Exploring Studies On New Application Of Fusarium Proliferatum Lactonase In Chiral Synthesis

Fusarium lactonase is a kind of efficient biocatalyst and its most important capacity is the catalytic hydrolysis of lactones into hydroxyl acids. Due to its great specificity and enantioselectivity, Fusarium lactonase has now been used widely in the industrialized production of D-pantolactone. In past twenty years, reports have been emerging regarding its screening, catalysis, purification, cloning and expression. However, all of these researches focused on the production of D-pantolactone, and only isolated reports are on Fusarium lactonase-mediated hydrolysis of other substrates.One fungus, Fusarium proliferatum (Matsushima) Nirenberg ECU2002, has been isolated from soil samples. It shows great activity and stereoselectivity in hydrolyzing 2-hydroxy-4-butyrolactones. In this paper, we carry out further researches on the Fusarium lactonase-catalyzed hydrolysis of chiral lactones, focusing on new application in asymmetric synthesis, especially in asymmetric catalysis.This paper is consisted of three parts. In the first part, we describe the cloning and expression of the lactonase gene from Fusarium proliferatum ECU2002 in E. coli JM109 (DE3) (reFPL). By optimizing expression conditions, the lactonase production was significantly enhanced making reFPL an easy-to-make biocatalyst. The reFPL was then purified with the aid of His tags before the N-terminal of mutual enzyme. Furthermore, biocatalytic properties of reFPL were also investigated, particularly its hydrolytic activity towards lactones, and we found that 4-substituted 2-hydroxy-4-butyrolactone was also suitable substrate for reFPL.The second part of this paper focused on the reFPL-catalyzed hydrolysis of 4-substituted 2-hydroxy-4-butyrolactones.4-Substituted 2-hydroxy-4-butyrolactones was synthesized via classical Claisen condensation, and in most cases cis-and trans-lactones were separable via chromatography on silica gel. Enzymatic resolution was then carried out on these cis-and trans-racemic mixtures. Three out of four possible stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones could be prepared in good optical purity. ReFPL shows hydrolytic activity towards a broad substrate spectrum against high substrate concentration (up to 200 mM), and it is also recyclable (at least three times). In the end, the configuration of the enzymatic hydrolyzed product was also assigned using different measures. The relative configuration was assigned according to the1H NMR and Nuclear Overhauser Effect (NOE) experiments and confirmed by X-ray diffraction. As for the absolute structure, it was assigned by different methods including X-ray diffraction and circular dichroism. In the third part, a new route to chiral 2-hydroxy-4-phenylbutyrates, precursors to the angiotensin converting enzyme inhibitors, was developed on the basis of reFPL-catalyzed hydrolysis of 2-hydroxy-4-phenyl-4-butyrolactone. The enantioselective hydrolysis of cis-and trans-lactones could be completed within the same time. Hydrogenation of this mixture over Pd/C affords chiral 2-hydroxy-4-phenylbutyrates. This route has great potential for industrial application.