Stereospecific production of double chiral compounds by novel enzymatic processes

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Chiral compounds are useful intermediates for the production of pharmaceuticals, fine chemicals, and so on. Because of high stereospecificity and environmental aspects, biocatalytic production of chiral compounds has been attracting considerable attention. On the other hand, double chiral compounds, which have two asymmetric carbons in their molecules, are also important for the synthesis of many useful compounds, such as pharmaceuticals and agrochemicals. There are several ways to enzymatically introduce two chiral centers into organic compounds.Two-step introduction of double chiral centers into the prochiral compound, ketoisophorone, forming (4R,6R)-actinol through two separate enzyme reactions were performed. By combination of old yellow enzyme and levodione reductase, production of doubly chiral compound, (4R,6R)-actinol, which is a useful chiral intermediate for the synthesis of naturally-occurring optically active compounds, such as zeaxanthin and xanthoxin, was investigated. E.coli transformant cells co-expressing old yellow enzyme or levodione reductase gene with cofactor regeneration enzyme gene were used as the catalysts. 100 g/l of ketoisophorone was almost stoichiometrically and stereospecifically converted to (4R,6R)-actinol by two-step reaction Ephedrine is also one of the useful double chiral compounds, and d-ψ-isomer has pharmacological effects as a decongestant or antiasthmatic compound. We found that Rhodococcus erythropolis produces a novel NADP^+-dependent amino alcohol dehydrogenase (AADH), which catalyze the asymmetric reduction of l-isomer in the racemic mixture of the corresponding amino ketone, MAK, to d-ψ-ephedrine. Using E.coli transformant cells coexpressing the genes of AADH and cofactor regeneration enzyme, biocatalytic conversion of racemic MAK to d-ψ-ephedrine by whole cell reaction was performed. Cosequently, 178 mM of d-ψ-ephedrine was formed in 24 h reaction with a molar yield of 89%.