A novel synthetic process for (R)-phenylephrine production using enantioselective enzymatic catalysis and Walden inversion reaction

Abstract

(R)-phenylephrine [(R)-PE] is an α-1-adrenergic receptor agonist that is widely used in over-the-counter drugs to treat the common cold. In this study, three bacteria strains were able to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-PE. And the other two yeast strains can convert 2-[benzyl-(methyl)amino]- 1-(3-hydroxyphenyl)ethanone (BMAHPE) to BMA-PE. We found that Rhodococcus erythropolis BCRC10909 can convert detectable level of HPMAE to (S)-PE by high performance liquid chromatography tandem mass spectrometry analysis. An amino alcohol dehydrogenase gene (RE_AADH) which possesses the ability to convert HPMAE to (S)-PE was then isolated from R. erythropolis BCRC10909 and expressed in Escherichia coli NovaBlue. The purified RE_AADH, tagged with 6'His, had a molecular mass of approximately 30 kDa and exhibited a specific activity of 0.19 mU/mg to HPMAE in the presence of NADPH, indicating this enzyme could be categorized as NADP+-dependent short-chain dehydrogenase reductase. E. coli NovaBlue cell expressing the RE_AADH gene was able to convert HPMAE to (S)-PE with more than 99% enantiomeric excess (ee), 78% yield and a productivity of 3.9 mmol (S)-PE/L h in 12 h at 30°C and pH 7. The (S)-PE, recovered from reaction mixture by precipitation at pH 11.3, could be converted to (R)-PE (ee > 99%) by Walden inversion reaction. This is the first reported biocatalytic process for the production of (S)-PE from HPMAE.

(R)-phenylephrine [(R)-PE]為一種類腎上腺素藥物，具有鬆弛支氣管、治療鼻黏膜腫脹的功效，因此常添加於傷風感冒及抗過敏藥物中。此藥物屬於對掌性化合物(chiral compound)，目前全部是以化學方法合成，在本研究中則要研發利用酵素法進行合成。本研究分別以1-(3-hydroxyphenyl)-2-(methylamino)ethanone (HPMAE)及2-[benzyl- (methyl)amino]-1-(3-hydroxyphenyl)ethanone (BMAHPE)為前驅物，篩選具有轉換成特定立體構型產物能力的微生物。經由小量的培養及反應，搭配HPLC-MS/MS方法分析測試，確認有三株細菌可將HPMAE還原成(S)-PE，另外兩株酵母菌則可以將BMAHPE還原成BMA-PE。利用PCR及選殖技術從其中一株Rhodococcus erythropolis BCRC 10909複製出一個amino alcohol dehydronase (RE_AADH)基因片段並在Escherichia coli NovaBlue中大量表現，並確認此RE_AADH具有轉換能力。經純化的RE_AADH分子量約30 kDa，需要NADPH協助才能進行，由序列比對其為NADP+-dependent short-chain dehydrogenase reductase的一種。在E. coli NovaBlue中表現RE_AADH，於30°C，pH 7條件下作用12小時，將HPMAE轉換成(S)-PE，轉換率可以達到78%，生產速率為3.9 mmol (S)-PE/L h，(S)-PE的光學純度ee值大於99%。接著將反應液的pH值調到pH 11.3就可以把(S)-PE沉澱出。將沉澱出之(S)-PE加入醋酸酐及硫酸，進行Wadern inversion反應改變旋光性，最後得到的產物為(R)-PE (ee > 99%)。這是第一個利用生物催化的方法從HPMAE合成出(S)-PE，未來將繼續尋找可以直接生成(R)-PE的菌株及酵素。