Stereoselective monoreduction of bulky 1,2-dicarbonyls catalyzed by a benzyl reductase from Pichia glucozyma (KRED1-Pglu)

Enantiomerically enriched hydroxyketones are well-established intermediates for the synthesis of several bioactive compounds [1] and can be chemically obtained by stereoselective reduction of one of the carbonyl moieties of the corresponding diketones. However, enzymatic strategies are characterized by higher catalytic efficiency, milder reaction conditions, higher stereo- and regioselectivity, and fewer numbers of synthetic steps. Therefore, they can be chosen as convenient and environmentally friendly alternatives.[2] A NADPH-dependent benzyl reductase from the non-conventional yeast Pichia glucozyma (KRED1-Pglu [3]) was over-expressed in E. coli, purified and exploited to catalyze the asymmetric monoreduction of bulky aromatic 1,2-dicarbonyl compounds. The cofactor was recycled by an enzyme-coupled system (glucose-glucose dehydrogenase (GDH) from Bacillus megaterium). The recombinant KRED1-Pglu showed a wide range of activity (24-97% conversion) and excellent stereoselectivity (ee ≥ 96% in all but one case). On the contrary, it proved either inactive or very poorly active towards most 1,3- and 1,4-dicarbonyls tested as potential substrates. In order to understand this peculiar behavior, the enzyme was crystallized (1.77 Å resolution) and its active site was investigated to identify the recognition residues involved in the desymmetrization reaction. QM and classical calculations also allowed for a proposal of the catalytic mechanism, along with an in silico reactivity prediction.[4] [1] G. Aullón; P. Romea; F. Urpí Synthesis, 2017, 49, 484-503. [2] P. Hoyos; J.-V. Sinisterra; F. Molinari; A.R. Alántara; P. Domínguez de María Acc. Chem. Res., 2010, 43, 288-299. [3] M.L. Contente; I. Serra; M. Brambilla; I. Eberini; E. Giannazza; V. De Vitis; F. Molinari; P. Zambelli; D. Romano Appl. Microbiol. Biotechnol., 2016, 100, 193-201. [4] M. Rabuffetti; P. Cannazza; M.L. Contente; A. Pinto; D. Romano; P. Hoyos; A.R. Alcántara; I. Eberini; T. Laurenzi; L. Gourlay; F. Di Pisa; F. Molinari Bioorg. Chem., 2021, 108, 104644.