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We report a novel and highly stereoselective electro-2-deoxyglycosylation from glycals. This method features excellent stereoselectivity, scope, and functional-group tolerance. This process can also be applied to the modification of a wide range of natural products and drugs. Furthermore, a scalable synthesis of glycosylated podophyllotoxin and a one-pot trisaccharide synthesis through iterative electroglycosylations were achieved.

Stereoselective β-mannosylation has been recognized as one of the greatest challenges of carbohydrate chemistry. Herein, we described a practical method for stereoselective construction of β-mannosides by using a 2,6-lactone-bridged thiomannosyl donor through the remote acyl-group participation as well as the steric effect of O-4 substituent. The two effects are enabled through the conversion of a regular mannopyranosyl 4 C1 conformation into a 2,6-lactone bridged conformation. The lactone donor could be readily prepared in three steps on a gram scale and the β-mannosylation proceeded smoothly with high stereoselectivity for primary, secondary and tertiary alcohol acceptors. In addition, this strategy was successfully applied to the synthesis of a naturally occurring trisaccharide.

Introduction Saffron stigmas from Crocus sativus L. (Iridaceae) are used as a drug in folk medicine, as a food additive and as a dying agent for at least 3500 years. Despite this long-term use the chemical composition of saffron seems still to be not fully known. Objective An analytical strategy for detailed investigations of aqueous saffron extract is developed based on reverse-phase high-performance liquid chromatography electrospray ionisation (HPLC-ESI) multistage mass spectrometry (MSn ) for crocins. Methods Commercially available stigmas are analysed by reverse-phase HPLC in combination with ESI/three-dimensional (3D)-ion trap mass spectrometry (MS) and MSn (n = 2 and 3). Sodium chloride is added to the analyte solution ready for injection to promote abundant [M + Na]+ adduct ions of crocins, being ideal precursor ions for low-energy collision-induced dissociation (CID)-MS2/3 . Results This strategy allows the detailed structural elucidation of known as well as previously unknown crocin derivatives (molecular mass of the aglycon, oligosaccharide chain length and linkage determination). The two isomeric trisaccharide substituents neapolitanose and gentiotriose are distinguished based on linkage-specific cross-ring cleavage for the first time. Furthermore, crocins containing up to six hexose units are also observed. Five novel crocin ester glycosides shifted by a mass difference of -40 Da indicate the presence of the here newly described C17 -aglycon, termed norcrocetin (crocetin = C20 ). Conclusions These findings indicate the action of at least two different carotenoid cleavage dioxygenases (CCD2 and tentatively CCD4) during biosynthesis of this new bis-apocarotenoid aglycon (norcrocetin) and the existence of even higher glycosylated crocin derivatives at trace level.

3D structural information was obtained from mono-, di- and trisaccharide formamide and isocyanide derivatives by analysis of their X-ray crystal structure and NMR spectroscopy. The isocyanide anomeric effect was observed. Data mining of the Cambridge Structural Database (CSD) was performed and statistically confirmed our findings. Application of the glycoside isocyanides in the synthesis of novel glycoconjugates as drug-like scaffolds by MCR chemistry underscores the usefulness of the novel building blocks.

Acyl group migration is a fundamental phenomenon in carbohydrate chemistry, recently shown to take place also between two non‐adjacent hydroxyl groups, across the glycosidic bond, in a β‐(1→4)‐linked mannan trisaccharide model compound. With the central mannoside unit containing acetyl groups at the O2 and O3 positions, the O2‐acetyl was in the earlier study shown to migrate to O6 of the reducing end. Potential implications of the general acyl migration process on cell signaling events and plant growth in nature are intriguing open questions. In the present work, migration kinetics in this original trisaccharide model system were studied in more detail together with potential interactions of the model compound and the migration products with DC‐SIGN lectin. Furthermore, we demonstrate here for the first time that similar migration may also take place in native polysaccharides, here represented by galactoglucomannan from Norway spruce., Kinetic calculations on the migration between saccharide units in a mannan trisaccharide model compounds and a galactoglucomannan polysaccharide were performed. Potential interactions of the model compounds with a lectin were also investigated.

A concise approach to a Neu5Ac-α-2,3-LacNPhth trisaccharide derivative was developed. First, the regio/stereoselective glycosylation between glycoside donors and glucoNPhth diol acceptors was investigated. It was found that the regioselectivity depends not only on the steric hindrance of the C2-NPhth group and the C6-OH protecting group of the glucosamine acceptors, but also on the leaving group and protecting group of the glycoside donors. Under optimized conditions, LacNPhth derivatives were synthesized in up to 92 % yield through a regio/stereoselective glycosylation between peracetylated-α-galactopyranosyl trichloroacetimidate and p-methoxyphenyl 6-O-tert-butyldiphenylsilyl-2-deoxy-2-phthalimido-β-d-glucopyranoside, avoiding the formation of glycosylated orthoesters and anomeric aglycon transfer. Then, the LacNPhth derivative was deacylated and then protected on the primary position by TBDPS to form a LacNPhth polyol acceptor. Finally, the Neu5Ac-α-2,3-LacNPhth derivative was synthesized in 48 % yield through the regio/stereoselective glycosylation between the LacNPhth polyol acceptor and a sialyl phosphite donor. Starting from d-glucosamine hydrochloride, the target Neu5Ac-α-2,3-LacNPhth derivative was synthesized in a total yield of 18.5 % over only 10 steps.

Rationale Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been used to authenticate and trace products such as honey, wine, and lemon juice, and compounds such as caffeine and pesticides. However, LC/IRMS has several disadvantages, including the high cost of the CO2 membrane and blocking by solidified sodium persulfate. Here, we developed an improved system for determining carbon isotope ratios using LC/IRMS. Methods The main improvement was the use of a post-column pump. Using the improved system, we determined δ13 C values for glucose with high accuracy and precision (0.1‰ and 0.1‰, respectively; n = 3). The glucose, fructose, disaccharide, trisaccharide, and organic acid constituents of honey samples were analyzed using LC/IRMS. Results The δ13 C values for glucose, fructose, disaccharides, trisaccharides, and organic acids ranged from -27.0 to -24.2‰, -26.8 to -24.0‰, -28.8 to -24.0‰, -27.8 to -22.8‰, and - 30.6 to -27.4‰, respectively. The analysis time was a third to a half of that required for analysis by previously reported methods. Conclusions The column flow rate could be arbitrarily adjusted with the post-column pump. We applied the improved method to 26 commercial honey samples. Our results can be expected to be useful for other researchers who use LC/IRMS.

Chondroitin sulfate (CS) is a sulfated polysaccharide that plays essential physiological roles. Here, we report to utilize an enzyme-based method to synthesize a library of CS oligosaccharides consisting of 15 different CS oligosaccharides. The library covers 4-O-sulfated and 6-O-sulfated oligosaccharides with the size ranging from trisaccharide to nonasaccharide. We also demonstrate the synthesis of unnatural 6-O-sulfated CS pentasaccharides containing either a 6-O-sulfo 2-azido galactosamine or a 6-O-sulfo galactosamine residue. The availability of structurally defined CS oligosaccharides offers a novel approach to investigate the biological functions of CS.

Regioselective differentiation of the hydroxy groups of mono- and oligosaccharide substrates is necessary to form building blocks that can be used for synthetic transformations. In this paper, we show that iron(III) chloride hexahydrate catalyses the tandem protection of mono- and disaccharides to give, in a one-pot procedure, selectively protected carbohydrate building blocks. This tandem protocol was successfully applied to a trisaccharide. The procedure is easy to carry out; for optimal results, each carbohydrate substrate required a fine tuning of the one-pot reaction conditions.

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was employed successfully to prepare a series 4 beta-triazole-linked picropodophyllotoxin glycoconjugates (25 - 34). Maltose residue, 1,6-beta-D-diglucose residue, and several triazole-linked disaccharide and trisaccharide residues were coupled to 4 beta-azido-podophyllotoxin derivatives through click glycosylation strategy. The initial click glycosylation products were treated with catalytic amount of NaOCH3 to facilitate global deacylation and epimerization at C-2 position to yield the cis-gamma-lactone moiety in the picropodophyllotoxin glycoconjugates. Most of these picropodophyllotoxin glycoconjugates show weak cytotoxicity (IC50 > 40 mu M) against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) as indicated by in vitro MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. However, compound 27 that contains a 1,6-beta-D-di-glucose residue displays strong anticancer activity against all cancer cell lines tested, with IC50 values ranging from 0.67 to 7.41 mu M, which is significantly more potent than the control drug etoposide against four of the five cancer cells tested. Structure activity relationship analysis suggests that the 4'-O-methyl group on the E ring of podophyllotoxin scaffold is perhaps important for the anticancer activity of glycosylated picropodophyllotoxin derivatives with a cis-g-lactone moiety.

An alginate lyase producing bacterial strain, Cobetia sp. WG-007, was isolated and identified from rotting seaweed. The alginate lyase, Aly-W02, was purified by procedures of ultrafiltration, Q-Sepharose Fast Flow, Phenyl Sepharose 6 Fast Flow, and Superdex-G100 with specific activity of 21,285.5 U/mg. Aly-W02 had an apparent molecular mass of 35 kDa. It exhibited maximum activity at 45 °C in 50 mM sodium phosphate buffer (pH 8.5). This alginate lyase was stable in the pH range of 6.0-8.5. Among the tested metal ions, the addition of K+ , Na+ , and Mg2+ ions can enhance the enzyme activities, while Ba2+ , Ni+ , Cu2+ , Mn2+ , Zn2+ , Ag+ , and ethylenediaminetetraacetic acid decreased the activities. It displayed high salt-tolerant ability; 0.8 M NaCl or 1.5 M KCl significantly enhanced the enzyme activity. Furthermore, Aly-W02 mainly released disaccharide, trisaccharide, and tetrasaccharid from alginate. It showed potential in producing low molecular weight alginate oligosaccharides.

Raffinose, a functional trisaccharide of α-d-galactopyranosyl-(1???6)-α-d-glucopyranosyl-(1???2)-?-d-fructofuranoside, is a prebiotic that shows promise for use as a food ingredient. RESULTS In this study, the production of raffinose from melibiose and sucrose was studied using whole recombinant Escherichia coli cells harboring the levansucrase from Clostridium arbusti SL206. The reaction conditions were optimized for raffinose synthesis. The optimal pH, temperature and washed cell concentration were pH 6.5 (sodium phosphate buffer, 50?mmol?L?1), 55?°C and 3% (w/v), respectively. High substrate concentrations, which led to low water activity and thus reduced levansucrase hydrolysis activity, strongly favored the production of raffinose through the fructosyl transfer reaction. Additionally, high concentrations of excess acceptor and donor glycosides favored raffinose production. When 30% (w/v) sucrose and 30% (w/v) melibiose were catalyzed using 3% (w/v) whole cells at pH 6.5 (sodium phosphate buffer, 50?mmol?L?1) and 55?°C, the highest raffinose yield was 222?g?L?1 after a 6?h reaction. The conversion ratio from each substrate to raffinose was 50%. CONCLUSION Raffinose could be effectively produced with melibiose as an acceptor and with sucrose as a fructosyl donor by whole recombinant E. coli cells harboring C. arbusti levansucrase. The yield from E. coli was significantly higher than those of the previously reported Bacillus subtilis levansucrase and fungal α-galactosidases. ? 2016 Society of Chemical Industry

Six new partially acylated resin glycosides were isolated from convolvulin of Ipomoea purga, Ipomoea stans, and Ipomoea murucoides (Convolvulaceae). The structures of compounds 1-6 were elucidated by a combination of NMR spectroscopy and mass spectrometry. The structure of jalapinoside B (1) consists of a hexasaccharide core bonded to an 11-hydroxytetradecanoic (convolvulinic) acid forming a macrolactone acylated by a 2-methylbutanoyl, a 3-hydroxy-2-methylbutanoyl, and a quamoclinic acid B units. Purginoic acid A (2) contains a hexasaccharide core bonded to a convolvulinic acid acylated by a 3-hydroxy-2-methylbutanoyl unit. Stansin A (4) is an ester-type heterodimer, and consists of two stansoic acid A (3) units, acylated by 2-methylbutanoic and 3-hydroxy-2-methylbutanoic acids. The site of lactonization was located at C-3 of Rhamnose, and the position for the ester linkage of the monomeric unit B on the macrolactone unit A was established as C-4 of the terminal rhamnose. Compounds 5 and 6 are glycosidic acids. Murucinic acid II (5) is composed of a pentasaccharide core bonded to an 11-hydroxyhexadecanoic (jalapinolic) acid, acylated by an acetyl unit. Stansinic acid I (6) is a tetrasaccharide core bonded to a jalapinolic acid, acylated by 2-methylbutanoyl and 3-hydroxy-2-methylbutanoyl units. Preliminary testing showed the cytotoxicity of compounds 1-6 toward OVCAR and UISO-SQC-1 cancer cell lines. In addition, compound 1 showed an antiproliferative activity on glioma C6 and RG2 tumor cell lines. Copyright © 2016 John Wiley & Sons, Ltd.

A new and efficient approach for direct and stereoselective synthesis of β-mannopyranosides by anomeric O-alkylation has been developed. This anomeric O-alkylation of mannopyranose-derived lactols is proposed to occur under synergistic control of a kinetic anomeric effect and metal chelation. The presence of a conformationally flexible C6 oxygen atom in the sugar-derived lactol donors is required for this anomeric O-alkylation to be efficient, probably because of its chelation with cesium ion. In contrast, the presence of a C2 oxygen atom plays a minor role. This glycosylation method has been successfully utilized for the synthesis of the trisaccharide core of complex N-linked glycans.

Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep-3, an eliminase from Flavobacterium heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.

The first total synthesis of the branched oligosaccharide OSE-1 of Mycobacterium gordonae (strain 990) is reported. An intramolecular aglycon delivery approach was used for constructing the desymmetrized 1,1'-α,α-linked trehalose moiety. A [3+2] glycosylation of the trisaccharide donor and trehalose acceptor furnished the right hand side pentasaccharide. Regioselective O3 glycosylation of L-rhamnosyl 2,3-diol allowed expedient synthesis of the left hand side tetrasaccharide. The nonasaccharide was assembled in a highly convergent fashion through a [4+5] glycosylation.

A highly efficient and practical total synthesis of the sulfated ganglioside SM1a, a kind of human epithelial carcinoma antigen identified in mammalian kidney, has been accomplished for the first time. The characteristic sequence of SM1a, β-D-Galp-(13)-β-D-NHAcGalp-(14)-β-D-(3-O-sulfate)-Galp-(14)-β-D-Glcp-ceramide was assembled by a [3+2] convergent approach. A key trisaccharide building block was formed from a new galactose acceptor 7 containing a potential sulfated site, GalNHTroc donor 6, and galactose donor 4. The cyclic glucosyl ceramide was glycosylated with trisaccharide trichloroacetimidate 2 to give the protected ganglioside backbone in good yield. Selective sulfonation at the 3-OH of the Gal residue followed by global deprotection gave the target molecule SM1a.

A scalable approach towards high-yielding and (stereo)selective glycosyl donors of the 2-ulosonic acid Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) is a fundamental requirement for the development of vaccines against Gram-negative bacteria. Herein, we disclose a short synthetic route to 3-iodo Kdo fluoride donors from Kdo glycal esters that enable efficient α-specific glycosylations and significantly suppress the elimination side reaction. The potency of these donors is demonstrated in a straightforward, six-step synthesis of a branched Chlamydia-related Kdo-trisaccharide ligand without the need for protecting groups at the Kdo glycosyl acceptor. The approach was further extended to include sequential iteration of the basic concept to produce the linear Chlamydia-specific α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo trisaccharide in a good overall yield.

Background Monoclonal (MoAb) reagents are routinely used and are usually very reliable for the serologic determination of ABO blood types. However, the fine specificity and cross-reactivity of these reagents are often unknown, particularly against synthetic antigens used in some diagnostic assays. If nonserologic assays or very sensitive techniques other than those specifically prescribed by the manufacturer are used, then there is a risk of incorrect interpretation of results. Study Design and Methods Forty-seven MoAbs and two polyclonal ABO reagents were tested against red blood cell (RBC) kodecytes prepared with A trisaccharide, A Type 1, A Type 2, A Type 3, A Type 4, B trisaccharide, B Type 1, B Type 2, acquired B trisaccharide, and Lea trisaccharide function-spacer-lipid (FSL) constructs. Natural RBCs were tested in parallel. In addition these FSL constructs were printed onto paper with a desktop inkjet printer and used in a novel immunoassay that identifies reactivity through the appearance of alphanumeric characters. Results Mapping of MoAbs with kodecytes and printed FSL constructs revealed a series of broad recognition patterns. All ABO MoAbs tested were reactive with the RBC dominant Type 2 ABO antigens. Unexpectedly some anti-A reagents were reactive against the B Type 1 antigen, while others were poorly reactive with trisaccharide antigens. Conclusions All ABO MoAbs detect the RBC dominant Type 2 ABO antigens; however, some reagents may show minor reactivity with inappropriate blood group antigens, which needs to be considered when using these reagents in alternative or highly sensitive analytic systems.

Chemical synthesis of a trisaccharide related to the cytotoxic triterpenoid saponins isolated from the bark of Albizia procera has been accomplished through a concise stepwise glycosylation strategy starting from commercially available D-xylose, 2-acetamido-2-deoxy-D-glucose and L-arabinose. The target trisaccharide was designed with a 4-methoxyphenyl (MP) aglycone to extend the scope of conversion to suitable glycoconjugates via selective removal of 4-methoxyphenyl (MP) group. An unexpected phenomenon, i.e., the arabinosyl residue assumed the 1C4 conformation instead of the typical 4C1 form, was observed. Deprotection could restore the normal conformation.

A new method for cleaving methyl ethers off protected saccharides using Co 2 (CO) 8 /Et 3 SiH/CO system was developed. The method showed regioselectivity in different protected monosaccharides with various anomeric groups. The primary and equatorial secondary methyl ethers were preferentially removed. This method was successfully applied to the synthesis of the DEF trisaccharide segment of Idraparinux, which is highly methylated.

Guided by the known molecular recognition interactions between N-acetylglucosaminyltransferase V (GnT-V) and certain synthetic substrates, we synthesized a radiolabeled double-stranded glycolipid composed of a long-chain alkyl unit and a radioiodinated phenylalkyl unit, [(125)I]-2-[N-(2-hydroxy-3-hexadecyloxy)propyl-15-(4-iodophenyl)pentadecanecarboxamido]ethyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)-β-D-glucopyranoside ([(125)I]2), as a novel intravital glycolipid mimic substrate of GnT-V. The radioactive iodine ((125)I) was incorporated via iododestannylation of the phenyltributyltin derivative, 2-[N-(2-acetoxy-3-hexadecyloxy)propyl-15-(4-tributylstannylphenyl)pentadecanecarboxamido]ethyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-3,4,6-O-acetyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-acetyl-β-D-glucopyranoside (26). Subsequent deacetylation at the final step afforded [(125)I]2.

RATIONALE Glycosphingolipids (GSLs) are important components of the cell membrane; however, little is known about GSLs in microalgae. We analyzed GSLs in three strains of Skeletonema microalgae by liquid chromatography coupled with mass spectrometry. METHODS Total lipid was extracted from Skeletonema microalgae. Separation of lipids was achieved via reversed-phase liquid chromatography, and mass spectrometric analysis of the lithium adduct of GSLs was conducted to determine their structures. RESULTS Two types of novel glycosphingolipids were identified from three strains of Skeletonema microalgae. The N-acyl groups were primarily long-chain saturated or monoenoic fatty acids (monounsaturated fatty acids) with 16C and 20 to 24C. The sphingoid long-chain bases (LCB) were sphingosine, sphingadienine and sphingatrienine. The saccharide polar head groups of glycosphingolipids were disaccharide (heptose-hexose) or trisaccharide (heptose-hexose-hexose). Semi-quantitative analysis of GLSs in the three strains of microalgae showed that GSL contents ranged from 0.09 to 8.79 nmol/mg dry microalgae powder. CONCLUSIONS A qualitative method was developed for the identification of GSLs in microalgae. Two types of novel GSLs were identified from three strains of Skeletonema, which might have important biological functions. It could also provide a reliable tool for chemotaxonomy of microalgae. Copyright © 2013 John Wiley & Sons, Ltd.

The first total synthesis of the O-antigen pentasaccharide repeating unit from Gram-negative bacteria Escherichia coli O111 was achieved starting from four monosaccharide building blocks. Key to the synthetic approach was a bis-glycosylation reaction to combine trisaccharide 10 and colitose 5. The colitose building block (5) was obtained de novo from non-carbohydrate precursors. The pentasaccharide was equipped at the reducing end with an amino spacer to provide a handle for subsequent conjugation to a carrier protein in anticipation of immunological studies.

Based on the combined use of dimethylformamide (DMF) modulation and neighboring group participation, three iterative one-pot α-glycosylation methods, i.e., one-pot (α,α)-, one-pot (β,α)-, and one-pot (α,β)-glycosylations, were developed. These methods are applicable to a range of thioglycosyl donors, confer stereocontrol in α-/β-glycosidic bond formation, and thus provide for rapid access to oligosaccharides with various permutations of anomeric configurations. The utility of these one-pot glycosylation methods is demonstrated in the synthesis of eight non-natural and natural oligosaccharide targets, including the core 1 serine conjugate, core 8 serine conjugate, the D-Gal-α(1→3)-D-Glc-α(1→3)-L-Rha trisaccharide unit of the cell wall component in Streptococcus pneumoniae, and the D-Glc-α(1→2)-D-Glc-α(1→3)-D-Glc trisaccharide terminus of the N-linked glycan precursor. Confirmation of the anomeric configurations of these oligosaccharides is evidenced by 1H, 13C, 13C-non-proton decoupling, and heteronuclear correlation 2D NMR experiments. Global deprotection of selected oligosaccharide targets is illustrated.

A modular synthesis of complex-type N-glycans containing the core fucosyl motif was optimized. The core trisaccharide building block was protected by a methoxyphenyl group for convenient core fucosylation. The trisaccharide was obtained on a large scale from the glycosylation of the corresponding chitobiosyl azide with a glucosyl donor followed by intramolecular inversion. Improved methods were established for the synthesis of the monosaccharide building blocks and for their couplings. The inversion to the β-mannoside was accompanied by previously unnoticed side-reactions resulting in the hydrolytic ring-opening of the iminocarbonate intermediate. The benzylidene-protected core trisaccharide was elongated into a biantennary N-glycan heptasaccharide by two regio- and stereoselective couplings. The final fucosylation also gave some of the β anomer, which could be removed by HPLC to give an α1,6-fucosylated N-glycan octasaccharide.

Certain carbohydrate structures are recognized as cancer antigens, and identification of these and relevant epitopes are essential in fighting the disease. The trisaccharide beta-D-GlcpNAc-(1?3)-be ...

O-Polysaccharides (O-antigens) are highly diverse glycopolymers present on the cell surface of Gram-negative bacteria, including the human opportunistic pathogen Providencia alcalifaciens. They define serospecificity of strains and are used for the serotyping of bacteria. In this work, a phosphorylated O-polysaccharide was isolated from P. alcalifaciens O22 and analyzed by chemical methods, ESI-MS, and 1H, 13C, and 31P NMR spectroscopy. It was found to contain two unusual components, 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N) and D-glyceramide 2-phosphate (D-GroAN-2-P), the latter being identified for the first time in bacterial polysaccharides. The structure of the trisaccharide repeating unit of the O-polysaccharide was established to be 4)-(D-GroAN-2-P-3-)--D-GalNAc-(14)--D-Gal-(13)--D-FucNAc4N-(1.

The first total synthesis of the hybrid ganglioside X2, which consisted of a highly branched octasaccharide and ceramide moieties, was accomplished by using a glucosyl ceramide cassette approach. With a disaccharyl donor, the heptasaccharide could not be constructed by glycosylation of the C4 hydroxy group of galactose at the reducing end of the pentasaccharide. In contrast, through an alternative approach with two branched glycan units, a GM2-core trisaccharide, and a lacto-ganglio tetrasaccharide, the heptasaccharyl donor could be prepared and subsequently joined with a glucosyl ceramide cassette to afford the protected ganglioside, X2. Finally, global deprotection completed the synthesis, thus affording the pure ganglioside X2.

Disaccharides that contain 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and d-glycero-d-talo-oct-2-ulosonic acid (Ko) substituted at the 8-position by 4-amino-4-deoxy-β-l-arabinopyranosyl (Ara4N) residues have been prepared. Coupling an N-phenyltrifluoroacetimidate-4-azido-4-deoxy-l-arabinosylglycosyl donor to acetyl-protected allyl glycosides of Kdo and Ko afforded anomeric mixtures of disaccharide products in 74 and 90 % yield, respectively, which were separated by chromatography. Further extension of an intermediate Ara4N-(1→8)-Kdo disaccharide acceptor, which capitalized on a regioselective glycosylation with a Kdo bromide donor under Helferich conditions, afforded the branched trisaccharide α-Kdo-(2→4)[β-l-Ara4N-(1→8)]-α-Kdo derivative. Deprotection of the protected di- and trisaccharide allyl glycosides was accomplished by TiCl4-promoted benzyl ether cleavage followed by the removal of ester groups and reduction of the azido group with thiol or Staudinger reagents, respectively. The reaction of the anomeric allyl group with 1,3-propanedithiol under radical conditions afforded the thioether-bridged spacer glycosides, which were efficiently coupled to maleimide-activated bovine serum albumin. The neoglycoconjugates serve as immunoreagents with specificity for inner core epitopes of Burkholderia and Proteus lipopolysaccharides.

We report the convergent preparation of four hexasaccharides to be used towards the synthesis of a safe anticancer vaccine based on the tumor-associated carbohydrate antigen dimLex. A common trisaccharide intermediate was first synthesized as a precursor to the tetrasaccharide backbones. These tetrasaccharides were then converted into diol acceptors and fucosylated at O-3 of both glucosamine residues. The conditions required to promote fucosylation at O-3 of the reducing end glucosamine units led to some loss of the fucosyl units already introduced at O-3 of the nonreducing end glucosamine residues. Despite this hurdle, optimization of the glycosylation conditions provided the desired hexasaccharides in sufficient quantities to prepare the final analogues. Propyl glycosides and cysteamine adducts were prepared easily in two steps from the protected allyl glycoside hexasaccharides, and a one-step deprotection under metal-dissolving conditions was key to our deprotection strategy.

The total synthesis of 2d-deoxy Lewisx pentasaccharide is described. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-α,β-D-galactopyranoside was condensed with a diol of glucosamine to regio- and stereoselectively give the disaccharide, the C-2′ position was then reduced after stereoselective fucosylation to afford a Lewisx trisaccharide analogue. Regioselective glycosylation of a known lactoside diol with this trisaccharide provided a pentasaccharide that, after deprotection, gave the target pentasaccharide.

A (2+1) glycosylation approach was employed to construct the core structure of a trisaccharide found in the soil bacterium Geobacillus stearothermophilus. The phospholipid was assembled by the reaction of a peracetylated trisaccharide bearing an anomeric hydroxy group with phosphoramidite and subsequent oxidation to the phosphate. The final coupling step with undecaprenyl monophosphate followed by global acetate deprotection completed the total synthesis of the highly amphiphilic target molecule.

An array of NMR spectroscopy experiments have been carried out to obtain conformationallydependent 1H,13C- and 13C,13C-spin-spin coupling constants in the trisaccharide a-L-Rhap-(1®2)[a-LRhap-(1®3) ...

A stereodivergent synthesis of differently configured C2-branched 4-amino sugar derivatives was accomplished. The Lewis acid mediated rearrangement of phenylthio-substituted 1,2-oxazines delivered glycosyl donor equivalents that can directly be employed in glycosidation reactions. Treatment with methanol provided internally protected amino sugar equivalents that have been transformed into the stereoisomeric methyl glycosides 28, ent-28, 29, ent-29 and 34 in two simple reductive steps. Reaction with natural carbohydrates or bicyclic amino sugar precursors allowed the synthesis of homo-oligomeric di- and trisaccharides 44, 46 and 47 or a hybrid trisaccharide 51 with natural carbohydrates. Access to a bivalent amino sugar derivative 54 was accomplished by reaction of rearrangement product 10 with 1,5-pentanediol. Alternatively, when a protected L -serine derivative was employed as glycosyl acceptor, the glycosylated amino acid 60 was efficiently prepared in few steps. In this report we describe the synthesis of unusual amino sugar building blocks from enantiopure 1,2-oxazines that can be attached to natural carbohydrates or natural product aglycons to produce new natural product analogues with potential applications in medicinal chemistry.

The synthesis of analogue 14f (Scheme 2) of a fragment of the S. pneumoniae type 1 C-substance antigen (Fig. 1, Ia) is presented. The protected disaccharide 5 and trisaccharide 7 (Scheme 1) were prepared by silver trifluoromethanesulfonate-catalyzed α-glycosylation of 2c with 4b (54%) and 2d with 6b (59%), respectively. A silver-silicate-catalyzed β-glycosylation of ribitol derivative 9 with trisaccharide bromide 8b gave 10a (55%). The selectively deprotected trisaccharideribitol 10b was coupled with spacer 11 (Scheme 2) using phosphorylating reagent 12 to give the fully protected phosphotriester derivative 14a (98%). The stepwise deprotection of 14a, including conversion of the azide functions into acetamido groups, turned out to be troublesome and afforded 14f in low yield.

Lauryl glycoside of β-D-Gicp-(1 3)-[β-D-Glcp-(1 6)-] a-D-Glcp-(1 3)-β-D-Glcp-(1 3)-[β-D-Glcp-(1 6)-] a-D-Glcp-(1 3)-β-D-Glcp-(1 3)-[β-D-Glcp-(1 6)-]β-D-Glcp was synthesized through 3 + 3 + 3 strategy. 3-O-Allyl-2, 4, 6-tri-O-benzoyl-β-D-glucopyranosyl-(1 3)-[2, 3, 4, 6-tetra-O-benzoyl-β-D-glucopyranosyl-(1 6)-] 1, 2-O-isopropylidene-a-D-glucofuranose was used as the key intermediate which was converted to the corresponding trisaccharide donor and acceptor readily.

β-D-Glcp( 13)-[β-D-Glcp-( 16)-/α-D-Manp-( 13)-β-D-Glcp( 13)-[β-D-Glcp-(16)-]D-Glcp (18) and β-D-Glcp( 13)-[β-D-Glcp-( 16)-] α-D-Manp-( 13)-β-D-Glcp-( 1 3)-[β-D-Glcp-( 16)-]β-D-Glcp-D-(13)-Glcp-1OMe (29) were synthesized as the analogues of the immunomodulator β-D-Glcp-(13)-[β-D-Glcp-(16)-]α-D-Glcp-(13)-β-D-Glcp-(13)-(β-D-Glcp-(16)-]D-Glcp through coupling of trisaccharide donors 9 with trisaccharide acceptor 16 and terrasaccharide acceptor 27 followed by deprotection. respectively.

A new triterpenoid saponin with novel sugar moiety (1), a new trisaccharide derivative (2); as well as a known monosaccharide derivative (3), were isolated from the dried stem of Epigynum aurilum. The structures of compounds 1 and 2 were determined by MS and NMR spectram analyses.

The phytoalexin elicitor β-(13)-branched β-(16)-linked glucohexatose has been regio-and stereospecifically synthesized by coupling of the 3, 6-branched gluco-trisaccharide Schmidt reagent 10 with a mixture of multiol) 3, 6-branched gluco-trisaccharides 13 which consists of free 5, 6′-OH trisaccharide, free 5,2′, 6′-OH trisaccharide, free 5,3′,6′-OH trisaccharide and so on. The compounds 10 and 13 were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, 2, 3, 4, 6-tetra-O-ben-zoyl-α-D-glucopyranosyl trichloroacetimidate, and 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl trichloroacetimidate through regio-and stereoselective manners.

A highly concise and effective synthesis of 1 2 α-linked mannopyranosyl oligosaccharides was achieved via TMSOTf promoted condensation of the corresponding benzoylated monosaccharide alkyl orthoester. 12 α-Linked mannosyl di, trisaccharide, antigenic factor 34, and dominant of antigenic factor 13 were readily synthesized by the new method.

A concise synthesis of α-Man1 2-α-Man1 3-(α-Man1 6)-α-Man1 2-α-Man1 2-α-Man1 2-Man, the antigenic factor 4 existing in Candida albicans, was achieved via TMSOTf promoted condensation of the corresponding acylated tetrasaccharide donor with the trisaccharide acceptor.

The beta-selectivity of mannosylation has been found to be dependent on the addition rate of the mannosyl trichloroacetimidate donor in an inverse-addition (I-A) procedure. This rate dependent I-A procedure can improve the selectivity of direct beta-mannosylation and is applicable to orthogonal glycosylations of thioglycoside acceptors. Further elaboration of this novel procedure enables the development of the contiguous sequential glycosylation strategy, which streamlines the preparation of oligosaccharides invoking beta-mannosidic bond formation. The synthetic utility of the contiguous glycosylation strategy was demonstrated by the preparation of the trisaccharide core of human N-linked glycoproteins and the trisaccharide repeating unit of the O-specific polysaccharide found in the cellular capsule of Salmonelle bacteria.

Zwitterionic polysaccharides (ZPSs) from Bacteroides fragilis and Streptococcus pneumoniae display unique T-cell activities. The first synthesis of a hexasaccharide representing two repeating units of the zwitterionic capsular polysaccharide from S. pneumoniae type 1 (Sp1) is reported. Key elements of the approach are stereoselective construction of 1,4-cis-alpha-galactose linkages based on a reactive trichloroacetimidate donor that incorporates a 6-O-acetyl group, which may contribute to the high alpha selectivity in glycosylation. After assembly of the fully protected hexasaccharide from five monosaccharide synthons 2-4, 24 and 25, selective deprotection of the primary hydroxyl groups of the four galactose residues followed by oxidation to the corresponding uronic acids provides hexasaccharide 19. The trisaccharide counterpart 1 was synthesized in similar fashion from three synthons, 2-4. This approach employed both conventional and dehydrative glycosylation methodologies and avoids the use of poorly reactive uronic acid derived glycosyl donors and acceptors.

A modular set of oligosaccharide building blocks was developed for the synthesis of multiantennary N-glycans of the complex type, which are commonly found on glycoproteins. The donor building blocks were laid out for the elongation of a core trisaccharide acceptor (beta-mannosyl chitobiose) conveniently protected with a single benzylidene moiety at the beta-mannoside. Through two consecutive regio- and stereoselective couplings the donors gave N-glycans with three to five antennae in high yields. Due to the consistent protection group pattern of the donors the deprotection of the final products can be performed by using a general reaction sequence.

A neuritegenic ganglioside from sea cucumber, HLG-2 (see figure), has been synthesized for the first time. The unique tandem of sialic acids, Neu5Gc-alpha(2,4)-NeuAc, was established by the combination of a reactive N-Troc sialyl donor and a 1,5-lactamized sialyl acceptor. The ceramide counterpart was assembled in a stereoselective manner. Direct connection of the trisaccharide and the ceramide successfully afforded a precursor of HLG-2, which was converted to ganglioside HLG-2 in pure form.A first synthesis of the neuritegenic ganglioside HLG-2, which was identified in extracts of the sea cucumber Holothuria leucospilota, is described. The characteristic sequence of the trisaccharide part, alpha-N-glycolylsialyl-(2,4)-alpha-N-acetylsialyl-(2,6)-glucoside, was efficiently assembled by coupling of a highly active N-2,2,2-trichloroethoxycarbonyl (Troc)-protected sialyl donor and a 1,5-lactamized sialyl acceptor with high stereoselectivity. The corresponding trisaccharyl imidate donor was directly glycosidated with the primary hydroxyl group of the ceramide part, producing protected HLG-2 in relatively high yield, global deprotection of which furnished ganglioside HLG-2 in highly pure form.

A new anhydroribotrisaccharide monomer, A2B3LR (1), was synthesized and ROP was carried out to elucidate the polymerizability and to obtain oligosaccharide-branched polysaccharides with defined structures. The new trisaccharide monomer was found to be polymerized readily with BF(3) . OEt(2) as a catalyst at -40 degrees C to give a lactose-branched polymer. Copolymerization with ADBR gave the corresponding copolymers in good yields. After removal of protective benzyl groups, D-lactose-branched ribofuranans with free hydroxyl groups were obtained in good yields. The structure of polymers was analyzed by (1)H, 13C, and two-dimensional NMR measurements, suggesting that D-lactose-branched ribofuranans had (1 --> 5)-alpha stereoregularity.

9 páginas, 7 figuras, 2 tablas, 1 esquema -- PAGS nros. 511-519, Weak recognition processes: Weak calcium-mediated carbohydrate–carbohydrate interactions have been detected by DOSY and TRNOESY NMR methods by employing a gold glyconanoparticle as a multivalent system. In addition, 3D models of trisaccharide-CaII-trisaccharide complexes based on results from molecular dynamics simulations are proposed. Diffusion-ordered NMR spectroscopy (DOSY-NMR) and TR-NOESY-NMR experiments are used to detect ligand binding to macromolecular receptors. These techniques have been applied to detect weak carbohydrate–carbohydrate self-recognition in solution, making use of sugar-decorated gold nanoparticles as the “macromolecule” and the same carbohydrate as the ligand. Changes in the diffusion coefficient of the free carbohydrate in the presence of the glyconanoparticle (only with CaII ions in the sample solution), as well as changes in the sign of the sugar NOE peaks—positive for the free sugar (in the presence or absence of CaII) and negative for the sugar only in the simultaneous presence of the glyconanoparticle and CaII ions—have been taken as proof of weak CaII-mediated carbohydrate–carbohydrate interactions in solution. Although different methods such as SPR, TEM, and AFM have been used in the past to detect carbohydrate–carbohydrate interactions with the aid of gold nanoparticles and gold selfassembled monolayers, they are restricted to high-affinity ranges. The methods used in this study allow expansion of the number of techniques to tackle this relevant biological problem, also for approaching ligand–receptor interactions below the high-affinity range. Additionally, 3D models of trisaccharide-CaII-trisaccharide complexes based on results from molecular dynamics simulations are proposed, The group at CIB-CSIC thanks MEC/MCINN for financial support (Grant CTQ2006–10874-C02–01). S.M.-S. also thanks MEC for her Ramón y Cajal contract. The group at Utrecht University thanks the European Commission for financial support (Glycogold project; Contract No: MRTN-CT-2004–005645)

To gain insight into the molecular details and hydration of amylopectin, the five constituting trisaccharides have been chemically synthesized as their methyl alpha-glycosides. All five trisaccharides were subjected to 950 MHz NMR spectroscopy for complete assignment and nanosecond molecular dynamics trajectories were calculated to study the structure and dynamics of the trisaccharides in aqueous solution. Systematic analysis of the simulation data revealed several examples of bridging water molecules playing an important role in the stabilization of specific amylopectin conformations, which was also supported by the experimental NMR data such as interresidue NOE's and heteronuclear scalar couplings between nuclei from neighboring residues. Although alpha-maltotriose, alpha-iso-maltotriose, alpha-panose and alpha-isopanose are relatively well characterized structures, the study also includes one less characterized trisaccharide with the structure alphaGlcp(1-->4)alphaGlcp(1-->6)alphaGlcp. This trisaccharide, tentatively labelled alpha-forkose, is located at the branch point of amylopectin, forking the amylopectin into two strands that align into double-helical segments. The results show that the conformation of alpha-forkose takes a natural bend form which fits well into the structure of the double-helical segment of amylopectin. As the only trisaccharide in this study the structure of alpha-forkose is not significantly influenced by the hydration. In contrast, alpha-isopanose takes a restricted, but rather extended form due to an exceptionally strong localized water density. The two homo-linkage oligomers, alpha-maltotriose and alpha-iso-maltotriose, showed to be the most extended and the most flexible trimers, respectively, providing regular structure for crystalline domains and maximum linker flexibility for amorphous domains.

The wild-type seed lima bean lectin (LBL), and recombinant LBL expressed in Escherichia coli show specificity for the human blood group A immunodominant trisaccharide GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-R. We have generated four site-specific mutants of LBL, two of which show altered specificity for extended carbohydrate structures. Four mutants, [C127Y]LBL, [H128P]LBL, [H128R]LBL and [W132F]LBL were expressed in E. coli. Two mutants show altered specificity for the substituent at the C2 hydroxy group of the penultimate Gal in the wild-type ligand which is alpha-L-fucose in the A trisaccharide. The mutant [C127Y]LBL showed specificity for the A disaccharide (GalNAc alpha 1-3Gal) and GalNAc alpha 1-4Gal, with free hydroxyl groups at the C2 position of Gal. The mutant [H128P]LBL bound the Forssman disaccharide structure GalNAc alpha 1-3GalNAc, in which the C2 hydroxyl group is substituted with an acetamido group. The third and fourth mutants, [H128R]LBL and [W132F]LBL, exhibited wild-type specificities, both recognizing the A trisaccharide. All of these mutant lectins bound the terminal GalNAc residues exposed on asialoovine submaxillary mucin, thus indicating that the monosaccharide-binding site had not been altered. We also determined that all but one mutant ([C127Y]LBL) retained the high-affinity binding site for N6 derivatives of adenine, indicative of tetramer formation; each mutant also expressed the low-affinity binding site for 8-anilinonaphthalene 1-sulfonate (1/monomer). Thus, by targeting two residues in LBL, we have identified a region of the protein that is part of the extended carbohydrate-binding site and which is specifically involved in the binding/recognition of substituents at the C2 position of the penultimate Gal of the A disaccharide. We have determined, by site-directed mutagenesis, that an essential Cys residue is involved in the specificity of LBL for the A trisaccharide.