Abstract
The recombinant carbonyl reductase from Rhodococcus erythropolis WZ010 (ReCR) demonstrated strict (S)-stereoselectivity and catalyzed the irreversible reduction of N-Boc-3-piperidone (NBPO) to (S)-N-Boc-3-hydroxypiperidine [(S)-NBHP], a key chiral intermediate in the synthesis of ibrutinib. The NAD(H)-specific enzyme was active within broad ranges of pH and temperature and had remarkable activity in the presence of higher concentration of organic solvents. The amino acid residue at position 54 was critical for the activity and the substitution of Tyr54 to Phe significantly enhanced the catalytic efficiency of ReCR. The kcat/Km values of ReCR Y54F for NBPO, (R/S)-2-octanol, and 2-propanol were 49.17 s−1 mM−1, 56.56 s−1 mM−1, and 20.69 s−1 mM−1, respectively. In addition, the (S)-NBHP yield was as high as 95.92% when whole cells of E. coli overexpressing ReCR variant Y54F catalyzed the asymmetric reduction of 1.5 M NBPO for 12 h in the aqueous/(R/S)-2-octanol biphasic system, demonstrating the great potential of ReCR variant Y54F for practical applications.
Highlights
Many natural products and active pharmaceutical ingredients share a common piperidine core, and the introduction of a chiral hydroxyl group on the C3-position of the piperidine ring may alter the bioactivity of the molecule [1,2,3]. (S)-N-Boc-3-hydroxypiperidine ((S)-NBHP) is a key chiral intermediate in the synthesis of ibrutinib as the inhibitor of Bruton’s tyrosine kinase [4]
Coenzymes are required in carbonyl reductase-catalyzed reactions, and well-established approaches for coenzyme regeneration include the use of a second enzyme and a second substrate, and the use of the second substrate catalyzed by the same enzyme (i.e., 2-propanol) [10]
In the presence of 10% (v/v) 2-propanol, the bioreduction of 0.5 M NBPO catalyzed by whole cells presence of 10% (v/v) 2-propanol, the bioreduction of 0.5 M NBPO catalyzed by whole cells overexpressing reductase from Rhodococcus erythropolis WZ010 (ReCR) Y54F gave a (S)-NBHP yield of 98.08% after 12 h, which was 1.34 times higher overexpressing ReCR Y54F gave a (S)-NBHP yield of 98.08% after 12 h, which was 1.34 times higher than that of ReCR (72.15%)
Summary
Many natural products and active pharmaceutical ingredients share a common piperidine core, and the introduction of a chiral hydroxyl group on the C3-position of the piperidine ring may alter the bioactivity of the molecule [1,2,3]. (S)-N-Boc-3-hydroxypiperidine ((S)-NBHP) is a key chiral intermediate in the synthesis of ibrutinib as the inhibitor of Bruton’s tyrosine kinase [4]. In the chemical synthesis of (S)-NBHP, employed strategies include the synthesis of racemic 3-hydroxypiperidine followed by chiral resolution and the enantiospecific synthesis of (S)-NBHP from chiral precursors. The former only achieves a maximum yield of 50%, making the process economically unviable, while the latter appears to be limited because of the lengthy procedure, rather poor yields of the products, and the use of potentially hazardous reagents [1,5,6]. An NADPH-dependent carbonyl reductase from Saccharomyces cerevisiae (YDR541C) was employed for the efficient synthesis of (S)-NBHP from NBPO by adopting a biphasic system to alleviate product inhibition and using glucose/glucose dehydrogenase to achieve coenzyme regeneration [8]. The enzyme KR-110 was heat-sensitive and the substrate inhibition was obviously observed at a substrate concentration of
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