Patel R N, Banerjee A, Ko R Y, Howell J M, Li W S, Comezoglu F T, Partyka R A, Szarka F T
Bristol-Myers Squibb Pharmaceutical Research Institute, New Brunswick, NJ.
Biotechnol Appl Biochem. 1994 Aug;20(1):23-33.
A key chiral intermediate [(3R-cis)-3-(acetyloxy)-4-phenyl-2-azetidinone (2)] for the semi-synthesis of paclitaxel (taxol; 5), an anti-cancer compound, was prepared by an enzymic process. The stereoselective enzymic hydrolysis of cis-3-(acetyloxy)-4-phenyl-2-azetidinone (1) to the corresponding (S)-(-)-alcohol (3) was carried out using various lipases. Lipase PS-30 (Pseudomonas cepacia) and BMS (Bristol-Myers Squibb) lipase (Pseudomonas sp. SC13856) catalysed hydrolysis of the undesired enantiomer of racemic compound 1, producing the (S)-(-)-alcohol (3) and the desired (R)-(+)-acetate (2). Reaction yields of > 96% and optical purities of > 99.5% were obtained. For a very efficient enzyme source (BMS lipase), a lipase fermentation using Pseudomonas sp. SC13856 was developed. In a fed-batch process using soybean oil, the fermentation resulted in 1500 units of extracellular lipase activity/ml. Crude BMS lipase (1.7 kg, containing 140,000 units/g) was recovered from the filtrate by ethanol precipitation. BMS lipase and commercially available lipase PS-30 were independently immobilized on Accurel polypropylene. These immobilized lipases were re-used (ten cycles) without loss of enzyme activity, productivity or optical purity of the product. The enzymic reaction process was scaled up to 75 and 150 litres using immobilized BMS lipase and lipase PS-30 respectively. From the reaction mixture, compound 2 was isolated in 88-90 mol% yield and 99.5% optical purity. A purity of 99.9 (area %) was demonstrated by g.c. for isolated compound 2.
一种用于半合成抗癌化合物紫杉醇（泰素；5）的关键手性中间体[(3R-顺式)-3-(乙酰氧基)-4-苯基-2-氮杂环丁烷酮(2)]通过酶促过程制备。使用各种脂肪酶将顺式-3-(乙酰氧基)-4-苯基-2-氮杂环丁烷酮(1)立体选择性酶促水解为相应的(S)-(-)-醇(3)。脂肪酶PS-30（洋葱假单胞菌）和BMS（百时美施贵宝）脂肪酶（假单胞菌属SC13856）催化外消旋化合物1的不需要的对映体水解，生成(S)-(-)-醇(3)和所需的(R)-(+)-乙酸酯(2)。反应产率>96%，光学纯度>99.5%。对于一种非常高效的酶源（BMS脂肪酶），开发了使用假单胞菌属SC13856的脂肪酶发酵方法。在使用大豆油的补料分批过程中，发酵产生1500单位/毫升的细胞外脂肪酶活性。通过乙醇沉淀从滤液中回收粗BMS脂肪酶（1.7千克，含140,000单位/克）。将BMS脂肪酶和市售脂肪酶PS-30分别固定在Accurel聚丙烯上。这些固定化脂肪酶可重复使用（十个循环），而不会损失酶活性、生产率或产物的光学纯度。酶促反应过程分别使用固定化BMS脂肪酶和脂肪酶PS-30放大至75升和150升。从反应混合物中，以88 - 90摩尔%的产率和99.5%的光学纯度分离出化合物2。通过气相色谱法证明分离出的化合物2的纯度为99.9（面积%）。
