Complex Oligosaccharides Research Articles

Synthetic Glycopeptides from the Mucin Family as Potential Tools in Cancer Immunotherapy

Compared to glycoproteins of healthy cells, glycoproteins of tumor cells are often aberrantly glycosylated. Thus, glycopeptide fragments of surface glycoproteins of tumor cells are of interest as tumor-associated antigens for the distinction between normal and tumor cells. Cancer immunotherapy directed at selectively targeting these tumor-associated glycoprotein structure alterations--deficient glycosylation and, thus, exposure of peptide epitopes which are masked in normal cells--is considered a promising approach for the treatment of cancer. For this purpose, glycoproteins from the mucin family are of particular interest. Mucins belong to a class of heavily O-glycosylated, high-molecular weight glycoproteins present on the surface of many epithelial cells. The mucin core protein consists of numerous tandem repeats rich in serine, threonine and proline. In their tumor-associated forms, epithelial mucins carry cryptic saccharide structures such as T(N)-, T-, sialyl-T(N)- and sialyl-T antigens and more complex oligosaccharides (e.g. Lewis(y)). In contrast to glycoproteins isolated from natural sources, synthetic glycopeptides can be obtained in high purity and with exactly defined structure. In this review, methodologies for the synthesis of mucin-type glycopeptides containing complex tumor-associated antigen structures are described. Due to the low immunogenicity often exhibited by synthetic tumor-associated glycopeptide antigens, their conjugation to carrier proteins or suitable T-cell epitopes is essential for the development of anti-tumor vaccines. The results of immunological evaluations of synthetic (glyco)peptides and oligosaccharides are described. Some of these synthetic vaccines show promising activities inducing proliferation of T-cells and cytotoxic T-cell responses.

Mechanistic Studies and Methods To Prevent Aglycon Transfer of Thioglycosides

Thioglycosides have been employed extensively for the synthesis of complex oligosaccharides, carbohydrate libraries, and mimetics of O-glycosides. While very useful, aglycon transfer is a problematic side reaction with thioglycosides. In this paper, a series of mechanistic studies are described. The aglycon transfer process is shown to affect both armed and disarmed thioglycosides, cause anomerization of the carbon-sulfur bond of a thioglycoside, and destroy the product of a glycosylation reaction. The results indicate that the aglycon transfer process can be a major problem for a wide range of thioglycosides. This side reaction is especially important to consider when carrying out complex reactions such as solid-phase glycosylations, one-pot or orthogonal multicomponent glycosylations, and construction of carbohydrate libraries. To prevent transfer, a number of modified aglycons were examined. The 2,6-dimethylphenyl (DMP) aglycon was found to effectively block transfer in a variety of model studies and glycosylation reactions. The DMP group can be installed in one step from a commercially available thiol (2,6-dimethylthiophenol) and is useable as a glycosyl donor. On the basis of these features, the DMP group is proposed as a convenient and improved aglycon for thioglycosides.

N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

Epithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N-glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E-cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N-glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N-glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin-catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with gamma-catenin and vinculin compared with the endogenous E-cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization.

Synthesis of a Library of Xylogluco-Oligosaccharides for Active-Site Mapping of Xyloglucanendo-Transglycosylase

Complex oligosaccharides containing alpha-D-xylosyl-(1-->6)-beta-D-glucosyl residues and unsubstituted beta-(1-->4)-linked D-glucosyl units were readily synthesized using enzymatic coupling catalyzed by the Cel7B E197A glycosynthase from Humicola insolens. Constituting this library required four key steps: (1) preparing unprotected building blocks by chemical synthesis or enzymatic degradation of xyloglucan polymers; (2) generating the donor synthon in the enzymatic coupling by temporarily introducing a lactosyl motif on the 4-OH of the terminal glucosyl units of the xylogluco-oligosaccharides; (3) synthesizing the corresponding alpha-fluorides, followed by their de-O-acetylation and the glycosynthase-catalyzed condensation of these donors onto various acceptors; and (4) enzymatically releasing lactose or galactose from the reaction product, affording the target molecules in good overall yields. These complex oligosaccharides proved useful for mapping the active site of a key enzyme in plant cell wall biosynthesis and modification: the xyloglucan endo-transglycosylase (XET). We also report some preliminary enzymatic results regarding the efficiency of these compounds.

Preparation and Evaluation of Unimolecular Pentavalent and Hexavalent Antigenic Constructs Targeting Prostate and Breast Cancer: A Synthetic Route to Anticancer Vaccine Candidates

Several novel, fully synthetic, carbohydrate-based antitumor vaccines have been assembled. Each construct consists of multiple cancer-related antigens displayed on a single polypeptide backbone. Recent advances in synthetic methodology have allowed for the incorporation of a complex oligosaccharide terminating in a sialic acid residue (i.e., GM2) as one of the carbohydrate antigens. Details of the vaccine synthesis as well as the results of preliminary immunological investigations are described herein.

Iterative one-pot syntheses of chitotetroses

Rapid syntheses of chitotetrose derivatives were achieved in good yields using the newly developed reactivity independent iterative one-pot strategy. The protective groups on donors and acceptors were independently evaluated allowing matching of the two partners in glycosylation. No anomeric reactivity adjustments or intermediate purification were necessary thus significantly improving the overall synthetic efficiency. Only near stoichiometric amounts of building blocks were required for the assembly of target molecules further highlighting the potential of the iterative one-pot method in complex oligosaccharide synthesis.

Oligomannoside mimetics by glycosylation of ‘octopus glycosides’ and their investigation as inhibitors of type 1 fimbriae-mediated adhesion of Escherichia coli

The glycocalyx of eukaryotic cells is composed of glycoconjugates, which carry highly complex oligosaccharide portions. To elucidate the biological role and function of the glycocalyx in cell-cell communication and cellular adhesion processes, glycomimetics have become targets of glycosciences, which resemble the composition and structural complexity of the glycocalyx constituents. Here, we report about the synthesis of a class of oligosaccharide mimetics of a high-mannose type, which were obtained by mannosylation of spacered mono- and oligosaccharide cores. These carbohydrate-centered cluster mannosides have been targeted as inhibitors of mannose-specific bacterial adhesion, which is mediated by so-called type 1 fimbriae. Their inhibitory potencies were measured by ELISA and compared to methyl mannoside as well as to a series of mannobiosides, and finally to the polysaccharide mannan. The obtained results suggest a new interpretation of the mechanisms of bacterial adhesion according to a macromolecular rather than a multivalency effect.

Biologically Active Glycosides from Asteroidea, 42. Isolation and Structure of a New Biologically Active Ganglioside Molecular Species from the Starfish Asterina pectinifera

A ganglioside molecular species GP-3 (1) has been obtained from the water-soluble lipid fraction of the chloroform/methanol extract of the starfish Asterina pectinifera. The structure of the ganglioside has been determined on the basis of chemical and spectroscopic evidence. Compound 1 represents new ganglioside molecular species possessing two moles of sialic acids at the inner part of the sugar moiety. Partial hydrolysis by hot water and an enzymatic hydrolysis by means of endoglycoceramidase (EGCase) have proved useful for structure elucidation of the complex oligosaccharide moiety. Moreover, 1 exhibits neuritogenic activity toward the rat pheochromocytoma cell line, PC-12 cells, in the presence of nerve growth factor (NGF).

A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a need for larger amounts of different carbohydrate structures. Leloir glycosyltransferases catalyze the synthesis of complex oligosaccharides. However they are difficult or expensive to obtain, and require expensive nucleotide activated sugars. In contrast non-Leloir pathway enzymes use sucrose, which is known to be a high energy donor of d-glucose for glucosyltransferases like dextransucrase, or a donor of d-fructose for fructosyltransferases like inulin- and levansucrases for the synthesis of polysaccharides. Here we present the synthesis and kinetic studies of oligosaccharides using non-Leloir glycosyltransferases and sucrose analogues as new substrates, like β-d-fructofuranosyl-α-d-galactopyranoside (Gal-Fru) by a fructosyltransferase (FTF) from B. subtilis NCIMB 11871. The sucrose analogues carry a high binding energy in the glycosidic bond similar to that of sucrose. Thus, β-d-Fructofuranosyl-α-d-galactopyranoside (Gal-Fru) and β-d-Fructofuranosyl-α-d-fucopyranoside (d-Fuc-Fru) have been shown to be substrates for fructosyltransferases, which produce oligo- or polysaccharides, also in the presence of acceptors.

Rapid analysis of oligosaccharides derived from glycoproteins by microchip electrophoresis

A novel method for fast profiling of complex oligosaccharides released from glycoproteins based on microchip electrophoresis (μ-CE) is presented here. The characterization of separation conditions, i.e., the composition, concentration and pH of running buffer as well as the applied voltage, has been performed using maltose (G 2), cellobiose ( G ′ 2 ), maltriose (G 3) and panose ( G ′ 3 ) as oligosaccharide isomer models. In μ-CE, much better separation of oligosaccharide isomers and oligosaccharide ladder was obtained in phosphate buffer than in borate buffer over a wide pH range. Under optimal conditions, high-performance separation of the N-linked complex oligosaccharides released from ribonuclease B, fetuin, α 1-acid glycoprotein (AGP) and IgG was achieved using polymethylmethacrylate (PMMA) microchips with an effective separation channel of 30 mm. These results represent the first reported analysis of the N-linked oligosaccharides derived from glycoproteins by μ-CE, indicating that the present μ-CE-based method is a promising alternative for characterization of the N-linked oligosaccharides in glycoproteins.

The Role of Mannosylated Enzyme and the Mannose Receptor in Enzyme Replacement Therapy

Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal-/-) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide-type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)-positive J774E cells. After intraperitoneal injection into lal-/- mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal-/- mice produced normalization of hepatic color, decreased liver weight (50%-58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal-/-;MMR-/-) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor-independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR.

Heparan Sulfate Mimicry: A SYNTHETIC GLYCOCONJUGATE THAT RECOGNIZES THE HEPARIN BINDING DOMAIN OF INTERFERON-γ INHIBITS THE CYTOKINE ACTIVITY

Cell-associated heparan sulfate (HS) is endowed with the remarkable ability to bind numerous proteins. As such, it represents a unique system that integrates signaling from circulating ligands with cellular receptors. This polysaccharide is extraordinary complex, and examples that define the structure-function relationship of HS are limited. In particular, it remains difficult to understand the structures by which HS interact with proteins. Among them, interferon-gamma (IFNgamma), a dimeric cytokine, binds to a complex oligosaccharide motif encompassing a N-acetylated glucosamine-rich domain and two highly sulfated sequences, each of which binds to one IFNgamma monomer. Based on this template, we have synthesized a set of glycoconjugate mimetics and evaluated their ability to interact with IFNgamma. One of these molecules, composed of two authentic N-sulfated octasaccharides linked to each other through a 50-Angstroms-long spacer termed 2O(10), displays high affinity for the cytokine and inhibits IFNgamma-HS binding with an IC(50) of 35-40 nm. Interestingly, this molecule also inhibits the binding of IFNgamma to its cellular receptor. Thus, in addition to its ability to delocalize the cytokine from cell surface-associated HS, this compound has direct anti-IFNgamma activity. Altogether, our results represent the first synthetic HS-like molecule that targets a cytokine, strongly validating the HS structural determinants for IFNgamma recognition, providing a new strategy to inhibit IFNgamma in a number of diseases in which the cytokine has been identified as a target, and reinforcing the view that it is possible to create"tailor-made"sequences based on the HS template to isolate therapeutic activities.

Coexpression of β1,6-N-Acetylglucosaminyltransferase V Glycoprotein Substrates Defines Aggressive Breast Cancers with Poor Outcome

Beta1,6-n-acetylglucosaminyltransferase-V (GnT-V) catalyzes the addition of complex oligosaccharide side chains to glycoproteins, regulating the expression and function of several proteins involved in tumor metastasis. We analyzed the expression of five cell-surface glycoprotein substrates of GnT-V, matriptase, beta1-integrin, epidermal growth factor receptor, lamp-1, and N-cadherin, on a tissue microarray cohort of 670 breast carcinomas with 30-year follow-up. Phaseolus vulgaris leukocytic phytohemagglutinin (LPHA), a lectin specific for beta1,6-branched oligosaccharides, was used to assay GnT-V activity. Our results show a high degree of correlation of the LPHA staining with matriptase, lamp-1, and N-cadherin expressions, but not with epidermal growth factor receptor or beta1-integrin expressions. In addition, many of the GnT-V substrate proteins exhibited strong coassociations. Elevated levels of GnT-V substrates were correlated with various markers of tumor progression, including positive node status, large tumor size, estrogen receptor negativity, HER2/neu overexpression, and high nuclear grade. Furthermore, LPHA and matriptase showed significant association with disease-related survival. Unsupervised hierarchical clustering of the GnT-V substrate protein expression and LPHA revealed two distinct clusters: one with higher expression of all markers and poor patient outcome and one with lower expression and good outcome. These clusters showed independent prognostic value for disease-related survival when compared with traditional markers of tumor progression. Our results indicate that GnT-V substrate proteins represent a unique subset of coexpressed tumor markers associated with aggressive disease.

KEY OLIGOSACCHARIDE OF CELL WALL PREPARED

A COMPLEX OLIGOSACCHARIDE that makes up part of the cell wall of the tuberculosis-causing bacterium has been prepared by total synthesis. The work makes the cell-wall component—a lipomannan—available in pure form for studies of its role in the disease. It also demonstrates the growing practicality of a long-recognized way to accelerate carbohydrate synthesis by omitting some protection and deprotection steps. The work was carried out by carbohydrate chemist Bert Fraser-Reid and postdocs K.N. Jayaprakash and Jun Lu at the Natural Products & Glycotechnology Research Institute, a nonprofit organization with labs at North Carolina State University's Centennial Campus ( Angew. Chem. Int. Ed . 2005, 44 ,5894). The researchers created the lipomannan component from simple precursors by using donor-acceptor matching. This technique can eliminate many of the tedious protecting-group manipulations that make carbohydrate synthesis difficult and time-consuming. properties of material isolated from natural sources is...

Glycosylation with Glycosyl p‐Bromophenyl Phthalates as New Efficient Glycosyl Donors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Characterization of caprine herpesvirus 1 glycoprotein D gene and its translation product

Caprine herpesvirus 1 (CpHV-1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main immunogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD shares most characteristics with other alphaherpesviruses and especially BoHV-1.

Methyl 3,4-O-dibenzoyl-2-O-benzyl-1-thio-β-L-fucopyranoside

The title compound, C28H28O6S, is a protected fucose donor, synthesized as part of our investigations into the preparation of complex oligosaccharides. The benzoyl groups are equatorial and axial, while the thio­ether and ether groups are equatorial. The carboxyl­ate O atoms form a number of intra- and inter­molecular C—H⋯O inter­actions.

Characterization of Disulfide Bonds in Human Nucleoside Triphosphate Diphosphohydrolase 3 (NTPDase3): Implications for NTPDase Structural Modeling

Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants constituted five distinct phenotypes, thus, strongly suggesting disulfide bonds between C92-C116 (first bond), C261-C308 (second bond), C289-C334 (third bond), C347-C353 (fourth bond), and C399-C422 (fifth bond). Due to conservation of the 10 cysteines, the identified five disulfide bonds are likely to exist in all cell-surface NTPDases. The third and fifth bonds are also present in the soluble NTPDases and are critical for processing, trafficking, and enzymatic activity. The fourth bond has minimal effect on processing and function, while the first and second bonds are of intermediate importance. Most of the N-linked glycosylation sites in the wild-type enzyme are processed to complex oligosaccharides, but at least one site is high-mannose or hybrid in structure. Interestingly, disruption of the first disulfide bond resulted in some enzyme that lost sensitivity to endoglycosidase H, suggesting that the first disulfide bond in the wild-type enzyme shields some high-mannose glycans from terminal glycosylation. Comparative modeling by threading and homology modeling of the NTPDase3 sequence revealed a high degree of structural fold similarity with a bacterial exopolyphosphatase (PDB ). The resultant theoretical 3-D model of the extracellular portion of NTPDase3, based on homology with this exopolyphosphatase, is consistent with the assignment of the disulfide bonds occurring in regions of good fold similarity between NTPDase3 and the exopolyphosphatase. The 3-D model obtained for NTPDase3 also suggests the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.

Glycosylation with Glycosyl p-Bromophenyl Phthalates as New Efficient Glycosyl Donors

The development of an efficient and stereoselective glycosylation methodology has been a major concern in synthetic organic chemistry in the past a decade due to the important biological functions of complex oligosaccharides and glycoconjugates. Devising new glycosyl donors and developing new activation systems for the existing donors resulted in major advances in this area. For example, several glycosylation methodologies using the efficient glycosyl donors such as thioglycosides, glycosyl sulfoxides, glycals, glycosyl trichloroacetimidates, n-pentenyl glycosides, and glycosyl fluorides have been available. In addition, there have been recent reports regarding the development of glycosylation methods via new glycosyl donors and the application of existing glycosyl donors in novel activation systems. However, there still remains a need for more efficient and generally applicable new glycosylation methodology. In our previous endeavors, we reported a novel type of glycosyl donors, the 2’-(benzyloxycarbonyl)benzyl (BCB) glycoside and the 2’-carboxybenzyl (CB) glycoside A, as shown in Figure 1 for the stereoselective β-mannopyranosylation and the 2-deoxyglycosylation. We applied this methodology to the synthesis of a trisaccharide and tetrasaccharide. More recently, we have also reported the synthesis of the glycosyl benzyl phthalate C and its use as a new type of glycosyl donor. Although the glycosyl benzyl phthalate C proved to be the efficient glycosyl donor, there still remains a need for improvement. Thus, it is desirable to conduct the glycosylation more efficiently and at lower temperature in order to control the stereochemistry of the product and to keep acid-labile reactants or products intact under the acidic condition resulting from the use of TMSOTf. We envisaged that certain glycosyl aryl phthalates D might be more reactive than C so that the glycosylation might be conducted at lower temperature. Herein we describe the synthesis of a new type of glycosyl donors, the glycosyl aryl phthalate D and report that the glycosylation with glycosyl p-bromophenyl phthalates could be conducted under milder condition than that with glycosyl benzyl phthalates.

Direct one-pot conversion of acylated carbohydrates into their alkylated derivatives under heterogeneous reaction conditions using solid NaOH and a phase transfer catalyst

A convenient one-pot protocol for the direct conversion of acyl-protected carbohydrates into their alkylated counterparts has been developed by using alkyl halides in the presence of solid sodium hydroxide and a phase transfer catalyst. These economically convenient, mild, two-phase reaction conditions allow the preparation of a variety of monosaccharide intermediates for use in the synthesis of complex oligosaccharides.