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.     

硬脂酸和月桂酸在Novozym 435催化下对芦丁酯化及分子筛对酯化率的影响

为了增加芦丁的脂溶性从而使其具有更优秀的抗氧化活性,以硬脂酸和月桂酸为酰基供体,在脂肪酶Novozym 435催化下对芦丁选择性酯化.经色谱柱提纯,得到两种带不同长度烃基的芦丁脂肪酸酯.用红外光谱和核磁共振波谱对芦丁硬脂酸进行了结构鉴定,表明该类酯化物的酯化反应位为鼠李糖的C4′″位羟基.以高效液相色谱监测酯化反应进程,分子筛添加时间对酯化率的研究结果显示,分子筛对酯化率和反应速率有提高的作用.分子筛添加时间对酯化率有影响.对于硬脂酸为酰基供体的情况,反应24h后添加分子筛的酯化反应可以得到最大的酯化转化率46%.以月桂酸为酰基供体的酯化反应,反应11 h后添加分子筛可以得到最大的酯化转化率64.5%.     

Enhancement of ethyl propionate synthesis by poly (AAc-co-HPMA-cl-MBAm)-immobilized Pseudomonas aeruginosa MTCC-4713, exposed to Hg2+ and NH4+ ions

A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa MTCC-4713 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel. The hydrogel-bound lipase achieved 93.6% esterification of ethanol and propionic acid (300 mM: 100 mM) into ethyl propionate at temperature 65 degrees C in 3 h in the presence of a molecular sieve (3 angstroms). In contrast, hydrogel-immobilized lipase pre-exposed to 5 mM of HgCl2 orNH4Cl resulted in approximately 97% conversion of reactants in 3 h into ethyl propionate under identical conditions. The salt-exposed hydrogel was relatively more efficient in repetitive esterification than the hydrogel-bound lipase not exposed to any of the cations. Moreover, bound lipase exposed Hg2+ or NH4+ ions showed altered specificity towards p-nitrophenyl esters and was more hydrolytic towards higher C-chain p-nitrophenyl esters (p-nitrophenyl laurate and p-nitrophenyl palmitate with C 12 and C 16 chain) than the immobilized lipase not exposed to any of the salts. The later showed greater specificity towards p-nitrophenyl caprylate (C 8).     

Facile synthesis of novel mutual derivatives of nucleosides and pyrimidines by regioselectively chemo-enzymatic protocol

We established a facile regioselectively chemo-enzymatic synthesis procedure for the preparation of mutual derivatives of nucleosides and pyrimidines by sequential Markovnikov addition and acylation. Firstly, pyrimidine derivatives containing vinyl ester group were synthesized from pyrimidines and divinyl esters through Markovnikov addition catalyzed by K₂CO₃ in DMSO at 80 °C, and the yields were ranged from 50% to 87%. Then regioselective acylation of ribavirin and cytarabine with pyrimidine vinyl ester was catalyzed by CAL-B (immobilized lipase from Candida antarctica) in anhydrous acetone. Reaction conditions of enzymatic acylation including enzyme resource and solvents were optimized. A series of mutual derivatives of nucleosides and pyrimidines were synthesized successfully and characterized with NMR, IR, and HRMS. This chemo-enzymatic protocol involving sequential Markovnikov addition and acylation provided a novel way of synthesizing complicated functional compounds regioselectively which was hard to be achieved either by chemical or by enzymatic methods.     

Lipase-catalyzed regioselective acylation of sucrose in two-solvent mixtures.

The enzymatic synthesis of 6-O-lauroylsucrose and 6-O-palmitoylsucrose was performed by transesterification of sucrose with the corresponding vinyl esters in a medium constituted by two solvents. More specifically, the acylation was carried out in 2-methyl-2-butanol (tert-amyl alcohol) containing a low percentage (not higher than 20%) of dimethyl sulfoxide. Several lipases were able to catalyze the transesterification, but that from Humicola lanuginosa (adsorbed on diatomaceous earth) was particularly useful. We optimized the synthesis of 6-O-lauroylsucrose varying the percentage and nature of the cosolvent, the molar ratio sucrose/vinyl laurate, the nature of bulk solvent and the enzyme content. Under the best conditions (2-methyl-2-butanol/DMSO 4:1 v/v), a sucrose conversion of 70% to 6-O-lauroylsucrose was achieved in 24 h using 50 mg biocatalyst/mL. As a side process, a low percentage (<5% in 24 h) of the initial sucrose is converted into the diesters 6,1'-di-O-lauroylsucrose and 6,6'-di-O-lauroylsucrose. The above methodology was also extended to the synthesis of 6-O-palmitoylsucrose. The acylation process was even faster, giving rise to an 80% conversion to monoester in 48 h using 25 mg biocatalyst/mL. This study shows that the use of two-solvent mixtures may become a feasible alternative for the synthesis of sucrose esters, allowing to exploit the catalytic potential of lipases.     

Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications.

Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.     

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.     

The synthesis of amphipathic prodrugs of 1,2-diol drugs with saccharide conjugates by high regioselective enzymatic protocol

A facile, high regioselective enzymatic synthesis approach for the preparation of amphipathic prodrugs with saccharides of mephenesin and chlorphenesin was developed. Firstly, transesterification of two drugs with divinyl dicarboxylates with different carbon chain length was performed under the catalysis of Candida antarctica lipase acrylic resin and Lipozyme in anhydrous acetone at 50 degrees C, respectively. A series of lipophilic derivatives with vinyl groups of mephenesin and chlorphenesin were prepared. The influences of different organic solvents, enzyme sources, reaction time, and the acylation reagents on the synthesis of vinyl esters were investigated. And then, protease-catalyzed high regioselective acylation of D-glucose and D-mannose with vinyl esters of mephenesin and chlorphenesin gave drug-saccharide derivatives in good yields. The studies of lipophilicity and hydrolysis in vitro of prodrugs verified that drug-saccharide derivatives had amphipathic properties, and both lipophilic and amphipathic drug derivatives had obvious controlled release characteristics.     

Acylation of sucrose with vinyl esters using immobilized hydrolases: demonstration that chemical catalysis may interfere with enzymatic catalysis

The acylation of sucrose with vinyl laurate in dimethylsulfoxide was catalyzed by Celite (28% conversion in 24 h at 40 °C, 150 mg catalyst ml^–1), Eupergit C (11% conversion in 24 h at 60 °C, 150 mg catalyst/ml), and even the simple Na₂HPO₄ (17% conversion in 24 h at 40 °C, 20 mg catalyst ml^–1). These chemical acylations must therefore be taken into account in acylations of hydroxyl-containing compounds with enol esters in polar solvents using immobilized enzymes.     

Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO₂) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO₂. The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45 °C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO₂ conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO₂ conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.     

Effect of the Immobilization Method of Lipase from Thermomyces lanuginosus on Sucrose Acylation

Lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized using granulation by incubating low-particle-size silica with the lipase. Granules with a particle diameter in the range 0.3-1 &#117 mm were obtained. The immobilized lipase was tested in the acylation of sucrose with vinyl laurate in mixtures of tert -amyl alcohol: dimethyl sulfoxide. Results were compared with immobilization of enzyme by adsorption on polypropylene (Accurel EP100), deposition on Celite by precipitation, and covalent attachment to Eupergit C. Granulated lipase converted >95% of sucrose into 6- O -lauroylsucrose in 6 &#117 h. Accurel-lipase was also very active, converting 70% of sucrose into monoester in 2 &#117 h. The residual activity of granules after five reaction cycles under the best reaction conditions was 72%; this value was considerably higher than the one observed for the same lipase adsorbed on Accurel (15% residual activity after five cycles).     

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.     

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.     

Novel enzymatic approach to the synthesis of flavonoid glycosides and their esters

Flavonoids such as (+)catechin can be efficiently solubilised in supersaturated solutions prepared with donor glycosides, e.g., p-nitrophenyl glycosides, di- and higher oligosaccharides, and poly(ethylene glycol) dimethyl ether in sufficiently high concentration for their efficient enzymatic glycosylation. Under these conditions several glycosidases readily accept (+)catechin as substrate and the target glycosides were prepared in one step in up to 26% yields. The regioselectivity of the reaction depends on the enzyme and substrate combination used; three positions, 5, 7, and 4', in the flavonoid can be glycosylated. The resulting and similar flavonoid glycosides were further modified by regioselective acylation with vinyl esters of arylpropenoic acids using lipases as biocatalyst. The efficiency of acylation was found to diminish in the order of vinyl cinnamate > vinyl ferulate > vinyl coumarate. This work demonstrates the feasibility of assembling complex flavonoid glycoside esters in just two steps by sequential use of commercially available glycosidases and lipases.     

A spectrophotometric transesterification-based assay for lipases in organic solvent

A new method to evaluate lipase activities in nonaqueous conditions using vinyl ester absorbance at ultraviolet (UV) wavelengths is described. The model reaction is the transesterification between vinyl stearate and pentanol in hexane at 30 °C or in decane at 50 °C. The conversion of vinyl stearate into pentyl stearate is monitored through decreasing UV absorbance at 200 nm. Six commercial lipases were tested with this method, and results were compared with gas chromatography (GC) quantification and a classical spectrophotometric method using p-nitrophenyl palmitate. Results from the new spectrophotometric assay are similar both to results from GC quantification (R² = 0.999) and to results from p-nitrophenyl palmitate (R² = 0.989). The proposed method is able to evaluate both high activity from immobilized lipases such as immobilized Candida antarctica B lipase (3060 ± 350 U g⁻¹) and low activity from crude enzymatic extracts such as Carica papaya dried latex (0.1 ± 0.04 U g⁻¹). The method has also been used to measure kinetic parameters of C. antarctica B lipase for vinyl stearate and the correlation between its synthesis activity and its concentration. The method has also proved to be effective in studying the acyl selectivity of a lipase by comparing its activities with increasing chain lengths of vinyl esters.     

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.     

Two-step sequential synthesis of pyrimidine derivatives containing a sugar branch via combining of enzymatic Michael addition/acylation

A new strategy for the enzymatic synthesis of pyrimidine derivatives containing a sugar branch was developed via combining of Michael addition and acylation. The first-step reaction of pyrimidines and vinyl 3-propionyloxy propionate was catalyzed by Amano lipase M from Mucor javanicus in DMSO. The initial reaction rates of different pyrimidines decreased in the order of fluorouracil, uracil, thymine, in agreement with their nucleophilicity. The succeeding regioselective acylation of d-glucose and d-mannose with the Michael adducts was catalyzed by alkaline protease from Bacillus subtilis in pyridine. The d-glucose and d-mannose were all acylated at C-6 position. Moderate yield was obtained for each step.     

Novel mannitol based non-ionic surfactants from biocatalysis: Part two: improved synthesis

The 1-O-lauroyl- -mannitol, a non-ionic surfactant, was synthesised via a chemo-enzymatic pathway starting from the 1,2:4,5-di-O-isopropylidene- -mannitol and vinyl laurate as acylation agent. The high hydrophobicity of the substrates allowed the enzymatic reaction to occur both in n-hexane and in solvent free conditions. The immobilised Candida antarctica lipase B was used as the catalyst of the enzymatic step. This enzyme acts differently depending on the position of the hydroxyls with respect to the isopropylidene groups. The acid selective hydrolysis of the isopropylidene groups gave the non-ionic surfactant without the presence of isomers.     

Lipase-catalyzed esterification of hydrophilic diols in organic solvents

Summary The direct, lipase-catalyzed esterification of hydrophilic diols in organic solvents was achieved by first adsorbing the hydrophilic, solvent immiscible substrate onto a solid support with high internal surface, namely silica gel and reacting the solid mixture with fatty acid vinyl esters in an appropriate organic solvent and in presence of an immobilized lipase fromMucor miehei (Lipozyme). Quantitative conversions of the acyl donors and very high reaction rates were observed in these transformations. Furthermore, mono- or diesters of these diols could be selectively produced by this method.     

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity (a(w)). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a(w) variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.