In vitro biocatalytic pathway design: orthogonal network for the quantitative and stereospecific amination of alcohols

Abstract

The direct and efficient conversion of alcohols into amines is a pivotal transformation in chemistry. Here, we present an artificial, oxidation-reduction, biocatalytic network that employs five enzymes (alcohol dehydrogenase, NADP-oxidase, catalase, amine dehydrogenase and formate dehydrogenase) in two concurrent and orthogonal cycles. The NADP-dependent oxidative cycle converts a diverse range of aromatic and aliphatic alcohol substrates to the carbonyl compound intermediates, whereas the NAD-dependent reductive aminating cycle generates the related amine products with >99% enantiomeric excess (R) and up to >99% conversion. The elevated conversions stem from the favorable thermodynamic equilibrium (K' = 1.88 × 10 and 1.48 × 10 for the amination of primary and secondary alcohols, respectively). This biocatalytic network possesses elevated atom efficiency, since the reaction buffer (ammonium formate) is both the aminating agent and the source of reducing equivalents. Additionally, only dioxygen is needed, whereas water and carbonate are the by-products. For the oxidative step, we have employed three variants of the NADP-dependent alcohol dehydrogenase from Thermoanaerobacter ethanolicus and we have elucidated the origin of the stereoselective properties of these variants with the aid of in silico computational models.

Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell‐free system

“…Seventy‐one percent conversion was obtained at 12 hr, giving 14.1 mM ( R )‐ 3a in 99% ee , with a TTN for NADH recycling of 1410. To the best of our knowledge, this was the highest TTN ever reported in ADH–AmDH‐catalyzed cascade transformation of alcohols to amines (TTN, <40; Knaus et al, ; Mutti et al, ; Thompson & Turner, ). The incomplete conversion of ketone 2a to amine 3a was mainly due to the low catalytic efficiency of the AmDH ( k cat / K m : 10 2 –10 3 ; Abrahamson et al, ; Ye et al, ).…”

“…AmDH: amine dehydrogenase; GDH: glucose dehydrogenase; MNP: magnetic nanoparticle; NOX: NADH oxidase; TTN: total turnover number to 3a is illustrated in Figure 1b. During the biotransformation, the Knaus et al, 2017;Mutti et al, 2015;. The immobilized AmDH and GDH for the cascade transformation of alcohol 1a enhanced the TTN for NADH recycling to 1410-1260 with the formation of (R)-3a in 99% ee and 71-84% conversion.…”

Sequential Two‐Step Stereoselective Amination of Allylic Alcohols through the Combination of Laccases and Amine Transaminases

“…Biocatalytic methods have emerged as a sustainable solution for the synthesis of chiral amines by means of the single action of enzymes of several classes, including lipases, amine transaminases (ATAs), amine oxidases, amine dehydrogenases, imine reductases or reductive aminases, but, interestingly, great efforts have in recent years been devoted to the synthesis of chiral amines through chemo‐, photo‐ and multienzymatic approaches . As an example, the selective amination of racemic sec ‐alcohols through elegant cascades based on the combination of alcohol‐dehydrogenase‐catalysed oxidation to ketones with subsequent bioamination by using ATAs or amine dehydrogenases has been described. In spite of these efforts, the biocatalytic synthesis of (3 E )‐4‐arylbut‐3‐en‐2‐amines has received little attention, the activity of commercially available amine transaminases for the bio‐transamination of (3 E )‐4‐phenylbut‐3‐en‐2‐one into optically active (3 E )‐4‐phenylbut‐3‐en‐2‐amine being low (<30 %) …”

Efficient synthesis of enantiopure amines from alcohols using restingE. colicells and ammonia

“…The bioamination of alcohols using purified ADH and AmDH in vitro required a higher molar concentration of the latter enzyme in order to achieve elevated conversions. 11,18,25 Although expression levels in vivo might theoretically match the optimal molar ratio found for in vitro experiments, we illustrate in this work that many additional dynamics affect the productivity of the bioamination using engineered E. coli resting cells. Since altering the relative expression levels of ADH and AmDH would also impact the effect of these other factors, further tuning of the expression levels was not considered necessary at this stage.…”