Biocatalytic Synthesis of α,β-Unsaturated 2-Keto Acids and Derivatives Using the Promiscuous Aldolase, NahE

Abstract

Type I aldolases catalyze carbon-carbon bond-forming reactions to form a diverse set of products in nature but often display high selectivity for their natural substrates. One such aldolase, NahE, is known to catalyze the condensation of pyruvate with a wide range of aldehydes to give trans-4-phenyl-2-oxo-3-butenoic acids under mild aqeuous conditions. These ,-unsaturated 2-oxo acids are versatile intermediates for synthetic transformations. NahE has also been used for the synthesis of α-fluoro-β-hydroxy esters, β-hydroxy esters, and quinaldic acids. However, a thorough study of the substrate scope on a practical scale has not been performed for the native NahE-catalyzed aldol condensation reaction. Here we report that NahE can accept >35 (hetero)aromatic and aliphatic aldehydes. Most condensation products derived from substituted benzaldehydes were isolated in >95% yield without need for further purification, while non-benzaldehyde substrates gave the corresponding products in isolated yields between 26 and 98%. Reactions could be performed on gram scale. These products could be converted to α,β-unsaturated carboxylic acids in up to 93% yield over two steps. This reaction sequence was also performed using whole cells in up to 79% yield. This work demonstrates that NahE is a robust, efficient, and versatile catalyst for organic synthesis.

Expanding Synthetic Applications of Δ1‐Piperidine‐2‐carboxylate/Δ1‐pyrroline‐2‐carboxylate Reductase from Pseudomonas syringae (DpkAPsyrin). Biocatalytic Asymmetric Synthesis of (S,E)‐2‐hydroxy‐4‐arylbut‐3‐enoic Acid Derivatives

“…13 C NMR (101 MHz, DMSO) δ 186.6, 164.9, 148.0, 134.3, 132.0, 129.6, 122.0. The spectral properties of this product agreed with those reported in the literature [5–6] …”

“…[4] Thus, we envision a straightforward enzymatic cascade process for the asymmetric synthesis of 2-hydroxy-4-arylbut-3-enoic acid derivatives. The strategy consisted of an aldol condensation reaction between pyruvate and aryl aldehydes catalyzed by trans-o-hydroxybenzylidene pyruvate hydratase-aldolase from Pseudomonas putida (HBPA Pputida , EC 4.1.2.45) [5] and an ensuing asymmetric reduction of the carbonyl group of the adduct catalyzed by DpkA Psyrin (Scheme 2B).…”

A Type 1 Aldolase, NahE, Catalyzes a Stereoselective Nitro‐Michael Reaction: Synthesis of β‐Aryl‐γ‐nitrobutyric Acids

“…NahE is a promiscuous enzyme that has been adapted for biocatalytic aldol condensation reactions, especially using the recombinant NahE from Pseudomonas putida . The reaction is thought to proceed by enamine‐activation of a ketone, pyruvate, that can react with an array of aldehydes to form trans ‐α,β‐unsaturated 2‐keto acids in near quantitative yields [28] . When alkyl aldehydes are used the R aldol products are formed with moderate to excellent enantioselectivity (80–96 % ee) [29, 30] .…”

“…The resulting products could serve as chiral building blocks, including direct precursors to GABA analogues through oxidative decarboxylation (Scheme 3e). [33] Using reaction conditions previously established by us [28,32] and others, [31] we tested the ability of NahE to catalyze the Michael addition of pyruvate to β-nitrostyrene to form 5-nitro-2-oxo-4-phenylpentanoic acid (1; Scheme 3e). With no further reaction optimization, the predicted product was isolated in 79 % yield, representing the first instance, to our knowledge, of promiscuous Michaelase activity of a wild-type aldolase.…”

“…The reaction is thought to proceed by enamine-activation of a ketone, pyruvate, that can react with an array of aldehydes to form trans-α,β-unsaturated 2-keto acids in near quantitative yields. [28] When alkyl aldehydes are used the R aldol products are formed with moderate to excellent enantioselectivity (80-96 % ee). [29,30] Howard et al also saw that elimination did not occur when a pyruvate analogue, βfluoropyruvate, was used as the substrate.…”