Study On Catalytic Synthesis Of Ibrutinib Chiral Intermediate By Recombinant E.coli Co-expressing Alcohol Dehydrogenase And Glucose Dehydrogenase

Optically pure secondary alcohols are a class of important chiral synthetic blocks,because of their active hydroxyl groups.It is possible to obtain different chiral synthons by simple modifications of secondary alcohols,such as natural products,pesticides,flavors and fragrances,materials,drugs.Ibrutinib is a targeted anti-cancer drug that inhibits Bruton tyrosine kinase for the treatment of mantle cell lymphoma.(S)-N-boc-3-hydroxy piperidine is a key chiral intermediate for the synthesis of Ibrutinib.In this paper,the gene of alcohol dehydrogenase from Thermoanaerobacter brockii(ADH)and glucose dehydrogenase from Bacillus subtilis(GDH)were ligated into the multiple cloning sites of pRSFDuet plasmid and constructed recombinant E.coli for co-expressing ADH and GDH.The regeneration of coenzyme NADPH can be achieved by the use of the constructed E.coli.Polyacrylamide gel electrophoresis showed that alcohol dehydrogenase and glucose dehydrogenase were soluble in the culture conditions and the molecular weight of adh and gdh respectively were 38 and 27 kDa,which were accordance with that reported in the literature.Then,the medium and culture conditions of the recombinant E.coli were optimized by single factor optimization method from the aspects of carbon source,nitrogen source,metal ion,induction condition,initial pH and culture temperature.The final optimized medium composition was(g/L):soluble starch 30,yeast powder 15,sodium chloride 5.The initial pH of the medium was adjusted to 6.0.The optimized culture conditions:IPTG(final concentration 0.025 mM)was added after incubation at 37~oC for 3 h,and then the culture was incubated at 40~oC for another 10 h.After optimization,the enzyme activity and cell mass reached 139.8 U/L and 6.6 g/L,which were approximately 11 times and 3.6 times of the original activity and cell mass,respectively.Equivalent amounts of cell from different media were compared according to asymmetric reduction 1-boc-3-piperidinone.The cells cultivated in the optimized medium reduced 1-boc-3-piperidinone which concentration is 50 mM,the conversion reached 95%after 8 h,while,the conversion was only 56%for the cells cultivated from initial LB media.At last,the synthesis of(S)-N-boc-3-hydroxypiperidine catalyzed by the recombinant E.coli whole cell was studied.The effect of substrate concentration,pH,co-solvent,the amount of cofactor and glucose on the target reaction were investigated.The optimized reaction conditions are as follows:at 50~oC,the reaction system contained 1.5 times the substrate molar equivalent of glucose as the secondary substrate,5%methanol as the co-solvent,co-enzyme addition of 0.1 mM,the conversion is higher.The conversion of 50 mM substrate in the system of 1 mL is 97.8%in 2 h.The substrate was raised to 250 mM and the pH was controlled by dilution of aqueous ammonia during the reaction,conversion is 98.5%within 1h.Further,the substrate concentration was increased to 500 mM,conversion is 96.2%within3 h without adding cofactor.