Reprogramming ThDP Enzymes for Z-Alkenes: Overriding Thermodynamic Preference via Noncovalent Controls

Area of Science:

• Biocatalysis
• Organic Chemistry
• Enzyme Engineering

Background:

• Conventional alkene synthesis favors thermodynamically stable E-isomers, making direct Z-alkene synthesis challenging.
• Thiamine diphosphate (ThDP)-dependent enzymes are crucial in C-C bond formation but not typically used for alkene synthesis.
• Existing methods for Z-alkene synthesis often require complex strategies like substrate control or directing groups.

Purpose of the Study:

• To reprogram a ThDP-dependent enzyme for the direct and selective synthesis of Z-α,β-unsaturated carboxylic acids.
• To achieve kinetic control over alkene stereochemistry using enzyme active site engineering.
• To develop a sustainable biocatalytic alternative to conventional Z-alkene synthesis methods.

Main Methods:

• Reprogramming a ThDP-dependent enzyme via active site engineering.
• Catalyzing a dehalogenative elimination reaction to override thermodynamic bias.
• Utilizing noncovalent interactions within the enzyme's active site for stereochemical control.
• Investigating a homoenolate-mediated pathway via a diverted Breslow intermediate.

Main Results:

• Achieved direct and selective synthesis of Z-α,β-unsaturated carboxylic acids.
• Demonstrated kinetic control for Z-alkene formation, overriding thermodynamic preference.
• Engineered the enzyme for stereodivergent synthesis, enabling access to both E and Z isomers.
• Established a biocatalytic platform for Z-alkene synthesis using noncovalent interactions.

Conclusions:

• The reprogrammed enzyme provides a fundamentally distinct and sustainable approach to Z-alkene synthesis.
• This work expands the catalytic capabilities of ThDP-dependent enzymes.
• The biocatalytic platform addresses a critical need for efficient Z-alkene synthesis methodologies.