Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research

Abstract

Synthetic oligosaccharides and glycoconjugates are increasingly used as probes for biological research and as lead compounds for drug and vaccine discovery. These endeavors are, however, complicated by a lack of general methods for the routine preparation of this important class of compounds. Recent development such as one-pot multi-step protecting group manipulations, the use of unified monosaccharide building blocks, the introduction of stereoselective glycosylation protocols, and convergent strategies for oligosaccharide assembly, are beginning to address these problems. Furthermore, oligosaccharide synthesis can be facilitated by chemo-enzymatic methods, which employ a range of glycosyl transferases to modify a synthetic oligosaccharide precursor. Glycosynthases, which are mutant glycosidases, that can readily form glycosidic linkages are addressing a lack of a wide range glycosyltransferases. The power of carbohydrate chemistry is highlighted by an ability to synthesize glycoproteins.

Figure 1. Examples of biologically active synthetic oligosaccharide and glycopeptide constructs

a, Structure of the anticoagulant drug Arixtra^®. b, Structure of the promising anticoagulant candidate drug Idraparinux. c, Synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. d, Synthesis of the erythropoietin (EPO; 78–166) glycopeptide through sequential fragment condensation using auxiliary-based cysteine-free native chemical ligation (NCL). Glycans are shown in red and the ligation sites are shown in bold. DIPEA, N, N-diisopropylethylamine; Et, ethyl; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; Me, methyl; TCEP-HCl, tris(2-carboxyethyl)phosphine hydrochloride.

Figure 2. Orthogonally protected mono- and disaccharide building blocks for oligosaccharide synthesis

a, One-pot protection of glucosides; with only four different types of one-pot reactions a total of 152 different glucose building blocks can be reached. b, The 15 most abundant monosaccharide units and their linkage types found in mammalian oligosaccharides are shown in the table. To the right is shown examples of four orthogonally protected building blocks that can be used to synthesize some of the units in the table. c, Disaccharide building blocks that can be used for a modular synthesis of heparan sulfate. Ac, acetyl; All, allyl; Bn, benzyl; Bz, benzoyl; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone; Et₃SiH, triethylsilane; GlcNAc, N-acetyl glucosamine; Lev, levolinoyl; Me, methyl; Piv, pivaloyl; TBAF, tetrabutylammonuium fluoride; TCA, trichloroacetyl; TMS, trimethylsilyl; TMSOTf, trimethylsilyl trifluoromethanesulfonate; Tol, toluene.

Figure 3. Common methods for the stereoselective formation of glycosidic linkages

a, Different types of anomeric linkages. b, Preparation of 1,2-trans glycosides by C-2 neighbouring group participation. c, Chiral auxiliary controlled installation of 1,2-cis-glycosides. A stable β-sulfonium ion shields the β-face, forcing the incoming alcohol to attack from the α-face. d, Synthesis of β-mannosides by intramolecular aglycon delivery. The alcohol is tethered to the C-2 position; subsequent activation of this adduct forces the alcohol to be delivered from the β-face. e, Introduction of β-mannosidic linkages by in situ formation of an α-triflate that is displaced in a S_N2-like manner to form a β-mannoside. f, Formation of an α-galactoside by steric constraints induced by a 4,6-O-silyl acetal. g, Introduction of β-arabinosidic linkages by locking the five-membered ring in a conformation that favors an attack of the incoming nucleophile from the 1,2-cis face. The right hand panel shows the Newman projection of the locked conformer. h, Introduction of α-sialosides by appropriate protection of the N-acetamido moiety. i, Formation of α-linked 2-amino-2-deoxy-glucosides using 2,3-oxazolidone protected glycoside donors. BSP, 1-benzenesulfinyl piperidine; CBz, benzyloxycarbonyl; MeCN, acetonitrile; NIS, N-iodosuccinimide; P, protecting group; Ph, phenyl; Phth, phthalimido; R, glycosyl, alkyl or aryl; TFA, trifluoroactyl; Tf₂O, trifluoromethanesulfonic anhydride; TfOH, trifluoromethanesulfonic acid; Troc, 2,2,2-trichloroethoxycarbonyl; TTBP, tri-tert-butylpyrimidine.

Figure 4. One-pot chemical synthesis of the tumor-associated antigen Globo-H by iterative preactivation of p-tolyl thioglycosides

AgOTf, silver trifluoromethanesulfonate; ROH, sugar alcohol.

Figure 5. Polymer supported synthesis of the tumor-associated KH-1 antigen

a, The linker-modified polystyrene resin and orthogonally protected monosaccharide building blocks used for the synthesis. Fmoc carbonates and Lev esters were used as temporary protecting groups; phosphates were used as anomeric leaving groups. b, The fully protected, resin released, nonasaccharide antigen. Bu, butyl; Pent, pentenyl.

Figure 6. Enzymatic and chemoenzymatic synthesis of oligosaccharides

a, Synthesis of a sialoside library using promiscuous enzymes, chemically modified (R¹ and R²) mannosamine and mannose, and modified galactose (R³) acceptors in a one-pot, three-enzyme approach. b, Mechanism of inverting glycosidases for the degradation of oligosaccharides. c, Mechanism for glycosylation by a glycosynthase using activated glycosyl fluorides as substrates. CMP, cytidine-5'-monophosphate; CTP, cytidine-5'-triphosphate.