Engineering an Alcohol Dehydrogenase from Kluyveromyces polyspora for Efficient Synthesis of Ibrutinib Intermediate

Abstract

S)-N-Boc-3-hydroxypiperidine [(S)-NBHP] is a key intermediate for the synthesis of mantle cell lymphoma drug, ibrutinib. Here, KpADH, an alcohol dehydrogenase from Kluyveromyces polyspora, exhibits evolutionary potential in the asymmetric reduction of N-Boc-3-piperidone (NBPO) to (S)-NBHP. By screening key residues in substrate binding pocket of KpADH, an excellent variant Y127W was obtained with 6-fold improved activity of 119.3 U mg À 1 , 1.8-fold enhanced half-life of 147 h and strict S-stereoselectivity (> 99% ee). When catalyzed by Y127W, a complete conversion of 600 g L À 1 NBPO was achieved at a substrate to catalyst ratio (S/C) of 30 in 10 h. Based on crystal-structure of Y127W, molecular docking and dynamic simulations reveal hydrogen bonds formed between W127 and Boc group of NBPO, as well as improved structural stability mainly contribute to the increased catalytic activity and stereoselectivity of Y127W. This study offers guidance for engineering ADHs for biosynthesis of chiral heterocyclic alcohols, and provides insights into mechanisms in catalytic activity and stereoselectivity toward carbonyl-containing heterocyclic substrates.

Sustainable Route Toward N‐Boc Amines: AuCl3/CuI‐Catalyzed N‐tert‐butyloxycarbonylation of Amines at Room Temperature

“…[16] N-Boc amine derivatives are key intermediates in synthesizing a number of commercially available drugs. For examples, N-Boc piperidine (3 a), N-Boc 4-chloropiperidine (3 e), N-Boc 4hydroxypiperidine (3 k), and N-Boc 3-hydroxypiperidine (3 q) are key intermediates for Ritalin, [17] Bepotastine, [18] Crizotinib, [19] and Ibrutinib, [20] respectively. Thus, we further illustrated the gramscale synthesis of various medically important N-Boc amines.…”

Semirational engineering of an aldo–keto reductase KmAKR for overcoming trade‐offs between catalytic activity and thermostability

“…Consequently, the higher substrate‐enzyme binding affinity mainly contributes to the increased catalytic efficiency ( k cat / K m ) of Km AKR M13 , attributing to the formation of more stable hydrogen bonds between Km AKR M13 and (5 S )‐CHOH. Similarly, the significant activity enhancement of the alcohol dehydrogenase from Kluyveromyces polyspora ( Kp ADH) was due to the additional hydrogen bonds formed between the Boc group of the substrate N‐Boc‐3‐piperidone (NBPO) and W127, which made the substrate‐enzyme binding more stable (Y. F. Wu et al, 2020).…”

“…Structural analysis showed that the hydrogen bonds between Y64/H122 and (5S)-CHOH are shortened from 2.9 to 2.6Å to 2.5 and 2.2Å, respectively (Figure 8a binding more stable (Y. F. Wu et al, 2020).…”

Recent advances in structure‐based enzyme engineering for functional reconstruction

“…Additional hydrogen bonds were introduced by the Y127W mutation between the indole group of W127 and the Boc group of NBPO, favoring the reach of the catalytic conformation for nucleophilic attack in the reaction. The Kp ADH‐Y127W shows strict S ‐stereoselectivity (>99% ee ), and a 5.03‐fold higher specific activity (119.3 U mg −1 ) than that of the wild‐type (Wu et al, 2021).…”