Hydroxyl Derivatives Research Articles

In vitro characterization of Synechocystis CYP120A1 revealed the first nonanimal retinoic acid hydroxylase

Retinoids are C(20) apocarotenoids that have various important functions in metazoans. In addition, several findings suggest their occurrence in eubacteria, including cyanobacteria. It has been shown that the Synechocystis cytochrome P450 enzyme CYP120A1 is a retinoic acid-binding polypeptide. In this work, we determined the reaction catalyzed by CYP120A1 and investigated its substrate specificity in vitro. CYP120A1-containing microsomes generated in yeast converted all-trans-retinoic acid into a compound exhibiting higher polarity in HPLC analysis. Liquid chromatography-MS analysis suggested the introduction of a single hydroxyl group, and NMR analysis of the purified product revealed C16 or C17 as the reaction site. Incubations with cis-retinoic acids, retinal, 3(R)-OH-retinal, retinol, beta-apo-13-carotenone (C(18)) and beta-apo-14'-carotenal (C(22)) resulted in the formation of the corresponding hydroxyl derivatives, as suggested by HPLC and liquid chromatography-MS analyses. Comparisons of the relative product amounts revealed the highest conversion rate for all-trans-retinoic acid, followed by beta-apo-13-carotenone (C(18)). As shown by real-time RT-PCR, CYP120A1 is expressed under normal growth conditions and is slightly induced by high-intensity light. Our work provides the first enzymatic study of a cyanobacterial cytochrome P450, showing it to be the first nonanimal retinoic acid-metabolizing enzyme characterized so far. Moreover, the CYP120A1-catalyzed reaction represents a novel modification of retinoids.

Unique Carotenoids in the Terrestrial Cyanobacterium Nostoc commune NIES-24: 2-Hydroxymyxol 2′-Fucoside, Nostoxanthin and Canthaxanthin

Cyanobacteria produce some carotenoids. We identified the molecular structures, including the stereochemistry, of all the carotenoids in the terrestrial cyanobacterium, Nostoc commune NIES-24 (IAM M-13). The major carotenoid was beta-carotene. Its hydroxyl derivatives were (3R)-beta-cryptoxanthin, (3R,3'R)-zeaxanthin, (2R,3R,3'R)-caloxanthin, and (2R,3R,2'R,3'R)-nostoxanthin, and its keto derivatives were echinenone and canthaxanthin. The unique myxol glycosides were (3R,2'S)-myxol 2'-fucoside and (2R,3R,2'S)-2-hydroxymyxol 2'-fucoside. This is only the second species found to contain 2-hydroxymyxol. We propose possible carotenogenesis pathways based on our identification of the carotenoids: the hydroxyl pathway produced nostoxanthin via zeaxanthin from beta-carotene, the keto pathway produced canthaxanthin from beta-carotene, and the myxol pathway produced 2-hydroxymyxol 2'-fucoside via myxol 2'-fucoside. This cyanobacterium was found to contain many kinds of carotenoids and also displayed many carotenogenesis pathways, while other cyanobacteria lack some carotenoids and a part of carotenogenesis pathways compared with this cyanobacterium.

Enzymatically fabricated and degradable microcapsules for production of multicellular spheroids with well-defined diameters of less than 150 μm

Microcapsules with a single, spherical hollow core less than 150μm in diameter were developed to obtain multicellular spheroids with well-defined sizes of less than 150μm in diameter. An aqueous solution of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) containing human hepatoma cell line (HepG2) cells and horse radish peroxidase (HRP) was injected into a coflowing stream of liquid paraffin, containing H2O2, resulting in cell-enclosing CMC-Ph microparticles, 135μm in diameter, via a peroxidase-catalyzed crosslinking reaction. The CMC-Ph microparticles were then coated with a phenolic hydroxyl derivative of alginate (Alg-Ph) gel membrane several dozen micrometers in thickness, crosslinked via the same enzymatic reaction process, followed by further crosslinking between the carboxyl groups of alginate by Sr2+. A hollow core structure was achieved by immersing the resultant microcapsules in a medium containing cellulase, which degrades the enclosed CMC-Ph microparticles. The HepG2 cells in the microcapsules then grew and completely filled the hollow core. Multicellular spheroids the same size as the CMC-Ph microparticles, with living cells at their outer surface, were collected within 1min by soaking them in a medium containing alginate lyase to degrade the Alg-Ph gel microcapsule membrane.

Oxidation of bovine serum albumin: identification of oxidation products and structural modifications

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long-lived proteins. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off-line nano-liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time-points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time-course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step-ladder mechanism.

Enzymatic reduction of polyunsaturated lysophosphati‐dylcholine hydroperoxides by glutathione peroxidase‐1

AbstractSome lipid peroxides are known to be converted to their corresponding alcohols in cells containing glutathione peroxidase (GPx). In this respect, we examined the enzymatic conversion of lysophosphatidylcholine (lysoPC) hydroperoxides to hydroxyl derivatives using RBL‐2H3 cells and erythrocyte GPx‐1. First, the incubation of RBL‐2H3 cells with arachidonoyl lysoPC led to the formation of a major product, with maximal UV absorbance at 234 nm and m/z [M+H]+ at 560.2, corresponding to monohydroxyeicosatetraenoyl lysoPC. Similarly, linoleoyl lysoPC was also converted to its hydroxyl derivative in RBL‐2H3 cells. Separately, lysoPC hydroperoxide, generated from soybean lipoxygenase 1‐catalyzed oxygenation of linoleoyl lysoPC, arachidonoyl lysoPC or docosahexaenoyl lysoPC, was converted by GPx‐1 to the corresponding hydroxyl derivatives. When the kinetic values were determined, the Km values (3.1–32.3 µM) of the polyunsaturated lysoPC hydroperoxides increased with decreasing number of double bonds, in contrast to a similar value of Vm among them. Moreover, the catalytic efficiency of docosahexaenoyl lysoPC hydroperoxide was much greater than that of H2O2 as substrate of GPx‐1. In related experiments, where phosphatidylcholine hydroperoxides were incubated with phospholipase A2 and GPx‐1, the complete conversion of phosphatidylcholine hydroperoxides to hydroxyl derivatives was confirmed by LC/MS. Taken together, it is proposed that GPx‐1‐type enzymes may participate in the conversion of polyunsaturated lysoPC hydroperoxides to hydroxyl derivatives in cell systems.

Metal Trifluoromethanesulfonate‐Catalyzed Regioselective Reductive Ring Opening of Benzylidene Acetals

AbstractA systematic study of various metal trifluoromethanesulfonates as efficient catalysts in the regioselective reductive ring opening of benzylidene acetals is described, including the effects of solvents, reducing agents, and temperature. These catalysts are found to be effective in cleaving the 4,6‐O‐acetal rings of hexopyranosides at either O4 or O6, respectively. When used in conjunction with a 1 M solution of BH3·THF in THF without extra addition of any solvent, it affects the ring fission at the O6 position to generate the corresponding primary alcohols, whereas O4‐opening takes place in acetonitrile in the presence of dimethylethylsilane as the reductant leading to the secondary hydroxyl derivatives in high selectivity and yields. These methodologies can be applied to a wide range of substrates containing various functional groups.

Human Cytochrome P450 1A2 Involvement in the Formation of Reactive Metabolites from a Species-Specific Hepatotoxic Pyrazolopyrimidine Derivative, 5-n-Butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine

5-n-Butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine) (OT-7100) is a pyrazolopyrimidine derivative with potential analgesic effects. Exclusively limited elevations in serum levels of aspirate- and alanine-aminotransferase were abnormally observed in a clinical study, in contrast to no toxicological potential to experimental animals. The aim of this study was to clarify the mechanism responsible for species-specific hepatotoxicity of this model compound. OT-7100 was primarily metabolized to a carboxylic acid derivative and an amino derivative (5-n-butyl-pyrazolo[1,5-a]pyrimidine, M-5) by hydrolysis in humans and rats. In human liver, pyrazolo[1,5-a]pyrimidine derivative M-5 was further metabolized to mainly M-23OH (a C-3-position hydroxyl derivative, 3-hydroxy-5-n-butyl-pyrazolo[1,5-a]pyrimidine). Studies with recombinant cytochrome P450s (P450s), correlation analysis using a panel of human liver microsomes as well as immunoinhibition with anti-P450 antibodies collectively suggested that human liver microsomal P450 1A2 preferentially metabolized M-5 to predominantly M-23OH. Human liver microsomes were capable of activating M-5 to a covalently bound metabolite faster than rat liver microsomes: reduced glutathione prevented the bindings. A cysteine adduct derivative of M-23OH at the C-6-position was structurally confirmed. On the contrary, rat liver microsomal P450 1A2 could metabolize M-5 to equally M-23OH, M-22OH (a C-6-position hydroxyl derivative, 6-hydroxy-5-n-butyl-pyrazolo[1,5-a]pyrimidine), or an unknown metabolite. These results suggest that differences in the regiospecific metabolic function of human and rat P450 1A2 would be responsible for the human-specific metabolic activation of the primary metabolite of OT-7100 to a proximate form. It is presumed that hepatotoxicity associated with OT-7100 could be likely related to the formation of a human-specific reactive metabolite from M-23OH. OT-7100 activation by inducible P450 1A2 may therefore exhibit marked individual differences.

Radical scavenging and antimicrobial activity of horsetail (Equisetum arvense L.) extracts

SummaryA reversed‐phase high performance liquid chromatography (RP‐HPLC) separation on C8 column and quantitative method were developed to analyse hydroxyl derivatives of benzoic and cinnamic acid and flavonoids in horsetail (Equisetum arvense L.) extracts. Total phenolic content of n‐butanol, ethyl acetate and water extracts, determined by the Folin‐Ciocalteu method, was 96.4, 26.4 and 15.4 mg g−1 of dry extracts, respectively. The antioxidative activity of horsetail extracts was tested by measuring their ability to scavenge stable 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and reactive hydroxyl radicals by electron spin resonance spectroscopy. The results demonstrated that the free radical scavenging activity (versus both DPPH and hydroxyl radicals) depended on the type and concentration of applied extracts; the highest DPPH (EC50 = 0.65 mg mL−1) and hydroxyl radical scavenging activities (EC50 = 0.74 mg mL−1) were obtained in the case of n‐butanol extract. The radical scavenging activity of extracts significantly correlated with total phenolic content. The antimicrobial tests showed that ethyl acetate and n‐butanol extracts inhibited the growth of tested bacteria.

Whole body hyperthermia reduces oxidative stress in the striatum of rats in an animal model of mitochondrial toxicity with 3-nitropropionic acid

Purpose: The goal of this study was to determine whether whole body hyperthermia (WBH) could reduce oxidative stress in the striatum produced by 3-nitropropionic acid (3-NP), a mitochondrial toxin that irreversibly inhibits succinate dehydrogenase (SDH), causing impairment of energy metabolism, oxidative stress and a selective degeneration of striatal cells.Methods: Rats were subjected to WBH (42°C) or normothermia control conditions for 30 min and then treated with 3-NP. Striatum samples were processed and the levels of protein carbonyl groups, biogenic amines, Hsp72 and salicylate hydroxylation (to probe the hydroxyl radical (OH•) intervention) were determined.Results: WBH significantly reduced oxidative stress in the striatum of animals treated with 3-NP, as judged by reductions in protein carbonyl and salicylate hydroxylation derivative levels, whereas striatal Hsp72 expression was significantly increased. The groups treated with 3-NP presented an increased in the dopamine (DA) derivatives 2,3-dihydroxyphenylacetic acid (DOPAC) and norepinephrine (NE) concentration, whereas the striatal relation DOPAC/DA concentration indicate a reduced dopamine turnover.Conclusions: These studies show, for the first time, that a heat shock pretreatment can ameliorate the oxidative stress produced by a metabolic toxin (3-NP) capable of impairing energy supply and produce selective striatal degeneration. These data contribute to a better understanding of the potential for thermal stress to modulate the type of oxidative stress usually present in neurodegenerative disorders associated with metabolic defects.

Characterization of metabolites of sibutramine in primary cultures of rat hepatocytes by liquid chromatography–ion trap mass spectrometry

Liquid chromatography-ion trap mass spectrometry was used for the detection and structural characterization of metabolites of the anti-obesity drug sibutramine. Metabolites were profiled from incubations of sibutramine in primary cultures of rat hepatocytes. In addition, enantioselectivity of sibutramine metabolism was investigated by carrying out separate incubations with (R)- and (S)-sibutramine. As a result, biotransformation profile for sibutramine with rat hepatocytes is proposed. Nineteen metabolites and several of their isomers formed via demethylation, hydroxylation, dehydrogenation, acetylation, attachment of CO(2), and glucuronidation were identified in MS(2) and MS(3) experiments, though the exact position of the functionality, mostly hydroxylation, could not always be determined from the mass spectrometric information. However, clear enantioselective formation was observed for two hydroxyl derivatives and two glucuronide conjugates, indicating that the hydroxyl/glucuronic acid moiety in those structures is close to the chiral center. Most of the metabolites found in this study are new metabolites of sibutramine, which were not previously reported.

A conceptually new approach to the asymmetric synthesis of 3-aryl and alkyl poly-substituted isoindolinones

A conceptually new and efficient asymmetric synthesis of C-3 arylated or alkylated poly-substituted isoindolinones is reported. The key step is the diastereoselective reduction of an N-acylhydrazonium species derived from the previously assembled corresponding hydroxyl derivative bearing the ( S)-2-methoxymethylpyrrolidine (SMP) auxiliary.

Microbial transformation of alantolactone by Mucor polymorphosporus

Two new transformed sesquiterpenes of alantolactone by Mucor polymorphosporus were obtained. They were characterized as 3β-hydroxy-11βH-eudesm-5-en-8β,12-olide (2) and 3β,11α-dihydroxy-eudesm-5-en-8β,12-olide (3), on the basis of spectral methods including 2D NMR. And product 3 was an unusual hydroxylation derivative of alantolactone at C-11.

Synthesis and stability of S‐(2‐[18F]fluoroethyl)‐L‐homocysteine for potential tumour imaging

AbstractThe F‐18 labelled methionine derivative S‐(2‐[18F]fluoroethyl)‐L‐homocysteine ([18F]FEHCys) was prepared by a one‐pot two‐step synthesis via the protected S‐(2‐bromoethyl)‐L‐homocysteine 1 and S‐(2‐chloroethyl)‐L‐homocysteine 2 precursors. The bromoethyl derivative 1 gave higher radiochemical yields (40% at 5 min) at 100°C compared with the chloro‐analogue (22% at 100°C in 30 min). However, [18F]FEHCys was found to be unstable in aqueous systems being transformed to the corresponding hydroxyl derivative within 20 min. Copyright © 2008 John Wiley & Sons, Ltd.

Selectins Ligand Decorated Drug Carriers for Activated Endothelial Cell Targeting

New active particulate polymeric vectors based on branched polyester copolymers of hydroxy-acid and allyl glycidyl ether were developed to target drugs to the inflammatory endothelial cell surface. The hydroxyl and carboxyl derivatives of these polymers allow grafting of ligand molecules on the polyester backbones at different densities. A known potent nonselective selectin ligand was selected and synthesized using a new scheme. This synthesis allowed the grafting of the ligand to the polyester polymers, preserving its binding activity as assessed by docking simulations. Selectin expression on human umbilical cord vascular endothelial cells (HUVEC) was induced with the pro-inflammatory bacterial lipopolysaccharide (LPS) or with the nonselective inhibitor of nitric oxide synthase L-NAME. Strong adhesion of the ligand decorated nanoparticles was evidenced in vitro on activated HUVEC. Binding of nanoparticles bearing ligand molecules could be efficiently inhibited by prior incubation of cells with free ligand, demonstrating that adhesion of the nanoparticles is mediated by specific interaction between the ligand and the selectin receptors. These nanoparticles could be used for specific drug delivery to the activated vascular endothelium, suggesting their application in the treatment of diseases with an inflammatory component such as rheumatoid arthritis and cancer.

2,2'- -Hydroxylase (CrtG) is Involved in Carotenogenesis of Both Nostoxanthin and 2-Hydroxymyxol 2'-Fucoside in Thermosynechococcus elongatus Strain BP-1

We identified the molecular structures, including the stereochemistry, of all carotenoids in Thermosynechococcus elongatus strain BP-1. The major carotenoid was beta-carotene, and its hydroxyl derivatives of (3R)-beta-cryptoxanthin, (3R,3'R)-zeaxanthin, (2R,3R,3'R)-caloxanthin and (2R,3R,2'R,3'R)-nostoxanthin were also identified. The myxol glycosides were identified as (3R,2'S)-myxol 2'-fucoside and (2R,3R,2'S)-2-hydroxymyxol 2'-fucoside. 2-Hydroxymyxol 2'-fucoside is a novel carotenoid, and similar carotenoids of 4-hydroxymyxol glycosides were previously named aphanizophyll. Ketocarotenoids, such as echinenone and 4-ketomyxol, which are unique carotenoids in cyanobacteria, were absent, and genes coding for both beta-carotene ketolases, crtO and crtW, were absent in the genome. From a homology search, the Tlr1917 amino acid sequence was found to be 41% identical to 2,2'- beta-hydroxylase (CrtG) from Brevundimonas sp. SD212, which produces nostoxanthin from zeaxanthin. In the crtG disruptant mutant, 2-hydroxymyxol 2'-fucoside, caloxanthin and nostoxanthin were absent, and the levels of both myxol 2'-fucoside and zeaxanthin were higher. Therefore, the gene has a CrtG function for both myxol to 2-hydroxymyxol and zeaxanthin to nostoxanthin. This is the first functional identification of CrtG in cyanobacteria. We also investigated the distribution of crtG-like genes, and 2-hydroxymyxol and/or nostoxanthin, in cyanobacteria. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in T. elongatus, we propose a biosynthetic pathway of the carotenoids and the corresponding genes and enzymes.

Hydroxylated Sclerosporin derivatives from a cytotoxic Wardomyces inflatus extract

The marine-derived fungus Wardomyces inflatus was isolated from the green alga Enteromorpha sp. Cultivation on a biomalt medium with added sea salt yielded an extract cytotoxic toward cancer cells. To date, we have isolated from this extract sclerosporin and four new hydroxylated sclerosporin derivatives, namely 8-hydroxy sclerosporin, 11-hydroxy sclerosporin, 12-hydroxy sclerosporin and 15-hydroxy sclerosporin. Sclerosporin is a rare antifungal and sporogenic cadinane-type sesquiterpene [1,2].

Tocochromanols, plastoquinol, and other biological prenyllipids as singlet oxygen quenchers—determination of singlet oxygen quenching rate constants and oxidation products

Singlet oxygen quenching rate constants for tocopherol and tocotrienol homologues have been determined in organic solvents of different polarities, as well as for other biological prenyllipids such as plastoquinol, ubiquinol, and α-tocopherolquinol. The obtained results showed that the quenching activity of tocochromanols was mainly due to the chromanol ring of the molecule and the activity increased with the number of the methyl groups in the ring and solvent polarity. Among prenylquinols, α-tocopherolquinol was the most active scavenger of singlet oxygen followed by ubiquinol and plastoquinol. The oxidation products of tocopherols were identified as 8a-hydroperoxy-tocopherones which are converted to the corresponding tocopherolquinones under acidic conditions. The primary oxidation products of prenylquinols, containing unsaturated side chains, were the corresponding prenylquinones that were further oxidized to hydroxyl side-chain derivatives. In the case of plastochromanol, the γ-tocotrienol homologue found in some seed oils, mainly the hydroxyl derivatives were formed, although 8a-hydroperoxy-γ-tocopherones were also formed to a minor extent, both from plastochromanol and from its hydroxyl, side-chain derivatives. The obtained results were discussed in terms of the activity of different prenyllipids as singlet oxygen scavengers in vivo.

ChemInform Abstract: An Efficient Synthesis of Resveratrol and a Hydroxyl Derivative via the Perkin Reaction: cis to trans Isomerization in a Demethylation Process.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Identification of intermediates formed during anaerobic benzene degradation by an iron‐reducing enrichment culture

Anaerobic benzene degradation is an important process in contaminated aquifers but is poorly understood due to the scarcity of microbial cultures for study. We have enriched a ferric iron-reducing culture that completely mineralizes benzene to CO(2). With (13)C(6)-labelled benzene as the growth substrate, ring-labelled benzoate was identified as a major intermediate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of culture supernatants. With increasing incubation time, (13)C(7)-labelled benzoate appeared, indicating that the carboxyl group of benzoate derived from CO(2) that was produced from mineralization of labelled benzene. This was confirmed by growing the culture in (13)C-bicarbonate-buffered medium with unlabelled benzene as the substrate, as the label appeared in the carboxyl group of benzoate produced. Phenol was also identified as an intermediate at high concentration. However, it was clearly shown that phenol was formed abiotically by autoxidation of benzene during the sampling and analysis procedure as a result of exposure to air. The results suggest that, in our culture, anaerobic benzene degradation proceeds via carboxylation and that caution should be exercised in interpreting hydroxylated benzene derivatives as metabolic intermediates of anaerobic benzene degradation.

Dimethylthallium(III) complexes with picolinic acid and its hydroxyl derivatives

The reaction of dimethylthallium(III) hydroxide with picolinic acid (Hpic), 3-hydroxypicolinic acid (H 23hpic) and 6-hydroxypicolinic acid (H 26hpic) in an aqueous/methanol mixture afforded the complexes [TlMe 2(pic)] ( 1), [TlMe 2(H3hpic)] ( 2) and [TlMe 2(H6hpic)] ( 3), respectively. Complex 3′, [NaTlMe 2(6hpic) 2] n , was obtained as a minor product from a methanolic solution of 3. Compounds 1– 3 were characterized by IR and Raman spectroscopy and, in the cases of 1, 2 and 3′, by single-crystal X-ray diffraction. Complex 3′ is the first example of an H6hpic − heterobimetallic compound to be isolated. The 1H and 13C NMR spectra of 1 and 2 are also discussed.