Regioselective Enzymatic Acylation Research Articles

Efficient regioselective enzymatic acylation of troxerutin: difference characterization of in vitro cellular uptake and cytotoxicity.

In this study, we developed a multi-site acylation strategy to improve the lipophilicity and cellular uptake of troxerutin, a natural flavonoid with many health-promoting bioactivities. By clarifying the acylation properties of troxerutin catalyzed by lipases from different sources, a series of troxerutin ester derivatives acylated at different sites was synthesized, including troxerutin dipropyl (TDP), tripropyl (TTP), tetrapropyl (TEP), dibutyl (TDB), monohexyl (TMH), monooctyl (TMO) and monodecyl (TMD) esters. Interestingly, the troxerutin esters acylated at multiple sites with shorter fatty chains (TDP, TTP and TEP) had similar lipophilicity to the mono-acylated esters bearing longer fatty chains (TMH, TMO and TMD, respectively) and meanwhile demonstrated surprisingly lower cytotoxicity than that of the long fatty-chain mono-esters. In particular, the multi-acylated esters with shorter fatty chains showed remarkably higher cellular uptake than the mono-esters with long fatty chains. In vitro gastrointestinal digestion suggested that the multi-acylated esters of troxerutin were more resistant to gastrointestinal degradation than the mono-esters. These results indicated that multi-site acylation with short fatty chains could be an effective alternative to introducing one-site mono-acylation for the modification of troxerutin and other flavonoid compounds.

Lipase-catalyzed selective esterification of catechin

Catechins show strong antioxidant, antitumoral, antiviral, and anti-inflammatory activities. The uses of catechins in food, cosmetic, and pharmaceutical formulations seem very attractive. Unfortunately, solubility and stability of catechins are poor in apolar media, which limits their efficient uses. In order to improve the solubility of catechins in the oil phase and maintain their oxidation resistance, a regioselective enzymatic acylation was investigated. The effects of reaction medium, water content, carbon chain length of acyl donor and other factors on the acylation reaction were studied, catechins were enzymatically esterified with an aliphatic acid (stearic acid) using an immobilized lipase Novozym 435 in n-butanol. The results show that when the ratio between catechins and stearic acid was 1:5, adding molecular sieves 4A after 11 h of reaction and the temperature of 60 °C led to the maximum conversion yield of 60.36%. Studies have shown that catechin stearate has a higher antioxidant activity than vitamin E and dibutyl hydroxytoluene (BHT).

Novel flavonolignan hybrid antioxidants: From enzymatic preparation to molecular rationalization

A series of antioxidants was designed and synthesized based on conjugation of the hepatoprotective flavonolignan silybin with l-ascorbic acid, trolox alcohol or tyrosol via a C12 aliphatic linker. These hybrid molecules were prepared from 12-vinyl dodecanedioate-23-O-silybin using the enzymatic regioselective acylation procedure with Novozym 435 (lipase B) or with lipase PS. Voltammetric analyses showed that the silybin-ascorbic acid conjugate exhibited excellent electron donating ability, in comparison to the other conjugates. Free radical scavenging, antioxidant activities and cytoprotective action were evaluated. The silybin-ascorbic acid hybrid exhibited the best activities (IC50 = 30.2 μM) in terms of lipid peroxidation inhibition. The promising protective action of the conjugate against lipid peroxidation can be attributed to modulated electron transfer abilities of both the silybin and ascorbate moieties, but also to the hydrophobic C12 linker facilitating membrane insertion. This was supported experimentally and theoretically by density functional theory (DFT) and molecular dynamics (MD) calculations. The results presented here can be used in the further development of novel multipotent antioxidants and cytoprotective agents, in particular for substances acting at an aqueous/lipid interface.

Chemo-Enzymatic Synthesis of Oligoglycerol Derivatives.

A cleaner and greener method has been developed and used to synthesize 14 different functionalized oligomer derivatives of glycerol in moderate 29%–39% yields over three steps. After successive regioselective enzymatic acylation of the primary hydroxyl groups, etherification or esterification of the secondary hydroxyl groups and chemoselective enzymatic saponification, the target compounds can efficiently be used as versatile building blocks in organic and supramolecular chemistry.

Efficient regioselective acylation of quercetin using Rhizopus oryzae lipase and its potential as antioxidant

The present investigation describes the regioselective enzymatic acylation of quercetin with ferulic acid using Rhizopus oryzae lipase. Optimization of reaction parameters resulted in 93.2% yield of the ester synthesized using 750IU of lipase in cyclo-octane at a temperature of 45°C. The reaction was successfully carried out upto 25g scale. The ester synthesized was analyzed by 1H Nuclear magnetic resonance spectroscopy. The ester synthesized (quercetin ferulate) showed higher antiradical activity as compared to ascorbic acid using the 2,2-diphenyl-1-picrylhydrazyl radical method. These results on enzyme-catalyzed acylation of quercetin might be used to prepare and scale-up other flavonoids derivatives.

A magnetic biocatalyst based on mussel-inspired polydopamine and its acylation of dihydromyricetin

A support made of mussel-inspired polydopamine-coated magnetic iron oxide nanoparticles (PD-MNPs) was prepared and characterized. The widely used Aspergillus niger lipase (ANL) was immobilized on the PD-MNPs (ANL@PD-MNPs) with a protein loading of 138 mg/g and an activity recovery of 83.6% under optimized conditions. For the immobilization, the pH and immobilization time were investigated. The pH and thermal and storage stability of the ANL@PD-MNPs significantly surpassed those of free ANL. The ANL@PD-MNPs had better solvent tolerance than free ANL. The secondary structure of free ANL and ANL@PD-MNPs was analyzed by infrared spectroscopy. A kinetic study demonstrated that the ANL@PD-MNPs had enhanced enzyme-substrate affinity and high catalytic efficiency. The ANL@PD-MNPs was applied as a biocatalyst for the regioselective acylation of dihydromyricetin (DMY) in DMSO and gave a conversion of 79.3%, which was higher than that of previous reports. The ANL@PD-MNPs retained over 55% of its initial activity after 10 cycles of reuse. The ANL@PD-MNPs were readily separated from the reaction system by a magnet. The PD-MNPs is an excellent support for ANL and the resulting ANL@PD-MNPs displayed good potential for the efficient synthesis of dihydromyricetin-3-acetate by enzymatic regioselective acylation.

Fatty acid esters of phloridzin induce apoptosis of human liver cancer cells through altered gene expression.

Phloridzin (phlorizin or phloretin 2′-O-glucoside) is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin) using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA) ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2), growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR) and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK), cell cycle machinery (CDKs, TERT, TOP2A, TOP2B) as well as epigenetics regulators (HDACs). These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects mediated through the attenuated expression of several key proteins involved in cell cycle regulation, DNA topoisomerases IIα activity and epigenetic mechanisms followed by cell cycle arrest and apoptosis.

Tandem glycosyl iodide glycosylation and regioselective enzymatic acylation affords 6-O-tetradecanoyl-α-d-cholesterylglycosides.

A generalized synthesis of α-d-cholesterylglycosides has been achieved using one-pot per-O-trimethylsilyl glycosyl iodide glycosidation. Both cholesteryl α-d-glucopyranoside (αCG) and cholesteryl α-d-galactopyranoside were prepared in high yield. These compounds were further esterified using regioselective enzymatic acylation with tetradecanoyl vinyl ester to afford 6-O-tetradecanoyl-α-d-cholesteryl glucopyranoside (αCAG) of Helicobacter pylori and the corresponding galactose analogue in 66–78% overall yields from free sugars. The tandem step-economy sequence provides novel analogues to facilitate glycolipidomic profiling.

Phloridzin fatty acid esters induce apoptosis and alters gene expression in human liver cancer cells (261.2)

Phloridzin Fatty Acid Esters Induce Apoptosis and Alters Gene Expression in Human Liver Cancer Cells Sandhya VG Nair, Ziaullah, HP Vasantha Rupasinghe*Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada Abstract Phloridzin (phloretin 2‐O‐glucoside) is a dihydrochalcone, which is specifically found in apple and well known for its beneficial biological effects. To date, there is no report of antiproliferative effect of phloridzin on human cancer cell lines. We have synthesized novel acylated derivatives of phloridzin with six different long chain saturated, mono‐, and poly‐unsaturated fatty acids by regioselective enzymatic acylation. We investigated the effect of these six phloridzin fatty acid esters and their parent compounds on cell proliferation of hepatocellular carcinoma cells (HepG2) and its potential molecular mechanisms. Phloridzin fatty acid esters inhibited HepG2 cells growth in a concentration‐dependent manner by inducing apoptosis, as evidenced by decreased cell viability, formation of apoptotic bodies, decreased mitochondrial membrane potential, activation of caspase 3 and inhibition of DNA topoisomerases IIα activity that might induce G0/G1 phase arrest. However, phloridzin fatty acid esters did not significantly affect the growth of normal hepatocytes. Antiproliferative effect of phloridzin docosahexaenoic acid (DHA) ester was also associated with the down regulation of anti‐apoptotic genes, growth factor receptors, PI3k/AKT/mTOR as well as epigenetics regulators. Results reveal that phloridzin fatty acid esters possess potential as therapeutic agents for treating hepatocellular carcinoma.

An efficient microwave-assisted enzyme-catalyzed regioselective synthesis of long chain acylated derivatives of flavonoid glycosides

This work describes a method of microwave-assisted regioselective enzymatic acylation, using Novozyme 435®, of quercetin-3-O-glucoside (isoquercitrin) and phloretin-2′-glucoside (phloridzin) with different long chain saturated, mono-, and poly-unsaturated fatty acids. The synthesized esters were analyzed by 1H NMR, and 13C NMR spectroscopy. In this investigation, microwave irradiation under solvent free condition has been found to be more efficient and economical than conventional conditions both, in terms of time and energy.

Highly efficient and regioselective acylation of pharmacologically interesting cordycepin catalyzed by lipase in the eco-friendly solvent 2-methyltetrahydrofuran

A total of nine lipases and three proteases were tested for enzymatic regioselective acylation(s) of cordycepin with vinyl acetate in organic media. The highest conversion with better initial reaction rate was achieved with immobilized Candida antarctica lipase B (Novozym 435). An eco-friendly solvent 2-methyltetrahydrofuran (MeTHF) was thought to be the most suitable reaction medium. Novozym 435 was found to be a useful biocatalyst for the 25-g scale syntheses of cordycepin acetate (96.2% isolated yield), and the biocatalyst displayed excellent regioselectivity and high operational stability during the transformation. The 5′-substituted cordycepin derivative was the sole detectable product from each acylation reaction. Novozym 435 could be recycled for the synthesis of cordycepin derivative on a 25-g scale and 63% of its original activity was maintained after being reused for 7 batches. MeTHF could be considered as an eco-friendly solvent for the large scale use in biotransformation.

Enzymatic regioselective acylation of nucleosides in biomass-derived 2-methyltetrahydrofuran: Kinetic study and enzyme substrate recognition

Enzymatic regioselective acylation of pyrimidine nucleosides was mediated by immobilized lipase from Penicillium expansum in 2-methyltetrahydrofuran (MeTHF), a bio-solvent derived from biomass. Despite of the moderate dissolution ability of MeTHF toward nucleosides, the initial enzymatic reaction rate was much higher in this eco-friendly solvent than in other commonly used organic solvents. This could be explained by the lower apparent activation energy of the enzymatic reaction (24.5 vs. 43.3–57.1kJ/mol) and the higher catalytic efficiency of the enzyme (Vmax/Km, 5.8 vs. 1.1–2.9h−1) in MeTHF. The enzymatic acylation of a group of ribonucleosides afforded the desirable 5′-esters with the conversions of 96–99% and 5′-regioselectivities of 96 to >99%. In enzymatic acylation of 2′-deoxynucleosides, however, 5′-regioselectivities showed a clear dependence on the 5-substituents present in the base moiety although the substrate conversions reached >98% within 1–3h. In the cases of 2′,3′-dideoxynucleoside analogs, the reaction rate decreased markedly due to the lack of 3′-hydroxyl.

Biocatalytic synthesis, structural elucidation, antioxidant capacity and tyrosinase inhibition activity of long chain fatty acid acylated derivatives of phloridzin and isoquercitrin

Our present investigation describes the regioselective enzymatic acylation of two series of acylated derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-unsaturated fatty acids. The biocatalytic synthesis was optimized to achieve 81–98% yields, using immobilized lipase B, from Candida antarctica (Novozym 435®), in acetone at 45°C. The synthesized esters have been analyzed by 1H NMR, 13C NMR spectroscopy and evaluated for their antioxidant capacity and tyrosinase inhibition, using in vitro assays. Among all the phloridzin and isoquercitrin derivatives, the greatest potential for inhibition of tyrosinase activity (p ⩽0.05) was exhibited by the α-linolenic acid ester of isoquercitrin.

Regioselective Enzymatic Acylation of Aureolic Acids to Obtain Novel Analogues with Improved Antitumor Activity

A variety of aureolic acids was efficiently obtained by lipase-catalyzed regioselective acylation reactions of mithramycin and its analogues mithramycin SK and mithramycin SDK. The acylation took place in the aglycone moiety or in the B sugar unit, depending on the substrate, lipase and acyl donor considered. Most of the novel acylated derivatives showed antitumor activity at double digit nanomolar concentrations, in some cases significantly improving the activity of the parent drugs.

ChemInform Abstract: Regioselective Enzymatic Acylation of Complex Natural Products: Expanding Molecular Diversity

AbstractReview: 80 refs.

Chemoenzymatic synthesis of cholesteryl-6-O-tetradecanoyl-α-d-glucopyranoside: a product of host cholesterol efflux promoted by Helicobacter pylori

In a three-step protocol involving regioselective enzymatic acylation, per-O-trimethylsilylation, and a one-pot α-glycosidation-deprotection sequence, cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside (α-CAG) of Helicobacter pylori is afforded starting from glucose in an overall yield of 45%. The production of CAG can be scaled to make purified quantities available to the biological community for the first time.

Regioselective enzymatic acylation of complex natural products: expanding molecular diversity

Enzymatic catalysis has become a common tool in both academia and industrial chemistry. The efforts of chemists over recent decades have led to the rationalization of the mechanism of action of biocatalysts, which have been routinely incorporated into many synthetic sequences. Nowadays, a further step consists in expanding the application of enzymes to the modification of complex molecular scaffolds common to many pharmaceutical leads isolated from nature. Regioselective enzymatic acylation is a process which has been profitably applied for this purpose in recent times, leading to new drugs with improved activity, stability and pharmacokinetic properties. This tutorial review provides an overview of this subject employing two classes of enzymes, hydrolases and acyltransferases, in the recently concluded decade although some representative older studies are commented upon, if required. We shall place special emphasis on those examples in which the novel acylated derivatives have improved the activity or properties of the parental molecules.

ChemInform Abstract: Enzymatic Regioselective Acylation of Polyhydroxylated Natural Compounds in Organic Solvents.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Application of organic solvent system for lipase-catalyzed regioselective benzoylation of 1-β-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.

Efficient synthesis of 5′-O-laurate of 1-β-d-arabinofuranosylcytosine via highly regioselective enzymatic acylation in binary solvent mixtures

Regioselective enzymatic acylations of 1-β-d-arabinofuranosylcytosine (ara-C) with vinyl laurate (VL) in binary organic solvents were explored for the preparation of 5′-O-laurate of ara-C. Among the nine kinds of enzymes, Novozym 435 showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. This lipase showed higher catalytic activity in hexane–pyridine than in other tested solvent mixtures. The most suitable VL to ara-C molar ratio, initial water activity, and reaction temperature were shown to be 15:1, 0.07, and 50°C, respectively, under which the initial reaction rate and the maximum substrate conversion were as high as 84.0mmolL−1h−1 and 98.1%, respectively. The product of Novozym 435-catalyzed acylation was characterized by 13C NMR and confirmed to be 5′-O-laurate of ara-C.