Noncovalent microarrays from synthetic amino-terminating glycans: Implications in expanding glycan microarray diversity and platform comparison.

Glycan microarrays have played important roles in detection and specificity assignment of glycan recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and noncovalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune receptor Dectin-1 toward ß-glucans and the interaction of rotavirus P[19] adhesive protein with mucin O-glycan cores.

Efficient Chemoenzymatic Synthesis of N-Glycans with a ß1,4-Galactosylated Bisecting GlcNAc Motif.

In human serum immunoglobulin G (IgG), a rare modification of biantennary complex N-glycans lead to a ß1,4-galactosylated bisecting GlcNAc branch. We found that the bisecting GlcNAc on a biantennary core-fucosylated N-glycan was enzymatically galactosylated under stringent reaction conditions. Further optimizations led to an efficient enzymatic approach to this particular modification for biantennary substrates. Notably, tri- and tetra-antennary complex N-glycans were not converted by bovine galactosyltransferase. An N-glycan with a galactosylated bisecting GlcNAc was linked to a lanthanide binding tag. The pseudo-contact shifts (PCS) obtained from the corresponding Dy-complex were used to calculate the conformational preferences of the rare N-glycan. Besides two extended conformations only a single folded conformation was found.

A Single Route to Mammalian N-Glycans Substituted with Core Fucose and Bisecting GlcNAc.

The occurrence of α1,6-linked core fucose on the N-glycans of mammalian glycoproteins is involved in tumor progression and reduces the bioactivity of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC). Since core-fucosylated N-glycans are difficult to isolate from natural sources, only chemical or enzymatic synthesis can provide the desired compounds for biological studies. A general drawback of chemical α-fucosylation is that the chemical assembly of α1,6-linked fucosides is not stereospecific. A robust and general method for the α-selective fucosylation of acceptors with primary hydroxy groups in α/ß ratios exceeding 99:1 was developed. The high selectivities result from the interplay of an optimized protecting group pattern of the fucosyl donors in combination with the activation principle and the reaction conditions. Selective deprotection yielded versatile azides of all mammalian complex-type core-fucosylated N-glycans with 2-4 antennae and optional bisecting GlcNAc.

A Semi-synthetic Oligosaccharide Conjugate Vaccine Candidate Confers Protection against Streptococcus pneumoniae Serotype 3 Infection.

The identification of immunogenic glycotopes that render glycoconjugate vaccines protective is key to improving vaccine efficacy. Synthetic oligosaccharides are an attractive alternative to the heterogeneous preparations of purified polysaccharides that most marketed glycoconjugate vaccines are based on. To investigate the potency of semi-synthetic glycoconjugates, we chose the least-efficient serotype in the current pneumococcal conjugate vaccine Prevnar 13, Streptococcus pneumoniae serotype 3 (ST3). Glycan arrays containing synthetic ST3 repeating unit oligosaccharides were used to screen a human reference serum for antibodies and to define the recognition site of two ST3-specific protective monoclonal antibodies. The glycan array screens identified a tetrasaccharide that was selected for in-depth immunological evaluation. The tetrasaccharide-CRM197 carrier protein conjugate elicited protective immunity as evidenced by opsonophagocytosis assays and protection against pneumonia caused by ST3 in mice. Formulation of the defined protective lead candidate glycotope has to be further evaluated to elicit optimal long-term immunity.

Automated glycan assembly of a S. pneumoniae serotype 3 CPS antigen.

Vaccines against S. pneumoniae, one of the most prevalent bacterial infections causing severe disease, rely on isolated capsular polysaccharide (CPS) that are conjugated to proteins. Such isolates contain a heterogeneous oligosaccharide mixture of different chain lengths and frame shifts. Access to defined synthetic S. pneumoniae CPS structures is desirable. Known syntheses of S. pneumoniae serotype 3 CPS rely on a time-consuming and low-yielding late-stage oxidation step, or use disaccharide building blocks which limits variability. Herein, we report the first iterative automated glycan assembly (AGA) of a conjugation-ready S. pneumoniae serotype 3 CPS trisaccharide. This oligosaccharide was assembled using a novel glucuronic acid building block to circumvent the need for a late-stage oxidation. The introduction of a washing step with the activator prior to each glycosylation cycle greatly increased the yields by neutralizing any residual base from deprotection steps in the synthetic cycle. This process improvement is applicable to AGA of many other oligosaccharides.

Highly Efficient Synthesis of Multiantennary Bisected N-glycans Based on Imidates.

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.

Automated solid-phase synthesis of a ß-(1,3)-glucan dodecasaccharide.

ß-Glucans are a group of structurally heterogeneous polysaccharides found in bacteria, fungi, algae and plants. ß-(1,3)-D-Glucans have been studied in most detail due to their impact on the immune system of vertebrates. The studies into the immunomodulatory properties of these glucans are typically carried out with isolates that contain a heterogeneous mixture of polysaccharides of different chain lengths and varying degrees of branching. In order to determine the structure-activity relationship of ß-(1,3)-glucans, access to homogeneous, structurally-defined samples of these oligosaccharides that are only available through chemical synthesis is required. The syntheses of ß-glucans reported to date rely on the classical solution-phase approach. We describe the first automated solid-phase synthesis of a ß-glucan oligosaccharide that was made possible by innovating and optimizing the linker and glycosylating agent combination. A ß-(1,3)-glucan dodecasaccharide was assembled in 56 h in a stereoselective fashion with an average yield of 88% per step. This automated approach provides means for the fast and efficient assembly of linker-functionalized mono- to dodecasaccharide ß-(1,3)-glucans required for biological studies.

Glycosylation efficiencies on different solid supports using a hydrogenolysis-labile linker.

Automated oligosaccharide assembly requires suitable linkers to connect the first monosaccharide to a solid support. A new hydrogenolysis-labile linker that is stable under both acidic and basic conditions was designed, synthesized and coupled to different resins. Glycosylation and cleavage efficiencies on these functionalized solid supports were investigated, and restrictions for the choice of solid support for oligosaccharide synthesis were found.

Progress toward developing a carbohydrate-conjugate vaccine against Clostridium difficile ribotype 027: synthesis of the cell-surface polysaccharide PS-I repeating unit.

Clostridium difficile strain ribotype 027 is a hypervirulent pathogen that is responsible for recent, severe outbreaks of serious nosocomial infections. As a foundation for the development of a preventative carbohydrate-based vaccine, we have synthesized a pentasaccharide cell wall repeating unit from PS-I unique to this strain, by the linear assembly of four monosaccharide building blocks.

Solid phase synthesis of oligosaccharides.

Oligosaccharides are involved in many fundamental biological processes. However, relatively little is known about the precise molecular mechanism of action of these macromolecules, because the complexity of these structures impeded their synthesis by chemical methods analogous to those employed to create oligonucleotides and peptides. Herein, we describe recently developed techniques for solid-supported oligosaccharide synthesis. Several key aspects of solid phase synthesis are highlighted and examined, including the choice of resin and the challenge of real-time reaction monitoring. Recent examples of manual and automated solid-supported syntheses of complex oligosaccharides are given and the automated solid phase synthesis of the tumor-associated carbohydrate antigen Globo-H is highlighted.