JP2005170794A - Optically active 2-alkyl-d-cysteineamide or its salt and method for producing these compounds - Google Patents
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本発明は、一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩、及びこれらの化合物を製造する方法に関する。詳しくは、一般式１で示される光学活性２−アルキル−Ｄ−システインアミド又はその塩と、一般式３で示される光学活性２−アルキル−Ｌ−システインアミド又はその塩とからなる、一般式２で示される２−アルキルシステインアミド又はその塩に、一般式３で示される光学活性２−アルキル−L−システインアミド又はその塩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生化学的に不斉加水分解し、一般式４で示される光学活性２−アルキル−L−システイン又はその塩に変換した後、未反応の一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩を分離回収することを特徴とする、光学活性２−アルキル−D−システインアミド又はその塩、及びこれらの化合物を製造する方法に関する。光学活性２−アルキル−D−システインアミド又はその塩は、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。 The present invention relates to an optically active 2-alkyl-D-cysteine amide represented by the general formula 1 or a salt thereof, and a method for producing these compounds. Specifically, the optically active 2-alkyl-D-cysteine amide represented by the general formula 1 or a salt thereof, and the optically active 2-alkyl-L-cysteine amide represented by the general formula 3 or a salt thereof, A microbial cell or microbial cell having an activity of stereoselectively hydrolyzing the optically active 2-alkyl-L-cysteine amide represented by the general formula 3 or a salt thereof to the 2-alkyl cysteine amide represented by the formula (1) or a salt thereof A chemical product is asymmetrically hydrolyzed by the action of the treated product, converted to an optically active 2-alkyl-L-cysteine or a salt thereof represented by the general formula 4, and then an optical activity represented by the unreacted general formula 1. Optically active 2-alkyl-D-cysteine amide or a salt thereof, and a method for producing these compounds, characterized by separating and recovering 2-alkyl-D-cysteine amide or a salt thereof About. Optically active 2-alkyl-D-cysteine amide or a salt thereof is an important substance as a production intermediate for various industrial chemicals, agricultural chemicals, and pharmaceuticals.
一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩は、分子内にメルカプト基、アミノ基及びカルボキシルアミド基を有し、かつ自然界にまれにしか存在しないD体であることから、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る化合物であり、産業上、非常に有用な化合物である。アミノ酸アミドからアミノ酸を製造するにあたって微生物の菌体又は菌体処理物を利用する方法については多く報告されているが（例えば、特許文献１参照）、α位にアルキル基を有するアミノ酸アミドの製造方法はあまり知られておらず、特に、一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩、及びこれらの化合物を製造する方法は知られていない。
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１で示される光学活性２−アルキル−D−システインアミド又はその塩、及びこれらの化合物の製造方法を提供することにある。 The object of the present invention is an optically active 2-alkyl-D-cysteine represented by the general formula 1, 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. An object of the present invention is to provide an amide or a salt thereof and a method for producing these compounds.
かかる実状に鑑み、本発明者らは光学活性２−アルキル−D−システインアミド又はその塩の工業的に実施可能な製造方法について鋭意研究を行ったところ、一般式２で示される２−アルキルシステインアミド又はその塩中に含まれる一般式３で示される光学活性２−アルキル−Ｌ−システインアミド又はその塩を生化学的に不斉加水分解して、一般式４で示される光学活性２−アルキル−L−システイン又はその塩に変換した後、未反応の光学活性２−アルキル−D−システインアミド又はその塩を分離回収することにより、光学活性２−アルキル−D−システインアミド又はその塩を製造する方法を見出し、本発明に到達した。 In view of this situation, the present inventors have conducted extensive research on an industrially feasible production method of optically active 2-alkyl-D-cysteine amide or a salt thereof. As a result, 2-alkylcysteine represented by the general formula 2 is obtained. An optically active 2-alkyl represented by the general formula 4 is obtained by biochemical asymmetric hydrolysis of the optically active 2-alkyl-L-cysteine amide represented by the general formula 3 contained in the amide or a salt thereof. -After conversion into L-cysteine or a salt thereof, unreacted optically active 2-alkyl-D-cysteine amide or a salt thereof is separated and recovered to produce optically active 2-alkyl-D-cysteine amide or a salt thereof. The present inventors have reached the present invention.
即ち、本発明は、以下の（１）〜（４）に示す、一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩、及びこれらの化合物を製造する方法に関するものである。
（１）一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩。
（一般式１中のＲは炭素数１〜４の低級アルキル基を示す。）
（２）一般式１で示される光学活性２−アルキル−Ｄ−システインアミド又はその塩と、一般式３で示される光学活性２−アルキル−Ｌ−システインアミド又はその塩とからなる、一般式２で示される２−アルキルシステインアミド又はその塩に、一般式３で示される光学活性２−アルキル−L−システインアミド又はその塩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、一般式４で示される光学活性２−アルキル−L−システイン又はその塩に変換した後、該反応液より未反応の一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩を分離回収することを特徴とする、光学活性２−アルキル−D−システインアミド又はその塩の製造方法。
（一般式２〜４中のＲは炭素数１〜４の低級アルキル基を示す。一般式２は、一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩と一般式３で示される光学活性２−アルキル−L−システインアミド又はその塩との混合物である２−アルキルシステインアミド又はその塩を示す。）
（３）一般式３で示される光学活性２−アルキル−Ｌ−システインアミド又はその塩を立体選択的に加水分解し、一般式４で示される光学活性２−アルキル−L−システイン又はその塩に変換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、ミコバクテリウム属、ミコプラナ属に属する細菌である、（２）に記載の光学活性２−アルキル−D−システインアミド又はその塩の製造方法。
（４）一般式１〜４に示すＲがメチル基である、（１）〜（３）に記載の光学活性２−アルキル−D−システインアミド又はその塩の製造方法。
That is, the present invention relates to an optically active 2-alkyl-D-cysteine amide represented by the general formula 1 or a salt thereof shown in the following (1) to (4), and a method for producing these compounds. .
(1) An optically active 2-alkyl-D-cysteine amide represented by the general formula 1 or a salt thereof.
(R in general formula 1 represents a lower alkyl group having 1 to 4 carbon atoms.)
(2) General formula 2 comprising optically active 2-alkyl-D-cysteine amide or a salt thereof represented by general formula 1 and optically active 2-alkyl-L-cysteine amide or a salt thereof represented by general formula 3 A microbial cell or microbial cell having an activity of stereoselectively hydrolyzing the optically active 2-alkyl-L-cysteine amide represented by the general formula 3 or a salt thereof to the 2-alkyl cysteine amide represented by the formula (1) or a salt thereof The treated product is allowed to act to convert to an optically active 2-alkyl-L-cysteine or a salt thereof represented by the general formula 4, and then the optically active 2-alkyl-D represented by the general formula 1 unreacted from the reaction solution. A method for producing optically active 2-alkyl-D-cysteine amide or a salt thereof, characterized by separating and recovering cysteine amide or a salt thereof.
(R in general formulas 2 to 4 represents a lower alkyl group having 1 to 4 carbon atoms. General formula 2 represents optically active 2-alkyl-D-cysteine amide represented by general formula 1 or a salt thereof and general formula 3 And 2-alkylcysteine amide or a salt thereof which is a mixture with the optically active 2-alkyl-L-cysteine amide or a salt thereof represented by
(3) The optically active 2-alkyl-L-cysteine amide or a salt thereof represented by the general formula 3 is stereoselectively hydrolyzed to form an optically active 2-alkyl-L-cysteine or a salt thereof represented by the general formula 4. The optically active 2-alkyl-D-cysteine amide according to (2) or a bacterium belonging to the genus Xantobacter, Protaminobacter, Mycobacterium, Mycoplana Method for producing salt.
(4) The method for producing an optically active 2-alkyl-D-cysteine amide or a salt thereof according to (1) to (3), wherein R shown in the general formulas 1 to 4 is a methyl group.
各種工業薬品、農薬、及び医薬品の製造原料として幅広い応用用途が期待され、産業上、高い利用価値を有する一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩、及びこれら化合物の製造方法を提供することができる。 Optically active 2-alkyl-D-cysteine amide or a salt thereof represented by the general formula 1, which is expected to have a wide range of applications as a raw material for producing various industrial chemicals, agricultural chemicals, and pharmaceuticals, and has high industrial utility value, and compounds thereof The manufacturing method of can be provided.
以下、本発明をさらに詳細に説明する。本発明の目的物質である一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩に於いて、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。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−ブチル及びt−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。 Hereinafter, the present invention will be described in more detail. In the optically active 2-alkyl-D-cysteine amide or a salt thereof represented by the general formula 1, which is the target substance of the present invention, R in the formula may be any lower alkyl group having 1 to 4 carbon atoms, and is particularly limited. There is no. For example, linear or branched lower alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and t-butyl are preferred, and the methyl group is particularly preferred.
また、一般式１で示される光学活性２−アルキル−Ｄ−システインアミドは塩を形成することもできるが、その種類は、実用上許容できる塩であればよく特に制限はない。例えば塩酸や硫酸等の無機酸、ギ酸や酢酸等の有機酸等の塩が挙げられる。 The optically active 2-alkyl-D-cysteine amide represented by the general formula 1 can also form a salt, but the type thereof is not particularly limited as long as it is a salt that is practically acceptable. Examples thereof include salts of inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as formic acid and acetic acid.
本発明の一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩は、一般式２で示される２−アルキルシステインアミド又はその塩の中に含まれている一般式３で示される光学活性２−アルキル−Ｌ−システインアミド又はその塩を生化学的に不斉加水分解して、一般式４で示される光学活性２−アルキル−L−システイン又はその塩に変換した後、残された未反応の光学活性２−アルキル−D−システインアミド又はその塩を分離回収することによって製造することができる。 The optically active 2-alkyl-D-cysteine amide or a salt thereof represented by the general formula 1 of the present invention is represented by the general formula 3 contained in the 2-alkyl cysteine amide or a salt thereof represented by the general formula 2. An optically active 2-alkyl-L-cysteine amide or a salt thereof is biochemically asymmetrically hydrolyzed to convert it into an optically active 2-alkyl-L-cysteine or a salt thereof represented by the general formula 4; It can be produced by separating and recovering the unreacted optically active 2-alkyl-D-cysteine amide or its salt.
本発明で使用する一般式２で示される２−アルキルシステインアミド又はその塩の製法
及び品質等に特に制限はないが、これを原料として行うそれ以降の反応が好適に進むものであればよい。そのような２−アルキルシステインアミド又はその塩は、例えばＪｕｓｔｕｓ Ｌｉｅｂｉｇｓ Ａｎｎ．Ｃｈｅｍ．（１９６６），６９７，１４０−１５７に記載された方法を応用することによって合成される４−アルキルチアゾリジンカルボン酸アミド又はその塩を加水分解することによって得ることができる。
Although there is no restriction | limiting in particular in the manufacturing method and quality, etc. of 2-alkyl cysteine amide or its salt shown by General formula 2 used by this invention, The subsequent reaction performed using this as a raw material should just advance suitably. Such 2-alkylcysteine amides or salts thereof are described, for example, in Justus Liebigs Ann. Chem. (1966), 697, 140-157, and can be obtained by hydrolyzing a 4-alkylthiazolidinecarboxylic acid amide or a salt thereof synthesized.
本発明の一般式２で示される２−アルキルシステインアミド又はその塩中に含まれる、一般式３で示される２−アルキル−L−システインアミド又はその塩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−システインアミド又はその塩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、プロタミノバクター属、ミコバクテリウム属、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）ＮＣＩＢ１００７１Ｔ、プロタミノバクター アルボフラバス（Ｐｒｏｔａｍｉｎｏｂａｃｔｅｒ ａｌｂｏｆｌａｖｕｓ）ＡＴＣＣ８４５８、ミコバクテリウム メタノリカ（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｍｅｔｈａｎｏｌｉｃａ）ＢＴ−８４（ＦＥＲＭ Ｐ８８２３）、ミコバクテリウム メタノリカ（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｍｅｔｈａｎｏｌｉｃａ）Ｐ−２３（ＦＥＲＭ Ｐ８８２５）、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、或いは細胞融合又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。 It is used for biochemical asymmetric hydrolysis of 2-alkyl-L-cysteine amide represented by the general formula 3 or a salt thereof contained in the 2-alkyl cysteine amide represented by the general formula 2 or a salt thereof according to the present invention. Any microorganism may be used as long as it has an activity of stereoselectively hydrolyzing 2-alkyl-L-cysteine amide or a salt thereof. Examples of such microorganisms include Protaminobacter and Mycobacterium. , And microorganisms belonging to the genus Mycoplana, such as Xanthobacter Flabus NCIB10071T, Protaminobacter alboflavus ATCC 8458, Mycobacterium metallica (Mycobacterium medium) lica) BT-84 (FERM P8823), Mycobacterium methanolica P-23 (FERM P8825), Mycoplana ramosa NIB9440, Mycoplana dimorpha (Myco42 It is not something. 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、栄養素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の処理物、例えば菌体培養液、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, nitrogen source, inorganic salts essential to each microorganism, nutrients, and the like that can be assimilated. The pH during culture is in the range of 4-10, and the temperature is 20-50 ° C. The culture is performed aerobically for about 1 day to 1 week. The microorganisms cultured in this way are used for the reaction as viable cells or treated products of the viable cells, for example, cell culture broth, separated cells, cell disrupted products, and purified enzymes. In addition, cells or enzymes can be immobilized and used according to a conventional method.
一般式３で示される２−アルキル−Ｌ−システインアミド又はその塩を含んだ一般式２で示される原料の２−アルキルシステインアミドを、微生物の菌体又は菌体処理物を用いて生化学的に不斉加水分解する際の反応条件は以下の通りである。原料である２−アルキルシステインアミド又はその塩の濃度は、該生体触媒あたりの反応効率及び操作性の容易さの面から０．１〜４０ｗｔ％の範囲が好ましく、０．５〜２０ｗｔ％の範囲がより好ましい。２−アルキルシステインアミド又はその塩の濃度が０．１ｗｔ％を下回る場合は反応液の容量が単に増えるだけで、生産性の面から不利となる。一方、濃度が４０ｗｔ％を超える場合は、基質阻害が生じ、菌体又は菌体処理物当たりの生産性の面から不利となるうえ、場合によっては反応産物である２−アルキル−L−システイン又はその塩が反応途中で析出するようになるため、反応後に行う菌体又は菌体処理物を遠心分離や濾過分離する際の随伴ロスとして失われる原因ともなり、貴重な原料を有効に活用するうえで不利となる。 The raw material 2-alkylcysteine amide represented by the general formula 2 containing the 2-alkyl-L-cysteine amide represented by the general formula 3 or a salt thereof is biochemically obtained by using a microbial cell or a treated microbial cell. The reaction conditions for the asymmetric hydrolysis are as follows. The concentration of the raw material 2-alkylcysteine amide or a salt thereof is preferably in the range of 0.1 to 40 wt%, and in the range of 0.5 to 20 wt% from the viewpoint of the reaction efficiency per biocatalyst and ease of operation. Is more preferable. When the concentration of 2-alkylcysteine amide or a salt thereof is less than 0.1 wt%, the volume of the reaction solution simply increases, which is disadvantageous in terms of productivity. On the other hand, when the concentration exceeds 40 wt%, substrate inhibition occurs, which is disadvantageous in terms of productivity per cell or treated cell, and in some cases, the reaction product 2-alkyl-L-cysteine or Since the salt precipitates during the reaction, it may be lost as a concomitant loss when centrifuging or treating the microbial cells after the reaction is centrifuged or filtered. Is disadvantageous.
一般式２で示される原料の２−アルキルシステインアミド又はその塩に対する微生物の菌体又は菌体処理物の使用量は、供給源の培養菌体レベルでの重量が乾燥菌体換算で重量比０．０００１〜３の範囲になるように添加することが好ましく、０．００１〜１の範囲になるようにすることがより好ましい。重量比が０．０００１を下回る場合は反応速度が遅いため処理に長時間を要することになり、３を超える場合は、処理時間は短くなるものの、微生物菌体の利用の面から効率的とは言えず、しかも反応後の菌体又は菌体処理物の分離に労力を要することとなり不利となる。なお、原料の２−アルキルシステインアミド又はその塩の反応液中における濃度が高い場合には、前記の菌体又は菌体処理物の使用量比が、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。 The amount of the microbial cell or the treated product of the microorganism relative to the raw material 2-alkylcysteine amide or a salt thereof represented by the general formula 2 is such that the weight at the culture cell level of the source is 0 by weight in terms of dry cells. It is preferable to add so that it may become the range of 0.0001-3, and it is more preferable to make it become the range of 0.001-1. If the weight ratio is less than 0.0001, the reaction rate is slow, so it takes a long time to process. If it exceeds 3, the treatment time is shortened, but it is efficient from the viewpoint of the use of microbial cells. In addition, it is disadvantageous because it requires labor to separate the cells or treated cells after the reaction. In addition, when the concentration of the raw material 2-alkylcysteine amide or a salt thereof is high in the reaction solution, the use amount ratio of the cells or the treated cells is 3 or less, which is the upper limit of the preferred range. The ratio at which the reaction can be suitably carried out may be appropriately selected.
反応温度は１０〜７０℃の範囲が好ましく、２０〜４０℃の範囲がより好ましい。反応温度が１０℃を下回る場合は反応速度が遅いため反応時間が長くなり不利となる。一方、反応温度が７０℃を超える場合は、菌体又は菌体処理物の触媒活性が失活により低下するとともに、目的物質である２−アルキル−D−システインアミド又はその塩の非酵素的な分解も伴うようになり、反応収率及び選択率の面で不利となる。また、工程間で必要となる反応溶液の昇温・冷却に多くのエネルギーを要することとなりコスト的に不利となる。 The reaction temperature is preferably in the range of 10 to 70 ° C, more preferably in the range of 20 to 40 ° C. When the reaction temperature is lower than 10 ° C., the reaction rate is slow and the reaction time becomes long, which is disadvantageous. On the other hand, when the reaction temperature exceeds 70 ° C., the catalytic activity of the microbial cell or the microbial cell treated product decreases due to deactivation, and non-enzymatic activity of the target substance 2-alkyl-D-cysteine amide or a salt thereof. Decomposition is also accompanied, which is disadvantageous in terms of reaction yield and selectivity. In addition, a large amount of energy is required to raise and cool the reaction solution required between the processes, which is disadvantageous in cost.
反応溶液はｐH４〜１３の水溶液が好ましく、ｐH５〜１０の範囲がより好ましい。よりさらには、ｐH７〜９の中性から比較的穏和な塩基性条件が特に好ましい。ｐH４を下回る場合は、菌体又は菌体処理物の触媒活性が低下し、ｐH１３を上回る場合も同様に触媒活性が低下する。そのうえさらに、反応液中に含まれる２−アルキル−Ｄ−システインアミド又はその塩、２−アルキル−Ｌ−システインアミド又はその塩、生成物である２−アルキル−Ｌ−システイン又はその塩同士が、ジスルフィド結合を形成し二量化するため好ましくない。また、２−アルキル−D−システインアミド又はその塩の非酵素的な加水分解反応も起こり易くなりこれも好ましくない。反応液のｐHを調整するに当たっては、水酸化ナトリウムや水酸化カリウム等の無機塩基類、及びアンモニア等を用いればよい。また、その他の物質を溶解してなる緩衝液を用いてもよい。さらに、触媒活性を増大させる目的で、Mg、Cu、Zn、Fe、Mn、Ｎｉ、Co等の金属イオンを存在させることにより反応速度を速めてもよい。 The reaction solution is preferably an aqueous solution having a pH of 4 to 13, and more preferably a pH of 5 to 10. Furthermore, neutral to relatively mild basic conditions of pH 7-9 are particularly preferred. When the pH is lower than 4, the catalytic activity of the microbial cells or the processed microbial cells is decreased, and when the pH is higher than 13, the catalytic activity is similarly decreased. Furthermore, 2-alkyl-D-cysteine amide or a salt thereof, 2-alkyl-L-cysteine amide or a salt thereof contained in the reaction solution, or 2-alkyl-L-cysteine or a salt thereof as a product, A disulfide bond is formed and dimerization is not preferable. In addition, non-enzymatic hydrolysis reaction of 2-alkyl-D-cysteine amide or a salt thereof easily occurs, which is not preferable. In adjusting the pH of the reaction solution, inorganic bases such as sodium hydroxide and potassium hydroxide, ammonia and the like may be used. Moreover, you may use the buffer solution which melt | dissolves another substance. Furthermore, for the purpose of increasing the catalytic activity, the reaction rate may be increased by the presence of metal ions such as Mg, Cu, Zn, Fe, Mn, Ni and Co.
生成した２−アルキル−L−システイン又はその塩と、本発明の目的物質である未反応の２−アルキル−D−システインアミド又はその塩との分離は、反応終了液から、例えば遠心分離や濾過膜などの通常の固液分離手段によって微生物菌体又は菌体処理物を除いた後、さらに必要に応じて活性炭を用いる等の処理を施して不純物を除いたうえ、その分離母液をｐH７〜１３、好ましくはｐH８〜１２に調整し、適当な有機溶媒で抽出しこれを濃縮することによって達成される。分離母液のｐHが７を下回る場合は、ｐＨが低いほど有機溶媒へ溶解しにくい２−アルキル−D−システインアミドの酸塩の構成比率が増加するため、抽出収率が低下し好ましくない。一方、ｐHが１３を上回る場合は、上記同様に二量体の形成や２−アルキル−D−システインアミド又はその塩の非酵素的な加水分解反応が起こり好ましくない。なお、抽出に用いる有機溶媒の種類は特に限定されず、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すれば良く、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、メチルイソプロピルケトンやメチルイソブチルケトン等のケトン類、或いは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。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。 Separation of the produced 2-alkyl-L-cysteine or a salt thereof from unreacted 2-alkyl-D-cysteine amide or a salt thereof which is the target substance of the present invention is performed by, for example, centrifuging or filtering from the reaction end solution. After removing microbial cells or treated cells by ordinary solid-liquid separation means such as a membrane, the impurities are removed by further treatment such as using activated carbon as necessary, and the separated mother liquor is adjusted to pH 7 to 13 , Preferably adjusted to pH 8-12, extracted with a suitable organic solvent and concentrated. When the pH of the separated mother liquor is less than 7, the constituent ratio of the acid salt of 2-alkyl-D-cysteine amide that is less soluble in an organic solvent increases as the pH is lower, which is not preferable because the extraction yield decreases. On the other hand, when the pH exceeds 13, formation of a dimer or non-enzymatic hydrolysis reaction of 2-alkyl-D-cysteine amide or a salt thereof occurs as described above, which is not preferable. The type of organic solvent used for the extraction is not particularly limited, and may be determined in view of the solubility of the target 2-alkyl-D-cysteine amide. Ethers such as diethyl ether and diisopropyl ether that are usually used Further, ketones such as methyl isopropyl ketone and methyl isobutyl ketone, or halogenated hydrocarbons such as methylene chloride and chloroform can be used. Of these, ethers such as diethyl ether and diisopropyl ether, and halogenated hydrocarbons such as methylene chloride are preferably used from the viewpoint of solubility of 2-alkyl-D-cysteine amide.
得られた光学活性２−アルキル−Ｄ−システインアミドを含む有機溶媒溶液は、その物性に合った方法を適宜用いて濃縮した後、メタノールやエタノールを溶媒に用いた再結晶等の精製方法を講じることによって精製できる。 The obtained organic solvent solution containing the optically active 2-alkyl-D-cysteine amide is concentrated using a method suitable for its physical properties, and then subjected to a purification method such as recrystallization using methanol or ethanol as a solvent. Can be purified.
なお、原料の２−アルキルシステインアミド又はその塩、反応後の２−アルキル−L−システイン又はその塩や２−アルキル−D−システインアミド又はその塩は構造内にメルカプト基を有することから酸化を受けやすく、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）となる。これを防止するため、菌体又は菌体処理物を用いた不斉加水分解反応から濃縮・精製に至る一連の製造工程は、窒素、アルゴン等の不活性ガス雰囲気下で行なうことが好ましく、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も可能である。また、反応に用いる全ての溶媒は、反応を実施前に脱気することにより、副生物の生成を防ぎ、より好適に反応を進めることができる。 The raw material 2-alkylcysteine amide or a salt thereof, 2-alkyl-L-cysteine or a salt thereof after reaction, or 2-alkyl-D-cysteine amide or a salt thereof has a mercapto group in the structure, so that it is oxidized. It is susceptible to dimerization (2,2′-dialkylcystine) when left in the presence of oxygen. In order to prevent this, a series of production steps from asymmetric hydrolysis reaction using bacterial cells or processed bacterial cells to concentration / purification is preferably performed in an inert gas atmosphere such as nitrogen or argon. A method in which a reducing substance such as 2-mercaptoethanol coexists is also possible. In addition, all the solvents used in the reaction can be degassed before the reaction, thereby preventing generation of by-products and allowing the reaction to proceed more suitably.
以下、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。
実施例１
１）菌体の調製
下記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て滅菌した後、キサントバクター フラバス（Ｘａｎｔｈｏｂａｃｔｅｒ ｆｌａｖｕｓ）ＮＣＩＢ １００７１Ｔを接種し、３０℃で４８時間振とう培養を行った。この培養液を遠心分離したところ、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
２）光学活性２−メチル−Ｄ−システインアミドの製造
ラセミ体の２−メチルシステインアミド塩酸塩１０．０ｇ（０．０６mol）を水３００ｍLに溶解した後、５００ｍLフラスコに入れ、乾燥菌体１．０ｇに相当する上記生菌体を加えて、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、遠心分離によって菌体を除去し、得られた上清液に、脱気したうえ窒素ガスで置換した活性炭２ｇを加えて2時間攪拌し不純物の吸着除去を行った。活性炭を濾別し、得られた濾液に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。集めた有機相に無水硫酸ナトリウムを加えて脱水をした後、これを濾別し、得られた濾液をエバポレーターで濃縮し白色のペースト状固体を得た。このペースト状濃縮物に、１Ｎ塩酸水溶液を３０ｍＬ加えて攪拌混合した後、再度エバポレーターを用いて濃縮乾固させた。得られた白色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、２−メチル−Ｄ−システインアミド塩酸塩の結晶を３．２ｇ（０．０２ｍｏｌ）濾取した。反応に仕込んだラセミ体混合物中の２−メチル−D−システインアミド塩酸塩からの単離収率は８１mol％、２−メチル−システインアミド塩酸塩からの単離収率は４１mol％であった。また、この結晶を光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９５％e.e.以上であった。
２−メチル−Ｄ−システインアミド塩酸塩；無色ガラス状固体（潮解性）、
１Ｈ−ＮＭＲ（９０MHｚ，Ｄ２Ｏ）δ［ｐｐｍ］ ３．１９（１Ｈ，ｄ，Ｊ１５．３Ｈｚ），２．９５（１Ｈ，ｄ，Ｊ１５．３Ｈｚ），１．６４（３Ｈ，ｓ）
１３Ｃ−ＮＭＲ（２２．６MHｚ，Ｄ２Ｏ）δ［ｐｐｍ］ １７３．７８（ｓ），６２．３１（ｓ），３１．７３（ｔ），２２．２１（ｑ）
ＩＲ［ｃｍ−１］（ＫＢｒ）１７０３，１６２４，１５７４，１５０６，１３７７，１２７９，１２３０，１１２４
元素分析 （測定値）C；２８．０１，H；６．６０，N；１６．３３，S;；１８．７２，Ｃｌ；２０．７５，（計算値）Ｃ；２８．１５，Ｈ；６．５０，Ｎ；１６．４１，Ｏ；９．３７，Ｓ；１８．７９，Ｃｌ；２０．７７
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
Example 1
1) Preparation of bacterial cells A medium having the following composition was prepared, and 200 mL of this medium was sterilized by placing it in a 1 L Erlenmeyer flask. Shaking culture was performed. When this culture solution was centrifuged, live cells corresponding to 1.0 g of dry cells were obtained.
Medium composition (pH 7.0)
Glucose 10g
Polypeptone 5g
Yeast extract 5g
KH2PO4 1g
MgSO4 ・ 7H2O 0.4g
FeSO4 ・ 7H2O 0.01g
MnCl2 ・ 4H2O 0.01g
1L of water
2) Production of optically active 2-methyl-D-cysteine amide After dissolving 10.0 g (0.06 mol) of racemic 2-methylcysteine amide hydrochloride in 300 mL of water, the solution was placed in a 500 mL flask and dried cells. The above viable cells corresponding to 0 g were added, and a hydrolysis reaction was performed by stirring at 40 ° C. for 24 hours under a nitrogen stream. After the reaction, the cells were removed by centrifugation, and 2 g of activated carbon that had been deaerated and replaced with nitrogen gas was added to the resulting supernatant and stirred for 2 hours to remove impurities by adsorption. Activated carbon was filtered off, and 30 mL of 4N aqueous sodium hydroxide solution was added to the resulting filtrate to adjust the pH to 10, followed by extraction with 200 mL of diethyl ether 5 times. The collected organic phase was dehydrated by adding anhydrous sodium sulfate and then filtered off, and the obtained filtrate was concentrated by an evaporator to obtain a white pasty solid. 30 mL of 1N hydrochloric acid aqueous solution was added to this paste-like concentrate, mixed with stirring, and then concentrated again to dryness using an evaporator. The obtained white solid was recrystallized using a small amount of ethanol under a nitrogen atmosphere, and 3.2 g (0.02 mol) of crystals of 2-methyl-D-cysteine amide hydrochloride was collected by filtration. The isolated yield from 2-methyl-D-cysteine amide hydrochloride in the racemic mixture charged in the reaction was 81 mol%, and the isolated yield from 2-methyl-cysteine amide hydrochloride was 41 mol%. Moreover, as a result of analyzing the crystal by liquid chromatography using an optical isomer separation column, the optical purity was 95% ee or higher.
2-methyl-D-cysteine amide hydrochloride; colorless glassy solid (deliquescent),
1 H-NMR (90 MHz, D 2 O) δ [ppm] 3.19 (1H, d, J15.3 Hz), 2.95 (1H, d, J15.3 Hz), 1.64 (3H, s)
13 C-NMR (22.6 MHz, D 2 O) δ [ppm] 173.78 (s), 62.31 (s), 31.73 (t), 22.21 (q)
IR [cm −1 ] (KBr) 1703, 1624, 1574, 1506, 1377, 1279, 1230, 1124
Elemental analysis (measured value) C; 28.01, H; 6.60, N; 16.33, S ;; 18.72, Cl; 20.75, (calculated value) C; 28.15, H; .50, N; 16.41, O; 9.37, S; 18.79, Cl; 20.77
実施例２
実施例１と同様にして各種微生物を培養し生菌体を得た。ラセミ体の２−メチルシステイン１０ｇ（０．０６ｍｏｌ）を含む水溶液に各種微生物の生菌体を加え、実施例１と同様に酵素反応を行い、除菌した後、得られた上清液を液体クロマトグラフィーで分析した。結果を表１に示す。なお、該上清液を光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、光学純度は何れも９５％e.e.以上であった。
収率２；ラセミ体の２−メチルシステインアミド中に含まれる２−メチル−Ｄ−システインアミドに対する、２−メチル−Ｄ−システインアミドの収率（ｍｏｌ％）
菌体１；プロタミノバクター アルボフラバス（Ｐｒｏｔａｍｉｎｏｂａｃｔｅｒ ａｌｂｏｆｌａｖｕｓ）ATCC8458
菌体２；ミコバクテリウム メタノリカ（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｍｅｔｈａｎｏｌｉｃａ）ＢＴ−８４（ＦＥＲＭ Ｐ８８２３）
菌体３；ミコバクテリウム メタノリカ（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｍｅｔｈａｎｏｌｉｃａ）Ｐ−２３（ＦＥＲＭ Ｐ８８２５）
菌体４；ミコプラナ ラモサ（Ｍｙｃｏｐｌａｎａ ｒａｍｏｓｅ）ＮＣＩＢ９４４０
菌体５；ミコプラナ ディモルファ（Ｍｙｃｏｐｌａｎａ ｄｉｍｏｒｐｈａ）ＡＴＣＣ４２７９
Example 2
In the same manner as in Example 1, various microorganisms were cultured to obtain viable cells. After adding viable cells of various microorganisms to an aqueous solution containing 10 g (0.06 mol) of racemic 2-methylcysteine, and performing sterilization by enzymatic reaction in the same manner as in Example 1, the resulting supernatant is liquid. Analyzed by chromatography. The results are shown in Table 1. The supernatant was analyzed by liquid chromatography using an optical isomer separation column, and as a result, the optical purity was 95% ee or higher.
Yield 2; Yield of 2-methyl-D-cysteine amide (mol%) with respect to 2-methyl-D-cysteine amide contained in racemic 2-methylcysteine amide
Cell 1; Protaminobacter alboflavus ATCC 8458
Cell 2; Mycobacterium methanolica BT-84 (FERM P8823)
Mycelium 3; Mycobacterium methanolica P-23 (FERM P8825)
Mycelium 4; Mycoplana ramosa NCIB 9440
Mycelium 5; Mycoplana dimorpha ATCC 4279
Claims (4)
（一般式１中のＲは炭素数１〜４の低級アルキル基を示す。） An optically active 2-alkyl-D-cysteine amide represented by general formula 1 or a salt thereof.
(R in general formula 1 represents a lower alkyl group having 1 to 4 carbon atoms.)
（一般式２〜４中のＲは炭素数１〜４の低級アルキル基を示す。一般式２は、一般式１で示される光学活性２−アルキル−D−システインアミド又はその塩と一般式３で示される光学活性２−アルキル−L−システインアミド又はその塩との混合物である２−アルキルシステインアミド又はその塩を示す。） The optically active 2-alkyl-D-cysteine amide or a salt thereof represented by the general formula 1 and the optically active 2-alkyl-L-cysteine amide or a salt thereof represented by the general formula 3 are represented by the general formula 2. A microbial cell or a treated product of a microorganism having an activity of stereoselectively hydrolyzing the optically active 2-alkyl-L-cysteine amide represented by general formula 3 or a salt thereof to 2-alkyl cysteine amide or a salt thereof. An optically active 2-alkyl-D-cysteine amide represented by the general formula 1 unreacted from the reaction solution after converting into an optically active 2-alkyl-L-cysteine or a salt thereof represented by the general formula 4 Or a method for producing an optically active 2-alkyl-D-cysteine amide or a salt thereof, wherein the salt is separated and recovered.
(R in general formulas 2 to 4 represents a lower alkyl group having 1 to 4 carbon atoms. General formula 2 represents optically active 2-alkyl-D-cysteine amide represented by general formula 1 or a salt thereof and general formula 3 And 2-alkylcysteine amide or a salt thereof which is a mixture with the optically active 2-alkyl-L-cysteine amide or a salt thereof represented by
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006000005A (en) * | 2004-06-15 | 2006-01-05 | Mitsubishi Gas Chem Co Inc | Method for producing optically active 2-alkyl-d-cysteine or its salt |
| JP2013518897A (en) * | 2010-02-03 | 2013-05-23 | エムイーエイチ アソシエイツ，インコーポレイテッド | Multiple substituted fluoromethanes as selective and bioactive isosteres |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006000005A (en) * | 2004-06-15 | 2006-01-05 | Mitsubishi Gas Chem Co Inc | Method for producing optically active 2-alkyl-d-cysteine or its salt |
| JP2013518897A (en) * | 2010-02-03 | 2013-05-23 | エムイーエイチ アソシエイツ，インコーポレイテッド | Multiple substituted fluoromethanes as selective and bioactive isosteres |
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| JP4484027B2 (en) | 2010-06-16 |
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