We report the first rationally designed (S)-selective MenD from E. coli for the synthesis of functionalized α-hydroxy ketones. By mutation of two amino acids in the active site stereoselectivity of the (R)-selective EcMenD (ee > 93%) was inverted giving access to (S)-5-hydroxy-4-oxo-5-phenylpentanoate derivatives with stereoselectivities up to 97% ee.

The catalytic asymmetric synthesis of chiral 2-hydroxy ketones by using different thiamine diphosphate dependent enzymes, namely benzaldehyde lyase from Pseudomonas fluorescens (PfBAL), a variant of benzoylformate decarboxylase from Pseudomonas putida (PpBFD-L461A), branched-chain 2-keto acid decarboxylase from Lactococcus lactis (LlKdcA) and a variant of pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC-E469G), was studied. Starting with the same set of substrates, substituted benzaldehydes in combination with different aliphatic aldehydes, PfBAL and PpBFD-L461A selectively deliver the (R)- and (S)-2-hydroxy-propiophenone derivatives, respectively. The (R)- and (S)-phenylacetylcarbinol (1-hydroxy-1-phenylacetone) derivatives are accessible in a similar way using LlKdcA and ApPDC-E469G, respectively. In many cases excellent stereochemical purities (>98 % enantiomeric excess) could be achieved. Hence, the regio- and stereochemistry of the product in the asymmetric aliphatic-aromatic cross-benzoin reaction can be controlled solely by choice of the appropriate enzyme or enzyme variant.

The thiamine diphosphate (ThDP)‐dependent enzyme 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase from Escherichia coli (EcMenD, E.C. 2.2.1.9) catalyzes the carboligation of α‐ketoglutarate (α‐KG) and various benzaldehyde derivatives with excellent chemo‐ as well as high R‐selectivity (enantiomeric excess (ee) >93 %) to yield chiral α‐hydroxy ketones. Based on the recently developed S‐pocket concept, we engineered S‐selective EcMenD variants by optimizing the steric properties and stabilization of the acceptor substrate in the S‐pocket. Moreover, the moderate S‐selectivity of the EcMenD variant I474A/F475G described recently for the carboligation of α‐KG and benzaldehyde (ee=75 %) could be improved by selective destabilization of the R‐pathway, which resulted in the variant I474A/F475G/R395Y (ee=85 % S). Subsequent investigation of the acceptor substrate range of this new variant revealed high S‐selectivity especially with meta‐substituted benzaldehydes, which gave access to 5‐hydroxy‐4‐oxo‐5‐arylpentanoates with excellent enantioselectivities of up to 99 % ee S. Thus, opening the S‐pocket and simultaneous destabilization of the R‐pathway provides a potential general new strategy to enhance the S‐selectivity of ThDP‐dependent enzymes.

Asymmetric mixed carboligation reactions of α-ketoglutarate with different aldehydes were explored with the thiamine diphosphate dependent enzymes SucA from E. coli, Kgd from Mycobacterium tuberculosis, and MenD from E. coli. All three enzymes proved to be efficient biocatalysts to selectively deliver chiral δ-hydroxy-γ-keto acids with moderate to excellent stereoselectivity. The high regioselectivity is due to the preserved role of α-ketoglutarate as acyl donor for these enzyme-catalyzed reactions.