Biocatalytic synthesis of fluorinated polyesters

The biocatalytic synthesis of fluorinated polyesters from activated diesters and fluorinated diols has been investigated. The effects of time, continuous enzyme addition, enzyme concentration, and diol chain length were studied to determine the factors that would limit chain extension, such as enzyme inactivation, enzyme specificity, the equilibrium position for the reaction, hydrolytic side reactions, and polymer precipitation. An enzyme screen demonstrated that only Novozym 435, an immobilized lipase from Candida antarctica, was effective in producing the fluorinated polyester. Molecular weight and polydispersity analyses were performed by means of gel permeation chromatography. End group analysis was accomplished through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Polymer molecular weight steadily increased and then leveled off after approximately 30 h, with a weight average molecular weight of approximately 1773. The majority of the polymer chains were terminated with either hydroxyl or vinyl groups. Polymers that were synthesized from bulk monomers had higher molecular weights, but high enzyme concentrations were required. Enzyme specificity toward shorter chain fluorinated diols appeared to be the governing factor in limiting chain growth. However, polymer molecular weight increased further (M(w) = 8094) when a fluorinated diol that contained an additional methylene spacer between the fluorine atoms and hydroxyl groups was used.     

Enzymatic synthesis and curing of biodegradable epoxide-containing polyesters from renewable resources

Epoxide-containing polyesters were enzymatically synthesized via two routes using unsaturated fatty acids as starting substrate. Lipase catalysis was used for both polycondensation and epoxidation of the unsaturated fatty acid group. One route was synthesis of aliphatic polyesters containing an unsaturated group in the side chain from divinyl sebacate, glycerol, and the unsaturated fatty acids, followed by an epoxidation of the unsaturated fatty acid moiety in the side chain of the resulting polymer. In another route, epoxidized fatty acids were prepared from the unsaturated fatty acids and hydrogen peroxide in the presence of lipase catalyst, and subsequently the epoxidized fatty acids were polymerized with divinyl sebacate and glycerol. The polymer structure was confirmed by NMR and IR, and for both routes, the high epoxidized ratio was achieved. Curing of the resulting polymers proceeded thermally, yielding transparent polymeric films with high gloss surface. Pencil scratch hardness of the present films improved, compared with that of the cured film obtained from the polyester having an unsaturated fatty acid in the side chain. The obtained film showed good biodegradability, evaluated by BOD measurement in an activated sludge.     

Laboratory and field evaluation of biodegradable polyesters for sustained release of isometamidium and ethidium

An overview is presented of the results obtained with biodegradable sustained release devices (SRDs) containing a mixture of polymers and either isometamidium (ISMM) or ethidium. Under controlled laboratory conditions (monthly challenge with tsetse flies infected with Trypanosoma congolense) the protection period in SRD treated cattle could be extended by a factor 2.8 (for ethidium) up to 4.2 (for ISMM) as compared to animals treated intramuscularly with the same drugs. Using a competitive drug ELISA ISMM concentrations were detected up to 330 days after the implantation of the SRDs, whereas after i.m. injection the drug was no longer present three to four months post treatment. Two field trials carried out in Mali under heavy tsetse challenge showed that the cumulative infection rate was significantly lower in the ISMM-SRD implanted cattle than in those which received ISMM intramuscularly. Using ethidium SRD, however, contradictory results were obtained in field trials in Zambia and in Mali. The potential advantages and inconvenients of the use of SRDs are discussed and suggestions are made in order to further improve the currently available devices.     

Biocatalytic synthesis of vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.     

Development of a dialysis in vitro release method for biodegradable microspheres

The purpose of this research was to develop a simple and convenient in vitro release method for biodegradable microspheres using a commercially available dialyzer. A 25 KD MWCO Float-a-Lyzer was used to evaluate peptide diffusion at 37°C and 55°C in different buffers and assess the effect of peptide concentration. In vitro release of Leuprolide from PLGA microspheres, having a 1-month duration of action, was assessed using the dialyzer and compared with the commonly used “sample and separate” method with and without agitation. Peptide diffusion through the dialysis membrane was rapid at 37°C and 55°C in all buffers and was independent of peptide concentration. There was no detectable binding to the membrane under the conditions of the study. In vitro release of Leuprolide from PLGA microspheres was tri-phasic and was complete in 28 days with the dialysis technique. With the sample and separate technique, linear release profiles were obtained with complete release occurring under conditions of agitation. Diffusion through the dialysis membrane was sufficiently rapid to qualify the Float-a-Lyzer for an in vitro release system for microparticulate dosage forms. Membrane characteristics render it useful to study drug release under real-time and accelerated conditions. Published: October 6, 2005     

Biocatalytic synthesis of oligosaccharides

Tremendous advances in biocatalytic approaches to oligosaccharide synthesis have taken place in the past two years. The use of isolated enzymes, both glycosyltransferases and glycosidases, or engineered whole cells allows the preparation of natural oligosaccharides and analogs required for glycobiology research.     

A novelin vitro release technique for peptide-containing biodegradable microspheres

The purpose of this study was to develop and evaluate a dialysisin vitro release technique for peptide-containing poly(d, 1-lactide-coglycolide) (PLGA) microspheres (ms) that would correlate within vitro data. Using a luteinizing hormone- releasing hormone analogue (LHRH), Orntide acetate, solubility and stability were determined in 0.1 M phosphate buffer (PB), pH 7.4, and in 0.1 M acetate buffer (AB), pH 4.0, with highperformance liquid chromotography (HPLC), and peptide permeability through a dialysis membrane (molecular weight cut-off 300,000) was determined. Orntide ms were prepared by a dispersion/solvent extraction/evaporation method and characterized for drug content (HPLC), particle size distribution (laser diffraction method), and surface morphology (scanning electron microscopy).In vitro release was studied in PB using a conventional extraction method and with a new dialysis method in AB. Gravimetric analyses of polymer mass loss and matrix hydration, and peptide adsorption to blank PLGA ms (50∶50, M_w 28 022) were carried out in PB and AB upon incubation at 37°C. Serum Orntide and testosterone levels in rats after administration of Orntide ms were determined by radioimmunoassay. Orntide acetate solubility was influenced by pH; approximately 2.3 mg/mL dissolved in PB and >18 mg/mL in AB. Stability was pH- and temperature-dependent. The peptide was very stable at pH 4.0, 4°C, but degraded rapidly at pH 7.4,37°C. Peptide permeability through the dialysis membrane was accelerated by agitation and>95% equilibrium was reached within 48 hours. The overall release rate was higher with the dialysis method. Mass loss of the Orntide ms was faster in AB (50% loss in 3 weeks: 95% in 35 days) than in PB (65% in 35 days). In contrast, hydration after 35 days was 4-fold higher in PB. The nonspecific adsorption to blank ms was greater in PB (128 μ g Orntide/10 mg PLGA) compared with AB (<5 μ g Orntide/10 mg PLGA). Administration of 30-day Orntide PLGA ms to rats resulted in an initial serum Orntide level of 21 ng/mL after 6 hours and a C_max of 87 ng/mL after 6 days. Testesterone levels were suppressed immediately after ms administration (3 mg Orntide/Kg) from 5.2 ng/mL to 0.3 ng/mL (after 24 hours) and remained suppressed for 38 days. Orntide acetate solubility and degradation kinetics were markedly influenced by pH of the buffer systems and mass loss; matrix hydration, as well as the nonspecific adsorption to blank ms, was pH-dependent. Thein vitro release profile obtained with the dialysis method in AB correlated well with thein vivo data, therepy providing a more reliable prediction ofin vivo performance.     

Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.     

Biocatalytic synthesis of atorvastatin intermediates

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and this drug leads to decreased levels of low density lipoprotein (LDL) cholesterol. Lower LDL cholesterol has direct relationship in reducing mortality from coronary heart diseases. Lipitor® (atorvastatin calcium) was the first drug to reach the annual sales of 10 billion dollars in USA and currently is the top selling pharmaceutical product globally. Atorvastatin has a side chain containing two chiral centers as its pharmacophore and it can be synthesized either from chiral pool precursors, by using metal catalysts; or more preferably by the application of free or immobilized enzymes and whole cell biocatalysts for carrying out either asymmetric synthesis or racemic resolution. Biocatalytic synthesis methods for chiral atorvastatin intermediates employ a wide variety of biocatalysts such as alcohol dehydrogenase, 2-deoxy-d-ribose 5-phosphate aldolase, nitrilase, lipase, etc. and each of these biocatalytic processes is discussed in detail in this paper.     

Stereoselective behavior of a novel biodegradable racemic ketoprofen injectable implant in rats

The stereoselectivity of release of ketoprofen (KET) enantiomers from a biodegradable injectable implant containing racemic KET (rac-KET) was investigated in vivo. A pre-column chiral derivatization RP-HPLC method was employed to assay diastereoisomeric derivatives of R- and S-KET. The rac-KET injectable implant, once injected subcutaneously in rats, produced long-lasting plasma levels of S-KET, which were always greater than those of R-KET. The difference in enantiomer concentration was to be related to stereoselective release, due to stereoselective interaction between D,L-PLG in the implant and KET enantiomers, as well as the chiral inversion of KET in vivo. The rac-KET injectable implant provided the sustained release of S-KET with effective plasma levels maintained for about 8 wk after a single injection.     

Lipase-catalyzed synthesis of aromatic polyesters

The enzymatic synthesis of aromatic polyesters by direct polyesterification between a diacid and a diol is described. The effects of the type of substrate, type and quantities of lipase, temperature, vacuum, and reaction time on the synthesis of aromatic polyesters were studied in detail. Among three lipases investigated, only Novozym 435 worked well for aromatic polyester synthesis. Temperature and vacuum played an important role in obtaining a high molar mass of the aromatic polyesters. Furthermore, with isophthalic acid and 1,6-hexanediol as substrates, the mass average molar mass of the polyester obtained increased with an increase in the lipase quantity up to 0.375 g (11.7%, w/w of total reactor contents). The mass average molar mass of the polyester was as high as 50000 g mol⁻¹ in 168 h, with a polydispersity of PD ≈ 1.4. Received 27 January 1998/ Accepted in revised form 19 May 1998     

脂肪酶催化的末端官能化聚酯合成研究进展

脂肪酶催化的末端官能化聚酯合成是高分子合成领域中极具吸引力且发展非常迅速的重要技术：重点介绍引发剂法、终止剂法以及酶促化学偶联合成末端官能化聚酯研究所取得的主要进展。     

Biocatalytic synthesis of optically active tertiary alcohols

The enzymatic preparation of optically pure tertiary alcohols under sustainable conditions has received much attention. The conventional chemical synthesis of these valuable building blocks is still hampered by the use of harmful reagents such as heavy metal catalysts. Successful examples in biocatalysis used esterases, lipases, epoxide hydrolases, halohydrin dehalogenases, thiamine diphosphate-dependent enzymes, terpene cyclases, -acetylases, and -dehydratases. This mini-review provides an overview on recent developments in the discovery of new enzymes, their functional improvement by protein engineering, the design of chemoenzymatic routes leading to tertiary alcohols, and the discovery of entirely new biotransformations.     

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.     

Biocatalytic synthesis of intermediates for the synthesis of chiral drug substances

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivity. Chiral intermediates and fine chemicals are in high demand, both from the pharmaceutical and agrochemical industries, for the preparation of bulk drug substances and agricultural products. Biocatalytic processes have been described for the synthesis of chiral intermediates for beta3- and beta2-receptor agonists, antihypertensive drugs, antiviral agents, melatonin receptor agonists, anticholesterol and anticancer drugs, and drugs to treat Alzheimer's disease.     

Biocatalytic synthesis and antitumor activities of novel silybin acylated derivatives with dicarboxylic acids

Novel silybin acylated derivatives with dicarboxylic acids were prepared in various organic solvents using immobilized Candida antarctica lipase B (Novozym 435(?)). The reaction parameters affecting the silybin conversion, such as the nature of the organic solvent and the acyl donor used were investigated. The antiproliferative effects of silybin monoesters, and their ability to modulate the secretion of vascular endothelial growth factor (VEGF) were estimated using K562 human lymphoblastoma cells and compared to the parental compound. The synthesized esters retained the biological function of silybin and in some cases were more effective, indicating that target biotransformation may generate novel compounds with improved antitumor and antiangiogenic activities.     

Prolonged release biodegradable vesicular carriers for rifampicin--formulation and kinetics of release

An attempt has been made to design suitable liposome and niosome-encapsulated drug delivery system for rifampicin and evaluated the same in vitro and in vivo. A modified lipid layer hydration method was employed to prepare these vesicular carriers. The formulated systems were characterized in vitro for size distribution analysis, drug entrapment, drug release profiles and vesicular stability at different conditions of storage. In vivo drug kinetics was evaluated in normal, healthy albino rats for niosomal formulation upon subcutaneous injection and various pharmacokinetic parameters were determined. Niosomes and liposomes exhibited mean diameter of 9.73 and 11.87 microns with entrapment efficiencies of 30.5 and 34.2% respectively. Both the products exhibited sustained release characteristics in vitro with zero order drug release kinetics up to initial 10 hr. Stability evaluation indicated that both formulations were not significantly leaky over a period of one month. Niosomal formulation elevated plasma elimination half life and decreased elimination rate constants for rifampicin in vivo suggested that encapsulation retarded the removal of the drug from circulation compared to free drug due to slow drug release into systemic circulation. A five-fold increase in the area under plasma rifampicin concentration-time curve for niosomal rifampicin as compared to free drug indicated better bioavailability of encapsulated drug. It is evident from this study that niosomes and liposomes could be promising delivery systems for rifampicin with prolonged drug release profiles and reasonably good stability characteristics.     

Lipase-catalyzed synthesis of aliphatic polyesters and properties characterization

A novel lipase-catalyzed synthesis of aliphatic polyesters has been achieved successfully by using Candida sp.99-125 with diethyl sebacate and 1,4-butanediol as starting substrates in absence of organic solvents. The lipase from Candida sp.99-125 was employed for the first time to catalyze synthesis of poly(butylene sebacate) and showed high catalytic activity for bulkpolymerization under mild reaction conditions. The eco-friendly processes, without any environmental pollution, avoided both phase separation of the reactants and the use of toxic solvents effectively. The polycondensation reactions were performed in two stages: first stage reaction under atmospheric pressure to convert the monomers to oligomers followed by second stage reaction under vacuum process, with a highest molecular weight of 15,800 obtained in molar ratio of 1:1 at 70 °C. The corresponding polyesters determined by thermogravimetric analysis (TGA) were much thermally stable up to 300 °C. Differential scanning calorimetry (DSC) and wide-angle X-ray diffraction (WAXS) analysis demonstrated the polymers had a strong ability to crystallize with the degree of crystallinity up to 66%.     

Copolymers from unsaturated macrolactones: toward the design of cross-linked biodegradable polyesters

The enzymatic synthesis of a series of random copolyesters by ring-opening polymerization of unsaturated macrolactones like globalide and ambrettolide with 1,5-dioxepan-2-one (DXO) and 4-methyl caprolactone (4MeCL) was investigated. (13)C NMR diad analysis confirmed the randomness of all copolymers irrespective of the comonomer ratios. Thermal investigation showed that incorporating the comonomers lowered the melting points of the polymers as compared with the macrolactone homopolymers. The decrease was dependent on the comonomer ratio. The unsaturated copolymers were thermally cross-linked using dicumyl peroxide, which resulted in completely amorphous insoluble networks. It was found that 10% incorporation of the unsaturated macolactone was sufficient to obtain a gel content of 95 wt %. Preliminary degradation tests confirm that the cross-linked copolymers are enzymatically degradable and that the incorporation of hydrophilic comonomers like DXO enhances degradation.