Alcohol dehydrogenase and application thereof in synthesis of chiral heterocyclic alcohol

Abstract

The invention discloses an efficient method for preparing a heterocyclic drug intermediate by an enzyme method. Alcohol dehydrogenase and glucose dehydrogenase are coupled to catalyze a heterocyclic ketone substrate to generate a chiral heterocyclic alcohol drug intermediate. The alcohol dehydrogenase disclosed by the invention can relieve the product inhibition effect in a single water phase system without adding any cosolvent, and the conversion rate can reach 99% or above within 8 hours. Alcohol dehydrogenase and glucose dehydrogenase are coupled, gram-level preparation with the substrate concentration as high as 200 g.L <-1 > is achieved in a single aqueous phase system without adding any exogenous coenzyme and organic cosolvent, and the catalyst loading capacity is 8% (m / m). The optical purity of the final product (R)-N-Boc-3-hydroxypiperidine is as high as 99.5%, and the product purity is 99.3%.

Description

technical field [0001] The invention belongs to the technical field of biochemical engineering, in particular to an alcohol dehydrogenase and its application in synthesizing chiral heterocyclic alcohols. Background technique [0002] N-tert-butoxycarbonyl-3-hydroxypiperidine [NBHP], as a typical organic heterocyclic amine, is widely used as the basis and reagent for the synthesis of organic compounds (including drugs) in the fields of lowering blood pressure, anti-tumor, and anti-coccidian Diseases and other high value-added medical fields have been applied. A pair of enantiomers of NBHP have different physiological activities, (S)-NBHP is the key chiral drug intermediate of ibrutinib, the top five commercially available lymphoma drug for many years, and (R)-NBHP It is widely used in the synthesis of benidipine for the treatment of hypertension and a variety of potential drugs, such as JAK inhibitors and Chk1 inhibitors. The preparation methods of NBHP mainly include chemi...

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Problems solved by technology

[0008] (6) In 2017, Li-Feng Chen et al. isolated NADPH-dependent reductase (YGL039W) from Kluyveromyces marxense ATCC748 to produce (R)-NBHP, which showed excellent catalytic activity and utilized GDH for cyclic coenzyme regeneration. Adding co-solvent isopropanol to the reaction system achieved a substrate concentration of 400g L -1 biotransformation, however its catalyst addition is higher (10%, m / v)

[0014] (12) Patent CN201310173088.2 discloses a method of using recombinant ketoreductase (KRED) enzyme powder to asymmetrically reduce N-BOC-3-piperidone, but does not disclose the gene sequence or amino acid of ketone reductase (KRED) sequence

Patent CN201310054684.9 discloses an asymmetric synthesis of (S)-1-Boc-3-hydroxypiperidine using alcohol dehydrogenase PAR, but the organic reagent isopropanol is used to carry out the coenzyme cycle, and the organic reagent has a negative effect on the enzyme activity. There is a large degree of damage, and there is obvious inhibitory effect

Patent CN201610132936.9 discloses an asymmetric reductase ketone reductase (KRED) using carbonyl reductase RECR enzyme, but the enzyme needs to be purified by Ni-NTA, and needs to use 2-octanol-water two-phase reaction, which is not conducive to large-scale production or higher production costs

The Pichia sp.SIT2014 reported in patent CN108220358A can be used as a biocatalyst for preparing (S)-NBHP, but too much catalyst addition increases the production cost

CN10822061A reports that ketoreductase MT-KRED is used for preparing (S)-NBHP, but needs to add expensive coenzyme in reaction process

[0015] Although the ketoreductase reported above can be used to prepare (R)- or (S)-NBHP, the reaction process needs to use expensive coenzymes, or more enzymes, organic solvents, etc., especially (R)-NBHP , is still in its infancy, which is not conducive to the production application in the actual industry

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