Chemical Synthesis and Antigenicity Evaluation of an Aminoglycoside Trisaccharide Repeating Unit of Pseudomonas aeruginosa Serotype O5 O-Antigen Containing a Rare Dimeric-ManpN3NA.

Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous SN2 substitution of azide groups at C2' and C2″ enabled the efficient synthesis of 1,2-cis-ß-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The ß-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.

Stereoselective Synthesis of the Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc Trisaccharide: a new Ligand for DCAR and Mincle C-Type Lectin Receptors.

In this work, we have discovered that the Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc trisaccharide, a fragment of the B antigen Type-1, is a new ligand of two C-type lectin receptors (CLRs) i. e. DCAR and Mincle which are key players in different types of autoimmune diseases. Accordingly, we report here on a straightforward methodology to access pure Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc trisaccharide. A spacer with a terminal primary amine group was included at the reducing end of the GlcNAc residue thus ensuring the further functionalization of the trisaccharide Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc.

Trisaccharide Sulfate and Its Sulfonamide as an Effective Substrate and Inhibitor of Human Endo-O-sulfatase-1.

Human endo-O-sulfatases (Sulf-1 and Sulf-2) are extracellular heparan sulfate proteoglycan (HSPG)-specific 6-O-endosulfatases, which regulate a multitude of cell-signaling events through heparan sulfate (HS)-protein interactions and are associated with the onset of osteoarthritis. These endo-O-sulfatases are transported onto the cell surface to liberate the 6-sulfate groups from the internal d-glucosamine residues in the highly sulfated subdomains of HSPGs. In this study, a variety of HS oligosaccharides with different chain lengths and N- and O-sulfation patterns via chemical synthesis were systematically studied about the substrate specificity of human Sulf-1 employing the fluorogenic substrate 4-methylumbelliferyl sulfate (4-MUS) in a competition assay. The trisaccharide sulfate IdoA2S-GlcNS6S-IdoA2S was found to be the minimal-size substrate for Sulf-1, and substitution of the sulfate group at the 6-O position of the d-glucosamine unit with the sulfonamide motif effectively inhibited the Sulf-1 activity with IC50 = 0.53 µM, Ki = 0.36 µM, and KD = 12 nM.

An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors.

The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors.

New synthesis of 6″-[18 F]fluoromaltotriose for positron emission tomography imaging of bacterial infection.

6″-[18 F]fluoromaltotriose is a positron emission tomography tracer that can differentiate between bacterial infection and inflammation in vivo. Bacteria-specific uptake of 6″-[18 F]fluoromaltotriose is attributed to the targeting of maltodextrin transporter in bacteria that is absent in mammalian cells. Herein, we report a new synthesis of 6″-[18 F]fluoromaltotriose as a key step for its clinical translation. In comparison with the previously reported synthesis, the new synthesis features unambiguous assignment of the fluorine-18 position on the maltotriose unit. The new method utilizes direct fluorination of 2″,3″,4″-tri-O-acetyl-6″-O-trifyl-α-D-glucopyranosyl-(1-4)-O-2',3',6'-tri-O-acetyl-α-D-glucopyranosyl-(1-4)-1,2,3,6-tetra-O-acetyl-D-glucopyranose followed by basic hydrolysis. Radiolabeling of the new maltotriose triflate precursor proceeds using a single HPLC purification step, which results in shorter reaction time in comparison with the previously reported synthesis. Successful synthesis of 6″-[18 F]fluoromaltotriose has been achieved in 3.5 ± 0.3% radiochemical yield (decay corrected, n = 7) and radiochemical purity above 95%. The efficient radiosynthesis of 6″-[18 F]fluoromaltotriose would be critical in advancing this positron emission tomography tracer into clinical trials for imaging bacterial infections.

Total Synthesis of the Congested, Bisphosphorylated Morganella morganii Zwitterionic Trisaccharide Repeating Unit.

Zwitterionic polysaccharides (ZPSs) activate T-cell-dependent immune responses by major histocompatibility complex class II presentation. Herein, we report the first synthesis of a Morganella morganii ZPS repeating unit as an enabling tool in the synthesis of novel ZPS materials. The repeating unit incorporates a 1,2-cis-α-glycosidic bond; the problematic 1,2-trans-galactosidic bond, Gal-ß-(1 → 3)-GalNAc; and phosphoglycerol and phosphocholine residues which have not been previously observed together as functional groups on the same oligosaccharide. The successful third-generation approach leverages a first in class glycosylation of a phosphoglycerol-functionalized acceptor. To install the phosphocholine unit, a highly effective phosphocholine donor was synthesized.

Total Synthesis of the Trisaccharide Antigen of the Campylobacter jejuni RM1221 Capsular Polysaccharide via de Novo Synthesis of the 6-Deoxy-d- manno-heptose Building Blocks.

A de novo approach utilizing the d-proline-catalyzed and LDA-promoted aldol reactions as key steps for the preparation of differentiated-protected 6-deoxy-d- manno-heptose building blocks was developed. PPh3AuBAr4F-catalyzed glycosylation with the 6-deoxy-d- manno-heptosyl o-hexynylbenzoate as donor was demonstrated as a direct and practical method for the stereoselective synthesis of the ß-linked 6-deoxy-d- manno-heptoside as the major product. Coupling of the 6-deoxy-α-d- manno-heptosyl H-phosphonate with the 3-hydroxyl disaccharide acceptor based on H-phosphonate chemistry was described for the construction of the trisaccharide skeleton with the acid-labile phosphodiester linkage. Finally, first total synthesis of the unique trisaccharide antigen of the capsular polysaccharide of Campylobacter jejuni RM1221 that belongs to HS:53 serotype complex was accomplished for further evaluation as vaccine candidate against C. jejuni RM1221 infection.

A chemo-enzymatic approach for the synthesis of human milk oligosaccharide backbone structures.

The ability of an engineered ß-N-acetylhexosaminidase to utilize a reactive oxazoline as donor molecule for transglycosylation reaction to synthesize human milk oligosaccharide backbone structures was studied. The human milk oligosaccharide precursor lacto-N-triose II and three regioisomers could be synthesized using the oxazoline, which was either in situ-generated resulting in a chemo-enzymatic sequential cascade or was used as a purified compound. The highest observed concentration of overall transglycosylation products in a cascade reaction was 13.7 mM after 18.5 h, whereas the use of purified oxazoline resulted in 25.0 mM of transglycosylation products after 6.5 h. Remarkably, the in situ-generated oxazoline could be used without any further purification and it was shown that the used enzyme tolerated significant amounts of reagents such as triethylamine, which is reported for the first time for an enzyme from the glycoside hydrolase family 20.

Synthesis of a Di-Mycoloyl Tri-Arabinofuranosyl Glycerol Fragment of the Mycobacterial Cell Wall, Based on Synthetic Mycolic Acids.

Fragments of mycobacterial cell walls such as arabinoglycerol mycolate and dimycoloyl diarabinoglycerol, comprising complex mixtures of mycolic acids, have immunostimulatory and antigenic properties. A related di-mycoloyl tri-arabinofuranosyl glycerol fragment has been isolated from cell wall hydrolysates. An effective stereoselective synthesis of tri-arabinofuranosyl glycerol, followed by coupling with stereochemically defined mycolic acids of different structural classes, to provide unique di-mycoloyl tri-arabinofuranosyl glycerols is now described.

Development of α-Gal-Antibody Conjugates to Increase Immune Response by Recruiting Natural Antibodies.

Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.

Total Synthesis of a Densely Functionalized Plesiomonas shigelloides Serotype 51 Aminoglycoside Trisaccharide Antigen.

Plesiomonas shigelloides, a pathogen responsible for frequent outbreaks of severe travelers' diarrhea, causes grave extraintestinal infections. Sepsis and meningitis due to P. shigelloides are associated with a high mortality rate as antibiotic resistance increases and vaccines are not available. Carbohydrate antigens expressed by pathogens are often structurally unique and are targets for developing vaccines and diagnostics. Here, we report a total synthesis of the highly functionalized trisaccharide repeating unit 2 from P. shigelloides serotype 51 from three monosaccharides. A judicious choice of building blocks and reaction conditions allowed for the four amino groups adorning the sugar rings to be installed with two N-acetyl (Ac) groups, rare acetamidino (Am), and d-3-hydroxybutyryl (Hb) groups. The strategy for the differentiation of amino groups in trisaccharide 2 will serve well for the syntheses of other complex glycans.

4,6- O-Pyruvyl Ketal Modified N-Acetylmannosamine of the Secondary Cell Wall Polysaccharide of Bacillus anthracis Is the Anchoring Residue for Its Surface Layer Proteins.

The secondary cell wall polysaccharide (SCWP) of Bacillus anthracis plays a key role in the organization of the cell envelope of vegetative cells and is intimately involved in host-guest interactions. Genetic studies have indicated that it anchors S-layer and S-layer-associated proteins, which are involved in multiple vital biological functions, to the cell surface of B. anthracis. Phenotypic observations indicate that specific functional groups of the terminal unit of SCWP, including 4,6- O-pyruvyl ketal and acetyl esters, are important for binding of these proteins. These observations are based on genetic manipulations and have not been corroborated by direct binding studies. To address this issue, a synthetic strategy was developed that could provide a range of pyruvylated oligosaccharides derived from B. anthracis SCWP bearing base-labile acetyl esters and free amino groups. The resulting oligosaccharides were used in binding studies with a panel of S-layer and S-layer-associated proteins, which identified structural features of SCWP important for binding. A single pyruvylated ManNAc monosaccharide exhibited strong binding to all proteins, making it a promising structure for S-layer protein manipulation. The acetyl esters and free amine of SCWP did not significantly impact binding, and this observation is contrary to a proposed model in which SCWP acetylation is a prerequisite for association of some but not all S-layer and S-layer-associated proteins.

Loop Protein Engineering for Improved Transglycosylation Activity of a ß-N-Acetylhexosaminidase.

Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of ß-N-acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto-N-triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild-type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG , had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild-type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate-binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG .

Synthesis of Fucosylated Chondroitin Sulfate Glycoclusters: A Robust Route to New Anticoagulant Agents.

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan with excellent anticoagulant activity. Studies show that FuCS and its depolymerized fragments exhibit a different anticoagulant mechanism from that of heparin derivatives, with decreased risks of adverse effects and bleeding. However, further exploitation has been hindered by the scarcity of structurally defined oligosaccharides. Herein, facile method is reported for the synthesis of the repeating trisaccharide unit of FuCS based on the degradation of chondroitin sulfate polymers. A series of simplified FuCS glycomimetics that have highly tunable structures, controllable branches, and defined sulfation motifs were generated by copper-catalyzed alkyne-azide cycloaddition. Remarkable improvement in activated partial thromboplastin time (APTT) assay activities was observed as the branches increased, but no significant influences were observed for prothrombin time (PT) and thrombin time (TT) assay activities. Further FXase inhibition tests suggested that glycoclusters 33 b-40 b selectively inhibited intrinsic anticoagulant activities, but had little effect on the extrinsic and common coagulation pathways. Notably, glycoclusters with the 2,4-di-O-sulfated fucosyl residue displayed the most potency, which was in consistent with that of natural polysaccharides. These FuCS clusters demonstrated potency to mimic linear glycosaminoglycans and offer a new framework for the development of novel anticoagulant agents.

Synthesis and biological evaluation of a trisaccharide repeating unit derivative of Streptococcus pneumoniae 19A capsular polysaccharide.

Streptococcus pneumoniae (SP) is a common human pathogen associated with a broad spectrum of diseases and it is still a leading cause of mortality and morbidity worldwide, especially in children. Moreover, SP is increasingly associated with drug resistance. Vaccination against the pathogen may thus represent an important strategy to overcome its threats to human health. In this context, revealing the molecular determinants of SP immunoreactivity may be relevant for the development of novel molecules with therapeutic perspectives as vaccine components. Serogroup 19 comprises the immune-cross reactive types 19F, 19A, 19B and 19C and it accounts for a high percentage of invasive pneumococcal diseases, mainly caused by serotypes 19F and 19A. Herein, we report the synthesis and biological evaluation of an aminopropyl derivative of the trisaccharide repeating unit of SP 19A. We compare two different synthetic strategies, based on different disconnections between the three monosaccharides which make up the final trisaccharide, to define the best approach for the preparation of the trisaccharide. Synthetic accessibility to the trisaccharide repeating unit lays the basis for the development of more complex biopolymer as well as saccharide conjugates. We also evaluate the binding affinity of the trisaccharide for anti-19A and anti-19F sera and discuss the relationship between the chemical properties of the trisaccharide unit and biological activity.

A novel synthesis of 6''-[18 F]-fluoromaltotriose as a PET tracer for imaging bacterial infection.

The aim of this study was to develop a positron emission tomography (PET) tracer to visualize and monitor therapeutic response to bacterial infections. In our continued efforts to find maltose based PET tracers that can image bacterial infections, we have designed and prepared 6''-[18 F]fluoromaltotriose as a second generation PET imaging tracer targeting the maltodextrin transporter of bacteria. We have developed methods to synthesize 6''-deoxy-6''-[18 F]fluoro-α-D-glucopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-4)-O-D-glucopyranose (6''-[18 F]-fluoromaltotriose) as a bacterial infection PET imaging agent. 6''-[18 F]fluoromaltotriose was prepared from precursor, 2'',3'',4''-tri-O-acetyl-6''-O-nosyl-α-D-glucopyranosyl-(1-4)-O-2',3',6'-tri-O-acetyl-α-D-glucopyranosyl-(1-4)-1,2,3,6-tetra-O-acetyl-D-glucopyranose (per-O-acetyl-6''-O-nosyl-maltotriose 4). This method utilizes the reaction between precursor 4 and anhydrous [18 F]KF/Kryptofix 2.2.2 in dimethylformamide (DMF) at 85°C for 10 minutes to yield per-O-acetyl-6''-deoxy-6-'' [18 F]-fluoromaltotriose (7). Successive acidic and basic hydrolysis of the acetyl protecting groups in 7 produced 6''-[18 F]fluoromaltotriose (8). Also, cold 6''- [19 F]fluoromaltotriose was prepared from per-O-acetyl-6''-hydroxymaltotriose via a diethylaminosulfur trifluoride reaction followed by a basic hydrolysis. A successful synthesis of 6''-[18 F]-fluoromaltotriose has been accomplished in 8 ± 1.2% radiochemical yield (decay corrected). Total synthesis time was 120 minutes. Serum stability of 6''-[18 F]fluoromaltotriose at 37°C indicated that 6''-[18 F]-fluoromaltotriose remained intact up to 2 hours. In conclusion, we have successfully synthesized 6''-[18 F]-fluoromaltotriose via direct fluorination of an appropriate precursor of a protected maltotriose.

Aqueous Glycosylation of Unprotected Sucrose Employing Glycosyl Fluorides in the Presence of Calcium Ion and Trimethylamine.

We report a synthetic glycosylation reaction between sucrosyl acceptors and glycosyl fluoride donors to yield the derived trisaccharides. This reaction proceeds at room temperature in an aqueous solvent mixture. Calcium salts and a tertiary amine base promote the reaction with high site-selectivity for either the 3'-position or 1'-position of the fructofuranoside unit. Because nonenzymatic aqueous oligosaccharide syntheses are underdeveloped, mechanistic studies were carried out in order to identify the origin of the selectivity, which we hypothesized was related to the structure of the hydroxyl group array in sucrose. The solution conformation of various monodeoxysucrose analogs revealed the co-operative nature of the hydroxyl groups in mediating both this aqueous glycosyl bond-forming reaction and the site-selectivity at the same time.

The targeted recognition of Lactococcus lactis phages to their polysaccharide receptors.

Each phage infects a limited number of bacterial strains through highly specific interactions of the receptor-binding protein (RBP) at the tip of phage tail and the receptor at the bacterial surface. Lactococcus lactis is covered with a thin polysaccharide pellicle (hexasaccharide repeating units), which is used by a subgroup of phages as a receptor. Using L. lactis and phage 1358 as a model, we investigated the interaction between the phage RBP and the pellicle hexasaccharide of the host strain. A core trisaccharide (TriS), derived from the pellicle hexasaccharide repeating unit, was chemically synthesised, and the crystal structure of the RBP/TriS complex was determined. This provided unprecedented structural details of RBP/receptor site-specific binding. The complete hexasaccharide repeating unit was modelled and found to aptly fit the extended binding site. The specificity observed in in vivo phage adhesion assays could be interpreted in view of the reported structure. Therefore, by combining synthetic carbohydrate chemistry, X-ray crystallography and phage plaquing assays, we suggest that phage adsorption results from distinct recognition of the RBP towards the core TriS or the remaining residues of the hexasacchride receptor. This study provides a novel insight into the adsorption process of phages targeting saccharides as their receptors.

Detection and quantitative analysis of two independent binding modes of a small ligand responsible for DC-SIGN clustering.

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor (CLR) present, mainly in dendritic cells (DCs), as one of the major pattern recognition receptors (PRRs). This receptor has a relevant role in viral infection processes. Recent approaches aiming to block DC-SIGN have been presented as attractive anti-HIV strategies. DC-SIGN binds mannose or fucose-containing carbohydrates from viral proteins such as the HIV envelope glycoprotein gp120. We have previously demonstrated that multivalent dendrons bearing multiple copies of glycomimetic ligands were able to inhibit DC-SIGN-dependent HIV infection in cervical explant models. Optimization of glycomimetic ligands requires detailed characterization and analysis of their binding modes because they notably influence binding affinities. In a previous study we characterized the binding mode of DC-SIGN with ligand 1, which shows a single binding mode as demonstrated by NMR and X-ray crystallography. In this work we report the binding studies of DC-SIGN with pseudotrisaccharide 2, which has a larger affinity. Their binding was analysed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol and molecular modelling. These studies demonstrate that in solution the complex cannot be explained by a single binding mode. We describe the ensemble of ligand bound modes that best fit the experimental data and explain the higher inhibition values found for ligand 2.

Investigating Glycol-Split-Heparin-Derived Inhibitors of Heparanase: A Study of Synthetic Trisaccharides.

Heparanase is the only known endoglycosidase able to cleave heparan sulfate. Roneparstat and necuparanib, heparanase inhibitors obtained from heparin and currently being tested in man as a potential drugs against cancer, contain in their structure glycol-split uronic acid moieties probably responsible for their strong inhibitory activity. We describe here the total chemical synthesis of the trisaccharide GlcNS6S-GlcA-1,6anGlcNS (1) and its glycol-split (gs) counterpart GlcNS6S-gsGlcA-1,6anGlcNS (2) from glucose. As expected, in a heparanase inhibition assay, compound 2 is one order of magnitude more potent than 1. Using molecular modeling techniques we have created a 3D model of 1 and 2 that has been validated by NOESY NMR experiments. The pure synthetic oligosaccharides have allowed the first in depth study of the conformation of a glycol-split glucuronic acid. Introducing a glycol-split unit in the structure of 1 increases the conformational flexibility and shortens the distance between the two glucosamine motives, thus promoting interaction with heparanase. However, comparing the relative activities of 2 and roneparstat, we can conclude that the glycol-split motive is not the only determinant of the strong inhibitory effect of roneparstat.