Enzymatic Preparation Of Atazanavir Chiral Intermediate (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol

(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-2-butanol,prochiralpharmaceutical intermediateofAtazanavir,canbepreparedbycatalyticreductionof(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-(2R)-butanone.Due to the participation of coenzyme in the reaction,such as NADH?NADPH and FAD,the expensive coenzyme is the limited factor to industrial application.To solve the problem of coenzyme regeneration,a genetic engineering method to express ketoreductase which are utilized inenzymepreparationwithultrasonication,couldcatalyze(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-2-butanolinto(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-(2R)-butanonewas established in this eassay.The gene fragment of keto reductase(KRED)were obtained by gene synthesis,which is deriving from short-chain dehydrogenase(SDP)of New sphingobacillus.The fragment was inserted into pET-28a and obtained pET-28a-kred which was transformed into host bacterial E.coli BL21(DE3).Then we obtained recombinant strain E.coli BL21-KRED,inducing with IPTG,and observed the SDS-PAGE showing that the site 34KD have a apparant band,in constrast to the concrol,E.coli BL21(DE3)without inducing.The optimize expression results show that E.coli BL21-KRED have the highest expression under the temperature 25?within 0.2mmol/L IPTG induced for 8 hours.With construction of the recombinant strain E.coli BL21-KRED,we realize the expressionofketoreductase,whichcouldcatalyze(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-2-butanolinto(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-(2R)-butanone.Afteroptimizingthe reaction condition,we utilized NAD~+as Hydrogen ion delivery toll,with cosolvent and reducingpowersuppliedbyisopropanol,andcatalyze(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-2-butanolinto(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-(2R)-butanone.Ourresultshowsthat underthetemperature27?,pH10.0andreactiontime15hours,(3S)-3-(N-Boc-aMino)-1-chloro-4-phenyl-(2R)-butanonegetsahighest yield.The reaction's percent conversion would almost arrive 100%,when the charging rate is under 15%.(2R)-butanone.Then we have utilized the absorbtion within diatomite and productino and optimized the technology of absorbtion,which includes temperature,time and frequency of absorbtion.Finally we get the conclution;in 40?,processing once or twice,2 hours each time,product will almost be absorbed..And then,ethyl acetate will elution the product down.By evaporation,we get the crude product.Through recrystal,crude product will be purified to be sterling with purity of 99.5%.The final yield of this technology is 82~85%.