Robert Westphal

Engineering stereoselectivity of ThDP-dependent enzymes

Thiamine diphosphate‐dependent enzymes are broadly distributed in all organisms, and they catalyse a broad range of C–C bond forming and breaking reactions. Enzymes belonging to the structural families of decarboxylases and transketolases have been particularly well investigated concerning their substrate range, mechanism of stereoselective carboligation and carbolyase reaction. Both structurally different enzyme families differ also in stereoselectivity: enzymes from the decarboxylase family are predominantly R‐selective, whereas those from the transketolase family are S‐selective. In recent years a key focus of our studies has been on stereoselective benzoin condensation‐like 1,2‐additions. Meanwhile, several S‐selective variants of pyruvate decarboxylase, benzoylformate decarboxylase and 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate (SEPHCHC) synthase as well as R‐selective transketolase variants were created that allow access to a broad range of enantiocomplementary α‐hydroxyketones and α,α′‐dihydroxyketones. This review covers recent studies and the mechanistic understanding of stereoselective C–C bond forming thiamine diphosphate‐dependent enzymes, which has been guided by structure–function analyses based on mutagenesis studies and from influences of different substrates and organic co‐solvents on stereoselectivity.

(S)-Selective MenD variants from Escherichia coli provide access to new functionalized chiral α-hydroxy ketones.

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.

Tailoring the S‐Selectivity of 2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate Synthase (MenD) from Escherichia coli

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.

A Tailor-Made Chimeric Thiamine Diphosphate Dependent Enzyme for the Direct Asymmetric Synthesis of (S)-Benzoins

Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion.

MenD from Bacillus subtilis: A Potent Catalyst for the Enantiocomplementary Asymmetric Synthesis of Functionalized α‐Hydroxy Ketones

The thiamine diphosphate‐dependent enzyme 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase (MenD) catalyzes a Stetter‐like 1,4‐addition of α‐ketoglutarate to isochorismate in the biosynthesis of menaquinone (vitamin K). Here, we describe the carboligation potential of MenD from Bacillus subtilis (BsMenD) for the nonphysiological 1,2‐addition of decarboxylated α‐ketoglutarate (succinylsemialdehyde) and various benzaldehyde derivatives. Furthermore, we engineer BsMenD variants for the enantiocomplementary asymmetric synthesis of functionalized α‐hydroxy ketones. Wild type BsMenD shows an excellent chemo‐ as well as high (R)‐selectivity for the carboligation of α‐ketoglutarate as the donor, and different benzaldehyde derivatives as acceptor yielding (R)‐α‐hydroxy ketones with up to >99 % ee. By engineering (S)‐selective BsMenD variants, based on the recently developed S‐pocket concept, we provide access to most of the corresponding (S)‐α‐hydroxy ketones with up to 98 % ee. In particular, benzaldehyde and meta‐substituted derivatives were converted with high enantioselectivities (ee of 91–98 % (S)). The significantly higher (S)‐selectivity of BsMenD variants than recently published MenD variants from Escherichia coli, could be attributed to a second‐shell residue next to the S‐pocket. A glycine residue, adjacent to the major S‐pocket residues I476 and F477 (standard numbering), is assumed to result in higher structural flexibility in the S‐pocket region of BsMenD, which in turn could result in improved stabilization of the antiparallel orientation of the acceptor.

Asymmetric synthesis of (S)-phenylacetylcarbinol – closing a gap in C–C bond formation

(S)-Phenylacetylcarbinol [(S)-PAC] and its derivatives are valuable intermediates for the synthesis of various active pharmaceutical ingredients (APIs), but their selective synthesis is challenging ...