Chien-Tai Ren

Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer

Cancer-associated carbohydrate antigens are often found on the surface of cancer cells. Understanding their roles in cancer progression will lead to the development of new therapeutics and high-sensitivity diagnostics for cancers. Globo H is a member of this family, which is highly expressed on breast cancer cells. Here, we report the development of a glycan microarray of Globo H and its analogs for measurement of the dissociation constants on surface (KD,surf) with three different monoclonal antibodies (VK-9, Mbr1, and anti-SSEA-3), to deduce their binding specificity. The glycan microarray was also used to detect the amount of antibodies present in the plasma of breast cancer patients and normal blood donors. It was shown that the amount of antibodies against Globo H from breast cancer patients were significantly higher than normal blood donors, providing a new tool for possible breast cancer diagnosis. Compared with the traditional ELISA method, this array method required only atto-mole amounts of materials and is more effective and more sensitive (5 orders of magnitude). The glycan microarray thus provides a new platform for use to monitor the immune response to carbohydrate epitopes after vaccine therapy or during the course of cancer progression.

Highly Alpha‐Selective Sialyl Phosphate Donors for Efficient Preparation of Natural Sialosides

N-Acetyl neuraminic acid (Neu5Ac) is most frequently found at the terminal end of glycoconjugates on the cell surface. This terminally exposed position allows Neu5Ac-containing conjugates to be exploited as receptors for viruses and bacteria, in addition to governing a wide variety of biological processes, such as tumor metastasis, cell differentiation, and cell–cell interactions. In naturally occurring sialosides, Neu5Ac is found linked to galactosides through the aACHTUNGTRENNUNG(2!3) or aACHTUNGTRENNUNG(2!6) linkage in Nand O-linked glycoproteins, and also to N-acetylgalactosamine through the aACHTUNGTRENNUNG(2!6) linkage in O-linked glycoproteins. In addition, polysialosides formed by the aACHTUNGTRENNUNG(2!8) or a ACHTUNGTRENNUNG(2!9) linkages are constituents of glycoproteins and glycolipids. The biological significance of sialoside receptors has driven research for their efficient synthesis. This has included significant efforts directed toward the development of sialic acid donors for efficient a-sialylation, including the use of anomeric leaving groups, such as halides, phosphites, sulfides, xanthates, and phenyltrifluoroacetimidates, the introduction of an auxiliary group at C-1 and C-3, the modification of the Nacetyl functional group at C-5, or the optimized combinations of the leaving group with positional modification. However, high yielding a-selective sialylation is still problematic owing to the presence of the C-1 electron-withdrawing carboxyl group at the tertiary anomeric center and the lack of a participating group at C-3 to direct the stereochemical outcome of glycosylation. The development of new strategies for the convergent synthesis of saccharides is a major focus in carbohydrate chemistry. Approaches such as armed–disarmed; onepot; reactivity-based, programmable one-pot; solidphase; orthogonal; and pre-activation methods have been developed to improve efficiency, with the ultimate goal of developing an automated method for oligosaccharide synthesis. The basic concept of these strategies involves the selective activation of one donor in the presence of an acceptor with the same or different leaving groups, so the newly formed product can be directly elongated without further aglycon leaving group adjustment. However, these approaches are not applicable to all glycosides. For example, sialic acid thioglycosides have limitations in our programmable reactivity-based, one-pot strategy due to their poor and narrow range of anomeric reactivity values (RRVs). To resolve the problem, sialylated disaccharides have been used as building blocks. However, application of this strategy is limited by the lack of an efficient and a-selective sialic acid donor that possesses a leaving group orthogonal to the thioglycoside. Herein, we report a new sialylation reagent 1 that employs an N-acetyl-5-N,4-O-carbonyl protection with dibutyl phosphate as the leaving group. Furthermore, the new donor was applied to develop efficient strategies to use to create natural sialosides. Many researchers have focused on the modification of the N-acetyl functional group of sialic acid for highly efficient a-sialylation. However, there is no general donor for different acceptors. The choice of C-5 modification to be used is highly dependent on protecting groups and the nature of glycosyl acceptors. There is an excellent review on C-5 modification by De Meo. Crich and co-workers have shown that the N-acetyl-5-N,4-O-carbonyl thiosialoside can [a] C.-H. Hsu, Dr. K.-C. Chu, Y.-S. Lin, Dr. J.-L. Han, Y.-S. Peng, Dr. C.-T. Ren, Prof. C.-Y. Wu, Prof. C.-H. Wong The Genomics Research Center, Academia Sinica (Taiwan) 128 Academia Road, Section 2, Nankang, Taipei 115 (Taiwan) Fax: (+886)2-2785-3852 E-mail : cyiwu@gate.sinica.edu.tw chwong@gate.sinica.edu.tw [b] C.-H. Hsu, Prof. C.-H. Wong Department of Chemistry, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) [c] C.-H. Hsu, Prof. C.-Y. Wu, Prof. C.-H. Wong Chemical Biology and Molecular Biophysics Taiwan International Graduate Program, Academia Sinica 128 Academia Road, Section 2, Nankang, Taipei 115 (Taiwan) [d] C.-H. Hsu Institute of Bioinformatics and Structural Biology National Tsing-Hua University, Hsin-Chu (Taiwan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200903035.

Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer

Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GH–keyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.

Differential Receptor Binding Affinities of Influenza Hemagglutinins on Glycan Arrays

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.

Efficient convergent synthesis of bi-, tri-, and tetra-antennary complex type N-glycans and their HIV-1 antigenicity.

The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.

Efficient and Stereoselective Synthesis of α(2→9) Oligosialic Acids: From Monomers to Dodecamers

most common a(2!8) polysialic acid (1) is found in mammalian tissues and bacteria (Neisseria meningitidis B, Escherichia coli K1, Morexella nonliquefaciens, and Mannheimia haemolytica A2), and the less common a(2!9) polysialic acid (2) and alternating a(2!8)/a(2!9) polysialic acids (3) were discovered to form extracellular capsules of N. meningitidis C and E. coli K92, respectively. Human pathogens encapsulated with polysialic acids cause invasive diseases such as meningitis and urinary tract infections. In pathogenic bacteria, these acidic polysaccharides serve as extracellular shields against the defense systems of their mammalian host. Therefore, polysialic acids are considered good targets for the development of bactericidal agents and antibacterial vaccines. For example, the current vaccines against meningococcal group C diseases are glycoconjugates of isolated a(2!9) polysialic acids and a carrier protein such as diphtheria or tentanus toxoid. However, these kinds of vaccines are often heterogeneous or contaminated with other antigenic components because of the difficulty of purifying polysialic acids from natural sources. An effective method to synthesize pure polysialic acids having a well-defined structure will not only simplify the complexities of vaccines but also provide a better understanding of the structure– activity relationships of polysialic acids in various biological events. Chemical sialylation is complicated as a result of the intrinsic structural features of sialic acid, thus resulting in poor yields or stereoselectivities. Even though notable progress toward the development of sialic acid donors for efficient a sialylation have been reported in the last decade, 12] the synthesis of poly/oligo sialic acid with satisfactory yields and excellent a selectivity is still very challenging. The advancement of donor development led to many approaches for the synthesis of a-specific oligosialic acids, including the synthesis of a(2!9) trisialic acid using C5-azido sialyl phosphite as donor, the synthesis of a(2!9) oligosialic acid using C5-TFA sialyl phosphite as a donor and C5-TFA thiosialoside as an acceptor, and the synthesis of a(2!8) tetrasialoside, a(2!9) trisialoside, and a(2!9) tetrasialoside using 5N,4O-carbonyl-protected thiosialosides. When using 5N,4O-carbonyl-protected thiosialosides as donors, the sequence of assembly starts from the reducing end to the nonreducing end, thus providing an opportunity to stereoselectively elongate the sugar chain one residue at a time. However, this approach has not successfully been used to synthesize an a-specific oligosialic acid polymer that is longer than a tetramer. In principle, convergent block synthesis is an intrinsically better strategy for the preparation of oligomers or polymers and has been applied to the synthesis of some carbohydrate polymers. However, this strategy is hindered by the limited choice of leaving groups to ensure a proper reactivity and Figure 1. Structures of polysialic acids.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Cancer-associated carbohydrate antigens as potential biomarkers for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ∼60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.

Modular synthesis of N -glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

Desorption Ionization of Biomolecules on Metals

Direct desorption ionization of various types of biomolecules on metal substrates without the need of matrices was observed by a time-of-flight mass spectrometer. It provides a new convenient method for detection of small biomolecules without the confusion of ion peaks from matrix compounds. Simple commercial Al foil can be used as the substrate to obtain mass spectra of biomolecules without the need of an etching process to produce a porous surface such as with direct ionization on silicon (DIOS). The desorption and ionization mechanism is also discussed.