JP2005306782A - New method for synthesizing l-carnosine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for synthesizing L-carnosine, by which L-histidine is acylated with unprotected β-alanine chloride to give high-purity L-carnosine in a high yield. <P>SOLUTION: The method for synthesizing L-carnosine by acylation of L-histidine comprises using an acid salt of unprotected β-alanine chloride as an acylation reagent. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel synthesis method of L-carnosine, that is, β-alanyl-L-histidine.

  L-carnosine is a natural dipeptide found in muscles, and has an activation and antioxidant effect on muscle ATPase, and its complex with zinc is commercially available as an anti-ulcer agent. Recently, it has spread in Europe and America as a health food supplement.

  In general, a method for synthesizing a dipeptide comprises acylating the amino group of the C-terminal amino acid with a reactive derivative of the amino terminal-protected N-terminal amino acid, for example, acid chloride, and deprotecting the amino group. Most of the methods for synthesizing L-carnosine reported so far follow this method, and phthalimide, benzyloxycarbonyl and the like are used as protecting groups for β-alanine, and acid chloride, acid azide, Succinimidyl, ethoxycarbonyl and the like are used. Since β-alanine does not have a chiral carbon atom, a method has been reported in which L-histidine is acylated with a reactive derivative of β-nitro or iodopropionic acid to give L-carnosine by reduction or reaction with ammonia. In addition, a method of using β-alanine lactam or thiolactam as an acylating agent for L-histidine has also been reported. Recently, a method for synthesizing L-carnosine by acylating L-histidine with cyanate acetate and catalytically reducing the resulting N-cyanoacetyl-L-histidine was disclosed. Further, when acylating L-histidine in the state of free base, a large amount of by-products are formed and the yield of L-carnosine is lowered. A method has also been reported.

  Prior art L-carnosine synthesis methods include the protection and deprotection of the amino group of β-alanine, conversion to an amino group if a precursor having a group that can be subsequently converted to an amino group, reduction of the cyano group Such an additional process was required, and the process was long and the yield was not satisfactory.

[References]
Baumann, L.M. et al. , J .; Biol. Chem. , 35, 263 (1918);
Barger, G .; et al. Biochem. , 12, 402 (1918);
Losse, G.M. et al. Ber. , 94, 2768 (1961);
Turner, R.A. A. , J .; Am. Chem. Soc. , 75, 2388 (1953);
Shferd, R.A. H. et al. , J .; Biol. Chem. , 108, 733 (1935);
Kroll, H .; et al. , J .; Am. Chem. Soc. 75, 2511 (1953);
Rinder Knecht, H.C. et al. , J .; Org. Chem. 29, 1968 (1964);
RU2030222;
Tetrahedron Lett. , 29, 5859-5862 (1988);
J. et al. Org. Chem. 48, 392-393 (1983);
WO2001 / 064638 (Special table 2004-509834).

  The present inventors conceived a method of synthesizing L-carnosine by reacting an acid chloride of β-alanine and L-histidine without protection, and this was achieved by using an acid salt of β-alanine chloride as an acylating reagent. I found it possible. When unprotected β-alanine is activated in the form of acid chloride, self-condensation between molecules due to nucleophilicity of the amino group can be considered, but in order to suppress nucleophilicity, unprotected β-alanine chloride is converted into acid chloride. Side reactions could be suppressed by quaternizing the amino group in the form of a salt.

  Accordingly, the present invention provides a method for synthesizing L-carnosine, which comprises using unprotected β-alanine chloride as an acylating reagent in a method for synthesizing L-carnosine by acylation of L-histidine. .

  In a preferred embodiment, L-histidine is also used in the acylation reaction in the form of an acid salt. Thereby, the production | generation and racemization of a by-product can be suppressed. Alternatively, acid may be added to the L-histidine solution.

  The acid that forms the salt is hydrochloric acid, acetic acid, methanesulfonic acid, trifluoroacetic acid, p-toluenesulfonic acid, etc., with hydrochloric acid and p-toluenesulfonic acid being particularly preferred. The acylation reaction is carried out by adding the powdered or solid acylating reagent to a solution of L-histidine under stirring and continuing stirring until most of the L-histidine is consumed. In this case, it is preferable to add an acylating reagent in excess, for example, at least 1.5 times the theory, and to carry out the reaction at a temperature below room temperature, for example, 0 ° C. After completion of the reaction, for example, the reaction solution is diluted with water, impurities are removed by ion-exchange resin treatment, and then an alcohol such as methanol or ethanol or a water-containing alcohol is added to crystallize to thereby improve the chemical and optical purity. High L-carnosine can be obtained in high yield.

  According to the present invention described here, high-purity L-carnosine is synthesized in high yield from β-alanine and L-histidine in a short process without the protection and deprotection of amino groups and other addition steps. It becomes possible to do.

Preferred embodiment

  In the present invention, an acid addition salt of unprotected β-alanine chloride (3-aminopropionic acid chloride) is used as an acylating reagent for L-histidine. This acylating reagent can be produced by reacting an acid addition salt of β-alanine produced in advance with thionyl chloride. The acylation reaction is performed by adding the solid or powdery acylating reagent to a solution of L-histidine and stirring. At this time, it is preferable to add the acylating reagent at least 1.5 times, particularly 2.0 times in excess so that as much L-histidine as possible is acylated.

  L-histidine is also preferably reacted in the form of an acid salt, whereby the amount of by-products can be reduced. Alternatively, an acid may be added to the L-histidine solution.

  Preferred reaction solvents are water, acetic acid, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone and the like, but an acid may be added to these solvents instead of the acid salt of L-histidine. For example, 1N hydrochloric acid, phosphate buffer (pH 2.8), trifluoroacetic acid / dimethylformamide mixed solution, methanesulfonic acid / dimethylformamide mixed solution, methanesulfonic acid / dimethylacetamide mixed solution, methanesulfonic acid / N-methyl-2- Pyrrolidone mixed solution.

  In the reaction between an acid chloride and an amine, a base that binds the generated hydrogen chloride is generally used, but in the acylation reaction of the present invention, the use of an acid-bonded base can be omitted. If used, tertiary amines such as triethylamine, N-methylmorpholine, N, N-dimethylaniline can be used.

  The acid that forms salts with β-alanine chloride or L-histidine includes a number of inorganic and organic acids, but in practice, hydrochloric acid, acetic acid, methanesulfonic acid, trifluoroacetic acid, and p-toluenesulfonic acid are preferred. Most preferred are hydrochloride and p-toluenesulfonic acid. The most preferred combination of acid and solvent that forms a salt with the acylating reagent and L-histidine is the dissolution of β-alanine chlorad p-toluenesulfonate and L-histidine p-toluenesulfonate in dimethylacetamide. This is a method of condensing. Unprotected reactions tend to racemize part of the product and reduce optical purity, or to produce tripeptide by-products that are predominantly β-alanyl-alanyl-histidine. The combination can minimize racemization and by-product formation.

  The preferred reaction temperature is room temperature or lower, most preferably -10 ° C to 0 ° C. It is also preferred to carry out the reaction in an inert gas such as an argon atmosphere. The reaction time is a time during which at least a major part of L-histidine, for example, 90% or more is consumed in the reaction. This would take at least 6 hours, for example, near a reaction temperature of 0 ° C.

  After completion of the reaction, L-carnosine can be recovered from the reaction solution according to a conventional method. For example, the reaction solution is diluted with water, adsorbed on a strongly acidic ion exchange resin, and then eluted with ammonia water. The eluate is concentrated and passed through a weakly acidic ion exchange resin. After concentration, ethanol is added to add L-carnosine. Crystallize.

The following examples illustrate the invention. The meanings of the abbreviations used in the examples are as follows.
β-Ala: β-alanine β-AlaCl: β-alanine chloride Ac: acetyl AcOH: acetic acid CAS: L-carnosine DMA: dimethylacetamide His: L-histidine MsOH: methanesulfonic acid NMP: N-methyl-2- Pyrrolidone p-TsOH: p-toluenesulfonic acid

Reference Example 1 Synthesis of β-AlaCl · HCl 50 g (0.56 mol) of β-Ala was suspended in a mixture of 490 g of toluene and 2.8 g of water, and hydrogen chloride gas was blown in over 8 hours while stirring vigorously. -Generate Ala.HCl. Thereafter, 245 ml (3.37 mol) of thionyl chloride is added dropwise to the reaction solution at room temperature, followed by stirring for 1 hour. Thereafter, the internal temperature is raised to 50 ° C. and the mixture is vigorously stirred for 38 hours. After the reaction, the reaction solution was quickly filtered, and the obtained crystals were washed with 200 ml of toluene. The washed crystals were suspended in 200 ml of toluene, concentrated by an evaporator, and 73.36 g (90 of brown powdery β-AlaCl · HCl (90 8%). IR (Nujol): 1794 cm ⁻¹ (C = 0)

[Reference Example 2] Synthesis of β-AlaCl · p-TsOH 50 g (0.56 mol) of β-Ala was dissolved in 100 ml of water, and 106.8 g (0.56 mol) of p-TsOH · H ₂ O was added thereto at room temperature. Stir for 30 minutes. Thereafter, the solvent is distilled off, 100 ml of toluene is added, and when the solvent is distilled off again, β-Ala · p-TsOH is quantitatively obtained. Next, 245 ml (3.37 mol) of thionyl chloride in toluene is added dropwise thereto at room temperature, and the mixture is stirred for 1 hour. Thereafter, the temperature was raised to 50 ° C., and the mixture was vigorously stirred for 38 hours. The solvent was distilled off under reduced pressure, toluene was added, the solvent was distilled off again, and 155.9 g (99.3% of powdery β-AlaCl · p-TsOH) was obtained. ) IR (Nujol): 1790 cm ^-1 (C = 0)

[Reference Example 3] Synthesis of His · 2p-TsOH 155.15 g (1.0 mol) of His was dissolved in 250 ml of water, and 380.42 g (2.0 mol) of p-TsOH · H ₂ O was added at room temperature, followed by stirring for 30 minutes. To do. Thereafter, the reaction solution is concentrated, and 200 ml × 2 of toluene is added and concentrated to obtain 495.2 g (98.9%) of His · 2p-TsOH. IR (KBr): 1730 cm ⁻¹ (C = 0), 1629 cm ⁻¹ (aromatic)

[Example 1] Use of β-AlaCl · HCl 0.5 g of His is dissolved in 10 ml or 20 ml of AcOH, and 2 equivalents of β-AlaCl · HCl is added at room temperature and stirred for 30 minutes. Unreacted His, produced CAS and by-produced AcHis were quantified in the reaction solution, respectively, and the percentage relative to the starting His was calculated.

AcOH reaction time HIS CAS AcHis
10ml 30min 30.5% 68.0% 1.6%
20ml 30min 34.6% 64.7% 0.7%

[Example 2] Use of β-AlaCl · HCl Dissolve 0.78 g of His in a solvent shown in the following table to 10 ml, add 2 equivalents of β-AlaCl · HCl at 5 ° C under an argon atmosphere, and add 4 equivalent at room temperature. Stir until time. After 2 hours and 4 hours of reaction time, unreacted His, CAS and by-produced Ala-Ala-His in the reaction solution were quantified, and the percentage relative to the starting His was calculated. Further, the optical purity (% ee) of the crude CAS precipitated with ethanol was measured.

Experiment No. Solvent Time His CAS% ee By-product
h%%
───── ───── ─── ───── ───── ───── ─────
1 1.0M 2 4.1 80.2 91.2 9.7
MsOH / DMA 4 1.4 79.4 86.6 11.8
───────────────────────────────────────
2 2.0M 2 7.0 79.7 86.3 7.7
MsOH / DMA 4 2.2 79.7 81.6 9.7
───────────────────────────────────────
3 2.0M 2 11.0 75.2 92.6 11.3
MsOH / DMA 4 4.2 78.5 85.8 11.3
───────────────────────────────────────

Example 3 Use of β-AlaCl · pTsOH and His · 2pTsOH 100 g (0.2 mol) of His · 2pTsOH was dissolved in 400 ml of DMA and 112 g (0.4 mol) of β-AlaCl · pTsOH at −10 ° C. in an argon atmosphere. Is added over 1 hour, and the temperature is gradually raised to 0 ° C. and stirred. After completion of the reaction, water is added to hydrolyze the remaining β-AlaCl. Thereafter, the reaction solution is diluted with water, adsorbed on the strong acid ion exchange resin (SK-1B), and then eluted with 2N ammonia water. The eluate is concentrated and passed through the weak acid ion exchange resin (WK-11). The mixture was concentrated, crystallized by adding 80% ethanol, and dried to obtain 29.3 g (64.7%) of CAS.
Chemical purity 99.2% (using ULTRON VX-ODS, Shinwa Chemical Industries, Ltd.), optical purity 99.3% ee (using SUMICHIRAL OA-5000, Sumitomo Chemical Co., Ltd.)
¹ H-NMR (D ₂ O, 200 MHz): δ 2.62-2.69 (2H, m, COCH ₂ ); 2.69 (1H, dd, C H ₂ CHCOOH); 2.98 (1H, dd, C H ₂ CHCOOH); 3.23 (2H, t, NCH ₂ ); 4.49 (1H, dd, C H COOH); 6.96 (1H, d, NHC H ); 7.71 (1H, d) , NCH)
IR (KBr): 1647 cm ⁻¹ , 1585 cm ⁻¹

Claims (10)

  A method for synthesizing carnosine, wherein an unprotected β-alanine chloride acid salt is used as an acylating reagent in a method for synthesizing L-carnosine by acylation of L-histidine.   The method according to claim 1, wherein the acid forming a salt with β-alanine chloride is selected from hydrochloric acid, acetic acid, methanesulfonic acid, trifluoroacetic acid, or p-toluenesulfonic acid.   The method of claim 1 or 2, wherein the acylating reagent is used at least 1.5 times the theory for L-histidine.   4. The method according to claim 1, wherein the acylating reagent is added as a solid or a powder to a solution of an acid salt of L-histidine or a solution of L-histidine containing an acid.   The method of claim 4, wherein the acid is selected from hydrochloric acid, acetic acid, methanesulfonic acid, trifluoroacetic acid, or p-toluenesulfonic acid.   The method according to any one of claims 1 to 5, wherein the acylation reaction is carried out at a temperature below room temperature.   An excess of β-alanine chloride / p-toluenesulfonate is added as a solid to a solution of L-histidine / p-toluenesulfonate in a polar solvent to acylate L-histidine. -Carnosine synthesis method.   8. The method of claim 7, wherein the polar solvent is dimethylacetamide.   The method according to claim 7 or 8, wherein the acylation reaction is performed at a temperature of -10 ° C to 0 ° C in an inert gas atmosphere.   The method according to any one of claims 7 to 9, wherein β-alanyl chloride · p-toluenesulfonate is added at least 1.5 times the theory to L-histidine.