The Synthesis Of(R)-3-Hydroxy-3-Ethyl Phenylpropinate By Asymmetric Reduction Of 3-Carbonyl-3-Ethyl Phenylpropinate With Interface-assembled Carbonyl Reductase As Catalyst

(R)-3-hydroxy-3-ethyl phenylpropinate is a key chiral intermediate of Atomoxetine which is a selective norepinephrine reuptake inhibitor.In February 2002,Atomoxetine became the first non-central stimulant drug approved by the US Food and Drug Administration(FDA)for the treatment of attention deficit hyperactivity disorder(ADHD).With Candida tropicalis CGMCC NO.15016 that was preserved by the laboratory as catalyst,(R)-3-hydroxy-3-ethyl phenylpropinate was prepared by asymmetric reduction of 3-carbonyl-3-ethyl phenylpropinate in phosphate buffer/organic solvent diphasic system.DNS method was used to study the tolerance mechanism of organic solvent to cells.At the same time,the optimum reaction system which was determined by gas chromatography is as follows:phosphate buffer/dibutyl phthalate(volume ratio 1:1)diphasic system,8%ethanol as co-substrate,cell concentration 30 g/L(Dry cell weight/volume of reaction),phosphate buffer pH 6.0,36 ?,120 r/min for 48 h.0.050 mol/L substrate can be converted to(R)-3-hydroxy-3-ethyl phenylpropinate.And the conversion and enantiometric excess of(R)-3-hydroxy-3-ethyl phenylpropinate can reach 100%.In order to further improve the utilization of 3-carbonyl-3-ethyl phenylpropinate,the carbonyl reductase which exists in the cells of Candida tropicalis CGMCC NO.15016 was isolated and purified.In toluene/Tris-HCl diphasic system,0.8 g/L carbonyl reductase(180 U/mg)was conjugated with 5.00 g/L polystyrene to obtain interfacial self-assembled carbonyl reductase(155 U/mg).The self-assembled carbonyl reductase was used as a catalyst to prepare(R)-3-hydroxy-3-ethyl phenylpropinate by asymmetric reduction of 3-carbonyl-3-ethyl phenylpropinate.The optimal reaction system was finally determined:toluene/Tris-HCl(volume ratio 12.5:10)diphasic system,10%ethanol as co-substrate,0.10 mol/L NADH,30 ?,140 r/min for 8 h.0.056 mol/L substrate can be converted to(R)-3-hydroxy-3-ethyl phenylpropinate.And the conversion and enantiometric excess of(R)-3-hydroxy-3-ethyl phenylpropinate were all up to 100%.The kinetic model of asymmetric reduction of 3-carbonyl-3-ethyl phenylpropinate using IACR was established in toluene/Tris-HCl(volume ratio 12.5:10)diphasic system.The experimental data of the change of product and substrate concentration during the reaction process were measured.The parameters in model are as follows::Vm=3×10-3 moL·L-1·h-1,KB=9×10-3 moL·L-1·h-1,KA=7×10-6 moL·L-1·h-1.The calculation curve from this kinetic model can be a good fit with the experimental data.