Electronic Effect‐Guided Rational Design of Candida antarctica Lipase B for Kinetic Resolution Towards Diarylmethanols

Abstract

Herein, we developed an electronic effect-guided rational design strategy to enhance the enantioselectivity of Candida antarctica lipase B (CALB) mutants towards bulky pyridyl(phenyl) methanols. Compared to W104A mutant previously reported with reversed S-stereoselectivity toward sec-alcohols, three mutants (W104C, W104S and W104T) displayed significant improvement of Senantioselectivity in the kinetic resolution (KR) of various phenyl pyridyl methyl acetates due to the increased electronic effects between pyridyl and polar residues. The electronic effects were also observed when mutating other residues surrounding the stereospecificity pocket of CALB, such as T42A, S47A, A281S or A281C, and can be used to manipulate the stereoselectivity. A series of bulky pyridyl(phenyl) methanols, including S-(4-chlorophenyl)(pyridin-2-yl) methanol (S-CPMA), the intermediate of bepotastine, were obtained in good yields and ee values.

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase

“…The engineering of catalytic‐tetrad residues can increase the enzyme catalytic efficiency (as shown in this study) and reconstruct the active sites for new enzymatic functions in Candida antarctica lipase B (Branneby et al, 2003; Li et al, 2021), halohydrin dehalogenase (Fox et al, 2007), and squalene‐hopene cyclase (Hammer et al, 2015). Together, these suggested that engineering the catalytic tetrad is an alternative strategy to tune enzymatic properties.…”

Ruthenium(II)‐Catalyzed Selective C(sp2)−H Acyloxylation of 2‐Aroyl‐Pyridine Derivatives with Sodium Carboxylate

“…[18] Additionally, the ketone functional group could be efficiently reduced into a methylene or hydroxyl group as demonstrated by examples 5 e and 5 f in 90% and 81% yield, respectively. [19] In conclusion, we have developed a rutheniumcatalyzed direct CÀ H acyloxylation of 2-aroyl pyridines with readily accessible sodium carboxylates under mild conditions. A wide range of aliphatic and aromatic sodium carboxylate coupling partners are well suited substrates in this ruthenium-catalyzed system, offering a variety of substituted acyloxylated 2-aroyl pyridines in 45%-84% yields.…”

“…Among a variety of additives that were examined (AgBF 4 , AgNTf, KSbF 6 , KPF 6 , NaPF 6 ), AgSbF 6 proved to be superior. Notably, the desired oxygenation reaction did not occur when [RuCl 2 (p-cymene)] 2 was used as the catalyst in the absence of an additive, which can be rationalized by the in situ generation of a cationic ruthenium(II) catalyst (entries [17][18][19][20][21][22]. The yield increased to 60% upon the addition of 40 mol% AgSbF 6 as an additive (entry 23).…”

Rational design of enzyme activity and enantioselectivity

“…Three polar amino acids including Cys, Ser and Thr with similar size as alanine were introduced at the 104 position to reconstruct the substrate binding pocket with increased polarity, thereby increasing the interactions between polar side chains of residues at 104 and N atom of pyridine-4-yl in the substrate. As a result, the mutants W104C and W104T improved the S -enantioselectivity from 91% to 99% and 98%, respectively, with the similar yield compared to the mutant W104A ( Li et al, 2021 ). Similarly, the substrate binding was rationally modified by simultaneously tuning electronic interactions and steric effects, leading to up to 22-fold enhancement in the enantioselectivity of an esterase BioH towards methyl ( S )-o-chloromandelate ( S -CMM) ( Gu et al, 2015 ).…”

“…The molecular dynamics simulations showed that, as expected, the mutant V328N formed a stable hydrogen bond with the ester group of the LAC, which was exactly placed at the opposite side in the catalytic pocket compared to Ser264 in the S -selective variant ( Calvó-Tusell et al, 2022 ). Strengthening electronic interactions between substrate and enzyme active site was also used to improve the enantioselectivity of Candida antarctica lipase B (CALB) ( Li et al, 2021 ). It was difficult for CALB to catalyze the hydrolytic kinetic resolution (KR) of bulky racemic phenyl(pyridin-4-yl) methyl acetate due to the steric effect of Trp104.…”