JP6126634B2 - アロマトレウム・アロマチカムEbN1(アゾアルカス・エスピーEbN1)のアルコールデヒドロゲナーゼを用いたL−フェニレフリンの製造方法 - Google Patents
アロマトレウム・アロマチカムEbN1(アゾアルカス・エスピーEbN1)のアルコールデヒドロゲナーゼを用いたL−フェニレフリンの製造方法 Download PDFInfo
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Description
Cycは、単核又は多核、特に単核の、4〜7員、特に5又は6員の、飽和又は不飽和、特に不飽和の、主として芳香族、炭素環式又は複素環式環、特に炭素環式環であり、少なくとも1つの遊離ヒドロキシル基を有し、場合により1回以上置換されていてもよく、6員環の場合には、ヒドロキシル基は、特にアミノ基を含むCycの側鎖に対してメタ位に位置し;
R1及びR2は、互いに独立して、H、又は場合により1回以上置換されていてもよい同一若しくは異なるアルキル残基である。〕
又は該化合物の塩、例えば、特に無機酸(HClなど)の酸付加塩
の製造方法であって、
いずれの場合も、純粋な立体異性体形態、例えば(R)若しくは(S)形態、又は立体異性体の混合物、例えばラセミ体であり、
(a)式Iのケトン:
を、アルコール、特に脂肪族アルコールの存在下で、ハロゲン化、例えば特に塩素化させるが、特に塩化スルフリルと反応させて、式IIのハロゲン化合物、特に塩素化合物:
を取得し、
(b)得られた式IIの化合物を、場合により事前の単離又は濃縮後、酵素的に還元して、式IIIのアルコール:
を取得し、
(c)得られた式IIIのアルコールを、場合により事前の単離又は濃縮後、式HNR1R2のアミン(R1及びR2は前記と同じ意味である)と反応させて式IVの化合物を取得し、場合により該化合物を反応混合物から、場合により純粋な立体異性体形態で単離する、
上記方法に関する。
(i)配列番号2、あるいは
(ii)配列番号2に対してアミノ酸残基の25%以下、例えば1〜24%、2〜20%、3〜15%若しくは4〜10%が、付加、欠失、挿入、置換、逆位若しくはこれらの組合せにより改変されている、及び/又は少なくとも50%、例えば少なくとも60%、70%、80%、90%、95%、96%、97%、98%、99%、100%若しくは100%以上、例えば1〜20倍、若しくは2〜10倍若しくは3〜5倍の、配列番号2の酵素の酵素活性の活性を依然として有する配列
から選択されるポリペプチド配列を有する酵素から選択される。
をハロゲン化、特に塩素化するが、特に好適なハロゲン化剤、特に塩化スルフリルと、脂肪族アルコールの存在下で反応させて、式IIのハロゲン化合物、特に塩素化合物を取得する、
上記方法に関する。
を酵素的に還元して、式IIIのアルコールを取得する、上記方法に関する。この間、前述のように酵素反応を実施する。
2.1 一般的な用語
特に示さない限り、以下の一般的意義が適用される。
ee%=[XA-XB]/[XA+XB]*100
(XA及びXBは、鏡像異性体A及びBのモル分率を意味する)
を用いて決定される。
「単核」若しくは「多核」残基は、1個以上の環状基を含む残基であり、多核残基の場合には、該環状基は、互いに直接、若しくは一般的架橋基を介して結合してもよいし、又は、互いに縮合してもよい。
前述のスキーム1に示す多段階反応を参照しながら、本発明の別の実施形態を以下に説明する。これに基づき、ここで具体的に説明される方法の改変は、当業者の能力の範囲内である。
ケトンのα塩素化のために塩化スルフリルを用いる原理はそれ自体公知であり、例えばD.P. Wymanら、J. Organic. Chem. Vol.29, 1964, p.1956〜1960に記載されている。
2の還元は、酵素により触媒される。この酵素は、(アゾアルカス・エスピー)アロマトレウム・アロマチカムEbN1由来のデヒドロゲナーゼEbN1であり、これは、特定の場合では、大腸菌において組換えにより調製される。
4.1 アルコールデヒドロゲナーゼ
本発明において使用する酵素は、特にアルコールデヒドロゲナーゼ(E.C. 1.1.1.1)から選択される。
マルチプルアライメントパラメータ:
ギャップ開始ペナルティ 10
ギャップ伸長ペナルティ 0.05
ギャップ分離ペナルティ範囲 8
ギャップ分離ペナルティ オフ
アライメントディレイに対する同一性% 40
残基特異的ギャップ オフ
親水性残基ギャップ オフ
トランジション重み付け 0
ペアワイズアライメントパラメータ:
FASTアルゴリズム オフ
K-tupleサイズ 1
ギャップペナルティ 3
ウインドウサイズ 5
最良対角線数(Number of best diagonals) 5
本発明においては、「発現」又は「過剰発現」という用語は、微生物における、対応DNAによりコードされる1以上の酵素の細胞内活性の産生又は増加を意味する。このため、例えば、遺伝子を生物内に挿入し、既存の遺伝子を別の遺伝子により置換し、遺伝子のコピー数を増加させ、強力なプロモーターを使用し、又は高い活性を有する対応酵素をコードする遺伝子を使用することが可能であり、場合によりこれらの手段を組合せることが可能である。
本発明では、本発明の酵素をコードする核酸配列を調節核酸配列の遺伝的制御下に含む発現構築物、及び該発現構築物の少なくとも1つを含むベクターを使用する。
本発明のベクターを使用して、例えば本発明の少なくとも1つのベクターで形質転換された組換え微生物を製造することが可能であり、それを使用して本発明において用いるポリペプチドを製造したり又は本発明に従って酵素反応を行うことが可能である。
本発明において使用する酵素は、組換え製造方法により得ることができる。該製造方法においては、酵素を産生する微生物を培養し、適当な場合にはポリペプチドの発現を誘導し、それを該培養から単離する。所望により、このようにして工業的規模でポリペプチドが製造されうる。
本発明の方法のステップにおいては、酵素を遊離又は固定化酵素として使用することが可能である。
実験の部:
インペラ撹拌器、バッフル、温度計及び滴下漏斗を備えた6,000 mlミニプラント(Miniplant)反応器に、410.11 g(12.80モル)のメタノール中435.68 g(3.20モル)の3-ヒドロキシアセトフェノンと、1,200 mlの酢酸エチルを導入する。20〜30℃で冷却しながら、691.05 g(5.12モル)の塩化スルフリルを上記溶液に2時間以内で滴下する。滴下した後、混合物を室温でさらに1時間撹拌する。次に、混合物を室温で1,600 mlのH2Oを用いて加水分解した後、得られた二相混合物を分離させる。水相を800 mlの酢酸エチルで1回以上抽出する。蒸留ブリッジ(distillation bridge)を用いて、一緒にした有機相からメタノール及び酢酸エチルを蒸留する。同時に、1,880 mlのイソプロパノールを蒸留液溜めに滴下する。有益な(valuable)生成物の17.3%イソプロパノール溶液2462.5 g(これは、含量426 g(2.51モル)に相当する)を取得した。従って、収率は78%である。
インペラ撹拌器、バッフル、温度計及び滴下漏斗を備えた2,000 mlミニプラント反応器に、192.24 g(6.00モル)のメタノール中204.23 g(1.50モル)の3-ヒドロキシアセトフェノンと、1,050 mlのCH2Cl2を導入する。20〜30℃で冷却しながら、283.44 g(2.10モル)の塩化スルフリルを上記溶液に2時間以内で滴下する。滴下した後、混合物を室温でさらに1時間撹拌する。次に、混合物を室温にて400 mlのH2Oで加水分解した後、得られた二相混合物を分離させる。相分離後、標準圧力で蒸留ブリッジを用いて、有機相からメタノール及びCH2Cl2を蒸留する。同時に、880 mlのイソプロパノールを同じ流速で滴下する。有益な生成物の25.7%イソプロパノール溶液837.78 g(これは、含量215 g(1.26モル)に相当する)を取得した。従って、収率は84%である。
実施例1又は2と同様に調製したケトン2を生体触媒により還元して、R-3を取得する。このために、好適な撹拌容器において、1 mM MgCl2、0.02 mMニコチンアミドアデニンジヌクレオチド(NAD)及び282 gのイソプロパノール(これも、補因子再生のための犠牲アルコールとして機能する)を1.44 Lの水性リン酸カリウムバッファー(50 mM、pH 7)中に溶解させる。立体選択性デヒドロゲナーゼ(E.C 1.1.1.1)を過剰産生する組換え大腸菌の細胞(生体乾燥重量3.75 gに相当する)を触媒として用いる。好適な生体触媒の製造については、WO 2005/108590号の実施例1〜3に記載されており、本明細書においてこの文献を明示的に参照する。水相を1.3 kgのMtBEで被覆する。292.8 gの2(イソパノール溶液として)を反応混合物に添加する。反応混合物中の2の濃度は、50 mMを超えてはならない。反応は、アキラル又はキラルクロマトグラフィーによりモニターすることができる。
85 mlのTHF中に15 g(86.9 mmol)の化合物R-3を溶解させ、圧力オートクレーブにおいて90℃で13.5 g(435mmol)のメチルアミンと反応させる。遊離物が完全に変換されるまで(約5時間)、反応させる。次に、室温まで冷却し、得られた懸濁液を蒸発により濃縮する。100 gの水を添加して、有益な生成物の遊離塩基を沈殿させ、これを単離する。12.85 g(76.8mmol、88%)のフェニレフリン遊離塩基を取得した。
Claims (17)
- 式IVの光学活性置換アルコール:
Cycは、単核又は多核の、飽和又は不飽和の、炭素環式又は複素環式の、1若しくは複数回置換されているか置換されていない環であり、該環は、少なくとも1個の遊離ヒドロキシル基を有し;
R1及びR2は、互いに独立して、H、又は1若しくは複数回置換されているか置換されていないアルキル残基である。〕
又は該化合物の塩
の製造方法であって、
いずれの場合も、純粋な立体異性体形態又は立体異性体の混合物であり、
(a)式Iのケトン:
を、脂肪族アルコールの存在下で、ハロゲン化剤と反応させて、式IIのハロゲン化合物:
を取得し、
(b)得られた式IIの化合物を酵素的に還元して、式IIIのアルコール:
を取得し、
(c)得られた式IIIのアルコールを、式HNR1R2のアミン(R1及びR2は前記と同じ意味である)と反応させて式IVの化合物を取得し、
ここで段階(b)が、アルコールデヒドロゲナーゼ(ADH)(E.C 1.1.1.1)から選択される酵素により触媒され、かつ段階(b)が、二相液体反応培地中で実施され、前記アルコールデヒドロゲナーゼが、アロマトレウム属の微生物に由来する酵素から選択される、上記方法。 - 段階(a)における反応が、式Iのアルカノンのモル当たり1〜10モル当量のアルコールの存在下で実施される、請求項1に記載の方法。
- 段階(c)における化学反応が、開鎖又は環状エーテル中の溶液中で実施される、請求項1または2のいずれか一項に記載の方法。
- 前記アルコールデヒドロゲナーゼが、アロマトレウム・アロマチカムEbN1菌に由来する酵素から選択される、請求項3に記載の方法。
- 段階(b)における反応が、還元等価体の添加により実施される、請求項1〜4のいずれか一項に記載の方法。
- 還元等価体がNADH又はNADPHである、請求項5に記載の方法。
- 反応中に消費された還元等価体が再生される、請求項5又は6に記載の方法。
- 前記再生が、酵素的、電気化学的、又は電気−酵素的に実施される、請求項7に記載の方法。
- 前記再生が、酵素的に実施され、再生酵素がADH、およびADHとは異なるデヒドロゲナーゼから選択される、請求項8に記載の方法。
- ADHとは異なるデヒドロゲナーゼが、グルコースデヒドロゲナーゼ、ギ酸デヒドロゲナーゼ、亜リン酸デヒドロゲナーゼから選択される、請求項9に記載の方法。
- 段階(b)における反応が、ADHを天然に若しくは組換えにより発現する微生物の存在下、又は該微生物に由来するADH含有画分の存在下、又は該微生物に由来するADH含有抽出物の存在下で実施される、請求項1〜10のいずれか一項に記載の方法。
- 段階(b)における反応が、エンテロバクター科(Enterobacteriaceae)、シュードモナス科(Pseudomonadaceae)、リゾビウム科(Rhizobiaceae)、ラクトバチルス科(Lactobacillaceae)、ストレプトマイセス科(Streptomycetaceae)、ロドコッカス科(Rhodococcaceae)、ロドシクルス科(Rhodocyclaceae)及びノカルディア科(Nocardiaceae)の細菌から選択される、ADH産生微生物の存在下、又はこれら微生物に由来するADH含有画分若しくは抽出物の存在下で実施される、請求項1〜11のいずれか一項に記載の方法。
- 前記微生物が、請求項1及び4のいずれか一項に定義されるアルコールデヒドロゲナーゼをコードする核酸構築物で形質変換されている組換え微生物である、請求項12に記載の方法。
- 水性有機反応培地が用いられ、式IIの遊離物及び式IIIの生成物の両方が水相よりも有機相における溶解度が高い、請求項1〜13のいずれか一項に記載の方法。
- Cycが、3-ヒドロキシフェニル残基であり、Halが塩素原子である、請求項1〜14のいずれか一項に記載の方法。
- 3-[(1R)-1-ヒドロキシ-2-メチルアミノ-エチル]-フェノールの製造のための、請求項16に記載の使用。
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