Reactivity of Cartilage and Selected Carbohydrates with Hydroxyl Radicals: An NMR Study to Detect Degradation Products

It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by γ-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosac-charides as well as carbon resonances in ¹³C-enriched glucose were continuously diminished upon γ-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) γ-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysac-charides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.     

Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme.     

Generation of 5-(2'-deoxycytidyl)methyl radical and the formation of intrastrand cross-link lesions in oligodeoxyribonucleotides

Hydroxyl radical is one of the major reactive oxygen species (ROS) formed from γ-radiolysis of water or Fenton reaction, and it can abstract one hydrogen atom from the methyl carbon atom of thymine and 5-methylcytosine to give the 5-methyl radical of the pyrimidine bases. The latter radical can also be induced from Type-I photo-oxidation process. Here, we examined the reactivity of the independently generated 5-(2′-deoxycytidyl)methyl radical (I) in single- and double-stranded oligodeoxyribonucleotides (ODNs). It was found that an intrastrand cross-link lesion, in which the methyl carbon atom of 5-methylcytosine and the C8 carbon atom of guanine are covalently bonded, could be formed from the independently generated radical at both GmC and mCG sites, with the yield being much higher at the former site. We also showed by LC-MS/MS that the same cross-link lesions were formed in mC-containing duplex ODNs upon γ irradiation under both aerobic and anaerobic conditions, and the yield was ~10-fold higher under the latter conditions. The independently generated radical allows for the availability of pure, sufficient and well-characterized intrastrand cross-link lesion-bearing ODN substrates for future biochemical and biophysical characterizations. This was also the first demonstration that the coupling of radical I with its 5′ neighboring guanine can occur in the presence of molecular oxygen, suggesting that the formation of this and other types of intrastrand cross-link lesions might have important implications in the cytotoxic effects of ROS.     

Enzymatic synthesis of β-D-2', 3'-unsaturated-5-fluorocytidine from β-D-2',3'-unsaturated thymidine

We have used crude preparations of N-deoxyribosyl transferases (NdRT-II) from Lactobacillus helveticus to catalyze the transfer of a glycosyl moiety from a donor nucleoside to an acceptor base. Optimal conditions for the transglycosylation reaction to make D-D4FC starting from D-D4T and 5-FC were determined after the analysis of several experimental parameters including reaction time, concentration of substrate, pH and the type of buffer. For the first time, a practical procedure for enzymatic synthesis of β-D-2',3'-unsaturated-5-fluorocytidine (β-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, D-D4FC) from β-D-2',3'-unsaturated thymidine (D-D4T) has been established. This method will be useful in the manufacture of important nucleoside analogues for anti-viral therapy.     

8-Hydroxy-2'-deoxyguanosine formation during the catalytic oxidation of hydrazines in the presence of 2'-deoxyguanosine.

The reaction of 2'-deoxyguanosine (1), substituted hydrazines (Hy) and oxygen, in the presence of Cu(II) as catalyst, yields 8-hydroxy-2'-deoxyguanosine (2). The rate of formation of 2 was found to be dependent upon the oxidation potential of the Hy and on structural factors of the Hy. The conversion of 1 to 2 under these conditions suggested that a Haber-Weiss/Fenton type of process was involved with Hy acting as reductant. However, the dependence of the rate of the conversion of 1 to 2 upon the structure of Hy suggested that the Hy substrates studied may be more directly involved in the process than that of a reducing agent. Additional studies of this reaction system, including the oxygen consumption, radical trapping studies and direct ESR measurements suggest that the conversion of 1 to 2, under the conditions used, involves the intermediacy of complex composed of the metal catalyst, 1, Hy and oxygen. The rate data for the conversion of 1 to 2 appear to be correlated with the carcinogenicity of Hy and therefore 2 may be an important DNA adduct in the carcinogenesis of hydrazines.     

Isolation, structural elucidation, and partial synthesis of lutein dehydration products in extracts from human plasma

All-E-(3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (anhydrolutein I) and all-E-(3R,6′R)-3-hydroxy-2′,3′-didehydro-β,ε-carotene (2′,3′-anhydrolutein II) have been isolated and characterized from extracts of human plasma using semipreparative high-performance liquid chromatography (HPLC) on a C₁₈ reversed-phase column. The identification of anhydroluteins was accomplished by comparison of the UV-Vis absorption and mass spectral data as well as HPLC-UV-Vis-mass spectrometry (MS) spiking experiments using fully characterized synthetic compounds. Partial synthesis of anhydroluteins from the reaction of lutein with 2% H₂SO₄ in acetone, in addition to anhydrolutein I (54%) and 2′,3′-anhydrolutein II (19%), also gave (3′R)-3′-hydroxy-3,4-dehydro-β-carotene (3′,4′-anhydrolutein III, 19%). While anhydrolutein I has been shown to be usually accompanied by minute quantities of 2′,3′-anhydrolutein II (ca. 7–10%) in human plasma, 3′,4′-anhydrolutein III has not been detected. The presence of anhydrolutein I and II in human plasma is postulated to be due to acid catalyzed dehydration of the dietary lutein as it passes through the stomach. These anhydroluteins have also been prepared by conversion of lutein diacetate to the corresponding anhydrolutein acetates followed by alkaline hydrolysis. However, under identical acidic conditions, loss of acetic acid from lutein diacetate proceeded at a much slower rate than dehydration of lutein. The structures of the synthetic anhydroluteins, including their absolute configuration at C(3) and C(6′) have been unambiguously established by ¹H NMR and in part by ¹³C NMR, and circular dichroism.     

Purification and characterization of 3β-hydroxysteroid-dehydrogenase/isomerase from bovine adrenal cortex

The formation of 4-ene-3-ketosteroids from 3β-hydroxy-5-ene precursors is an obligatory step in the biosynthesis of hormonal steroids such as glucocorticoids, mineralocorticoids, estrogens and androgens. In the adrenal cortex, pregnenolone, 17-hydroxy-pregnenolone and dehydroisoandrosterone are converted to progesterone, 17-hydroxy-progesterone and androstenedione, respectively, by the enzymatic system 3β-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase (3β-HSD/I).

The present work reports a two step purification procedure which yields an homogenous preparation of 3β-HSD/I from bovine adrenal cortex. It uses solubilization of the microsomal proteins followed by two chromatographic steps, i.e. DEAE-cellulose and heparine-sepharose columns. The enzyme was obtained as an homogeneous protein exhibiting an apparent molecular size of 45 kDa upon SDS-gel electrophoresis and of 81 kDa upon gel filtration. The purified enzyme exhibits both the 5-ene-3β-ol steroid dehydrogenase and isomerase activities in contrast to previous work using a more complex procedure which yielded a final preparation having lost its isomerase activity [Hiwatashi et al., Biochem. J. 98 (1985) 1519–1525]. N-terminal aminoacid (29 residues) sequence of the purified protein was determined and was found identical to that predicted from the nucleic acid sequence of the recently identified enzyme cDNA [Zhas et al. FEBS Lett. 259 (1989) 153–157].     

Effective anomerisation of 2'-deoxyadenosine derivatives during disaccharide nucleoside synthesis

The formation of a disaccharide nucleoside (11) by O3'-glycosylation of 5'-O-protected 2'-deoxyadenosine or its N6-benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding alpha-anomeric product (12). The latter reaction can be explained by instability of the N-glycosidic bond of purine 2'-deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2'-deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3'-O-beta-D-ribofuranosyl-2'deoxyadenosines and its alpha-anomer.     

Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement

Oxidation reactions that involve several oxygen and nitrogen reactive species together with nucleobase radical cations give rise among various classes of lesions to modified bases. About 70 of oxidized nucleosides that include diastereomeric forms have been characterized in mechanistic studies involving isolated DNA and related model compounds. However, only eight modified bases have been accurately measured within cellular DNA upon exposure to either gamma or UVA radiations. Emphasis is placed in this survey on recent developments of HPLC associated with tandem mass spectrometry (MS/MS) operating in the mild electrospray ionization mode. Interestingly, the HPLC-MS/MS assay in the multiple reaction monitoring mode appears to be the more sensitive and accurate method currently available for singling out several oxidized nucleosides including 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl-2'-deoxyuridine, 5-hydroxy-2'-deoxyuridine, and the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine within isolated and cellular DNA. However, one limitation of the assay that also applied to all chromatographic methods is the slight side-oxidation of normal bases during DNA extraction and subsequent work-up. This explains why the combined use of DNA repair glycosylases with either the comet assay or the alkaline elution technique is a better alternative to monitor the formation of low levels of oxidized bases within cellular DNA.     

Galactosylation of antibiotics using the β-galactosidase from Aspergillus oryzae

The β-galactosidase from Aspergillus oryzae has been shown to catalyze the synthesis of β-galactosides of antibiotics such as chlorphenisin and chloramphenicol using β-lactose as the galactosyl donor. Among the water-miscible organic solvents tested, 20% (v/v) acetonitrile in the reaction mixture gave the highest yield in galactoside synthesis. The products obtained were purified by preparative TLC and liquid chromatography and analyzed by ¹H-and ¹³C-NMR, and MS (FAB). Chlorphenisin and chloramphenicol were galactosylated exclusively at their primary hydroxy groups. The pH optimum for the transgalactosylation reaction was between pH 4–5. Increasing concentrations of galactosyl donor and aglycon caused increasing yields of galactosides. When the resulting galactosylated antibiotic was withdrawn from the sample, further synthesis was observed. This could be accelerated either by withdrawing the resulting monosaccharides (glucose and galactose) or exchanging them for mannose.     

Free radical oxidation of 15-(S)-hydroxyeicosatetraenoic acid with the Fenton reagent: characterization of an epoxy-alcohol and cytotoxic 4-hydroxy-2E-nonenal from the heptatrienyl radical pathway

The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent (Fe2+/EDTA/H2O2) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after 1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-(2)H)-15-HETE (1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of 1a as a potential precursor of the cytotoxic gamma-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between 1a and the epoxy-alcohol 2a, previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid.     

Aromatic ring cleavage of a β-biphenyl ether dimer catalyzed by lignin peroxidase of phanerochaete chrysosporium

Under aerobic conditions homogeneous lignin peroxidase catalyzed the oxidation of 1-(4'-methoxyphenyl)-2-(2″,5′-dimethoxy-4″-phenylphenoxy)-1,3-dihydroxypropane (I) to yield four products: 1-(4'-methoxy-phenyl)-1,2,3-trihydroxypropane (X), 4-[-hydroxy--(4'-methoxyphenyl)-methyl]-1,3-dioxolane-2-one (V), 4-(4'-methoxyphenyl)-5-hydroxymethyl-1,3-dioxolane-2-one (VI) and 5-hydroxy-5-carbomethoxy-4-phenyl-oxol-3-en-2-one (VIII). V, VI and VIII are all products of ring opening reactions. When the reaction was conducted under anaerobic conditions, the substrate was oxidized but no ring-cleaved products were detected. During the oxidation of I, 4 atoms of ¹⁸O from ¹⁸O₂ were incorporated into the lactol product VIII.     

Synthèse de nucléosides pyrimidiques non protégés en O-2′ à partir de sulfites cycliques en C-1—C-2

Treatment of 3,5,6-tri-O-benzoyl-- -glucofuranose 1,2-sulfite with an excess of bis(trimethylsil) uracil, in fusion processes without any catalyst, afforded an excellent yield of 1-(3,5,6-tri-O-benzoyl-2-O-trimethylsilyl-β- -glucofuranosyl)uracil, which was readily hydrolyzed in slightly acid conditions to give in almost quantitative yield 1-(3,5,6-tri-O-benzoyl-β- -glucofuranosyl)uracil. This new synthetic method for nucleosides unprotected at O-2′ was also tested in other sugar series. In some cases, only the 1′,2′-trans-nucleosides were obtained, but in others, small yields (3–10%) of 1′,2′-cis-nucleosides were detected. The -to-β ratio seems to be dependent on the reaction temperature. 2,4-Dimethoxypyrimidine also reacted with sugar 1,2-sulfites and 4-O-methyl-1-(3,5,6-tri-O-benzyl-β- -glucopyranosyl)-2-pyrimidinone was prepared in 85% yield from 3,5,6-tri-O-benzyl-- -glucopyranose 1,2-sulfite.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

Circulating levels of pregnanolone isomers during the third trimester of human pregnancy

This study addresses the question of whether changes in the biosynthesis and metabolism of neuroactive pregnanolone isomers (PIs) might participate in the timing of parturition in humans. The time profiles of unconjugated allopregnanolone (3-hydroxy-5-pregnan-20-one, P35), pregnanolone (3-hydroxy-5β-pregnan-20-one, P35β), isopregnanolone (3β-hydroxy-5-pregnan-20-one, P3β5) and epipregnanolone (3β-hydroxy-5β-pregnan-20-one, P3β5β), pregnenolone, their polar conjugates, progesterone, 5-dihydroprogesterone (P5), and 5β-dihydroprogesterone (P5β) were monitored in the plasma of 30 healthy women during the third trimester of pregnancy, at 1-week intervals from the 30th week of gestation using GC–MS. Changes in the steroid levels were evaluated by two-way ANOVA with gestational age and subject as independent factors. The mean concentrations of free PIs ranged from 2 to 50 nmol/L, while the mean levels of their polar conjugates were 40–100× higher. The ratio of 5-PIs to progesterone significantly but inconspicuously culminated in the 35th week. The decelerating biosynthesis of free 5β-PIs from the 31st week and their escalating sulfation was found from the 30th week. The changes were particularly evident in the second most abundant PI pregnanolone that may, like the allopregnanolone, sustain the pregnancy via attenuation of hypothalamic GABA_A-receptors and prevent uterine contractility via binding to nuclear pregnane X receptor.     

Cooperative erythropoietic assay of several steroid metabolites in polycythemic mice

A blinded cooperative assay of several androstane and pregnane steroid metabolites has been carried out in order to determine whether 5β-H derivatives are as active as testosterone in stimulating in vivo erythropoiesis. The steroids tested were: testosterone, 5-dihvdrotestosterone, 5β-dihydrotestosterone, 5β-pregnane-3,20-dione, 3-dihydroxy-5β-pregnàne-11,20-dione and 3β-hydroxy-5β-pregnan-20-one. The incorporation of radioactive iron into newly formed red cells in exhypoxic polycythemic mice was used to compare the effects of the steroids. Testosterone and 5-dihydrotestosterone both produced significant increases in ⁵⁹Fe incorporation. 5β-dihydrotestosterone, 5β-pregnane-3,20-dione, 3-hydroxy-5β-pregnane-11,20-dione and 3β-hydroxy-5β-pregnan-20-one were all devoid of significant erythropoietic activity in polycythemic mice in almost all instances. Thus, under the conditions chosen, this study failed to demonstrate that 5β-steroids increase radioactive iron incorporation in red cells of exhypoxic polycythemic mice.