Lipases catalysed hydrolysis/acylation

Reaction in organic solvent can sometimes provide superior selectivity to that observed in aqueous solution. For example, Keeling et al recently produced enantioenriched a-trifluoromethyl-a-tosyloxymethyl epoxide, a key intermediate in the synthetic route to a series of nonsteroidal glucocorticoid receptor agonist drug candidates, through the enan-tioselective acylation of a prochiral triol using the hpase from Burkholderia cepacia in vinyl butyrate and TBME (Scheme 1.59). In contrast, attempts to access the opposite enantiomer by desymmetrization of the 1,3-diester by lipase-catalysed hydrolysis resulted in rapid hydrolysis to triol under a variety of conditions. 

Candida antractica lipase B- and immobilised Mucor miehei lipase- catalysed alcoholysis and C-rugosa lipase- catalysed hydrolysis have been successfully used for the highly effective synthesis of optically active trifluoromethylated 1-and 2-hydroxyalkane-phosphonates (268) and (269) from their racemic O-acylated precursors (270) and (271) (Scheme 68). ... 

Process and reaction monitoring. We have used TLC to monitor the time required for completion of the methylation reaction (Shantha, Decker and Hennig, 1993) and to study the formation of artifacts due to methylation of phthalates present in lipid samples (Shantha and Ackman, 1991b). TLC has been used to study the extent of hydrolysis of lipase-catalysed hydrolysis of triglycerides, phospholipase-catalysed hydrolysis of phospholipids and to study the activity of the enzymes. We have used TLC to study the extent of synthesis of acylated amino acids, and TLC has also been used to study the success of radiosynthesis. 

Scheme 4.1 Mechanisms of lipase-catalysed hydrolysis (R = H) or acylation (R H).

The mechanism of lipase-catalysed esterification or hydrolysis is shown in Scheme 4.1. The mechanism involves the formation of two tetrahedral intermediates, the first formed by nucleophilic attack of the serine residue of the catalytic triad onto the substrate. The tetrahedral intermediate loses water (R = H) or an alcohol (R H) to give an acyl enzyme complex that is either attacked by water (R = H) for hydrolysis or an alcohol (R H) for acylation. A second tetrahedral intermediate is formed that dissociates from the enzyme to give an ester or acid, thus regenerating the Hpase in its native form. Both of the tetrahedral intermediates involved in the mechanism are stabilized by hydrogen bonds to the oxyanion hole. 

Lipases are able to catalyse the acylation of alcohols in addition to the hydrolysis of esters. For acylations, the reactions are typically carried out in low-water systems (water activity (a ) < 1)), to minimize hydrolysis, and with a suitably reactive acyl donor to ensure high rates of reaction and efficient conversions. Suitable acyl donors include oximes, vinyl esters and anhydrides (Scheme 4.5). 

Lipase-catalysed interesterification has found many applications in production of edible and specialty lipids due to mild reaction conditions, high catalytic efficiency, the inherent selectivity of natural catalysts and production of much purer products as compared to chemical methods (Sonnet, 1988). Lipases (hydrolases) are used for hydrolysis and ester synthesis. They are classified as non-specific or random, positional specific or 1,3-specific and acyl group- or structure-specific, depending on their activity towards fatty acids... 

For 1,3-spedfic lipase catalysed interesterification reactions, Kyotani et al. (1988a) have provided elaborate kinetic models of the reactions using biphasic and microaqueous conditions. Four models were studied (a) first order kinetics (b) hydrolysis followed by resynthesis (c) reaction via the glyceride-enzyme complex (d) reactions via the acyl-enzyme complex. Details of the models are beyond the scope of this chapter. Interested readers are referred to Kyotani et al. (1988a) for an in-depth treatment of each model. For illustration purposes, the mathematical treatment of the simplest model is presented here. 

Excellent yields were achieved in the selective acylation at 0-3 of 6-0-acelyl-l,5-anhydro-2-deoxy-D-arafe/no-hex-l-enitol (25) by lipase mediated acyltransfer from several vinylesters. As shown in Scheme 7, the starting material (25) could be recovered in 80% yield from two of the products (26) by enzymatic hydrolysis. Reports have been published on the lipase mediated selective synthesis of 2-functionalised 3-monoesters (27) of methyl 5-0-decyl-a-D-arabinofuranoside, on the regioselective, lipase-catalysed acylation of methyl a- and -D-arabino- and -xylo-pyranoside, on the regioselective acylation and deacylation of 2 -deoxynucleoside derivatives by use of a Pseudomonas fluorescens lipase and a Bacillus subtilis protease, respectively, and on the regioselective acylation of castanospermin with a variety of enzymes in pyridine. 

Figure 8.4 Alkanolamide synthesis by lipase-catalysed acylation of ethanolamine with a fatty acid. O-Acyl ester is formed as an intermediate product, which is immediately converted to the amide by acyl migration. When add Is In excess, the amide will react further with the acid, yielding the amide ester. The amide ester can be converted back into the amide through hydrolysis or aminoiysis in the latter case two moles of amide will be formed from one mole of amide ester.

More recently enzymes have been used to resolve related intermediates also used in prostaglandin synthesis.38 Acylation with vinyl acetate catalysed by twelve lipases was tried and the best was Amano PS from Pseudomonas cepacia. At 55% conversion the alcohol (+)-156 was obtained in 40% yield and 91% ee and the acetate (+)-157 in 78% ee. This low enantiomeric purity could be enhanced to 95% by hydrolysis to (-)-156 and reacetylation with the enzyme. Note that the alcohol and acetate of the same series have opposite signs of rotations. 

A more positive discrimination between the two primary OHs might be made if a reagent-dominated reaction were used and an enzyme is ideal for this job. Various lipases could be used either in the acylation of the triol 210 or in the hydrolysis of the diester 212. In either case, the required monoester 211 could be formed in excellent ee. Novozyme (Novo Nordisk) SP 435 catalyses the monoacetylation of 210 in MeCN solution to give 211 R = Me in 95% yield and 96.6% ee after only 55 minutes. 

A highly enantioselective enzymatic acylation was observed on N-hydroxymethylated P-lactam 50, which was prepared from ( )-14 with paraformaldehyde by sonication in tetrahydrofuran [88]. Lipase AK-catalysed butyrylation with vinyl butyrate gave the readily separable azetidinones 51 and 52. Hydrolysis of 51 and 52 resulted in the 2-ACPC hydrochlorides 53 and 54, respectively (Scheme 8) [88]. 

A schematic representation of enzyme catalysed interesterification processes and products is shown in Figure 12.6. These processes generally involve hydrolysis and re-synthesis. Under restricted water conditions, interesterification is found to be predominant (Matsuo et aL, 1980, 1981 Coleman and Macrae, 1980). Chemically catalysed interesterification processes lead to randomization of the acyl groups along the glycerol chain. Using lipases with regio-specificity, the acyl transfers are restricted to the fatty acids located at the precise positions specific to the enzyme. 

A number of selective acylations and deacylations catalysed by enzymes, especially by lipases in organic solvents, have been reported. Methyl pentofuranosides could be selectively acetylated at 0-5 on a preparative scale by use of a lipase and 2,2,2-trifluoroethyl acetate as acyl donor in THF, while selective hydrolysis could be effected at the primary positions of peracetylated methyl pentofuranosides and hexopyranosides, and at the anomeric centres of peracetylated D-rilao- and D-xylo-furanoses and -pyranoses with lipases in aqueous DMF. ° Methyl -D-glucopyranoside, D-mannose, and 2-acetamido-2-deoxy-D-mannose were all substituted selectively at 0-6 by lipase-mediated... 

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