Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

Enzymatic resolution of 2-aryloxy-1-propanols via lipase-catalyzed enantioselective acylation using acid anhydrides as acyl donors

Pseudomonas sp. lipase-catalyzed enantioselective acylation procedure using acid anhydrides as acyl donors was exploited for the resolution of 2-aryloxy-1-propanols carrying different substituents on the benzene ring. These primary alcohols, which belong to primary alcohols with an oxygen atom at the stereocenter, were resolved generally with moderate to good enantioselectivity (E of up to 55) through the acylation with hexanoic anhydride in diisopropyl ether at 25 °C in a short reaction time. With the alcohol substrate, which gave a low enantioselectivity in the acylation at ordinary temperature, the selectivity proved to be enhanced by conducting the reaction at low temperature (−10 °C). By this acylation procedure employing the acid anhydride, enantiomerically pure (R)-2-phenoxy-1-propanol was prepared in a gram-scale reaction.     

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3'-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3',6'-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3'-O-acetylasperulosidic acid (2a), 3',6'-O-diacetylasperulosidic acid (2b), 4'-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6'-O-acetylpuerarin (3a), 6'-O-decanoylpuerarin (3b), 6'-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

Donor specificity and regioselectivity in Lipolase mediated acylations of methyl α-d-glucopyranoside by vinyl esters of phenolic acids and their analogues

Methyl α-d-glucopyranoside as a model acceptor was acylated by several phenolic and non-phenolic vinyl esters using immobilised Lipolase. Donor specificity and regioselectivity of reaction were investigated. Conversion and rate of acylation by structurally varied donors indicates that the synthetic reactivity of Lipolase corresponds to the hydrolytic activity of feruloyl esterase type A. Lipolase exhibited remarkable regioselectivity for primary position of methyl α-d-glucopyranoside. The acylation occurred exclusively at 6-O primary position when vinyl esters of phenolic acids (hydroxybenzoates, hydroxyphenylalkanoates and hydroxycinnamates) served as acyl donors (5–77%). In addition to the major 6-O-acyl products (52–79%), 2,6-di-O-acylated derivatives were isolated from reaction mixtures (2–13%) when non-phenolic donors were used (vinyl esters of fully methoxylated derivatives of phenolic acids, along with vinyl benzoates, cinnamates or some heterocyclic analogues).     

Methods for the immobilization of lipases and their use for ester synthesis

The lipase from Pseudomonas fluorescens was immobilized onto five different carriers: celite, octyl-silica, aminopropyl-silica, gluterdialdehyde-activated silica and Eupergit C250L. Activities and operational stabilities of the prepared catalysts were compared using the enantioselective acylation of (R,S)-1-phenylethanol by vinyl acetate as acyl donor and t-butylmethyl ether with variable water content (0.038-0.97% v/v) as reaction medium. The above carriers provide catalysts with widely different specific activities ranging from excellent 25 mmol/h mg protein (celite) to 0.07 mmol/h mg protein (glutardialdehyde-activated silica) on the lower end. The lipase immobilized onto Eupergit C250L exhibited the best operational stability among the catalysts studied. It retained 30% of its initial activity after 11 cycles of application, each with a duration between 2 and 6 h.     

Asymmetric synthesis and biological evaluation of the enantiomeric isomers of the immunosuppressive FTY720-phosphate

A practical asymmetric synthesis of both enantiomers of the immunosuppressive FTY720-phosphate (2) was accomplished, and the enantiomers were pharmacologically evaluated. Several lipases showed considerable activity and enantioselectivity for O-acylation of N-acetyl FTY720 (3) or N-benzyloxycarbonyl FTY720 (7) in combination with vinyl acetate or benzyl vinyl carbonate as the acyl donors. The synthesis using the lipase-catalyzed acylation as the key step produced the enantiomerically pure (>99.5% ee) enantiomers of 2 in multigram quantities. (S)-Isomer of 2 had more potent binding affinities to S1P(1,3,4,5) and inhibitory activity on lymphocyte migration toward S1P than (R)-2, suggesting that (S)-isomer of 2 is responsible for the immunosuppressive activity after administration of 1. Severe bradycardia was observed in anesthetized rats when (S)-2 was administered intravenously, while (R)-2 had no clear effect on heart rate up to 0.3 mg/kg.     

Use of ionic liquids as media for the biocatalytic preparation of flavonoid derivatives with antioxidant potency

Biocatalytic preparation of acylated derivatives of flavonoid glycosides was performed using various immobilized lipases in two different ionic liquids, namely 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF(4)) and 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]PF(6)). The influence of various reaction parameters on the performance and the regioselectivity of the biocatalytic process was pointed out, using as model reaction the acylation of naringin and rutin with vinyl butyrate, catalyzed by immobilized Candida antarctica lipase at 60 degrees C. The biocatalytic modification of flavonoids strongly depended on the ionic liquid used, the molar ratio of substrates, as well as the acyl donor chain length. The highest conversion yield (about 65% after 96 h of incubation) was obtained with short chain acyl donors (up to four carbon atoms), at a relatively high molar ratio (10-15) in both ionic liquids used. The amount of monoacylated flavonoid derivatives produced in a single-step biocatalytic process in [bmim]BF(4) was up to 5.5 g/L for monoacylated rutin and 30 g/L for monoacylated naringin. The regioselectivity of the process was higher in [bmim]BF(4) than in [bmim]PF(6) or organic solvents. Reaction rates observed in ionic liquids were up to four times higher than those reported for organic media. The acylation of sugar moiety of rutin with various acyl donors affected its antioxidant potential towards both isolated LDL and total serum model in vitro. A significant increase of antioxidant activity was observed for rutin-4'-O-oleate.     

Simple and efficient solid support scavenging of excess acyl donors after enzymatic acylations in organic solvents

A simple and efficient method for removing excess acyl donors following enzymatic acylations in organic solvents was developed. This method is based on selective chemical scavenging of acyl donors using an amino-functionalized solid support, and does not affect the desired acylated product. A wide variety of different acyl donors, including vinyl and trifluoroethyl esters and vinyl carbonates, can be quantitatively removed by this method, thus providing a simple and highly efficient tool for purification of reaction products after enzymatic acylation.     

Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH

Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF₄] or 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF₆]) and t-butanol as organic solvent was investigated. The best enzyme was commercially available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF₆] in the presence of 40% t-BuOH with CAL-B at 60 °C.     

Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid

Immobilised 1,3-specific lipase from Rhizopus arrhizus was used as catalyst for the esterification of -glycero-3-phosphate and fatty acid or fatty acid vinyl ester in a solvent-free system. With lauric acid vinyl ester as acyl donor, a_w<0.53 favored the synthesis of lysophosphatidic acid (1-acyl-rac-glycero-3-phosphate, LPA1) and the spontaneous acyl migration of the fatty acid on the molecule. Subsequent acylation by the enzyme resulted in high phosphatidic acid (1,2-diacyl-rac-glycero-3-phosphate, PA) formation and high total conversions (>95%). With oleic acid, maximum conversions of 55% were obtained at low water activities. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/(PA+LPA)]. Thus, LPA was the only product with lauric acid vinyl ester as acyl donor at 25°C. Increased substrate ratio ( -glycero-3-phosphate/fatty acid) from 0.05 to 1 resulted in a higher ratio of LPA to PA formed, but a lower total conversion of -glycero-3-phosphate. Increased amounts of enzyme preparation did not result in higher esterification rates, probably due to high mass-transfer limitations.     

Novel and Highly Efficient Regioselective Route to Helicid Esters by Lipozyme TLL

Highly regioselective acylation of helicid with fatty acid vinyl esters catalyzed by the lipase from Thermomyces lanuginosus has been successfully performed for the first time. For the enzymatic caproylation of helicid, under the optimal conditions, initial reaction rate was 33.2 mM/h, and substrate conversion and regioselectivity were greater than 99%. In addition, the acyl recognition of the enzyme in the regioselective acylation of helicid was investigated. The results showed that although 6’-O-acyl derivatives of helicid were exclusively obtained with all the tested acyl donors, the enzymatic reaction rate varied widely with different acyl donors, presumably owing to their different interactions with the active site of the lipase. It is also interesting that the different configuration of only one hydroxyl group at C-3 in helicid couldn’t affect the lipase-catalyzed esterification and helicid has the same regioselectivity as that of D-glucose and arbutin.     

Esterification of streptol — a cyclitol derivative — by Candida rugosa lipase: influence of the acyl donor on regioselectivity

The influence of the nature of acyl donors on the regioselectivity of Candida rugosa lipase for the esterification of streptol — a cyclitol derivative — was investigated. Excellent regioselectivity for the formation of 3,7-disubstituted derivatives was observed for vinyl butyrate (100% 3,7-derivative, 68% yield) and vinyl propionate (100% 3,7-derivative, 46% yield) as acyl donors. In contrast, for vinyl methacrylate as acyl donor, a mixture of 71% 3,7-derivative and 29% 1,7-derivative was obtained. Varying the chain length, a certain dependency of regioselectivity on the acyl donor was observed, however, no logical correlation satisfying all cases was found. Mono-substituted streptol derivatives were obtained by employing Novozym 243.     

Novel and highly regioselective route for synthesis of 5-fluorouridine lipophilic ester derivatives by lipozyme TL IM

For the first time, lipozyme TL IM, an inexpensive lipase from Thermomyces lanuginosa, was successfully applied to the regioselective synthesis of lipophilic 5-fluorouridine ester derivatives. The ESI-MS and (13)C NMR analysis confirmed that the end products of the acylation were 5'-O-acyl 5-fluorouridines, more powerful anti-tumor drugs than 5-fluorouridine itself. Notably, the chain length of acyl donors had an obvious effect on the initial rate and the maximum substrate conversion of the regioselective acylation. The acylation of 5-fluorouridine with vinyl laurate was used as a model to explore the influence of various factors on the reaction with respect to the initial rate, the maximum substrate conversion and the regioselectivity. The optimum water activity, the molar ratio of vinyl laurate to 5-fluorouridine, reaction temperature and shaking rate were 0.07, 15/1, 45 degrees C and 200rpm, respectively, under which the maximum substrate conversion and the regioselectivity were as high as 98.4 and >99%, respectively, after a reaction time of around 6h.     

Effect of ultrasound on enzymatic acylation of konjac glucomannan

A comparative study was made of enzymatic acylation of konjac glucomannan with vinyl esters under ultrasonic irradiation and shaking in organic solvent tert-butanol. Among the 13 enzymes selected, Novozym 435 exhibited the highest acylation activity towards KGM whether under ultrasonic irradiation or shaking. The application of ultrasonic irradiation instead of shaking during the acylation led to improvement in the initial reaction rate, yield and degree of substitution of the modified KGM. Appropriate ultrasound power (100 W) and water activity (0.75) were found to accelerate enzymatic reaction. The acceleration effect of ultrasound on Novozym 435-catalyzed acylation decreased with an increase in the chain length of the acyl donors from C2 to C18. Moreover, the acylation of KGM in tert-butanol was proved to be a regioselective one, with C6-OH being acylated. Compared with shaking, ultrasound did not change regioselectivity of Novozym 435 in the acylation.     

Studies on the enantioselectivity in the lipase-catalyzed synthesis of monoacylglycerols from isopropylidene glycerol

Summary The regioselective lipase-catalyzed acylation of isopropylidene glycerol using different vinyl esters as acyl donors in toluene was studied. Reaction progress and enantioselectivity were monitored by gas chromatography using a permethylated -cyclodextrin phase. All vinyl esters were completely converted after 20 to 24 h and it was found that the S-enantiomer reacted faster. Lower enantiomeric excess were found using e. g. vinyl palmitate (18 %ee) compared to e. g. vinyl butyrate (42 %ee) with crude lipase from Pseudomonas cepacia. Immobilization using the sol-gel method resulted in higher remaining activities (up to 69%) and increased enantioselectivity E (up to 8.5).     

Resolution of 2-octanol by SBA-15 immobilized Pseudomonas sp. lipase

Lipase from Pseudomonas sp. (PSL) was immobilized on SBA-15 (a highly ordered hexagonal array mesoporous silica molecular sieve) through physical adsorption and the immobilized PSL was used in resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed for the immobilized PSL compared with those of the free one. The effects of reaction conditions, such as solvents, temperature, water activity and substrate ratio were investigated. Under the optimum conditions, the residual (S)-2-octanol was recovered with 99% enantiomeric excess at 52% conversion. The results also indicated that the immobilized PSL maintained 90% of its initial activity even after reusing it five times.     

Biocatalytic synthesis of ascorbyl esters and their biotechnological applications

Ascorbyl fatty acid esters act both as antioxidants and surfactants. These esters are obtained by acylation of vitamin C using different acyl donors in presence of chemical catalysts or lipases. Lipases have been used for this reaction as they show high regioselectivity and can be used under mild reaction conditions. Insolubility of hydrophilic ascorbic acid in non-polar solvents is the major obstacle during ascorbic acid esters synthesis. Different strategies have been invoked to address this problem viz. use of polar organic solvents, ionic liquids, and solid-phase condensation. Furthermore, to improve the yield of ascorbyl fatty acid esters, reactions were performed by (1) controlling water content in the reaction medium, (2) using vacuum to remove formed volatile side product, and (3) employing activated acyl donors (methyl, ethyl or vinyl esters of fatty acids). This mini-review offers a brief overview on lipase-catalyzed syntheses of vitamin C esters and their biotechnological applications. Also, wherever possible, technical viability, scope, and limitations of different methods are discussed.     

Thioethyl-, Vinyl-, Ethyl Octanoate Esters and Octanoic Acid as Acyl Donors in Lipase Catalysed Acyl Transfer Reactions

S-Ethyl thiooctanoate, vinyl octanoate, ethyl octanoate and octanoic acid were studied as acyl donors in a lipase catalysed acyl transfer reaction with 2-octanol as acyl acceptor. The reaction conditions had a pronounced effect on the equilibrium displacement and the apparent enantioselectivity. The thioethyl and vinyl esters proved to be efficient acyl donors under atmospheric pressure and 39 d`C, affording a high apparent enantiomeric ratio. Under these reaction conditions the apparent enantioselectivity seemed to be enhanced by water, which was present in the reaction system and caused the production of octanoic acid, by hydrolysis of me acyl enzyme.     

Sol–gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents

Lipase PS from Burkholderia cepacia (formerly Pseudomonas cepacia) was successfully immobilized in sol–gels under low methanol conditions using lyophilization in order to dry the gel. The enzyme was also cross-linked with glutaraldehyde to CLEAs without any additives. These immobilized enzyme preparations were employed for the highly enantioselective acylations of 1-phenylethanol (1), 1-(2-furyl)ethanol (2) and N-acylated 1-amino-2-phenylethanol (3) with vinyl acetate in organic solvents. Enzymatic hydrolysis of the obtained ester product was observed as a side reaction of the acylation of 3 in the presence of lipase PS powder. Hydrolysis was suppressed when the immobilized preparations of lipase PS were used.