Constrained sialic acid donors enable selective synthesis of α-glycosides

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Science  17 May 2019:
Vol. 364, Issue 6441, pp. 677-680
DOI: 10.1126/science.aaw4866

Sweet spot for making oligosaccharides

Sugars pose a challenge for chemists: how to string together functional group–rich building blocks that can adopt multiple conformations. Two papers in this issue used sugar building blocks constrained by a macrocyclic linker to encourage formation of a specific glycosidic linkage (see the Perspective by Pohl). Ikuta et al. used glucose building blocks containing a linker that changes the sugar conformation to synthesize cyclic oligomers with only three or four units. The linker changes the conformation of the glucose monomers, enabling them to come together despite the strain in the final structure. Komura et al. prepared sialic acid building blocks with a linker that allows for selective formation of the α-anomeric linkage with a range of nucleophiles. They synthesized dimers of sialic acid with many different linkages and a pentamer with four α(2,8) linkages. This method enabled chemical synthesis of components of mammalian glycans involved in brain development, cell adhesion, and immune response.

Science, this issue p. 674, p. 677; see also p. 631


Sialic acid is a sugar residue present in many biologically significant glycans of mammals, commonly as a terminal α-glycoside. The chemical structure of sialic acid, which features an anomeric center with carboxyl and methylene substituents, poses a challenge for synthesis of the α-glycoside, thus impeding biological and therapeutic studies on sialic acid–containing glycans. We present a robust method for the selective α-glycosidation of sialic acid using macrobicyclized sialic acid donors as synthetic equivalents of structurally constrained oxocarbenium ions to impart stereoselectivity. We demonstrate the power of our method by showcasing broad substrate scope and applicability in the preparation of diverse sialic acid–containing architectures.

Stereoselective α-glycosidation of sialic acid (α-sialidation) is a long-standing challenge in carbohydrate chemistry. Studies on sialic acid–containing glycans (sialoglycans) have focused not only on carbohydrate chemistry but also on glycobiology, owing to the key roles of these species in various biological processes, including brain development, immune response, and microbial attachment (14). Sialoglycans often contain sialic acid groups incorporated as terminal residues through α-glycosidic linkages, are structurally diverse (5), and thus are difficult to obtain in sufficient quantity in a single form from natural sources.

α-Sialidation is more difficult than the glycosidation of aldohexoses (e.g., glucose and galactose), owing to the specific chemical structure of sialic acid, a 3-deoxy-2-ketoaldonic acid (6, 7). An α-sialoside is formed by nucleophilic substitution at the hindered tertiary C2 anomeric center, rather than substitution of the secondary C1 anomeric center of aldohexoses. The electron-withdrawing carboxyl group flanking the anomeric center makes the oxocarbenium ion of sialic acid unstable and thus more prone to 2,3-elimination, generating the 2,3-ene derivative. The lack of an equatorial hydroxyl group at the C3 position also precludes neighboring group participation in the α-sialidation; thus, anomeric effects control β-isomer production. The glycerol moiety at C6 also sterically hinders glycoside formation. Nitrile solvent–assisted α-sialidation (8) has been employed in combination with sialic acid donors (sialyl donors) (6, 7, 9, 10) (Fig. 1 and fig. S1). Auxiliaries at C1 and C3 have been installed to favor the α-sialoside (1119), and recently, O4,N5-carbonyl-7,8-O-picoloyl–substituted and 3-fluorinated sialyl donors have been developed as α-selective donors independent of nitrile solvent assistance (2023) (fig. S1). Unlike stereospecific α-sialidation by enzymes (24, 25), the stereoselectivity of the reported methods is affected by the steric and electronic properties of the coupling partners as well as those of the sialyl donors and the activation conditions, making the chemical synthesis of α-sialoglycans a grand challenge. Herein, we report a simple and comprehensive method for stereocontrolled α-sialidation.

Fig. 1 Strategies for synthesizing the α-glycoside of sialic acid.

Abbreviations: MP, p-methoxyphenyl; Pg, protecting group; Lg, leaving group; SR, alkyl- or arylsulfenyl; SeR, alkyl- or arylselenyl; Me, methyl; Gc, glycolyl; d.e., diastereomeric excess.

We drew inspiration from the bridgehead carbocation (anti-Bredt carbocation) chemistry reported by Kraus (26) and envisioned that an anti-Bredt oxocarbenium ion of sialic acid would restrict the attack of the nucleophile on the posterior (α-face) of the bicyclic system. The ring strain in the bridgehead oxocarbenium ion could also provide a driving force for glycosidic bond formation. We designed a bicyclic α-thioglycoside of sialic acid with tethers at the C1-carboxy group and the C5-amino group as a glycosyl donor. The viability of bridgehead oxocarbenium ions of highly substituted aza- and oxa-rings is difficult to predict based on the proposed rules for bridgehead olefins [S value (27) and olefin strain energy (28, 29)]. We therefore synthesized the C1 to C5 tethered sialyl donors with bicyclo[8.2.2] to bicyclo[13.2.2] systems (1 to 6, respectively) from free sialic acid in nine steps (26 to 33% overall yield), i.e., more steps than for the synthesis of a representative O4,N5-carbonyl–substituted sialyl donor (seven steps; 42% overall yield) (21) (Fig. 2 and figs. S2 to S4). Glycosidation of each donor (except for 1 and 2) with acceptor 7 produced an isolable amount of the corresponding α-glycoside as a single stereoisomer (table S1). Sialyl donor 5 with a bicyclo[12.2.2] system provided the highest yield (83%) of α-glycoside 12 at −40°C. Although the reactivity of 6 was comparable to that of 5, 6 was much more prone to 2,3-elimination, decreasing the yield of α-glycoside. These results may be affected by the flexibility of the bicyclic structure. The high tolerance of the more flexible 6 to a planar structure around the anomeric center may enhance 2,3-elimination of the oxocarbenium cation. The stereochemistry of the anomeric position of the glycosidated products was determined to be α by using reported nuclear magnetic resonance methods (figs. S5 to S10).

Fig. 2 Evaluation of bicyclic sialic acid donors.

Isolated yields are reported. Ac, acetyl; Bn, benzyl; equiv., equivalents; MP, p-methoxyphenyl; MS, molecular sieves; NIS, N-iodosuccinimide; Ph, phenyl; TfOH, trifluoromethanesulfonic acid.

Taking 5 as a model bicyclic sialyl donor, we next sought to embed a 2,2-dichloroethoxycarbonyl moiety in the tether to ensure the selective reductive cleavage at the C5 amine group and subsequent diversification to natural congeners. We prepared sialyl donors 14 and 15, bearing a phenylsulfenyl moiety and a dibenzylphosphate moiety (30) as a leaving group, respectively (figs. S11 to S13). Examination of their glycosidation revealed the high reactivity and broad substrate scope of these sialyl donors (Fig. 3A, tables S2 to S4, figs. S14 to S17). The glycosidations of primary to tertiary hydroxyl groups linked to nonsugar backbones, including simple (16 and 17) and bulky (18 and 19) derivatives, afforded very high yields of α-sialosides 27 to 30. Sialyl donor 14 underwent coupling reactions with glycosyl acceptors 20 to 23, producing the glycosidic linkages of natural sialic acids 31 to 34. Donor 14 also allowed the double sialylation of tetrasaccharide 24 in a regioselective manner, furnishing the full sequence of the glycan moiety of ganglioside GD1α35. The scope of the method was expanded with 15, and the oxidant-free reaction conditions allowed couplings with unsaturated substrates (25 and 26). Conditions similar to those for O-glycosidations could also be used for C-glycosidations with sialyl donor 15 (Fig. 3B and figs. S18 to S20). With methallyltrimethylsilane (38) and silylenol 39, corresponding C-sialosides 41 and 42 were produced as single α-glycosides. Donor 15 also underwent an aromatic electrophilic substitution with protected catechin 40 to give 43.

Fig. 3 Evaluation of the substrate scope and deprotection.

Isolated yields are reported. (A) O-Glycoside formation. All glycosides were obtained as single α-glycosides. Sia, sialic acid; Lg, leaving group; Ph, phenyl; Ac, acetyl; TMS, trimethylsilyl; TESOTf, triethylsilyl trifluoromethanesulfonate; TMSOTf, trimethylsilyl trifluoromethanesulfonate; NIS, N-iodosuccinimide; MS, molecular sieves; Piv, pivaloyl; Cbz, benzyloxycarbonyl; Bz, benzoyl; Bn, benzyl; SE, 2-(trimethylsilyl)ethyl; TFA, trifluoroacetyl; MP, p-methoxyphenyl. (B) C-glycoside formation. All glycosides were obtained as single α-glycosides. (C) Synthesis of sialyl glycolipids. RT, room temperature; TBDMS, tert-butyldimethylsilyl; TBB, p-tert-butylbenzoyl; DMAP, 4-dimethylaminopyridine; EDC; 1-ethyl-3-(dimethylamino)propyl carbodiimide; Pyr, pyridine; TBAF, tetra-n-butylammonium fluoride; THF, tetrahydrofuran, Fuc, fucosyl; HOBt, 1-hydroxy-1H-benzotriazole.

We next synthesized sialyl glycolipids with various modifications on the sialic acid residue. Late-stage sialidation and C5 amine modifications demonstrate the feasibility of our method for the assembly of highly complex molecules (Fig. 3C). Using phosphate donor 15, unsaturated glucosyl lipid 44 was α-sialylated at the 6-OH, giving 45. The treatment of 45 with zinc in acetic acid selectively opened the 16-membered ring at the carbamoyl moiety to give the C5-amino sialoside, which then underwent selective acylations to generate the C5-modified sialic acid congeners. Treatment with acetic anhydride, acetoxyacetyl chloride, and (2,3,4-tri-O-acetyl-α-l-fucosyl)oxyacetic acid (46) yielded N-acetyl, N-glycolyl, and N-fucosylglycolyl derivatives 47 to 49, respectively. Global deprotection delivered glycolipids 50 to 52 (31), which are congeners of gangliosides relevant to the function of sea urchin sperm. These molecules would be useful in the study of the fertilization mechanism.

We next investigated the construction of the tandem sequences of sialic acid, which are involved in biologically relevant glycans. We performed glycosylations using 14 and sialyl acceptors 53 to 57 (Fig. 4A, figs. S21 to S23). Donor 14 could glycosylate the primary hydroxyl groups of 53 and 54 to produce α(2,9)- and α(2,11)-linked dimers 58 and 59, respectively. The formation of an α(2,8)-linkage between sialic acids is of particular interest, because glycans with this linkage are relevant in brain development (2). This reaction is synthetically challenging, because hydrogen bonding with the adjacent acetamide group results in poor reactivity of 8-OH (6, 7, 32, 33) (fig. S27). We carried out the glycosylation of a known acceptor, 55 (34), which gave a low yield of the glycoside, indicating the need for a highly reactive coupling partner for 14. For this purpose, we developed macrocyclized acceptor 56, which is less prone to hydrogen bonding between the 8-OH and C5 amino groups. As expected, the enhanced reactivity of 56 was demonstrated by an increase in the yield of sialyl-α(2,8)-sialyl glycoside 61. Density functional theory (DFT) calculations indicated that 8-OH within 56 is exposed in the lowest-energy conformation (figs. S24-S26). The poor reactivity of 4-OH (33) is alleviated in 57, yielding sialylated product 62.

Fig. 4 Synthesis of oligomeric sialic acids.

(A) Evaluation of disialic acid synthesis. Isolated yields are reported. All glycosides were obtained as single α-glycosides. Abbreviations: NIS, N-iodosuccinimide; SE, 2-(trimethylsilyl)ethyl; TfOH, trifluoromethanesulfonic acid; Me, methyl; Ac, acetyl. (B) Synthesis of pentasialoside. All glycosides were obtained as single α-glycosides. Ph, phenyl; Bn, benzyl; Bz, benzoyl; CAc, chloroacetyl; RT, room temperature.

To demonstrate the utility of the bicyclic sialic acid donor and acceptor, we synthesized an oligomer of α(2,8)-linked sialic acid (Fig. 4B, figs. S28 to S33). To complete the glycosylation and subsequent conversion to the glycosyl acceptor, a chloroacetyl group was used as a transient protecting group for both the C7 and C8 hydroxyl groups of the sialyl donor. We started with the glycosylation of sialyl galactoside acceptor 63 with donor 64, giving a disialoside, which was then selectively deprotected at C7 and C8 by 1-selenocarbamoylpiperidine (65) to afford the corresponding glycosyl acceptor. Repeating this cycle three times led to pentasialoside 66 in high yield. Finally, global deprotection of 66 delivered α(2,8)-linked pentasialoside 67.

We have shown that a substituent of sialic acid, the C1 carboxyl group, which is generally detrimental to the glycosidation reaction, can instead be harnessed as the key to solving the long-standing issue of α-sialidation. Direct α-sialylation of oligosaccharides has been previously underutilized because the difficulty of separating the stereoisomers makes the synthetic process rather complicated. Our method can streamline the synthesis of sialoglycans. In addition, as the substituents on the C5 amino moiety, N-acetyl and N-glycolyl, are strongly associated with the biological function of sialoglycans (5), this method will be valuable for producing glycans for biological studies. Because the difficult separation of the stereoisomers of the sialylated products is a bottleneck in automated oligosaccharide synthesis, this method would contribute toward expanding the scope of this synthetic process (35).

Supplementary Materials

Materials and Methods

Figs. S1 to S39

Tables S1 to S4

References (3688)

NMR Spectra

Data Files S1 to S7

References and Notes

Acknowledgments: A portion of the computations was performed at Research Center for Computational Science (RCCS), Okazaki. The iCeMS is supported by a World Premier International Research Center Initiative (WPI), Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We thank all the past and current members of the Bioactive Molecule Science Laboratory (Kassei lab) of Gifu University for their sincere efforts in our “Sialidation Quest” project. We dedicate this paper to the memory of the late Professor Akira Hasegawa, who pioneered sialic acid chemistry at Gifu University. Funding: This work was supported in part by JSPS KAKENHI grant nos. JP18K05028 (T.U.), JP18K05461 (H.-N.T.), JP15K07409 (H.I.), JP15H04495 (H.A.), and JP18H03942 (H.A.); JST CREST grant no. JPMJCR18H2 (H.A.); and the Mizutani Foundation for Glycoscience (H.A.). Author contributions: N.K. and H.A. conceived this research and designed the experiments; N.K., K.K., S.A., A.I., and H.A. performed the experiments; T.U. performed the global reaction route mapping and DFT calculations; N.K. H.-N.T., A.I., H.I., M.K., and H.A. analyzed the data; N.K. and H.A. wrote the paper; and all authors participated in the revisions of the manuscript. Competing interests: The authors declare no competing interests. Data and materials availability: All data are available in the manuscript or the supplementary materials.
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