophan and its salts, which can find application in the pharmaceutical industry. L-glutamyl-L-tryptophan, known

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also called "Timogen" (Temporary Pharmacopeia. VFS N 42-1981-90), is one of the regulatory peptides and finds application, for example, as an immunostimulating agent for the treatment of patients with immunodeficiency (RF patent N 1582393, class A 61 K 37/02). Unlike other peptides isolated from thymus (thymalin, timopoetinov I and II, and others), timogen can be synthesized by chemical means.

The basis for the synthesis of L-glutamyl-L-tryptophan are glutamic acid

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and tryptophan

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Synthesis of L-glutamyl-L-tryptophan by conventional methods of peptide chemistry, multistage and must include protection phase-carboxyl and a-amino group of glutamic acid, the production of activated ester with a carboxyl group of glutamic acid, which interacts tryptophan, and the release of the protective groups and video, V. Doi, M. Inoue, T. Ishida // Biochem. Biophys. Res. Commun. 1988. Vol. 154, N 1. P. 199-204: H. Iyo, H. Hitoshi, J. Isami, I. Kafuku, M. Doi, M. Inoue, T. Ishida // Chem. Pharm. Bull. 1991. Vol. 39, N 1. P. 2483-2486: I. D. Riabinina, I. C. Miroshnichenko, A. A. Yarylo. Immunology. 1991. N 6. S. 20-23].

Also known methodology for individual stages. So, to protect the amino group of glutamic acid take it-benzyl ether, which is obtained by the esterification of glutamic acid, benzyl alcohol, followed by hydrolysis of dimensional ether in the presence of copper salts [R. L. Prestidge, D. R. K. Hardino, J. E. Battersby, W. S. Hancock // J. Org. Chem. 1975. Vol. 40, N 22. P. 3287-3288], or benzylalkonium salt of the copper complex glutamic acid benzylchloride [R. Ledger, F. H. S. Stewart // Austral. J. Chem. 1965. Vol. 18 N 9. P. 1477-1484; W. A. R. van Heesvijk, M. J. D. Eenink, J. Feijen // Synth. 1982. N 9. P. 744-747].

Conventional methods of peptide chemistry, expensive, multi-stage and give a low yield of the target product.

L-glutamyl-L-tryptophan very little is soluble in water and isotonic sodium chloride (to dissolve the suspension is treated with ultrasound for 2 min) and very soluble in 0.01 M sodium hydroxide solution (VFS 42-1981-90). Sonication leads to the partial destruction of the molecule L-glutamyl-L-tryptophan, and 0.01 M sodium hydroxide solution has Reparata.

The closest to the essential features of the claimed method is a method of obtaining L-glutamyl-L-tryptophan and its monosodium salt, which includes stages: (1) protection of the amino group of glutamic acid, (2) activation-carboxyl group of glutamic acid by formation of a complex of activated ester, (3) the complex interaction of the activated ester with tryptophan, (4) removal of the protective group from the product obtained in stage (3), by hydrogenation in the presence of a palladium catalyst, substance-donor hydrogen and sodium bicarbonate, (5) the selection of the target product [A. P. Erugin, S. C. Kulikov. J. of General chemistry. 1995. So 65, Vol. 10. C. 1735-1738].

According to the prototype stage of protection of the amino group of glutamic acid is used glutamic acid with a protected carboxyl group, obtained from the potassium salt diglutamate copper interaction with benzylbromide in the presence of triethylamine; -benzyl ester of glutamic acid interacts with O-benzyloxycarbonyl-N-hydroxysuccinimide in the presence of triethylamine and network-benzyl ester N-benzylamino acid with a protected amino group. The compound obtained vzaimodeistvie with N-hydroxysuccinimide oxycarbonyl-- benzylglycine-tryptophan, obtained from the previous stage is dissolved in alcohol and hydronaut on palladium catalyst in spirtovodnogo mixture in the presence of sodium bicarbonate and ammonium formate (hydrogen donor). The resulting release of L-glutamine-L-tryptophan is isolated in the form of a monosodium salt. To obtain L-glutamine-L-tryptophan solution of its monosodium salt is acidified, for example, hydrochloric acid to pH 3.5. L-glutamine-L-tryptophan, poorly soluble in water, is precipitated in the form of sediment.

The known method has a number of disadvantages. The multistage method; the reaction of obtaining the source-benzyl ester of glutamic acid is output not exceeding 45% by weight; at the stage of obtaining the substance protected a-amino group of flows adverse reaction, the product which is not suitable for further transformations and should be separated. All this leads to a low yield of L-glutamine-L-tryptophan or its monosodium salt, based on the source of glutamic acid. A multi-stage, low intermediate output, adverse reaction to one of the stages, as well as the high cost of reagents used, especially imides lead to very high cost L-glutamine-L-tryptophan and its monosodium salt, obtained singing and cheaper process for L-glutamine-L-tryptophan, and in achieving higher yield of the target product in the calculation of the original glutamic acid.

This technical result is achieved in that in a method of producing L-glutamine-L-tryptophan and its salts, including the stage of protection of the amino group of glutamic acid activation-carboxyl group of glutamic acid by formation of a complex of the activated ester with tryptophan, removal of protective groups by the hydrogenation product of the interaction of tryptophan with a palladium catalyst and a hydrogen donor and selection of the target product, protection of the amino group of glutamic acid is carried interaction directly glutamic acid with an excess of carboxymethylated in an alkaline environment. Activation-carboxyl group of glutamic acid is performed by the interaction obtained in the previous phase of benzyloxycarbonyl-L-glutamic acid and excess polyformaldehyde with 3-(N-benzyloxycarbonyl-oxazolidin-5-one-4-yl)propionic acid, the hydrogenation product in spirtovodnogo the solution is treated with an equivalent amount of acid or inorganic compounds of alkali or alkaline earth metal.

As connected to the potassium in the form of potassium bicarbonate, compounds of magnesium as magnesium hydroxide or calcium in the form of calcium hydroxide and other Acid or inorganic compound take in the equivalent amount in order to get one-deputizing salt.

According to the claimed method glutamic acid reacts with excess carboxymethylated in an alkaline environment, giving N-benzyl-oxycarbonyl-L-glutamic acid (protected-amino group). N-benzyl-oxycarbonyl-L-glutamic acid reacts with polyformaldehyde in benzene in the presence of, for example, paratoluenesulfonyl, turning into 3-(N-benzyloxycarbonyl-oxazolidin-5-one-4-yl)propionic acid. The latter condenses with tryptophan in the presence of triethylamine or sodium acetate or potassium carbonate and tetrabutylammonium bromide. As a result of condensation is obtained N-benzyloxycarbonyl-N-hydroxymethyl-L-glutamyl-L-tryptophan. The last release is performed by treatment with ammonium formate (or formate, methylamine or dimethylamine) in spirtovodnogo mixture in the presence of palladium catalyst (5-10% Pd on coal or palladium mobile). Product release in spirtovodnogo mixture is treated with an equivalent amount of acid or sodium bicarbonate, bikar the L-glutamyl-L-tryptophan General formula:

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where

Me = Na, K, Mg, Ca, etc.

Magnesium and calcium coordinate two anion L-glutamyl-L-tryptophan.

The method is illustrated by examples.

Example 1 (control prototype).

a) Receiving-benzyl ester of glutamic acid.

of 58.8 g (400 mM) glutamic acid are dissolved in 1500 ml of water. The solution is heated to 75 - 80oC and with stirring add 36 ml of acetic acid, and then in small portions to 88.4 g (400 mM) basic carbonate of copper. Suspension visiteuses diglutamate copper intensively stirred until cooling and leave for two days. The precipitate is filtered, washed with water and dried in a vacuum desiccator over pjatiokisi phosphorus.

59,7 g (406 mM) glutamic acid are dissolved in 300 ml of water and cooling to the solution was added with 44.8 g (800 mM) KOH. To the resulting solution was added 300 ml of dimethylformamide and 98 g (200 mM) obtained diglutamate copper and stirred until dissolution. To the resulting mixture of 11.2 ml (80 mM) of triethylamine, then 105 ml (882 mM) benzylbromide, stirred for 5 hours at a temperature of 18 - 20oC and leave for 5 days. The precipitation is filtered and washed. Output-benzyl ester of glutamic acid - 85,5 g (45% of whom are in 432 ml of dioxane, add and 47.5 g (339 mM) of triethylamine and of 85.4 g (343 mM) o-benzylcarbamoyl-N-hydroxysuccinimide. The mixture is left under stirring at a temperature of 18-20oC at night. After that, the dioxane is distilled off in vacuum. The residue is dissolved in ethyl acetate; -benzyl ester N-benzyloxycarbonylamino acid and extracted from an ethyl acetate solution with water and twice with saturated solution of NaHCO3. The aqueous layer is acidified with sulfuric acid to pH 2.0 and the product extracted with ethyl acetate. The organic layer was washed with a saturated solution of sodium chloride, dried with magnesium sulfate and evaporated. The output 114 g (90,6%).

C) Activation-carboxyl group of glutamic acid.

of 27.0 g (264 mM) -benzyl ester N-benzyloxycarbonylamino acid dissolved in 267 ml of dry dimethylformamide, add 30,4 g (264 mM) of N-hydroxysuccinimide, cooled with ice and added dropwise with vigorous stirring a solution of 54.3 g (264 mM) dicyclohexylcarbodiimide in 200 ml of dimethylformamide. The mixture is stirred for one hour at a temperature of 0oC and left under stirring at room temperature for 16 hours. Spin-off dicyclohexylphosphino separated by filtration. Activated N-hydroxysuccinimidyl ether N-gasoline the P> g) the Condensation of tryptophan.

To a solution of N-hydroxysuccinimide ether N-benzyloxycarbonyl-benzylguanine acid in dimethylformamide obtained in stage (b), add the 64.1 g (314 mM) tryptophan and 44.3 ml (316 mM) of triethylamine. The mixture is stirred for 16 hours at a temperature of 18-20oC. the Reaction mass is filtered, diluted with 2.5 l of water and N-benzyloxycarbonyl--benzylglycine-tryptophan extracted 2 times with 700 ml of ethyl acetate. The organic solution is washed with water, 2 H a solution of sulfuric acid and a saturated solution of sodium chloride, then dried with sodium sulfate and distilled off the ethyl acetate under vacuum. Output - 132,5 g (93%) and N-benzyloxycarbonyl-benzylpenicillin.

d) the release and allocation of monosodium salt of L-glutamyl-L-tryptophan.

135,5 g (251 mM) and N-benzyloxycarbonyl-- benzylpiperidine dissolve when heated in 450 ml of ethanol and cooled to room temperature. To the solution add 250 ml of water and with vigorous stirring, 21.1 g (251 mM) of sodium bicarbonate or 39.6 g (629 mM) of ammonium formate. Then slowly add the slurry 22% palladium on coal in 200 ml of water. The mixture is stirred for 5 hours, the catalyst is separated by filtration, sleep is diluted with 1 l of acetone, mix and leave for 16 hours at a temperature of 0-5oC. the Product is filtered, washed with acetone, dried, recrystallized from a mixture of water with ethanol and receive of 83.6 g (97%) of the monosodium salt of L-glutamyl-L-tryptophan.

When researching product identify:

- ultraviolet range of 0.01% solution in water in the region of 250 nm;

- range proton nuclear resonance of 0.05 mol/l solution in deuterated phosphate buffer;

- transparency on GF XI, vol. 1, S. 198;

- the color on GF XI, vol. 1, S. 194;

- solubility in GF XI, vol. 1, S. 175.

Output salts of L-glutamyl-L-tryptophan in the calculation of the original glutamic acid is 36%.

Example 2 (the proposed method).

a) Protection of the amino group.

44 g (299 mM) glutamic acid are suspended in a solution of 50 g (595 mM) NaHCO3in 230 ml of water with vigorous stirring; the suspension is cooled with ice and add 2 H NaOH solution to pH 9.

Get solution of sodium glutamate, which at a temperature of 5oC for 1.5 hour added dropwise 48 ml (338 mM) carboxymethylated, the pH of the reaction mixture support equal to 9 by addition of 2 H NaOH solution. After adding stirred for 2 hours at room temperature is about pH 2, fallen benzyloxycarbonyl-L-glutamic acid is separated by filtration, washed with water and dried. Yield 78 g (93%).

b) Activation-carboxyl group of glutamic acid.

of 28.1 g (100 mM) benzyloxycarbonyl-L-glutamic acid, 6 g (200 mM) polyformaldehyde ("Paraform" company SERVA, Germany) and 1.2 g (6 mM) of para-toluenesulfonic acid are dissolved in 400 ml of benzene and boiled for 1 hour with a nozzle Dean-stark. Add 100 ml of ethyl acetate, washed with 0.3 M aqueous solution of potassium carbonate and three times with water, dried with magnesium sulfate, evaporated and dried in a vacuum desiccator over KOH/P2O5. Received 27.8 g (95%) of 3-(N-benzyloxycarbonyl-oxazolidin-5-one-4-yl)propionic acid.

Individual according to thin-layer chromatography in the system chloroform-methanol-acetic acid (17:2:1).

C) Condensation of tryptophan.

27.8 g (95 mM) 3-(N-benzyloxycarbonyl-oxazolidin-5-one-4-yl)propionic acid are dissolved in 50 ml of dimethylformamide, add to 20.9 g (102/5 mM) tryptophan and 28.6 ml (205 mM) of triethylamine. The mixture is stirred at a temperature of 18-20oC, then filtered, diluted with 500 ml of water, washed with ether, the aqueous layer separated, acidified, layered sodium chloride and the product extracted with hatom sodium and evaporated under vacuum. The resulting oily product is dried under vacuum. Received a 43.4 g (92%) of N-benzyloxycarbonyl-N-hydroxymethyl-L-glutamyl-L-tryptophan.

Individual according to thin-layer chromatography in the system chloroform-methanol-25% ammonia-water (15:10:2:3) and chloroform-methanol-acetic acid (17:2:1).

g) the release and allocation of monosodium salt of L-glutamyl-L-tryptophan.

to 43.4 g (85,5 mM) of N-benzyloxy-carbonyl-N-hydroxymethyl-L-glutamyl-L-tryptophan dissolved in 150 ml of isopropyl alcohol. To the resulting solution was added a solution of 13.5 g (214 mM) of ammonium formate in 85 ml of water, then a suspension of 10% palladium on coal in 68 ml of water. The mixture is stirred at a temperature of 18-20oC for 5 hours, after which the catalyst is separated by filtration. To the filtrate is added 7.2 g (85,5 mM) NaHCO3stir to dissolve and the solution is distilled isopropanol. The aqueous solution is filtered through 20 g of silica gel and evaporated to crystallization mass. The crystals are mixed with 340 ml of acetone, incubated for 16 h at a temperature of 4oC and the product is separated by filtration. The product is dissolved in 100 ml of water, adjusted the pH to 6.5 and filtered through 200 g of alumina (pH sorbent to 9.5). The filtrate is evaporated and paracrystalline-L-tryptophan.

The product is examined as in example 1. Yield per source glutamic acid is 80%.

Example 3.

The synthesis are as in example 2, but at the stage of release after hydrogenation and separation of the catalyst by filtration the filtrate add to 85.5 ml of 1 M HCl solution (85,5 mM). The precipitated L-glutamyl-L-tryptophan is separated, washed on the filter and dried.

Example 4.

The synthesis are as in example 2, but at the stage of release, after hydrogenation and separation of the catalyst by filtration, to the filtrate is added 8.6 g (85,5 mM) KHCO3. Next, as in example 2.

Example 5.

The synthesis are as in example 2, but at the stage of release, after hydrogenation and separation of the catalyst by filtration, to the filtrate is added 2.5 g (42,75 mM) Mg(OH)2. Next, as in example 2.

Example 6.

The synthesis are as in example 2, but at the stage of release, after hydrogenation and separation of the catalyst by filtration, to the filtrate is added 3.2 g (42,75 mM) Ca(OH)2. Next, as in example 2.

As seen from the above examples, the inventive method for L-glutamyl-L-tryptophan and its monisola simpler, does not contain any stage with low output of the product. In the synthesis does not use costly imides, which, together with the reduction and simplification of the process and increase the yield of the product leads to a reduction in the cost of product in 3-5 times.

1. The method of obtaining L-glutamyl-L-tryptophan and its salts, comprising the successive stages of protection of the amino group of glutamic acid activation-carboxyl group of glutamic acid by formation of a complex of activated ester, complex interaction of the activated ester with tryptophan, removal of protective groups by the hydrogenation product of interaction with tryptophan in the presence of a palladium catalyst and a hydrogen donor and selection of the target product, characterized in that the protection of the amino group of glutamic acid is performed by the interaction of glutamic acid with an excess of carbobenzoxy in an alkaline medium, activation-carboxyl group of glutamic acid is performed by the interaction obtained in the previous phase of menthoxycarbonyl-L - glutamic acid with an excess of polyformaldehyde with 3- (N-benzyloxycarbonyloxy-5-one-4-yl)propionic acid, and the product of hydrogenation in spirtovodnogo the solution is treated with an equivalent amount of acid or n is fact, as the inorganic compounds of the alkali metal used sodium bicarbonate.

3. The method according to p. 1, characterized in that the inorganic compound of an alkali metal use bicarbonate of potassium.

4. The method according to p. 1, wherein as the inorganic compounds of the alkaline earth metal is used magnesium hydroxide.

5. The method according to p. 1, wherein as the inorganic compounds of the alkaline earth metal is used calcium hydroxide.

 

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