Efficient synthesis of the key chiral alcohol intermediate of Crizotinib using dual-enzyme@CaHPO4 hybrid nanoflowers assembled by mimetic biomineralization

BACKGROUND To develop an environment-friendly approach for the synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethyl alcohol, an intermediate of anti-cancer drug Crizotinib, aldehyde ketone reductase and alcohol dehydrogenase were overexpressed in Escherichia coli Rosetta (DE3) and purified via chromatography. Subsequently, they were co-crystalized with CaHPO4 at 4 degrees C to form dual-enzyme@CaHPO4 hybrid nanoflowers (hNFs) which was then used to catalyze the synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethyl alcohol along with the evaluation of its thermal stability and recycling stability. RESULTS At optimum pH of 7.0, the activities of AKR and ADH confined in the dual-enzyme@CaHPO4 hybrid nanoflowers were 3.3- and 2.1-fold that of the corresponding free one. The thermos-stability of confined enzymes was also significantly improved: both enzymes within the hNFs remained more than 80% of initial activities after incubation at 60 degrees C for 8 h, while free enzymes only retained 20% of initial activities under the same treatment conditions. Moreover, AKR and ADH immobilized with a mole ratio of 3:1 confined in hybrid nanoflowers exhibited the highest catalytic activity for the synthesis of chiral ethyl alcohol with a yield up to 90.8% after 12 h. Besides, the final product (S)-1-(2,6-dichloro-3-fluorophenyl) ethyl alcohol showed a high ee value of 99.99%. Further, the hybrid nanoflowers retained their initial activity after 16 recycling cycles of synthesis reaction. CONCLUSION The dual-enzyme@CaHPO4 hybrid nanoflowers efficiently catalyzed synthesis of the chiral compound (S)-1-(2,6-dichloro-3-fluorophenyl) ethyl alcohol. The method can also be applied to other multi-enzyme systems and facilitate their cascade reactions and substrate channeling. (c) 2018 Society of Chemical Industry