Biodegradable amphiphilic triblock copolymer bearing pendant glucose residues: preparation and specific interaction with Concanavalin A molecules

A novel biodegradable amphiphilic block copolymer PLGG-PEG-PLGG bearing pendant glucose residues is successfully prepared by the coupling reaction of 3-(2-aminoethylthio)propyl-alpha-D-glucopyranoside with the pendant carboxyl groups of PLGG-PEG-PLGG in the presence of N,N'-carbonyldiimidazole. The polymer PLGG-PEG-PLGG, i.e., poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}-block-poly(ethylene glycol)-block- poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}, is prepared by ring-opening copolymerization of L-lactide (LLA) with (3s)-benzoxylcarbonylethylmorpholine-2,5-dione (BEMD) in the presence of dihydroxyl PEG with molecular weight of 2000 as macroinitiator and Sn(Oct)2 as catalyst, and then by catalytic hydrogenation. The glucose-grafted copolymer shows a lower degree of cytotoxicity to ECV-304 cells and improved specific recognition and binding with Concanavalin A (Con A). Therefore, this kind of glucose-grafted copolymer may find biomedical applications.     

A convenient preparation of 6-oligo(lactic acid)cyclomaltoheptaose as kinetically degradable derivative for controlled release of amoxicillin

6-Oligo(lactic acid)cyclomaltoheptaose (6-OLA-βCD) with an average substitution of about 7.0 lactic acid units was prepared as a new water-soluble cyclomaltoheptaose (βCD) derivative (solubility of about 70.7-fold that of βCD), based on the ring-opening polymerization of 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide). The product was characterized by ¹H NMR, ¹³C NMR, IR, and MS spectroscopy. The complexation of amoxicillin with 6-OLA-βCD was found to be much stronger than that with βCD at first, and then 6-OLA-βCD was shown to decompose moderately into βCD and lactic acid. 6-OLA-βCD might be greatly valuable in a controlled release system for Amoxicillin (AMX).     

1,4-Dioxane-2,5-dione-type monomers derived from l-ascorbic and d-isoascorbic acids. Synthesis and polymerisation

l-Ascorbic and d-isoascorbic acids have been used as the starting materials for the preparation of (3R,4′S)-3-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPTA), (3R and S, 4′S,6R)-3-methyl-6-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPTP) and (3R,4′R)-3-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPEA), three novel 1,4-dioxane-2,5-dione-type monomers. Ring-opening homopolymerisation and copolymerisation of the IPTA monomer, derived from l-ascorbic acid, with d,l-lactide have been performed. The polymers were characterised by elemental microanalysis, as well as IR and ¹H and ¹³C NMR spectroscopies. GPC was used to estimate product molecular weights, and thermal studies (DSC and TGA) revealed that all the polymers were amorphous, being stable up to 250 °C under nitrogen.     

Identification of genes involved in inversion of stereochemistry of a C-12 hydroxyl group in the catabolism of cholic acid by Comamonas testosteroni TA441

Comamonas testosteroni TA441 degrades steroids such as testosterone via aromatization of the A ring, followed by meta-cleavage of the ring. In the DNA region upstream of the meta-cleavage enzyme gene tesB, two genes required during cholic acid degradation for the inversion of an α-oriented hydroxyl group on C-12 were identified. A dehydrogenase, SteA, converts 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α-hydroxyandrosta-1,4-diene-3,12,17-trione, and a hydrogenase, SteB, converts the latter to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione. Both enzymes are members of the short-chain dehydrogenase/reductase superfamily. The transformation of 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione is carried out far more effectively when both SteA and SteB are involved together. These two enzymes are encoded by two adjacent genes and are presumed to be expressed together. Inversion of the hydroxyl group at C-12 is indispensable for the subsequent effective B-ring cleavage of the androstane compound. In addition to the compounds already mentioned, 12α-hydroxyandrosta-1,4,6-triene-3,17-dione and 12β-hydroxyandrosta-1,4,6-triene-3,17-dione were identified as minor intermediate compounds in cholic acid degradation by C. testosteroni TA441.     

Physiological and Chemical Investigations into Microbial Degradation of Synthetic Poly(cis-1,4-isoprene)

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6,10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of β-oxidation, (iii) oxidation of the conjugated double bond resulting in a β-keto acid, and (iv) decarboxylation.     

Physiological and chemical investigations into microbial degradation of synthetic Poly(cis-1,4-isoprene)

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6, 10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of beta-oxidation, (iii) oxidation of the conjugated double bond resulting in a beta-keto acid, and (iv) decarboxylation.     

Systems Metabolic Engineering Strategies for Non‐Natural Microbial Polyester Production

Plastics, used everyday, are mostly synthetic polymers derived from fossil resources, and their accumulation is becoming a serious concern worldwide. Polyhydroxyalkanoates (PHAs) are naturally produced polyesters synthesized and intracellularly accumulated by many different microorganisms. PHAs are good alternatives to petroleum‐based plastics because they possess a wide range of material properties depending on monomer types and molecular weights. In addition, PHAs are biodegradable and can be produced from renewable biomass. Thus, producing PHAs through the development of high‐performance engineered microorganisms and efficient bioprocesses gained much interest. In addition, non‐natural polyesters comprising 2‐hydroxycarboxylic acids as monomers have been produced by fermentation of metabolically engineered bacteria. For example, poly(lactic acid) and poly(lactic acid‐co‐glycolic acid), which have been chemically synthesized using the corresponding monomers either fermentatively or chemically produced, can be produced by metabolically engineered bacteria by one‐step fermentation. Recently, PHAs containing aromatic monomers could be produced by fermentation of metabolically engineered bacteria. Here, metabolic engineering strategies applied in developing microbial strains capable of producing non‐natural polyesters in a stepwise manner are reviewed. It is hoped that the detailed strategies described will be helpful for designing metabolic engineering strategies for developing diverse microbial strains capable of producing various polymers that can replace petroleum‐derived polymers.     

Degradation of 1,4-dioxane and cyclic ethers by an isolated fungus

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d8 and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.     

1,4-diazepine-2,5-dione ring formation during solid phase synthesis of peptides containing aspartic acid beta-benzyl ester.

The Fmoc-based SPPS of H-Xaa-Asp(OBzl)-Yaa-Gly-NH(2) sequences results in side reactions yielding not only aspartimide peptides and piperidide derivatives, but also 1,4-diazepine-2,5-dione-peptides. Evidence is presented to show that the 1,4-diazepine-2,5-dione derivative is formed from the aspartimide peptide. The rate of this ring transformation depends primarily on the tendency to aspartimide and piperidide formation, which is influenced by the nature of the amino acid following the aspartic acid beta-benzyl ester (Xaa). However the bulkiness of the amino acid side chain preceeding the aspartic acid beta-benzyl ester (Yaa) is also important. Under certain conditions the 1,4-diazepine-2,5-dione peptide derivative may even be formed dominantly, which is a highly undesirable side reaction in peptide synthesis, but which provides a new way for the synthesis of diazepine peptide derivatives with targeted biological or pharmacological activity.     

Degradation of 1,4-Dioxane and Cyclic Ethers by an Isolated Fungus

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d₈ and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.     

Identification of 6-substituted 4-arylsulfonyl-1,4-diazepane-2,5-diones as a novel scaffold for human chymase inhibitors

A novel series of 6-substituted 4-sulfonyl-1,4-diazepane-2,5-diones were designed, synthesized and evaluated as human chymase inhibitors. Structure-activity relationship studies led to the identification of a potent inhibitor, (6S)-6-(5-chloro-2-methoxybenzyl)-4-[(4-chlorophenyl)sulfonyl]-1,4-diazepane-2,5-dione, with an IC(50) of 0.027 microM.     

Impact of lactic acid on cell proliferation and free radical‐induced cell death in monolayer cultures of neural precursor cells

Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic‐acid‐based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices. Biotechnol. Bioeng. 2009;103: 1214–1223. © 2009 Wiley Periodicals, Inc.     

Hydroxyl Radical Formation in Skeletal Muscle of Rats with Glucocorticoid-Induced Myopathy

Steroid myopathy is a well-known adverse effect of glucocorticoids that causes muscle weakness and atrophy; however, its pathogenic mechanism is still unclear. Recently, oxidative stress was reported to contribute to steroid myopathy, but there is no report that actually attempts to measure hydroxyl radical. I developed an animal model of steroid myopathy in rat with dexamethasone (9-Fluoro−11β,17, 21-trihydroxy−16α-methylpregna−1,4-diene−3,20-dione), and measured hydroxyl radical using the salicylate trapping method. There was significant dose-dependent relation between both 2,5- and 2,3-dihydroxybenzoic acids and dexamethasone in the treated group, compared to the control group. These results suggest that hydroxyl radical plays a role in the pathogenesis of steroid myopathy.     

Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli

The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.     

Biodegradation of aliphatic homopolyesters and aliphatic-aromatic copolyesters by anaerobic microorganisms

The anaerobic degradability of natural and synthetic polyesters is investigated applying microbial consortia (3 sludges, 1 sediment) as well as individual strains isolated for this purpose. In contrast to aerobic conditions, the natural homopolyester polyhydroxybutyrate (PHB) degrades faster than the copolyester poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV). For the synthetic polyester poly(epsilon-caroplacton) (PCL), microbial degradation in the absence of oxygen could be clearly demonstrated; however, the degradation rate is significantly lower than for PHB and PHBV. Other synthetic polyesters such as poly(trimethylene adipate) (SP3/6), poly(tetramethylene adipate) (SP4/6), and aliphatic-aromatic copolyesters from 1,4-butanediol, terephthalic acid, and adipic acid (BTA-copolymers) exhibit only very low anaerobic microbial susceptibility. A copolyester with high amount of terephthalic acid (BTA 40:60) resisted the anaerobic breakdown even under thermophilic conditions and/or when blended with starch. For the synthetic polymers, a number of individual anaerobic strain could be isolated which are able to depolymerize the polymers and selected strains where identified as new species of the genus Clostridium or Propionispora. Their distinguished degradation patterns point to the involvement of different degrading enzymes which are specialized to depolymerize either the natural polyhydroxyalkanoates (e.g., PHB), the synthetic polyester PCL, or other synthetic aliphatic polyesters such as SP3/6. It can be supposed that these enzymes exhibit comparable characteristics as those described to be responsible for aerobic polyester degradation (lipases, cutinases, and PHB-depolymerases).     

Selective synthesis of 3-hydroxy acids from Meldrum's acids using SmI2-H2O

The single-step synthesis of 3-hydroxy carboxylic acids from readily available Meldrum's acids involves a selective monoreduction using a SmI(2)-H(2)O complex to give products in high crude purity, and it represents a considerable advancement over other methods for the synthesis of 3-hydroxy acids. The protocol includes a detailed guide to the preparation of a single electron-reducing SmI(2)-H(2)O complex and describes two representative examples of the methodology: monoreduction of a fully saturated Meldrum's acid (5-(4-bromobenzyl)-2,2-dimethyl-1,3-dioxane-4,6-dione) and tandem conjugate reduction-selective monoreduction of α,β-unsaturated Meldrum's acid (5-(4-methoxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione). The protocol for selective monoreduction of Meldrum's acids takes ～6 h to complete.     

Screening of microorganisms for biodegradation of poly(lactic-acid) and lactic acid-containing polymers.

The ability of some microorganisms to use lactic acid stereocopolymers and copolymers with glycolic acid as sole carbon and energy sources was studied under controlled or natural conditions. First, 14 filamentous fungal strains were tested in liquid cultures, adopting total lactic acid consumption, nitrogen source exhaustion, and maximal biomass production as selection criteria. Two strains of Fusarium moniliforme and one strain of Penicillium roqueforti were able to totally assimilate DL-lactic acid, partially soluble racemic oligomers (MW = 1,000), and the nitrogen source. Only one strain of F. moniliforme was able to grow on a poly(lactic acid)-glycolic acid copolymer (MW = 150,000) after 2 months of incubation at 28 degrees C on synthetic agar medium. Mycelium development was examined by scanning electron microscopy. F. moniliforme filaments were observed to grow not only at the copolymer surface but also through the bulk of the copolymer. In a second approach, plates made of a racemic poly(lactic acid) were buried in the soil before being incubated in petri dishes containing mineral agar medium under controlled conditions. Five strains of different filamentous fungi were isolated, and their ability to assimilate racemic poly(lactic acid) oligomers was tested in liquid cultures.     

Synthesis and properties of malic acid-containing functional polymers.

Poly-L-lactides containing beta-alkyl alpha-malate-units were prepared by ring-opening copolymerizations of L-lactide with 3-(s)-[(benzyloxycarbonyl)methyl]- (BMD) and 3-(s)-[(dodecyloxycarbonyl)methyl]-1,4-dioxane-2,5-diones (DMD). The solution-cast films of these copolymers were alkali-treated to form a carboxyl-functionalized surface on which cell-binding Arg-Gly-Asp tripeptide (RGD) was immobilized with dicyclohexylcarbodiimide as coupling agent. For the copolymer of L-lactide and BMD the benzyl groups were removed by catalytic hydrogenolysis to obtain a fully carboxyl-functionalized copolymer (PLGM), and RGD was immobilized on the surface of its cast film. All the RGD-immobilized films thus prepared exhibited improved cell attachment compared with the original films. The cell attachment increased with increasing amount of immobilized RGD, which depended on the composition of the alpha-malate units in the copolymer. The RGD-immobilized PLGM films were degraded rapidly during the cell culture, while the RGD-immobilized films of the beta-alkyl alpha-malate-containing polymers survived the cell culture with little degradation. The rate of hydrolysis increased with increasing content of alpha-malate units for both series, depending on the structure of the protecting groups of the beta-carboxyl. These results suggest that the RGD-immobilized polymers could be a new class of functional bioresorbable polymer having improved cell-attachment and adjustable hydrolysis rate.     

Enantiospecific enzymatic preparation of (2S,3S)-3-alkylaspartic acids of current interest in the synthesis of stereoregular poly[β-(2S,3S)-3-alkylmalic acids] as new optically active functional polyesters

(2S,3S)-3-methyl- and 3-isopropylaspartic acids were synthesized by bioconversion of the corresponding alkylfumarates (mesaconate and 3-isopropylfumarate) using β-methylaspartase from cell-free extracts of Clostridium tetanomorphum. Optically pure (2S,3S)-3-alkylaspartic acids were transformed in several steps to benzyl (3S,4R)-3-alkylmalolactonates without any racemization of the two chiral centers. These optically active α,β-substituted-β-lactones were polymerized by anionic ring opening polymerization yielding optically active semi-crystalline polyesters. ¹³C NMR analysis of poly[benzyl β-3-isopropylmalate] in CDCl₃ has shown that only the iso-type stereosequence is present in the polymer, indicating that the macromolecular chain is constituted by the only units of benzyl β-(2S,3S)-3-isopropylmalate monomer. The polymerization reaction was done without any racemization of the two stereogenic centers as in the case of benzyl (3S,4R)-3-methylmalolactonate. © 1996 Wiley-Liss, Inc.     

The degradation of beta-sitosterol by Pseudomonas sp. NCIB 10590 under aerobic conditions

The bacterial degradation of beta-sitosterol by Pseudomonas sp NCIB 10590 has been studied. Major biotransformation products included 24-ethylcholest-4-en-3-one, androsta-1,4-diene-3,17-dione, 3-oxochol-4-en-3-one-24-oic acid and 3-oxopregn-4-en-3-one-20-carboxylic acid. Minor products identified were 26-hydroxy-24-ethylcholest-4-en-3-one, androst-4-ene-3,17-dione, 3-oxo-24-ethylcholest-4-en-26-oic acid, 3-oxochola-1,4-dien-3-one-24-oic acid, 3-oxopregna-1,4-dien-3-one-20 carboxylic acid and 9 alpha-hydroxyandrosta-1,4-diene-3,17-dione. Studies with selected inhibitors have enabled the elucidation of a comprehensive pathway of beta-sitosterol degradation by bacteria.