JP5295758B2 - Process for producing 1β-methylcarbapenem - Google Patents
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Description
技術分野
本発明は、微生物を用いた医薬品または医薬品原料として有用な１β−メチルカルバペネム系化合物の製造法に関し、さらに詳しくはカルバペネム系化合物を生産する微生物を用い、カルバペネム系化合物の１β位炭素原子上にメチル基を供与するような前駆体、すなわち（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体を含む培地で培養し、１β−メチルカルバペネム系化合物を製造する方法に関する。 TECHNICAL FIELD The present invention relates to a method for producing a 1β-methylcarbapenem compound useful as a pharmaceutical or a pharmaceutical raw material using a microorganism. More specifically, the present invention relates to a method for producing a carbapenem compound on a 1β-position carbon atom of a carbapenem compound. The present invention relates to a method for producing a 1β-methylcarbapenem compound by culturing in a medium containing a precursor that donates a methyl group, that is, a (2S, 4S) -4-methylglutamic acid analog.
背景技術
カルバペネムは、放線菌の生産するチエナマイシンとして初めて発見された抗菌活性物質の総称であり、それは黄色ブドウ球菌を含むグラム陽性菌から緑膿菌を含むグラム陰性菌にわたり、広範囲の菌種に対して強い抗菌力を示すことが知られている。代表的なチエナマイシンの生産菌は、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｃａｔｔｌｅｙａであるが、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｐｅｎｅｍｉｆａｃｉｅｎｓもまたチエナマイシンを生産することが知られている。この他、Ｎ−アセチルチエナマイシン、Ｎ−アセチルデヒドロチエナマイシン、オリバン酸、エピチエナマイシン、ｃａｒｐｅｔｉｍｙｃｉｎ、ｐｌｕｒａｃｉｄｏｍｙｃｉｎ、または１−ｃａｒｂａｐｅｎ −２−ｅｍ−３−ｃａｒｂｏｘｙｌｉｃ ａｃｉｄなどのさまざまなカルバペネムも微生物により生産される（Expert Opin. Investig. Drugs,11(4)529 (2002)）。 Background Art Carbapenem is a generic name of antibacterial active substances first discovered as thienamycin produced by actinomycetes. It ranges from Gram-positive bacteria including Staphylococcus aureus to Gram-negative bacteria including Pseudomonas aeruginosa. It is known to exhibit strong antibacterial activity. A typical thienamycin-producing bacterium is Streptomyces cattleya, but Streptomyces penemifaciens is also known to produce thienamycin. In addition, various carbapenems such as N-acetylthienamycin, N-acetyldehydrothienamycin, olivanic acid, epithienamycin, carpetimicin, pluracidomycin, or 1-carbapen-2-em-3-carboxylic acid are also microorganisms. (Expert Opin. Investig. Drugs, 11 (4) 529 (2002)).
しかし従来より、カルバペネムは化学的、生化学的に不安定であり、また発酵法による生産量が非常に少ないことが知られていた。一方、チエナマイシンの腎デヒドロペプチダーゼ−１（DHP−１）に対する安定性は、カルバペネム骨格の１位にβ位のメチル基を導入することで改善されるとともに（Heterocycles,21.29(1984)）、安定性が向上した１β−メチルチエナマイシンはチエナマイシンと同等以上の抗菌力を有することが報告されている（J. Antibiotics,46.10.1629(1993））。この知見を元に、近年では１β位にメチル基の入ったカルバペネム、すなわち１β−メチルカルバペネム系化合物が抗菌薬として広く用いられるようになった。 Conventionally, however, it has been known that carbapenem is chemically and biochemically unstable, and the amount produced by fermentation is very small. On the other hand, the stability of thienamycin against renal dehydropeptidase-1 (DHP-1) is improved by introducing a methyl group at the 1-position of the carbapenem skeleton (Heterocycles, 21.29 (1984)). It has been reported that 1β-methylthienamycin with improved antibacterial activity equal to or better than thienamycin (J. Antibiotics, 46.10.1629 (1993)). Based on this knowledge, in recent years, carbapenem having a methyl group at the 1β-position, that is, 1β-methylcarbapenem-based compound has been widely used as an antibacterial agent.
１β−メチルカルバペネム系化合物は、培養、発酵等による生産の報告はなく、化学合成によって製造される。１β−メチルチエナマイシンについても合成方法の報告があるのみである（Tetrahedron Lett.,26.5.587(1985)、薬学研究の進歩,2.214(1986)）。しかしながら、１β−メチルカルバペネム系化合物の合成による製造は不斉炭素が多いため、生産性やコスト面での課題が多く、培養、発酵による製造法の開発が強く望まれている。 The 1β-methylcarbapenem-based compound is produced by chemical synthesis, with no reports of production by culture, fermentation or the like. Only 1β-methylthienamycin has been reported for synthesis (Tetrahedron Lett., 26.5.587 (1985), Advances in pharmaceutical research, 2.214 (1986)). However, since production by synthesis of 1β-methylcarbapenem-based compounds has many asymmetric carbons, there are many problems in productivity and cost, and development of production methods by culture and fermentation is strongly desired.
一方、チエナマイシンの生合成経路は既に解明されており、γ−グルタミルリン酸が初発物質となることが知られている（J. Biol. Chem.,260.4637(1985)）。この生合成経路を改変することにより、１β−メチルチエナマイシンを生物学的に製造する手法の開発が期待されているが、そのような手法は未だ本発明者らが知る限りでは、報告されていない。 On the other hand, the biosynthetic pathway of thienamycin has already been elucidated, and it is known that γ-glutamyl phosphate is the first substance (J. Biol. Chem., 260.4637 (1985)). By modifying this biosynthetic pathway, it is expected to develop a method for biologically producing 1β-methylthienamycin. However, as far as the present inventors know, such a method has not been reported. Not.
本発明者らは、今般、１β−メチルカルバペネム系化合物を培養または発酵により、低コストで生産可能であることを見出した。本発明はかかる知見に基づくものである。 The present inventors have now found that 1β-methylcarbapenem compounds can be produced at low cost by culturing or fermentation. The present invention is based on such knowledge.
従って、本発明は、微生物の培養により、１β−メチルカルバペネム系化合物を製造する方法の提供をその目的としている。 Accordingly, an object of the present invention is to provide a method for producing a 1β-methylcarbapenem compound by culturing microorganisms.
そして、本発明は、カルバペネム系化合物の生産能を有する微生物を、（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体を含む培地で培養することを含んでなる、１β−メチルカルバペネム系化合物の製造方法である。 The present invention also relates to a method for producing a 1β-methylcarbapenem compound comprising culturing a microorganism capable of producing a carbapenem compound in a medium containing a (2S, 4S) -4-methylglutamic acid analog. It is.
微生物の寄託
Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５ Ｐ３−２８株は、２００７年２月１４日付で独立行政法人産業技術総合研究所 特許生物寄託センター(〒305‐8566 日本国 茨城県つくば市東1丁目1番地1 中央第6)に寄託された。受託番号は、ＦＥＲＭ ＢＰ−１０７８３である。 Deposit of microorganisms Streptomyces sp. The SF2205 P3-28 strain was deposited on February 14, 2007 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki, 305-8566, Japan) . The accession number is FERM BP-10784.
カルバペネム系化合物の生産能を有する微生物
本発明におけるカルバペネム系化合物の生産能を有する微生物は、カルバペネム系化合物を生産する能力を有している微生物であれば特に限定されないが、例えばチエナマイシン類縁体生産菌が挙げられる。チエナマイシン類縁体生産菌として、好ましくはＳｔｒｅｐｔｏｍｙｃｅｓ属の菌株が挙げられ、さらに好ましくはＳｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株またはその変異体、Ｓｔｒｅｐｔｏｍｙｃｅｓ Ｃａｔｔｌｅｙａ ＭＡ４２９７（ＦＥＲＭ Ｐ−３３０９）、またはＳｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．Ａ２７１（ＦＥＲＭ Ｐ−３９８４）が挙げられ、さらに一層好ましくはＳｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株またはその変異体が挙げられる。前記Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株の変異体の例としては、寄託されたＳｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５ Ｐ３−２８株が挙げられる。 Microorganisms capable of producing carbapenem compounds The microorganisms capable of producing carbapenem compounds in the present invention are not particularly limited as long as they are microorganisms capable of producing carbapenem compounds. For example, thienamycin analog-producing bacteria Is mentioned. The thienamicin analog-producing bacterium preferably includes a strain of the genus Streptomyces, and more preferably, Streptomyces sp. SF2205 strain or a mutant thereof, Streptomyces Cattleya MA4297 (FERM P-3309), or Streptomyces sp. A271 (FERM P-3984), and even more preferably Streptomyces sp. The SF2205 strain or a variant thereof can be mentioned. The Streptomyces sp. Examples of mutants of the SF2205 strain include the deposited Streptomyces sp. And SF2205 P3-28 strain.
ここで、チエナマイシン類縁体とは、微生物の生産するカルバペネム系化合物のうち、チエナマイシンの６位のヒドロキシエチル基の立体構造と８位のヒドロキシル基の立体構造とが同じである物質を表す。前記チエナマイシン類縁体は、例えばチエナマイシン（例えば、Ｓｔｒｅｐｔｏｍｙｃｅｓ Ｃａｔｔｌｅｙａ ＭＡ４２９７（ＦＥＲＭ Ｐ−３３０９）、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株、またはその変異体により生産される)、Ｎ−アセチルチエナマイシン（例えば、Ｓｔｒｅｐｔｏｍｙｃｅｓ Ｃａｔｔｌｅｙａ ＭＡ４２９７（ＦＥＲＭ Ｐ−３３０９）、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．Ａ２７１（ＦＥＲＭ Ｐ−３９８４）により生産される）、Ｎ−アセチルデヒドロチエナマイシン（例えば、Ｓｔｒｅｐｔｏｍｙｃｅｓ Ｃａｔｔｌｅｙａ ＭＡ４２９７（ＦＥＲＭ Ｐ−３３０９）により生産される）が挙げられる。1β−メチルチエナマイシン類縁体とは、前記チエナマイシン類縁体の1β位にメチル基を有する物質を表す。 Here, the thienamycin analog represents a substance having the same three-dimensional structure of the hydroxyethyl group at the 6-position and the three-dimensional structure of the hydroxyl group at the 8-position among the carbapenem compounds produced by the microorganism. The thienamycin analog may be, for example, thienamycin (eg, produced by Streptomyces Catleyya MA4297 (FERM P-3309), Streptomyces sp. SF2205 strain, or a variant thereof), N-acetylthiemycin FERM P-3309), Streptomyces sp.A271 (produced by FERM P-3984), N-acetyl dehydrothienamycin (eg produced by Streptomyces Cattley MA4297 (FERM P-3309)). The 1β-methylthienamycin analog represents a substance having a methyl group at the 1β position of the thienamycin analog.
（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体
本明細書において、（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体とは、カルバペネム系化合物の１β位の炭素原子上にメチル基を供与する物質またはその前駆体であり、例えば（２Ｓ，４Ｓ）−４−メチルグルタミン酸、（２Ｓ，４Ｓ）−４−メチルプロリン、（２Ｓ，４Ｓ）−５−ヒドロキシロイシン、（３Ｓ，５Ｓ）−３−メチル−１−ピロリン−５−カルボン酸、（２Ｓ，４Ｓ）−４−メチルグルタミン酸−５−セミアルデヒド、または（２Ｓ，４Ｓ）−４−メチル−５−グルタミルリン酸が挙げられる。好ましくは、（２Ｓ，４Ｓ）−４−メチルグルタミン酸である。また、前記前駆体は、一般的に知られている代謝系において（２Ｓ，４Ｓ）−４−メチルグルタミン酸となり得る物質であることを意味する。 (2S, 4S) -4-methylglutamic acid analog In the present specification, the (2S, 4S) -4-methylglutamic acid analog is a substance that donates a methyl group on the 1β-position carbon atom of a carbapenem compound or For example, (2S, 4S) -4-methylglutamic acid, (2S, 4S) -4-methylproline, (2S, 4S) -5-hydroxyleucine, (3S, 5S) -3-methyl- Examples include 1-pyrroline-5-carboxylic acid, (2S, 4S) -4-methylglutamic acid-5-semialdehyde, or (2S, 4S) -4-methyl-5-glutamylphosphoric acid. Preferred is (2S, 4S) -4-methylglutamic acid. The precursor means a substance that can be (2S, 4S) -4-methylglutamic acid in a generally known metabolic system.
１β−メチルカルバペネム系化合物
本明細書において、１β−メチルカルバペネム系化合物とは、メロペネム、ビアペネム、エルタペネム、ドリペネム、または１β−メチルチエナマイシンあるいはその類縁体などが挙げられ、好ましくは１β−メチルチエナマイシンあるいはその類縁体（例えば、Ｎ−アセチル−１β−メチルチエナマイシン、Ｎ−アセチルデヒドロ−１β−メチルチエナマイシンなどが挙げられる）が挙げられ、さらに好ましくは１β−メチルチエナマイシンが挙げられる。 1β-methylcarbapenem-based compound In this specification, the 1β-methylcarbapenem-based compound includes meropenem, biapenem, ertapenem, doripenem, 1β-methylthienamycin or its analogs, preferably 1β-methylthie Namycin or an analog thereof (for example, N-acetyl-1β-methylthienamycin, N-acetyldehydro-1β-methylthienamycin and the like), and more preferably 1β-methylthienamycin is used. Can be mentioned.
培養
１β−メチルカルバペネム系化合物は、カルバペネム系化合物の生産能を有する微生物を（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体を含む培地で培養することにより、製造することができる。１β−メチルカルバペネム系化合物を含む培養物はそのまま用いられてもよいが、好ましくは、この１β−メチルカルバペネム系化合物は培養物から単離されて、場合により精製されて用いられる。 The cultured 1β-methylcarbapenem compound can be produced by culturing a microorganism capable of producing a carbapenem compound in a medium containing a (2S, 4S) -4-methylglutamic acid analog. The culture containing the 1β-methylcarbapenem compound may be used as it is, but preferably the 1β-methylcarbapenem compound is isolated from the culture and optionally purified before use.
本発明の好ましい態様によれば、前記（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体が（２Ｓ，４Ｓ）−４−メチルグルタミン酸であり、かつ前記カルバペネム系化合物の生産能を有する微生物がチエナマイシン類縁体生産菌、好ましくはチエナマイシンを生産するＳｔｒｅｐｔｏｍｙｃｅｓ属、さらに好ましくはチエナマイシン生産菌Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株またはその変異体とされる。 According to a preferred aspect of the present invention, the (2S, 4S) -4-methylglutamic acid analog is (2S, 4S) -4-methylglutamic acid and the microorganism capable of producing the carbapenem compound is a thienamycin analog. A body-producing bacterium, preferably a genus Streptomyces that produces thienamycin, more preferably a thiemycin-producing bacterium Streptomyces sp. The SF2205 strain or a variant thereof is used.
本発明のさらに好ましい態様によれば、前記（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体が（２Ｓ，４Ｓ）−４−メチルグルタミン酸であり、かつ前記カルバペネム系化合物の生産能を有する微生物がチエナマイシン類縁体生産菌であり、かつ前記１β−メチルカルバペネム系化合物が１β−メチルチエナマイシン類縁体、好ましくは１β−メチルチエナマイシンとされる。 According to a further preferred aspect of the present invention, the (2S, 4S) -4-methylglutamic acid analog is (2S, 4S) -4-methylglutamic acid, and the microorganism capable of producing the carbapenem-based compound is thienamicin. It is an analog-producing bacterium, and the 1β-methylcarbapenem compound is a 1β-methylthienamycin analog, preferably 1β-methylthienamycin.
本発明のさらに好ましい態様によれば、（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体が（２Ｓ，４Ｓ）−４−メチルグルタミン酸であり、かつカルバペネム系化合物の生産能を有する微生物がチエナマイシンを生産するＳｔｒｅｐｔｏｍｙｃｅｓ属であり、かつ前記１β−メチルカルバペネム系化合物が１β−メチルチエナマイシン類縁体、好ましくは１β−メチルチエナマイシンとされる。 According to a further preferred embodiment of the present invention, a microorganism having the ability to produce a carbapenem compound, wherein the (2S, 4S) -4-methylglutamic acid analog is (2S, 4S) -4-methylglutamic acid and produces carbapenem compounds. And the 1β-methylcarbapenem compound is a 1β-methylthienamycin analog, preferably 1β-methylthienamycin.
本発明のさらに好ましい態様によれば、（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体が（２Ｓ，４Ｓ）−４−メチルグルタミン酸であり、かつカルバペネム系化合物の生産能を有する微生物がチエナマイシン生産菌Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株またはその変異体であり、かつ前記１β−メチルカルバペネム系化合物が１β−メチルチエナマイシン類縁体、好ましくは１β−メチルチエナマイシンとされる。 According to a further preferred embodiment of the present invention, the (2S, 4S) -4-methylglutamic acid analog is (2S, 4S) -4-methylglutamic acid, and the microorganism capable of producing a carbapenem compound is a thienamycin-producing bacterium. Streptomyces sp. The SF2205 strain or a variant thereof, and the 1β-methylcarbapenem compound is a 1β-methylthienamycin analog, preferably 1β-methylthienamycin.
本発明のさらに好ましい態様によれば、（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体が（２Ｓ，４Ｓ）−４−メチルグルタミン酸であり、かつカルバペネム系化合物の生産能を有する微生物がＳｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５ Ｐ３−２８株であり、かつ前記１β−メチルカルバペネム系化合物が１β−メチルチエナマイシン類縁体、好ましくは１β−メチルチエナマイシンとされる。 According to a further preferred embodiment of the present invention, a microorganism having (2S, 4S) -4-methylglutamic acid analog (2S, 4S) -4-methylglutamic acid and capable of producing a carbapenem-based compound is selected from Streptomyces sp. SF2205 P3-28 strain, and the 1β-methylcarbapenem compound is a 1β-methylthienamycin analog, preferably 1β-methylthienamycin.
本発明による１β−メチルカルバペネム系化合物の製造法において、微生物の培養は、放線菌の培養に用いられる慣用の成分（例えば、炭素源、窒素源、無機塩、ビタミン、金属などの増殖因子成分など）を含む液体培地で、好気的条件での培養法、振盪培養法、電気撹拌培養法、または深部培養法により行うことができる。 In the method for producing a 1β-methylcarbapenem compound according to the present invention, microorganisms are cultured using conventional components used for culturing actinomycetes (for example, growth factor components such as carbon sources, nitrogen sources, inorganic salts, vitamins, metals, etc.) ) Can be carried out by a culture method under aerobic conditions, a shaking culture method, an electric stirring culture method, or a deep culture method.
前記炭素源としては、グルコース、スクロース、水飴、でんぷん、アルコール、グリセリン、または油脂などが挙げられる。 Examples of the carbon source include glucose, sucrose, starch syrup, starch, alcohol, glycerin, and fats and oils.
前記窒素源としては、アミノ酸、アルカリ金属の硝酸塩や亜硝酸塩、ペプチド、ポリペプトンやカザミノ酸などのタンパク分解物、コーンスティープリカー、脱脂大豆、小麦胚芽、酵母エキス、または酵母粉末などが挙げられる。 Examples of the nitrogen source include amino acids, alkali metal nitrates and nitrites, peptides, protein degradation products such as polypeptone and casamino acid, corn steep liquor, defatted soybeans, wheat germ, yeast extract, or yeast powder.
前記無機塩としては、アルカリ金属やアルカリ土類金属のリン酸塩、硫酸塩、硝酸塩、亜硝酸塩、または塩酸塩などが挙げられる。 Examples of the inorganic salt include phosphates, sulfates, nitrates, nitrites, and hydrochlorides of alkali metals and alkaline earth metals.
前記ビタミンとしては、パントテン酸、ニコチン酸、ビオチン、シアノコバラミン、チアミン、リボフラビン、ｐ−アミノ安息香酸、または塩化コリンなどが挙げられる。 Examples of the vitamin include pantothenic acid, nicotinic acid, biotin, cyanocobalamin, thiamine, riboflavin, p-aminobenzoic acid, or choline chloride.
前記金属としては、前記無機塩の他、鉄、銅、コバルト、マンガン、モリブデン、亜鉛、またはセレンなどが挙げられる。 Examples of the metal include iron, copper, cobalt, manganese, molybdenum, zinc, and selenium in addition to the inorganic salt.
前記培地のｐＨは特に限定されないが、好ましくはｐＨ４〜８程度である。 The pH of the medium is not particularly limited, but is preferably about pH 4-8.
前記培養は、それぞれの微生物に応じた慣用されている条件で行うことができる。例えば、微生物がＳｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株もしくはその変異株である場合には、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．の培養は、例えば培養温度が１５℃〜４５℃、好ましくは２５℃〜３５℃、培養時間が７２〜２４０時間程度で行うことができる。 The culture can be performed under commonly used conditions according to each microorganism. For example, if the microorganism is Streptomyces sp. In the case of SF2205 strain or a mutant strain thereof, Streptomyces sp. The culture can be performed, for example, at a culture temperature of 15 ° C to 45 ° C, preferably 25 ° C to 35 ° C, and a culture time of about 72 to 240 hours.
（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体は、培地1Ｌ中に10〜5000mg、好ましくは100〜1000mgを添加することができる。添加する時期は特に限定されないが、好ましくは、培養初日〜培養４日目の間に添加することができる。 The (2S, 4S) -4-methylglutamic acid analog can be added in an amount of 10 to 5000 mg, preferably 100 to 1000 mg, in 1 L of the medium. Although the time to add is not particularly limited, it can be preferably added between the first day of culture and the fourth day of culture.
本発明によって得られる１β−メチルカルバペネム系化合物の培養物からの採取に当たっては、その性状を利用した慣用された分離手段（例えば、固層抽出法、イオン交換樹脂法、吸着または分配カラムクロマト法、ゲル濾過法、透析法、沈殿法、または再結晶化法など）を単独でまたは適宜組合せることにより採取することができる。例えば、培養物に含まれる１β−メチルチエナマイシンを陰イオン交換樹脂に吸着させ、水で洗浄した後、適当なイオンを含む水溶液で溶離する。この溶離液を濃縮し、逆相シリカゲルまたはアンバーライトＸＡＤ−２を充填したカラムに供してカラムクロマトグラフィーを行い、１β−メチルチエナマイシンを精製する。さらに、必要に応じて陽イオン交換樹脂、陰イオン交換樹脂、またはゲル濾過担体に供して１β−メチルチエナマイシンを精製してもよい。 In collecting from the culture of the 1β-methylcarbapenem compound obtained by the present invention, conventional separation means utilizing the properties (for example, solid layer extraction method, ion exchange resin method, adsorption or distribution column chromatography method, Gel filtration method, dialysis method, precipitation method, recrystallization method and the like) can be collected alone or in appropriate combination. For example, 1β-methylthienamycin contained in the culture is adsorbed on an anion exchange resin, washed with water, and eluted with an aqueous solution containing appropriate ions. The eluent is concentrated and applied to a column packed with reverse phase silica gel or Amberlite XAD-2 to perform column chromatography to purify 1β-methylthienamycin. Furthermore, 1β-methylthienamycin may be purified by subjecting to a cation exchange resin, an anion exchange resin, or a gel filtration carrier as necessary.
本発明を以下の実施例によって詳細に説明するが、本発明はこれら実施例に限定されるものではない。すなわち、１β−メチルチエナマイシンの生産に用いる菌株、添加する（２Ｓ，４Ｓ）−４−メチルグルタミン酸類縁体および１β−メチルチエナマイシンの性状に基づき、１β−メチルチエナマイシンの製造法を種々改変することができるが、そのような方法も本発明による製造法に包含される。 The present invention will be described in detail by the following examples, but the present invention is not limited to these examples. That is, the method for producing 1β-methylthienamycin is based on the strain used for the production of 1β-methylthienamicin, the added (2S, 4S) -4-methylglutamic acid analog and the properties of 1β-methylthienamycin. Although various modifications can be made, such a method is also included in the production method according to the present invention.
実施例１
（２Ｓ，４Ｓ）−４−メチルグルタミン酸（トロント・リサーチ・ケミカル社製）１００ｍｇを脱イオン水１０ｍＬに溶解し、水酸化ナトリウム水溶液でｐＨ８．０に調整した。調整後の溶液は使用するまで−２０℃で保存した。 Example 1
100 mg of (2S, 4S) -4-methylglutamic acid (manufactured by Toronto Research Chemical Company) was dissolved in 10 mL of deionized water and adjusted to pH 8.0 with an aqueous sodium hydroxide solution. The adjusted solution was stored at −20 ° C. until use.
チエナマイシン生産菌Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５株の変異株Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＳＦ２２０５ P３−２８株を、ＴＳＬ培地（２．５％溶性でんぷん、２．０％グルコース、０．７％ポリペプトン、０．６％小麦胚芽、０．４５％酵母エキス、０．３％脱脂大豆粕、０．３％Ｌａｂ−Ｌｅｍｃｏ−Ｐｏｗｄｅｒ、および０．３％炭酸カルシウム含有、ｐＨ７．１）中、２８℃で振とう培養した。２日間培養後、うち１ｍＬをＴｈ培地（２．５％グルコース、１．５％コーンスティープリカー、０．５％綿実粕、０．１％酵母エキス、０．００１％塩化コバルト、０．０００５％硫酸亜鉛、および０．３％炭酸カルシウム含有、ｐＨ７．２）５０ｍＬへ移植し、２８℃で振とう培養した。培養途中の２日目に、終濃度１００μｇ／ｍＬとなるように（２Ｓ，４Ｓ）−４−メチルグルタミン酸溶液を添加し、７日間培養した。 Thienamycin-producing bacteria Streptomyces sp. A variant of the SF2205 strain, Streptomyces sp. SF2205 P3-28 strain was added to TSL medium (2.5% soluble starch, 2.0% glucose, 0.7% polypeptone, 0.6% wheat germ, 0.45% yeast extract, 0.3% defatted soybean meal , 0.3% Lab-Lemco-Powder, and 0.3% calcium carbonate, pH 7.1) at 28 ° C. with shaking. After culturing for 2 days, 1 mL of Th medium (2.5% glucose, 1.5% corn steep liquor, 0.5% cottonseed meal, 0.1% yeast extract, 0.001% cobalt chloride, 0.0005) It was transplanted to 50 mL containing% zinc sulfate and 0.3% calcium carbonate, pH 7.2), and cultured with shaking at 28 ° C. On the second day during the culture, a (2S, 4S) -4-methylglutamic acid solution was added to a final concentration of 100 μg / mL, and the cells were cultured for 7 days.
得られた培養物４５０ｍＬの遠心沈降上清（３５００ｒｐｍ、１０分間）を１００ｍＬ容量のＤｏｗｅｘ１−Ｘ２カラム (１００−２００ｍｅｓｈ、ＨＣＯ３ -フォーム、室町ケミカル) にアプライし、水８００ｍＬおよび飽和炭酸水３００ｍＬでカラムを洗浄した後、飽和炭酸水１００ｍＬで１β−メチルチエナマイシンを溶出した。溶出液は凍結乾燥し、得られた残渣７３ｍｇを１ｍＬのリン酸緩衝液（５０ｍＭ、ｐＨ７．０）に溶解してＨＰＬＣ（カラム：イナートシルＯＤＳ−２、２０×２５０ｍｍ、５μｍ、ジーエルサイエンス社製、温度：室温、検出波長：３０５ｎｍ）に注入した。注入後３０分間、０．５％アセトニトリル−２ｍＭリン酸緩衝液（ｐＨ６．７）を流速７ｍＬ／分で通液し、続いて移動相を５％アセトニトリル−２ｍＭリン酸緩衝液（ｐＨ６．７）に換え、流速７ｍＬ／分で１７．３分後に溶出された成分を集めた。The centrifugal sedimentation supernatants of the resulting cultures 450 mL (3500 rpm, 10 minutes) Dowex 1-X2 column 100mL volume (100-200 mesh, HCO 3 - form, Muromachi Chemical) was applied to, water 800mL and saturated carbonated water 300mL After washing the column, 1β-methylthienamycin was eluted with 100 mL of saturated carbonated water. The eluate was lyophilized, and 73 mg of the obtained residue was dissolved in 1 mL of phosphate buffer (50 mM, pH 7.0) and HPLC (column: inertosyl ODS-2, 20 × 250 mm, 5 μm, manufactured by GL Sciences Inc., (Temperature: room temperature, detection wavelength: 305 nm). 30% after injection, 0.5% acetonitrile-2 mM phosphate buffer (pH 6.7) was passed at a flow rate of 7 mL / min, and then the mobile phase was 5% acetonitrile-2 mM phosphate buffer (pH 6.7). The components eluted after 17.3 minutes at a flow rate of 7 mL / min were collected.
得られた溶出液を減圧下で濃縮し、次に示す条件でＬＣ／ＭＳおよびＬＣ／ＮＭＲによる分析を行った。その結果、保持時間および各種スペクトルデータに、Ｓｈｉｈらの方法（J. Antibiotics,46.10.1629(1993）)に従って合成した１β−メチルチエナマイシンの標品と一致するピークが検出され、１β−メチルチエナマイシンが生産されていることを確認した。また、そのピーク高を標品と比較して、１β−メチルチエナマイシンの収量を２８μｇと算出した。 The obtained eluate was concentrated under reduced pressure, and analyzed by LC / MS and LC / NMR under the following conditions. As a result, peaks corresponding to the standard of 1β-methylthienamycin synthesized according to the method of Shih et al. (J. Antibiotics, 46.10.1629 (1993)) were detected in the retention time and various spectral data, and 1β-methyl was detected. It was confirmed that thienamycin was produced. Further, the peak height was compared with the standard product, and the yield of 1β-methylthienamycin was calculated to be 28 μg.
ＬＣ／ＭＳ測定条件
ＵＶ検出器：２９９６フォトダイオードアレイ検出器（ウォーターズ社製）
ＭＳ検出器：Ｍｉｃｒｏｍａｓｓ ＺＱ（ウォーターズ社製）
カラム：Ｓｙｍｍｅｔｒｙ Ｃ１８、４．６×２５０ｍｍ、５μｍ（ウォーターズ社製）
移動相：１０％アセトニトリル−０．１％ＴＦＡ水溶液
流速：０．８ｍＬ／分、 保持時間：１４．５分 LC / MS measurement conditions UV detector: 2996 photodiode array detector (manufactured by Waters)
MS detector: Micromass ZQ (Waters)
Column: Symmetry C18, 4.6 × 250 mm, 5 μm (manufactured by Waters)
Mobile phase: 10% acetonitrile-0.1% aqueous TFA flow rate: 0.8 mL / min, retention time: 14.5 minutes
ＬＣ／ＮＭＲ測定条件
ＮＭＲ：ＡＶＡＮＣＥ ５００（ブルカー・バイオスピン社製）
カラム：イナートシルＯＤＳ−２、４．６×２５０ｍｍ、５μｍ（ジーエルサイエンス社製）
移動相：５％重アセトニトリル−５０ｍＭリン酸重水緩衝液（ｐＨ６．５）
流速：１．０ｍＬ／分、保持時間：１１．２分 LC / NMR measurement conditions NMR: AVANCE 500 (Bruker BioSpin)
Column: Inertsil ODS-2, 4.6 × 250 mm, 5 μm (manufactured by GL Sciences Inc.)
Mobile phase: 5% deuterated acetonitrile-50 mM deuterated phosphate buffer (pH 6.5)
Flow rate: 1.0 mL / min, retention time: 11.2 minutes
スペクトルデータ
UV λmax: 313 nm
ES-MS positive: m/z 287 [M+H]+, 309 [M+Na]+, 325 [M+K]+
1H-NMRスペクトル（500 MHz）δ（ppm）： 1.06 (3H, d, J=7.3 Hz), 1.15 (3H, d, J=6.3 Hz), 2.82 (H, ddd, J=5 .7, 8.7, 14.2 Hz), 3.00 (H, ddd, J=5.0, 8.7, 12.8 Hz), 3.09 (H, ddd, J=4.5, 5.0, 14.2 Hz), 3.16 (H, ddd, J=4.5, 5.7, 12.8 Hz), 3.26 (H,qd, J=7.3, 8.3 Hz), 3.31 (H, dd, J=2.3, 6.0 Hz), 4.07 (H, dd, J=2.3, 8.3 Hz), 4.09 (H, qd, J=6.0, 6.3 Hz) Spectral data
UV λmax: 313 nm
ES-MS positive: m / z 287 [M + H] + , 309 [M + Na] + , 325 [M + K] +
1 H-NMR spectrum (500 MHz) δ (ppm): 1.06 (3H, d, J = 7.3 Hz), 1.15 (3H, d, J = 6.3 Hz), 2.82 (H, ddd, J = 5.7, 8.7, 14.2 Hz), 3.00 (H, ddd, J = 5.0, 8.7, 12.8 Hz), 3.09 (H, ddd, J = 4.5, 5.0, 14.2 Hz), 3.16 (H, ddd, J = 4.5, 5.7, 12.8 Hz), 3.26 (H, qd, J = 7.3, 8.3 Hz), 3.31 (H, dd, J = 2.3, 6.0 Hz), 4.07 (H, dd, J = 2.3, 8.3 Hz), 4.09 (H, qd, J = 6.0, 6.3 Hz)
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| JPS58152476A (en) * | 1982-03-05 | 1983-09-10 | Sanraku Inc | Novel bacterium |
| JPH0751092A (en) * | 1993-08-10 | 1995-02-28 | Tanabe Seiyaku Co Ltd | Production of optically active pyrrolidine derivative |
| JPH07196660A (en) * | 1993-12-28 | 1995-08-01 | Lederle Japan Ltd | 2-(heterocycloalkenylalkyl)thiocarbapenem derivative |
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| JPS58152476A (en) * | 1982-03-05 | 1983-09-10 | Sanraku Inc | Novel bacterium |
| JPH0751092A (en) * | 1993-08-10 | 1995-02-28 | Tanabe Seiyaku Co Ltd | Production of optically active pyrrolidine derivative |
| JPH07196660A (en) * | 1993-12-28 | 1995-08-01 | Lederle Japan Ltd | 2-(heterocycloalkenylalkyl)thiocarbapenem derivative |
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