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anel Discussion

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trategy for functional analysis ofglycans based on synthesis:
preparation of homogeneous glycans
and reconstruction ofglycan environment mimics

K oichi Fukase

Department of Chemistry, Graduate School of Science,
Osaka University

Koichi Fukase was graduated from Department of Chemistry, School of Science,
Osaka University in 1982 and received his Ph. D. in 1987 from Osaka University (Prof.
Tetsuo Shiba). After spending 1 year as a JSPS Postdoctoral Fellow (Prof. TetsuoShiba), he obtained the position as an Assistant Professor (1988–1996) at Department of Chemistry, School of Science, Osaka University (Prof. Shoichi Kusumoto). In
1994, he joined the group of Prof. W. Clark Still at Columbia University as a Ministryof Education Research Fellow. After going back to Osaka in 1995, he was promoted
to Lecturer (1996), Associate Professor (1998), and became a full Professor at Department of Chemistry, Graduate School of Science, Osaka University in 2004.
He has been a board member of the Japanese Society of Carbohydrate Researchfrom 2007 and became a President of the Japan Society of Carbohydrate in 2017.
His research interest is in the field of carbohydrate chemistry and glycobiology, synthetic organic chemistry, bio-imaging, and innate immunity.

Diversity is the essence of glycans
Glycans are the most abundant biomass on the earth. Innovationin glycoscience will encourage social system transformation using
natural glycans as renewable material feedstocks for production of
biofuels, bioplastics, new fabrics, medical materials, and etc. Glycoscience is also indispensable for the comprehensive understanding of life and the medical technology advances, since glycans are
one of the three fundamental elements (nucleic acids, proteins,glycans) of life and participate in many key biological processesincluding molecular trafficking and clearance, receptor activation,
signal transduction, endocytosis, and various recognition processes such as cell adhesion, cell differentiation and proliferation,infection, inflammation, immunity, cancer progression and metastasis, fertilization, and etc.

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the 25th Anniversary of Mizutani Foundation for Glycoscience

Structural complexity and heterogeneity are major features ofglycans. Most classes of glycans exist as complex glycoconjugatessuch as glycoproteins, glycolipids, glycosaminoglycans, and etc.
Glycans even at specific glycosylation site on glycoproteins areheterogeneous. These diversities are called glycoforms, which perturb and regulate the function of glycoconjugates via various glycan recognition proteins. Abnormal cell surface glycoforms and/or glycan profiles are associated with diseases such as cancer, atherosclerosis, neurodegenerative diseases, and etc. Therefore, reliable biomarkers have been developed for the disease diagnosis1).
Bioinformatics will help the development of reliable biomarkers,
although the complexity of glycan-related information restricted
the utilization of bioinformatics in glycan biomarker research. Theauthor expects that the biomarker researches will be dramaticallyaccelerated by the use of “artificial intelligence” in conjunctionMEMORATIVE SYMPOSIUM
with glycomics research using mass spectrometry, lectin microarrays, glycan microarrays, surface plasmon resonance (SPR) analysis,
and etc 2),3).
On the other hand, the biological functions of glycans including
bio-functional role of glycoforms have not yet been sufficiently
elucidated due to their inherent complexity. Synthetic studies ofglycans have greatly contributed to the functional studies of glycans by supplying homogeneous glycans. It is often difficult to obtain homogeneous glycans from nature, and the possibility of contamination of other active substances cannot be excluded evenafter extensive purification, when glycans from natural source areused. Therefore, the use of chemically synthesized compounds
without contamination of other active substances is essential inorder to determine the active principle or identify “glycocode” asshown later. In recent years, the synthesis of glycan has made great
progress. It has been becoming possible to investigate the physiological significance of glycoforms by using synthetic glycoproteinsand glycolipids as a homogeneous form.
Multivalent interaction between glycans and glycans recognition
molecules such as lectins is also important feature in glycan function. Recognition via multiple sites can allow for the high affinity
and high selectivity. In addition, interaction between multiple
glycans and multiple lectins leads to pattern recognition of cells; inother words, glycans add a certain personality to cells, extracellular
microparticles, glycoproteins, and etc. Synthetic studies can also
contribute to the analysis of such complex systems by providing
the reconstruction models such as synthetic glyco-clusters, glyconanoparticles, and model glycoconjugates. Furthermore, reconstruction of glycan environment on living cells by using chemicalprobes can give opportunities for in vivo functional analysis basedon live cell imaging 4),5).

glycosidases and carbohydrate transferases. The glycocode is deciphered by glycan-binding proteins such as lectins. The lengthsof the glycocodes generally are ca. 1 to 5 residues per one carbohydrate recognition domain. These are actually appropriate sizes
for the synthesis, considering the synthetic easiness and industrial
applications. Therefore, chemical synthesis has contributed theidentification of the minimal structure required for recognition(glycocode) by lectins and other glycan recognition proteins. There
have been a number of reports regarding the identification of glycocode by using synthetic or naturally derived glycans. Unravelingthe glycocode has been getting more and more important but still
difficult task considering the diversity and complexity of glycans.
Therefore, the efficient methods for the synthesis of various-typeof glycans will be continuously developed including stereoselective glycosylation, protection-deprotection strategy for regioselective control in glycosylation, solid-phase synthesis, automatedsynthesis, microflow synthesis, enzymatic and chemoenzymaticsynthesis, and so on 7).
One of the remarkable successes is the development of Fondaparinux,
which is an anticoagulant related to low molecular weight heparins and is marketed by GlaxoSmithKline (Figure 1). Many synthetic
studies of have been carried out by Seeberger, Hung, Boons,Bonnaffé, Tamura, Suda, and so on to investigate protein interactions with synthetic glycosaminoglycan fragments 8). For example,
Hung identified GlcNS–IdoA2S as the minimum requirement for
fibroblast growth factor-1 (FGF-1) interaction with 48 disaccharide
heparan sulfate fragment library.

Elucidation of the glycocode is the main object ofglycan synthesis
The glycan chain encodes the glycocode (glycan code) that includes crucial information about the recognition, localizationin cell, distribution and circulation in the body and tissues, andage of glycoproteins 6). The glycocode is encoded by a series of

Figure 1

The structure of Fondaparinux

Figure 2

Peptidoglycan partial structures required for binding with the recognition proteins

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Our group, Boons, and Mobashery have synthesized various partial structures and derivatives of peptidoglycan (PGN), which is amajor component of cell wall and has been well-known stimulant
of innate immunity. Interaction of PGN with various proteins wasrevealed by using the synthetic fragments (Figure 2) 9),10).
Asparagine-linked oligosaccharides (N-glycans) on glycoproteins
have high diversity and complexity and are involved in a varietyof important physiological events. Various syntheses of N-glycans
have been studied to investigate their biological functions 11) .
α-Sialylation and β-mannosylation have been the key issues forthe synthesis of N-glycans. Kinetic solvent effect of nitrile found byKanie, Kiso, and Hasegawa has been mainly used for α-sialylation,
whereas intramolecular aglycon delivery developed by Ito, Ishiwata, Stork, and Hindsgaul or Crich’s S N2-like glycosylation has
been used for β-mannosylation. Danishefsky succeeded in synthesizing various types of N-glycans, including a core-fucosylated
glycan and a triantennary glycan. Ito synthesized high-mannose/
complex-type glycans and has clearly showed the biological roleof N-glycan in protein folding and protein quality control in ER.
Unverzagt et al. reported the syntheses of N-glycans with various
structures, including core fucose. Chemoenzymatic approaches
using a variety of glycosidases or glycosyltransferases have been
employed for the synthesis of N-glycans by Ito, Boons, Wang, andWong. Kajihara reported the semi-synthetic approach for thesynthesis of triantennary glycan from natural biantennary glycan.
Schmidt et al. carried out the synthesis of complex-type N-glycans
not only in liquid phase but also on solid phase. Our researchgroup has reported the solid-phase synthesis of a sialic acid con-taining glycan as well as the solution synthesis of core-fucosylated
N-glycan. In the latter, glycosyl-asparagine was first synthesizedby N-glycosylation, and the oligosaccharide chains were thenelongated. We reinvestigated α-sialylation, β-mannosylation, andN-glycosylation by using microflow system to reveal that precise
temperature control was essential for these glycosylations. Intermolecular hydrogen bonds involving acetamide groups were
found to reduce the reactivity in glycosylations: the protection ofNHAc as NAc2 dramatically improved the reactivity.
Reconstruction of glycan environments
It has been difficult to elucidate the bio-functions of glycans because of the structural diversity and heterogeneity of glycans aswell as the multivalent recognition of glycans with various recognition proteins as mentioned above. Reconstructing glycan environments using homogenous synthesized glycans can facilitate
the investigation and control of the glycan functions. A numberof glycoconjugates or clusters have reported on dendrimers, liposomes, nanoparticles, and protein templates for biodistributionstudies. Glycoclusters containing N-glycan structures have beenused for bio-distribution studies. André, Gabius, and Unverzagt
have prepared albumin connecting various di-, tri-, and tetraantennary N-glycans, but introducing ratio of N-glycan to albumin
were not high enough to observe the cluster effects. Our group reported the synthesis of polylysine-based glycodendrimers with 16
molecules of biantennary N-glycans via the Cu-mediated Huisgencycloaddition reaction, and we have successfully mimicked thecluster effects of N-glycans and visualized the sialic acid-dependent circulatory residence by Positron Emission Tomography (PET) 12).

Figure 3

Fluorescent imaging of albumin conjugated with a series of N-glycans

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the 25th Anniversary of Mizutani Foundation for Glycoscience

MEMORATIVE SYMPOSIUM
However, the instability of N-glycan dendrimers in the serum aswell as the difficulty of the synthesis hampered the further studies.
Tanaka performed the systematic studies using human serum albumin (HSA), which is conjugated with a series of N-glycans, eachsequentially trimmed from biantennary sialoglycans by using amethod that combined strain-promoted alkyne-azide cyclization
and 6π-azaelectrocyclization. Fluorescent imaging and dissection analysis revealed a glycan-dependent shift from urinary togall bladder excretion mediated by sequential trimming of nonreducing end sialic acids. Albumins conjugated with GlcNAc- andhybrid biantennary-terminated congeners, were selectively taken
up by sinusoidal endothelial and stellate cells in the liver. Tanaka
also succeeded the preparation of glycoclusters having different
N-glycans, which showed very interesting bio-distribution (Figure3, See page 77). They also demonstrated the pattern recognitionof cells by using the conjugates of N-glycans with other ligands onthe cell surface.
Monodisperse glycooligomers carrying different sugar ligands(mannose, galactose, and glucose) at well-defined positions weresynthesized 14). Interestingly, all heteromultivalent structures
showed high affinities toward Concanavalin A in comparison totheir homomultivalent analogues.
The glycoclusters have the potential in various areas, e.g. as glycoprobes and novel biosensors for diagnosis and therapeutics suchas tumor-targeting agents, anti-infectious drugs, vaccines, and etc.
Conclusions
As described above, chemical synthesis has contributed to un-cover the glycan functions by supplying homogeneous bio-probes
or reconstructing glycan recognition systems. Another importantfeature of chemical synthesis is that chemical synthesis can easily
supply various partial structures, derivatives, and analogues. Thesechemically synthesized compounds have high potential as glycanrelated drugs, since they can target specific proteins responsible
for the pharmacological action.
Anti-infective glycan-related drugs such as anti-influenza drugs
Tamiflu and Relenza have been realized, since glycans responsiblefor intercellular communication at the cell surface also serves as
the window of infection of viruses and bacteria. It Is important tocontinue anti-infective drug discovery in order to overcome thedrug-resistance, prevent and treat neglected tropical diseases, anddeal with emerging and re-emerging infectious diseases. The α2,6sialyl-gal glycan is necessary for human-type influenza virus infection, and ABO · Lewis blood group glycans for Norovirus, Lewis
blood group type glycans for Helicobacter pylori, and etc.
Synthetic studies of glycans can also contribute to the development of preventive medicine such as vaccine. Carbohydrate
antigens from pathogens such as bacteria, viruses, protozoans
are promising vaccine candidates. Cancer vaccines using tumorassociated carbohydrate antigens (TACAs) have been extensively
studied, but they have not been approved for the practical use.
The low antigenicity of the carbohydrate antigens must be overcome for the effective immunization. Therefore, the use of carrierproteins or adjuvants is necessary to induce the effective immune
responses. Development of new safe carbohydrate based vaccines
as well as adjuvants will be realized in the near future.

References1)	Taniguchi, N., Kizuka, Y. (2015) Glycans and cancer: role of N-glycans in cancer biomarker, progression and metastasis, and therapeutics. Adv. Cancer Res., 126, 11-51.
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3)	Puvirajesinghe, T.M., Turnbull, J.E., (2016) Glycoarray technologies: deciphering interactions from proteins to live cell responses. Microarrays, 5, 3.
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5)	Son, S.H., Daikoku, S., Ohtake, A., Suzuki, K., Kabayama, K., Ito, Y., Kanie, O. (2014) Syntheses of lactosyl ceramide analogues carrying novel bifunctional BODIPY
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6)	Kasai, K., Hirabayashi, J. (1996) Galectins: a family of animal lectins that decipher glycocodes. J. Biochem., 119, 1-8.
7)	Seeberger, P.H., Finney, N., Rabuka, D., Bertozzi, C.R. (2009) Chemical and enzymatic synthesis of glycans and glycoconjugates. In: Varki A, Cummings RD, Esko JD,Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory
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9)	Wang, N., Huang, C.Y., Hasegawa, M., Inohara, N., Fujimoto, Y., Fukase, K. (2013) Glycan sequence-dependent Nod2 activation investigated by using a chemicallysynthesized bacterial peptidoglycan fragment library. Chembiochem., 14, 482-488.
10)	Fujimoto, Y., Pradipta, A.R., Inohara, N., Fukase, K. (2012) Peptidoglycan as Nod1 ligand; fragment structures in the environment, chemical synthesis, and their innate immunostimulation. Nat. Prod. Rep., 29, 568-579.
11)	Nagasaki, M., Manabe, Y., Minamoto, N., Tanaka, K., Silipo, A., Molinaro, A., Fukase, K. (2016) Chemical synthesis of a complex-type N-glycan containing a corefucose. J. Org. Chem., 81, 10600-10616.
12)	Tanaka, K., Siwu, E.R., Minami, K., Hasegawa, K., Nozaki, S., Kanayama, Y., Koyama, K., Chen, W.C., Paulson, J.C., Watanabe, Y., Fukase, K. (2010) Noninvasive
imaging of dendrimer-type N-glycan clusters: in vivo dynamics dependence on oligosaccharide structure. Angew. Chem. Int. Ed. Engl., 49, 8195-8200.
13)	Ogura, A., Tahara, T., Nozaki, S., Morimoto, K., Kizuka, Y., Kitazume, S., Hara, M., Kojima, S., Onoe, H., Kurbangalieva, A., Taniguchi, N., Watanabe, Y., Tanaka,K. (2016) Visualizing Trimming Dependence of Biodistribution and Kinetics with Homo- and Heterogeneous N-Glycoclusters on Fluorescent Albumin. Sci Rep., 6,21797.
14)	Ponader, D., Maffre, P., Aretz, J., Pussak, D., Ninnemann, N.M., Schmidt, S., Seeberger, P.H., Rademacher, C., Nienhaus, G.U., Hartmann, L. (2014) Carbohydratelectin recognition of sequence-defined heteromultivalent glycooligomers. J. Am. Chem. Soc., 136, 2008-2016.

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