(3R,5R)-tert-butyl 3,5-dimethylpiperazine-1-carboxylate

Background: Aldo-keto reductases-catalyzed transformations of ketones to chiral alcohols have become an established biocatalytic process step in the pharmaceutical industry. Previously, we have discovered an aldo-keto reductase (AKR) from Kluyveromyces marxianus that is active to the aliphatic tert-butyl 6-substituted (5R/S)-hydroxy-3-oxohexanoates, but it is inactive to aromatic ketones. In order to acquire an excellent KmAKRmutant for ensuring the simultaneous improvement of activity-thermostability toward tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) and broadening the universal application prospects toward more substrates covering both aliphatic and aromatic ketones, a fluorescence-based high-throughput (HT) screening technique was established. Main methods and major results: The directed evolution of KmAKR variant M5 (KmAKR-W297H/Y296W/K29H/Y28A/T63M) produced the "best" variant M5-Q213A/T23V. It exhibited enhanced activity-thermostability toward (5R)-1, improved activity toward all 18 test substrates and strict R-stereoselectivity toward 10 substrates in comparison to M5. The enhancement of enzymatic activity and the extension of substrate scope covering aromatic ketones are proposed to be largely attributed to pushing the binding pocket of M5-Q213A/T23V to the enzyme surface, decreasing the steric hindrance at the entrance and enhancing the flexibility of loops surrounding the active center. In addition, combined with 0.94 g dry cell weight (DCW) L-1 glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration, 2.81 g DCW L-1 M5-Q213A/T23V completely converted (5R)-1 of up to 450 g L-1 at 120 g g-1 substrates/catalysts (S/C), yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2, > 99.5% d.e.p ) with a space-time yield (STY) of 1.08 kg L-1 day-1 . Conclusions: A fluorescence-based HT screening system was developed to tailor KmAKR's activity, thermostability and substrate scope. The "best" variant M5-Q213A/T23V holds great potential application for the synthesis of aliphatic/aromatic R-configuration alcohols.

Three strongly emissive Cu(i) complexes [Cu(tBupzmpy)(POP)]BF4(1), [Cu(Phpzmpy)(POP)]BF4(2) and [Cu(Adpzmpy)(POP)]BF4(3) (tBupzmpy = 2-(5-(tert-butyl)-1H-pyrazol-3-yl)-6-methylpyridine, Phpzmpy = 2-methyl-6-(5-phenyl-1H-pyrazol-3-yl)pyridine, Adpzmpy = 2-(5-((3R,5R)-adamantan-1-yl)-1H-pyrazol-3-yl)-6-methylpyridine, and POP = bis[2-(diphenylphosphino)phenyl]ether) were synthesized and characterized. These complexes exhibit bright bluish-green photoluminescence in the solid state with quantum yields of 91% (1), 71% (2) and 77% (3) and lifetimes of 13.4 μs (1), 32.9 μs (2) and 34.1 μs (3) at room temperature. The results of theoretical calculations, coupled with the temperature dependence of the spectroscopic properties and emission decay behaviors, reveal that the title Cu(i) complexes emit efficient thermally activated delayed fluorescence (TADF) from excited states involving metal-to-ligand charge transfer (MLCT) transitions and ligand-to-ligand charge transfer (LLCT) transitions. The emissive-state characteristics and emission properties of the investigated Cu(i) complexes were tuned effectively by changing the steric and electronic structures of the diimine ligands.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The Km values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate vmax was 55.56 μmol/min/mg. Compared with M1, the catalytic efficiency kcat/Km of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e.P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.