| Aprepitant,as a new neurokinin-1（NK-1）receptor antagonist,is widely administered to cancer patients with nausea and vomiting that are induced by chemotherapy.（R）-[3,5-bis（trifluoromethyl）phenyl]ethanol（（R）-BTPE）is a key chiral intermediate for the preparation of Aprepitant.Both the processes of distillation and column chromatography were developed to separate（R）-BTPE and prochiral substrate BTAP,and a Aspen Plus method was simultaneously used to simulate the distillation separation process.Finally,the high purity and yield of（R）–BTPE were received.（R）-EHB is a key chiral intermediate during the synthesis ofβ-lactamase inhibitors.This research also proposed the asymmetric reduction of prochiral carbonyl compound EAA to its corresponding alcohol（R）-EHB by immobilized whole cells of recombinant Escherichia coli;the immobilization conditions and its biocatalytic application process were also optimized.Firstly,both the experiment and simulation method were used during distillation to separate（R）-BTPE and BTAP.The deviation between experiment and simulation data was less than 9%,indicating that Aspen Plus software could simulate the separation process.The optimal simulation parameters for distillation procedure were as follows:reflux ratio 3.5,the theoretical plate number 18,feed location 7,feed and the recovery ratio of feed liquid 0.8.Finally,a purity of 99.6%,recovery of99.4%for BTAP was received;and a purity of 97.6%,yield of 98.1%for（R）-BTPE was also realized.Secondly,a silica gel column chromatography method was used to purify the product from extract of biotransformation.The optimized results of column chromatography process were as follows:petroleum ether/dichloromethane 4:1 as eluent;mesh of silica gel was 200-300,sample volume（g/mg）was1:5.5,elution rate 12s/mL.Under the optimized conditions,the purity and recovery rate of BTAP were 99.8%,and 98.8%respectively;for（R）-BTPE,the purity and recovery rate were99.8%and 96.8%respectively.Finally,the preparation of（R）-EHB was conducted by immobilized cells.The optimized immobilization conditions were as follows:fixed recombinant engineering cells with sodium alginate,pH 12 of glycine-NaOH buffer（50mM）,the amount of entrapped cells was 0.4 g/ml,the concentration of CaCl2 was 4%,the concentration of sodium alginate was3%.By using the immobilized cells as biocatalysts,the yield of（R）-EHB reached 92.8%.Compared with free cells,the immobilized cells showed better temperature adaptability during bioreduction and the relative enzyme activity of immobilized cells still maintained at 23.2%after three batches reactions,whereas the free cells can be used only once in reaction. |
