Markedly improving lipase-mediated asymmetric ammonolysis of d,l-p-hydroxyphenylglycine methyl ester by using an ionic liquid as the reaction medium

Abstract

A comparative study was made of lipase-catalyzed asymmetric ammonolysis of D,L-p-hydroxyphenylglycine methyl ester (D,L-HPGME) with ammonium carbamate as the ammonia donor in nine ionic liquids (ILs) and four organic solvents. An obvious enhancement in the enantioselectivity (E value) of the ammonolysis was observed using ILs as the reaction media when compared to the organic solvents tested. However, the rate of ammonolysis in the IL 1-hexyl-3-methylimidazolium tetrafluoroborate (C₆MIm·BF₄), which was the best IL medium for the reaction, was much lower than that achieved in tert-butanol. It was also found that both the cation and the anion of ILs have a significant effect on the reaction. Candida antarctica lipase B immobilized on an acrylic resin (CAL-B, i.e., Novozym 435) displayed no ammonolysis activity toward D,L-HPGME in two ILs, 1-butyl-3-methylimidazolium bromide (C₄MIm·Br) and 1-butyl-3-methyl-imidazolium nitrate (C₄MIm·NO₃). When an IL–tert-butanol co-solvent system was adopted as the solvent for the enzymatic ammonolysis, the initial rate and enantioselectivity were enhanced markedly. It was noticed that the ammonolysis was dependent on the water activity (a_w) in the co-solvent systems, and an a_w of 0.75 was optimal. Among the six co-solvent systems examined, the lowest apparent K_m and activation energy (E_a), and the highest V_max of the ammonolysis were achieved with the co-solvent mixture of C₆MIm·BF₄ and tert-butanol (20 : 80, v/v), in which the lipase CAL-B also exhibited good stability.