Asymmetric Reductive Amination of Structurally Diverse Ketones with Ammonia Using a Spectrum-Extended Amine Dehydrogenase

Abstract

Amine dehydrogenase-catalyzed reductive amination of prochiral ketones with ammonia is a promising method for the synthesis of optically pure amines in the pharmaceutical and fine chemical industries. However, previously reported amine dehydrogenases show restricted catalytic capacity toward bulky ketones, which limits their widespread applications toward the production of chiral amines. Herein, we expanded the substrate scope of an engineered amine dehydrogenase GkAmDH from Geobacillus kaustophilus via laboratory evolution for the reductive amination of an extensive set of ketones. Several beneficial mutants were identified with a up to 2.2 U mg–1 activity toward bulky benzylacetone, 110-fold higher than that of M0. Using the engineered M3 and M8, structurally diverse bulky chiral amines could be synthesized with up to >99% conversion, >99% ee, and up to 18,900 TON. Among them, two key chiral intermediates used in the synthesis of the drugs medroxalol and dilevalol were produced on a gram scale in up to 85% yield and >99% ee. Additionally, the engineered enzymes M3 and M8 displayed considerable thermostability with a half-life of more than three days at 50 °C. These results demonstrate that these engineered amine dehydrogenases are promising biocatalysts for the synthesis of chiral amines. Molecular dynamics simulations provide insights into how mutations improve the amination activity toward bulky ketones and the thermostability.

Impact of aqueous micellar media on biocatalytic transformations involving transaminase (ATA); applications to chemoenzymatic catalysis

“…The highly lipophilic ketone 2 shown in Fig. 2E was most receptive to the presence of additional TPGS-750-M 13 in the aqueous medium. Thus, while 2 wt% TPGS-750-M afforded an enhancement of 19% to form 14 over a 24-hour period (i.e., from 22 to 41%), increasing the amount to 6 wt% resulted in furthering the extent of conversion to 56%.…”

“…In recognition of the importance of amines, together with increasingly available opportunities to access them, 8 studies were envisioned involving transaminases (aka aminotransferases, ATAs) 13,14 known to convert ketones to nonracemic primary amines. Key to these efforts was the potentially beneficial impact on the extent of conversion, albeit unknown, that a surfactant might have simply due solely to its presence in the reaction medium.…”

Artemisinin Targets Transcription Factor PDR1 and Impairs Candida glabrata Mitochondrial Function

“…To investigate the mechanism of the decreased artemisinin sensitivity in PDR1 mutants, molecular dynamics simulations were performed for both the wild type PDR1 and mutant PDR1 with artemisinin following a previously described method [ 23 ]. Tleap module in Amber14 was used for building the force field parameter for each system, and the force field parameters were generated through ff14SB and GAFF2 force fields.…”

Cover Feature: Expanding the Substrate Scope of Native Amine Dehydrogenases through In Silico Structural Exploration and Targeted Protein Engineering (ChemCatChem 22/2022)

“…In addition to X-ray crystallography, MD simulations were run to understand the influence of W145A mutation on protein flexibility and specificity towards cofactors and substrates. [8,23] Accordingly, the study was carried out on an extensive set of Enzyme-Cofactor-Ligand systems where both CfusAmDHÀW145A (M) or CfusAmDH (WT) were considered, in complex with NAD + (NAD) or NADP + (NADP) cofactors, and 2 a (PEN) or 2 c (HEP) ligands.…”

“…[6] To extend the applicability of these ORs in synthetic processes, further protein engineering work has been reported to design enzymes that offer higher conversions of longer or more hindered carbonyl compounds. Key examples are the improved L-AmDH-TV L39A/ A112G, [7] GkAmDHÀM0/M3/M8 [8] and L-BcAmDHÀM5 which displayed activity towards nonan-2-one, dioxolo-benzylacetone and the even more bulky ketones 6-phenylpentan-2-one and 6phenylhexan-2-one, [9] respectively. As the main effort was directed towards the production of chiral amines, only very few enzyme activities towards long aliphatic aldehydes have been reported thus far.…”