Biosynthesis of Novel Aromatic Copolyesters from Insoluble 11-Phenoxyundecanoic Acid by Pseudomonas putida BM01

Two types of novel aromatic copolyesters were synthesized from 11-phenoxyundecanoic acid (11-POU) as the sole carbon source and the cosubstrates 11-POU and octanoate, respectively, by isolated Pseudomonas putida BM01 that is known to accumulate high concentrations of medium-chain-length polyesters. Insoluble 11-POU was recrystallized in situ in buffer by alkaline treatment and pH adjustment, followed by autoclaving. The resulting microcrystals, whose structure was different from that of the commercially available crystalline powder, suspended in media were rapidly consumed by the bacterium. Synthesized polymers were characterized by gas chromatography, nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. The aromatic copolyesters synthesized from 11-POU were composed of two monomer units consisting of 3-hydroxy-5-phenoxyvalerate (5POHV) as the major component (72 to 85 mol%) and 3-hydroxy-7-phenoxyheptanoate (7POHH) as the minor component (15 to 28 mol%). The aromatic copolyesters showed a crystalline melting transition at 70(deg)C. When the bacterium was grown on the cosubstrates 11-POU and octanoate, the bacterium synthesized the copolyesters composed of aromatic and aliphatic monomers poly(5POHV-co-7POHH-co-3-hydroxy-9-phenoxynonanoate-co-3-hydroxyalkanoates) . The addition of octanoate in the feed shifted the major monomer unit in the polymer from 5POHV to 7POHH. A further-fragmented metabolite, 3-phenoxypropionate, whose concentration reached a steady state at the time of greatest polyester accumulation, was detected in the medium. The metabolic pathway of 11-POU is suggested.     

Novel thermosensitive 5-fluorouracil-cyclotriphosphazene conjugates: synthesis, thermosensitivity, degradability, and in vitro antitumor activity

A novel thermosensitive macromolecular prodrug of 5-fluorouracil (5-FU) was synthesized using cyclotriphosphazene, and its thermosensitivity, degradability, and in vitro antitumor activity were studied. A series of alpha-substituted glycine derivatives of 5-FU containing carboxylic groups were prepared, and cyclotriphosphazenes with amino groups were synthesized via the stepwise substitution of hexachlorocyclotriphosphazene (NPCl(2))(3) with methoxy-poly(ethylene glycol) (MPEG) or alkoxy ethylene oxide and lysine ethyl ester (LysOEt). The coupling reaction of the two derivatives, and their subsequent deprotection, yielded a thermosenstive 5-FU-cyclotriphosphazene conjugate, which exhibited a unique octopus-shaped molecular structure, in which the three hydrophilic PEG groups (or alkoxy ethylene oxides) were oriented in one direction, opposing the other three hydrophobic groups containing 5-FU, with respect to the trimer ring plane. This conjugate exhibited a reversible and thermosensitive phase transition in an aqueous medium, from soluble to insoluble states. The lower critical solution temperature (LCST) of the conjugate was controlled by substitution with different hydrophilic/hydrophobic side groups, and a few of the conjugates displayed LCSTs which were just below body temperature. This, of course, implies possible applications for local drug delivery by direct intratumoral injection. The conjugate exhibited gradual degradation at 37 degrees C in both neutral and acidic buffer solutions, and high temperature significantly facilitated its hydrolytic degradation. All of the conjugates displayed dose-dependent cytotoxicity against the leukemia L1210 cell line and exhibited more pronounced cytotoxic effects than did 5-FU.     

Biodegradation of polyesters containing aromatic constituents

Polymers, which undergo a controlled biological degradation by micro-organisms came to remarkable interest during the last years. Composting for instance could so be established as an alternative waste management system for parts of the plastic waste. Within this group of innovative polymer, polyesters play a predominant role, due to their potentially hydrolyzable ester bonds. While aromatic polyesters such as poly(ethylene terephthalate) exhibit excellent material properties but proved to be almost resistant to microbial attack, many aliphatic polyesters turned out to be biodegradable but lack in properties, which are important for application. To combine good material properties with biodegradability, aliphatic-aromatic copolyesters have been developed as biodegradable polymers for many years. This article reviews the attempts to combine aromatic and aliphatic structures in biodegradable plastics and work, which has been done to evaluate the degradation behaviour and environmental safety of biodegradable polyesters, containing aromatic constituents.     

Thermodynamics of interactions between amino acid side chains: experimental differentiation of aromatic-aromatic, aromatic-aliphatic, and aliphatic-aliphatic side-chain interactions in water

A stationary phase for high-pressure liquid chromatography has been prepared by derivatizing microparticulate silica gel with functionality mimicking the side chain of isoleucine. The chromatographic retentions of a series of hydrophobic and amphiphilic amino acid analytes on this stationary phase (Ile MSP) using an aqueous mobile phase were measured as a function of temperature from 273 K to 323 K. Observed temperature dependencies are consistent with a constant change in heat capacity, DeltaC degrees P, upon binding of the analyte to the stationary phase. The curvatures of plots of retention data versus temperature (related to the magnitude of DeltaC degrees P) are distinctly different for retention of aromatic and aliphatic analytes, with retention of aliphatic analytes Val, Ile, and Leu exhibiting the characteristic signature of the hydrophobic effect, i.e., a large negative DeltaC degrees P upon desolvation from water and a maximum of retention around room temperature. Retention of aromatic analytes (Trp, Phe, and Tyr) involves smaller heat capacity changes and pronounced negative enthalpies of interaction with the stationary phase. Estimates of DeltaC degrees P for the interactions of analyte side chains with the Ile side chain were obtained by fitting the temperature dependence of retention to an expression derived from thermodynamic considerations and chromatographic theory. Similar estimates were made for interactions with the Phe side chain, using previously published data for a phenylalanine mimic stationary phase (Phe MSP) (. Protein Sci. 1:786-795). As with the Ile MSP, the retentions of aliphatic analytes show temperature dependencies markedly different from those of aromatic analytes. Data from both phases indicate that a realistic differentiation can be made between the interactions of various types of amino acid side chains tested (i.e., aliphatic/aliphatic, aliphatic/aromatic, and aromatic/aromatic) by comparison of the corresponding thermodynamic functions for pairwise interactions. The retention of leucine on the Phe MSP and that of phenylalanine on the Ile MSP showed similar DeltaC degrees P values, suggesting that the aromatic-aliphatic interaction is reasonably independent of the residue attached to the stationary phase. This result is consistent with a one-to-one interaction and suggests a simple way to estimate the column-dependent phase factor, making it possible to compare entropies and free energies of interaction obtained using different MSPs. The possibilities for using MSP-derived interaction potentials in folding simulations are discussed.     

Exploring mild enzymatic sustainable routes for the synthesis of bio‐degradable aromatic‐aliphatic oligoesters

The application of Candida antarctica lipase B in enzyme‐catalyzed synthesis of aromatic‐aliphatic oligoesters is here reported. The aim of the present study is to systematically investigate the most favorable conditions for the enzyme catalyzed synthesis of aromatic‐aliphatic oligomers using commercially available monomers. Reaction conditions and enzyme selectivity for polymerization of various commercially available monomers were considered using different inactivated/activated aromatic monomers combined with linear polyols ranging from C₂ to C₁₂. The effect of various reaction solvents in enzymatic polymerization was assessed and toluene allowed to achieve the highest conversions for the reaction of dimethyl isophthalate with 1,4‐butanediol and with 1,10‐decanediol (88 and 87% monomer conversion respectively). M_w as high as 1512 Da was obtained from the reaction of dimethyl isophthalate with 1,10‐decanediol. The obtained oligomers have potential applications as raw materials in personal and home care formulations, for the production of aliphatic‐aromatic block co‐polymers or can be further functionalized with various moieties for a subsequent photo‐ or radical polymerization.     

Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

Effects of urea and guanidine hydrochloride on peptide and nonpolar groups

The free energy transfer of several N-acetyl(glycine)n ethyl esters (n = 1-3) and side chain derivatives (Ala, Val, Nva, Leu, Nle, and Phe) from water to urea and guanidine hydrochloride solutions has been determined from the solubility and distribution coefficients of these compounds between aqueous and nonaqueous phases. These uncharged model peptides, unlike the amino acids used for a similar study, avoid complication due to charge effects for the transfer process. The compounds with an increase in the number of glycyl groups show additivity of the group free energy toward the transfer from water to urea solution but not to guanidine hydrochloride solution. The derivatives with a side chain show that the principle of group additivity does not hold true for the aliphatic side chains for the transfer to either urea or guanidine hydrochloride solutions. In fact, the free energy of transfer of the side chains, viz., aliphatic ones, is found to be energetically unfavorable in moderately high denaturant concentration. Phenylalanyl, the only aromatic side chain studied here, showed a favorable free energy of transfer to the denaturant solutions. In addition, the values of the favorable free energy obtained in this study are much smaller than the values obtained from the study of the amino acids. The transfer of the glycyl group to the denaturant solutions is exothermic whereas the transfer of the side chains is endothermic in nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bromine K-edge EXAFS studies of bromide binding to bromoperoxidase from Ascophyllum nodosum.

Bromine K-edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris-buffered) bromide and vanadium model compounds containing Br-V, Br-C(aliphatic) and Br-C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine.     

Design,synthesis, and properties of new biodegradable aromatic/aliphatic liquid crystalline copolyesters

Liquid crystalline copolyesters of high molecular weight were obtained by polycondensation of aromatic diols, diacyl dichlorides, oligolactides, and poly(ethylene glycol)s. Hydrophilicity of the copolyesters was controlled by the content of ethyleneoxy moieties as verified by contact angle measurements. Copolyesters with ethyleneoxy moieties showed significant enhancement of degradability under physiological conditions in comparison to copolyester without ethyleneoxy moieties, which makes these copolyesters promising materials for bone tissue engineering as also verified by hardness testing and mechanical testing.     

Design and synthesis of self-degradable antibacterial polymers by simultaneous chain- and step-growth radical copolymerization

Self-degradable antimicrobial copolymers bearing cationic side chains and main-chain ester linkages were synthesized using the simultaneous chain- and step-growth radical polymerization of t-butyl acrylate and 3-butenyl 2-chloropropionate, followed by the transformation of t-butyl groups into primary ammonium salts. We prepared a series of copolymers with different structural features in terms of molecular weight, monomer composition, amine functionality, and side chain structures to examine the effect of polymer properties on their antimicrobial and hemolytic activities. The acrylate copolymers containing primary amine side chains displayed moderate antimicrobial activity against E. coli but were relatively hemolytic. The acrylate copolymer with quaternary ammonium groups and the acrylamide copolymers showed low or no antimicrobial and hemolytic activities. An acrylate copolymer with primary amine side chains degraded to lower molecular weight oligomers with lower antimicrobial activity in aqueous solution. This degradation was due to amidation of the ester groups of the polymer chains by the nucleophilic addition of primary amine groups in the side chains resulting in cleavage of the polymer main chain. The degradation mechanism was studied in detail by model reactions between amine compounds and precursor copolymers.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

平菇(Pleurotus ostreatus)对稻草中木质素的生物降解及降解产物分析

变色圈试验证明平菇可以选择性优先降解稻草中的木质素,培养15d后,平菇对稻草中的木质素降解率为17.86%,对综纤维素降解率为2.44%,选择性指数为9.79。生料栽培平菇后,稻草中的木质素被降解50.24%。用气—质色谱(GC/MS)和红外光谱(IR)对木质素降解产物分析结果表明,平菇对稻草中木质素的降解效果十分明显,降解产物中检测出了大量含有苯环的小分子,证明木质素聚合体的降解首先发生在单体的侧链及单体间的连键上,发生Cα-Cβ、β-O-4等断裂,形成了单体。在进一步的降解过程中,平菇表现了其自身特有的降解机制,取代苯环单体上的甲氧基为甲基,而后发生苯环的开裂,这与报道的白腐菌降解过程有所不同。红外光谱分析中,平菇对木质素的降解明显,降解产物中含有很多木质素单体所特有的基团,如紫丁香基、愈创木基等,说明木质素的降解首先发生的是侧链的氧化反应。     

Polymers of malic acid and 3-alkylmalic acid as synthetic PHAs in the design of biocompatible hydrolyzable devices.

Poly(beta-malic acid) and poly(beta-3-alkylmalic acid) derivatives, as synthetic polyhydroxyalkanoates (PHAs), present several advantages as macromolecular materials for temporary biomedical applications. Indeed, such polymers, which can be synthesized through different chemical and biological routes, have cleavable ester bonds in their backbone for hydrolytic degradation, stereogenic centres in the monomers units for controlling the macromolecular structure. bioassimilable or non-toxic repeating units and lateral chemical functions which can be adapted to specific requirements. The strategy for building such complex architectures, with one or several specific pendant groups, is based on the anionic ring-opening polymerization or copolymerization of the large family of malolactonic and 3-alkylmalolactonic acid esters. Because we are able to control the monomer synthesis and the polymerization step, we have been able to prepare different degradable materials for the biomedical field, such as: degradable associating networks made up by the association of random copolyesters containing a small percentage of hydrophobic moieties and beta-cyclodextrin copolymers; degradable macromolecular micelles constituted by degradable amphiphilic block copolymers of poly(beta-malic acid) as hydrophilic segments and poly(beta-alkylmalic acid alkyl esters) as hydrophobic blocks; and degradable nanoparticles made up by hydrophobic poly(beta-malic acid alkyl esters) derivatives. We have also prepared a terpolymer which exhibits growth factor-like properties in vivo. Finally, poly(beta-malic acid) has been used as an additive in the preparation of peritoneal dialysis bags.     

Synthesis and characterization of biodegradable hyperbranched poly(ester-amide)s based on natural material

A series of novel AB3-type monomers were prepared from nontoxic natural gallic acid and amino acids. These monomers were then melt-polycondensed in the presence of MgO as a catalyst via a transesterification process at 170-190 degrees C to yield the hyperbranched poly(ester-amide)s bearing terminal acetyl groups. FTIR and NMR spectra confirmed the structures of all the monomers and polymers. The degrees of branching, estimated from 1H NMR and quantitative 13C NMR spectra, were 0.50-0.68. These hyperbranched polymers displayed moderately high molecular weights. Hydrolytic and enzymatic degradation studies were carried out in vitro at 37.5 degrees C in NaOH hydrotropic solution and in Tris-HCl buffer (pH = 8.6) containing proteinase K, respectively. The results indicate that the hyperbranched poly(ester-amide)s are degradable hydrolytically as well as enzymatically, and the rate of hydrolytic degradation increases with the pH value of the solution.     

A well-defined amphipathic conformation for the calcium-free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution

Daptomycin is a 13-residue cyclic lipopeptide with Ca2+-dependent bactericidal activity against a variety of high-risk pathogens. Ring closure in daptomycin is via an ester linkage between the side chain of Thr4 and the C-terminal carboxyl of the main chain; the N-terminal residue is capped by a decanoyl aliphatic chain. Extensive NMR data obtained under solution conditions that minimize aggregation have provided constraints for a detailed conformational analysis of daptomycin in aqueous solution, which should facilitate the rational design of improved analogs and enhance understanding of its mode of action. Transannular and shorter-range nuclear Overhauser effects (NOEs) as well as amide temperature shifts and 3J(NH alpha) coupling constants indicate that daptomycin adopts a well-defined conformation containing a distorted hairpin formed by Gly5-D-Ala6 type II' beta-turn. A number of hydrophobic moieties (the lipid N-cap and the Trp1 and Kyn13 side chains) are clustered at one end of the hairpin, while neutral polar and anionic residues are localized on the other end, leading to amphipathicity in the molecule. These features suggest a mode of action in which the large hydrophobic cluster of the peptide interacts with the acyl chain region of a membrane. This interaction may be facilitated by Ca2+ ions, both by neutralizing the anionic charges and by favoring association with the membrane head groups. Interestingly, our findings differ from two recent articles in which the aqueous conformation of Ca2+-free daptomycin is reported to lack a well-defined conformation (D. Jung, A. Rozek, M. Okron, and R. E. W. Hancock, Chemistry & Biology, 2004, Vol. 11, pp. 949-957) or is suggested to populate an alternate conformation (L.-J. Ball, C. M. Goult, J. A. Donarski, J. Micklefield, and V. Ramesh, Organic & Biomolecular Chemistry, 2004, Vol. 2, pp. 1872-1878).     

Protease-catalyzed incorporation of non-protein amino acids into peptides via the kinetically controlled approach

Through the kinetically controlled approach, employing the methyl ester as an acyl donor, non-protein aliphatic amino acids were incorporated into peptides using papain in an aqueous buffer, while the incorporation of non-protein aromatic amino acids was achieved by exploiting -chymotrypsin in an aqueous buffer or acetonitrile with low water content. © Rapid Science Ltd. 1998     

Hydrocarbon emulsification and enhanced crude oil degradation by lauroyl glucose ester

Enzymatically synthesized lauroyl glucose emulsified different hydrophobic substrates when assayed spectrophotometrically. Stable emulsions were formed with triglycerides as well as with hydrocarbons. There was a linear relation between the concentration of lauroyl glucose (50-450 microg) and emulsification activity under the assay conditions when tested with aromatic and aliphatic hydrocarbons. This sugar ester was able to emulsify the aromatic hydrocarbons benzene, toluene and xylene. Long chain alkanes (n-decane and n-hexadecane) as well as brominated long chain alkanes (1-bromodecane and 1-bromohexadecane) were efficiently emulsified. The effect of lauroyl glucose ester on degradation of crude oil by a known oil-degrading Rhodococcus species was also investigated. The culture showed enhanced degradation of crude oil when lauroyl glucose ester was used as an emulsifier. It degraded 70% of the aliphatic fraction of Bombay High crude oil in the presence of the sugar ester at a concentration of 200mg l(-1) as compared to 50% without the emulsifier.     

Surface hydrolysis of polyacrylonitrile with nitrile hydrolysing enzymes from Micrococcus luteus BST20

A new Micrococcus luteus strain BST20 was isolated with ability to metabolize PAN polymers as sole carbon source. Out of seven synthetized PAN copolymers containing different moieties of acrylic acid and/or vinyl acetate the polymer with lowest crystallinity (PAN with 5% vinyl acetate) was most easily metabolized. (13)C labelled PAN was completely converted to the acrylic acid by this strain. M. luteus BST20 produced membrane-bound nitrile hydrolysing enzymes able to convert nitrile groups on PAN powder surface to the corresponding acids. Similarly, nitrile groups on PAN fabrics were transformed to the corresponding acid as indicated by an K/S increased after dying with Methylene blue and the released ammonia. On small soluble substrates the enzyme system showed a preference for aliphatic and aromatic substituted aliphatic nitriles.     

Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity.

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.     

The chemical composition of suberin in apoplastic barriers affects radial hydraulic conductivity differently in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays L. cv. Helix)

Apoplastic transport barriers in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays L. cv. Helix) were isolated enzymatically. Following chemical degradation (monomerization, derivatization), the amounts of aliphatic and aromatic suberin monomers were analysed quantitatively by gas chromatography and mass spectrometry. In corn, suberin was determined for isolated endodermal (ECW) and rhizo-hypodermal (RHCW) cell walls. In rice, the strong lignification of the central cylinder (CC), did not allow the isolation of endodermal cell walls. Similarly, exodermal walls could not be separated from the rhizodermal and sclerenchyma cell layers. Suberin analyses of ECW and RHCW of rice, thus, refer to either the entire CC or to the entire outer part of the root (OPR), the latter lacking the inner cortical cell layer. In both species, aromatic suberin was mainly composed of coumaric and ferulic acids. Aliphatic suberin monomers released from rice and corn belonged to five substance classes: primary fatty acids, primary alcohols, diacids, omega-hydroxy fatty acids, and 2-hydroxy fatty acids, with omega-hydroxy fatty acids being the most prominent substance class. Qualitative composition of aliphatic suberin of rice was different from that of corn; (i) it was much less diverse, and (ii) besides monomers with chain lengths of C(16), a second maximum of C(28) was evident. In corn, C(24) monomers represented the most prominent class of chain lengths. When suberin quantities were related to surface areas of the respective tissues of interest (hypodermis and/or exodermis and endodermis), exodermal cell walls of rice contained, on average, six-times more aliphatic suberin than those of corn. In endodermal cell walls, amounts were 34 times greater in rice than in corn. Significantly higher amounts of suberin detected in the apoplastic barriers of rice corresponded with a substantially lower root hydraulic conductivity (Lp(r)) compared with corn, when water flow was driven by hydrostatic pressure gradients across the apoplast. As the OPR of rice is highly porous and permeable to water, it is argued that this holds true only for the endodermis. The results imply that some caution is required when discussing the role of suberin in terms of an efficient transport barrier for water. The simple view that only the quantity of suberin present is important, may not hold. A more detailed consideration of both the chemical nature of suberins and of the microstructure of deposits is required, i.e. how suberins impregnate wall pores.