N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide

A complex trisaccharide β-d-GalpNAcA-(1 → 4)-β-d-GlcpNAc-(1 → 4)-d-ManpNAc (3) was prepared in a good yield (35%) in a transglycosylation reaction catalyzed by β-N-acetylhexosaminidase from Talaromyces flavus using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galacto-hexodialdo-1,5-pyranoside (1) as a donor followed by the in situ oxidation of the aldehyde functionality by NaClO₂. The disaccharide β-d-GlcpNAc-(1 → 4)-d-ManpNAc (2) was used as galactosyl acceptor. A disaccharide β-d-GalpNAcA-(1 → 4)-d-GlcpNAc (4; 39%) originated as a by-product in the reaction. Oligosaccharides comprising a carboxy moiety at C-6 are shown to be very efficient ligands to natural killer cell activation receptors, particularly to human receptor CD69. Thus, oxidized trisaccharide 3 is the best-known oligosaccharidic ligand to this receptor, with IC₅₀ = 2.5 × 10⁻⁹ M. The presented method of introducing a β-d-GalpNAcA moiety into carbohydrate structures is versatile and can be applied in the synthesis of other complex oligosaccharides.     

Two novel oligosaccharides from Solanum nigrum

Phytochemical analysis of Solanum nigrum has resulted in the isolation of two novel disaccharides. Their structures were determined as ethyl β-d-thevetopyranosyl-(1→4)-β-d-oleandropyranoside (1) and ethyl β-d-thevetopyranosyl-(1→4)-α-d-oleandropyranoside (2), respectively, by chemical and spectroscopic methods.     

Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.     

Glycosylated nervogenic acid derivatives from Liparis condylobulbon (Reichb.f.) leaves

Three new nervogenic acid glycosides, 1-O-α-l-rhamnopyranosyl 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoate, 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoic acid, and bis{3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoyl} 1,2-O-β-d-glucopyranose, which we named condobulbosides A–C, were isolated from a methanol extract of the leaves of Liparis condylobulbon together with an apigenin C-glycoside, schaftoside. Their structures were established on the basis of spectral techniques, namely, UV, IR, HR-MS spectroscopy, both 1D and 2D NMR experiments, and chemical reactions.     

Hemiterpene glucosides and other constituents from Spiraea canescens

Five glycosides, 2-(trans-cinnamoyloxy-methyl)-1-butene-4-O-β-d-glucopyranoside (1), 4-(6′-O-trans-cinnamoyl)-(2-hydroxymethyl-4-hydroxy-butenyl-β-d-glucopyranoside (2), 6′′-O-trans-p-coumaroyl-(4-hydroxybenzoyl)-β-d-glucopyranoside (3), 6′-O-(4-methoxy-trans-cinnamoyl) α/β-d-glucopyranose (4) 6′-O-(4′′-methoxy-trans-cinnamoyl)-kaempferol-3-β-d-glucopyranoside (7) along with six known compounds, (+)-isolariciresinol 3a-O-β-d-glucopyranoside (8) (+)-lyoniresinol 3a-O-β-d-glucopyranoside (9), apigenin 7-O-β-d-glucopyranoside (10), quercetin 3-O-β-d-glucopyranoside (11), 6′-O-cinnamoyl-α/β-d-glucopyranose (6) 6’-O-p-coumaroyl-α/β-d-glucopyranose (5) were isolated from the whole plant of Spiraea canescens. Some of these compounds showed potent radical scavenging activity in relevant non-physiological assays. Their structures were determined by NMR spectroscopic and CID mass spectrometric techniques.     

Synthesis of monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside for epitope mapping of anti-Candida albicans antibodies

A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation–reduction sequence at C-2′. The β-manno configurations of the final deprotected congeners 2–7 were confirmed by measurement of ¹J_C1,H1 heteronuclear and ³J_1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the protective epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site.     

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

Morphological and structural characterization of a polysaccharide from Gynostemma pentaphyllum Makino and its anti-exercise fatigue activity

The crude polysaccharide was obtained from Gynostemma pentaphyllum Makino by water extraction followed by ethanol precipitation. The polysaccharide was successively purified by chromatography on DEAE-52 and SephadexG-150 column, and three polysaccharide fractions were obtained and termed GPP1-a, GPP2-b, and GPP3-a, respectively. The administration with GPP1-a markedly prolonged exhaustive exercise time of the mice. Structural features of GPP1-a were investigated by a combination of instrumental and chemical analyses, including atomic force microscope (AFM), scanning electron microscope (SEM), partial acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, gas chromatography–mass spectrometry (GC–MS) analysis and NMR spectroscopy. The results indicate that GPP1-a has a backbone of (1 → 4)-linked α-d-Glucose residues, which occasionally branches at O-6. The branches are mainly composed of (1 → 6)-linked α-d-Glucose, (1 → 3)-linked β-d-Galactose and (1 → 6)-linked α-d-Galactose residues, and terminated with β-d-Galactose residues and β-l-Arabinose residues.     

Unique transglycosylation potential of extracellular α-d-galactosidase from Talaromyces flavus

The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail.     

Purification, cDNA cloning and homology modeling of endo-1,3-beta-D-glucanase from scallop Mizuhopecten yessoensis

The retaining endo-1,3-β-d-glucanase (LV) with molecular mass of 36 kDa was purified to homogeneity from the crystalline styles of scallop Mizuhopecten yessoensis. The purified enzyme catalyzed hydrolysis of laminaran as endo-enzyme forming glucose, laminaribiose and higher oligosaccharides as products (K_m  600 μg/mL). The 1,3-β-d-glucanase effectively catalyzed transglycosylation reaction that is typical of endo-enzymes too. Optima of pH and temperature were at 4.5 and 45 °C, respectively. cDNA encoding the endo-1,3-β-d-glucanase was cloned by PCR-based methods. It contained an open reading frame that encoded 339-amino acids protein. The predicted endo-1,3-β-d-glucanase amino acid sequence included a characteristic domain of the glycosyl hydrolases family 16 and revealed closest homology with 1,3-β-d-glucanases from bivalve Pseudocardium sachalinensis, sea urchin Strongylocentrotus purpuratus and invertebrates lipopolysaccharide and β-1,3-glucan-binding proteins.The fold of the LV was more closely related to κ-carrageenase, agarase and 1,3;1,4-β-d-glucanase from glycosyl hydrolases family 16. Homology model of the endo-1,3-β-d-glucanase from M. yessoensis was obtained with MOE on the base of the crystal structure of κ-carrageenase from P. carrageonovora as template. Putative three-dimensional structures of the LV complexes with substrate laminarihexaose or glucanase inhibitor halistanol sulfate showed that the binding sites of the halistanol sulfate and laminarihexaose are located in the enzyme catalytic site and overlapped.     

The efficient total synthesis of bis-glycosyl apigenin from naringenin: a greener way

An efficient total synthesis of 7-O-β-d-glucopyranosyl-4′-O-α-l-rhamnopyranosyl apigenin (1) was developed in only four steps from naringenin. Compared with our previously reported first total synthesis route (six steps and 19.6% overall yield), this new route contained two steps of highly regioselective glycosylation without any selective protection steps. 7,4′-di-O-β-d-glucopyranosyl apigenin (2) was also prepared efficiently by this method. The method is environmentally friendly, economical, and provides a greener method for flavonoid synthesis starting from an inexpensive flavanone.     

New cell wall glycopolymers of the representatives of the genus Kribbella

Each of the cell walls of four representatives of the genus Kribbella (order Actinomycetales; suborder Propionibacterineae; family Nocardioidaceae) contains a neutral polysaccharide and an acidic polysaccharide with unusual structures. Common to all four strains studied is a mannan with the following repeating unit: In the cell wall of the strain VKM Ac-2541, a teichulosonic acid was identified with a monosaccharide component that has not hitherto been found in Gram-positive bacteria, viz., pseudaminic acid, and an unusual linkage type in the polymeric chain,

Full-size image (1K)

where R = Н (45%), α-d-Galp3OMe (37%) or α-d-Galp2,3OMe (18%).The anionic cell wall components of three other strains are represented by teichuronic acids with a rare constituent, viz., a diaminosugar, 2,3-diacetamido-2,3-dideoxyglucopyranose. The structures of their repeating units differ in the nature of the acidic components:→4)-β-d-Manp2,3NAcA-(1→6)-α-d-Glcp2,3NAc-(1→ (VKM Ас-2538 and VKM Ас-2540) and →4)-β-d-ManpNAcA-(1→6)-α-d-Glcp2,3NAc-(1→ (VKM Ас-2539).The structures of all the glycopolymers were established by chemical and NMR spectroscopic methods; they are identified in Gram-positive bacteria for the first time.     

Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea

Amylosucrase (ASase, EC 2.4.1.4) is a member of family 13 of the glycoside hydrolases that catalyze the synthesis of an α-(1→4)-linked glucan polymer from sucrose instead of an expensive activated sugar, such as ADP- or UDP-glucose. Transglycosylation reactions mediated by the ASases of Deinococcus geothermalis (DGAS) and Neisseria polysaccharea (NPAS) were applied to the synthesis of salicin glycosides with sucrose serving as the glucopyranosyl donor and salicin as the acceptor molecule. Two salicin glycoside transfer products were detected by TLC and HPLC analyses. The synthesis of salicin glycosides was very efficient with NPAS with a yield of over 90%. In contrast, DGAS specifically synthesized only one salicin transglycosylation product. The transglycosylation products were identified as α-d-glucopyranosyl-(1→4)-salicin (glucosyl salicin) and α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-salicin (maltosyl salicin) by NMR analysis. The ratio between donor and acceptor had a significant effect on the type of product that resulted from the transglycosylation reaction. With more acceptors present in the reaction, more glucosyl salicin and less maltosyl salicin were synthesized.     

Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against β-glycosidases

β-(1→4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(β-d-Xylp)-4-thiohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(β-d-Galp)-4-thiopentopyranosid-2-ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced with NaBH₄ or K-Selectride, and the stereochemical course of the reduction was highly dependent on the reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH₄–THF at 0 °C gave a 3-deoxy-4-S-(β-d-Xylp)-4-thio-α-d-ribo-hexopyranoside derivative (6) as major product (74% yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11 gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-β-d-threo-pentopyranoside derivative 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at −78 °C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides 19 and 18. They displayed strong inhibitory activity against the β-galactosidase from Escherichia coli. Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (K_i = 0.80 mM), whereas 19 was a mixed-type inhibitor (K_i = 32 μM).     

Screening strains for directed biosynthesis of β-d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production

One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β-d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β-d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β-d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml⁻¹ was obtained.     

The system of sulfated galactans from the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta): Location and structural analysis

Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low M_r (6–11.4 kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium M_r (18–45 kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which “pure” κ/ι-carrageenans were isolated.Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures.Alkaline treatment and alkaline dialysis of the main extracts gave “pure” κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low M_r agarans or agaran-dl-hybrids.     

Synthesis and glycogen phosphorylase inhibitory activity of N-(β-d-glucopyranosyl)amides possessing 1,4-benzodioxane moiety

A series of N-(β-d-glucopyranosyl)amides 5d–i were synthesized by PMe₃ mediated Staudinger reaction of O-peracetylated β-d-glucopyranosyl azide (1) followed by acylation with carboxylic acids 3d–i and subsequent Zemplén deacetylation. The new compounds were tested for their inhibitory activity against rabbit muscle glycogen phosphorylase and the structure–activity relationships of these compounds are also discussed in this paper.     

Ultracytochemistry of glycoproteins in the eccrine nasolabial glands of the North American raccoon (Procyon lotor)

The distribution and quality of glycoproteins was studied by means of electron microscopic cytochemical methods, particularly lectin cytochemistry, in the secretory cells of the eccrine nasolabial glands of the North American raccoon (Procyon lotor). In the dark and clear glandular cells, complex glycoconjugates were demonstrable, predominantly, in secretory granules, the cisternae of the Golgi apparatus, the surface coat of the plasma membrane, and as glycogen particles. Secretory granules found in the dark cells contained a variety of saccharide residues, such as α-d-mannose, β-d-galactose, β-N-acetyl-d-glucosamine and sialic acid. Several sugars were also detectable in the surface coat of the plasma membrane and the Golgi apparatus.The results obtained may be helpful to understand the specific functions of the glandular secretions of the raccoon nasolabial glands. These could be, particularly, binding of water on the snout surface and protection against microbial hazards, to maintain the structural and functional integrity of the relatively thin snout epidermis in carnivores.     

E versus Z geometry in β-d-arabino-hexopyranosidulose oximes

Koenigs–Knorr-type glycosidations of peracylated 2Z-benzoyloxyimino-glycopyranosyl bromides invariably proceed with retention of the Z-geometry. Accordingly, the many β-d-hexosidulose oximes in literature which were prepared in this way and for which the oxime geometry has not been addressed explicitly, are the Z-oximes throughout. By contrast, oximation of β-d-hexopyranosid-2-uloses leads to mixtures of E and Z oximes readily separable and structurally verifiable by ¹H and ¹³C NMR. Configurational assignments rested on comparative evaluation of NMR data of E and Z isomers, and, most notably on an X-ray structural analysis of the pivaloylated isopropyl 2E-benzoyloxyimino-2-deoxy-β-d-arabino-hexopyranoside revealing the unusual ¹S₅^1,4B conformation for the pyranoid ring.     

Growth inhibitory activity of extracts and compounds from Cimicifuga species on human breast cancer cells

The purpose of this report is to explore the growth inhibitory effect of extracts and compounds from black cohosh and related Cimicifuga species on human breast cancer cells and to determine the nature of the active components. Black cohosh fractions enriched for triterpene glycosides and purified components from black cohosh and related Asian species were tested for growth inhibition of the ER⁻ Her2 overexpressing human breast cancer cell line MDA-MB-453. Growth inhibitory activity was assayed using the Coulter Counter, MTT and colony formation assays.Results suggested that the growth inhibitory activity of black cohosh extracts appears to be related to their triterpene glycoside composition. The most potent Cimicifuga component tested was 25-acetyl-7,8-didehydrocimigenol 3-O-β-d-xylopyranoside, which has an acetyl group at position C-25. It had an IC₅₀ of 3.2 μg/ml (5 μM) compared to 7.2 μg/ml (12.1 μM) for the parent compound 7,8-didehydrocimigenol 3-O-β-d-xylopyranoside. Thus, the acetyl group at position C-25 enhances growth inhibitory activity.The purified triterpene glycoside actein (β-d-xylopyranoside), with an IC₅₀ equal to 5.7 μg/ml (8.4 μM), exhibited activity comparable to cimigenol 3-O-β-d-xyloside. MCF7 (ER⁺Her2 low) cells transfected for Her2 are more sensitive than the parental MCF7 cells to the growth inhibitory effects of actein from black cohosh, indicating that Her2 plays a role in the action of actein. The effect of actein on Her2 overexpressing MDA-MB-453 and MCF7 (ER⁺Her2 low) human breast cancer cells was examined by fluorescent microscopy. Treatment with actein altered the distribution of actin filaments and induced apoptosis in these cells.These findings, coupled with our previous evidence that treatment with the triterpene glycoside actein induced a stress response and apoptosis in human breast cancer cells, suggest that compounds from Cimicifuga species may be useful in the prevention and treatment of human breast cancer.