Rational Design and Synthesis of Methyl-ß-d-galactomalonyl Phenyl Esters as Potent Galectin-8N Antagonists.

Galectin-8 is a ß-galactoside-recognizing protein having an important role in the regulation of bone remodeling and cancer progression and metastasis. Methyl ß-d-galactopyranoside malonyl aromatic esters have been designed to target and engage with particular amino acid residues of the galectin-8N extended carbohydrate-binding site. The chemically synthesized compounds had in vitro binding affinity toward galectin-8N in the range of 5-33 µM, as evaluated by isothermal titration calorimetry. This affinity directly correlated with the compounds' ability to inhibit galectin-8-induced expression of chemokines and proinflammatory cytokines in the SUM159 breast cancer cell line. X-ray crystallographic structure determination revealed that these monosaccharide-based compounds bind galectin-8N by engaging its unique arginine (Arg59) and simultaneously cross-linking to another arginine (Arg45) located across the carbohydrate-binding site. This structure-based drug design approach has led to the discovery of novel monosaccharide galactose-based antagonists, with the strongest-binding compound (Kd 5.72 µM) holding 7-fold tighter than the disaccharide lactose.

Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

Stereoselective Phenylselenoglycosylation of Glycals Bearing a Fused Carbonate Moiety toward the Synthesis of 2-Deoxy-ß-galactosides and ß-Mannosides.

A phenylselenoglycosylation reaction of glycal derivatives mediated by diphenyl diselenide and phenyliodine(III) bis(trifluoroacetate) under mild conditions is described. Stereoselective glycosylation has been achieved by installing fused carbonate on those glycals. 3,4-O-Carbonate galactals and 2,3-O-carbonate 2-hydroxyglucals are converted into corresponding glycosides in good yields with excellent ß-selectivity, resulting in 2-phenylseleno-2-deoxy-ß-galactosides and 2-phenylseleno-ß-mannosides which are good precursors of 2-deoxy-ß-galactosides and ß-mannosides, respectively.

Prophylactic Antiviral Activity of Sulfated Glycomimetic Oligomers and Polymers.

In this work, we investigate the potential of highly sulfated synthetic glycomimetics to act as inhibitors of viral binding/infection. Our results indicate that both long-chain glycopolymers and short-chain glycooligomers are capable of preventing viral infection. Notably, glycopolymers efficiently inhibit Human Papillomavirus (HPV16) infection in vitro and maintain their antiviral activity in vivo, while the glycooligomers exert their inhibitory function post attachment of viruses to cells. Moreover, when we tested the potential for broader activity against several other human pathogenic viruses, we observed broad-spectrum antiviral activity of these compounds beyond our initial assumptions. While the compounds tested displayed a range of antiviral efficacies, viruses with rather diverse glycan specificities such as Herpes Simplex Virus (HSV), Influenza A Virus (IAV), and Merkel Cell Polyomavirus (MCPyV) could be targeted. This opens new opportunities to develop broadly active glycomimetic inhibitors of viral entry and infection.

6-Deoxy-6-fluoro galactofuranosides: regioselective glycosylation, unexpected reactivity, and anti-leishmanial activity.

Selective glycosylation of the C-6 fluorinated galactofuranosyl acceptor 2 was studied with four galactofuranosyl donors. It was highlighted that this electron-withdrawing atom strongly impacted the behavior of the acceptor, thus leading to unprecedented glycosylation pathways. Competition between expected glycosylation of 2, ring expansion of this acceptor and furanosylation, and intermolecular aglycon transfer was observed. Further investigation of the fluorinated synthetic compounds showed that the presence of fluorine atom contributed to increase the inhibition of the growth of Leishmania tarentolae, a non-pathogenic strain of Leishmania.

Discrimination between Human Colorectal Neoplasms with a Dual-Recognitive Two-Photon Probe.

Colorectal cancer is a major cause of cancer-related deaths worldwide. Histologic diagnosis using biopsy samples of colorectal neoplasms is the most important step in determining the treatment methods, but these methods have limitations in accuracy and effectiveness. Herein, we report a dual-recognition two-photon probe and its application in the discrimination between human colorectal neoplasms. The probe is composed of two monosaccharides, d-glucosamine and ß-d-galactopyranoside, in a fluorophore for the monitoring of both glucose uptake and ß-gal hydrolysis. In vitro/cell imaging studies revealed the excellent selectivity and sensitivity of the probe for glucose transporter-mediated glucose uptake and ß-gal activity. Cancer-specific uptake was monitored by increased fluorescence intensity, and additional screening of cancer cells was achieved by changes in emission ratio owing to the higher activity of ß-gal. Using human colon tissues and two-photon microscopy, we found that the plot of intensity versus ratio can accurately discriminate between colorectal neoplasms in the order of cancer progression (normal, adenoma, and carcinoma).

Perfluorophenylboronic acid-catalyzed direct α-stereoselective synthesis of 2-deoxygalactosides from deactivated peracetylated d-galactal.

Perfluorophenylboronic acid 1c catalyzes the direct stereoselective addition of alcohol nucleophiles to deactivated peracetylated d-galactal to give 2-deoxygalactosides in 55-88% yield with complete α-selectivity. The unprecedented results reported here also enable the synthesis of disaccharides containing the 2-deoxygalactose moiety directly from the deactivated peracetylated d-galactal. This convenient and metal-free glycosylation method works well with a wide range of alcohol nucleophiles as acceptors and tolerates a range of functional groups without the formation of the Ferrier byproduct and without the need for a large excess of nucleophiles or additives. The method is potentially useful for the synthesis of a variety of α-2-deoxygalactosides.

Novel hydrophilic galactose-conjugated chlorin e6 derivatives for photodynamic therapy and fluorescence imaging.

We synthesized new hydrophilic chlorin e6 derivatives with two and four galactose fragments conjugated to the macrocycle via carbon atom in position 6 of the galactose fragment. Galactose fragments were inserted by alkylation of the amino groups of chlorin e6 amides with one and two ethylene diamine fragments on the macrocycle periphery with triflate of diacetone galactose, followed by removal of diisopropylidene protection by 70% aqueous trifluoroacetic acid. The synthesized compounds were shown to be capable of penetrating the membrane of HeLa cells; they have intense red fluorescence inside the cell and have phototoxic properties towards HeLa cells (upon LED irradiation at 660 nm and light exposure value of 12 J/cm2). These properties, along with water solubility, allow us to consider the synthesized compounds to be promising as potential antitumor PSs and diagnostic compounds for visualizing malignant tumors and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases.

Tn antigen analogues: the synthetic way to "upgrade" an attracting tumour associated carbohydrate antigen (TACA).

In the last two decades, the paramount importance of Tumor Associated Carbohydrate Antigens (TACAs) as targets for anticancer vaccine development has been firmly assessed. The Tn antigen is an ideal target for immunotherapy, in that it is masked on normal cells, but exposed on cancer cells. However, it is difficult to elicit an effective and long-lasting response against Tn antigen and other TACAs. Here we report on the Tn antigen analogues developed to boost the latent Tn immune response. Hopefully, the results reported herein will be of help for the rational design of effective TACA-based immunostimulants.

Targeting Bacterial Biofilm: A New LecA Multivalent Ligand with Inhibitory Activity.

Biofilm formation by bacterial pathogens is a hallmark of chronic infections and is associated to increased antibiotic tolerance that makes pathogens difficult to eradicate with conventional antibiotic therapies. Infections caused by Pseudomonas aeruginosa are of great concern, especially for immunocompromised and cystic fibrosis patients. P. aeruginosa lectins LecA and LecB are virulence factors and play a key role in establishing biofilm; therefore, inhibition of the function of these proteins has potential in dismantling the bacterium from the protective biofilm environment and in restoring the activity of antibiotics. Here, we report the NMR characterization of the binding of a galactose-based dendrimer (Gal18) to LecA. Moreover, we demonstrate the activity of the Gal18 molecule in inhibiting P. aeruginosa biofilm formation in vitro.

Structure-Based Design of a Monosaccharide Ligand Targeting Galectin-8.

Galectin-8 is a ß-galactoside-recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3-O-[1-carboxyethyl]-ß-d-galactopyranoside (compound 6) as a ligand to target the N-terminal domain of galectin-8 (galectin-8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3'-O-sialylated lactose (3'-SiaLac), with galectin-8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin-8N for 6 was 32.8 µm, whereas no significant affinity was detected for the C-terminal domain of galectin-8 (galectin-8C). The crystal structure of the galectin-8N-6 complex validated the predicted binding conformation and revealed the exact protein-ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin-8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide-based scaffold as a binder of galectin-8.

Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection.

Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone-usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29ß-NAc, is a biphenyl N-acetyl-ß-galactosaminoside with a Ki of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate-lectin interactions. 29ß-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29ß-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29ß-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29ß-NAc-inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

Synthesis, kinetics and inhibition of Escherichia coli Heptosyltransferase I by monosaccharide analogues of Lipid A.

Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.

Development of Pseudomonas aeruginosa Lectin LecA Inhibitor by using Bivalent Galactosides Supported on Polyproline Peptide Scaffolds.

LecA is a galactose-binding tetrameric lectin from Pseudomonas aeruginosa involved in infection and biofilm formation. The emergent antibiotic resistance of P. aeruginosa has made LecA a promising pharmaceutical target to treat such infections. To develop LecA inhibitors, we exploit the unique helical structure of polyproline peptides to create a scaffold that controls the galactoside positions to fit their binding sites on LecA. With a modular scaffold design, both the galactoside ligands and the inter-ligand distance can be altered conveniently. We prepared scaffolds with spacings of 9, 18, 27, and 36 Šfor ligand conjugation and found that glycopeptides with galactosides ligands three helical turns (27 Å) apart best fit LecA. In addition, we tested different galactose derivatives on the selected scaffold (27 Å) to improve the binding avidity to LecA. The results validate a new multivalent scaffold design and provide useful information for LecA inhibitor development.

GSH-Responsive supramolecular nanoparticles constructed by ß-d-galactose-modified pillar[5]arene and camptothecin prodrug for targeted anticancer drug delivery.

Supramolecular construction of a targeted and stimuli-responsive drug delivery system is still a challenging task. Herein, GSH-responsive supramolecular prodrug nanoparticles were constructed by the host-guest complexation between a ß-d-galactose-functionalized water-soluble pillar[5]arene (GalP5) and a disulfide bond containing camptothecin prodrug (G). The obtained prodrug nanoparticles were stable under physiological conditions, whereas efficient drug release was triggered in a simulated tumor environment with high GSH concentration. In vitro studies revealed that these prodrug nanoparticles preferentially entered asialoglycoprotein receptor-overexpressing HepG2 cells due to the active targeting effect of galactose units. This active targeting effect resulted in the maximization of anticancer efficacy and reduction of the undesirable side effects to normal cells.

Synthesis and biological properties of galactofuranosyl-containing fluorescent dyes.

Two fluorescent galactofuranosides were synthesized and their biological activities evaluated on non-infected and Leishmania infected macrophages. Both tagged scaffolds were able to penetrate macrophages. Compared to the activity of the parent octyl galactofuranoside used as a reference, the fluorescein-conjugate showed altered biological properties while the rhodamine 6G one synergistically acted with the lipid chain to significantly increase antiparasitic activity.

A Selective Galactose-Coumarin-Derived Galectin-3 Inhibitor Demonstrates Involvement of Galectin-3-glycan Interactions in a Pulmonary Fibrosis Model.

Synthesis of doubly 3-O-coumarylmethyl-substituted thiodigalactosides from bis-3-O-propargyl-thiodigalactoside resulted in highly selective and high affinity galectin-3 inhibitors. Mutant studies, structural analysis, and molecular modeling revealed that the coumaryl substituents stack onto arginine side chains. One inhibitor displayed efficacy in a murine model of bleomycin-induced lung fibrosis similar to that of a known nonselective galectin-1/galectin-3 inhibitor, which strongly suggests that blocking galectin-3 glycan recognition is an important antifibrotic drug target.

Structural Insights into Solid-to-Solid Phase Transition and Modulated Crystal Formation in Octyl-ß-d-Galactoside Crystals.

Despite the significance of synthetic monotailed ß-linked galactolipids, for a detailed understanding of natural galactolipids, many aspects of these ß-linked galactolipids' crystal structures such as temperature-dependence and hydration characteristics remain inadequately understood. In this manuscript, we demonstrated detailed insight of crystal characteristics of one of the simplest monotailed galactolipids, octyl-ß-d-galactoside (MOß-Gal), using thermal analyses, Fourier-transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD) measurements and grazing-incidence wide-angle X-ray diffraction (GI-WAXD) analysis. As a result, it was revealed that the MOß-Gal anhydrous crystal showed a continuous structural change from the high-symmetry structure to low-symmetry crystal lattice via the strengthened hydrogen bonding interaction as the temperature decreased. In addition, the hemihydrate crystal was found to be in the modulated "ribbon phase". These insights strongly suggest that ß-linked galactolipids possess intrinsic characteristics necessary to form a modulated structure even in the crystal state and demonstrate the importance of the presence of tiny amounts of water as cushioning media for preventing order parameter evolution.

Synthesis of the copper chelator TGTA and evaluation of its ability to protect biomolecules from copper induced degradation during copper catalyzed azide-alkyne bioconjugation reactions.

One of the most successful bioconjugation strategies to date is the copper(I)-catalyzed cycloaddition reaction (CuAAC), however, the typically applied reaction conditions have been found to degrade sensitive biomolecules. Herein, we present a water soluble copper chelator which can be utilized to protect biomolecules from copper induced degradation.

Synthesis and immunodetection of 6-O-methyl-phosphoramidyl-α-D-galactose: a Campylobacter jejuni antigenic determinant.

Campylobacter jejuni is a leading cause of traveler's diarrhea. Previously, we have shown that a C. jejuni capsule polysaccharide (CPS) conjugate vaccine can fully prevent C.jejuni diarrhea in non-human primates. C.jejuni CPSs are decorated with non-stoichiometric amounts of O-methyl phosphoramidate (MeOPN) units that are key serospecific markers. In the case of C.jejuni serotype complex HS23/36, the MeOPN are at positions 2 and 6 of the CPS galactose (Gal). We describe here the synthesis of the p-methoxyphenyl glycoside of MeOPN→6-α-D-Galp, and its immunodetection by antisera raised by C.jejuni CPS conjugates with MeOPN at primary positions. The synthetic approach in this work served as the foundation for a similar MeOPN→6-Gal construct used in a conjugate vaccine, whose synthesis, immunogenicity and efficacy will be described elsewhere.