Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

Stereoselective synthesis of a C-glycosylic compound (a "methyl C-glycoside") through a regioselective free-radical ring-opening reaction. A single-crystal X-ray structure determination

Readily available 3,4,6-tri-O-acetyl-D-glucal was converted to 2,6-anhydro-5,7-O-benzylidene-1,3,4-trideoxy-D-arabino-hept-3-enitol, a methyl C-glycosylic compound. Cyclopropanation of 4,6-O-benzylidene-D-glucal, followed by tributylstannyl radical-mediated regioselective ring opening of the 1,2-cyclopropano sugar led to a 2,6-anhydro-1-deoxyheptose, (a "methyl C-beta-D-glycoside"). The stereochemistry of the 1,2-cyclopropano sugar and the "methyl C-glycoside" were confirmed by single-crystal X-ray diffraction studies.     

Convenient preparation of 3,5-anhydro- and 2,5-anhydropentofuranosides, and 5,6-anhydro-D-glucofuranose by use of the Mitsunobu reaction

Methyl 3,5-anhydro-alpha-D-xylofuranosides are obtained by use of the Mitsunobu reaction from 2-O-protected methyl alpha-D-xylofuranosides, which are easily prepared from D-xylose. The Mitsunobu reaction of methyl 3-N-benzylamino-3-deoxy- and 3-azido-3-deoxyarabinofuranosides, which are prepared from the conveniently available methyl 2,3-anhydro-alpha-D- and 2,3-anhydro-alpha-l-lyxofuranosides by nucleophilic ring opening, yields the corresponding methyl 2,5-anhydro-alpha-D- and 2,5-anhydro-alpha-l-arabinofuranosides. Ring opening of 3,5-anhydro-1,2-O-isopropylidene-alpha-D-xylofuranose with azide yields the corresponding 5-azido derivative. The structure and configuration of the products is confirmed by NMR spectroscopy. 5,6-Anhydro-1,2-O-isopropylidene-alpha-D-glucofuranose is formed by the Mitsunobu reaction of 1,2-O-isopropylidene-alpha-D-glucofuranose. Its structure is verified by single-crystal X-ray diffraction analysis.     

DBU assisted expeditious synthesis of glycosyl dienes via glycosylated beta-hydroxy esters

DBU catalyzed condensation of 3-O-benzyl(methyl)-5,6-dideoxy-1,2-O-isopropylidene-beta-L-threo-hept-4-enofuranuronates with different aldehydes produces the corresponding 3-O-benzyl(methyl)-6-carbethoxy-5,6-dideoxy-1,2-O-isopropylidene-7-phenyl-beta-L-threo-hept-4-enofuranoses. The latter on treatment with methanesulfonyl chloride followed by DBU catalyzed E2 reaction of the methanesulfonyloxy intermediates gave the respective 3-O-benzyl(methyl)-6-carbethoxy-5,6,7-trideoxy-1,2-O-isopropylidene-7-phenyl-beta-L-threo-hept-4,6-dienofuranose in moderate to good yields.     

Acyloxy neighboring-group participation in the acid-catalyzed cleavage of methyl 2,3-anhydro-beta-D-ribofuranoside.

The reaction of methyl 2,3-anhydro-beta-D-ribofuranoside with hydrogen bromide in an acetic acid-acetic anhydride solution leads to the formation of methyl 2,3-di-O-acetyl-5-bromo-5-deoxy-alpha,beta-D-xylofuranoside. Similar treatment of methyl 2,3-anhydro-5-O-benzoyl-beta-D-robofuranoside provided methyl 2-O-acetyl-3-O-benzoyl-5-bromo-5-deoxy-alpha,beta-D-xylofuranosides. The position of halogen substitution was ascertained by hydrogenolysis to the resultant 5-deoxy sugars, which were characterized by their n.m.r. spectra. Confirmation of the structural assignment for methyl 2-O-acetyl-3-O-benzoyl-5-deoxy-alpha,beta-D-xylofuranoside was obtained by synthesis from 1,2-O-isopropylidene-alpha-D-xylofuranose. The formation of the 5-bromo derivatives under the reported conditions probably occurred through the intermediacy of the 3,5-acyloxonium ions. Similar conversions were observed when the starting compound was treated with hydrogen chloride, acetyl bromide, or acetyl chloride in acetic acid-acetic anhydride solutions.     

Stereochemistry of a methyl-group rearrangement during the biosynthesis of lanosterol.

1. (3RS,6R)-[6-2H1,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3H1,6-14C] and (3RS)-[6-3H1,6-14C]mevalonolactones were synthesised from R-[2H1,3H1,2-14C], S-[2H1,3H1,2-14C] and [3h1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-1,4-diene-3,17-dione by incubation with Mycobacterium phlei in the presence of 2,2'.dipyridyl. Each specimen of androsta-1,4-diene-3,17-dione was converted into androsta-1,4-dien-3-one-17-ethylene ketail. 4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-14C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -- 5 were tested with [3H1,2-14C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2S-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2H1,6-3H1,6-14C]mevalonate gave acetic acid which was converted into 2S-malate that retained 68.6% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-E16-2H1,6-3H1,6-14C]mevalonate was oxidised to acetic acid which was converted into 2S-malate that retained 31.9% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 10. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is cncluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.     

Mass spectra of methyl sterculate and malvalate and 1,2-dialkylcyclopropenes

The mass spectra of 1,2-dipropyl-, 1,2-dipentyl-, 1,2-dihexyl-, 1,2-diheptyl-, and 1,2-dioctyl-cyclopropene, methyl malvalate, methyl sterculate, malvalyl alcohol, 1,2-dipropyl-, 1,2-dipentyl-, and 1,2-dihexylcyclopropene-3-carboxylic acid, and methyl-9,10-(carbethoxymethano)-9-octadecenoate are presented. A noticable feature of the 1,2-disubstituted cyclopropene spectra is the total absence of a cyclopropenium ion. The cyclopropenes with a carboxyl group in the 3-position yield cyclopropenium ions in the mass spectra. β-Cleavage to a allylic ion appears to be important.     

Multiply 13C-substituted monosaccharides: synthesis of D-(1,5,6-13C3)glucose and D-(2,5,6-13C3)glucose.

D-(1,5,6-13C3)Glucose (7) has been synthesized by a six-step chemical method. D-(1,2-13C2)Mannose (1) was converted to methyl D-(1,2-13C2)mannopyranosides (2), and 2 was oxidized with Pt-C and O2 to give methyl D-(1,2-13C2)mannopyranuronides (3). After purification by anion-exchange chromatography, 3 was hydrolyzed to give D-(1,2-13C2)mannuronic acid (4), and 4 was converted to D-(5,6-13C2)mannonic acid (5) with NaBH4. Ruff degradation of 5 gave D-(4,5-13C2)arabinose (6), and 6 was converted to D-(1,5,6-13C3)glucose (7) and D-(1,5,6-13C3)mannose (8) by cyanohydrin reduction. D-(2,5,6-13C3)Glucose (9) was prepared from 8 by molybdate-catalyzed epimerization.     

Synthesis of derivatives of C-nucleoside analogues using 'push-pull' functionalized monosaccharides.

7-Deoxy-1,2:3,4-di-O-isopropylidene-alpha-D-galacto-heptopyranos-6-ulose (1) reacted with carbon disulphide and methyl iodide in the presence of a base to furnish 7,8-dideoxy-1,2:3,4-di-O-isopropylidene-8,8-[bis(methylthio)]-alpha-D-galacto-oct-7-enopyranos-6-ulose (2). This 'push-pull' activated unsaturated monosaccharide underwent a ring closure reaction with hydrazine hydrate to give the 'inversed' C-nucleoside analogue 3. Compound 1 and malononitrile yielded the 7-cyano-6,7-dideoxy-1,2:3,4-di-O-isopropylidene-6-methyl-alpha-D-galacto-oct-6-enopyranurononitrile (4). Treatment of 4 with carbon disulphide and methyl iodide in the presence of a base afforded the sugar 'push-pull' butadiene 5 which was transformed into the pyridine nucleoside analogue 6.     

Synthesis of 3-C-(methyl beta-D-xylofuranosid-3-yl)-5-phenyl-1,2,4-oxadiazole.

Nucleophilic addition of KCN to 5-O-benzoyl-1,2-O-isopropylidene-alpha-D-erythro-pentofuranos-3-++ +ulose gave the xylo cyanohydrin stereoselectively. Several xylos-3-yl-1,2,4-oxadiazole derivatives were synthesized from this cyanohydrin and were converted into 3-C-(methyl beta-D-xylofuranosid-3-yl)-5-phenyl-1,2,4-oxadiazole.     

Substrate specificity of coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator.

The substrate specificity of adenosylcobalamin-dependent diol dehydrase was further studied in detail using an enzyme preparation that appears homogeneous by ultracentrifugal and gel electrophoretical criteria. Besides 1,2-propanediol and 1,2-ethanediol, glycerol, 1,2- and 2,3-butanediol were found to serve as substrate for the enzyme, whereas 1,3-propanediol was not. Of the substrate analogs tested, glycerol displayed some striking features: it was dehydrated to β-hydroxypropionaldehyde with concomitant inactivation of the enzyme. Although the initial velocity with glycerol was comparable to that with 1,2-propanediol, the dehydration reaction ceased almost completely within 3 min accompanying rapid, irreversible inactivation of the holoenzyme. 1,2- and 2,3-Butanediol were converted to butyraldehyde and methyl ethyl ketone, respectively, at a rate much lower than that with 1,2-propanediol. 2,3-Butanediol is the only compound, other than 1,2-diols, known at present to show a considerable substrate activity.     

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.     

Synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 14. 2. Synthesis of methyl-6-O-acetyl-4-O-(2,3,46-tetra-O-benzoyl -beta-D-galactosylpyranosyl)-2-deoxy-2-phthalimido-beta-D-gluco- pyranoside--a lactosamine precursor in the monomer synthesis for polycondensation

Glycosylation of methyl 6-O-acetyl-3-O-benzoyl-2-deoxy-phthalimido-beta-D-glucopyranoside and its 4-trityl ether by benzobromogalactose, 1-O-acetyl-2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranose, 1,2-[(alpha-p-tolylthio)benzylidene]- and 1,2-O-[(alpha-cyano)benzylidene]-3,4,6-tri-O-benzoyl-alpha-D- galactopyranoses proceeds non-stereospecifically. The best yield of beta-linked disaccharide was obtained upon glycosylation by benzobromogalactose in the presence of silver triflate and tetramethylurea in nitromethane.     

Formation of novel nitrofuran metabolites in xanthine oxidase-catalyzed reduction of methyl 5-nitro-2-furoate

After methyl 5-nitro-2-furoate was incubated with milk xanthine oxidase, three reduction products were isolated from the incubation mixture. Among them, two reduction products were new types of nitrofuran metabolites, i.e., metabolites 1 and 2 were identified as the dihydroxyhydrazine derivative (1,2-dihydroxy-1,2-di(5-methoxycarbonyl-2-furyl) hydrazine) and the hydroxylaminofuran derivative (methyl 5-hydroxylamino-2-furoate), respectively. Metabolite 3 was also identified as the aminofuran derivative (methyl 5-amino-2-furoate) by comparison with a synthetic sample.     

Biodegradation of Halogenated Hydrocarbon Fumigants by Nitrifying Bacteria

Three species of nitrifying bacteria were tested for the ability to degrade the halocarbon fumigants methyl bromide, 1,2-dichloropropane, and 1,2-dibromo-3-chloropropane. The soil nitrifiers Nitrosomonas europaea and Nitrosolobus multiformis degraded all three fumigants, while the marine nitrifier Nitrosococcus oceanus degraded only methyl bromide under the conditions tested. Inhibition of biodegradation by allylthiourea and acetylene, specific inhibitors of ammonia monooxygenase, suggests that ammonia monooxygenase is the enzyme which catalyzes fumigant degradation.     

Antileukemic effects of Didemnum psammatodes (Tunicata: Ascidiacea) constituents

Chemical investigation of the methanolic extract of the ascidian Didemnum psammatodes has led to the identification of fourteen known compounds: three methyl esters (methyl myristate, methyl palmitate and methyl stearate), four steroids (cholesterol, campesterol, stigmasterol and beta-sitosterol), two fatty acids (palmitic acid and stearic acid), three glyceryl ethers {(1,2-propanediol, 3-(heptadecyloxy), batyl alcohol and 1,2-propanediol, 3-[(methyloctadecyl)oxy]} and two nucleosides (thymidine and 2'-deoxyguanosine). Their structures were proposed by NMR and comparison with literature data and GC analysis in comparison with authentic sample. The cytotoxic activity of these compounds was evaluated against human leukemia cell line panel using the MTT assay. The mixture of the three methyl esters was the most active group of compounds, showing antiproliferative and cytotoxic effects. Further studies on their mode of action suggest that these activities are connected with inhibition of DNA synthesis and induction of both necrosis and apoptosis.     

Specificity of the Westerfeld adaptation of the Voges-Proskauer test.

Aliphatic chain compounds at least four carbons long with vicinal carbonyl groups in the 2,3 positions were detected by the Westerfeld test. Acetoin, which has one carbonyl group and an adjacent hydroxyl group, gave positive results, but methyl action (3-hydroxy-3-methyl-2-butanone) was negative, and subsequent tests supported the conclusion that acetoin is oxidized to diacetyl by alpha-naphthol during the Westerfeld test in the absence or presence of air. 2,3-Pentanedione and 2,3-heptanedione gave positive results, but equimolar concentrations of these compounds gave maximal absorbancy readings that were only 35% (2,3-pentanedione) and 31% (2,3-heptanedione) of those obtained with diacetyl or acetoin. Negative results were obtained with pyruvic acid, 2,3-butylene glycol, and carbon ring compounds (1,2-cyclohexanedione, alloxan, and 3,4-dihydroxy-3-cyclobutene-1,2-dione). alpha-Naphtho could not be replaced in the test by beta-naphthol, 1,2,3,4,-tetrahydroxy-1-naphthol, or 5,6,7,8-tetrahydroxy-1-naphthol. Creatine could not be replaced by arginine, guanidine . HCl, or guanidinoacetic acid.     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Synthesis of an L-rhamnose tetrasaccharide, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci.

A tetrasaccharide, alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-L-Rhap, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci was synthesized as its methyl and octyl glycosides. Selective 3-O-glycosylation of allyl alpha-L-rhamnopyranoside with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl trichloroacetimidate gave allyl 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranoside (3). Benzoylation, deallylation, and trichloroacetimidation afforded 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (6). Self condensation of 3,4-di-O-benzoyl-beta-L-rhamnopyranose 1,2-methyl orthoester or 1,2-octyl orthoester gave methyl or octyl 2-O-acetyl-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranoside (16 or 17), and subsequent selective deacetylation gave the disaccharide acceptor (18 or 19). Coupling of 6 with 18 (or 19), followed by deacylation in ammonia-saturated methanol, produced the target tetrasacharide.     

The effect of acid proteinase inhibitors on chicken pepsin.

1. The activity of chicken pepsin was partially inhibited by dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide, but was unaffected by p-bromophenacyl bromide. 2. In the presence of Cu2+, diazoacetylnorleucine methyl ester completely inactivated chicken pepsin with the incorporation of 1 mol/mol. The mechanism of the reaction was similar to that with pig pepsin. 3. Chicken pepsin was completely inactivated by 2-diazo-4-bromoacetophenone in the presence of Cu2+. 4. Chicken pepsin was almost completely inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 25 degrees C, 3-4mol of inhibitor/mol being incorporated. The reaction at 10 degrees C was investigated briefly. 5. Calf chymosin was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 10 degrees C, the incorporation of 1 mol/mol being required for complete inhibition. 6. The characteristics of the reactions of chicken pepsin with the above compounds were compared with those of other acid proteinases.