Improving the Long-term Enantioselectivity of a Silicon-Carbon Bond-Forming Enzyme

Abstract

Enantioselectivity is a key advantage of enzymatic catalysis. Understanding the most important factors influencing enantioselectivity necessitates thorough investigation for each specific enzyme. In this study, we explore various approaches to optimize reaction conditions for organosilicon production using an immobilized Cytochrome C recently tailored via directed evolution. Over extended reactions, this enzyme experiences a loss of enantioselectivity. Mass spectrometry (MS) revealed covalent modifications on the enzyme, but mutating the respective amino acids did not restore enantioselectivity. Nuclear magnetic resonance (NMR), along with a detailed comparison of the influence of reaction components such as cosolvents and reducing agents, indicated significant conformational changes in the presence of the diazo ester substrate. Additionally, we identified sodium ascorbate as a suitable and milder reducing agent compared to the previously used sodium dithionite, ensuring anaerobic conditions for silicon-carbon bond formation. Ultimately, maintaining a high enzyme-to-substrate ratio in the reaction was found to be crucial for achieving high enantiomeric purity of the organosilicon product over four days in sequential, repetitive batch reactions, thus improving the previously established reaction system. The methods and findings presented here are particularly valuable for addressing enantioselectivity issues in other enzymes that operate with diazo compounds as the substrates in carbene-transfer reactions.

Keywords: Anaerobic synthesis; Cytochromes; enantioselectivity; organosilicon; protein NMR.