Gold nanoparticles coated with a pyruvated trisaccharide epitope of the extracellular proteoglycan of Microciona prolifera as potential tools to explore carbohydrate-mediated cell recognition

The species-specific cell adhesion in the marine sponge Microciona prolifera involves the interaction of an extracellular proteoglycan-like macromolecular complex, otherwise known as aggregation factor. In the interaction, two highly polyvalent functional domains play a role: a cell-binding and a self-interaction domain. The self-recognition has been characterized as a Ca(2+)-dependent carbohydrate-carbohydrate interaction of repetitive low affinity carbohydrate epitopes. One of the involved epitopes is the pyruvated trisaccharide beta-d-Galp4,6(R)Pyr-(1-->4)-beta-d-GlcpNAc-(1-->3)-l-Fucp. To evaluate the role of this trisaccharide in the proteoglycan-proteoglycan self-recognition, beta-d-Galp4,6(R)Pyr-(1-->4)-beta-d-GlcpNAc-(1-->3)-alpha-l-Fucp-(1-->O)(CH(2))(3)S(CH(2))(6)SH was synthesized, and partially converted into gold glyconanoparticles. These mimics are being used to explore the self-interaction phenomenon for the trisaccharide epitope, via TEM aggregation experiments (gold glyconanoparticles) and atomic force microscopy (AFM) experiments (self assembled monolayers; binding forces).     

The 2-naphthylmethyl (NAP) group in carbohydrate synthesis: first total synthesis of the GlyCAM-1 oligosaccharide structures

Total syntheses of the GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) oligosaccharide structures: [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-(6-O-SO3Na)-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (1) and [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (2) through a novel sialyl LewisX tetrasaccharide donor are described. Employing sequential glycosylation strategy, the starting trisaccharide was regio- and stereoselectively constructed through coupling of a disaccharide imidate with the monosaccharide acceptor phenyl-6-O-naphthylmethyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside with TMSOTf as a catalyst without affecting the SPh group. The novel sialyl Lewisx tetrasaccharide donor 3 was then obtained by alpha-L-fucosylation of trisaccharide acceptor with the 2,3,4-tri-O-benzyl-1-thio-beta-L-fucoside donor. The structure of the novel sialyl Lewisx tetrasaccharide was established by a combination of 2D DQF-COSY and 2D ROESY experiments. Target oligosaccharides 1 and 2 were eventually constructed through heptasaccharide which was obtained by regioselective assembly of advanced sialyl Lewisx tetrasaccharide donor 3 and a sialylated trisaccharide acceptor in a predictable and controlled manner. Finally, target heptasaccharides 1 and 2 were fully characterized by 2D DQF-COSY, 2D ROESY, HSQC, HMBC experiments and FAB mass spectroscopy.     

Synthesis of the glycopeptidolipid of Mycobacterium avium Serovar 4: first example of a fully synthetic C-mycoside GPL

The preparation of the glycopeptidolipid (GPL) present in the cell wall of Mycobacterium aviumSerovar 4, namely 3,4-di-O-Me-alpha-L-Rhap-(1-->1)[R-C(21)H(43)CH(OH)CH(2)CO-D-Phe-[4-O-Me-alpha-L-Rhap-(1-->4)-2-O-Me-alpha-L-Fucp-(1-->3)-alpha-L-Rhap-(1-->2)-6-deoxy-alpha-L-Talp-(1-->3)]-D-allo-Thr-D-Ala-L-Alaol] (1), is described. The synthesis was based on the disconnection of the final structure into four building blocks, an L-rhamnosyl pseudodipeptide, a 6-deoxy-L-talosyl dipeptide, a trisaccharide donor, and a 3-hydroxyalkanoic acid. The key steps are the creation of the glycosidic linkage between the trisaccharide donor, used as a pentenyl glycoside, and the 6-deoxy-L-talose unit of an appropriate D-Phe-O-(6-deoxy-L-talosyl)-D-allo-Thr derivative and the final coupling of the two glycodipeptide fragments. Pentenyl glycosides were shown to provide useful donors in several glycosylation steps. This work constitutes the first synthesis of the full structure of a so-called "polar mycoside C" GPL.     

Efficient synthesis of 3, 6-dideoxy-beta-D-arabino-hexopyranosyl-terminated LacdiNac glycan chains of the Trichinella spiralis parasite

The synthesis of a linear trisaccharide epitope of the Trichinella spiralis N-linked glycan, in a form amenable to glycoconjugate formation, is reported. The trisaccharide contains the synthetically challenging LacdiNAc [beta-GalpNAc(1-->4)-beta-GlcpNAc] element, as well as a terminal 3,6-dideoxy-beta-D-arabino-hexopyranose (tyvelose) residue. An orthogonal protection strategy is described, which permits the protection and manipulation of three amino groups present in the disaccharide beta-GalNAc(1-->4)-beta-GlcNAc and the tether used to prepare neoglycoconjugates. The beta-linked dideoxyhexose was generated in excellent yield by the introduction of the dideoxyhexose unit as a beta-D-ribo-hexopyranoside (paratose) followed by an oxidation-reduction sequence to generate the beta-D-arabino configuration in high diastereomeric excess. The required dideoxyhexose donor was synthesized in a series of high-yielding steps from glucose utilizing the p-methoxyphenyl glycoside.     

Synthesis of various sulfoforms of the trisaccharide beta-d-GlcpA-(1-->3)- beta-d-Galp-(1-->3)-beta-d-Galp-(1-->OMP) as probes for the study of the biosynthesis and sorting of proteoglycans

A straightforward preparation of various sulfoforms of the trisaccharide 4-methoxyphenyl O-(sodium beta-d-glucopyranosyluronate)-(1-->3)-( beta-d-galactopyranosyl)-(1-->3)-beta-d-galactopyranoside (1), namely its 6a- and 4a-monosulfate, 6b- and 4b-monosulfate and 6a,6b-disulfate derivatives, is reported for the first time. These compounds, which are partial structures of the linkage region of proteoglycans, will serve as probes for the study of the biosynthesis and sorting of these macromolecules. A key trisaccharide derivative, in which the two similar d-Gal units were differentiated at C-4,6 with 4,6-benzylidene and 4,6-di-tert-butylsilylene acetals, respectively, was used as a common intermediate. Both acetal groups showed excellent orthogonality, and allowed the preparation of all target compounds in high yield. Noteworthy is the possibility to prepare the 6a- and 6b-monosulfated and the 6a,6b-disulfated species through a one-pot regioselective procedure starting from a tetrol precursor.     

Synthesis of tri-, hexa-, and nonasaccharide subunits of the atypical O-antigen polysaccharide of the lipopolysaccharide from Danish Helicobacter pylori strains

Synthesis of trisaccharide repeating unit, -->3)-alpha-D-Rhap-(1-->2)-alpha-D-Manp3CMe-(1-->3)-alpha-L-Rha p-(1-->, and its dimeric hexa- and trimeric nonasaccharide subunits of the atypical O-antigen polysaccharide of the lipopolysaccharide from Danish H. pylori strains D1, D3, and D6 has been accomplished. Successful synthesis of the hexasaccharide and the nonasaccharide was possible by dimerization and trimerization of the suitably protected trisaccharide repeating unit, in which three monosaccharide moieties were arranged in a proper order by placing the sterically demanding 3-C-methyl-D-mannose moiety in between D- and L-rhamnoses. Key steps include the coupling of three monosaccharide moieties and dimerization and trimerization of the trisaccharide unit by glycosylations employing the 2'-carboxybenzyl glycoside method. Also presented is a method for the synthesis of the novel branched sugar, 3-C-methyl-D-mannose moiety by elaboration of its equatorial hydroxyl and axial methyl groups at C-3' in the disaccharide stage.     

黑果枸杞叶多糖LRLP3的结构、抗氧化活性及免疫活性

黑果枸杞叶经水提醇沉, 离子交换柱层析和凝胶柱层析分离纯化, 得到平均分子量为79400的均一多糖组分LRLP3. 对该多糖的理化性质、 结构、 抗氧化活性及免疫活性的研究结果表明, LRLP3为多分支结构, 主链为(1→3)βGalp, 大部分半乳糖6位存在分支; 支链由(1→6)βGalp, (1→4)βGalp, (1→3)βAraf, (1→3)αArap, (1→5)βAraf和(1→2,4)αRhap组成, 非还原末端由αAraf, βGalp和βGlcp组成. LRLP3具有较强的还原能力, 可显著清除1,1-二苯基-2-三硝基苯肼(DPPH)自由基、 羟自由基和超氧阴离子自由基, 有效抑制Cu²⁺/H₂O₂诱导的蛋白氧化损伤和H₂O₂诱导的细胞氧化损伤. LRLP3在体外对未经诱导和经刀豆蛋白(ConA)或脂多糖(LPS)诱导的小鼠脾细胞增殖均有促进作用.     

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of neogala-series glycolipid analogues containing a mannose residue from the earthworm Pheretima hilgendorfi.

Two kinds of glycosphingolipid analogues from the earthworm Pheretima hilgendorfi were synthesized as follows: the trisaccharide 2-(tetradecyl)hexadecyl alpha-D-mannopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->6)-beta-D- galactopyranoside (13) and the tetrasaccharide 2-(tetradecyl) hexadecyl alpha-D-galactopyranosyl-(1-->6)-[alpha-D-mannopyranosyl-(1-->4)]-beta-D - galactopyranosyl-(1-->6)-beta-D-galactopyranoside (20) were synthesized by stepwise condensation of suitably protected monosaccharide units. A 2-(trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside derivative (5) was used as the glycosyl acceptor and thiophenyl derivatives of D-galactose and D-mannose were used as donors respectively.     

Solid-phase oligosaccharide synthesis: preparation of complex structures using a novel linker and different glycosylating agents

[formula: see text] A beta-(1-->4)-linked trisaccharide was prepared in 53% yield on a polymer support using glycosyl phosphates and released by cross-metathesis of a novel linker to reveal the anomeric n-pentenyl glycoside. Heptasaccharide 33 was prepared in 9% yield in 14 steps.     

Highly efficient oligosaccharide synthesis on water-soluble polymeric primers by recombinant glycosyltransferases immobilised on solid supports

Recombinant beta-1,4-galactosyltranferase (beta 1,4-GalT) and alpha-2,6-sialytransferase (alpha 2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical synthesis of a trisaccharide derivative, Neu-5Ac alpha(2-->6)Gal beta(1-->4)GlcNAc beta-O-(CH2)6-NH2, on a water-soluble primer having GlcNAc residues through a alpha-chymotrypsin-sensitive linker.     

Synthesis of a common tetrasaccharide motif of Haemophilus influenzae LPS inner core structures

A conserved tetrasaccharide structure, L-glycero-alpha-D-manno-heptopyranosyl-(1-->2)-(6-O-aminoethylphosphono-L-glycero-alpha-D-manno-heptopyranosyl)-(1-->3)-[beta-D-glucopyranosyl-(1-->4)]-L-glycero-alpha-D-manno-heptopyranose, from the LPS inner core of Haemophilus influenzae has been synthesised as its ethylamino glycosides to allow later conjugations. Starting from a previously synthesised suitably protected trisaccharide intermediate, the third heptose and subsequently the spacer were introduced using thioglycoside donor chemistry. The phosphoethanolamine was formed employing a Boc-protected phosphoamidite. Final deprotection and conjugation to biotin gave conjugates that will be used to study the specificity of MAbs raised against native LPS structures.     

Chemical synthesis of CD52 glycopeptides containing the acid-labile fucosyl linkage

Glycopeptide 1 with the fucosylated trisaccharide, beta-d-GlcNAc(1-->4)[alpha-l-Fuc(1-->6)]-beta-d-GlcNAc, linked to the Asn of CD52 peptide was prepared by two methods, both of which used the free glycosyl Asn 12 and glycotripeptide 21 as key intermediates. Thus, after the trisaccharide was prepared and linked to Asn, the carbohydrate moiety was deprotected to give 12. From 12, 21 was constructed in homogeneous NMP solutions by elongating the peptide chain alone the N-terminus. Though the glycopeptides were easily soluble in NMP, they were barely soluble in diethyl ether, because of the free trisaccharide. Consequently, addition of diethyl ether to the reaction mixtures could precipitate the glycopeptides, and the products were conveniently isolated and purified in the solid form. The coupling of 21 with a free nonapeptide 24 in NMP afforded 1. 1 was also prepared by solid-phase synthesis, using the acid-sensitive 2-chlorotrityl resin. In this case, 21 was attached to the nonapeptide on the resin, and the resulting glycopeptide was then released with dilute acetic acid. Deprotection of the peptide under moderate acidic conditions gave 1. The acid-labile alpha-fucose was not affected in these syntheses.     

Facile synthesis of trisaccharide moiety corresponding to antitumor activity in triterpenoid saponins isolated from Pullsatilla roots

A trisaccharide found in triterpenoid saponins isolated from Pullsatilla roots appears as an important promoiety for the enhancement of anticancer activity of their aglycones. Thus a facile synthetic method for a trisaccharide moiety, allyl-2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->4)]-3-O-benzoyl-beta-L-arabinopyranoside (3), has been firstly developed through the regio- and stereoselective glycosylations from arabinose in total 16% yield via route 2 (eight steps). In this synthetic procedure, the protection of anomeric -OH of L-arabinose with equatorially oriented allyl group unlike with the axial 4-methoxybenzyl protecting group well promoted glycosyl bond formation between alpha-L-rhamnopyranosyl trichloroacetimidate and 2-OH of arabinose. As expected, the synthesized trisaccharide moiety 3 has no cytotoxicity (ED50: >100 microM) against three human cancer cell lines (A-549, SK-OV-3, and SK-MEL-2), respectively.     

Weak calcium-mediated interactions between Lewis X-related trisaccharides studied by NMR measurements of residual dipolar couplings

The Lewis X (LeX) determinant, a trisaccharide with the carbohydrate sequence Galbeta(1-->4)[Fucalpha(1-->3)]GlcNAcbeta, is believed to be responsible for Ca2+-mediated cell-cell interactions. In partly oriented phases composed of mixtures of penta(ethyleneglycol)monododecyl ether HO(CH2CH2O)5C12H25 and n-hexanol in the presence of Ca2+ ions, the variation of the residual dipolar couplings 1DCH of various CiHi vectors in LeX as a function of the concentration of the trisaccharide demonstrates the existence of very weak LeX-Ca2+-LeX complexes in solution. Synthetic 3-, 4-, and 6-deoxy-LeX variants were also shown to form complexes in the presence of calcium ions, despite the replacement of one of their hydroxyl groups by hydrogen atoms. This is the first direct observation in solution of a calcium-mediated interaction between LeX molecules.     

Synthesis, inhibitory effects on nitric oxide and structure-activity relationships of a glycosphingolipid from the marine sponge Aplysinella rhax and its analogues

The novel glycosphingolipid, β-D-GalNAcp(1-->4)[α-D-Fucp(1-->3)]-β-D-GlcNAcp(1-->)Cer (A), isolated from the marine sponge Aplysinella rhax has a unique structure, with D-fucose and N-acetyl-D-galactosamine moieties attached to a reducing-end N-acetyl-D-glucosamine through an α1-->3 and β1-->4 linkage, respectively. We synthesized glycolipid 1 and some non-natural di- and trisaccharide analogues 2-6 containing a D-fucose residue. Among these compounds, the natural type showed the most potent nitric oxide (NO) production inhibitory activity against LPS-induced J774.1 cells. Our results indicate that both the presence of a D-Fucα1-3GlcNAc-linkage and the ceramide aglycon portion are crucial for optimal NO inhibition.     

Direct chemical synthesis of the beta-D-mannans: the beta-(1-->2) and beta-(1-->4) series

The direct syntheses of a beta-(1-->2)-mannooctaose and of a beta-(1-->4)-mannohexaose are reported by means of 4,6-O-benzylidene-protected beta-mannosyl donors. The synthesis of the (1-->2)-mannan was achieved by means of the sulfoxide coupling protocol, whereas the (1-->4)-mannan was prepared using the analogous thioglycoside/sulfinamide methodology. In the synthesis of the (1-->4)-mannan, the glycosylation yields and stereoselectivities remain approximately constant with increasing chain length, whereas those for the (1-->2)-mannan consist of two groups with the formation of the tetra- and higher saccharides giving yields and selectivities consistently lower than those of the lower homologues. The decrease in yield after the trisaccharide in the (1-->2)-mannan synthesis is attributed to steric interference by the n-3 residue and is consistent with the collapsed, disordered structure predicted by early computational work. The consistently high yields and selectivities seen in the synthesis of the (1-->4)-mannan are congruent with the more open, ordered structure originally predicted for this polymer. The lack of order in the structure of the (1-->2)-mannan, as compared to the high degree of order in the (1-->4)-mannan, is also evident from a comparison of the NMR spectra of the two polymers and even from their physical nature: the (1-->2)-mannan is a gum and the (1-->4)-mannan is a high melting solid.     

Cap-and-tag solid phase oligosaccharide synthesis

A new "cap-and-tag" strategy is applied to solid phase oligosaccharide synthesis. Acetyl-capping and fluorous-tagging allowed for the facile separation of the desired F-tagged oligosaccharide from the acetyl-capped deletion sequences using fluorous solid phase extraction. To illustrate this approach, a protected Glc-beta-(1-->6)-Man-alpha-(1-->6)-Glc-beta-1-->pentenyl trisaccharide was synthesized.     

Mirror-image carbohydrates: synthesis of the unnatural enantiomer of a blood group trisaccharide

Methyl D-glucoside and d-glucose pentaacetate are transformed, respectively, into methyl alpha-O-glucuronide 3 and hydroxymethyl beta-C-glucuronide 9, which undergo decarboxylative elimination efficiently to produce 4-deoxypentenoside 4 and L-glucal 10. These unsaturated pyranosides provide an expeditious entry into mirror-image oligosaccharides, as demonstrated in the synthesis of the unnatural enantiomer of the H-type II blood group determinant trisaccharide (D-Fuc-(alpha1-->2)-L-Gal-(beta1-->4)-L-GlcNAc-beta-OMe). This work illustrates that D-glucose, a common starting material in the synthesis of naturally occurring carbohydrates, can also be used to prepare their mirror-image analogues.     

Triterpenoid saponins from the roots of Sophora koreensis

From the roots of Sophora koreensis (Fabaceae), three new oleanene-type triterpene glycosides, echinosophorosides A(1) (1) and B (2), and acetyl-subproside II (5), were isolated as their methyl esters, together with the four known ones sophoraflavoside I, kudzusaponin SA(3), subproside II, and azukisaponin V. The structures of the new saponins were elucidated to be 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl kudzusapogenol A 22-O-alpha-L-arabinopyranoside (1), 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl-(1-->2)-beta-D-glucuronopyranosyl abrisapogenol C 22-O-alpha-L-arabinopyranoside (2), and 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl kudzusapogenol A 22-O-acetate (5), respectively. It is noteworthy that two arabinopyranosyl moieties in the same molecule, echinosophoroside B (2), have different conformations. The conformation of the arabinopyranosyl moiety existing in the trisaccharide moiety was determined to be (1)C(4), whereas that of the arabinopyranosyl unit at C-22 was identified as (4)C(1).     

Transsialidase from Trypanosoma cruzi for regio- and stereoselective synthesis of N-acyl-modified sialylated oligosaccharides and measurement of transfer rates

Recombinant transsialidase from Trypanosoma cruzi (TcTS) was used for the sialylation with natural and non-natural derivatives of neuraminic acid. Neu5Ac-alpha(2-->3)-Gal-beta(1-->6)-Glc-alphaOMe was prepared in 80 % yield. Correspondingly, the modified trisaccharide derivatives Neu5Prop-alpha(2-->3)-Gal-beta(1-->6)-Glc-alphaOMe (32 %) and Neu5Gc-alpha(2-->3)-Gal-beta(1-->6)-Glc-alphaOMe (Prop=propanoyl, Gc=glycolyl) were obtained in 60 % yield, respectively.