Process for the production of glutamine derivatives

Abstract: The invention relates to a process for producing glutamine derivatives through formation of a peptide bond between the C-terminal of an amino acid or a peptide and the N-terminal of glutamine in accordance with the active esterification process, characterised by that said formation of a peptide bond is carried out in the presence of a weak base, and said glutamine is unprotected.

Description: The present invention relates to the synthesis of glutamine-containing peptides. More particularly, it relates to a process for the synthesis of glutaminecontaining peptides which comprises reacting an N-protected amino acid or peptide with N-hydroxysuccinimide or the like to form an active ester by the use of a condensing agent, such as cyclohexylcarbodiimide, and subjecting the ester to a reaction with unprotected glutamine to form a peptide bond, followed by eliminating the protective group therefrom. Glutamine is an indispensable component of culture media or infusions, but it is very difficult to use glutamine because of its low thermal stability. Glutamine-containing dipeptides exhibit a good thermal stability and can be handled, even at temperature conditions of sterilization. Glutamine-containing dipeptides are highly useful as ingredients for serum-free media. They have also been employed as ingredients for infusions. Their practical importance would therefore be apparent. It is known that in the prior processes for producing glutamine-containing peptides, various side reactions take placed due to the instability of the β-amido group contained therein, thus making their synthesis quite difficult (see, e.g., J. Rudinger, "Angew. Chem.", 71 , 742 (1959)). Other known literature concerning the synthesis of glutamine containing peptides is given hereinbelow. (1) Y. Shimonishi, S. Sakakibara and S. Akabori, Bull. Chem. Soc. Jap., 35 , 1966-1970. (2) Y. Shimonishi, Bull. Chem. Soc. Jap., 37 , 200-203. (3) S. Akabori, S. Sakakibara and Y. Shimonishi, Bull. Chem. Soc. Jap., 34 , 739. However, processes described in the above literature involve significant difficulties if they are to be used as a commercial process. In addition, final products obtained by the processes are contaminated with impurities which could hardly be removed. Furthermore, the processes give only poor yields. For example, when applied to the synthesis of alanylglutamine, the process described in (1) above gives a yield of only ca 5%, and the process described in (3) above provides a yield of at best ca. 28%. The known processes are therefore by no means satisfactory from the commercial point of view. It was an object of the invention to develop a commercially advantageous process for producing glutamine-containing derivatives. The present inventors have found that highly pure glutamine-containing derivatives can be obtained in high yields through formation of a peptide bond between the C-terminal of an amino acid or a peptide and the N-terminal of glutamine in accordance with the active esterification process, if the formation of a peptide bond is carried out in the presence of a weak base, and if unprotected glutamine is used thereby. It is preferable to use an N-protected amino acid or peptide. Any of conventional protective groups, including carbobenzoxy, t-butyloxycarbonyl and benzyl groups, can be useful for the protection. It can however be preferable to utilize carbobenzoxy groups because of easiness of crystallization. As a weak base, there can be used carbonates (e.g., sodium carbonate), hydrogencarbonates (e.g., sodium hydrogencarbonate), or the like. Amino acids to be used in the process of the present invention can be D- or L-form, or a racemate. In the case where, e.g., Gly-Gln is to be produced according to the process of the present invention, Z-Gly is allowed to react with N-hydroxybenztriazole or N-hydroxysuccinimide and with a carbodiimide (preferably dicyclohexylcarbodiimide) in a water-miscible solvent, such as dioxane, tetrahydrofuran, acetone, acetonitrile and dimethylformamide (preferably in dioxane or tetrahydrofuran), to form an active ester, and then the ester is allowed to react with unprotected L-glutamine to give Z-Gly-L-Gln. The above reactions are usually carried out at a temperature of from 30 to 20 °C for a period of 1 to 30 hours, preferably from 5 to 10 hours. The thus obtained Z-Gly-L-Gln is subjected to a treatment for eliminating the protective group, for example, by hydrogen, using a palladium-carbon catalyst in a mixture of water and an alcohol, preferably methanol or ethanol. This elimination reaction is usually carried out at a temperature of from 10 to 40 °C, preferably from 15 to 30 °C, for a period of 1 to 3 hours. The elimination of protective groups utilizing hydrogen and palladium-carbon catalyst is almost free from side reactions and therefore can be an excellent technique in the production of glutamine-containing peptides. The progress of the reaction can be followed by thin layer chromatography. After the termination of the reaction has been confirmed, the catalyst is removed by filtration, and then an alcohol (preferably isopropanol or ethanol) is added to the reaction mixture so as to allow the desired product to crystallize in a highly pure state. In the case of alanylglutamine, for example, it can be produced in an yield of 58% when synthesized according to the process of the invention, whereas its yield can be at best 30% in the prior processes. Accordingly, the process of the present invention can be advantageous over the prior processes not only in purity but also in yield. Abbreviations used in this specification are as follows: Ala: Alanine; Gln: Glutamine; Gly: Glycine; and Z: Protective group, in particular carbobenzoxy group. The present invention will be illustrated in further detail by way of examples. Example 1 Synthesis of L-Alanyl-L-Glutamine (a) N-carbobenzoxy-L-alanyl-L-glutamine (Z-L-Ala-L-Gln) Into a 300 ml round bottom flask were charged 10 g (44.8 mmol) of Z-L-Ala and 5.2 g (45.2 mmol) of N-hydroxysuccinimide, and the contents were dissolved in 100 ml of dioxane. Then, a solution of 9.2 g (44.7 mmol) of dicyclohexylcarbodiimide in 40 ml of dioxane was added dropwise, during which the temperature of the reaction mixture was maintained at 25 to 20 °C. After the reaction had been allowed to proceed for 5 hours, the precipitated dicyclohexylurea was filtered off. On the other hand, 7.2 g (50 mmol) of L-Gln was dissolved in 70 ml of aqueous 10% sodium hydrogencarbonate solution, and the resulting solution was cooled to 15 to 10 °C. The dioxane solution prepared above was then added dropwise to the cooled solution, and the reaction was allowed to proceed for 5 hours. After the completion of the reaction, the pH of the reaction mixture was adjusted to 6.8 with 1N HCl, and the dioxane was distilled off. After insoluble substances were filtered off, its pH was adjusted to 3 to 3.5 with 1N HCl. Crystals precipitated within 1 to 2 minutes, and the crystals were collected by filtration, washed with water and dried under reduced pressure to give 7 g (20 mmol) of Z-L-Ala-L-Gln. The pH of the above mother liquor was adjusted to 2.0 with 1N HCl, so as to precipitate additional 5 g of Z-L-Ala-L-Gln. (b) L-alanyl-L-glutamine (L-Ala-L-Gln) Into a 200 ml three-necked flask was charged 5.0 g (14.2 mmol) of Z-L-Ala-L-Gln, and the peptide was dissolved in 100 ml of a mixture of methanol and water (methanol: water = 7:3) and stirred with a magnetic stirrer. After the air in the flask had been replaced with nitrogen, 0.8 g of 2% by weight of palladium-carbon was added thereto, and the protective group was eliminated in a stream of hydrogen. After 2 hours, the completion of the reaction was confirmed by thin layer chromatography, and then the palladium-carbon was filtered off. The filtrate was concentrated to 20 ml under reduced pressure, and 40 ml of isopropyl alcohol was added thereto. The resulting mixture was stirred for 30 minutes, during which crystals precipitated. The crystals were collected by filtration, washed with isopropyl alcohol and then dried under reduced pressure to give 2.8 g (12.9 mmol) (90%) of L-Ala-L-Gln. m.p.: 205 - 207 °C (decomp.) [α] 20 / D : +10.5° (C=2.0, H₂O) 3400, 3350, 1640, 1630, 1600, 1520, 1380, 1150 Rf: 0.20 (silica gel, phenol:water = 3:1) Example 2 Synthesis of Glycyl-L-Glutamine (a) N-carbobenzoxy-glycyl-L-glutamine (Z-Gly-L-Gln) Into a 300 ml round bottom flask were charged 10 g (47.8 mmol) of Z-Gly and 5.5 g (47.8 mmol) of N-hydroxysuccinimide, and the contents were dissolved in 100 ml of dioxane. Then, a solution of 9.9 g (48.0 mmol) of dicyclohexylcarbodiimide in 40 ml of dioxane was added thereto, during which the temperature of the reaction mixture was maintained at 25 to 20 °C. After 4 hours, the precipitated dicyclohexylurea was filtered off. On the other hand, 7.2 g (50 mmol) L-Gln was dissolved in 70 ml of aqueous 10% sodium hydrogencarbonate solution, and the resulting solution was cooled to 15 °C. The dioxane solution prepared above was then added dropwise to the cooled solution. After the completion of the reaction, the pH of the reaction mixture was adjusted to 7.0 with 1N HCl, and the dioxane was distilled off. After insoluble substances had been filtered off, the filtrate was adjusted to a pH of 2.0 with 1N HCl and then allowed to stand overnight in a refrigerator. Precipitated crystals were collected by filtration, washed with water and then dried under reduced pressure to give 13.2 g (39.2 mmol) of Z-Gly-L-Gln. (b) Glycyl-L-Glutamine (Gly-L-Gln) Into a 200 ml of three-necked flask was charged 10 g (29.7 mmol) of Z-Gly-L-Gln, and 100 ml of 70% aqueous methanol was added thereto with stirring by a mechanical stirrer. After the air in the flask had been replaced with nitrogen, 1.5 g of 2% by weight palladium-carbon was added thereto, and the protective group was eliminated in a stream of hydrogen. It was in the state of slurry in the initial stage of the reaction, but the Z-Gly-L-Gln dissolved with the progress of the reaction. After 3 hours, the completion of the reaction was confirmed by thin layer chromatography, and then the palladium-carbon was filtered off. After the filtrate had been concentrated to 30 ml, 40 ml of methanol was added thereto, and the resulting solution was allowed to stand overnight at 5 °C, so as to allow crystals to precipitate. The crystals formed were collected by filtration, washed with a small quantity of cold methanol and then dried under reduced pressure to give 4.9 g (22.2 mmol) (75%) of Gly-L-Gln·H₂O. m.p.: 198 °C (decomp.) [α] 20 / D : -1.6° (C=4.0, H₂O) 3520, 3400, 3250, 3100, 1690, 1570, 1410, 1340, 1280 Rf: 0.13 (silica gel, phenol:water = 3:1)

Claims: Process for producing glutamine derivatives through formation of a peptide bond between the C-terminal of an amino acid or a peptide and the N-terminal of glutamine in accordance with the active esterification process, characterised by that said formation of a peptide bond is carried out in the presence of a weak base, and said glutamine is unprotected. A process as defined in claim 1, wherein said amino acid is glycine or alanine. A process as defined in any of claims 1 or 2, wherein the N-terminal of said amino acid or peptide is protected by a protective group. A process as defined in claim 3, wherein said protective group is a carbobenzoxy group. A process as defined in any of claims 1 to 4, wherein said weak base is a carbonate or a hydrogen carbonate. A process as defined in any of claims 1 to 5, wherein an amino acid or peptide protected at the N-terminal by a protective group is reacted with a carbodiimide in a water miscible solvent to form an active ester, the ester is reacted with the unprotected glutamine, the resulting peptide is treated to eliminate the protective group, and the peptide is removed from the reaction medium and isolated in a pure state.

Inventors: KATOH TOSHIHISA, KURAUCHI MASAHIKO, KATOH, TOSHIHISA, KURAUCHI, MASAHIKO
Applicants: AJINOMOTO KK, AJINOMOTO CO., LTD.
Application year: 1988
Main classification: C07K1
IPC codes: C07K5, C07K1