Synthetic Study toward Saccharomicin Based upon Asymmetric Metal Catalysis.

Here, we report a de novo metal-catalyzed approach toward the stereoselective glycosidic bond formation in saccharomicin. The signature step is highlighted by the Pd-catalyzed asymmetric coupling of ene-alkoxyallenes and highly functionalized alcohol substrates. The reaction showed high chemo-, regio-, and ligand-driven diastereoselectivity. In combination with the ring-closing metathesis and late-stage functionalization, this method led to highly efficient synthesis of saccharosamine-rhamnose and rhamnose-fucose fragments.

Hexavalent thiofucosides to probe the role of the Aspergillus fumigatus lectin FleA in fungal pathogenicity.

Aspergillus fumigatus is a pathogenic fungus infecting the respiratory system and responsible for a variety of life-threatening lung diseases. A fucose-binding lectin named FleA which has a controversial role in A. fumigatus pathogenesis was recently identified. New chemical probes with high affinity and enzymatic stability are needed to explore the role of FleA in the infection process. In this study, we developed potent FleA antagonists based on optimized and non-hydrolysable thiofucoside ligands. We first synthesized a set of monovalent sugars showing micromolar affinity for FleA by isothermal titration calorimetry. The most potent derivative was co-crystallized with FleA to gain insights into the binding mode in operation. Its chemical multimerization on a cyclodextrin scaffold led to an hexavalent compound with a significantly enhanced binding affinity (Kd = 223 ± 21 nM) thanks to a chelate binding mode. The compound could probe the role of bronchial epithelial cells in a FleA-mediated response to tissue invasion.

An efficient synthetic route to O-(2-O-benzyl-3,4-di-O-acetyl-α/ß-l-fucopyranosyl)-trichloroacetimidate.

An efficient synthetic route to prepare O-(2-O-benzyl-3,4-di-O-acetyl-α/ß-l-fucopyranosyl)-trichloroacetimidate from l-fucose was developed by introducing the thiophenyl group at the anomeric center and the benzylidene functional group to protect the 3 and 4 positions. Although three approaches were considered, the best result was obtained when, after the 2-hydroxyl benzylation, both protective groups were simultaneously removed by using acetic anhydride and perchloric acid supported on silica as catalyst. Selective deacetylation of the obtained tri-O-acetate followed by the reaction of the resultant hemiacetal with trichloroacetonitrile and DBU afforded the trichloroacetimidate with an overall yield of 56% from the l-fucose.

Modified Galacto- or Fuco-Clusters Exploiting the Siderophore Pathway to Inhibit the LecA- or LecB-Associated Virulence of Pseudomonas aeruginosa.

Galacto- and fuco-clusters conjugated with one to three catechol or hydroxamate motifs were synthesised to target LecA and LecB lectins of Pseudomonas aeruginosa (PA) localised in the outer membrane and inside the bacterium. The resulting glycocluster-pseudosiderophore conjugates were evaluated as Trojan horses to cross the outer membrane of PA by iron transport. The data suggest that glycoclusters with catechol moieties are able to hijack the iron transport, whereas those with hydroxamates showed strong nonspecific interactions. Mono- and tricatechol galactoclusters (G1C and G3C) were evaluated as inhibitors of infection by PA in comparison with the free galactocluster (G0). All of them exhibited an inhibitory effect between 46 to 75 % at 100 µM, with a higher potency than G0. This result shows that LecA localised in the outer membrane of PA is involved in the infection mechanism.

Indirect synthetic route to α-l-fucosides via highly stereoselective construction of α-l-galactosides followed by C6-deoxygenation.

We developed an indirect synthetic method for α-l-fucosides. Based on the fact that l-fucose is 6-deoxy-l-galactose, our strategy consists of the stereoselective construction of α-l-galactoside and its conversion to α-l-fucoside via C6-deoxygenation. The formation of α-l-galactoside is strongly directed using 4,6-O-di-tert-butylsilylene(DTBS)-protected l-galactosyl donors. The DTBS-directed α-l-galactosylation showed broad substrate applicability along with excellent coupling yield and α-selectivity. In the C6-deoxygenation of α-l-galactosides, the Barton-McCombie reaction facilitated the conversion to l-fucosides with good yield. To demonstrate the applicability of our method, we synthesized naturally occurring α-l-fucosides.

Recognition of Complex Core-Fucosylated N-Glycans by a Mini Lectin.

The mini fungal lectin PhoSL was recombinantly produced and characterized. Despite a length of only 40 amino acids, PhoSL exclusively recognizes N-glycans with α1,6-linked fucose. Core fucosylation influences the intrinsic properties and bioactivities of mammalian N-glycoproteins and its level is linked to various cancers. Thus, PhoSL serves as a promising tool for glycoprofiling. Without structural precedence, the crystal structure was solved using the zinc anomalous signal, and revealed an interlaced trimer creating a novel protein fold termed ß-prism III. Three biantennary core-fucosylated N-glycan azides of 8 to 12 sugars were cocrystallized with PhoSL. The resulting highly resolved structures gave a detailed view on how the exclusive recognition of α1,6-fucosylated N-glycans by such a small protein occurs. This work also provided a protein consensus motif for the observed specificity as well as a glimpse into N-glycan flexibility upon binding.

Synthesis of α-l-Fucopyranoside-Presenting Glycoclusters and Investigation of Their Interaction with Photorhabdus asymbiotica Lectin (PHL).

Photorhabdus asymbiotica is a gram-negative bacterium that is not only as effective an insect pathogen as other members of the genus, but it also causes serious diseases in humans. The recently identified lectin PHL from P. asymbiotica verifiably modulates an immune response of humans and insects, which supports the idea that the lectin might play an important role in the host-pathogen interaction. Dimeric PHL contains up to seven l-fucose-specific binding sites per monomer, and in order to target multiple binding sites of PHL, α-l-fucoside-containing di-, tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol were chosen as multivalent scaffolds, and the fucoclusters were built from the above-mentioned cores by coupling with different oligoethylene bridges and propargyl α-l-fucosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between fucoside derivates and PHL was investigated by several biophysical and biological methods, ITC and SPR measurements, hemagglutination inhibition assay, and an investigation of bacterial aggregation properties were carried out. Moreover, details of the interaction between PHL and propargyl α-l-fucoside as a monomer unit were revealed using X-ray crystallography. Besides this, the interaction with multivalent compounds was studied by NMR techniques. The newly synthesized multivalent fucoclusters proved to be up to several orders of magnitude better ligands than the natural ligand, l-fucose.

Synthesis and evaluation of galacto-noeurostegine and its 2-deoxy analogue as glycosidase inhibitors.

An epimer of the known glycosidase inhibitor noeurostegine, galacto-noeurostegine, was synthesised in 21 steps from levoglucosan and found to be a potent, competitive and highly selective galactosidase inhibitor of Aspergillus oryzae ß-galactosidase. Galacto-noeurostegine was not found to be an inhibitor of green coffee bean α-galactosidase, yeast α-glucosidase and E. coli ß-galactosidase, whereas potent but non-competitive inhibition against sweet almond ß-glucosidase was established. The 2-deoxy-galacto-noeurostegine analogue was also prepared and found to be a less potent inhibitor of the same enzymes.

L-fucose from vitamin C with only acetonide protection.

Addition of human milk oligosaccharides (HMO) to baby foods may protect infants from disease. As many simple HMOs are fucosylated this is likely to increase the demand for L-fucose as a synthetic building block. Any chemical synthesis must be cheap to compete with a biotechnological process. Acetonide is the only protecting group we have used in this new synthesis of L-fucose from vitamin C in 27% overall yield (purification by recrystallization; no chromatography required in the entire sequence).

Convergent de novo synthesis of vineomycinone B2 methyl ester.

An efficient de novo synthesis of vineomycinone B2 methyl ester has been achieved. The longest linear route required only 14 steps from achiral commercially available starting materials (4.0% overall yield). The key transformations included the de novo asymmetric synthesis of two key fragments, which were joined by a convergent late stage Suzuki's glycosylation for the construction of the aryl ß-C-glycoside. A subsequent BBr3 one-pot debenzylation, demethylation and air oxidation provided vineomycinone B2 methyl ester.

De novo synthesis of the bacterial 2-amino-2,6-dideoxy sugar building blocks D-fucosamine, D-bacillosamine, and D-xylo-6-deoxy-4-ketohexosamine.

The cell-surface glycans on bacteria contain many monosaccharides that cannot be obtained by isolation from natural sources. Availability of differentially protected monosaccharides is therefore often limiting access to potential oligosaccharide vaccine antigens. D-Fucosamine, D-bacillosamine, and D-xylo-2,6-deoxy-4-ketohexosamine building blocks were prepared via a divergent de novo synthesis from L-Garner aldehyde. The route relies on a chelation-control assisted organometallic addition and an anti-selective dihydroxylation reaction.

Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel L-FucNAc and D-FucNAc synthons and immunochemical evaluation.

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-ß-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-ß-D-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile L- and D-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM(197). The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide-CRM(197) conjugate.

Synthesis of homo- and heterofunctionalized glycoclusters and binding to Pseudomonas aeruginosa lectins PA-IL and PA-IIL.

Homo- and heterofunctionalized glycoclusters with galactose and/or fucose residues targeting both PA-IL and PA-IIL lectins of Pseudomonas aeruginosa were synthesized using "Click" chemistry and DNA chemistry. Their binding to lectins (separately or in a mixture) was studied using a DNA Directed Immobilization carbohydrate microarray. Homoglycoclusters bind selectively to their lectin while the heteroglycocluster binds simultaneously both lectins with a slight lower affinity.

Syntheses and NMR characterizations of epimeric 4-deoxy-4-fluoro carbohydrates.

The synthesis and NMR characterizations of 1,2,3,6-tetra-O-benzoyl-4-deoxy-4-fluoro-ß-d-galactopyranoside, the 4-deoxy-4-fluoro epimer of an intermediate in the synthesis of a drug substance, needed for use as a potential impurity standard and to confirm the stereoselectivity of a key fluorination step, are described.

Nucleophilic radiosynthesis of 2-[18F]fluoro-2-deoxy-D-galactose from Talose triflate and biodistribution in a porcine model.

INTRODUCTION: The galactose analogue 2-[(18)F]fluoro-2-deoxy-D-galactose (FDGal) is a promising positron emission tomography (PET) tracer for studies of regional differences in liver metabolic function and for clinical evaluation of patients with liver cirrhosis and patients undergoing treatment of liver diseases. However, there is an unmet need for routine production of FDGal from readily available starting material. In this study, we present the preparation of FDGal with high radiochemical purity and in amounts sufficient for clinical investigations from commercially available Talose triflate (1,3,4,6-tetra-O-acetyl-2-O-trifluoromethanesulfonyl-ß-D-talopyranose). In addition, the biodistribution of FDGal in the pig is presented. METHODS: FDGal was prepared by nucleophilic fluorination of Talose triflate followed by basic hydrolysis. The entire synthesis was performed using the GE TRACERlab MX 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG) synthesizer and existing methods for quality control of FDG were applied. Biodistribution of FDGal was studied by successive whole-body PET recordings of two anaesthetized 37-kg pigs. RESULTS: Up to 3.7 GBq sterile, pyrogen-free and no-carrier-added FDGal was produced with a radiochemical yield of 3.8±1.2% and a radiochemical purity of 98±1% (42 productions; yield is decay corrected). The adopted quality control methods for FDG were directly applicable for FDGal. Biodistribution studies in the pig revealed the liver and the urinary bladder as critical organs in terms of radiation dose. CONCLUSION: Commercially available Talose triflate is a suitable starting material for routine productions of FDGal. The presented radiosynthesis and quality control methods allow for the production of pure, no-carrier-added FDGal in sufficient amounts for clinical PET-investigations of the liver.

The spirocyclopropyl moiety as a methyl surrogate in the structure of l-fucosidase and l-rhamnosidase inhibitors.

Nitrogen-in-the-ring analogues of l-fucose and l-rhamnose were prepared, which feature a spirocyclopropyl moiety in place of the methyl group of the natural sugar. The synthetic route involved a titanium-mediated aminocyclopropanation of a glycononitrile as the key step. Four new spirocyclopropyl iminosugar analogues were generated, which displayed some activity towards l-fucosidase and l-rhamnosidase.

Developing an asymmetric, stereodivergent route to selected 6-deoxy-6-fluoro-hexoses.

Free radical bromination and nucleophilic fluorination allows the conversion of methyl sorbate into the 6-fluoro analogue which undergoes sequential asymmetric dihydroxylation reactions. A range of 6-deoxy-6-fluorosugars were prepared by using different combinations of ligands. While the enantiomeric excesses obtained were comparable to those from other 6-substituted sorbates, the regioselectivity of dihydroxylation was moderate, with both 2,3- and 4,5-diols being obtained. A successful temporary persilylation strategy was evolved to convert the products of dihydroxylation rapidly to the fluorosugars 6-deoxy-6-fluoro-L-idose, 6-fluoro-L-fucose and 6-deoxy-6-fluoro-D-galactose, which were obtained in overall yields of 4%, 6% and 8% from methyl 6-fluoro-hexa-2E,4E-dienoate .

Synthesis and cytotoxicity evaluation of natural alpha-bisabolol beta-D-fucopyranoside and analogues.

alpha-Bisabolol beta-d-fucopyranoside, a cytotoxic naturally occurring compound, was efficiently synthesized along with five other alpha-bisabolol glycosides (beta-d-glucoside, beta-d-galactoside, alpha-d-mannoside, beta-d-xyloside and alpha-l-rhamnoside). Glycosidation of alpha-bisabolol was performed using Schmidt's inverse procedure and provided excellent yields (83-95%). Cytotoxicity was evaluated against a broad panel of cancerous cell lines including human and rat glioma (U-87, U-251 and GL-261) since the anticancer activity of alpha-bisabolol was previously demonstrated against brain tumor cell lines. The addition of a sugar moiety markedly increased alpha-bisabolol cytotoxicity in most cases. Among the synthesized glycosides, alpha-bisabolol alpha-l-rhamnopyranoside exhibited the strongest cytotoxic activity with IC(50) ranging from 40 to 64muM. According to ADME in silico predictions, this glycoside closely respects physicochemical parameters necessary to cross the blood-brain barrier passively.

Synthesis of novel DC-SIGN ligands with an alpha-fucosylamide anchor.

The dendritic cell-specific intercellular adhesion molecule (ICAM) 3-grabbing nonintegrin (DC-SIGN) is a C-type lectin that appears to perform several different functions. Besides mediating adhesion between dendritic cells and T lymphocytes, DC-SIGN recognizes several pathogens some of which, including HIV, appear to exploit it to invade host organisms. The intriguing diversity of the roles attributed to DC-SIGN and their therapeutic implications have stimulated the search for new ligands that could be used as biological probes and possibly as lead compounds for drug development. The natural ligands of DC-SIGN consist of mannose oligosaccharides or fucose-containing Lewis-type determinants. Using the known 3D structure of the Lewis-x trisaccharide, we have identified some monovalent alpha-fucosylamides that bind to DC-SIGN with inhibitory constants 0.4-0.5 mM, as determined by SPR, and have characterized their interaction with the protein by STD NMR spectroscopy. This work establishes for the first time alpha-fucosylamides as functional mimics of chemically and enzymatically unstable alpha-fucosides and describes interesting candidates for the preparation of multivalent systems able to block the receptor DC-SIGN with high affinity and with potential biomedical applications.