WO2011118450A1 - 光学活性n-メチルアミノ酸及び光学活性n-メチルアミノ酸アミドの製造方法 - Google Patents
光学活性n-メチルアミノ酸及び光学活性n-メチルアミノ酸アミドの製造方法 Download PDFInfo
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- WO2011118450A1 WO2011118450A1 PCT/JP2011/056046 JP2011056046W WO2011118450A1 WO 2011118450 A1 WO2011118450 A1 WO 2011118450A1 JP 2011056046 W JP2011056046 W JP 2011056046W WO 2011118450 A1 WO2011118450 A1 WO 2011118450A1
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- optically active
- methylamino acid
- acid amide
- methylamino
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
Definitions
- Optically active N-methylamino acids and optically active N-methylamino acid amides are widely used as raw materials for peptide pharmaceuticals, and specific uses include raw materials for rheumatic drugs and anticancer agents (Special Table 2001).
- No. 518515 International Publication No. 1999/17792
- International Publication No. 1999/17792 Japanese Patent Publication No. 2001-514659
- Japanese Patent Publication No. 2000-502092 International Publication No. 1997/22621
- Japanese Patent Laid-Open No. 2001-190298 discloses that an optically active N-methylamino acid can be produced by reacting an ⁇ -keto acid compound with an inexpensive methylamine using an aminotransferase. ing.
- ⁇ -keto acid compounds are usually difficult to obtain industrially, this method is not necessarily economically advantageous.
- substrates to which the aminotransferase can be applied are limited.
- Rhodococcus sp. A method of performing stereoselective hydrolysis using an amidase contained in AJ270 has also been reported (Tetrahedron: Asymmetry, 16, 2409 (2005)). By this method, an optically active N-methylamino acid can be obtained as a product of hydrolysis, and an optically active N-methylamino acid amide can be obtained as an unreacted product.
- this method has high stereoselectivity for N-methylphenylglycinamides and N-methylcyclohexylglycinamides, it has stereoselectivity for lower aliphatic N-methylamino acid amides. Low.
- microorganisms belonging to the genus Mycoplana or the genus Mycobacterium having a stereoselective hydrolysis activity toward an amide bond even in an organic solvent aqueous solution are known (Japanese Patent Laid-Open No. 2009-278914).
- the reaction involving them is a reaction using a poorly water-soluble aromatic amino acid amide as a substrate, and the solvent is also in an organic solvent aqueous solution.
- N-methylamino acid amide is a water-soluble substrate and is preferably reacted in an aqueous solution.
- enzymes have different reaction activities depending on the properties of the solvent and the substrate, and thus the above-mentioned microorganisms were not considered to have activity on N-methylamino acid amide.
- an object of the present invention is to provide an efficient method for producing optically active N-methylamino acid and / or optically active N-methylamino acid amide useful as a raw material for pharmaceuticals by using a biocatalyst.
- the method for producing an optically active N-methylamino acid and / or optically active N-methylamino acid amide according to the present invention can be obtained by treating a microbial cell belonging to the genus Mycoplana or a processed product thereof with -Reacting with a methylamino acid amide to obtain an optically active N-methylamino acid of general formula (2) and an optically active N-methylamino acid amide of general formula (3) as an enantiomer.
- acting a bacterial cell or a treated product thereof on the N-methylamino acid amide of the general formula (1) means that the bacterial cell or the treated product is subjected to, for example, an aqueous solution of N-methylamino acid amide Coexisting with and reacting by its catalytic action.
- microorganism of the present invention having the activity of stereoselectively hydrolyzing the N-methylamino acid amide of the general formula (1) or its treated product has the activity of stereoselectively hydrolyzing the amide bond of the N-methylamino acid amide. It is what has.
- Such microorganisms having biocatalytic activity are microorganisms belonging to the genus Mycoplana, and specifically, Mycoplana ramosa and Mycoplana dimorpha are preferable.
- microorganisms having amino acid amide hydrolysis activity for example, cells derived from microorganisms belonging to the genus Xanthobacter, Protaminobacter, Mycobacterium, Pseudomonas, Rhodococcus, Serratia, and Chromobacterium was used as a biocatalyst, it had almost no hydrolysis activity to N-methylamino acid amide.
- microorganisms are usually cultured using a medium containing an assimilating carbon source, nitrogen source, inorganic salts essential to each microorganism, nutrients, and the like.
- a medium containing an assimilating carbon source, nitrogen source, inorganic salts essential to each microorganism, nutrients, and the like In particular, when urea is added as a nitrogen source, the activity as a biocatalyst of microorganisms obtained by culturing is high, which is preferable.
- the pH during culture is in the range of 4 to 10, and the temperature is 20 to 50 ° C.
- the culture is aerobic for about 1 day to 1 week.
- the microorganism thus cultured is used for the reaction as a microbial cell or a processed product of the microbial cell, for example, a culture solution, a separated microbial cell, a crushed cell, or a purified biocatalyst.
- it can also be used as a biocatalyst by immobilizing
- the pH during the reaction is preferably in the range of 6 to 10, more preferably in the range of pH 7 to 8.
- an acid or a base may be added as appropriate.
- the pH may be adjusted by adding an acid such as hydrochloric acid, sulfuric acid or nitric acid.
- N-methylamino acid amide acid salt is used as a raw material, It is preferable to adjust the pH by adding a base such as sodium or potassium hydroxide.
- a base such as sodium or potassium hydroxide.
- the pH is lower than 6, the catalytic activity of the microbial cells or the processed microbial cells may decrease and the reaction may not proceed.
- the pH is higher than 10 since a non-enzymatic hydrolysis reaction with a base occurs, the apparent reaction rate increases, but the optical purity may decrease.
- the microbial cells and their processed products are removed from the reaction end solution by a normal solid-liquid separation means using, for example, a centrifugal separator or an ultrafiltration membrane.
- the optically active N-methylamino acid as a reaction product can be recovered by an extraction operation into a solvent that dissolves the optically active N-methylamino acid.
- An insoluble salt may be formed.
- the organic solvent used at this time is not particularly limited as long as it dissolves optically active N-methylamino acid, and examples thereof include alcohols, and examples thereof include methanol, ethanol, isopropanol, and isobutanol.
- the organic solvent from which the optically active N-methylamino acid has been extracted includes unreacted N-methylamino acid amide
- a method using the difference in solubility between the optically active N-methylamino acid and N-methylamino acid amide Unreacted N-methylamino acid amide can be separated.
- the solvent used here may be any solvent that has low solubility in optically active N-methylamino acid and high solubility in N-methylamino acid amide.
- ketones such as acetone and methylethylketone, or toluene and xylene And hydrocarbons.
- the optically active N-methylamino acid By adding the solvent to an organic solvent solution from which the optically active N-methylamino acid has been extracted, or a solution obtained by distilling the organic solvent from the organic solvent solution, a solid-liquid separation operation such as filtration or centrifugation is performed. By dissolving and removing the N-methylamino acid amide in the reaction, the optically active N-methylamino acid can be obtained as crystals.
- Comparative Example 2 (1) Culture of other strains Similar to pMCA1 / JM109 FERM BP-10334 in Comparative Example 1, microorganisms Xanthobacter Flavus NCIB10071T, Xanthobacter autotrophicus DSM431 TK0502 -8823, Pseudomonas putida, Rhodococcus erythropolis, Serratia marcescens, and Chromobacterium iodium were inoculated into a medium having the composition shown in Table 4 and cultured at 30 ° C. for 48 hours under shaking. The obtained culture solution was centrifuged to obtain a concentrated cell fluid (Table 5). The results were as shown in Table 5.
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Abstract
Description
(ii) Mycoplana属に属する微生物の菌体若しくはその処理物を、一般式(1)のN-メチルアミノ酸アミドに作用させて得られた、光学活性N-メチルアミノ酸と光学活性N-メチルアミノ酸アミドとを含む加水分解液から、光学活性アミノ酸を結晶として析出させ、光学活性N-メチルアミノ酸と光学活性N-メチルアミノ酸アミドとを分離することをさらに含む、前記(i)の方法。
(iii) 前記Mycoplanaに属する微生物が、Mycoplana ramosa又はMycoplana dimorphaである、前記(i)又は(ii)の方法。
(iv) 一般式(1)、(2)および(3)中のRがイソプロピル基である、前記(i)~(iii)のいずれかの方法。
基質のN-メチルアミノ酸アミド、反応生成物である光学活性N-メチルアミノ酸の量または光学純度は、以下のHPLC条件で測定した。
(1)反応率の測定
カラム:Lichrosorb RP-18(4.6φ×250mm)
カラム温度:30℃, 検出:RI
溶離液:過塩素酸50mM水溶液, 流速:0.5mL/min
(2)光学純度の測定
カラム:Sumichiral OA-5000(4.6φ×50mm)
カラム温度:30℃, 検出:260nm吸収
溶離液:1mM硫酸銅水溶液, 流速:1.0mL/min
Mycoplana ramosa ATCC49678の培養
N-メチルアミノ酸アミドを基質とした場合に、効率良く立体選択的に加水分解することが判明したMycoplana ramosa ATCC49678を、表1に示す組成を有する培地に接種し、30℃で69時間振盪培養した。得られた培養液を5℃にて遠心加速度1200×gにて15分間の遠心分離を行った。上清液を除去し、濃縮菌体液2.65gを得た。
N-メチルバリンアミドの加水分解
200mLフラスコでラセミ体のN-メチルバリンアミド塩酸塩1.0gを、水50gに溶解し、さらに20%水酸化ナトリウム水溶液でpHを8.0に調整した後に水を加え、基質溶液100gを調製した。各々の基質溶液に、参考例1で取得したMycoplana ramosa ATCC49678の濃縮菌体液を0.5g添加し、マグネチックスターラで撹拌下35℃にて1時間反応させた。反応液をHPLCで測定したところ、N-メチルバリンアミドの47.3%が加水分解した。また、加水分解により生成したN-メチルバリンはL体であり、99%ee以上の光学純度を示した。
その他菌株の培養
参考例1のMycoplana ramosa ATCC49678と同様に、スクリーニングによって選抜したMycoplana dimorpha ATCC4279、Mycoplana dimorpha NCIB9439Tの各菌株を、表1に示した組成の培地に接種し、振盪下30℃で70時間培養した。得られた培養液を遠心分離し濃縮菌体液を取得した。
結果は表2に示される通りであった。なお表中、濃縮菌体取得量とは、濃縮菌体液の量(g)を意味する。
N-メチルバリンアミドの加水分解
200mLフラスコでラセミ体のN-メチルバリンアミド塩酸塩1.0gを、水50gに溶解し、さらに20%水酸化ナトリウム水溶液でpHを8.0に調整した後に水を加え、基質溶液100gを調製した。各々の基質溶液に、参考例2で取得した各濃縮菌体液を0.5g添加し、マグネチックスターラで撹拌下35℃にて1時間反応させた。反応液をHPLCで測定したところ、いずれもN-メチルバリンアミドの加水分解が進行した。また、いずれも加水分解により生成したN-メチルバリンはL体であり、99%ee以上の光学純度を示した。
結果は表3に示される通りであった。なお表中、生成率とは、反応に用いた基質N-メチルバリンアミド塩酸塩に含まれるL-N-メチルバリンアミドについて、加水分解によりL-N-メチルバリンへ転化した割合を意味する。
(1)pMCA1/JM109 FERM BP-10334の培養
表4に示す組成を有する組成を有する培地を滅菌し、アンピシリン50mgを添加した後にアミノ酸アミド、特にフェニルグリシンアミドの様な難水溶性アミノ酸アミドに対し加水分解活性を有する遺伝子組み換え大腸菌pMCA1/JM109 FERM BP-10334を接種し、30℃で16時間振盪培養した。得られた培養液を遠心分離し濃縮菌体液1.63gを得た。
200mLフラスコでラセミ体のN-メチルバリンアミド塩酸塩1.0gを、水50gに溶解し、さらに20%水酸化ナトリウム水溶液でpHを8.0に調整した後に水を加え、基質溶液100gを調製した。基質溶液に、前記(1)で取得したpMCA1/JM109 FERM BP-10334の濃縮菌体液を0.5g添加し、マグネチックスターラで撹拌下35℃にて24時間反応させた。反応液をHPLCで測定したところ、N-メチルバリンアミドの加水分解は進行しなかった。
(1)その他菌株の培養
比較例1のpMCA1/JM109 FERM BP-10334と同様に、アミノ酸アミド加水分解活性を有する微生物Xanthobacter Flavus NCIB10071T、Xanthobacter autotrophicus DSM431 TK0502、Protaminobacter alboflavus NCIB8167、Mycobacterium methanolica BT-84 FERM P-8823、Pseudomonas putida、Rhodococcus erythropolis、Serratia marcescence、およびChromobacterium iodiumを、表4に示した組成を有する培地に接種し、振盪下30℃で48時間培養した。得られた培養液を遠心分離し濃縮菌体液を取得した(表5)。
結果は表5に示される通りであった。
200mLフラスコでラセミ体のN-メチルバリンアミド塩酸塩1.0gを、水50gに溶解し、さらに20%水酸化ナトリウム水溶液でpHを8.0に調整した後に水を加え、基質溶液100gを調製した。各々の基質溶液に、前記(1)で取得した各濃縮菌体液を0.5g添加し、マグネチックスターラで撹拌下35℃にて24時間反応させた。反応液をHPLCで測定したところ、Rhodococcus erythropolisを除くいずれの菌株もN-メチルバリンアミドの加水分解は進行しなかった。一方、Rhodococcus erythropolisはN-メチルバリンアミドの57.5%が加水分解したが、生成したN-メチルバリンの光学純度はD体過剰で23%eeに過ぎなかった。
実施例1のN-メチルバリンアミドの加水分解後の溶液へザルトリウス限外ろ過器VIVAFLOW200を用いて限外ろ過を行い、菌体を除去した。得られた限外ろ過液へ20%水酸化ナトリウムを1.12g加え、ロータリーエバポレータにて濃縮した。さらに50℃で濃縮物の真空乾燥を行った。濃縮乾燥物へメタノール60mLを加え、25℃にてマグネチックスターラで30分間撹拌した。撹拌後、スラリーの吸引ろ過により固体をろ別し、つづけて、ろ液をロータリーエバポレータにて濃縮した。得られた濃縮物へアセトン10mLを加え、25℃にてマグネチックスターラで30分間撹拌した。撹拌後、吸引ろ過によりアセトン溶液と固体に分離した。前記固体を回収し、50℃で真空乾燥を行った。乾燥後、白色粉末として0.45gのN-メチル-L-バリンを取得した。ラセミ体中のN-メチル-L-バリンアミド基準で回収率は90%であった。また、取得したN-メチル-L-バリンの光学純度は99%eeであった。
実施例3のアセトン溶液をロータリーエバポレータにて濃縮した。さらに、濃縮物は50℃で真空乾燥を行った。乾燥後、白色粉末として0.45gのN-メチル-D-バリンアミドを取得した。
ラセミ体中のN-メチル-D-バリンアミド基準で回収率は90%であった。また、取得したN-メチル-D-バリンアミドの光学純度は99%eeであった。
Claims (4)
- Mycoplana属に属する微生物の菌体若しくはその処理物を、一般式(1)のN-メチルアミノ酸アミドに作用させて得られた、光学活性N-メチルアミノ酸と光学活性N-メチルアミノ酸アミドとを含む加水分解液から、光学活性アミノ酸を結晶として析出させ、光学活性N-メチルアミノ酸と光学活性N-メチルアミノ酸アミドとを分離することをさらに含む、請求項1に記載の方法。
- 前記Mycoplanaに属する微生物が、Mycoplana ramosa又はMycoplana dimorphaである、請求項1又は2に記載の方法。
- 一般式(1)、(2)および(3)中のRがイソプロピル基である、請求項1~3のいずれか一項に記載の方法。
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JP2012506953A JPWO2011118450A1 (ja) | 2010-03-23 | 2011-03-15 | 光学活性n−メチルアミノ酸及び光学活性n−メチルアミノ酸アミドの製造方法 |
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JPH01277499A (ja) * | 1988-04-28 | 1989-11-07 | Mitsubishi Gas Chem Co Inc | L−α−アミノ酸の製造法 |
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JPH01277499A (ja) * | 1988-04-28 | 1989-11-07 | Mitsubishi Gas Chem Co Inc | L−α−アミノ酸の製造法 |
Non-Patent Citations (2)
Title |
---|
URAKAMI T. ET AL.: "Recharacterization and Emended Description of the Genus Mycoplana and Description of Two New Species, Mycoplana ramosa and Mycoplana segnis", INT. J. SYST. BACTERIOL., vol. 40, no. 4, 1990, pages 434 - 442 * |
WANG MX. ET AL.: "Synthesis of optically active a-methylamino acids and amides through biocatalytic kinetic resolution of amides", TETRAHEDRON ASYM., vol. 16, no. 14, 2005, pages 2409 - 2416 * |
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WO2014157651A1 (ja) * | 2013-03-29 | 2014-10-02 | 三菱瓦斯化学株式会社 | アミノ酸アミド化合物及びアミノ酸の製造方法、並びにイミダゾリジン化合物 |
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