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Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined glycans is key to establishing structure-activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydrate-based vaccines.

No abstract

Clostridium difficile is a leading cause of severe nosocomial infections. Cell-surface carbohydrate antigens are promising vaccine candidates. Here we report the first total synthesis of oligomers of the lipoteichoic acid antigen repeating unit. Synthetic glycan microarrays revealed anti-glycan antibodies in the blood of patients that help to define epitopes for vaccine development.

Dedicated to Professor Joachim Thiem on the occasion of his 65th birthdayRecombinant therapeutic glycoproteins contain mainly asparagine-linked oligosaccharides (N-glycans), which are often essential for the proper function of the glycoprotein. The heterogeneity of the glycans in natural glycoproteins is a large obstacle for the entire research in the field of glycobiology, [1] and despite recent advances in chemical synthesis of N-glycans, [2][3][4][5][6][7][8][9][10] the majority of N-glycans for biological studies are still isolated from natural sources.[11] We have developed modular building blocks for the most abundant complex N-glycans, [12] which after fortuitous results from the test of this modular system allowed the synthesis of complex N-glycans with the maximum number of branches and core substitutions (glycan F; Scheme 1).Previously we have developed modular building blocks for the synthesis of complex N-glycans with up to four antennae, [12] which contained a bisecting GlcNAc moiety [4,13,14] or a core fucose moiety. [15] As a result of the steric hindrance, bisected N-glycans with three or four antennae are especially difficult to obtain. [4] Encouraged by recent improvements [14] we investigated the complex N-glycan F (Scheme 1). A particularly high substitution pattern is found at the Mana1,6Manb unit of F where each mannose contains a total of four glycosidic partners. Pentaantennary N-glycans are found in ovomucoid, [16] fish hyosophorin, [17] CHO cells, [18] and HepG2 cells. [19] First, unsubstituted pentaantennary N-glycans were assembled to reduce the synthetic complexity of F. The synthesis of tetrasaccharide donor D began with the 3-Oallylation of benzylmannoside (2) via a stannylene acetal to give 3 (Scheme 2). [20,21] Threefold glycosylation of triol 3 with donor 1 (6 equiv) gave the tetrasaccharide 4 (77 %). After deallylation of 4, the acetylation of alcohol 5 required catalytic amounts of DMAP. After catalytic hydrogenation of 6 to remove the benzyl group, the hemiacetal was converted into imidate D and coupled with the hexasaccharide 12 to give the decasaccharide 13 in 65 % yield after optimization (Scheme 3).The high reactivity of donor D prompted us to incorporate a bisecting GlcNAc moiety and a core fucose residue. Thus, the branched trisaccharide B [12] was coupled to the core trisaccharide A (Scheme 4).[15] The resulting hexasaccharide (80 %) was acetylated and the benzylidene acetal was cleaved (75 % over 2 steps). After the selective chloroacetylation of 14, the hexasaccharide 15 was coupled with thioglycoside C [4] to give the bisected heptasaccharide 16. Dechloroacetylation yielded the acceptor 17, which was coupled with the disaccharide 18 (77 %). The nonasaccharide 19 was deprotected and fucosylated to give the triantennary decasaccharide 21 in 93 % yield.Next we investigated the coupling of the heptasaccharide 17 with the donor tetrasaccharide D (5 equiv; Scheme 5). After purification by flash chromatography and HPLC, only 9 % of the bisected pentaantennary compound 22 wa...

The occurrence of N-glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N-glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N-glycans of the complex type by employing modular TFAc-protected donors for all antennae. The TFAc-protected N-glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N-glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one-pot double glycosylation. With this new method, rare N-glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.

When using benzyl ethers as permanent protecting groups in oligosaccharide synthesis selective oxidative debenzylation with NaBrO(3) + Na(2)S(2)O(4) under biphasic conditions is efficient and compatible with anomeric azides and many other functions.

A building block approach for biantennary N-linked oligosaccharides from glycoproteins (N-glycans) has been developed. Starting from a core trisaccharide (beta-mannosyl chitobiose) containing a benzylidene-protected beta-mannoside, the attachment of the disaccharide building blocks for the antennae can be performed in a double regio- and stereoselective manner. A short synthesis of a GlcNPhtbeta1,2Man donor was developed. The benzylidene acetal moiety, as a minimal protection of the beta-mannoside, allows selective alpha-glycosylation at OH-3 of the 2,3-diol with GlcNbeta1,2Man trichloroacetimidate donors. Subsequent debenzylidenation leads to a 4,6-diol, which can be selectively extended at OH-6. Overreaction at OH-4 was generally low when phthalimido-protected donors were used. This general strategy represents a modular synthesis of N-glycans and their glycoconjugates.