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Synthetic approach toward complexity of sialic acidcontaining glycans ab
Hiromune Ando a
Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan
b
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto Published online: 11 Dec 2014.
Click for updates To cite this article: Hiromune Ando (2014): Synthetic approach toward complexity of sialic acid-containing glycans, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.990228 To link to this article: http://dx.doi.org/10.1080/09168451.2014.990228
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014
Award Review
Synthetic approach toward complexity of sialic acid-containing glycans Hiromune Ando1,2,* 1
Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan; 2Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto
Received October 4, 2014; accepted November 4, 2014
Downloaded by [University of Toronto Libraries] at 22:36 02 February 2015
http://dx.doi.org/10.1080/09168451.2014.990228
The biological relevance of sialic acid-containing glycans (sialo-glycans) to various interactions at the cell surface has increased the demand for the supply of structurally defined sialo-glycans. Due to their unique structures, the stereoselective synthesis of glycosides of sialic acid is inherently difficult, which makes the synthetic approach to synthesize diverse and complex sialo-glycans far from successful. However, the gap between the chemical and natural synthesis of complex sialo-glycans is narrowing through the emergence of synthetic methods. This review highlights recent progress made in the synthesis of complex sialo-glycans via cutting-edge synthetic methods. Key words: sialic acid; glycan; glycosylation; organic synthesis
The carbohydrates present in the outer leaflet of the cell membrane are generally smaller molecules than polysaccharides such as starch, glycogen, and cellulose, and a large number of them are conjugated with membrane proteins and lipids, forming glycoproteins and glycolipids (glycoconjugates). Their sugar chains, namely glycans, are comprised of various monosaccharide residues such as glucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, sialic acid, and so on and have different lengths and sequences, thus imparting remarkable heterogeneity. However, the structural diversity of glycans confers a substantial hindrance toward gaining an understanding of the multiple biological functions of glycoconjugates at the atomic or molecular level. The supply of structurally homogenous glycoconjugates is therefore a crucial issue in glycobiology and translational research. The isolation of sufficient quantities of fine glycoconjugates from natural resources is virtually impossible due to structural heterogeneity; hence, organic synthetic chemistry has been playing a vital role in the supply of fine glycans and glycoconjugates, and in the production of various glycoconjugate probes for functional research.
Among glycans, those containing sialic acid are the most in demand because of their important biological characteristics. Sialic acids are a diverse family of 3-deoxy-2-nonulosonic acid analogs. They contain over 50 analogs including three major molecules, N-acetyl- and N-glycolylneuraminic acid (Neu5Ac and Neu5Gc), and KDN, and their partially modified analogs (Fig. 1). In glycan chains on the cell surfaces of vertebrates, sialic acids are typically seen as monomeric or oligomeric residues attached at the non-reducing ends via α-glycosidic linkages and exposed to the outermost position of the cell surface. Therefore, sialic acids have crucial roles in a wide range of cell surface interactions with signaling molecules, other biomolecules, cells, and invading organisms.1–4) Furthermore, it has been found that in such cellular recognition, the structural diversity of sialic acid is properly regulated spatiotemporally, but the mechanism for this is not fully understood at the molecular level.5) Several sialic acid-containing glycosphingolipids (gangliosides; a representative example in mammals is shown in Fig. 1) are distributed specifically within the mammalian brain and are known to possess neuritegenic activity.6) Furthermore, highly neuritegenic gangliosides have been found in echinoderms such as sea cucumbers, sea urchins, starfish, and sea lilies and these have been the focus of much attention in medicinal chemistry.7) Most glycan structures in echinodermatous gangliosides differ remarkably from those in vertebrate gangliosides, which feature sialic acid-embedded structures. However, the biological significance of gangliosides in echinoderms and their relevance to neuritegenic activities remain unknown. Chemical construction of the α-glycoside of sialic acid (α-sialoside) remains a challenge. α-Sialoside can be synthesized by a reaction of oxocarbenium ion generated from a sialic acid donor (sialyl donor) with the hydroxyl group of a coupling partner (glycosyl acceptor). However, the absence of hydroxyl groups adjacent to anomeric positions in sialic acid complicates the introduction of stereo-directing functionality, destabilizes the oxocarbenium ion, and makes it prone to 2,3elimination in collaboration with a carboxyl group at
*Email: [email protected] This review was written in response to the author’s receipt of the Japan Society for Bioscience, Biotechnology, and Agrochemistry Award for the Encouragement of Young Scientists in 2012. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Fig. 1. Structures of sialic acids and ganglioside GM3. Note: Arrows point possible sites to be modified with various substituents indicated above or below each arrow in inter- or intramolecular manner.
the anomeric position. In addition, stereoselectivity and reaction yields are frequently affected by the mode of protection of the sialyl donor and the structure of the glycosyl acceptor. Therefore, the highly complex gangliosides have defied chemical synthesis based on conventional methodology. It is now crucial to broaden the scope of the synthetic chemistry of sialic acid-containing glycans to unveil the biological significance of sialo-glycans. This review focuses on recent advances made toward achieving the synthesis of complex sialoglycans.
products by widening the mobility differences on silica gel.12) More importantly, N-Troc-sialosides can be converted to various congeners in a facile manner (Fig. 2) Neu5Ac, Neu5Gc, and 1,5-lactamized-Neu congeners can be derived through selective deprotection of the Troc group (Zn in AcOH) followed by N-acylation. On the other hand, if the Troc group is removed under mild acidic conditions, the O-8 acetyl group migrates to the C-5 amino group generated to afford 8-OH intermediate in high yield. The 8-OH intermediate is used for the synthesis of O-8 partially modified congeners, such as 8-O-sulfonyl and -methyl.13,14) By taking advantage of the prominent features of the N-Troc-sialyl donor as a versatile synthetic unit, we synthesized the pentasaccharide moiety of ganglioside GAA-7, which contains unusually modified sialic acid residues (8-O-Me-Neu5Gc). (Fig. 3)15) Ganglioside GAA-7 is a neuritegenic glycolipid from the starfish Asterias amurensis versicolor Sladen.16) The terminal branch of the sialic acid residue stemming from N-acetyl-galactosamine (GalNAc) residue is the most characteristic and specific substructure in the GAA-7 glycan moiety. For the double glycosylation of the GalN residue with sialic acid, 8-O-Me-N-Troc-sialyl donor 3 was synthesized from 9-O-chloroacetyl-NTroc-sialic acid derivative 1 via selective removal of the chloroacetyl group with a selenourea analog, subsequent 8O to 9O acetyl migration, and methylation of the C-8 hydroxyl group. Examination of the double sialylation of various GalN-Gal acceptors with donor 3
I. Synthesis of partially modified sialic acid-containing glycans Many reliable methods are available for establishing α-glycosides of sialic acid (α-sialoside) with high stereoselectivity and high-yield glycosylation.8–11) Sialyl donors used in the reliable methods are fine- tuned for stereoselectivity and high-yield glycosylation via structural modifications. We have developed an N-Troc-sialyl donor, which offers high-yield glycosylation and high stereoselectivity, using various glycosyl acceptors and stereo-directing assistance with a nitrile solvent. In addition, an N-Troc-sialyl donor facilitates the chromatographic separation of anomers of sialylated
Fig. 2. Efficient conversion to sialic acid congeners for N-Trocsialoside.
Fig. 3. Synthesis of GAA-7 glycan. Notes: CAc, chloroacetyl; MS, molecular sieves; NIS, N-iodosuccinimide; TTBP, 2,4,6-tri-tert butylpyrimidine.
Glycan synthesis
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indicated that GalN3-Gal 6 was the most suitable coupling partner, in which C-2 acetamide was converted into azide, and C-4 hydroxyl group was protected as benzyl ether for boosting the reactivity of the C-3 hydroxyl group. Under optimized conditions, the acceptor underwent double sialylation with 3 to produce objective tetrasaccharide 7 in 41% yield, accompanied by other stereoisomers including β(2,3)/α(2,6)-disialyl (7%), α(2,3)/β(2,6)-disialyl (9%), and β(2,3)/β(2,6)-disialyl isomers (2%). All isomers were completely separable due to distinct mobility differences on silica gel. Next, tetrasaccharide 7 was converted into tetrasaccharyl donor via manipulation of the protecting groups. Finally, the tetrasaccharyl donor and a glucosyl acceptor 8 were coupled to produce the protected GAA-7 glycan, followed by global deprotection that delivered GAA-7 glycan 9.
II. Glycosylation of hydroxyl group in sialic acid for construction of complex sialo-glycans The complexity of sialo-glycans can be increased by glycosylation at 4-, 8-, 9-, or 11-OH of a sialic acid residue with sialic acid, fucose, galactose, and N-acetyl-galactosamine. A common issue in synthesizing sialic acid-embedded structures is the diminished reactivity of the hydroxyl groups of sialic acid. In particular, C-4 and C-8 hydroxyl groups have low reactivity, which is probably due to hydrogen bonding with the C-5 acetamide group.8) When the hydroxyl groups in sialyl acceptors 12 and 13 were reacted with N-Troc-sialyl donor 10, the yields were 10% and less than 1%, respectively. (Fig. 4) To resolve this issue, several sialyl acceptors with highly reactive C-4 or C-8 hydroxyl groups have been developed.17–20) These are usually equipped with special protection at the C-5 amino group, which probably prevents hydrogen bonding. Recently, we found that the reactivity of 4-OH and 8-OH was greatly ameliorated in 1,5-lactamized sialic acid by glycosylation with sialic acid or another glycosyl donor.13,21) For example, the glycosylation of 1,5-lactamized sialyl acceptors 14 and 15 with N-Troc-sialy donor 10 yielded 49 and 66% of the desired α(2,8)- or α(2,4)-linked disialic acids, respectively. Furthermore, it was demonstrated that repetition of the cycle of 1,5-lactamization and sialylation
Fig. 4. Elevated reactivity of C-4 and C-8 hydroxyl groups in 1,5lactamized sialic acid derivatives.
3
produced an α(2,8)-linked oligomer of sialic acid, which is a challenging structure to synthesize, in a highly stereoselective manner.22) Because 1,5-lactamization proceeds exclusively in the cis-configuration (α-anomer), α-sialoside was selectively transformed into the lactamized form in a mixture with the β-anomer and 2,3-dehydrated derivative. This reaction facilitated the chromatographic separation of the α-anomer from the glycosylation reaction mixture during each cycle. Ganglioside Hp-s6 was isolated from the sperm of the sea urchin Hemicentrotus pulcherrimus in 1996, and its biological function is considered relevant to fertilization.23) Although this molecule seems simpler than other complex gangliosides, the 8′-O-sulfonyl disialic acid structure has not yet been synthesized. For the parallel purposes of facilitating site-specific modification and stereoselective glycosylation at the 8-OH of the outer sialic acid residue, the N-Troc-sialyl donor was used as the coupling partner of the 1,5-lactamized sialyl thioglycoside acceptor.13) (Fig. 5(a)) Since the phenylsulfenyl group at the bridgehead anomeric center of acceptor 16 is stereoelectronically deactivated, the phenylsulfenyl group of sialyl donor 10 was selectively reacted with NIS in the presence of a catalytic amount of TfOH, affording the desired disialic acid derivative 17 as a single stereoisomer. Disialic acid 17 was converted to an active form by opening the 1,5-lactam moiety, and then the Troc group was deprotected under
Fig. 5. (a) Synthesis of Hp-s6 glycan. (b) Structures of HPG-1 and HPG-7 glycans.
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mild acidic conditions to yield the 8-OH-N–Ac derivative. The free hydroxyl group retrieved at C-8 was tentatively capped with a selectively removable levulinoyl (Lev) group for sulfonylation at a late stage, producing a suitably protected disialyl donor 18. The disialyl donor upon activation with NIS-TfOH in EtCN reacted with glucosyl acceptor 19 to generate a trisaccharide. Further manipulations of protecting groups yielded a fully acyl-protected trisaccharide, and 8-OH was sulfonated after selective deprotection of Lev. Finally, all protected functional groups were retrieved to deliver Hp-s6 glycan 20. Based on the synergic use of the N-Troc-sialyl donor and the 1,5-lactamized sialyl acceptor, syntheses of more complex glycan moieties of echinodermatous gangliosides have also been achieved, including those containing embedded disialic acid residues, involving HPG-124) and HPG-7 glycans.25) (Fig. 5(b))
3. Conclusion Development of a highly reactive synthetic equivalent of the oxocarbenium cation of sialic acid, a highly reactive sialyl acceptor, and an efficient approach to synthesize partially modified sialic acid congeners has collectively paved the way for tackling the wide complexity of sialo-glycans. However, complex varieties exist in living organisms, including mammals and echinoderms; thus the synthetic methodology for sialoglycans needs to be advanced and become more generalized to cultivate the biological potential of these compounds.
Acknowledgment I am grateful to start my research on carbohydrate chemistry under the tutelage of the late Prof. Akira Hasegawa. I also deeply appreciate the continuous, warm guidance and support of Prof. Makoto Kiso and Prof. Hideharu Ishida. I would like to thank the following contributors: Assistant Prof. Akihiro Imamura, Drs. Hidenori Tanaka, Kohki Fujikawa, Yuki Iwayama, Shinya Nakashima Hideki Tamai, and all past and present members in the laboratory of Bio-active Molecular Science.
References [1] Varki A. Glycan-based interactions involving vertebrate sialicacid-recognizing proteins. Nature. 2007;446:1023–1029. [2] Lopez PHH, Schnaar RL. Gangliosides in cell recognition and membrane protein regulation. Curr. Opin. Struct. Biol. 2009;19:549–557. [3] Wennekes T, van den Berg BHN, Boot RG, van der Marel GA, Overkleeft HS, Aerts LMFG. Glycosphingolipids – nature, function, and pharmacological modulation. Angew. Chem. Int. Ed. 2009;48:8848–8869. [4] Neu U, Bauer J, Stehle T. Viruses and sialic acids: rules of engagement. Curr. Opin. Struct. Biol. 2011;21:610–618. [5] Angata T, Varki A. Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective. Chem. Rev. 2002;102:439–470.
[6] Tsuji S, Yamashita T, Tanaka M, Nagai Y. Synthetic sialyl compounds as well as natural gangliosides induce neuritogenesis in a mouse neuroblastoma cell line (Neuro2a). J. Neurochem. 1988;50:414–423. [7] Kaneko M, Yamada K, Miyamoto T, Inagaki M, Higuchi R. Neuritogenic activity of gangliosides from echinoderms and their structure–activity relationship. Chem. Pharm. Bull. 2007;55: 462–463. [8] Boons GJ, Demchenko AV. Recent advances in O-Sialylation. Chem. Rev. 2000;100:4539–4566. [9] Ando H, Imamura A. Proceedings in synthetic chemistry of sialo-glycoside. Trends Glycosci. Glycotechnol. 2004;16:293–303. [10] De Meo C, Priyadarshani U. C-5 modifications in N-acetyl-neuraminic acid: scope and limitations. Carbohydr. Res. 2008;343:1540–1552. [11] Hanashima S. Recent strategies for stereoselective sialylation and their application to the synthesis of oligosialosides. Trends Glycosci. Glycotechnol. 2011;23:111–121. [12] Ando H, Koike Y, Ishida H, Kiso M. Extending the possibility of an N-Troc-protected sialic acid donor toward variant sialo-glycoside synthesis. Tetrahedron Lett. 2003;6883–6886. [13] Ando H, Koike Y, Koizumi S, Ishida H, Kiso M. 1,5-Lactamized sialyl acceptors for various disialoside syntheses: novel method for the synthesis of glycan portions of Hp-s6 and HLG-2 gangliosides. Angew. Chem. Int. Ed. 2005;44: 6759–6763. [14] Tamai H, Ando H, Tanaka H-N, Hosoda-Yabe R, Yabe T, Ishida H, Kiso M. The total synthesis of the neurogenic ganglioside LLG-3 isolated from the starfish Linckia laevigata. Angew. Chem. Int. Ed. 2011;50:2330–2333. [15] Tamai H, Ando H, Ishida H, Kiso M. First synthesis of a pentasaccharide moiety of ganglioside GAA-7 containing unusually modified sialic acids through the use of N-Troc-sialic acid derivative as a key unit. Org. Lett. 2012;14:6342–6345. [16] Higuchi R, Inukai K, Jhou JX, Honda M, Komori T, Tsuji S, Nagai Y. Biologically active glycosides from asteroidea, XXXI. glycosphingolipids from the StarfishAsterias amurensis versicolor sladen, 2. Structure and biological activity of ganglioside molecular species. Liebigs Ann. Chem. 1993;359–366. [17] Meo C, Demchenko AV, Boons GJ. A stereoselective approach for the synthesis of α-sialosides. J. Org. Chem. 2001;66: 5490–5497. [18] Tanaka H, Nishimura Y, Adachi M, Takahashi T. Synthetic study of α(2,8) oligosialoside using N-Troc sialyl N-phenyltrifluoroimidate. Hetereocycles. 2006;67:107–112. [19] Tanaka H, Nishiura Y, Takahashi T. Stereoselective synthesis of oligo-α-(2,8)-sialic acids. J. Am. Chem. Soc. 2006;128: 7124–7125. [20] Hanashima S, Sato K, Ito Y, Yamaguchi Y. Silylene/oxazolidinone double-locked sialic acid building blocks for efficient sialylation reactions in dichloromethane. Eur. J. Org. Chem. 2009;4215–4220. [21] H Ando, H Shimizu, Y Katano, Y Koike, S Koizumi, H Ishida, M. Kiso Studies on the α-(1,4)- and α-(1,8)-fucosylation of sialic acid for the total assembly of the glycan portions of complex HPG-series gangliosides. Carbohydr. Res. 2006;341:1522–1532. [22] Tanaka H, Ando H, Ishida H, Kiso M, Ishihara H, Koketsu M. Synthetic study on α(2, 8)-linked oligosialic acid employing 1,5-lactamization as a key step. Tetrahedron Lett. 2009;50: 4478–4481. [23] Ijuin T, Kitajima K, Song Y, Kitazume S, Inoue S, Haslam SM, Morris HR, Dell A, Inoue Y. Isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm. Glycocojugate J. 1996;13:01–413. [24] Shimizu H, Iwayama Y, Imamura A, Ando H, Ishida H, Kiso M. Synthesis of the disialic acid-embedded glycan part of ganglioside HPG-1. Biosci. Biotechol. Biochem. 2011;75:2079–2082. [25] Iwayama Y, Ando H, Tanaka H-N, Ishida H, Kiso M. Synthesis of the glycan moiety of ganglioside HPG-7 with an unusual trimer of sialic acid as the inner sugar residue. Chem. Commun. 2011;47:9726–9728.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Synthetic approach toward complexity of sialic acidcontaining glycans ab
Hiromune Ando a
Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan
b
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto Published online: 11 Dec 2014.
Click for updates To cite this article: Hiromune Ando (2014): Synthetic approach toward complexity of sialic acid-containing glycans, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.990228 To link to this article: http://dx.doi.org/10.1080/09168451.2014.990228
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014
Award Review
Synthetic approach toward complexity of sialic acid-containing glycans Hiromune Ando1,2,* 1
Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan; 2Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto
Received October 4, 2014; accepted November 4, 2014
Downloaded by [University of Toronto Libraries] at 22:36 02 February 2015
http://dx.doi.org/10.1080/09168451.2014.990228
The biological relevance of sialic acid-containing glycans (sialo-glycans) to various interactions at the cell surface has increased the demand for the supply of structurally defined sialo-glycans. Due to their unique structures, the stereoselective synthesis of glycosides of sialic acid is inherently difficult, which makes the synthetic approach to synthesize diverse and complex sialo-glycans far from successful. However, the gap between the chemical and natural synthesis of complex sialo-glycans is narrowing through the emergence of synthetic methods. This review highlights recent progress made in the synthesis of complex sialo-glycans via cutting-edge synthetic methods. Key words: sialic acid; glycan; glycosylation; organic synthesis
The carbohydrates present in the outer leaflet of the cell membrane are generally smaller molecules than polysaccharides such as starch, glycogen, and cellulose, and a large number of them are conjugated with membrane proteins and lipids, forming glycoproteins and glycolipids (glycoconjugates). Their sugar chains, namely glycans, are comprised of various monosaccharide residues such as glucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, sialic acid, and so on and have different lengths and sequences, thus imparting remarkable heterogeneity. However, the structural diversity of glycans confers a substantial hindrance toward gaining an understanding of the multiple biological functions of glycoconjugates at the atomic or molecular level. The supply of structurally homogenous glycoconjugates is therefore a crucial issue in glycobiology and translational research. The isolation of sufficient quantities of fine glycoconjugates from natural resources is virtually impossible due to structural heterogeneity; hence, organic synthetic chemistry has been playing a vital role in the supply of fine glycans and glycoconjugates, and in the production of various glycoconjugate probes for functional research.
Among glycans, those containing sialic acid are the most in demand because of their important biological characteristics. Sialic acids are a diverse family of 3-deoxy-2-nonulosonic acid analogs. They contain over 50 analogs including three major molecules, N-acetyl- and N-glycolylneuraminic acid (Neu5Ac and Neu5Gc), and KDN, and their partially modified analogs (Fig. 1). In glycan chains on the cell surfaces of vertebrates, sialic acids are typically seen as monomeric or oligomeric residues attached at the non-reducing ends via α-glycosidic linkages and exposed to the outermost position of the cell surface. Therefore, sialic acids have crucial roles in a wide range of cell surface interactions with signaling molecules, other biomolecules, cells, and invading organisms.1–4) Furthermore, it has been found that in such cellular recognition, the structural diversity of sialic acid is properly regulated spatiotemporally, but the mechanism for this is not fully understood at the molecular level.5) Several sialic acid-containing glycosphingolipids (gangliosides; a representative example in mammals is shown in Fig. 1) are distributed specifically within the mammalian brain and are known to possess neuritegenic activity.6) Furthermore, highly neuritegenic gangliosides have been found in echinoderms such as sea cucumbers, sea urchins, starfish, and sea lilies and these have been the focus of much attention in medicinal chemistry.7) Most glycan structures in echinodermatous gangliosides differ remarkably from those in vertebrate gangliosides, which feature sialic acid-embedded structures. However, the biological significance of gangliosides in echinoderms and their relevance to neuritegenic activities remain unknown. Chemical construction of the α-glycoside of sialic acid (α-sialoside) remains a challenge. α-Sialoside can be synthesized by a reaction of oxocarbenium ion generated from a sialic acid donor (sialyl donor) with the hydroxyl group of a coupling partner (glycosyl acceptor). However, the absence of hydroxyl groups adjacent to anomeric positions in sialic acid complicates the introduction of stereo-directing functionality, destabilizes the oxocarbenium ion, and makes it prone to 2,3elimination in collaboration with a carboxyl group at
*Email: [email protected] This review was written in response to the author’s receipt of the Japan Society for Bioscience, Biotechnology, and Agrochemistry Award for the Encouragement of Young Scientists in 2012. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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H. Ando
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Fig. 1. Structures of sialic acids and ganglioside GM3. Note: Arrows point possible sites to be modified with various substituents indicated above or below each arrow in inter- or intramolecular manner.
the anomeric position. In addition, stereoselectivity and reaction yields are frequently affected by the mode of protection of the sialyl donor and the structure of the glycosyl acceptor. Therefore, the highly complex gangliosides have defied chemical synthesis based on conventional methodology. It is now crucial to broaden the scope of the synthetic chemistry of sialic acid-containing glycans to unveil the biological significance of sialo-glycans. This review focuses on recent advances made toward achieving the synthesis of complex sialoglycans.
products by widening the mobility differences on silica gel.12) More importantly, N-Troc-sialosides can be converted to various congeners in a facile manner (Fig. 2) Neu5Ac, Neu5Gc, and 1,5-lactamized-Neu congeners can be derived through selective deprotection of the Troc group (Zn in AcOH) followed by N-acylation. On the other hand, if the Troc group is removed under mild acidic conditions, the O-8 acetyl group migrates to the C-5 amino group generated to afford 8-OH intermediate in high yield. The 8-OH intermediate is used for the synthesis of O-8 partially modified congeners, such as 8-O-sulfonyl and -methyl.13,14) By taking advantage of the prominent features of the N-Troc-sialyl donor as a versatile synthetic unit, we synthesized the pentasaccharide moiety of ganglioside GAA-7, which contains unusually modified sialic acid residues (8-O-Me-Neu5Gc). (Fig. 3)15) Ganglioside GAA-7 is a neuritegenic glycolipid from the starfish Asterias amurensis versicolor Sladen.16) The terminal branch of the sialic acid residue stemming from N-acetyl-galactosamine (GalNAc) residue is the most characteristic and specific substructure in the GAA-7 glycan moiety. For the double glycosylation of the GalN residue with sialic acid, 8-O-Me-N-Troc-sialyl donor 3 was synthesized from 9-O-chloroacetyl-NTroc-sialic acid derivative 1 via selective removal of the chloroacetyl group with a selenourea analog, subsequent 8O to 9O acetyl migration, and methylation of the C-8 hydroxyl group. Examination of the double sialylation of various GalN-Gal acceptors with donor 3
I. Synthesis of partially modified sialic acid-containing glycans Many reliable methods are available for establishing α-glycosides of sialic acid (α-sialoside) with high stereoselectivity and high-yield glycosylation.8–11) Sialyl donors used in the reliable methods are fine- tuned for stereoselectivity and high-yield glycosylation via structural modifications. We have developed an N-Troc-sialyl donor, which offers high-yield glycosylation and high stereoselectivity, using various glycosyl acceptors and stereo-directing assistance with a nitrile solvent. In addition, an N-Troc-sialyl donor facilitates the chromatographic separation of anomers of sialylated
Fig. 2. Efficient conversion to sialic acid congeners for N-Trocsialoside.
Fig. 3. Synthesis of GAA-7 glycan. Notes: CAc, chloroacetyl; MS, molecular sieves; NIS, N-iodosuccinimide; TTBP, 2,4,6-tri-tert butylpyrimidine.
Glycan synthesis
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indicated that GalN3-Gal 6 was the most suitable coupling partner, in which C-2 acetamide was converted into azide, and C-4 hydroxyl group was protected as benzyl ether for boosting the reactivity of the C-3 hydroxyl group. Under optimized conditions, the acceptor underwent double sialylation with 3 to produce objective tetrasaccharide 7 in 41% yield, accompanied by other stereoisomers including β(2,3)/α(2,6)-disialyl (7%), α(2,3)/β(2,6)-disialyl (9%), and β(2,3)/β(2,6)-disialyl isomers (2%). All isomers were completely separable due to distinct mobility differences on silica gel. Next, tetrasaccharide 7 was converted into tetrasaccharyl donor via manipulation of the protecting groups. Finally, the tetrasaccharyl donor and a glucosyl acceptor 8 were coupled to produce the protected GAA-7 glycan, followed by global deprotection that delivered GAA-7 glycan 9.
II. Glycosylation of hydroxyl group in sialic acid for construction of complex sialo-glycans The complexity of sialo-glycans can be increased by glycosylation at 4-, 8-, 9-, or 11-OH of a sialic acid residue with sialic acid, fucose, galactose, and N-acetyl-galactosamine. A common issue in synthesizing sialic acid-embedded structures is the diminished reactivity of the hydroxyl groups of sialic acid. In particular, C-4 and C-8 hydroxyl groups have low reactivity, which is probably due to hydrogen bonding with the C-5 acetamide group.8) When the hydroxyl groups in sialyl acceptors 12 and 13 were reacted with N-Troc-sialyl donor 10, the yields were 10% and less than 1%, respectively. (Fig. 4) To resolve this issue, several sialyl acceptors with highly reactive C-4 or C-8 hydroxyl groups have been developed.17–20) These are usually equipped with special protection at the C-5 amino group, which probably prevents hydrogen bonding. Recently, we found that the reactivity of 4-OH and 8-OH was greatly ameliorated in 1,5-lactamized sialic acid by glycosylation with sialic acid or another glycosyl donor.13,21) For example, the glycosylation of 1,5-lactamized sialyl acceptors 14 and 15 with N-Troc-sialy donor 10 yielded 49 and 66% of the desired α(2,8)- or α(2,4)-linked disialic acids, respectively. Furthermore, it was demonstrated that repetition of the cycle of 1,5-lactamization and sialylation
Fig. 4. Elevated reactivity of C-4 and C-8 hydroxyl groups in 1,5lactamized sialic acid derivatives.
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produced an α(2,8)-linked oligomer of sialic acid, which is a challenging structure to synthesize, in a highly stereoselective manner.22) Because 1,5-lactamization proceeds exclusively in the cis-configuration (α-anomer), α-sialoside was selectively transformed into the lactamized form in a mixture with the β-anomer and 2,3-dehydrated derivative. This reaction facilitated the chromatographic separation of the α-anomer from the glycosylation reaction mixture during each cycle. Ganglioside Hp-s6 was isolated from the sperm of the sea urchin Hemicentrotus pulcherrimus in 1996, and its biological function is considered relevant to fertilization.23) Although this molecule seems simpler than other complex gangliosides, the 8′-O-sulfonyl disialic acid structure has not yet been synthesized. For the parallel purposes of facilitating site-specific modification and stereoselective glycosylation at the 8-OH of the outer sialic acid residue, the N-Troc-sialyl donor was used as the coupling partner of the 1,5-lactamized sialyl thioglycoside acceptor.13) (Fig. 5(a)) Since the phenylsulfenyl group at the bridgehead anomeric center of acceptor 16 is stereoelectronically deactivated, the phenylsulfenyl group of sialyl donor 10 was selectively reacted with NIS in the presence of a catalytic amount of TfOH, affording the desired disialic acid derivative 17 as a single stereoisomer. Disialic acid 17 was converted to an active form by opening the 1,5-lactam moiety, and then the Troc group was deprotected under
Fig. 5. (a) Synthesis of Hp-s6 glycan. (b) Structures of HPG-1 and HPG-7 glycans.
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H. Ando
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mild acidic conditions to yield the 8-OH-N–Ac derivative. The free hydroxyl group retrieved at C-8 was tentatively capped with a selectively removable levulinoyl (Lev) group for sulfonylation at a late stage, producing a suitably protected disialyl donor 18. The disialyl donor upon activation with NIS-TfOH in EtCN reacted with glucosyl acceptor 19 to generate a trisaccharide. Further manipulations of protecting groups yielded a fully acyl-protected trisaccharide, and 8-OH was sulfonated after selective deprotection of Lev. Finally, all protected functional groups were retrieved to deliver Hp-s6 glycan 20. Based on the synergic use of the N-Troc-sialyl donor and the 1,5-lactamized sialyl acceptor, syntheses of more complex glycan moieties of echinodermatous gangliosides have also been achieved, including those containing embedded disialic acid residues, involving HPG-124) and HPG-7 glycans.25) (Fig. 5(b))
3. Conclusion Development of a highly reactive synthetic equivalent of the oxocarbenium cation of sialic acid, a highly reactive sialyl acceptor, and an efficient approach to synthesize partially modified sialic acid congeners has collectively paved the way for tackling the wide complexity of sialo-glycans. However, complex varieties exist in living organisms, including mammals and echinoderms; thus the synthetic methodology for sialoglycans needs to be advanced and become more generalized to cultivate the biological potential of these compounds.
Acknowledgment I am grateful to start my research on carbohydrate chemistry under the tutelage of the late Prof. Akira Hasegawa. I also deeply appreciate the continuous, warm guidance and support of Prof. Makoto Kiso and Prof. Hideharu Ishida. I would like to thank the following contributors: Assistant Prof. Akihiro Imamura, Drs. Hidenori Tanaka, Kohki Fujikawa, Yuki Iwayama, Shinya Nakashima Hideki Tamai, and all past and present members in the laboratory of Bio-active Molecular Science.
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