Semi-rational Design Of Fluoroacetate Dehalogenase RPA1163 For Kinetic Resolution Of α-Fluorocarboxylic Acids On Gram-scale

Since the incorporation of fluorine often induces remarkable changes of physical and biological properties of organic compounds,organofluorines have occupied a very important position in a variety of fields,especially in the field of agrichem and pharmacy,exceeded 35% and 20% of products in market are organofluorines.Among them,α-fluorocarboxylic acids(FCAs)have attracted considerable attention due to their inherent bioactivities and utility as building blocks in the construction of more complex pharmaceutical molecules.The demand for these compounds calls for the fast development of synthetic methodologies.Up to date,chiral FCAs are mainly synthesized by chemical methods.However,the traditional chemical methods show complicated operation and poor stereoselectivity,evenmore,they often cause environmental pollution as organic reagents or metal catalysts are inevitable.With the development of biocatalysis in recent years,biocatalysts have been considered as complement or even substitute of chemical catalysts due to their high-catalytic efficiencies,high selectivities,and environment friendly.Therefore,obtaining such compounds by biocatalysis is more in line with the requirements of modern chemistry.Here the synthetic utility of fluoroacetate dehalogenase RPA1163 is explored for the production of enantiomerically pure(R)-α-fluorocarboxylic acids and(R)-α-hydroxylcarboxylic acids via kinetic resolution of racemic α-fluorocarboxylic acids.This study focuses on three aspects:1.Firstly,the stability and substrate spectrum of fluoroacetate dehalogenase were studied.While wild-type(WT)RPA1163 shows high thermostability and fairly wide substrate-scope,many interesting,yet poorly or moderately accepted substrates exist.2.To improve the activity of some substrates and to develop up-scaled production,in silico calculations and semi-rational mutagenesis were employed.Residue W185 was engineered to alanine,serine,threonine or asparagine.The two best mutants W185 N and W185 T showed significantly improved performance in the reactions of these substrates,while in silico calculations shed light on the origin of these improvements.3.10 α-fluorocarboxylic acids and 10 α-hydroxycarboxylic acids were prepared on gram-scale via kinetic resolution enabled by WT,W185 T or W185 N.