Protein immobilization on poly-(N-substituted) acrylamides

A series of water-soluble polymers containing side chains derived from N-acryloyl-β-alanine, N-ethylacrylamide and N-[3-(N′,N′,N′-trimethylammonio)propyl] acylamide chloride has been prepared and characterized. A related series of insoluble gels was also prepared. Protein may be attached to these materials by means of amide bond formation between carboxyl groups on the polymers and amino groups of the protein; the preparation and characterization of conjugates formed with α-chymotrypsin are described. Polymers bearing negatively or positively charged side chains are attached to this enzyme at only a single amino acid and the integrity of the active site is largely preserved in these systems. The corresponding gels are not able to bind as much enzyme as are the soluble polymers and bound enzyme is less active in these cases.     

Rapid Accumulation of Anionic Peroxidases and Phenolic Polymers in Soybean Cotyledon Tissues following Treatment with Phytophthora megasperma f. sp. Glycinea Wall Glucan

Phytophthora megasperma Drechs. f. sp. glycinea Kuan & Erwin (PMG) cell wall glucan has been extensively characterized as an elicitor of the pterocarpan phytoalexins, the glyceollins in soybean (Glycine max L.). Just recently, this glucan was shown to be a potent elicitor of conjugates of the isoflavones, daidzein and genistein as well. Here we report that PMG wall glucan also induces a rapid and massive accumulation of phenolic polymers in soybean cotyledon cells proximal to the point of elicitor application. Deposition of phenolic polymers is over then times that in wounded controls within just 4 hours of elicitor treatment and reaches a maximum by 24 hours. In the same tissues, isoflavone conjugates begin to accumulate at 8 hours and glyceollin at 12 hours. By 24 hours, the total deposition of wall bound phenolics in elicitor-treated tissues is several times greater than the peak glyceollin and isoflavone responses combined. Histochemical stains and quantitation of phenolic residues released after saponification and nitrobenzene or copper oxide oxidation suggest that the covalently linked phenolics include both lignin- and suberin-like polymers as well as simple esterified coumaric and ferulic acid monomers. Accumulations of phenolic polymers are accompanied by equally rapid and massive increases in activity of a specific group of anionic peroxidases. Although increases in peroxidase activity are not strictly limited to cells immediately adjacent to the area of elicitor treatment, the deposition of phenolic polymers is significantly less extensive in distal cells.     

Choline conjugates of auxins. I. Direct evidence for the hydrolysis of choline-auxin conjugates by pea cholinesterase

We synthesized the choline conjugates of three derivatives of phenylacetic acid, four derivatives of phenoxyacetic acid, and of naphthalene-1-acetic acid and measured the effects of these conjugates on pea (Pisum sativum L. cv. Alaska) stem segment elongation. We also synthesized the thioester analogs of six choline conjugates and measured their rates of hydrolysis by pea cholinesterase and their rates of spontaneous hydrolysis. With one exception, conjugates that stimulated growth were hydrolyzed at high rates by pea cholinesterase, while conjugates that were hydrolyzed at low rates by pea cholinesterase did not stimulate growth. The results are consistent with a model in which cholinesterase releases active auxins from these conjugates.     

Synthesis of amino acid conjugates to 2-imino-3-methylene-5-carboxypyrrolidine and 2-imino-3-methylene-6-carboxypiperidine

The four stereomers of 2-imino-3-methylene-5-l(carboxy-l-valyl)pyrrolidine, a bacterial metabolite that is inhibitory to the fire blight bacterium Erwinia amylovora, were synthesised and compared for antibacterial activity. Several alternative amino acid conjugates with l,l-stereochemistry were also prepared, and the synthesis was extended to 3-methylenepiperidine-6-l-carboxylic acid and a selection of 2-imino-3-methylenepiperidine-6-l-carboxy-l-amino acid conjugates. All synthetic amino acid conjugates (l,l-stereomers) were inhibitory to the growth of E. amylovora. The likely participation of the conjugated iminomethylene moiety as a Michael acceptor is implicated.     

Anhydride Prodrug of Ibuprofen and Acrylic Polymers

The objective of this study was to synthesize anhydride prodrugs for carboxylic-acid-bearing agents such as non-steroidal anti-inflammatory drugs, shield the carboxylic acid group from irritative effects, and obtain sustained release patterns. Ibuprofen was used as a representative drug for anhydride derivatization. Conjugates of ibuprofen with carboxylic acid moieties of different acrylic polymers were prepared by dehydration reaction using acetic anhydride. Products were characterized by infrared spectroscopy, nuclear magnetic resonance, and scanning electron microscopy followed by preparation of microspheres with different sizes from the conjugate Eudragit® L-100-ibuprofen. The drug release was monitored by high-performance liquid chromatography. Ibuprofen was bound to the polymers via an anhydride bond in high reaction yields (75–95%) with drug loading of up to 30% (w/w). These anhydride derivatives hydrolyzed and release the drug at different periods ranging from 1 to 5 days, depending on the hydrophobicity and the cross-linking of the conjugates. The release of drug from the microspheres was correlated to their size and ranged from 2 to almost 8 days. This study demonstrates the promise of anhydride prodrug for extending drug action while shielding the carboxylic acid group.     

Fabrication of Surface-Active Antioxidant Food Biopolymers: Conjugation of Catechin Polymers to Egg White Proteins

The oxidation of unsaturated lipids commonly occurs at oil-water interfaces in heterogeneous foods (such as emulsions), and so there is a need for surface-active antioxidants to inhibit lipid oxidation. In this study, catechin was oxidatively polymerized by horseradish peroxidase to obtain catechin polymers (CTP). An amphiphilic antioxidant was then produced by conjugating CTP with egg white proteins (EWP) using a hydrogen peroxide-ascorbic acid pair as a radical initiator system. The covalent attachment of CTP to EWP was confirmed by electrophoresis, liquid chromatography-mass spectrometry, and fluorescence analyses. The antioxidant capacity of CTP-EWP conjugates was evaluated using several in vitro models. The conjugates exhibited strong scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (79% at 1 mg/mL), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (97% at 0.5 mg/mL), and had a high ferric reducing power. Furthermore, CTP-EWP conjugates exhibited an inhibitive effect on lipid peroxidation in linoleic acid oil-in-water emulsions, indicating that the conjugates may have potential applications in food, pharmaceutical and cosmetic industries.     

Stimuli-responsive materials prepared from carboxymethyl chitosan and poly(γ-glutamic acid) for protein delivery

The development of stimuli-responsive materials in response to the molecules involved in biological processes has gained increased attentions. In this work, carboxymethyl chitosan (CM-chitosan) and poly(γ-glutamic acid) (pGlu) were reacted with a naturally occurring compound, genipin, leading to the formation of genipin-crosslinked CM-chitosan/pGlu conjugates with fluorescence emissions. The genipin-conjugated polymers were sensitive to the oxidation product of glucose, gluconic acid and hydrogen peroxide (H₂O₂). Fluorescence emissions of the polymers were quenched by gluconic acid and H₂O₂. An increase in the hydrodynamic diameter together with the quenching of fluorescence indicated that the genipin-conjugated polymers were self-aggregated into nanoparticles, in response to the stimulus of gluconic acid (but not for H₂O₂). Bovine serum albumin (BSA) could be loaded in the self-aggregated nanoparticles, and the incorporated BSA slowly released from the nanoparticles under hyper-gluconic acid conditions. This material is hence proposed as a stimuli-responsive material for optical sensing and protein delivery purposes.     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-²H₄-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS². These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.     

Design,characterization, and in vitro antiproliferative efficacy of gemcitabine conjugates based on carboxymethyl glucan

Gemcitabine (GEM) is widely used in clinical practice in the treatment of cancer and several other solid tumors. Nevertheless, the antitumor effect of GEM is partially prevented by some limitations including short half life, and lack of tumor localizing. Carboxymethyl glucan (CMG), a carboxymethylated derivative of β-(1-3)-glucan, shows biocompatibility and biodegradability as well as a potential anticarcinogenic effect. To enhance the antiproliferative activity of GEM, four water soluble conjugates of GEM bound to CMG via diverse amino acid linkers were designed and synthesized. ¹H NMR, FT IR, elementary analysis and RP-HPLC chromatography were employed to verify the correct achievement of the conjugates. In vitro release study indicated that conjugates presented slower release in physiological buffer (pH 7.4) than acidic buffer (pH 5.5) mimicking the acidic tumor microenvironment. Moreover, A549, HeLa and Caco-2 cancer cell lines were used to evaluate the in vitro cytotoxicity of conjugates and the results showed that binding GEM to CMG significantly enhanced antiproliferative activity of GEM on A549 cells. Therefore, these conjugates may be potentially useful as a delivery vehicle in cancer therapy and worthy of further study on structure-activity relationship and antiproliferative activity in vitro and in vivo, especially for lung tumor.     

Metabolism of Auxin in Tomato Fruit Tissue: Formation of High Molecular Weight Conjugates of Oxindole-3-Acetic Acid via the Oxidation of Indole-3-Acetylaspartic Acid

High performance liquid chromatography of extracts of tomato (Lycopersicon esculentum Mill.) incubated with a relatively low concentration (4 μm) of [1-¹⁴C]indole-3-acetic acid (IAA) revealed the presence of two major polar metabolites. Hydrolysis of the two metabolites with 7 n NaOH yielded the same compound, which had a retention time similar to that of ring-expanded oxindole-3-acetic acid (OxIAA) on high performance liquid chromatography. The identity of the indolic moiety of these conjugates as OxIAA was further confirmed by gas chromatography-mass spectrometry. Chromatography of the two OxIAA conjugates on a calibrated Bio-Gel P-2 column indicated that their molecular weights are about 1200 and 1000. Aspartic acid and glutamic acid were the major amino acids detected in acid hydrolysates of the two conjugates. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of indole-3-acetylaspartic acid (IAAsp) with a concomitant decrease in the formation of the two OxIAA conjugates. Feeding experiments with labeled IAAsp and OxIAA showed that IAAsp and not OxIAA is the precursor of these conjugates. The data obtained indicate that exogenous IAA is converted in tomato pericarp tissue to high molecular weight conjugates, presumably peptides, of OxIAA via the oxidation of IAAsp. The oxidation of IAAsp seems to be a rate-limiting step in the formation of these conjugates from exogenous IAA.     

Determination of abscisic acid in Eucalyptus haemastoma leaves using gas chromatography/mass spectrometry and deuterated internal standards

Abscisic acid and 2-trans-abscisic acid each with three deuterium atoms in the C-3 methyl group, have been synthesized chemically and used as internal standards in selected ion monitoring experiments to establish the endogeneous concentrations of these compounds and their conjugates in turgid and wilted Eucalyptus haemastoma leaves. The analytical procedure used GC/CIMS(methane) to detect the methyl esters of abscisic acid, 2-trans-abscisic acid and their deuterated internal standards. A three-fold increase in the concentration of abscisic acid occurred on wilting and the amounts of 2-trans-abscisic acid and conjugates of both compounds were determined.     

Biohybrid Polymer-Antimicrobial Peptide Medium against Enterococcus faecalis

Antimicrobial peptides (AMPs) are conserved evolutionary components of the innate immune system that are being tested as alternatives to antibiotics. Slow release of AMPs using biodegradable polymers can be advantageous in maintaining high peptide levels for topical treatment, especially in the oral environment in which dosage retention is challenged by drug dilution with saliva flow and by drug inactivation by salivary enzymatic activity. Enterococcus faecalis is a multidrug resistant nosocomial pathogen and a persistent pathogen in root canal infections. In this study, four ultra-short lipopeptides (C16-KGGK, C16-KLLK, C16-KAAK and C16-KKK) and an amphipathic α-helical antimicrobial peptide (Amp-1D) were tested against E. faecalis. The antibacterial effect was determined against planktonic bacteria and bacteria grown in biofilm. Of the five tested AMPs, C16-KGGK was the most effective. Next C16-KGGK was formulated with one of two polymers poly (lactic acid co castor oil) (DLLA) or ricinoleic acid-based poly (ester-anhydride) P(SA-RA). Peptide-synthetic polymer conjugates, also referred to as biohybrid mediums were tested for antibacterial activity against E. faecalis grown in suspension and in biofilms. The new formulations exhibited strong and improved anti- E. faecalis activity.     

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: From chemical characterization to targeted analysis of phenolic metabolites in humans

In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism.     

Metabolism of Indole-3-Acetic Acid: III. Identification of Metabolites Isolated from Crown Gall Callus Tissue

The metabolism of labeled indole-3-acetic acid (IAA-2-¹⁴C) was investigated in Parthenocissus tricuspidata crown gall callus tissue. After 48 hours incubation, 85 to 90% of the supplied IAA was taken up by the tissue, and of that taken up, about 45% was conjugated with five amino acids. The conjugates found were aspartic and glutamic acid (minor ones) as well as glycine, alanine, and valine (major ones). The last four are being reported for the first time as metabolites of IAA. These conjugates were identified through their chromatographic properties, hydrolysis products, and their mass spectra. The possible significance of these amino acid conjugates is discussed.     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

Determination of alkylresorcinol metabolites in human urine by gas chromatography–mass spectrometry

Alkylresorcinols (ARs) are phenolic lipids present at high concentrations in the outer parts of rye and wheat kernels and have been proposed as biomarkers for intake of whole grain and bran products of these cereals. AR are absorbed in the small intestine and after hepatic metabolism two major metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), are excreted in urine either as such or as conjugates. Urine samples from nine individuals were incubated with different enzymes to assess type and extent of conjugates. In comparison with DHBA, which was mostly found in the free form, the less polar DHPPA was conjugated to a greater extent and the major conjugates were glucuronides. In this method, urine samples were hydrolyzed using β-glucuronidase from Helix pomatia and syringic acid was used as internal standard. Samples, silylated with BSTFA, were analyzed by GC–MS utilizing a BP-5 fused silica capillary column and single ion monitoring of molecular ions (m/z 370 [DHBA], m/z 398 [DHPPA]). Recoveries of DHBA and DHPPA were estimated to be 94% and 93%, respectively. The average intra-assay/inter-assay coefficients of variation were 4.9/5.7% for DHBA and 7.6/9.3% for DHPPA.     

Energy-dependent export of the 13-oxooctadecadienoic acid–glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, β,γ-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K_m of 325.5 μM and a V_max of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K_m of 169 μM and a V_max of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.     

In vitro and in vivo conjugation of dietary hydroxycinnamic acids by UDP-glucuronosyltransferases and sulfotransferases in humans

Hydroxycinnamic acids are a class of phenolic antioxidants found widely in dietary plants. Their biotransformation in the human organism primarily involves Phase II conjugation reactions. In this study, activities of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) towards major dietary hydroxycinnamic acids (caffeic, dihydrocaffeic, dihydroferulic, ferulic and isoferulic acids) were investigated. Conjugate formation was evaluated using human liver and intestinal S9 homogenates, and in vitro characterization was carried out using recombinant human UGTs and SULTs. Analysis of the kinetics of hydroxycinnamic acid conjugation in human S9 homogenates revealed that intrinsic clearance (V_max/K_m) is much greater for sulfation than for glucuronidation. Assessment of activity using a panel of recombinant human SULTs showed that SULT1A1 is most active in the sulfation of caffeic, dihydrocaffeic and isoferulic acids, while SULT1E1 is most active in the sulfation of ferulic and dihydroferulic acids. Only isoferulic acid was significantly glucuronidated by human liver S9 homogenates, explained by the high activity of liver-specific UGT1A9. Studies on the kinetics of active SULTs and UGTs demonstrated a markedly lower K_m for SULTs. To further corroborate our findings, we carried out an intervention study in healthy humans to determine the hydroxycinnamic acid conjugates in urine after consumption of hydroxycinnamate-rich coffee (200 ml). Analysis showed that sulfates are the main conjugates in urine, with the exception of isoferulic acid, which is mainly glucuronidated. These data suggest that sulfates are the predominant hydroxycinnamic acid conjugates in humans, and that SULT mediated sulfation is a major factor determining the bioavailability of hydroxycinnamic acids in vivo.     

Quinone-Amino Acid Conjugates Targeting Leishmania Amino Acid Transporters

The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1–15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.