JP4540568B2 - Method for producing L-carnosine - Google Patents

Description

  The present invention relates to a method for producing high-purity L-carnosine by purifying a crude L-carnosine containing impurities.

  L-carnosine is a dipeptide synthesized from β-alanine and L-histidine, and has a tissue repair promoting action, an immunomodulating action, and an anti-inflammatory action, so that demand for pharmaceuticals, health foods and the like is increasing. . In addition, since it easily forms a chelate bond with a metal, it has been applied to antiulcer drugs such as polaprezinc complexed with zinc and therapeutic agents for taste disorders. The L-carnosine used in these pharmaceuticals must have a high purity, and generally a purity of 99.5% or more is required.

  As described above, L-carnosine is synthesized using β-alanine or a derivative thereof and L-histidine or a derivative thereof as raw materials, but crystallization is generally employed for purification of the crude L-carnosine obtained by the reaction. Has been. (See Non-Patent Documents 1, 2, and 3.)

Chemical and Pharmaceutical Bulletin 38 (11) p3140-3146 (1990) Journal of Organic Chemistry 48p393-395 (1983) Journal of the American Chemical Society (75) p2511-2512 (1953)

  In general, impurities contained in the crude L-carnosine are very similar to L-carnosine in terms of polarity and are difficult to purify. For this reason, there was a problem that the yield would be low if purification to a sufficient purity was performed. For example, in Non-Patent Document 3, recrystallization is performed with a mixed solution of water and ethanol to precipitate purified L-carnosine crystals, but the yield at this time is as low as 65%.

  Accordingly, an object of the present invention is to provide a method for efficiently producing high-purity L-carnosine.

  The present invention solves the above-described problem, and is a method for producing L-carnosine including a purification step of purifying a crude L-carnosine containing impurities, wherein the purification step includes an aqueous solution of the crude body. A ripening step of adding a seed crystal of L-carnosine to a mixed solution obtained by mixing alcohol and ripening at 30 to 80 ° C. for 1 to 10 hours; and the mixed solution aged in the step A crystal precipitation step of adding an alcohol to precipitate highly purified L-carnosine crystals.

  According to the production method of the present invention, L-carnosine can be obtained with high purity and high yield.

  The production method of the present invention includes a purification step of purifying a crude L-carnosine containing impurities. As a crude material used as a raw material for the purification step, a conventional production method, that is, a crude product synthesized using β-alanine or a derivative thereof and L-histidine or a derivative thereof as raw materials can be used without particular limitation. However, the yield from histidine is very high, for example, 90%, and the purity of the crude product itself (where purity refers to the peak area in the chart obtained when performing high performance liquid chromatography (HPLC) analysis). Phthaloyl-L-carnosine obtained by reacting an acid chloride of phthaloyl-β-alanine with L-histidine protected with a trimethylsilyl group is high. It is preferable to use a crude product obtained by a method of deprotecting with hydrazine. This method is described in, for example, Dokladina na Bulgarska Akademiya na Naukite 44 (8) p53-56 (1991), the outline of which will be described below.

  The phthaloyl-β-alanine used in the above-mentioned crude production method may be a reagent or an industrial raw material, but is described in Journal of the American Chemical Society (75) p2388-2390 (1953). It is also possible to obtain it by reacting phthalic anhydride and β-alanine at high temperature.

  Acid chloride of phthaloyl-β-alanine can be obtained by reacting with oxalyl dichloride or thionyl chloride. In this case, 1 to 30 equivalents of oxalyl dichloride or thionyl chloride may be added dropwise to phthaloyl-β-alanine in a solvent such as toluene or dichloromethane, and the reaction may be performed under reflux of the solvent, or 20 to 20 in the absence of solvent. You may react at 100 degreeC. After the reaction, residual oxalyl dichloride and the like are distilled off to obtain phthaloyl-β-alanine acid chloride.

  Further, L-histidine protected with a trimethylsilyl group used in the above method is synthesized as follows. That is, the reaction was carried out under reflux in 3 to 10 equivalents of 1,1,1,3,3,3-hexamethyldisilazane under a sulfuric acid catalyst. After pH adjustment, a solvent such as xylene was added, and the solvent was distilled off. L-histidine protected with a trimethylsilyl group is obtained by recrystallization from a halogenated solvent such as dichloromethane or chloroform.

  In the crude production method, the acid chloride of phthaloyl-β-alanine obtained as described above and L-histidine protected with a trimethylsilyl group were mixed in a halogenated solvent such as dichloromethane or chloroform, To give phthaloyl-L-carnosine hydrochloride. The obtained phthaloyl-L-carnosine hydrochloride is neutralized with an alkali such as lithium hydroxide monohydrate in alcohol to obtain phthaloyl-L-carnosine, which is hydrazine hydrate in water. The reaction solution containing a crude product of L-carnosine can be obtained by deprotection by pH and adjusting pH after acetic acid treatment.

  The reaction solution (aqueous solution) containing the crude product thus obtained can be used for the purification step as it is or by adjusting the concentration by adding water, but in order to remove a large amount of by-products. It is preferable to isolate it once before subjecting it to the purification step. For example, the crude reaction solution (aqueous solution) obtained by the above-described method contains many by-products derived from hydrazine such as phthalhydrazide. After distilling off water from the reaction solution, the residue is removed with alcohol. By reprecipitation, a crude product of L-carnosine from which these by-products derived from hydrazine have been removed can be obtained. The crude body thus isolated usually has a purity of about 95 to 98%.

  In the production method of the present invention, the purity of the crude product is preferably as high as possible before performing the purification step by crystallization. In addition, since the crude product obtained by the reaction as described above may contain coloring components that are difficult to remove by crystallization, such coloring components are removed by activated carbon treatment before purification by crystallization. It is preferable to do this. The activated carbon treatment can be performed by removing the activated carbon after bringing the crude aqueous solution of L carnosine into contact with the activated carbon.

  At this time, activated carbon that can be used as a reagent or industrially can be used without particular limitation. The following can be mentioned if the activated carbon which can be used conveniently is illustrated concretely.

  Japan Norit PK, PKDA MESY / MRX, ELORIT, AZ0, DARCO, HYDRODARCO 3000/4000, DARCO 12X20LI, DARCO12X20DC, PETRODACO, DARCO MRX, GAC, GAC PLUS, GARCU VACURE, GARCU , CA, CN, CG, DARCO KB / KBB, S-51, S-51-HF, S-51-FF, PREMIUM darco, DARCO GFP, HDC / HDR / HDH, GRO SAFE, FM-1, DARCO TRS, Powdered activated carbons such as DARCO FGD, SX, SX ULTRA, SA, D-10, PN, ZN, SA-SW, W, GL, HB PLUS, ROW, RO, ROX, RB, R, R. Molded activated carbon and impregnated activated carbon such as EXTRA, SORBONORIT, GF 40/50, CNR, ROZ, RBAA, RBHG, RZN, RGM, etc., PICA granular activated carbon, spherical activated carbon, powdered activated carbon, Nippon Enviro Chemical's Molcybon , WHA, granular white birch (X2M, GM2X, GH2X, GHXUG, GS1X, GS3X, GTX, GTSX, G2X, GS2X, GAAX, MAC-W, GOC, GOX, GOX, APRC, TAC, MAC, XRC, NCC, SRCX) Functional activated carbon such as granular white birch (G2C, C2C, WH2C, W2C, WH5C, W5C, LGK-400, LGK-100, LH2C, KL, G2X, GH2X, WH2X, S2X, C2X, X7000H, X7100H, X700H- 3, X710 Granular activated carbons such as H-3, LGK-700, DX7-3), X-7000, X-7100, X-7000-3, X-7100-3, white birch (C, M, A, P, PHC) , FAC-10), carborafine, strong white birch, refined white birch, refined white birch, special white birch, white birch DO-2, white birch DO-5, white birch DO-11, etc., powdered activated carbon, honeycomb carbon white birch, molded carbon, carbon Activated carbon products such as paper, white birch C-DC, carborafine DC, granular white birch DC, aldenite, and aldenite SP Granular activated carbon such as SG and SGP from Nimura Chemical Industry, TA, TS, TG, TM, etc. Granular activated carbon, S, FC, SA1000, K, A, KA, AC, M, P, IC, IP, CB, GB, GLP, CLP, W, etc. Powdered activated carbon, CG48B, CG48BR, CW1 Crushed activated carbons such as 30B, CW130A, CW130BR, CW130AR, CW480SZ, CW6100SZ, GL130A, GL240A, GM130A, GM240A, GMC.

  Among these, CA, CN, CG, DARCO KB / KBB, S-51, S-51-HF, S-51-FF, PREMIUM DARCO, DARCO GFP, HDC / HDR / HDH, GRO SAFE, FM-1, DARCO TRS, DARCO FGD, SX, SX ULTRA, SA, D-10, PN, ZN, SA-SW, W, GL, HB PLUS, white birch (C, M, A, P, PHC, FAC-10), carbo Powdered activated carbons such as raffin, strong white birch, refined white birch, purified white birch 2, special white birch, white birch DO-2, white birch DO-5, white birch DO-11 are preferred, and white birch (C, M, A, P) in particular. Refined white birch, purified white birch 2, special white birch, CA, CN, CG are preferred.

  The amount of the activated carbon used is suitably 0.01 to 10 times, preferably 0.02 to 5 times the mass of L-carnosine, from the viewpoint of the removal of coloring components and the economical efficiency. is there.

  The contact between the crude aqueous solution and the activated carbon can be performed by a flow method or a batch method. In the distribution method, the crude aqueous solution may be distributed through a column or column packed with activated carbon. In the batch method, activated carbon and a crude aqueous solution may be mixed in a container.

  The activated carbon treatment temperature is preferably as the temperature is lower, but the activated carbon effect is more likely to occur, but L-carnosine may be crystallized, and thus is usually in the range of -5 ° C to 40 ° C, preferably 0 to 30 ° C. If the activated carbon treatment time is too short, a sufficient effect cannot be obtained, and even if it is too long, impurities may increase due to decomposition or the like, and therefore usually 0.1 hours to 10 hours, preferably 0.5 to 8 hours. It is done in the range. Moreover, the separation of the activated carbon after the activated carbon treatment when the batch method is employed can be performed by filtration.

  By performing such activated carbon treatment, a crude L-carnosine body (generally having a purity of 97 to 99%) from which colored components have been removed can be obtained.

  The manufacturing method of this invention refine | purifies the L-carnosine crude body which performed the activated carbon treatment as needed in the refinement | purification process which comprises the process of following (A) and (B).

(A) A maturing step of adding a seed crystal of L-carnosine to a mixed solution obtained by mixing an aqueous solution of an L-carnosine crude material and an alcohol, and aging at 30 to 80 ° C. for 1 to 10 hours, and B) A crystal precipitation step of adding alcohol to the mixed solution aged in the above step to precipitate highly purified L-carnosine crystals.

  In the case where L-carnosine crystals are precipitated by simply adding alcohol to the crude aqueous solution, or when ripening is carried out by adding seed crystals, the aging conditions deviate from the conditions defined in the present invention. It is difficult to obtain L-carnosine having a purity of 5% or more in high yield. Even if the seed crystal is added and ripened, if the crystal is precipitated by cooling as it is, the yield decreases.

  The concentration of L-carnosine in the aqueous solution of the crude L-carnosine used in the step (A) is 5 to 30% by mass based on the total mass of the aqueous solution, because it can be purified with high purity and high yield. It is preferable to set it as 10-20 mass%.

  As the alcohol mixed with the aqueous solution in the step (A), it is preferable to use a linear alcohol having 1 to 4 carbon atoms because it can be purified with high purity. Specific examples of the alcohol that can be suitably used include methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, and tert-butanol.

  The ratio of the mixed solution of water and alcohol when preparing the mixed solution in the step (A) is such that the ratio of water: alcohol is 1: 0.5 to 1:10, particularly 1: It is preferable that the ratio is 1: 1 to 1: 5. It is also possible to adjust the concentration by supplying water separately when mixing the crude aqueous solution and the alcohol. From the viewpoint of impurity removal property, ease of taking out crystals, yield, and yield per batch, the concentration of L-carnosine contained in the mixed solution is 1 to 15% by mass based on the total mass of the solution, particularly It is preferable to set it as 5-10 mass%.

  In preparing the mixed solution, alcohol is preferably added dropwise to the aqueous solution of the L-carnosine crude product. When a crude L-carnosine aqueous solution is dropped into alcohol, crystals may precipitate immediately after the dropping, and high purity may not be achieved. The liquid temperature when the alcohol is dropped is preferably kept at 0 to 100 ° C, particularly 5 to 80 ° C. If the liquid temperature is too low, crystallization is promoted, and crystallization occurs when alcohol is dripped, and L-carnosine having non-uniform purity may be formed. If it is too high, the solvent evaporates, And the ratio of alcohol may change, and the purity may decrease.

  In the step (A), it is necessary to add a seed crystal after preparing the mixed solution, and to age at 30 to 80 ° C. for 1 to 10 hours. By doing so, it becomes possible to obtain L-carnosine of higher purity. If the addition amount of the seed crystal is too small, secondary nuclei are generated, a uniform crystal cannot be obtained, and even if it is too much, it is not economical, and usually 0 based on the mass of L-carnosine. A seed crystal having a mass of 0.001 to 0.5 times, preferably 0.005 to 0.3 times is added. The aging temperature after seed crystal addition may be 30 to 80 ° C., but it is preferably 40 to 70 ° C. because it can be purified with higher purity. Although depending on the temperature, the ratio of water and alcohol, and the amount of solvent, a aging time of about 1 to 10 hours is sufficient.

  In the step (B), alcohol is added to the mixed solution thus aged to precipitate highly purified L-carnosine crystals. If crystals are precipitated without adding alcohol, the yield cannot be increased. As the alcohol to be added at this time, it is preferable to use the same kind of alcohol used in preparing the mixed solution. The amount of the alcohol to be added is 0.5 to 15 times, particularly 1 to 10 times the mass of the L-carnosine contained, from the viewpoints of purity, yield, economy, and yield per batch. It is preferable to do this.

  In the step (B), in order to increase the yield, it is preferable to cool the solution after dropping the alcohol. At this time, the cooling rate is preferably 0.01 ° C./min to 5 ° C./min, particularly 0.05 ° C./min to 2 ° C./min from the viewpoint of purity and production efficiency. The final temperature achieved by cooling is preferably −10 to 30 ° C., particularly preferably 0 to 25 ° C., and high purity L-carnosine is recovered in a high yield by maintaining the final achieved temperature for 1 to 2 hours. Can do.

  The crystals of L-carnosine thus precipitated are isolated by solid-liquid separation by filtration, centrifugation, or the like, and drying by natural drying, air drying, vacuum drying, or the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not restrict | limited at all by these Examples.

Production Example 1
Add 107 g (0.488 mol) of phthaloyl-β-alanine to a 1,000 ml four-necked flask equipped with a stirring blade, thermometer, and gas absorption device, and add 600 ml of oxalyl dichloride in the presence of 10 ml of N, N′-dimethylformamide. In addition, after stirring at room temperature for 2 hours, oxalyl dichloride was distilled off under vacuum to obtain yellow crystals (1). In another 1,000 ml four-necked flask, 62.08 g (0.40 mol) of L-histidine, 256 ml (1.2 mol) of 1,1,1,3,3,3-hexamethyldisilazane, 0.1 ml of concentrated sulfuric acid The mixture was further stirred for 30 minutes under reflux. After cooling, 150 ml of xylene was added, the solvent was distilled off at 75 ° C., and the residue was recrystallized from 80 ml of chloroform (2). 1 was dissolved in 400 ml of chloroform, added dropwise to (2), and stirred at room temperature for 8 hours. After stirring, 400 ml of ethanol was added and stirred for 30 minutes, and then the crystals were filtered to obtain 154 g of phthaloyl-L-carnosine hydrochloride.

  To a 1,000 ml four-necked flask were added 800 ml of methanol, 17.1 g of lithium hydroxide monohydrate and 154 g of phthaloyl-L-carnosine hydrochloride, and the mixture was stirred at room temperature for 30 minutes and stirred for 15 minutes under reflux. After cooling, the crystals were filtered and dried to obtain 138 g of phthaloyl-L-carnosine.

  A 1,000 ml four-necked flask was charged with 138 g of phthaloyl-L-carnosine, 23 ml (0.46 mol) of hydrazine hydrate, and 460 ml of water, and stirred for 1 hour under reflux. Thereafter, 31 ml of acetic acid was added dropwise, stirred for 10 minutes and cooled. The obtained solid was removed by filtration, the pH was adjusted, and then reprecipitated with ethanol. Further, water was added to obtain a yellowish L-carnosine crude aqueous solution (L-carnosine 81.2 g / water 406 g). It was. The HPLC purity of L-carnosine was 97.9%.

Example 1
After cooling the L-carnosine crude aqueous solution obtained in Production Example 1 to a 1,000 ml four-necked flask equipped with a stirring blade and a thermometer at 5 ° C., 8.1 g of activated carbon white lees A manufactured by Nippon Environment Chemical Co., Ltd. was added. Stir for 2 hours. After stirring, the activated carbon was filtered, and the activated carbon was washed with 20 ml of water to obtain a colorless and clear aqueous solution. The HPLC purity of L-carnosine contained in the aqueous solution was 99.0%.

  To a 2,000 ml four-necked flask equipped with a stirring blade and a thermometer, 505 g of a crude aqueous solution of L-carnosine after activated carbon treatment was added, the temperature was raised to 50 ° C., and 730 ml of methanol was added dropwise over 30 minutes. Next, 1.4 g of seed crystals were added to the mixed solution and aged at 50 ° C. for 2 hours. After confirming that the crystals were sufficiently grown, 406 ml of methanol was newly added dropwise over 30 minutes, cooled to 5 ° C. at a speed of 0.4 ° C./min, and kept at 5 ° C. for 1 hour. Thereafter, the precipitated crystals of L-carnosine were filtered and vacuum-dried. As a result, white L-carnosine with 73.6 g (purification yield: 90.7%) and HPLC purity of 99.87% was obtained.

Example 2
The same operation as in Example 1 was performed, except that an L-carnosine aqueous solution obtained by the same method as in Production Example 1 was used, and 8.1 g of CA manufactured by Japan Norit was used for the activated carbon. As a result, white L-carnosine having 73.2 g (purification yield: 90.1%) and HPLC purity of 99.85% was obtained.

Example 3
The same operation as in Example 1 was performed except that an aqueous L-carnosine solution obtained by the same method as in Production Example 1 was used and ethanol was used as the alcohol. As a result, white L-carnosine having 73.2 g (purification yield: 90.2%) and HPLC purity of 99.80% was obtained.

Example 4
The same operation as in Example 1 was performed except that an L-carnosine aqueous solution obtained by the same method as in Production Example 1 was used and isopropanol was used as the alcohol. As a result, white L-carnosine having 73.3 g (purification yield 90.3%) and HPLC purity 99.86% was obtained.

Comparative Example 1
The same operation as in Example 1 was performed except that the L-carnosine aqueous solution obtained by the same method as in Production Example 1 was used and the aging temperature was 20 ° C. As a result, L-carnosine having 73.2 g (purification yield: 90.2%) and HPLC purity of 99.13% was obtained and not very purified.

Comparative Example 2
The same operation as in Example 1 was performed, except that an aqueous L-carnosine solution obtained in the same manner as in Production Example 1 was used, seed crystals were added without aging, and then immediately dropped into methanol to precipitate crystals. It was. As a result, L-carnosine having 73.3 g (purification yield: 90.3%) and HPLC purity of 99.0% was obtained and hardly purified.

Comparative Example 3
The same operation as in Example 1 was performed, except that an aqueous L-carnosine solution obtained by the same method as in Production Example 1 was used, and methanol was not added after aging. As a result, white L-carnosine with 62.8 g (purification yield: 77.3%) and HPLC purity of 99.91% was obtained, and the yield was lowered.

Claims (3)

A method for producing L-carnosine comprising a purification step of purifying a crude L-carnosine containing impurities, wherein the purification step comprises adding an aqueous solution of the crude and an alcohol to a mixed solution obtained by mixing L -Adding a seed crystal of carnosine and aging for 1 to 10 hours at 30 ° C to 80 ° C, and adding alcohol to the mixed solution aged in the above step to precipitate highly purified L-carnosine crystals A crystal precipitation step. The method according to claim 1, further comprising a step of subjecting a crude aqueous solution of L-carnosine containing impurities to an activated carbon treatment as a previous step of the aging step. Obtaining a crude product of impure L- carnosine by deprotecting the protected L- histidine and phthaloyl -L- carnosine obtained by reacting an acid chloride with a trimethylsilyl group of phthaloyl -β- alanine with hydrazine The method according to claim 1, further comprising a step.