Acyl Transfer Reactions

In the reaction mechanism described above for lipase-catalyzed hydrolysis of lipids, deacylation involves nucleophilic attack of the acylenzyme by H2O. This step can also be accomplished by other nucleophiles, such as alcohols and amines, resulting in an acyl transfer instead of hydrolysis. In an aqueous environment, hydrolysis is the favorable route, whereas in organic media, the acylenzyme is exposed to nucleophiles without competition from H2O, and acyl transfer prevails. It has been shown that Rhizomucor miehei lipase retains high rates in esterification and transesterifica-tion at water activity < 0.0001 (76).

The acyl transfer reaction provides a novel use of lipases as catalysts in organic synthesis. Using amines as nucleophiles, esters can be converted to the corresponding amides in organic media at ambient temperature. For example, Candida antarctica lipase-catalyzed aminolysis of ethyl octanoate gives a 95% yield of octanamide (77). Peptide bonds can be formed

Figure 7 "Scooting" and "hopping" models of interfacial catalysis. (Adapted from Ref. 72, 73.)

between N-acetyl-L-(amino acid)-2-chloroethyl esters and amides of L or D amino acids (78). This reaction can be utilized in the production of optically active amides by taking advantage of the regio- and stereo-specificity of the enzyme (79). Using hydroxyacid esters, bifunctional molecules containing both a methyl ester and an OH group as substrates, lipases can catalyze intermolecular or intramolecular condensation to give corresponding oligomers or lactones as the product (80, 81). The preference for oligomerization or lactonization depends on the chain length and degree of substitution of the substrate. This reaction has been exploited for the synthesis of optically active y-substituted lactones from symmetrical hydroxy-carboxylic acid esters (82). Lipases have been used in the regioselective acylation of sugars, the process of which can rarely be effectively performed in organic synthesis and often requires multisteps of protection and deprotection of the various hydroxyl groups. Porcine pancreatic lipase catalyzes transesterification between monosaccharides (glucose, galactose, man-nose) and trichloroethyl carboxylates in pyridine resulting in the selective acylation at the C6-OH (83). Sucrose esters, used as emulsifiers in food, cosmetics, and medicines, can be synthesized by the lipase-catalyzed transesterification of sucrose with esters of fatty acids (84). In similar reactions, nucleosides and furanose and pyranose derivatives can be selectively acylated (85, 86).

One of the lipase-catalyzed reactions important to the food industry is the interesterification of triacylgly-cerols. In these reactions, a triacyglycerol reacts with a fatty acid, alcohol, or fatty acid alkyl ester, to produce a mixture of new triacylglycerols resulting from a rearrangement of the fatty acid moieties. Chemical catalysts such as sodium metal or sodium alkoxide can promote interesterification; however, acyl migration among the triacylglycerol molecules is nonspecific with random distribution of the acyl fatty acids. By exploiting lipases as catalysts, it is possible to generate products enriched with the new fatty acids at specific positions, thereby changing the physicochemical properties of triacylglycerols (87, 88). For example, in the interesterification of a triacylglycerol/fatty acid mixture catalyzed by 1,3-specific lipase, the product consists of novel triacylglycerols with the fatty acid selectively incorporated to the 1- and 3-positions, and no enrichment at the 2-position. Using Geotrichum candidum lipase which has specificity for cis-9 unsatu-rated fatty acids, linolenic acid, for example, can be selectively exchanged and enriched in an interestified triacylglycerol mixture. Miller et al. (89) studied the kinetics of interesterification of trilaurin and lauric acid catalyzed by Candida cylindracea lipase in cyclo-hexane. The reaction follows a two-step mechanism: initial hydrolysis of the trilaurin to dilaurin and lauric acid with Km = 7.8 x 10~3 M, and kcat = 0.9 sec , and subsequent reesterification of dilaurin and lauric acid with Km values of 1.7 x 10~2 M (lauric acid) and 0.16 M (dilaurin), and kcat = 3sec_1. The use of microbial lipases in nonconventional solvents, such as supercritical fluids, has also been investigated as a means of improving the activity and stability of the enzyme for promoting acyl transfer reactions in an anhydrous environment (90).

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