Degradation of ciprofloxacin by basidiomycetes and identification of metabolites generated by the brown rot fungus Gloeophyllum striatum

Ciprofloxacin (CIP), a fluoroquinolone antibacterial drug, is widely used in the treatment of serious infections in humans. Its degradation by basidiomycetous fungi was studied by monitoring 14CO2 production from [14C]CIP in liquid cultures. Sixteen species inhabiting wood, soil, humus, or animal dung produced up to 35% 14CO2 during 8 weeks of incubation. Despite some low rates of 14CO2 formation, all species tested had reduced the antibacterial activity of CIP in supernatants to between 0 and 33% after 13 weeks. Gloeophyllum striatum was used to identify the metabolites formed from CIP. After 8 weeks, mycelia had produced 17 and 10% 14CO2 from C-4 and the piperazinyl moiety, respectively, although more than half of CIP (applied at 10 ppm) had been transformed into metabolites already after 90 h. The structures of 11 metabolites were elucidated by high-performance liquid chromatography combined with electrospray ionization mass spectrometry and 1H nuclear magnetic resonance spectroscopy. They fell into four categories as follows: (i) monohydroxylated congeners, (ii) dihydroxylated congeners, (iii) an isatin-type compound, proving elimination of C-2, and (iv) metabolites indicating both elimination and degradation of the piperazinyl moiety. A metabolic scheme previously described for enrofloxacin degradation could be confirmed and extended. A new type of metabolite, 6-defluoro-6-hydroxy-deethylene-CIP, provided confirmatory evidence for the proposed network of congeners. This may result from sequential hydroxylation of CIP and its congeners by hydroxyl radicals. Our findings reveal for the first time the widespread potential for CIP degradation among basidiomycetes inhabiting various environments, including agricultural soils and animal dung.     

Degradation of Ciprofloxacin by Basidiomycetes and Identification of Metabolites Generated by the Brown Rot Fungus Gloeophyllum striatum

Ciprofloxacin (CIP), a fluoroquinolone antibacterial drug, is widely used in the treatment of serious infections in humans. Its degradation by basidiomycetous fungi was studied by monitoring ¹⁴CO₂ production from [¹⁴C]CIP in liquid cultures. Sixteen species inhabiting wood, soil, humus, or animal dung produced up to 35% ¹⁴CO₂ during 8 weeks of incubation. Despite some low rates of ¹⁴CO₂ formation, all species tested had reduced the antibacterial activity of CIP in supernatants to between 0 and 33% after 13 weeks. Gloeophyllum striatum was used to identify the metabolites formed from CIP. After 8 weeks, mycelia had produced 17 and 10% ¹⁴CO₂ from C-4 and the piperazinyl moiety, respectively, although more than half of CIP (applied at 10 ppm) had been transformed into metabolites already after 90 h. The structures of 11 metabolites were elucidated by high-performance liquid chromatography combined with electrospray ionization mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. They fell into four categories as follows: (i) monohydroxylated congeners, (ii) dihydroxylated congeners, (iii) an isatin-type compound, proving elimination of C-2, and (iv) metabolites indicating both elimination and degradation of the piperazinyl moiety. A metabolic scheme previously described for enrofloxacin degradation could be confirmed and extended. A new type of metabolite, 6-defluoro-6-hydroxy-deethylene-CIP, provided confirmatory evidence for the proposed network of congeners. This may result from sequential hydroxylation of CIP and its congeners by hydroxyl radicals. Our findings reveal for the first time the widespread potential for CIP degradation among basidiomycetes inhabiting various environments, including agricultural soils and animal dung.     

Degradation of the fluoroquinolone enrofloxacin by wood-rotting fungi.

The veterinary fluoroquinolone enrofloxacin was degraded in vitro by four species of wood-rotting fungi growing on wetted wheat straw containing carbonyl-14C-labeled drug. A maximum 14CO2 production of 17% per week was observed with the brown rot fungus Gloeophyllum striatum, resulting in up to 53% after 8 weeks. However, rates reached at most 0.2 and 0.9% per week, if enrofloxacin was preadsorbed to native or gamma ray-sterilized soil, respectively.     

Hydroxylated metabolites of 2,4-dichlorophenol imply a fenton-type reaction in Gloeophyllum striatum

While degrading 2,4-dichlorophenol, two strains of Gloeophyllum striatum, a basidiomycetous fungus causing brown rot decay of wood, simultaneously produced 4-chlorocatechol and 3,5-dichlorocatechol. These metabolites were identified by comparing high-performance liquid chromatography retention times and mass spectral data with those of chemically synthesized standards. Under similar conditions, 3-hydroxyphthalic hydrazide was generated from phthalic hydrazide, a reaction assumed to indicate hydroxyl radical formation. Accordingly, during chemical degradation of 2,4-dichlorophenol by Fenton's reagent, identical metabolites were formed. Both activities, the conversion of 2,4-[U-(14)C]dichlorophenol into (14)CO(2) and the generation of 3-hydroxyphthalic hydrazide, were strongly inhibited by the hydroxyl radical scavenger mannitol and in the absence of iron. These results provide new evidence in favor of a Fenton-type degradation mechanism operative in Gloeophyllum.     

Patterns of metabolites produced from the fluoroquinolone enrofloxacin by basidiomycetes indigenous to agricultural sites

Supernatants of mycelial cultures of seven basidiomycetous fungi indigenous to agricultural sites were evaluated for metabolites generated from the veterinary fluoroquinolone enrofloxacin (EFL) by employing high–performance liquid chromatography/high–resolution electrospray ionization mass spectrometry. From exact masses, molecular formulae were derived, and the most probable chemical structures were deduced. Patterns of major metabolites were surprisingly similar but differed greatly from that provided by Gloeophyllum striatum due to the absence of monohydroxylated EFL congeners and a greater variety of metabolites with a modified piperazine moiety. The structures of three metabolites were elucidated by ¹H–nuclear magnetic resonance spectroscopy. Of 61 compounds detected, 48 were new, while 13 were known from a pattern of 87 EFL metabolites identified for G. striatum. Ethylpiperazine moieties carrying oxido, hydroxy, oxo, and acetoxy groups, or showing partial degradation, were linked to the unmodified, oxidatively decarboxylated, or multiply hydroxylated core of EFL and to isatin– and anthranilic acid–type EFL congeners. Cleavage of the fluoro–aromatic bond was observed for two, ¹⁴CO₂ formation for six species. Metabolites with a hydroxylated aromatic part implied subsequent ring cleavage to be brought about by the formation of potentially four oxidizable ortho–aminophenol– and one catechol–type intermediates. EFL degradation appears to be a common activity among basidiomycetes.     

Hydroxylated Metabolites of 2,4-Dichlorophenol Imply a Fenton-Type Reaction in Gloeophyllum striatum

While degrading 2,4-dichlorophenol, two strains of Gloeophyllum striatum, a basidiomycetous fungus causing brown rot decay of wood, simultaneously produced 4-chlorocatechol and 3,5-dichlorocatechol. These metabolites were identified by comparing high-performance liquid chromatography retention times and mass spectral data with those of chemically synthesized standards. Under similar conditions, 3-hydroxyphthalic hydrazide was generated from phthalic hydrazide, a reaction assumed to indicate hydroxyl radical formation. Accordingly, during chemical degradation of 2,4-dichlorophenol by Fenton's reagent, identical metabolites were formed. Both activities, the conversion of 2,4-[U-¹⁴C]dichlorophenol into ¹⁴CO₂ and the generation of 3-hydroxyphthalic hydrazide, were strongly inhibited by the hydroxyl radical scavenger mannitol and in the absence of iron. These results provide new evidence in favor of a Fenton-type degradation mechanism operative in Gloeophyllum.     

Orally active cephalosporins. Part 2: synthesis, structure-activity relationships and oral absorption of cephalosporins having a C-3 pyridyl side chain

A series of 7beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamid o]cephalosporins having a pyridine ring connected through various spacer moieties at the C-3 position was designed and synthesized and evaluated for antibacterial activity and oral absorption in rats. All compounds showed potent antibacterial activity against Staphylococcus aureus, whereas antibacterial activity against gram-negative bacteria was markedly influenced by the spacer moiety between the pyridine and cephem nucleus. Oral absorption was influenced by the position of the pyridine nitrogen as well as by the spacer moiety. Among these compounds, FR86830 (14), having a 4-pyridylmethylthio moiety at the C-3 position, showed the most well balanced activity and moderate oral absorption.     

Outlines of an “exploding” network of metabolites generated from the fluoroquinolone enrofloxacin by the brown rot fungus Gloeophyllum striatum

Degradation of the veterinary fluoroquinolone antibiotic enrofloxacin (EFL) was studied with three strains of Gloeophyllum, basidiomycetous fungi thought to produce extracellular hydroxyl radicals. Metabolites generated in a mineral medium were analyzed by combined high-performance liquid chromatography/high-resolution electrospray ionization mass spectrometry. Their origin was inferred from peak doublets representing ¹²C and ¹⁴C isotopomers detected at a defined proportion. From each exact molecular mass, the molecular formula was derived for which the most probable chemical structure was postulated, using for guidance 18 known EFL metabolites. All supernatants provided similar metabolite patterns, with the most comprehensive consisting of 87 compounds. These metabolites belonged to five families headed by EFL, its oxidatively decarboxylated or defluorinated congeners, an isatin-, and an anthranilic acid-type derivative. Metabolites hydroxylated in the aromatic part suggested the formation of three catechols and two oxidizable ortho-aminophenol-type compounds. After oxidation to the respective ortho-quinones or ortho-quinone imines and oxidative ring cleavage at one of three alternative sites, the formation of various cis,cis-muconic acid-type derivatives is likely, one of which could be detected. Anthranilic acid-type compounds provided two additional sites for ortho-aminophenol formation and aromatic ring cleavage. An “exploding” network of diverse EFL congeners produced by Gloeophyllum suggests the broad utility of our model for studying biodegradation.     

Structural determinants of blockade of eosinophil activation, adhesion and secretion by synthetic analogs of phomactin

We examined the structural determinants of phomactin analogs to assess their efficacy as antagonist of PAF. Six analogs of phomactin were synthesized to determine their inhibitory effects on adhesion, superoxide release, leukotriene C4 (LTC4) synthesis and [3H]PAF binding in human eosinophils. Phomactin analogs inhibited both PAF- and IL-5-induced eosinophil adhesion. Analog A, which bears an alkene moiety between C-1 and C-14, a ketone at the C-2 position, and an alkyne moiety between C-3 and C-4, had the greatest anti-adhesive effect. Change of the alkene between C-1 and C-14 to an alkane (analog I) decreased the anti-adhesive effect by 2.5-4 fold, while substitution of ketone by hydroxyl (analog G) at the C-2 position caused an 11-fold decrease in the anti-adhesive effect. Substitution of the alkyne moiety between C-3 and C-4 by an alkene (B and E) or alkane (D) blocked completely the anti-adhesive effect. Analogs A and I completely blocked superoxide release from eosinophils caused by phorbol-12-myristate-13-acetate or PAF and LTC4-release caused by fMLP plus cytochalasin B. Change of the alkyne moiety between C-3 and C-4 to an alkene (B and E) or alkane (D) blocked completely these inhibitory effects of phomactin. Analog A decreased the maximal binding of [3H]PAF binding to eosinophils without change of the apparent dissociation constant. We conclude that phomactin analogs are specific non-competitive PAF antagonists and have exceptional efficacy in inhibiting adhesion, metabolic activity and leukotriene secretion in human eosinophils. We further define the structural alterations in the phomactin molecule that regulate its inhibitory functions.     

Non-enzymatic depolymerization of cotton cellulose by fungal mimicking metabolites

Small, low molecular weight, non-enzymatic compounds have been linked to the early stages of brown rot decay as the enzymes involved with holocellulose degradation are too large to penetrate the S3 layer of intact wood cells. We investigated the most notable of these compounds, i.e. hydrogen peroxide, iron, and oxalic acid. The former two are involved in the Fenton reaction in which they react to form hydroxyl radicals, which cause an accelerated depolymerization in cotton cellulose. We found the same reaction to be caused by both iron Fe³⁺ and Fe²⁺. A 10 mM oxalic acid solution showed significant depolymerization effect on cotton cellulose. An oxalic acid/sodium oxalate buffered pH gradient had an inhibitory effect on the reduction of cellulose polymers at increased pH values. The organic iron chelator, EDTA, was found to promote depolymerization of cellulose in combination with Fenton’s reagents, but inhibited the effect of oxalic acid in the absence of iron and hydrogen peroxide. Manganese was tested to see if metals other than iron could generate a significant impact on the degree of polymerization (DP) in cotton cellulose. Depolymerizing properties comparable to iron were seen. The results confirm that low molecular weight metabolites are capable of depolymerizing cellulose and suggest an importance of these mechanisms during incipient decay by brown rot fungi.     

The synthesis and antibacterial activity of totarol derivatives. Part 2: Modifications at C-12 and O-13

Alterations of the C-12 and C-13 aromatic ring substituents of totarol (1) afforded the series of derivatives 2-14, and introduction of substituents at C-12 gave exclusively 2a-14a. The majority of these analogues were tested in vitro against the following organisms: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. The results were evaluated in terms of structure-activity relationship which reveals that: (a) the phenolic moiety at C-13, in general, is essential for antibacterial activity at < 32 microg/mL against gram-positive species, and (b) derivatization at C-12 has an undesirable effect on the antibacterial activity of this class of compounds, while (c) all compounds tested are ineffective against the gram-negative Klebsiella pneumoniae.     

Hydroxylated metabolites of the antimalarial drug primaquine. Oxidation and redox cycling.

Oxidation and redox cycling of the hydroxylated metabolites of the antimalarial drug primaquine (i.e. 5-hydroxyprimaquine, 5-hydroxydemethylprimaquine, and 5,6-dihydroxy-8-aminoquinoline) were studied. The three metabolites readily oxidized under physiological conditions, forming hydrogen peroxide and the corresponding quinone-imine derivatives as the main products. The latter compounds were characterized by visible, NMR, and infrared spectroscopy. Concomitant formation of drug-derived radicals and hydroxyl radicals was attested by direct and spin-trapping EPR experiments, respectively. The use of the spin stabilization method indicated that the radicals derived from 5-hydroxydemethylprimaquine and 5,6-dihydroxy-8-aminoquinoline are of the o-semiquinone type. Tentative structures are proposed for the radicals based on product identification and computer simulation of the experimental EPR spectra. The quinone-imines obtained from the reduced metabolites did not react at appreciable rates with NADPH but underwent redox cycling upon addition of ferredoxin:NADP+ oxidoreductase, forming hydrogen peroxide and hydroxyl radicals. The effect of antioxidant enzymes on hydroxyl radical yield obtained during oxidation and redox cycling indicates that the main route for hydroxyl radical formation is the metal ion-catalyzed reaction between the drug-derived radicals and hydrogen peroxide. Taken together, the results indicate that hydrogen peroxide is the potential toxic product formed from the primaquine metabolites.     

Studies on the Biosynthesis of Kanamycins

Incorporation of the radioactive degradation products of kanamycin A or related metabolites into kanamycin A by growing cells of Streptomyces kanamyceticus was examined. ³H-Deoxystreptamine was incorporated into deoxystreptamine moiety of kanamycin, but neither ¹⁴C-3-amino-3-deoxy-d-glucose nor ¹⁴C-6-amino-6-deoxy-d-glucose was incorporated. ³H-Kanamycin A added to medium was modified and inactivated.     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

12S,19- and 12S,20-dihydroxyeicosanoids: novel 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid metabolites formed by hydroxylation and reduction in murine lymphocytes

Murine spleen cells and purified B lymphocytes oxidized arachidonic acid via the lipoxygenase pathway. The major metabolite of both the whole spleen and enriched B lymphocytes was 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. A novel metabolite was observed that did not have an absorbance from 210 to 400 nm, indicating the absence of a conjugated double bond system. The new metabolite was converted to the methyl ester, reduced by platinum oxide, derivatized to the trimethylsilyl ether, and analyzed by gas chromatography-mass spectrometry. A major and a minor component were observed in the analysis of the new compound. The major component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-19. The minor component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-20. The new metabolites are characterized as a mixture of 12S,19- and 12S,20-dihydroxyeicosanoids presumably formed by hydroxylation and reduction of one or more double bonds of 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. These metabolites were formed predominantly with whole spleen lymphocytes but could be detected at longer incubation times or by using 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid as the starting substrate with highly enriched B lymphocytes.     

Structure prerequisite for antioxidant activity of silybin in different biochemical systems in vitro.

Structural analogues (flavanone: 2-4 and flavone: 5 and 6, respectively) of silybin (1a) were synthesized and tested for inhibitory activity on O(2)(-) release and PKC translocation in PMA-stimulated neutrophils as well as xanthine oxidase activity in order to identify the molecular structures responsible for the antioxidant property of silybin. Concerning the prevention of hem-mediated oxidative modification of LDL by silybin, the hydroxyl radical scavenging activity of its structural analogues was also determined. We demonstrated that the basic skeleton of 1a (4) is responsible for its inhibitory activity on O(2)(-) release in PMA-stimulated neutrophils via inhibition of PKC translocation, since introduction of a double bound and hydroxyl groups at C-5 and C-7 position (5 and 6) did not result in further increase in inhibition of O(2)(-) release. It has been shown that the presence of the phenolic hydroxyl group at C-5 and C-7 of 1a is essential for the inhibition of xanthine oxidase activity. Moreover, introduction of a double bond into the C-ring of 2 and 3, resulting in flavone derivatives (5 and 6), markedly enhanced the antioxidant effect in all the tested systems. Finally, silybin (1a) and its flavon derivatives (5 and 6) directly scavenged hydroxyl radicals as well. On the basis of these results it might be concluded that different moiety of silybin is responsible for inhibition of overproduction of O(2)(-) in stimulated neutrophils, xanthine oxidase activity, and for prevention of hem-mediated oxidative modification of LDL.     

Orally active cephalosporins. Part 4: synthesis, structure--activity relationships and oral absorption of novel 3-(4-pyrazolylmethylthio)cephalosporins with various C-7 side chains

A series of 3-(4-pyrazolylmethylthio)cephalosporins with various C-7 side chains was designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity against Haemophilus influenzae was markedly increased by the C-7 oxime moiety. Deamination at the 2 position of, or introduction of a substituent such as halogen or methyl to, the 5 position of the (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) moiety improved oral absorption. Among these compounds, FR192752 having a (Z)-2-(2-amino-5-chlorothiazol-4-yl)-2-hydroxyiminoacetamido moiety, showed potent antibacterial activity against both Gram-positive and Gram-negative bacteria including H.influenzae and penicillin G-resistant Streptococcus pneumoniae (PRSP). Further, it showed higher oral absorption than CFDN and FK041.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Construction of a branched chain at C-3 of a hexopyranoside. Synthesis of miharamycin sugar moiety analogs

Synthesis of the conveniently protected epimer at C-3' of the miharamycin sugar moiety was accomplished starting from the corresponding 3,3'-spiroepoxide. Reaction of the epoxide with lithium cyanide, followed by hydrolysis and spontaneous cyclization, afforded the intermediate deoxylactone methyl 4,6-O-benzylidene-3-C-(carboxymethyl)-alpha-D-glucopyranoside-3',2-lacto ne (8). Stereoselective hydroxylation with MoO5 x py x HMPA, reduction with lithium aluminum hydride and cyclization with diethyl azodicarboxylate-triphenylphosphine gave the target molecule methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(R)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside (5). Direct reduction of 8 gave other analogs having no C-3' hydroxyl group together with having a C-3' hydroxyl group (hemiacetal). In addition, C-3' epimers were also synthesized through C-3', C-3' dihydroxy analogs. Wittig reaction of an appropriate ketosugar with [(ethoxycarbonyl)methylene]triphenylphosphorane leading to a 7:3 Z/E mixture, followed by hydroxylation with osmium tetroxide, reduction and cyclization afforded the target molecule 5 and the miharamycin sugar moiety methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(S)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside. Examination of X-ray data for 5 and its NMR spectroscopy data allowed us to explain a contradiction reported in the literature.