Synthesis and characterization of drug–saccharide conjugates by enzymatic strategy in organic media

A facile and efficient strategy to prepare drug derivatives with saccharides was developed, and 12 drug–saccharide conjugates were obtained by selective enzymatic synthesis methods. When the transesterification of chlorphenesin vinyl esters with glucose was chosen as a model reaction, the influence of reaction conditions was optimized. Then, we successfully prepared a series of chlorphenesin–saccharide derivatives of three monosaccharides (glucose, mannose and galactose) and three disaccharides (maltose, lactose and sucrose). In order to study the relationship between the solubility of drug–saccharide conjugates and the structure of parent drugs, five hydrophobic drugs (chlorphenesin, mephenesin, guaifenesin, propranolol and clorprenaline) were chosen as substrates to synthesize drug–saccharide conjugates. The results indicated that the aqueous solublity of drug–saccharide derivatives was greatly improved, which was changed by different saccharides and the structure of parent drugs.     

Synthesis and characterization of drug–saccharide conjugates by enzymatic strategy in organic media

A facile and efficient strategy to prepare drug derivatives with saccharides was developed, and 12 drug–saccharide conjugates were obtained by selective enzymatic synthesis methods. When the transesterification of chlorphenesin vinyl esters with glucose was chosen as a model reaction, the influence of reaction conditions was optimized. Then, we successfully prepared a series of chlorphenesin–saccharide derivatives of three monosaccharides (glucose, mannose and galactose) and three disaccharides (maltose, lactose and sucrose). In order to study the relationship between the solubility of drug–saccharide conjugates and the structure of parent drugs, five hydrophobic drugs (chlorphenesin, mephenesin, guaifenesin, propranolol and clorprenaline) were chosen as substrates to synthesize drug–saccharide conjugates. The results indicated that the aqueous solublity of drug–saccharide derivatives was greatly improved, which was changed by different saccharides and the structure of parent drugs.     

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).     

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.     

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.     

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 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.     

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.     

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).     

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).     

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.     

Enzyme catalyzed synthesis of some vinyl drug esters in organic medium

A series of vinyl drug esters was synthesized using acyclovir and chloramphenicol with different carbon chain length acyl donors by alkaline protease from Bacillus subtilis and Lipozyme respectively, in non-aqueous medium. The corresponding vinyl drug derivatives were confirmed by nuclear magnetic resonance and infrared spectrometry. The influences of different organic solvents, reaction time, temperature, and content of water on synthesis of vinyl chloramphenicol esters were studied.     

Controllable synthesis of polymerizable ester and amide prodrugs of acyclovir by enzyme in organic solvent

A facile control of the acylation position at the primary hydroxyl and amino of acyclovir, respectively, was achieved and five polymerizable acyclovir prodrugs were synthesized. Various reaction conditions were studied in detail. Thus, lipase acrylic resin from Candida antarctica (CAL-B) in pyridine or acetone showed high chemo-selectivity toward the primary hydroxyl of acyclovir. However, lipase PS 'Amano' (PS) in DMSO selectively acylated the amino group. The selectivity of PS could be adjusted by changing reaction solvents. The acyclovir vinyl derivatives obtained would be important monomers used for the preparation of macromolecular nucleoside drugs.     

Controllable Regioselective Acylation of Rutin Catalyzed by Enzymes in Non-aqueous Solvents

An efficient route to synthesize 3′′- and 4′′′-vinyl rutin esters has been developed by enzyme-catalyzed regioselective acylation of rutin with divinyl dicarboxylates in organic media. Alkaline protease from Bacillus subtilis provided 3′′-O-substituted vinyl rutin esters in pyridine, and Novozym 435 gave 4′′′-O-substituted vinyl rutin esters in tert-butanol.     

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.     

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.     

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.     

Acylation of Bowman–Birk Soybean Proteinase Inhibitor by Unsaturated Fatty Acid Derivatives

A procedure was developed for acylation of Bowman–Birk soybean proteinase inhibitor (BBI) by N-hydroxysuc-cinimide esters of oleic, linoleic, and -linolenic acids in a dimethyl sulfoxide–dioxane–pyridine mixture. BBI derivatives containing two acylated amino groups were prepared with high yield. The use of the reversible modifier citraconic anhydride in the first stage of synthesis permitted the synthesis of hydrophobized BBI derivatives retaining high antitrypsin and antichymotrypsin activities. It was found that the insertion of two long chain moieties in the BBI molecule decreases its thermostability.     

Controllable selective enzymatic synthesis of N-acyl and O-acylpropranolol vinyl esters and preparation of polymeric prodrug of propranolol

Controllable selective synthesis strategy of polymerizable N-acyl and O-acylpropranolol vinyl derivatives was developed by enzyme-catalyzed acylation of propranolol using divinyl dicarboxylates with different carbon chain length as acyl donor. The influence of parameters including enzyme, solvents and chain length of acyl donor on the reaction was investigated in detail. Lipase AY30 in diisopropyl ether demonstrated high selectivity towards the amino group of propranolol, while lipase M from Mucor javanicus in dioxane acylated selectively the hydroxyl group of propranolol. N-Acylpropranolol (3a–3c) and O-acylpropranolol vinyl (4a–4c) derivatives were obtained successfully, and can be used for preparing functional macromolecular prodrugs of beta-blockers drugs. N-(Vinyladipoyl)propranolol (NVAP) was copolymerized with methyl methacrylate (MMA) using AIBN as initiator. The obtained polymeric prodrug was characterized with IR, NMR and GPC. The poly(NVAP-co-MMA) has M_n of 3.23 × 10⁴, and M_w/M_n of 1.66.     

Synthesis and in vitro evaluation of S-acyl-3-thiopropyl prodrugs of Foscarnet

A new enzyme-labile group called S-acyl-3-thiopropyl group (SATP) has been synthesized from allylic esters of phosphonate. After demonstration of the enzyme-labile character of the SATP in cellular extracts, it has been introduced onto the phosphonate moiety of PFA (Foscarnet) to obtain potential lipophilic prodrugs. To ponder the lipophilicity of the triesters of PFA, esters of monomethylether of polyethyleneglycols and of thioglycerol were introduced on the PFA carboxylate moiety. The SATP groups were introduced in an attempt to deliver PFA after bioactivation inside the cells. The PFA prodrugs were evaluated in vitro for their activity against human immunodeficiency viruses (HIV-1 and HIV-2).