Nicola L. B. Pohl

Synthesis and Quantitative Evaluation of Glycero‐D‐manno‐heptose Binding to Concanavalin A by Fluorous‐Tag Assistance

tion was reported earlier in the first demonstration of a microarray fabrication strategy based on noncovalent fluorous interactions. Mannose and other monosaccharides tagged by single C8F17 chains were noncovalently immobilized to fluorocarbon-coated glass slide surfaces and shown to tolerate conditions necessary for identifying binding partners such as conA. More recently, this fluorous-based microarray approach has proven valuable for the probing of other classes of small molecules. In the case of histone deacetylase inhibitors with dissocation constants of less than 0.1 s , the hits found by fluorous microarrays were comparable to those found by techniques such as surface plasmon resonance (SPR) and solution-based biochemical assays. Ideally, of course, the relative quantification of these binding interactions could also be carried out within the same fluorous microarray screening format. ConA is a plant lectin that is widely used like antibodies as research tools and diagnostics to identify the presence of specific sugars, such as mannose, on cells; however, in reality the sugar specificities of lectins have not been tested broadly, especially against less readily available carbohydrates. ConA is the most-studied lectin and is usually considered to bind terminal alpha-linked mannose, glucose, and N-acetylglucosamine. Earlier inhibition data suggest that modifications at the C-3, C-4, and C-6 positions of the d-mannopyranose deter binding to conA. In particular, the loss of the hydroxy group in the C-6 position as in 6-deoxy-d-mannose and 1,6-anhydrob-d-manno-pyranose result in complete loss of activity. To date, no studies have been realized on the addition of another hydroxymethyl group at this same position. In fact, surprisingly little is known about how seven-carbon sugars might mimic six-carbon sugars or interact with proteins. The seven-carbon sugars l-glycero-d-mannoand dglycero-d-manno-heptose (Figure 1) are common constituents in pathogenic bacteria of lipopolysaccharides (LPS) that mediate numerous responses to bacterial infections. The increasing resistance of many bacterial strains against conventional antibiotics is prompting the need for a detailed understanding of the immunological responses against these outer carbohydrate coats to inform the development of new therapeutic agents such as bacterial cell wall biosynthesis inhibitors and vaccines. Although humans lack mannoheptoses, the success of a vaccine against this bacterial sugar would presume the lack of cross-reactivity of antibodies generated against this distinct heptose antigen with the many mannose residues found in humans. As lectins are often used as antibody replacements in cellular carbohydrate-binding experiments, we reasoned that a study of the feasibility of using manno-heptose motifs in vaccine design should begin with assessing the ability of a well-characterized mannosebinding protein, conA, to cross-react with heptopyranoside motifs. To test the ability of conA to bind to l-glycero-d-mannoand d-glycero-d-manno-heptoses, we first needed a route to readily access both diastereomers. Several methods have been reported. To start from a less expensive six-carbon sugar, we required an approach to differentiate the alcohol at C-2 from Figure 1. Structure of mannose and heptomannose, and the strategy for their incorporation into a microarray by noncovalent fluorous interactions for screening with concanavalin A.

Toward solution-phase automated iterative synthesis: fluorous-tag assisted solution-phase synthesis of linear and branched mannose oligomers

We report herein the first synthesis of linear and branched mannose oligosaccharides using fluorous-tag assistance with reagents and FSPE protocols that are amenable to automation. The particular fluorous linker proved to maintain solubility of the growing oligosaccharide chain such that identical reaction solvent conditions and purification protocols could be used between glycosylation and deprotection reactions, thereby rendering the procedures amenable to automation.

Synthesis of fluorous tags for incorporation of reducing sugars into a quantitative microarray platform.

Carbohydrate microarrays can map out key interactions of carbohydrates and proteins in a high-throughput manner, but require the inclusion of a range of sugars for their optimal use. Here we present the synthesis and use of a new hydroxylamine-modified fluorous tag that allows the facile incorporation of reducing sugars into a noncovalent fluorous-based microarray after simple purification by fluorous solid-phase extraction (FSPE). The microarray supports quantitative screening against carbohydrate-binding proteins.

Tailoring the immune response by targeting C-type lectin receptors on alveolar macrophages using " pathogen-like" amphiphilic polyanhydride nanoparticles

C-type lectin receptors (CLRs) offer unique advantages for tailoring immune responses. Engagement of CLRs regulates antigen presenting cell (APC) activation and promotes delivery of antigens to specific intracellular compartments inside APCs for efficient processing and presentation. In these studies, we have designed an approach for targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide pathogen-like properties. Two conserved carbohydrate structures often found on the surface of respiratory pathogens, galactose and di-mannose, were used to functionalize the surface of polyanhydride nanoparticles and target CLRs on alveolar macrophages (AMϕ), a principle respiratory tract APC. Co-culture of functionalized nanoparticles with AMϕ significantly increased cell surface expression of MHC I and II, CD86, CD40 and the CLR CIRE over non-functionalized nanoparticles. Di-mannose and galactose functionalization also enhanced the expression of the macrophage mannose receptor (MMR) and the macrophage galactose lectin, respectively. This enhanced AMϕ activation phenotype was found to be dependent upon nanoparticle internalization. Functionalization also promoted increased AMϕ production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Additional studies demonstrated the requirement of the MMR for the enhanced cellular uptake and activation provided by the di-mannose functionalized nanoparticles. Together, these data indicate that targeted engagement of MMR and other CLRs is a viable strategy for enhancing the intrinsic adjuvant properties of nanovaccine adjuvants and promoting robust pulmonary immunity.

Carbohydrate arrays: recent developments in fabrication and detection methods with applications.

Carbohydrate arrays can provide facile analyses of carbohydrate-binding proteins, antibodies in serum, and enzyme activities with minimal quantities of sugar samples. Recent developments in detection methods for carbohydrate arrays, especially surface plasmon resonance (SPR) and mass spectrometry, have enabled not only qualitative but in some cases quantitative data analysis in a variety of multivalent display formats. These advancements make diagnostic tools based on carbohydrate arrays more promising for infectious disease detection, cancer monitoring, and vaccine development.

Synthesis of Multivalent Tuberculosis and Leishmania-Associated Capping Carbohydrates Reveals Structure-Dependent Responses Allowing Immune Evasion

Mycobacterium tuberculosis and the protozoan parasites of the genus Leishmania are intracellular pathogens that can survive in macrophages--the very white blood cells of the immune system responsible for engulfing and ultimately clearing foreign invaders. The ability of these pathogens to hide within immune cells has made the design of effective therapies, including vaccines, to control tuberculosis and leishmaniasis particularly challenging. Herein we present the synthesis and development of carbohydrate-based probes to demonstrate that changes in pathogen-associated surface oligosaccharides are sufficient to alter cellular immune responses and thereby let a pathogen hide from immune surveillance.

Bacterial CMP-sialic acid synthetases: production, properties, and applications

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell–cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5′-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.

Monosaccharide Identification as a First Step toward de Novo Carbohydrate Sequencing: Mass Spectrometry Strategy for the Identification and Differentiation of Diastereomeric and Enantiomeric Pentose Isomers

De novo carbohydrate sequencing, including monosaccharide identification, largely remains a tremendous analytical challenge. A first step in the complete structural determination of any large polysaccharide is an accurate and robust method for analysis of the constituent monosaccharides. Herein, the first mass spectrometry-based method for the complete identification and absolute configuration determination of all 12 pentose isomers, including the d and l enantiomers for arabinose, lyxose, ribose, xylose, ribulose, and xylulose, is reported. As compared to earlier work to distinguish hexose isomers, the chiral separation of the pentose isomers was significantly more challenging. Specifically, the 12 pentoses are much more structurally similar to one another, with only the axial or equatorial orientation of two hydroxyl groups differentiating among these isomers in their five-membered ring furanose structure and smaller energetic differences between pentose conformations than between hexose conformations. Despite such inherently minimal energetic differences between the 12 pentoses, two unique fixed ligand kinetic method combinations were discovered to achieve chiral discrimination for this set of isomers. This assay can be readily applied to the identification of any isolated pentose monosaccharide using only microgram quantities and a commercial instrument and complements the method to distinguish hexose isomers. A workflow that incorporates this mass spectrometry-based method and thereby could achieve complete de novo identification of all monosaccharide building blocks in an oligo- or polysaccharide is proposed.

Multidimensional Analysis of 16 Glucose Isomers by Ion Mobility Spectrometry

Diastereomeric adducts comprising an enantiomerically pure monosaccharide analyte, a peptide, and/or an amino acid and a divalent metal ion (for 16 different monosaccharide isomers) are generated by electrospray ionization and analyzed by combined ion mobility spectrometry-mass spectrometry (IMS-MS) techniques. Mobility distributions of [l-Ser + M + H](+) (where l-Ser is l-serine and M is a given monosaccharide), [l-Phe-Gly + M + H](+) (where l-Phe-Gly is l-phenylalanine-glycine), and [Mn(II) + (l-Phe-Gly - H) + M](+) complex ions are used to determine collision cross sections (ccs in Å(2)), and groups of cross sections for different clusters are proposed as means of identifying the sugar isomers. Within one type of complex, variations in ccs do not always allow delineation between the 16 glucose isomers, but interestingly, when ccs of three different ions are combined as a spatial vector, enantiomers are partially resolved. As a result of this analysis, l-glucose, d-glucose, l-allose, d-allose, d-gulose, d-galactose, and l-mannose are delineated, and for all eight enantiomeric pairs, d and l entities display different coordinates. In addition, different combinations of amino acids, peptide, and metal ions are surveyed, and the potential for yielding unique coordinates for the generated diastereomeric complexes is assessed.

Complete Hexose Isomer Identification with Mass Spectrometry

AbstractThe first analytical method is presented for the identification and absolute configuration determination of all 24 aldohexose and 2-ketohexose isomers, including the D and L enantiomers for allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, and tagatose. Two unique fixed ligand kinetic method combinations were discovered to create significant enough energetic differences to achieve chiral discrimination among all 24 hexoses. Each of these 24 hexoses yields unique ratios of a specific pair of fragment ions that allows for simultaneous determination of identification and absolute configuration. This mass spectrometric-based methodology can be readily employed for accurate identification of any isolated monosaccharide from an unknown biological source. This work provides a key step towards the goal of complete de novo carbohydrate analysis. Graphical Abstractᅟ