Development of a Biocatalytic Process to Prepare (S)-N-Boc-3-hydroxypiperidine

Abstract

S)-N-Boc-3-hydroxypiperidine (S-NBHP) is a useful synthon for the synthesis of pharmaceutical intermediates including ibrutinib, the API of the newly approved drug Imbruvica, for the treatment of lymphoma. To our knowledge, there are no published biotransformation methods scalable to prepare S-NBHP. We report here the development of an efficient process catalyzed by recombinant ketoreductase (KRED) to reduce N-Boc-piperidin-3-one to obtain optically pure S-NBHP. The process has been optimized and demonstrated to have commercial potential with 100 g/L of substrate concentration, product of >99% ee with under 5% of enzyme loading (w/w). ■ INTRODUCTIONA large number of natural and unnatural bioactive molecules have one or more piperidine rings. 1 There are many active pharmaceutical ingredients (API) which also contain this moiety. 2 The hydroxyl group on the C3-position would introduce a chiral carbon atom and may significantly affect the bioactivity of the molecule. 3 As a result, chiral hydroxypiperidines and their derivatives are important synthons used in the pharmaceutical industry.Several methods have been reported for the synthesis of chiral piperidines including classic diastereomeric resolution, 4 asymmetric synthesis, 5 and asymmetric reduction, for example, of 4-oxo-piperidine-3-carboxylic acid esters followed by multistep conversions (Scheme 1). 6 All these methods suffered from low yields or lengthy synthesis. Lacheretz et al. reported the bioreduction of cyclic 3-oxo-amines using the tissue of Daucus carota, where six piperidin-3-one derivatives could be reduced to S-alcohols with varying enantioselectivity. 7 Among the substrates, the reduction of N-Boc-piperidin-3-one showed the highest enantioselectivity, giving (S)-N-Boc-3-hydroxypiperidine (S-NBHP) with 95% optical purity. Unfortunately the reaction is not practical due to low substrate concentration (3 mM), high catalyst concentration (23%, m/v), moderate chiral purity (95% ee) and low yield (73%).Ketoreductase (KRED)-mediated biotransformation has been applied more and more widely in pharmaceutical industry nowadays. 8 In general, there are two strategies to locate a KRED for practical application. On the one hand, the discovery of new natural KRED biocatalysts using activity screening and genome mining continues to enlarge the substrate spectrum. 9 On the other hand, enzyme engineering technologies including directed evolution and semirational and rational design can engineer KREDs with high efficiency and stability. 10 Directed evolution and rational design as the most important technique of protein engineering have emerged to be powerful tools for manipulating protein properties with numerous successful stories. 11 The availability of highly diversified KRED libraries makes it increasingly possible to discover biocatalysts to meet the metrics of industrial processes. Here we report such a work to screen and apply recombinant KREDs to catalyze the asymmetric preparation of S-NBHP on an industrial scale. ■ RESULTS AND DISCUSSIONThe target enz...

Efficient bioreductive production of (S)-N-Boc-3-hydroxypiperidine using ketoreductase ChKRED03

“…However, at a substrate concentration of 100 g/L, the system achieved a maximal conversion of 90.6% even after continuously removing acetone from the reactor, and no improvement was observed with prolonged reaction time. Only the fed-batch strategy of two 50 g/L batches afforded 97.7% conversion after 24 h [20]. Whereas, in our system, no significant substrate inhibition was observed for the tested concentration of 200 g/L, and the productivity was 16-fold of the previous process ( Table 1).…”

“…Up until now, the only practical solution to the biocatalytic production of (S)-NBHP has been reported by Ju et al [20]. It has the advantage of using NADH as the cofactor and isopropanol as the ultimate reducing agent, avoiding the use of a second enzyme.…”

“…Basic requirements [25] Previous work [20] Our work a Substrate was added in two 50 g/L batches. For one 100 g/L batch, a maximal conversion of 90.6% was reported.…”

“…The first bioreductive synthesis of (S)-NBHP was achieved together with other five hydroxypiperidines using the tissue of Daucus carota, resulting in moderate yield (73%) and good stereoselectivity (95% ee). However, the concentration of the substrate, N-Boc-piperidin-3-one (NBPO), was very low (3.3 mM) [19].mercially available KRED, which involves adding substrate in two 50 g/L batches at a substrate/catalyst ratio of 20 (w/w), and delivers enantiopure (S)-NBHP (>99% ee) with 99.8% conversion after 24 h, corresponding to a space-time yield of 100 g/L/d [20].…”

A Biocatalytic Route to Highly Enantioenriched β‐Hydroxydioxinones

“…[24] Specifically, ketoreductases have been utilized in the production of a variety of statins, [25] β-lactam antibiotics, [26] anti-inflammatory medicines, [27] cancer treatments [28] and other various pharmaceutical building blocks. [29] Whole cell systems for biocatalytic reductions have been employed, but these processes are traditionally plagued by low yields and low selectivity.…”