Synthesis of 5-epi-[6-(2)H(2)]valiolone and stereospecifically monodeuterated 5-epi-valiolones: exploring the steric course of 5-epi-valiolone dehydratase in validamycin A biosynthesis.

In validamycin A biosynthesis, as well as that of acarbose, the valienamine and validamine moieties are ultimately derived from a C(7) sugar, sedoheptulose 7-phosphate, which is cyclized to 2-epi-5-epi-valiolone by a cyclase that operates via a dehydroquinate (DHQ) synthase-like mechanism. 2-epi-5-epi-Valiolone is first epimerized at C-2 to give 5-epi-valiolone and then dehydrated between C-5 and C-6 to yield valienone. To probe the dehydration mechanism of 5-epi-valiolone to valienone, stereospecifically 6alpha- and 6beta-monodeuterated 5-epi-valiolones were synthesized. The key step in the synthesis was desulfurization of the tetrabenzyl-6,6-bis(methylthio)-5-epi-valiolone and introduction of the deuterium utilizing Zn, NiCl(2), ND(4)Cl/D(2)O, and THF. Extensive studies using various combinations of protio- and deuteroreagents and solvents probed the mechanism of the reductive desulfurization, which is crucial for the preparation of stereospecifically monodeuterated 5-epi-valiolones. Incorporation experiments with the labeled precursors in the validamycin A producer strain, Streptomyces hygroscopicus var. limoneus, revealed that the dehydration of 5-epi-valiolone to valienone occurs by a syn elimination of water.     

Biosynthesis of the validamycins: identification of intermediates in the biosynthesis of validamycin A by Streptomyces hygroscopicus var. limoneus.

To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in (2)H-, (3)H-, or (13)C-labeled form and fed to cultures of Streptomyces hygroscopicus var. limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintillation counting, (2)H- or (13)C NMR or selective ion monitoring mass spectrometry (SIM-MS) techniques. The results demonstrate that 2-epi-5-epi-valiolone (9) is specifically incorporated into 1 and labels both cyclitol moieties. This suggests that 9 is the initial cyclization product generated from an open-chain C(7) precursor, D-sedoheptulose 7-phosphate (5), by a DHQ synthase-like cyclization mechanism. A more proximate precursor of 1 is valienone (11), which is also incorporated into both cyclitol moieties. The conversion of 9 into 11 involves first epimerization to 5-epi-valiolone (10), which is efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of 2-epi-valienone (15) is also observed. Reduction of 11 affords validone (12), which is also incorporated specifically into 1, but labels only the reduced cyclitol moiety. The mode of introduction of the nitrogen atom linking the two pseudosaccharide moieties is not clear yet. 7-Tritiated valiolamine (8), valienamine (2), and validamine (3) were all not incorporated into 1, although each of these amines has been isolated from the fermentation, with 3 being most prevalent. Demonstration of in vivo formation of [7-(3)H]validamine ([7-(3)H]-3) from [7-(3)H]-12 suggests that 3 may be a pathway intermediate and that the nonincorporation of [7-(3)H]-3 into 1 is due to a lack of cellular uptake. We thus propose that 3, formed by amination of 12, and 11 condense to form a Schiff base, which is reduced to the pseudodisaccharide unit, validoxylamine A (13). Transfer of a D-glucose unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge between the two pseudosaccharide units are also discussed.     

Evolutionary divergence of sedoheptulose 7-phosphate cyclases leads to several distinct cyclic products

Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.     

In vitro characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa

Forosamine (4-dimethylamino)-2,3,4,6-tetradeoxy-beta-D-threo-hexopyranose) is a highly deoxygenated sugar component of several important natural products, including the potent yet environmentally benign insecticide spinosyns. To study D-forosamine biosynthesis, the five genes (spnO, N, Q, R, and S) from the spinosyn gene cluster thought to be involved in the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-D-forosamine were cloned and heterologously expressed, and the corresponding proteins were purified and their activities examined in vitro. Previous work demonstrated that SpnQ functions as a pyridoxamine 5'-monophosphate (PMP)-dependent 3-dehydrase which, in the presence of the cellular reductase pairs ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase, catalyzes C-3 deoxygenation of TDP-4-keto-2,6-dideoxy-D-glucose. It was also established that SpnR functions as a transaminase which converts the SpnQ product, TDP-4-keto-2,3,6-trideoxy-D-glucose, to TDP-4-amino-2,3,4,6-tetradeoxy-D-glucose. The results presented here provide a full account of the characterization of SpnR and SpnQ and reveal that SpnO and SpnN functions as a 2,3-dehydrase and a 3-ketoreductase, respectively. These two enzymes act sequentially to catalyze C-2 deoxygenation of TDP-4-keto-6-deoxy-D-glucose to form the SpnQ substrate, TDP-4-keto-2,6-dideoxy-D-glucose. Evidence has also been obtained to show that SpnS functions as the 4-dimethyltransferase that converts the SpnR product to TDP-D-forosamine. Thus, the biochemical functions of the five enzymes involved in TDP-D-forosamine formation have now been fully elucidated. The steady-state kinetic parameters for the SpnQ-catalyzed reaction have been determined, and the substrate specificities of SpnQ and SpnR have been explored. The implications of this work for natural product glycodiversification and comparative mechanistic analysis of SpnQ and related NDP-sugar 3-dehydrases E1 and ColD are discussed.     

Simultaneous determination of substrate and product in the process of preparation of valienamine by capillary zone electrophoresis

A simple and rapid CZE method was established for the simultaneous determination of valienamine, acarbose and validamycin A, using a 20‐kV CZE with the detection wavelength of 193 nm and 50 mM phosphoric acid–20 mM Tris (pH 5.3) as a running buffer. The calibration curves of valienamine, acarbose, and validamycin A showed a good linear relationship at a concentration range of 5–1000 μg/mL. The detection limits of valienamine, acarbose, and validamycin A were 0.3, 0.6, and 0.6 μg/mL, respectively, and the average recoveries of each of the above were 99.9, 99.5, and 100.3%. The method has been successfully applied for simultaneous determination of substrate and product in the process of preparation of valienamine.     

Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E. coli K12

WecE gene, encoding a sugar aminotransferase (SAT), has been cloned from E. coli K12 and expressed in E. coli BL21 (DE3). The enzyme was purified and characterized. WecE used TDP-4-keto-6-deoxy-D-glucose (TDP-D-Glc4O) and L-glutamate as a good amino acceptor and donor, respectively, leading to the production of TDP-4-amino-4,6-dideoxy-D-galactose (TDP-Fuc4N), which was identified by NMR studies. WecE also showed a similar activity for TDP-4-keto 6-deoxy-D-mannose (TDP-D-Man4O), but no activity for GDP-4-keto-6-deoxy-D-mannose (GDP-D-Man4O), suggesting that the nucleotide moiety would become a key determinant to the substrate specificity of amine acceptor for the activity of the SAT. Multiple alignments showed that SATs have four highly conserved motifs located around the active site and could be divided into three subgroups (VIalpha, VIbeta, and VIgamma) that might be closely related with their substrate specificities.     

Triterpene glycosides from the sea cucumber Eupentacta fraudatrix. Structure and biological action of cucumariosides A1, A3, A4, A5, A6, A12 and A15, seven new minor non-sulfated tetraosides and unprecedented 25-keto, 27-norholostane aglycone

Seven new minor triterpene glycosides, cucumariosides A1 (1), A3 (2), A4 (3), A5 (4), A6 (5), A12 (6) and A15 (7) have been isolated from the Far Eastern sea cucumber Eupentacta fraudatrix. Structures of the glycosides were elucidated by 2D NMR spectroscopy and MS. Glycosides 1-7 belong to the group of cucumariosides A, having linear tetrasaccharide carbohydrate moieties without any sulfate group and possessing 3-O-methyl-D-xylose as a terminal monosaccharide unit. The latter peculiarity is rare in sea cucumber glycosides, but typical for the glycosides from E. fraudatrix. Glycosides 1-7 differ from each other by side chain structures in the aglycone moieties; three of them have unique structural features. The first is the presence of a 25-butoxy-group in the side chain of cucumarioside A3 (2), the second is a 23E,25-diene system in cucumarioside A6 (5) and the third is a 25-keto-27-nor-holostane aglycone in cucumarioside A12 (6); these were never previously found in sea cucumber glycosides. Cytotoxic activity of glycosides 1-7 against mouse spleen lymphocytes and the cells of the ascite form of mouse Ehrlich carcinoma, along with hemolytic activity against mouse erythrocytes and antifungal activity were studied. Glycosides 1 and 5 were the most active in all the tests.     

Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes

The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.     

Total synthesis of the originally proposed and revised structures of palmerolide A and isomers thereof

Palmerolide A is a recently disclosed marine natural product possessing striking biological properties, including potent and selective activity against the melanoma cancer cell line UACC-62. The total syntheses of five palmerolide A stereoisomers, including the originally proposed (1) and the revised [ent-(19-epi-20-epi-1)] structures, have been accomplished. The highly convergent and flexible strategy developed for these syntheses involved the construction of key building blocks 2, 19-epi-2, 20-epi-2, ent-2, 3, ent-3, 4, and ent-4, and their assembly and elaboration to the target compounds. For the union of the building blocks, the Stille coupling reaction, Yamaguchi esterification, Horner-Wadsworth-Emmons olefination, and ring-closing metathesis reaction were employed, the latter being crucial for the stereoselective formation of the macrocycle of the palmerolide structure. The Horner-Wadsworth-Emmons olefination and the Yamaguchi lactonization were also investigated and found successful as a means to construct the palmerolide macrocycle. The syntheses were completed by attachment of the enamide moiety through a copper-catalyzed coupling process.     

Glycal glycosylation and 2-nitroglycal concatenation, a powerful combination for mucin core structure synthesis

A 3,4-O-unprotected galactal derivative having bulky 6-O-TIPS protection (compound 2) could be regioselectively 3-O-glycosylated with O-(galactopyranosyl) trichloroacetimidates; depending on the protecting group pattern stereoselectively alpha- and beta-linked disaccharides were obtained. With O-(2-azido-2-deoxyglucopyransyl) trichloroacetimidate as donor (compound 10A), glycosylation of 2 and of a 6-O-unprotected galactal derivative led in acetonitrile as solvent exclusively to a beta(1-3)- and a beta(1-6)-linked disaccharide, respectively. Nitration of the galactal moieties of the saccharides followed by Michael-type addition of serine and threonine derivatives (7a,b) installed the alpha-galacto-configuration, thus readily furnishing O-glycosyl amino acid building blocks for the incorporation of core 1, core 2, core 3, core 6, and core 8 structures into glycopeptides. 2-Nitrogalactal and 2-nitroglucal derivatives could be also successfully employed in glycoside bond formation via Michael-type addition in a reiterative manner, affording the corresponding core 5, core 7, and core 6 building blocks. In this approach, highly stereoselective glycoside bond formations were based exclusively on Michael-type addition to the nitro-enol ether moiety of the 2-nitroglycals. Hence, 2-nitroglycals are versatile intermediates for base-catalyzed glycoside bond formation.     

Hydroxy-1-aminoindans and derivatives: preparation, stability, and reactivity

The chemical stability and reactivity of hydroxy-1-aminoindans and their N-propargyl derivatives are strongly affected by the position of the OH group and its orientation relative to that of the amino moiety. Thus, the 4- and 6-OH regioisomers were found to be stable, while the 5-OH analogues were found to be inherently unstable as the free bases. The latter, having a para orientation between the OH and the amino moieties, could be isolated only as their hydrochloride salts. 7-Hydroxy-1-aminoindans and 7-hydroxy-1-propargylaminoindans represent an intermediate case; while sufficiently stable even as free bases, they exhibit, under certain experimental conditions, unexpected reactivity. The instability of the 5- and 7-hydroxy-aminoindans is attributed to their facile conversion to the corresponding, reactive quinone methide (QM) intermediates. The o-QM obtained from 7-hydroxy-aminoindans was successfully trapped with ethyl vinyl ether via a Diels-Alder reaction to give tricyclic acetals 32a,b.     

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase, TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce TDP-D-ravidosamine routinely for glycosylation studies.     

Synthesis and characterization of bioorganometallic conjugates composed of NCN-pincer platinum(II) complexes and uracil derivatives

The conjugation of the NCN-pincer platinum(II) complexes as an oraganometallic compound and the uracil derivatives as a nucleobase was demonstrated to give the corresponding bioorganometallics. The NCN-pincer ligands bearing the 6-ethynyl-1-octyluracil, 5-ethynyl-1-octyluracil, and the furanopyrimidine moiety were synthesized. In a crystal state, the NCN-pincer ligand bearing the 6-ethynyl-1-octyluracil moiety was found to form a hydrogen-bonded dimer through intermolecular hydrogen bonds between the uracil moieties, which was connected through π-π interaction between the uracil and benzene moieties of the NCN-pincer ligand. The reaction of the NCN-pincer ligand bearing the 6-ethynyl-1-octyluracil moiety with [Pt(tolyl-4)₂(SEt₂)]₂ led to the formation of the NCN-pincer platinum(II) complex bearing the 6-ethynyl-1-octyluracil moiety. The NCN-pincer platinum(II) complex bearing the furanopyrimidine moiety was obtained by the reaction of the NCN-pincer ligand bearing the furanopyrimidine moiety with [Pt(tolyl-4)₂(SEt₂)]₂. The single-crystal X-ray structure determination of the NCN-pincer platinum(II) complex bearing the furanopyrimidine moiety revealed the formation of the furanopyrimidine ring and the π stack dimer between the furanopyrimidine and benzene moieties of the NCN-pincer ligand in the crystal packing. The NCN-pincer platinum(II) complexes bearing the 6-ethynyl-1-octyluracil moiety or the furanopyrimidine moiety exhibited emission in both solution and solid states.     

Synthesis and Characterization of 5‐(1,2,4‐Triazol‐3‐yl)tetrazoles with Various Energetic Functionalities

In this contribution the synthesis and full structural as well as spectroscopic characterization of three 5‐(1,2,4‐triazol‐3‐yl)tetrazoles along with selected energetic moieties like nitro, nitrimino, and azido groups are presented. The main goal is a comparative study on the influence of those variable energetic moieties on structural and energetic properties. A complete characterization including IR and Raman as well as multinuclear NMR spectroscopy of all compounds is presented. Additionally, X‐ray crystallographic measurements were performed and reveal insights into structural characteristics as well as inter‐ and intramolecular interactions. The standard enthalpies of formation were calculated for all compounds at the CBS‐4M level of theory and reveal high positive heats of formation for all compounds. The calculated detonation parameters (using the EXPLO5.05 program) are in the range of 8000 m s^?1 (8097 m s^?1 ( 5 ), 8020 m s^?1 ( 6 ), 7874 m s^?1 ( 7 )). As expected, the measured impact and friction sensitivities as well as decomposition temperatures strongly depend on the energetic moiety at the triazole ring. The C? C connection of a triazole ring with its opportunity to introduce a large variety of energetic moieties and a tetrazole ring, implying a large energy content, leads to the selective synthesis of primary and secondary explosives.     

Enaminone substitutents attached to cyclopentadienes: 3E/3Z stereochemistry of 1-metalla-1,3,5-hexatriene intermediates (M = Cr, W) as a functional criterion for the formation of cyclopentadienes and six-membered heterocycles, respectively

Reactions of NH-enaminones 2 with [2-(1-cycloalkenyl)ethynyl]carbene complexes 7 (M=W, Cr) gave tetrahydropentalenes, tetrahydroindenes, and hexahydroazulenes 8a-i, in which the NH-enaminone moiety is attached to the cyclopentadiene unit. The reaction involved formation of (3E)-1-metalla-1,3,5-hexatriene intermediates, which underwent pi cyclization faster than 3E/3Z isomerization. Tungsten complexes 12 and 13 were characterized as reaction intermediates. Compounds 8 are potentially bidentate ligands with respect to coordination both of the cyclopentadienyl and the enaminone moieties.     

Synthesis and biological activity of kalkitoxin and its analogues

Total syntheses of kalkitoxin, isolated from the Caribbean Lyngbya majuscula, and its analogues, 3-epi-, 7-epi-, 8-epi-, 10-epi-, 10-nor-, and 16-nor-kalkitoxin, were achieved via oxazolidinone-based diastereoselective 1,4-addition reaction of a methyl group and efficient TiCl(4)-mediated thiazoline ring formation as the key steps. The biological activities of synthetic kalkitoxin and its analogues were evaluated with brine shrimp.     

Liquid-Crystalline Polyurethanes. 5. Polyurethanes Containing the Cholesterol Moiety in the Side Chain

In the preceding papers of this series, we reported the synthesis of liquid-crystalline polyurethanes containing mesomorphic moieties in the main chain and in side chains [1–4]. Liquid crystalline polymers with side groups containing the cholesterol moiety have also been studied [5, 6]. The main focus of attention has centered around the study of phase transitions of acrylic and methacrylic derivatives of cholesterol. This paper describes the synthesis of a new type of liquid-crystalline polyurethane containing the cholesterol moiety in side chains.     

Triterpene glycosides from the sea cucumber Eupentacta fraudatrix. Structure and cytotoxic action of cucumariosides A2, A7, A9, A10, an, A13 and A14, seven new minor non-sulfated tetraosides and an aglycone with an uncommon 18-hydroxy group

Seven new minor triterpene glycosides, cucumariosides A2 (1), A7 (2), A9 (3), A10 (4), A11 (5), A13 (6) and A14 (7) have been isolated from the Far Eastern sea cucumber Eupentacta fraudatrix. Structures of the glycosides were elucidated by 2D NMR spectroscopy and MS. Glycosides 1-7 belong to the group of cucumariosides A, having linear tetrasaccharide carbohydrate moieties without any sulfate group and possessing 3-O-methyl-D-xylose as a terminal monosaccharide unit. Glycosides 1, 2, 5-7 differ from each other in side chain structures in aglycone moieties, while cucumarioside A10 (4) has a 23,24,25,26,27-pentanorlanostane aglycone with 18(16)-lactone. Cucumarioside A9 (3), having an uncommon 18-hydroxy group, is the second representative of the unique metabolically active glycosides that are regarded as intermediates of glycoside biosynthesis in sea cucumbers. Cytotoxic activities of glycosides 1-7 and cucumarioside A8 (8) against mouse spleen lymphocytes and the cells of the ascite form of mouse Ehrlich carcinoma, along with hemolytic activity against mouse erythrocytes and antifungal activity were studied. Cucumariosides A2 (1), A8 (8) and A13 (6) demonstrated high hemolytic activities. Glycosides 1, 4 and 6 showed moderate cytotoxic activity. Only cucumarioside A8 (8), having an 18-oxymethylene group and a 24(25)-double bond, was very active in all the tests.     

Liquid chromatography coupled to nuclear magnetic resonance spectroscopy for the identification of isoflavone glucoside malonates in T. pratense L. leaves

Previous studies revealed that the main isoflavones in extracts of leaves of T. pratense L. are biochanin A and formononetin, their 7-O-glucosides, and two glucoside malonate isomers of each of them. Since LC-MS(/MS) did not provide sufficient information to distinguish the glucoside malonate isomers, in the present paper LC-NMR as well as off-line two-dimensional NMR were used to obtain further structural information. Matrix solid-phase dispersion (MSPD) was applied to obtain sufficiently high analyte concentrations to perform LC-NMR. Stop-flow reversed-phase LC-NMR was performed using a gradient of deuterated water and deuterated acetonitrile. Offline COSY and NOESY experiments were carried out to determine the positions of the glucose moiety on the flavonoid aglycone, and of the malonate moiety on the glucose. Based on the fragmentation patterns in MS/MS and the NMR spectra, the two formononetin glucoside malonate isomers were identified as 7-O-beta-D-glucoside 6"-O-malonate and 7-O-beta-D-glucoside 4"-O-malonate; i.e. they only differ in the substitution position of the malonate group on the glucoside ring. The biochanin A glucoside malonate isomers, however, have quite different structures. The main and later eluting isomer is biochanin A 7-O-beta-D-glucoside 6"-O-malonate, and the minor and earlier eluting isomer is 5-hydroxy-7-methoxyisoflavone 4'-O-beta-D-glucoside 4"-O-malonate: the positions of the methoxy group and the glucoside 6"-O-malonate group on the flavonoid skeleton are interchanged.     

Gas chromatographic determination of (phosphorylated) 2-keto-3-deoxyoctonic acid, heptoses and glucosamine in bacterial lipopolysaccharides after treatment with hydrofluoric acid, methanolysis and trifluoroacetylation

Quantification of phosphorylated sugar constituents of lipopolysaccharides has been performed by the following sequence: dephosphorylation by treatment with hydrofluoric acid, cleavage to monomeric constituents by methanolysis and analysis of the released sugars by capillary gas chromatography. Lipopolysaccharides of Salmonella minnesota Rd1P+, Bordetella pertussis NIH 114 and Vibrio cholerae, NAG and 95R strains, were used as model substances. Comparison of the chromatographic data obtained from hydrofluoric acid-treated and untreated lipopolysaccharide preparations indicated that all lipopolysaccharides examined contained one moiety of glucosamine bound to phosphate in a stable linkage. 2-Keto-3-deoxyoctonic acid appeared phosphorylated to a variable extent. Lipopolysaccharides of the two V. cholerae strains contained one moiety of fully phosphorylated 2-keto-3-deoxyoctonic acid, whereas in that of S. minnesota Rd1P+ only one of the three moieties was phosphorylated. Lipopolysaccharide of B. pertussis had one moiety of 2-keto-3-deoxyoctonic acid, ca. 70% phosphorylated. All four of the preparations examined contained L-glycero-D-manno-heptose in amounts varying from 2.6 to 5.2 moieties. In the lipopolysaccharides of B. pertussis and strain 95R of V. cholerae this sugar was unphosphorylated, whereas the two remaining strains contained one phosphorylated moiety of this sugar. Phosphorylated lipopolysaccharide constituents can be analysed by this approach on a 50-100 micrograms scale.