JP2006000005A - Method for producing optically active 2-alkyl-d-cysteine or its salt - Google Patents
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本発明は、ＤＬ体の混合物である一般式（１）で示される２−アルキルシステインアミドから、一般式（４）で示される光学活性２−アルキル−D−システイン又はその塩を製造する方法に関する。詳しくは、一般式（１）で示される２−アルキルシステインアミドに、２−アルキル−L−システインアミドのアミド結合を立体選択的に加水分解する活性を有する微生物の菌体又は菌体処理物を作用させて、反応生成物である一般式（２）で示される２−アルキル−L−システインと、未反応物である一般式（３）で示される光学活性２−アルキル−D−システインアミドの混合物となし、この混合物から光学活性２−アルキル−D−システインアミドを分取した後、そのアミド結合を加水分解して一般式（４）で示される光学活性２−アルキル−D−システイン又はその塩を製造する方法に関する。光学活性２−アルキル−D−システイン又はその塩（以下、単に光学活性２−アルキル−D−システインと記すことがある）は、各種工業薬品、農薬、及び医薬品の製造中間体として重要な物質である。
（一般式（１）、（２）、（３）、及び（４）中のＲは炭素数１〜４の低級アルキル基である。）
The present invention relates to a method for producing an optically active 2-alkyl-D-cysteine represented by the general formula (4) or a salt thereof from a 2-alkylcysteine amide represented by the general formula (1) which is a mixture of DL forms. . Specifically, a microbial cell or a treated product of a microorganism having an activity of stereoselectively hydrolyzing the amide bond of 2-alkyl-L-cysteine amide to the 2-alkyl cysteine amide represented by the general formula (1). The reaction product of 2-alkyl-L-cysteine represented by the general formula (2) as a reaction product and the optically active 2-alkyl-D-cysteine amide represented by the general formula (3) as an unreacted product After separating optically active 2-alkyl-D-cysteine amide from this mixture, the amide bond is hydrolyzed to give optically active 2-alkyl-D-cysteine represented by the general formula (4) or its The present invention relates to a method for producing a salt. Optically active 2-alkyl-D-cysteine or a salt thereof (hereinafter sometimes simply referred to as optically active 2-alkyl-D-cysteine) is an important substance as a production intermediate for various industrial chemicals, agricultural chemicals, and pharmaceuticals. is there.
(R in the general formulas (1), (2), (3), and (4) is a lower alkyl group having 1 to 4 carbon atoms.)
従来、光学活性２−アルキルシステインの製造方法としては、光学活性なL−システインメチルエステルを出発原料として、ピバルアルデヒドで環化、ホルムアルデヒドで保護し、リチウム試薬とヨウ化メチルでメチル化した後、塩酸で開環、脱保護して２−メチル−L−システインを塩酸塩として得る方法が知られている（例えば、特許文献１、非特許文献１参照）。しかし、この方法は出発原料が光学活性体であり、工程数が多く煩雑であり、高価な試薬を必要とするため、工業的に優れた方法とは言いがたい。 Conventionally, as a method for producing optically active 2-alkylcysteine, optically active L-cysteine methyl ester is used as a starting material, cyclized with pivalaldehyde, protected with formaldehyde, and methylated with lithium reagent and methyl iodide. A method is known in which 2-methyl-L-cysteine is obtained as a hydrochloride by ring opening and deprotection with hydrochloric acid (see, for example, Patent Document 1 and Non-Patent Document 1). However, this method is not an industrially excellent method because the starting material is an optically active substance, has many steps, is complicated, and requires an expensive reagent.
また、光学活性２−アルキルシステインの製造法として、L−システインエチルエステルを出発原料として、ニトリル化合物で環化、ヨウ化メチルなどのメチル化剤を用いてメチル化を行いラセミ体のエステルとし、リパーゼ等を用いた酵素加水分解反応による光学分割により目的とする光学活性４−メチル−チアゾリン−４−カルボン酸を得て、さらに加水分解を施すことにより光学活性２−メチルシステインを得る方法が報告されている（例えば、特許文献２、特許文献３参照）。しかし、この方法は光学活性体を原料とするにもかかわらず、工程途中における中間体がラセミ体となり再び光学分割を行う必要があるため、工程が煩雑となり工業的には適しているとは言いがたい。 As a method for producing optically active 2-alkylcysteine, L-cysteine ethyl ester is used as a starting material, cyclized with a nitrile compound, and methylated with a methylating agent such as methyl iodide to give a racemic ester, A method for obtaining optically active 2-methylcysteine by obtaining desired optically active 4-methyl-thiazoline-4-carboxylic acid by optical resolution by enzymatic hydrolysis using lipase or the like and further hydrolyzing is reported. (For example, see Patent Document 2 and Patent Document 3). However, although this method uses an optically active substance as a raw material, the intermediate in the process becomes a racemate, and it is necessary to perform optical resolution again. It ’s hard.
本発明の目的は、各種工業薬品、農薬及び医薬品の製造原料として広範な活用が期待され、産業上、非常に有用な化合物である一般式（４）で示される光学活性２−アルキル−D−システイン又はその塩の製造方法を提供することにある。 The object of the present invention is expected to be widely used as a raw material for production of various industrial chemicals, agricultural chemicals and pharmaceuticals, and is an optically active 2-alkyl-D- represented by the general formula (4) which is a very useful compound in industry. The object is to provide a method for producing cysteine or a salt thereof.
かかる実状に鑑み、本発明者らは鋭意研究を行ったところ、一般式（1）で示される２−アルキルシステインアミドのアミド結合を生化学的に不斉加水分解して一般式（２）で示される光学活性２−アルキル−L−システインを生成せしめ、一般式（３）で示される未反応の光学活性２−アルキル−D−システインアミドを回収した後に、これを加水分解することにより一般式（４）で示される光学活性２−アルキル−D−システインを製造する方法を見出し、本発明に到達した。即ち、本発明は、以下の１）から３）に示す、一般式（４）で示される光学活性２−アルキル−D−システイン又はその塩の製造法に関するものである。
１）一般式（１）で示される２−アルキルシステインアミドに、２−アルキル−L−システインアミドのアミド結合を立体選択的に加水分解する活性を有する微生物の菌体又は菌体処理物を作用させて、反応生成物である一般式（２）で示される２−アルキル−L−システインと、未反応物である一般式（３）で示される光学活性２−アルキル−D−システインアミドの混合物となし、この混合物から光学活性２−アルキル−D−システインアミドを分取した後、そのアミド結合を加水分解して一般式（４）で示される光学活性２−アルキル−D−システインを得ることを特徴とする、２−アルキルシステインアミドから光学活性２−アルキル−D−システイン又はその塩を製造する方法。
（一般式（１）、（２）、（３）、及び（４）中のＲは炭素数１〜４の低級アルキル基である。）
２）一般式（１）で示される２−アルキル−Ｌ−システインアミドのアミド結合を立体選択的に加水分解する活性を有する微生物が、キサントバクター属、プロタミノバクター属、ミコプラナ属に属する細菌である、１）に記載の２−アルキルシステインアミドから光学活性２−アルキル−D−システイン又はその塩を製造する方法。
３）一般式（１）、（２）、（３）、及び（４）においてＲがメチル基である、１）又は２）に記載の２−アルキルシステインアミドから光学活性２−アルキル−D−システイン又はその塩を製造する方法。
In view of this situation, the present inventors conducted extensive research and found that the amide bond of 2-alkylcysteine amide represented by the general formula (1) was biochemically asymmetrically hydrolyzed and represented by the general formula (2). The optically active 2-alkyl-L-cysteine shown is produced and the unreacted optically active 2-alkyl-D-cysteine amide shown by the general formula (3) is recovered and then hydrolyzed to give the general formula A method for producing the optically active 2-alkyl-D-cysteine represented by (4) has been found and the present invention has been achieved. That is, the present invention relates to a method for producing an optically active 2-alkyl-D-cysteine represented by the general formula (4) or a salt thereof shown in the following 1) to 3).
1) Acting on a 2-alkylcysteine amide represented by the general formula (1) with a microbial cell or a treated product of a microorganism having an activity of stereoselectively hydrolyzing the amide bond of 2-alkyl-L-cysteine amide A mixture of 2-alkyl-L-cysteine represented by general formula (2) as a reaction product and optically active 2-alkyl-D-cysteine amide represented by general formula (3) as an unreacted product. After separating the optically active 2-alkyl-D-cysteine amide from this mixture, the amide bond is hydrolyzed to obtain the optically active 2-alkyl-D-cysteine represented by the general formula (4). A process for producing optically active 2-alkyl-D-cysteine or a salt thereof from 2-alkylcysteine amide.
(R in the general formulas (1), (2), (3), and (4) is a lower alkyl group having 1 to 4 carbon atoms.)
2) Microorganisms having the activity of stereoselectively hydrolyzing the amide bond of 2-alkyl-L-cysteine amide represented by the general formula (1) belong to the genus Xantobacter, Protaminobacter, and Mycoplana A method for producing optically active 2-alkyl-D-cysteine or a salt thereof from the 2-alkylcysteine amide according to 1), which is a bacterium.
3) In the general formulas (1), (2), (3), and (4), R is a methyl group, from the 2-alkylcysteine amide according to 1) or 2) to an optically active 2-alkyl-D- A method for producing cysteine or a salt thereof.
各種工業薬品、農薬及び医薬品の製造原料として広範な活用が期待され、産業上、非常に有用な化合物である一般式（４）で示される光学活性２−アルキル−D−システイン又はその塩を製造する効率的に優れた方法を提供することが可能となる。 Produced optically active 2-alkyl-D-cysteine or a salt thereof represented by the general formula (4) which is expected to be widely used as a raw material for producing various industrial chemicals, agricultural chemicals and pharmaceuticals, and is an industrially very useful compound. It is possible to provide an efficient method.
以下に本発明の詳細について説明する。本発明の一般式（４）で示される光学活性２−アルキル−D−システインにおいて、式中のRは炭素数１〜４の低級アルキル基であればよく、特に制限はないが、例えば、メチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、secブチルおよびtertブチルなどの直鎖又は分枝した低級アルキル基が好適であり、メチル基が特に好適である。 Details of the present invention will be described below. In the optically active 2-alkyl-D-cysteine represented by the general formula (4) of the present invention, R in the formula is not particularly limited as long as it is a lower alkyl group having 1 to 4 carbon atoms. Straight or branched lower alkyl groups such as ethyl, propyl, isopropyl, butyl, isobutyl, sec butyl and tert butyl are preferred, and the methyl group is particularly preferred.
また、一般式（４）で示される光学活性２−アルキル−D−システインの塩の種類は、実用上許容できる塩であれば特に制限はないが、例えば塩酸や硫酸等の無機酸、ギ酸や酢酸等の有機酸、ナトリウムやカリウム等のアルカリ金属、マグネシウムやカルシウム等のアルカリ土類金属、トリメチルアミンやテトラメチルアンモニウム等の有機アルカリ、或いはアンモニア等が上げられる。得られる２−アルキルシステインの安定性という意味で特に塩酸塩、硫酸塩が好適である。 Further, the type of the salt of the optically active 2-alkyl-D-cysteine represented by the general formula (4) is not particularly limited as long as it is a practically acceptable salt. For example, inorganic acids such as hydrochloric acid and sulfuric acid, formic acid, Examples include organic acids such as acetic acid, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium and calcium, organic alkalis such as trimethylamine and tetramethylammonium, and ammonia. Hydrochloride and sulfate are particularly preferable in terms of the stability of the resulting 2-alkylcysteine.
本発明の一般式（４）で示される光学活性２−アルキル−D−システイン又はその塩は、一般式（１）で示される２−アルキルシステインアミドのアミド結合を生化学的に不斉加水分解して一般式（２）で示される光学活性２−アルキル−L−システインを生成せしめ、一般式（３）で示される未反応の光学活性２−アルキル−D−システインアミドを回収した後、さらにそのアミド結合の加水分解を行うことにより製造することができる。本発明で使用する一般式（１）で示される２−アルキルシステインアミドは、その製法及び品質等に特に制限はなく、例えば４−アルキルチアゾリジンカルボン酸アミド誘導体を加水分解する方法などによって得ることができる。 The optically active 2-alkyl-D-cysteine or a salt thereof represented by the general formula (4) of the present invention biochemically asymmetrically hydrolyzes the amide bond of the 2-alkylcysteine amide represented by the general formula (1). Then, an optically active 2-alkyl-L-cysteine represented by the general formula (2) was produced, and an unreacted optically active 2-alkyl-D-cysteine amide represented by the general formula (3) was recovered. It can be produced by hydrolysis of the amide bond. The 2-alkylcysteine amide represented by the general formula (1) used in the present invention is not particularly limited in its production method and quality, and can be obtained by, for example, a method of hydrolyzing a 4-alkylthiazolidinecarboxylic acid amide derivative. it can.
本発明の一般式（１）で示される２−アルキルシステインアミドの生化学的不斉加水分解に使用される微生物は、２−アルキル−L−システインアミドのアミド結合を立体選択的に加水分解する活性を有する微生物であればよく、このような微生物として例えば、キサントバクター属、プロタミノバクター属及びミコプラナ属等に属する微生物、具体的にはキサントバクター フラバス（Ｘａｎｔｈｏｂａｃｔｅｒ ｆｌａｖｕｓ）ＮＣＩＢ １００７１、プロタミノバクター アルボフラバス（Ｐｒｏｔａｍｉｎｏｂａｃｔｅｒ ａｌｂｏｆｌａｖｕｓ）ＡＴＣＣ８４５８、ミコプラナ ラモサ（Ｍｙｃｏｐｌａｎａ ｒａｍｏｓｅ）ＮＣＩＢ９４４０、ミコプラナ ディモルファ（Ｍｙｃｏｐｌａｎａ ｄｉｍｏｒｐｈａ）ＡＴＣＣ４２７９が挙げられるが、これらに限定されるものではない。また、これら微生物から人工的変異手段によって誘導される変異株、或いは細胞融合若しくは遺伝子組換え法等の遺伝学的手法により誘導される組換え株等の何れの株であっても上記能力を有するものであれば、本発明に使用できる。 The microorganism used for the biochemical asymmetric hydrolysis of 2-alkylcysteine amide represented by the general formula (1) of the present invention stereoselectively hydrolyzes the amide bond of 2-alkyl-L-cysteine amide. Any microorganism having activity may be used. Examples of such microorganisms include microorganisms belonging to the genus Xantobacter, Protaminobacter, and Mycoplana, such as Xantobacter flavus NCIB 10071, Pro Taminobacter alboflavus ATCC 8458, Mycoplana ramosa NCIB 9440, Mycoplana dimorpha ATCC 4279 However, it is not limited to these. In addition, any strains such as mutant strains derived from these microorganisms by artificial mutation means or recombinant strains derived by genetic techniques such as cell fusion or genetic recombination have the above-mentioned ability. Anything can be used in the present invention.
これらの微生物の培養は、通常資化し得る炭素源、窒素源、各微生物に必須の無機塩、栄養等を含有させた培地を用いて行われる。培養時のｐHは、４〜１０の範囲であり、温度は２０〜５０℃である。培養は1日〜１週間程度好気的に行われる。このようにして培養した微生物は、生菌体又は該生菌体処理物、例えば培養液、分離菌体、菌体破砕物、さらには精製した酵素として反応に使用される。また、常法に従って菌体又は酵素を固定化して使用することもできる。 These microorganisms are usually cultured using a medium containing a carbon source, a nitrogen source, an inorganic salt essential for each microorganism, nutrients, and the like that can be assimilated. The pH during the culture is in the range of 4 to 10, and the temperature is 20 to 50 ° C. The culture is performed aerobically for about 1 day to 1 week. The microorganisms cultured in this manner are used for the reaction as viable cells or treated products of the viable cells, such as culture broth, separated cells, disrupted cells, and purified enzymes. In addition, cells or enzymes can be immobilized and used according to a conventional method.
２−アルキルシステインアミドの生化学的不斉加水分解反応の条件は、２−アルキルシステインアミド濃度０．１〜４０ｗｔ％、２−アルキルシステインアミドに対する微生物の使用量は、乾燥菌体として重量比０．０００１〜３、反応温度１０〜７０℃、ｐH４〜１３の範囲であることが好ましい。なお、２−アルキルシステインアミドの濃度が高い場合には、前記微生物の使用量比が、好ましい範囲の上限である３以下であって、反応が好適に実施できる比率を適宜選択すればよい。本反応に用いる２−アルキルシステインアミドや生成する２−アルキルシステインは酸化を受けやすく、酸素存在下で放置すると2量化したジスルフィド（２，２’−ジアルキルシスチン誘導体）となる。これを防止するため、生化学的不斉加水分解反応は防酸化雰囲気で行なうのが好ましい。ここで言う防酸化雰囲気とは、窒素、アルゴン等の不活性ガス雰囲気、或いは反応系内に２−メルカプトエタノール等の還元性物質を共存させる方法を示す。反応を行う際、さらにＭｇ、Ｃｕ、Ｚｎ、Ｆｅ、Ｍｎ、Ｎｉ、Ｃｏ等の金属イオンを酵素触媒の活性化剤として添加してもよい。添加する量は使用する培養菌体の菌株の種類、添加する金属イオンの種類によって異なり一概には言えないが、通常は１〜５０ｐｐｍとなる濃度の金属イオンを添加することにより、不斉加水分解速度を向上させることができる。例えば、２価のＭｎイオンを５〜２０ｐｐｍ加えた場合、反応速度は、無添加の場合に比較して２〜５倍と大幅に向上する。 The conditions for the biochemical asymmetric hydrolysis reaction of 2-alkylcysteine amide were as follows: the concentration of 2-alkylcysteine amide was 0.1 to 40 wt%, and the amount of microorganisms used relative to 2-alkylcysteine amide was 0 by weight as dry cells. It is preferable that they are 0.0001-3, reaction temperature 10-70 degreeC, and the range of pH 4-13. When the concentration of 2-alkylcysteine amide is high, the use amount ratio of the microorganism is 3 or less, which is the upper limit of the preferred range, and the ratio at which the reaction can be suitably carried out may be appropriately selected. The 2-alkylcysteine amide used in this reaction and the 2-alkylcysteine produced are susceptible to oxidation, and when left in the presence of oxygen, dimerized disulfide (2,2'-dialkylcystine derivative) is obtained. In order to prevent this, the biochemical asymmetric hydrolysis reaction is preferably performed in an antioxidant atmosphere. The term “antioxidation atmosphere” as used herein refers to an inert gas atmosphere such as nitrogen or argon, or a method in which a reducing substance such as 2-mercaptoethanol coexists in the reaction system. When performing the reaction, metal ions such as Mg, Cu, Zn, Fe, Mn, Ni, and Co may be further added as an activator for the enzyme catalyst. The amount to be added varies depending on the type of strain of cultured cells used and the type of metal ions to be added, but it cannot be generally stated, but it is usually asymmetric hydrolysis by adding metal ions at a concentration of 1 to 50 ppm. Speed can be improved. For example, when divalent Mn ions are added in an amount of 5 to 20 ppm, the reaction rate is significantly improved to 2 to 5 times that in the case of no addition.
２−アルキルシステインアミドの生化学的不斉加水分解反応で生成した光学活性２−アルキル−L−システインと、本発明の目的物である未反応の光学活性２−アルキル−D−システインアミドとの分離は、反応終了液から、例えば遠心分離或いは濾過膜などの通常の固液分離手段により微生物菌体を除いた母液を、ｐH７〜１４、好ましくはｐH８〜１２に調整した後、非水溶性有機溶媒で抽出し、その抽出液を濃縮すると光学活性２−アルキル−D−システインアミドが得られる。この際に用いる非水溶性有機溶媒としては特に限定されず、目的とする２−アルキル−Ｄ−システインアミドの溶解性を見て決定されれば良く、通常に用いられるジエチルエーテルやジイソプロピルエーテル等のエーテル類、塩化メチレンやクロロホルム等のハロゲン化炭化水素等が使用可能であるが、中でも２−アルキル−Ｄ−システインアミドの溶解性の点でジエチルエーテルやジイソプロピルエーテル等のエーテル類、塩化メチレン等のハロゲン化炭化水素類が好適に用いられる。得られた２−アルキル−Ｄ−システインアミドは、その物性に合わせて再結晶やカラムクロマトグラフィー等の通常に行なわれる精製方法で精製できる。また、この抽出操作や精製操作においても酸化を防止するために、窒素、アルゴン等の不活性ガス雰囲気で操作することが好ましい。 An optically active 2-alkyl-L-cysteine produced by a biochemical asymmetric hydrolysis reaction of 2-alkylcysteine amide and an unreacted optically active 2-alkyl-D-cysteine amide which is an object of the present invention Separation is carried out by adjusting the mother liquor from which the microbial cells have been removed from the reaction-finished liquid to a pH of 7 to 14, preferably pH 8 to 12, by a normal solid-liquid separation means such as centrifugation or filtration membrane, and then to a water-insoluble organic solvent. Extraction with a solvent and concentration of the extract give optically active 2-alkyl-D-cysteine amide. The water-insoluble organic solvent used in this case is not particularly limited, and may be determined in view of the solubility of the target 2-alkyl-D-cysteine amide, such as commonly used diethyl ether and diisopropyl ether. Ethers, halogenated hydrocarbons such as methylene chloride and chloroform can be used. Among them, ethers such as diethyl ether and diisopropyl ether, methylene chloride and the like are particularly preferable in terms of solubility of 2-alkyl-D-cysteine amide. Halogenated hydrocarbons are preferably used. The obtained 2-alkyl-D-cysteine amide can be purified by a conventional purification method such as recrystallization or column chromatography according to its physical properties. In order to prevent oxidation in this extraction operation and purification operation, it is preferable to operate in an inert gas atmosphere such as nitrogen or argon.
次に、得られた2−アルキル−Ｄ−システインアミドの水溶液を窒素、アルゴン等の不活性ガス雰囲気下で、酸触媒を用いて加熱還流すると、ほぼ定量的に加水分解反応が進行して、対応する２−アルキル−Ｄ−システインが得られる。この際に用いられる酸としては、通常に加水分解触媒として用いられる一般的な酸で特に問題はないが、揮発性の酸である塩酸が反応後の処理が容易であり、コスト的にも安価であることから好適である。また酸の使用量は、２−アルキル−Ｄ−システインアミドに対して同等モル以上であればよく、上限は限定されないが、過剰の酸の後処理などに掛かるコスト的な観点から、好ましくは２〜１５倍モルの範囲が好適である。このようにして得られた反応液から過剰の酸、アンモニウム塩を除去すると目的とする２−アルキル−Ｄ−システインと使用した酸の塩が得られる。過剰の酸及びアンモニウム塩と、目的とする２−アルキル−Ｄ−システインの分離は、再結晶や脱塩透析、イオン交換樹脂等、一般的に用いられる手法で適宜実施されれば良く、その方法は限定されない。 Next, when the obtained aqueous solution of 2-alkyl-D-cysteine amide is heated and refluxed using an acid catalyst in an inert gas atmosphere such as nitrogen or argon, the hydrolysis reaction proceeds almost quantitatively. The corresponding 2-alkyl-D-cysteine is obtained. The acid used at this time is a general acid that is usually used as a hydrolysis catalyst, but there is no particular problem. However, hydrochloric acid, which is a volatile acid, is easy to process after the reaction, and is inexpensive in terms of cost. Therefore, it is preferable. The amount of acid used may be equal to or greater than that of 2-alkyl-D-cysteine amide, and the upper limit is not limited, but is preferably 2 from the viewpoint of cost for excessive acid post-treatment. A range of ˜15 times mole is preferred. When the excess acid and ammonium salt are removed from the reaction solution thus obtained, the target 2-alkyl-D-cysteine and the acid salt used are obtained. Separation of the excess acid and ammonium salt from the desired 2-alkyl-D-cysteine may be carried out as appropriate by commonly used techniques such as recrystallization, desalting dialysis, and ion exchange resin. Is not limited.
本発明の方法によって得られる光学活性２−アルキル-Dーシステインの具体例を挙げるならば、例えば２−メチル−D−システイン、２−エチル−D−システイン等の光学活性２−アルキル−D−システインがある。 Specific examples of the optically active 2-alkyl-D-cysteine obtained by the method of the present invention include optically active 2-alkyl-D-cysteine such as 2-methyl-D-cysteine and 2-ethyl-D-cysteine. There is.
次に実施例を挙げ、本発明について更に詳細に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
実施例１
次の組成を有する培地を調製し、この培地２００ｍＬを１Ｌの三角フラスコに入れ、滅菌後、キサントバクター フラバス（Ｘａｎｔｈｏｂａｃｔｅｒ ｆｌａｖｕｓ）ＮＣＩＢ １００７１を接種し、３０℃で４８時間振とう培養を行い、培養液から、遠心分離により乾燥菌体１．０ｇに相当する生菌体を得た。
培地組成（ｐH７．０）
グルコース １０ｇ
ポリペプトン ５ｇ
酵母エキス ５ｇ
KH2PO4 １ｇ
MgSO4・７H2O ０．４ｇ
FeSO4・７H2O ０．０１ｇ
MnCl2・４H2O ０．０１ｇ
水 １Ｌ
次いでラセミ体の２−メチルシステインアミド塩酸塩１０．０ｇ（０．０５９ｍｏｌ）を水３００ｍＬに溶かした後、５００ｍＬフラスコに入れ、乾燥菌体１．０ｇに相当する生菌体を加えて、窒素気流下、４０℃で２４時間攪拌して加水分解反応を行った。反応後、反応液から遠心分離によって菌体を除去して上清を得た。この上清液に、脱気後に不活性ガス置換した活性炭２ｇを加えて２時間攪拌した後活性炭を濾別した。濾液に４Ｎ水酸化ナトリウム水溶液３０ｍＬを加えて、ｐＨ１０とした後、２００ｍＬのジエチルエーテルで５回抽出した有機相を無水硫酸ナトリウムで５時間乾燥し、沈殿物を濾別した濾液をエバポレーターで濃縮して白色ペースト状固体を得た。このペースト状固体を少量のエタノールを用いて窒素雰囲気下で再結晶し、結晶３．５ｇ（０．０２６ｍｏｌ）濾取した。得られた２−メチル−Ｄ−システインアミド３．５ｇ（０．０２６ｍｏｌ）を濃塩酸（３６％）２０ｍＬに溶解し、窒素気流下で１時間加熱還流した。反応液をロータリーエバポレーターで濃縮乾固した後、得られた固体をエタノールで再結晶させた。得られた結晶は真空乾燥を行い、２−メチル−Ｄ−システイン塩酸塩 ４．３ｇ（０．０２５ｍｏｌ）の白色結晶を得た。反応に仕込んだラセミ混合物中の２−メチル−D−システインアミド塩酸塩からの単離収率は８３ｍｏｌ％、２−メチル−D−システインアミドのラセミ混合物からの単離収率は４２ｍｏｌ％であった。得られた結晶を学異性体分離カラムを用いた液体クロマトグラフィーによって分析した結果、光学純度は９７％ｅ．ｅ．以上であった。
Example 1
A medium having the following composition was prepared, 200 mL of this medium was placed in a 1 L Erlenmeyer flask, sterilized, inoculated with Xantobacter flavus NCIB 10071, and cultured with shaking at 30 ° C. for 48 hours. From the liquid, live cells corresponding to 1.0 g of dried cells were obtained by centrifugation.
Medium composition (pH 7.0)
Glucose 10g
Polypeptone 5g
Yeast extract 5g
KH 2 PO 4 1g
MgSO 4・ 7H 2 O 0.4g
FeSO 4・ 7H 2 O 0.01g
MnCl 2 · 4H 2 O 0.01g
1L of water
Next, 10.0 g (0.059 mol) of racemic 2-methylcysteine amide hydrochloride was dissolved in 300 mL of water, then placed in a 500 mL flask, and the viable cells corresponding to 1.0 g of dry cells were added, and a nitrogen stream was added. Then, the hydrolysis reaction was carried out by stirring at 40 ° C. for 24 hours. After the reaction, the cells were removed from the reaction solution by centrifugation to obtain a supernatant. To this supernatant liquid, 2 g of activated carbon substituted with inert gas after deaeration was added and stirred for 2 hours, and then the activated carbon was filtered off. The filtrate was adjusted to pH 10 by adding 30 mL of 4N aqueous sodium hydroxide solution, and the organic phase extracted five times with 200 mL of diethyl ether was dried over anhydrous sodium sulfate for 5 hours. The filtrate obtained by filtering the precipitate was concentrated with an evaporator. A white pasty solid was obtained. This pasty solid was recrystallized using a small amount of ethanol under a nitrogen atmosphere, and 3.5 g (0.026 mol) of crystals were collected by filtration. 3.5 g (0.026 mol) of the obtained 2-methyl-D-cysteine amide was dissolved in 20 mL of concentrated hydrochloric acid (36%), and heated to reflux for 1 hour under a nitrogen stream. The reaction solution was concentrated to dryness with a rotary evaporator, and the obtained solid was recrystallized with ethanol. The obtained crystals were vacuum-dried to obtain 4.3 g (0.025 mol) of white crystals of 2-methyl-D-cysteine hydrochloride. The isolated yield from 2-methyl-D-cysteine amide hydrochloride in the racemic mixture charged in the reaction was 83 mol%, and the isolated yield from the racemic mixture of 2-methyl-D-cysteine amide was 42 mol%. It was. As a result of analyzing the obtained crystals by liquid chromatography using a column for separation of isomers, the optical purity was 97% e.e. e. That was all.
Claims (3)
（一般式（１）、（２）、（３）、及び（４）中のＲは炭素数１〜４の低級アルキル基である。） The 2-alkylcysteine amide represented by the general formula (1) is allowed to act on a microbial cell or a treated product of a microorganism having an activity of stereoselectively hydrolyzing the amide bond of 2-alkyl-L-cysteine amide. And a mixture of 2-alkyl-L-cysteine represented by general formula (2) as a reaction product and optically active 2-alkyl-D-cysteine amide represented by general formula (3) as an unreacted product. The optically active 2-alkyl-D-cysteine amide is fractionated from this mixture, and then the amide bond is hydrolyzed to obtain the optically active 2-alkyl-D-cysteine represented by the general formula (4). A method for producing optically active 2-alkyl-D-cysteine or a salt thereof from 2-alkylcysteine amide.
(R in the general formulas (1), (2), (3), and (4) is a lower alkyl group having 1 to 4 carbon atoms.)
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