Structural revision of shatavarins I and IV, the major components from the roots of Asparagus racemosus

The two major steroidal saponins from the roots of Asparagus racemosus were isolated by RP-HPLC and their structure determined by extensive NMR studies. Their structures did not match those reported previously for shatavarins I and IV and were found to be 3-O-{[β-d-glucopyranosyl(1→2)][α-l-rhamnopyranosyl(1→4)]-β-d-glucopyranosyl}-26-O-(β-d-glucopyranosyl)-(25S)-5β-furostan-3β,22α,26-triol and 3-O-{[β-d-glucopyranosyl(1→2)][α-l-rhamnopyranosyl(1→4)]-β-d-glucopyranosyl}-(25S)-5β-spirostan-3β-ol.     

Thyonosides A and B, two new saponins isolated from the holothurian Thyone aurea

The structures of two saponins, thyonosides A and B, isolated from the holothurian Thyone aurea collected in Namibia, were elucidated by 1D and 2D NMR (¹H, ¹³C, ¹H-¹H COSY, ¹H-¹H J-resolved, TOCSY, HMQC, HMBC and NOESY). The two compounds have the same aglycon but different oligosaccharidic chains. Thyonoside A has a 3-O-methyl-β-d-xylopyranosyl-(1→3)-6-O-sodium sulphate-β-d-glucopyranosyl-(1→4)-β-d-quinovopyranosyl-(1→2)-4-O-sodium sulphate-β-d-xylopyranosyl chain, and thyonoside B a 3-O-methyl-β-d-xylopyranosyl-(1→4)-β-d-xylopyranosyl-(1→4)-β-d-quinovopyranosyl-(1→2)-4-O-sodium sulphate-β-d-xylopyranosyl chain. The holostane-type aglycon features an endocyclic double bond at position 7-8, a double bond at position 25-26 and a β-acetoxy group at C16.     

Isolation of a polysaccharide with anticancer activity from Auricularia polytricha using high-speed countercurrent chromatography with an aqueous two-phase system

Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000–K₂HPO₄–KH₂PO₄–H₂O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy (¹H, ¹³C). These results showed AAPS-2 is a polysaccharide with a backbone of (1 → 3)-linked-β-d-glucopyranosyl and (1 → 3, 6)-linked-β-d-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1→)-β-d-glucopyranosyl at the O-6 position of (1→3, 6)-linked-β-d-glucopyranosyl of the main chain.     

Cyclooxygenase-2 enzyme inhibitory withanolides from Withania somnifera leaves

Four novel withanolide glycosides and a withanolide have been isolated from the leaves of Withania somnifera. The structures of the novel compounds were elucidated as physagulin D (1→6)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (1), 27-O-β-d-glucopyranosyl physagulin D (2), 27-O-β-d-glucopyranosyl viscosalactone B (3), 4,16-dihydroxy-5β, 6β-epoxyphysagulin D (4), and 4-(1-hydroxy-2,2-dimethylcyclo-propanone)-2,3-dihydrowithaferin A (5) on the basis of 1D-, 2D NMR and MS spectral data. In addition, seven known withanolides withaferin A (6), 2,3-dihydrowithaferin A (7), viscosalactone B (8), 27-desoxy-24,25-dihydrowithaferin A (9), sitoindoside IX (10), physagulin D (11), and withanoside IV (12) were isolated. These withanolides were assayed to determine their ability to inhibit cycloxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes and lipid peroxidation. The withanolides tested, except compound 9, showed selective COX-2 enzyme inhibition ranging from 9 to 40% at 100 μg/ml. Compounds 4, 10 and 11 also inhibited lipid peroxidation by 40, 44 and 55%, respectively. The inhibition of COX-2 enzyme by withanolides is reported here for the first time.     

Agariblazeispirols A and B, an unprecedented skeleton from the cultured mycelia of the fungus, Agaricus blazei

Agariblazeispirols A and B, which have a unique steroidal skeleton, have been isolated from the cultured mycelia of Agaricus blazei (Agaricaceae). The absolute structure of Agariblazeispirol A was established to be (20S,22R,23R,24S)-13β,22:22,25-diepoxy-5-methoxy-14β-methyl-18-nor-des-A-ergosta-5,7,9,11-tetraen-23-ol by extensive 1D and 2D NMR spectral data, and X-ray analysis. The structure of Agariblazeispirol B was elucidated to be a stereoisomer of agariblazeispirol A at its carbon 22, (20S,22S,23R,24S)-13β,22:22,25-diepoxy-5-methoxy-14β-methyl-18-nor-des-A-ergosta-5,7,9,11-tetraen-23-ol by comparison of extensive 1D and 2D NMR spectral data with those of agariblazeispirol A. Both compounds showed a moderate circumvention of drug resistance on mouse leukemia P388/VCR cells.     

Structure of tecophilin,a tri-caffeoylanthocyanin from the blue petals of Tecophilaea cyanocrocus, and the mechanism of blue color development

The pigment, tecophilin, in blue flowers of Tecophilaea cyanocrocus was isolated and the structure was determined to be 3-O-(6-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-7-O-(6-O-(4-O-(2-O-(4-O-β-d-glucopyranosyl-(E)-caffeoyl)-6-O-(4-O-β-d-glucopyranosyl-(E)-caffeoyl)-β-d-glucopyranosyl)-(E)-caffeoyl)-β-d-glucopyranosyl)delphinidin. The reproduction experiment of the same color as petals according to the results of chemical analysis and measurement of vacuolar pH of blue cells clarified that the blue color solely develops by tecophilin without interaction of metal ions nor co-pigments. ¹H NMR analysis and CD spectrum indicate the co-existence of clockwise intermolecular self-association of the delphinidin nuclei and intramolecular π–π stacking between the chromophore and caffeoyl residues to derive bathochromic shift of the absorption spectrum and stabilize the color by preventing hydration reaction.     

Preparative isolation and purification of isobenzofuranone derivatives and saponins from seeds of Nigella glandulifera Freyn by high-speed counter-current chromatography combined with gel filtration

Although the medicinal plant and food Nigella glandulifera Freyn has been researched for decades, isobenzofuranones have never been isolated before. Two isobenzofuranone derivatives and two saponins were successfully separated and purified from seeds of N. glandulifera Freyn by high-speed counter-current chromatography (HSCCC) with the optimized two-phase solvent system, n-hexane-ethyl acetate–methanol–water (7:3:5:5, v/v). Salfredin B₁₁ (22.1 mg, HPLC purity 95.3%), 5, 7-dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one (18.9 mg, HPLC purity 97.3%) and crude sample 2 (555 mg) were separated from 600 mg of ethyl acetate extract of N. glandulifera Freyn. Following a cleaning-up step by chromatography on Sephadex LH-20, hederagenin (12 mg) and 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin (45 mg) were separated from sample 2. All of the fractions before peak II were collected and subjected to a Sephadex LH-20 column and eluted by methanol, two of triterpene saponins (12 mg of hederagenin and 45 mg of 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin) were isolated. The structures of peak fractions were identified by IR, electron ionization MS, ¹H NMR and ¹³C NMR. 5, 7-Dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one was isolated for the first time from higher plant and salfredin B11 was isolated for the first time in this plant.     

Synthesis of the Lewis b pentasaccharide and a HSA-conjugate thereof

Helicobacter pylori, a gastric pathogen, binds to various blood group antigens, including the Lewis types, present in the gastric tissue and a relation between the presentation of the ligands and the overall strength of binding has been assumed. Synthetic Lewis b tetra- and hexasaccharide conjugates are available but not the analogous pentasaccharide. An efficient synthesis of the amino spacer equipped Lewis b pentasaccharide, 3-aminopropyl α-l-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→3)-[α-l-fucopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→3)-β-d-galactopyranoside, is presented to enable further investigation of the carbohydrate recognition process of H. pylori.     

A general method for the synthesis of oligosaccharides consisting of α-(1→2)- and α-(1→3)-linked rhamnan backbones and GlcNAc side chains

A general method has been developed for the synthesis of oligosaccharides consisting of (1→2)- and (1→3)-linked rhamnans with GlcNAc side chains. As examples, highly effective and convergent syntheses of two decasaccharides in the O polysaccharide moiety of the lipopolysaccharide of the phytopathogenic bacterium Pseudomonas syringae pv. ribicola NCPPB 1010 were achieved. The two decasaccharides consist of O polysaccharide repeating units I+II and II+I, respectively. Allyl 3-O-acetyl-4-O-benzoyl-α-l-rhamnopyranoside, allyl 2-O-benzoyl-3-O-chloroacetyl-α-l-rhamnopyranoside, 2,4-di-O-benzoyl-3-O-chloroacetyl-α-l-rhamnopyranosyl trichloroacetimidate, and 3-O-acetyl-2,4-di-O-benzoyl-α-l-rhamnopyranosyl trichloroacetimidate, which were obtained by highly regioselective 3-O-acylations, were used as the key synthons to obtain the required α-(1→2)- and α-(1→3)-linked rhamnoocta saccharide acceptors with 3³- and 3⁷-free hydroxyl groups. Therefore, several disaccharides were synthesized, from which tetrasaccharides and hexasaccharides were then synthesized. Coupling of the hexasaccharide donors with the disaccharide acceptors gave the octasaccharide acceptors. Finally, the coupling of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl trichloroacetimidate with the octasaccharide acceptors, followed by deprotection, afforded the two target decasaccharides. A repeating hexasaccharide unit of the cell wall polysaccharide of β-hemolytic Streptococci Group A was also synthesized in a similar way.     

C-(β-d-Glucopyranosyl)formamidrazones,formic acid hydrazides and their transformations into 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazoles: a synthetic and computational study

Synthesis of O-perbenzoylated 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazoles, precursors of potent inhibitors of glycogen phosphorylase, was studied by ring closures of N¹-acyl-carboxamidrazone type intermediates. Reactions of C-(β-d-glucopyranosyl)formimidate or C-(β-d-glucopyranosyl)formamidine with acid hydrazides as well as acylation of C-(β-d-glucopyranosyl)formamidrazone by acid chlorides unexpectedly gave the corresponding 1,3,4-oxadiazoles instead of 1,2,4-triazoles. The desired triazoles were obtained in reactions of C-(β-d-glucopyranosyl)formamidine or C-(β-d-glucopyranosyl)formyl chloride with arenecarboxamidrazones, and also in acylations of N¹-tosyl-C-(β-d-glucopyranosyl)formamidrazone with acid chlorides. Theoretical calculations (B3LYP and M06-2X DFT with the standard 6-31G(d,p) basis set) on simple model compounds with methyl and phenyl substituents to understand the bifurcation of the ring closure of N¹-acyl-carboxamidrazones indicated that in general the reaction led to 1,2,4-triazoles. However, the probability of the 1,3,4-oxadiazole forming pathway was shown to be significantly higher with N¹-benzoyl-acetamidrazones, which were closest analogues of the intermediates resulting in C-glucosyl-1,3,4-oxadiazoles. It was thereby demonstrated that the substitution pattern of the N¹-acyl-carboxamidrazones played a fundamental role in determining the direction of the ring closing reaction.     

Justiciosides E-G, triterpenoidal glycosides with an unusual skeleton from Justicia betonica

Three new triterpenoidal glucosides, justiciosides E, F and G, were isolated from the aerial portion of Justicia betonica. Their structures were established through chemical and spectroscopic analyses, and showed an unusual A-nor-B-homo oleanan-12-ene skeleton type for the aglycone moiety as A-nor-B-homo-oleanan-10,12-diene-3β,11α,28-triol 28-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside, A-nor-B-homo-oleanan-10,12-diene-3β,11α,28-triol 28-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside, and 11α-methoxy-A-nor-B-homo-oleanan-10,12-diene-3β,11α,28-triol 28-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside, respectively.     

Highly glycosylated flavonoids from the pods of Bobgunnia madagascariensis

Methanolic extracts of the pods of Bobgunnia madagascariensis (Leguminosae) yielded four pentaglycosylated flavonoids, including the 3-O-α-l-rhamnopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-β-d-glucopyranoside-7-O-α-l-rhamnopyranosides of 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-benzopyran-4-one (kaempferol) and 3,5,7-trihydroxy-2-(3,4-dihydroxyphenyl)-4H-benzopyran-4-one (quercetin), which were characterized by a novel O-linked branched tetrasaccharide. Spectroscopic and chemical methods were used to determine the structures of the latter, which co-occurred with the corresponding β-d-galactopyranosyl isomers, and two saponins. Conformational isomerism of quercetin 3-O-α-l-rhamnopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-β-d-glucopyranoside-7-O-α-l-rhamnopyranoside was detected in solution by NMR, a phenomenon previously associated only with C-glycosylflavonoids.     

Synthesis of a common trisaccharide fragment of glycoforms of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide

The first synthesis of the common trisaccharide of glycoforms of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide is reported. A fully protected trisaccharide precursor was prepared via a highly efficient α-(1→4)-glucosylation of a β-(1→3)-linked 6-O-benzyl-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-α-d-galactopyranoside. In contrast, an alternative sequence of glycosylations, which involves β-glucosylation of an α-(1→4)-linked Glc-GalN₃ unit, did not lead to the target trisaccharide backbone. Further O-deacetylation, azido group reduction and debenzylation of the protected trisaccharide precursor gave the corresponding trisaccharide amine. The latter structure was used in the synthesis of a series of trisaccharides bearing an acetyl group, an l-alanine or an N-acetylated l-alanine residue on its amino group at C-2 of GalN.     

Neoverataline A and B, two antifungal alkaloids with a novel carbon skeleton from Veratrum taliense

Bioassay-guided fractionation of the ethanol extract of the roots and rhizomes of Veratrum taliense yielded two new and thirteen known steroidal alkaloids. The structures of the two new compounds, neoverataline A and B, were established by extensive spectroscopic analyses to be 3,4-secocevane-4,9-olid-14,15,16,20-tetra-ol-3-oic acid and 3,4-secocevane-4,9-olid-7,14,15,16,20-penta-ol-3-oic acid, respectively, and are a novel carbon skelton. All of the fifteen alkaloids were subjected to in vitro antifungal assays, which showed that the verazine- (veramitaline, stenophylline B, stenophylline B-3-O-β-d-glucopyranoside, veramiline-3-O-β-d-glucopyranoside) and jerveratrum-type (jervine, jervine-3-O-β-d-glucopyranoside) alkaloids exhibited strong antifungal activities against the phytopathogenic fungus Phytophthora capisis with MICs of 160, 120, 160, 80, 80 and 120 μg·L⁻¹, respectively. Furthermore, the verazine-type alkaloids stenophylline B, stenophylline B 3-O-β-d-glucopyranoside and veramiline 3-O-β-d-glucopyranoside were shown to also inhibit the growth of another fungal phytopathogen Rhizoctonia cerealis with MICs of 160, 120 and 120 μg mL⁻¹. The MICs of triadimefon (an antifungal agrochemical used herein as a positive control) against P. capisis and R. cerealis were 120 and 80 μg mL⁻¹, respectively. A preliminary structure-activity relationship regarding these alkaloids has been formulated.     

Über triterpene—XXIX : Zur struktur des avenacins

From the roots of oat (Avena sativa) a second antibiotic active glycoside, avenacine B, was isolated in addition to the main glycoside avenacine A. In the aglycone of A the double bond could not be established contrary to the statement in the literature but an aldehyde group in addition to the keto group was identified and therefore the proposed structure has to be revised. In the aglycone of B the —CH₂OH-group of A has been substituted by CH₃. The structures of the sugar chains in both glycosides have been determined, in avenacine A as [β-d-gluco-pyranosyl(1 → 4)-[β-d-glucopyranosyl (1 → 2)]-α-l-arabinosyl (1)- and in B as [β-d-glucopyranosyl (1 → 4)]-β-d-glucopyranosyl (1)-.     

Mildbraedin, a novel kaempferol tetraglycoside from the tropical forest legume Mildbraediodendron excelsum

Aqueous methanol extracts of the leaves of Mildbraediodendron excelsum yielded a novel flavonol glycoside characterized by an O-linked branched tetrasaccharide. The structure of the compound was determined by spectroscopic methods to be kaempferol 3-O-α-l-rhamnopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-β-d-galactopyranoside. This previously unrecorded natural product was the major phenolic component of leaf material obtained both from a living specimen of the plant and a historic collection made in the field in 1928.     

Synthesis of branched tri- to pentasaccharides representative of fragments of Shigella flexneri serotypes 3a and/or X O-antigens

Fragments of the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→}_n ((E)AB_AcCD)_n polymer were synthesized. D(E)A, CD(E)A, _AcCD(E)A were obtained according to a linear strategy, whereas BCD(E)A and B_AcCD(E)A were derived from the condensation of appropriate BC and D(E)A building blocks. Oligosaccharides were synthesized as their propyl glycoside, relying on (i) the efficient trichloroacetimidate chemistry, (ii) a common EA allyl glycoside, and (iii) a 2-trichloroacetamido-d-glucopyranose precursor to residue D. Final Pd/C-mediated deprotection, run under a high pressure of hydrogen, ensured O-acetyl stability. All targets are parts of the O-antigen of Shigella flexneri 3a, a prevalent serotype. Non-O-acetylated oligosaccharides are shared by the S. flexneri serotype X O-antigen.     

An efficient synthesis of a hexasaccharide—the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A

A general method for the synthesis of 2-xylose or glucuronic acid branched (1→3)-linked mannose oligosaccharides has been developed. As a typical example, the synthesis of the methyl glycoside of β-d-GlcpA-(1→2)-α-d-Manp-(1→3)-[β-d-Xylp-(1→2)-]α-d-Manp-(1→3)-[β-d-Xylp-(1→2)-]α-d-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A, was achieved in a regio- and stereoselective manner.     

Two new saponins from Anemarrhena asphodeloides Bge.

Two new saponins 3-O-β-D-glucopyranosyl (1→2)-β-D-mannopyranosyl sarsasapogenin, named timosaponin A IV(1) and (5β, 25S)-26-O-β-D-glucopyranosyl-furost-20(22)-en-3,26-diol-3-O-β-D-glucopyranosyl (1→4) glucopyranosyl (1→2)-β-D-galacopyranoside, named timosaponin B IV(2), were isolated by silica gel column chromatography and preparative HPLC from Anemarrhena asphodeloides Bge. Their structures were elucidated by chemical characters and spectroscopic analysis.