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The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.

Significance Antibodies are important therapeutic agents and have been used for the treatment of many diseases, including infectious and inflammatory diseases, and cancer. The therapeutic efficacy of an antibody is usually determined not only by the selectivity and affinity toward the target but also by the Fc-glycan structure interacting with the Fc receptors on immune cells. This study describes the preparation of various antibodies with different Fc-glycan structures as homogeneous glycoforms for the investigation of their effector activities. During this study, it was discovered that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimal structure that is able to enhance the activities of antibodies against cancer, influenza, and inflammatory diseases.

A novel total synthesis of laurencenone C, a chamigrene sesquitepenoid natural product, has been accomplished in 11 steps. In the synthetic sequence, the B-ring of the spirocyclic core was constructed by a one-pot operation involving a Knoevenagel condensation between ethyl cyanoacetate and paraformaldehyde combined with a Diels-Alder reaction of the resulting ethyl 2-cyanoacrylate with isoprene. Moreover, a lithium naphthalenide-induced reductive alkylation ofthe Diels-Alder adduct was employed to create the C-6 quaternary center and to set the stage for assembling the A-ring.

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.

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.