Highly Efficient and Enzymatic Regioselective Undecylenoylation of Gastrodin in 2-Methyltetrahydrofuran-Containing Systems

Highly efficient and regioselective acylation of pharmacologically interesting gastrodin with vinyl undecylenic acid has been firstly performed through an enzymatic approach. The highest catalytic activity and regioselectivity towards the acylation of 7′-hydroxyl of gastrodin was obtained with Pseudomonas cepacia lipase. In addition, it was observed the lipase displayed higher activity in the eco-friendly solvent 2-methyltetrahydrofuran-containing systems than in other organic solvents. In the co-solvent mixture of tetrahydrofuran and 2-methyltetrahydrofuran (3/1, v/v), the reaction rate was 60.6 mM/h, substrate conversion exceeded 99%, and 7′-regioselectivity was 93%. It was also interesting that the lipase-catalyzed acylation couldn’t be influenced by the benzylic alcohol in gastrodin. However, pseudomonas cepacia lipase displayed different regioselectivity towards gastrodin and arbutin.     

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.     

A lipase‐catalyzed process for green synthesis of temsirolimus

Temsirolimus is an intravenous drug for the treatment of renal cell carcinoma that can be prepared using enol acyl donors, which is not favorable in process development. An improved enzymatic process to prepare temsirolimus has been developed employing lipase‐catalyzed regioselective acylation of rapamycin with environmentally friendly acyl donors. After screening of common commercial lipases and none‐enol acyl donors, it was found that p‐nitrophenyl 2,2,5‐trimethyl‐1,3‐dioxane‐5‐carboxylate reacted as efficient acyl donor when catalyzed by immobilized Thermomyces lanuginose lipase. By optimizing the process conditions (i.e., reaction temperature, solvents, and additives), the reaction time was significantly shortened while the reaction conversion reached 95.4% in methyl tert‐butyl ether after 48 h at 50°C using the new acyl donors. This work demonstrated a cost‐effective, efficient, and scalable process to synthesize temsirolimus.     

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.     

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.     

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).     

Highly regioselective enzymatic synthesis of 5′-O-stearate of 1-β-D-arabinofuranosylcytosine in binary organic solvent mixtures

In this paper, highly regioselective enzymatic acylations of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5′-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS–ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50°C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol·L⁻¹h⁻¹, 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13(¹³C) NMR and confirmed to be 5′-O-stearate of ara-C.     

Synthesis of monosaccharide derivatives and polymeric prodrugs of 5-fluorouridine via two-step enzymatic or chemo-enzymatic highly regioselective strategy

Efficient protocols for the selective synthesis of monosaccharide derivatives and polymeric prodrugs of 5-fluorouridine (5-FUR) have been developed. Firstly, transesterification of 5-FUR and divinyl dicarboxylates ranging from 4 to 10 carbon atoms were performed under the catalysis of Candida antarctica lipase acrylic resin in anhydrous THF at 50 °C, respectively. A series of vinyl 5-FUR esters were prepared, with high acylation regioselectivity at 5′-OH. The influences of reaction parameters including enzyme, solvents, molar ratio of substrates, reaction time, the carbon length of acyl donor and reaction temperature were investigated in details. And then, protease-catalyzed highly regioselective acylation of d-glucose, d-mannose and d-galactose with vinyl esters of 5-FUR gave 5-FUR-saccharide derivatives successfully. Moreover, a series of polymeric prodrugs of 5-FUR with the different linker lengths were prepared by the chemo-polymerization of vinyl 5-FUR esters in DMF initiated by azobisisobutyronitrile (AIBN).     

Regio-selective acylation of biologically important iridoid glycosides by Candida antarctica lipase

Lipase catalyzed regio-selective acylation of five iridoid glycosides viz., picroside I&II, catalpol, agnuside and negundoside in the presence of various acyl donors such as vinyl acetate and p-nitrophenyl alkanoates was studied. The regio-selectivity of enzymatic acylation and yields were found to vary amongst different substrates. Monoacylated products were isolated with all the substrates under scrutiny indicating high regio-selective nature of such transformations. A series of acyl esters of picroside-I, picroside-II, catalpol, agnuside and negundoside have been synthesized by this enzymatic trans-esterification methodology.     

Efficient regioselective acylation of andrographolide catalyzed by immobilized Burkholderia cepacia lipase

For the first time, PSL-C, an immobilized lipase from Burkholderia cepacia, was successfully applied to the regioselective acylation of andrographolide by vinyl acetate in acetone. FT-IR spectra demonstrated the occurrence of acylation reaction. The ¹³C NMR, ESI-MS and elemental analysis confirmed that the 14-acetylandrographolide was formed exclusively. Water activity and reaction temperature had a significant effect on the initial rate and the substrate conversion, but little effect on the regioselectivity of the reaction. The optimal water activity and reaction temperature were 0.11 and 50 °C, respectively. Under these conditions, the initial rate and substrate conversion were 50.2 mM h⁻¹ and 99.0%, respectively, after a reaction time of around 4 h. Besides, immobilized lipase also displayed higher operational stability and 83.5% of its original activity was maintained after being reused for eight batches.     

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.     

A chemo-biocatalytic approach in the synthesis of β-O-naphtylmethyl-N-peracetylated lactosamine

A chemo-enzymatic approach combining an enzymatic regioselective hydrolysis of peracetylated N-acetyl-α-d-glucosamine (1) with a mild controlled acyl migration led to 2-acetamido-2-deoxy-1,3,6-tri-O-acetyl-α-d-glucopyranose, which was further used in a glycosylation reaction in the synthesis of β-O-naphtylmethyl-N-peracetylated lactosamine.Candida rugose lipase (CRL) immobilized on octyl-agarose and modified by covering it with polyethyleneimine was the best catalyst in terms of activity, stability and regioselectivity in the hydrolysis of 1, producing the deacetylation in C-6 in 95% overall yield. Other immobilized lipases were not specific or with a very low activity towards the hydrolysis of 1.An acyl chemical migration by incubation of the deacetylated C-6 derivative at pH 8.5, 4 °C, and 10–20% acetonitrile permitted to obtain up to 75% overall yield of the 4-OH derivative product. This molecule was successfully applied in a glycosylation reaction to get the peracetylated α-d-lactosamine and finally, the peracetyl-β-O-naphtylmethyl-lactosamine derivative in 20% overall yield.     

Application of organic solvent system for lipase-catalyzed regioselective benzoylation of 1-<Emphasis Type="Italic">β</Emphasis>-D-arabinofuranosylcytosine

In this paper, enzymatic regioselective acylation of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl benzoate (VB) using immobilized Candida antarctica lipase B in binary organic solvents was explored. It was found that the lipase showed high regioselectivity (> 99%) towards the 5′-OH of ara-C in the representative organic solvent mixture (hexane-pyridine). To understand the enzymatic processes and provide a fair comparison of hexane-pyridine with C₄MIm·PF₆-pyridine (the representative ionic liquid-containing system), the effect of each process variable on the reactions in hexane-pyridine was investigated. The results indicate that the optimum hexane content, initial a _w , molar ratio of VB to ara-C, and temperature were 28% (v/v), 0.11, 15, and 40°C, respectively. Under optimized conditions, the initial reaction rate in hexanepyridine (44.4 mM/h) was much higher than that in C₄MIm·PF₆-pyridine (29.4 mM/h) for each case. The maximum conversion yield, however, was increased when the reaction system was shifted from hexane-pyridine to C₄MIm·PF₆-pyridine. Further study revealed that the presence of an acidic by-product (benzoate acid, released during the acylation process) may cause rapid inactivation of the enzyme in hexane-pyridine, leading to a lower conversion rate, whereas the ionic liquid may have coating and protecting effects on the lipase during the reaction.     

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.     

A combinatorial biocatalysis approach to an array of cholic acid derivatives

A 39-member library of bile acid derivatives was prepared starting from 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid methyl ester using a combinatorial biocatalytic approach. A regioselective oxidation step, catalyzed by hydroxysteroid dehydrogenases, followed by an acylation step with a series of different acyl donors catalyzed by Candida antarctica lipase B, led to the modification of the bile acid scaffold. Each member of the library was obtained in high purity and good yield.     

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.     

Effect of different reaction parameters on the lipase-catalyzed selective acylation of polyhydroxylated natural compounds in ionic liquids

The effect of various reaction parameters on the enzymatic acylation of plant polyhydroxylated compounds, including phenolic and flavonoid glucosides (salicin, helicin, esculin and naringin), was investigated in imidazolium-based ionic liquids (1-butyl-3-methylimidazolium tetrafluoroborate [bmim]BF₄ and 1-butyl-3-methylimidazolium hexafluorophosphate [bmim]PF₆), using immobilized lipase B from Candida antarctica. The conversion yield, the regioselectivity and the reaction rate of the biocatalytic process strongly depended on the ionic liquid used, their water content, the incubation temperature, as well as the solubility and the concentration of substrates. For most glucosides tested, one major product (monoacylated derivative) was detected as a result of the acylation of the primary hydroxyl group of glucose moiety. The acylation rate and the regioselectivity of the process are higher in [bmim]BF₄, where the solubility of all glucosides is significantly higher than in [bmim]PF₆ or acetone. Response surface methodology (RSM) based on a five level-three variable central composite circumscribed design, was employed to evaluate the interactive effect of the molar ratio of substrates (MR), the initial concentration of glucoside (N) and the reaction time (RT), as well as for their optimization in [bmim]BF₄. At the optimal reaction conditions the maximum acylation yield was 87%. The amount of monoacylated derivatives produced in a single-step biocatalytic process reached values up to 31.6 g/l which is considerably higher than those reported for organic media.     

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.     

Enzyme catalyzed acylation and deacylation of the sugar moieties of nucleosides

We have found that a lipase from Pseudomonas fluorescens (PFL) accelerated regioselective acylation of 2'-deoxynucleosides with the use of acid anhydrides as acyl donor in dry polar solvents. Different regioselective deacylation of 3',5'-di-O-acyl-2'-deoxynucleosides was found to take place when a lipase (PFL) or a protease from Bacillus subtilis (Subtilisin) was used.     

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).