Room Temperature Solid-Phase Ionic Liquid-Immobilized Enzyme for Biocatalysis in Organic Solvent: Markedly Enhanced Enantioselectivity

Abstract

Nonaqueous biocatalysis provides a useful component of methodology in organic synthesis. For example, lipase catalysis in organic solvents is of great use for the synthesis of optically active compounds such as chiral alcohols, acids, and their esters. However, biocatalysis in nonaqueous media often suffers from reduced activity, selectivity or stability of enzymes. To overcome these limitations, many approaches have focused on the development of more efficient enzymes. Among them, the use of ionic liquids as an alternative solvent for the biotransformations in nonaqueous solvents enhanced the activity, selectivity, and stability of enzymes. And novel approach with ionic liquid-coated enzyme (ILCE), which was readily prepared by mixing the enzyme powder with a room temperature solid phase ionic liquid (RTSPIL) at elevated temperature, was previously reported showing enhanced enantioselectivity and stability in organic solvents. Also, we have recently reported that the enzyme coated by RTSPIL during lyophilization in aqueous medium exhibited better activity. Although it was difficult to clarify the reason that why ionic liquids increase significantly the catalytic activity as well as the selectivity of the lipase for enzyme-catalyzed transesterifications, we have speculated that the ionic environments by ionic liquid could improve activity and selectivity of the enzyme in biotransformations. On the other hands, numerous methods have been developed for the immobilization of enzymes onto various supports such as ceramic, polymer, gels, membranes, and microcapsules to increase the stability and recyclability of the biocatalyst in organic syntheses. To extend our knowledge of the application with ionic liquids for biocatalysis, we have expected that the immobilized enzyme in ionic environments could be more efficient during immobilizing of enzymes on supporting materials compared to that in non-ionic environments. Herein, we wish to report the synthesis of new RTSPIL composed of imidazolium cation modified with the methacryloyl group and hexafluorophosphate (PF6) anion and the biocatalysis in an organic solvent using lipase immobilized on this RTSPIL, which is an aggregate of small molecules. The novel RTSPIL, [MOPMIM]-[PF6], ([MOPMIM] + = 1-(3’ -methacryloyloxypropyl)-3-methylimidazolium) was synthesized according to the following procedure reported previously to immobilize the lipase, which was modified with methacryloyl group that can readily interact with the functional moieties such as thiol, amine, and alcohol on the surface of lipase.(Scheme 1). [MOPMIM]-[PF6] is very stable in moisture and melts at temperature of 70 C. The remarkable property of [MOPMIM]-[PF6] is low solubility in water due to hydrophobic character of PF6 anion, which lets us immobilize the lipase on RTSPIL in a buffer solution. Indeed, Burkholderia cepacia lipase (BCL, native) was immobilized on [MOPMIM]-[PF6] through interaction between [MOPMIM][PF6] and BCL in a phosphate buffer. The enantioselectivity of BCL/[MOPMIM]-[PF6] was examined in the transesterification reaction of bulky secondary alcohols (A-E) with vinyl acetate in toluene at 25 C (Scheme 1). For comparison, the same reactions were carried out with non-immobilized BCL. After the reaction reached 10-50% completion, the enzymes were removed by filtration and the resulting solution was concentrated. The organic residues were subjected to silica gel chromatography to obtain unreacted substrate and acetylated product. Their optical purities were then determined by high-performance liquid chromatography (HPLC) using a chiral column, which allowed us to measure the enantiomeric excess (ee) up to > 99.5%. The E values were calculated using the equation, E = ln[1-c(1+eep)]/ln[1c(1-eep)], were c = ees/(ees + eep). 14 The results are described in Table 1. The data from Table 1 indicate that the transesterification of A-E catalyzed by BCL/[MOPMIM]-[PF6] proceeded with better enantioselectivity in toluene than by BCL (Table 1, entry 1-10). The best enantioselectivity (E = 1194) was ob-