Thioarylation of 6-Amino-2,3,6-trideoxy-d-manno-oct-2-ulosonic Acid (IminoKdo): Access to 3,6-Disubstituted Picolinates and Mechanistic Insights.

In this work, we present a metal-free coupling protocol for the regio- and stereoselective C3-thioarylation of 6-amino-2,3,6-trideoxy-d-manno-oct-2-ulosonic acid (iminoKdo). The developed procedure enables the coupling of electron-rich, electron-deficient, and hindered arylthiols, providing a series of C3-modified iminoKdo derivatives in moderate to good yields. Elucidation of active species through controlled experimental studies and time-lapse 31 P NMR analysis provides insights into the reaction mechanism. We demonstrate that, following a tandem Staudinger/aza-Wittig reaction of an azido-containing keto ester, an inseparable equimolar mixture of imine/enamine is formed. The enamine then undergoes a Stork-like nucleophilic attack with the in situ-formed disulfide reagent, resulting in the formation of the coupling products. Additionally, we describe a rarely reported acid-promoted aromatization of the C3-thioarylated iminoKdo skeleton into 3,6-disubstituted picolinates, which are reminiscent of dichotomines.

Proton- versus Cation-Selective Transport of Saccharide Rim-Appended Pillar[5]arene Artificial Water Channels.

Transport of water across cell membranes is a fundamental process for important biological functions. Herein, we focused our research on a new type of symmetrical saccharide rim-functionalized pillar[5]arene (PA-S) artificial water channels with variable pore structures. To point out the versatility of PA-S channels, we systematically varied the nature of anchoring/gate keepers d-mannoside, d-mannuronic acid, or sialic acid H-bonding groups on lateral pillar[5]arene (PA) arms, known as good membrane adhesives, to best describe the influence of the chemical structure on their transport activity. The control of hydrophobic membrane binding-hydrophilic water binding balance is an important feature influencing the channels' structuration and efficiency for a proper insertion into bilayer membranes. The glycosylated PA channels' transport performances were assessed in lipid bilayer membranes, and the channels were able to transport water at high rates (∼106-107 waters/s/channel within 1 order of magnitude as for aquaporins), serving as selective proton railways with total Na+ and K+ rejection. Molecular simulation substantiates the idea that the PAs can generate supramolecular pores, featuring hydrophilic carbohydrate gate-keepers that serve as water-sponge relays at the channel entrance, effectively absorbing and redirecting water within the channel. The present channels may be regarded as a rare biomimetic example of artificial channels presenting proton vs cation transport selectivity performances.

Synthesis and evaluation of inhibitors of Mycobacterium tuberculosis UGM using bioisosteric replacement.

There is a dearth of tuberculosis (TB) drug development activity as current therapeutic treatments are inadequate due to the appearance of drug-resistant TB. The enzyme UDP-galactopyranose mutase (UGM) is involved in the biosynthesis of galactan which is essential for cell wall integrity and bacterial viability. Its inhibition has thus been featured as profitable strategy for anti-TB drug discovery. In this study, we report on the synthesis of amides derived from rosmarinic acid, their inhibitory effect towards purified UGM using three distinct biochemical assays: FP, HPLC and SPR. The rosmarinic amides generally showed a significantly higher affinity for UGM than the corresponding rosmarinic ester. In particular, compound 5h displayed interesting binding affinity values (Kd = 58 ± 7, 63 ± 9 µM towards KpUGM and MtUGM respectively). Furthermore, a new UGM SPR assay was established and confirmed that 5h binds to UGM with a dissociation constant of 104.8 ± 6.5 µM. Collectively, this study validates the amide bioisosteric strategy which has been successfully implemented to develop UGM inhibitors from rosmarinic acid, providing a substantial basis for further design of novel UGM inhibitors and anti-mycobacterial agents.

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan.

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-ß-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal ß-(1,5) and ß-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

Identification of inhibitors of UDP-galactopyranose mutase via combinatorial in situ screening.

An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.

Synthesis of a biotinylated heptose 1,7-bisphosphate analogue, a probe to study immunity and inflammation.

d-glycero-d-manno-Heptose-1ß,7-bisphosphate (HBP) is a bacterial metabolite that can induce a TIFA-dependent innate immune response in mammals. It was recently discovered that after HBP enters into the cytoplasm of the host cell, it is transformed into ADP-heptose-7-phosphate, which then leads to ALPK1-TIFA-dependent inflammatory response. In order to provide a molecular tool allowing the discovery of the proteins involved in this novel inflammatory pathway, we designed and synthesized a biotinylated analogue of HBP. This chemical probe displays an anomeric ß-phosphate and a phosphonate at the 7-position, and a d-configured 6-position to which is attached the biotin moiety. To do so, different synthetic strategies were explored and described in this report. Moreover, we demonstrated that the biotinylated version of HBP is still biologically active and can activate the NF-κB pathway in HEK293T cells.

Synthesis and biological evaluation of 3,4-dihydro-1H-[1,4] oxazepino [6,5,4-hi] indol-1-ones and 4,6-dihydrooxepino [5,4,3-cd] indol-1(3H)-ones as Mycobacterium tuberculosis inhibitors.

This study focuses on the synthesis of 1,7- and 3,4-indole-fused lactones via a simple and efficient reaction sequence. The functionalization of these "oxazepino-indole" and "oxepino-indole" tricycles is carried out by palladium catalysed CC coupling, nucleophilic substitution or 1,3-dipolar cycloaddition. The evaluation of their activity against Mycobacterium tuberculosis shows that the "oxazepino-indole" structure is a new inhibitor of M. tuberculosis growth in vitro.

Fluorinated carbohydrates as chemical probes for molecular recognition studies. Current status and perspectives.

This review provides an extensive summary of the effects of carbohydrate fluorination with regard to changes in physical, chemical and biological properties with respect to regular saccharides. The specific structural, conformational, stability, reactivity and interaction features of fluorinated sugars are described, as well as their applications as probes and in chemical biology.

Dynamic Constitutional Frameworks as Antibacterial and Antibiofilm Agents.

Dynamic constitutional frameworks (DCFs) were synthesized and screened for biofilm inhibition or disruption. They are composed of a trialdehyde core reversibly linked to a diamine PEG connector and to a variety of neutral, anionic, or cationic heads, to generate a library of DCFs to generate multivalent dendritic architectures in the presence of Pseudomonas aeruginosa and Staphylococcus aureus. The best DCFs were always polycationic and the nature of the cationic heads significantly impact the antibiofilm activity. The best antibiofilm activity was observed for DCF3B, displaying a polyethyleneimine head. A simple inactive guanidinium functional head strongly inhibited biofilm growth when assayed as a multivalent DCF3C. Using a more advanced in vitro biofilm model of chronic wound infection, DCF3C was found significantly superior than all other DCFs. These results demonstrate the versatility and effectiveness of DCFs as low cost and efficient systems for antibiofilm disruption.

Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling.

d-Glycero-d-manno-heptose-1ß,7-bisphosphate (HBP) and d-glycero-d-manno-heptose-1ß-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated in vivo at a low-nanomolar concentration (6 nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.

Synthesis and evaluation of heterocycle structures as potential inhibitors of Mycobacterium tuberculosis UGM.

In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis. In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an "oxazepino-indole" structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro.

Direct Evaluation of Live Uropathogenic Escherichia coli Adhesion and Efficiency of Antiadhesive Compounds Using a Simple Microarray Approach.

Many pathogens use host glycans as docking points for adhesion. Therefore, the use of compounds blocking carbohydrate-binding adhesins is a promising strategy for fighting infections. In this work, we describe a simple and rapid microarray approach for assessing the bacterial adhesion and efficiency of antiadhesive compounds targeting uropathogenic Escherichia coli UTI89, which displays mannose-specific adhesin FimH at the tip of fimbriae. The approach consisted in direct detection of live fluorescently labeled bacteria bound to mannan printed onto microarray slides. The utility of the arrays for binding/inhibition assays was first validated by comparing array-derived results for the model mannose-binding lectin concanavalin A with data obtained by isothermal titration calorimetry. Growth phase-dependent binding of UTI89 to the arrays was observed, proving the usefulness of the setup for detecting differences in FimH expression. Importantly, bacteria labeling and binding assays entailed minimal manipulation, helping to preserve the integrity of fimbriae. The efficiency of three different dodecamannosylated fullerenes as FimH-targeted antiadhesives was next evaluated in competition assays. The results revealed a superior activity of the mannofullerenes (5- to 18-fold per mannose residue) over methyl α-d-mannopyranoside. Moreover, differences in activity were detected for mannofullerenes differing in the structure/length of the spacer used for grafting mannose onto the fullerene core, further demonstrating the sensitivity of the assay. Overall, the approach combines straightforward and time-saving protocols for microarray preparation, bacteria labeling, and binding assays, and it can be easily tailored to other bacteria bearing carbohydrate-binding adhesins.

Pyruvate-Kinase-Coupled Glycosyltransferase Assays: Limitations, Struggles and Problem Resolution.

Enzyme assays involving coupled pyruvate kinase (PK) have been used for many years to monitor the activity of major classes of enzymes including glycosyltransferases. Numerous potent inhibitors have been discovered and kinetically characterized thanks to this technology. However, when inhibitors of these important enzymes are screened, PK inhibitors or activators are very often observed. In this study we report solutions to resolve the problems encountered either during the screening or during the kinetic characterization of glycosyltransferase inhibitors by means of PK-coupled assays. The enzyme under study-WaaC-is an important glycosyltransferase involved in the bacterial lipopolysaccharide (LPS) biosynthesis pathway. Firstly we showed that alternative kinases such as nucleoside 5-diphosphate kinase (NDPK), myokinase (MK), and ADPdependent hexokinase that catalyze similar reactions to PK are prone to the same troubles. Moreover, an ADP chemosensor was used as an alternative but the sensitivity was not sufficient to allow a proper screening. Finally, we found that a stepwise PK/luciferase assay resolved the problems encountered with PK inhibitors and that a WaaC HPLC assay allowed the identification of WaaC inhibitors acting as PK activators, thus allowing false positive and false negative results linked to the coupling to PK to be eliminated.

Natural and Synthetic Flavonoids as Potent Mycobacterium tuberculosis UGM Inhibitors.

This study reports a novel class of inhibitors of uridine 5'-diphosphate (UDP) galactopyranose mutase (UGM) derived from a screening of natural products. This enzyme is an essential biocatalyst involved in the cell wall biosynthesis of Mycobacterium tuberculosis. Flavonoids are potent inhibitors of UGM. The synthesis of novel methylated flavonoids allowed a structure-activity relationship analysis to be performed and which functional groups and structural elements were required for UGM inhibition could be determined. The binding mode of one of the best inhibitors was found to be noncompetitive. Docking simulations indicated that this molecule was likely to bind UGM in its open conformation, in a cavity recently identified as a "druggable" pocket. Importantly, two of the best inhibitors of the M. tuberculosis UGM displayed moderate activity against whole M. tuberculosis cells. This study reports the first natural products that act as inhibitor of UGM. Given the importance of natural products in medicinal chemistry, these results create new opportunities for the discovery of new antitubercular agents.

Force Nanoscopy as a Versatile Platform for Quantifying the Activity of Antiadhesion Compounds Targeting Bacterial Pathogens.

The development of bacterial strains that are resistant to multiple antibiotics has urged the need for new antibacterial therapies. An exciting approach to fight bacterial diseases is the use of antiadhesive agents capable to block the adhesion of the pathogens to host tissues, the first step of infection. We report the use of a novel atomic force microscopy (AFM) platform for quantifying the activity of antiadhesion compounds directly on living bacteria, thus without labeling or purification. Novel fullerene-based mannoconjugates bearing 10 carbohydrate ligands and a thiol bond were efficiently prepared. The thiol functionality could be exploited as a convenient handle to graft the multimeric species onto AFM tips. Using a combination of single-molecule and single-cell AFM assays, we demonstrate that, unlike mannosidic monomers, multivalent glycofullerenes strongly block the adhesion of uropathogenic Escherichia coli bacteria to their carbohydrate receptors. We expect that the nanoscopy technique developed here will help designing new antiadhesion drugs to treat microbial infections, including those caused by multidrug resistant organisms.

Debenzylative Cycloetherification: An Overlooked Key Strategy for Complex Tetrahydrofuran Synthesis.

Tetrahydrofuran (THF) is a major structural feature found in many synthetic and natural products displaying a variety of biological properties. This review summarizes the main synthetic approaches that have been developed to construct tetrahydrofuran moieties involving debenzylative cycloetherification reactions (DBCE). Interestingly, this reaction is regio- and stereoselective without the requirement of a selective protection/deprotection strategy. Many applications of this process have been reported, including carbafuranoside synthesis, regioselective deprotection of the benzyl group positioned γ to an alkene, and total synthesis of natural products. The stereochemical outcome and the mechanism of these interesting transformations are also discussed.

Biologically Active Heteroglycoclusters Constructed on a Pillar[5]arene-Containing [2]Rotaxane Scaffold.

A synthetic approach combining recent concepts for the preparation of multifunctional nanomolecules (click chemistry on multifunctional scaffolds) with supramolecular chemistry (self-assembly to prepare rotaxanes) gave easy access to a large variety of sophisticated [2]rotaxane heteroglycoclusters. Specifically, compounds combining galactose and fucose have been prepared to target the two bacterial lectins (LecA and LecB) from the opportunistic pathogen Pseudomonas aeruginosa.

Mechanistic Insight into Heptosyltransferase Inhibition by using Kdo Multivalent Glycoclusters.

The synthesis of unprecedented multimeric Kdo glycoclusters based on fullerene and calix[4]arene central scaffolds is reported. The compounds were used to study the mechanism and scope of multivalent glycosyltransferase inhibition. Multimeric mannosides based on porphyrin and pillar[5]arenes were also generated in a controlled manner. Twelve glycoclusters and their monomeric ligands were thus assayed against heptosyltransferase WaaC, which is an important bacterial glycosyltransferase that is involved in lipopolysaccharide biosynthesis. It was first found that all the multimers interact solely with the acceptor binding site of the enzyme even when the multimeric ligands mimic the heptose donor. Second, the novel Kdo glycofullerenes displayed very potent inhibition (Ki =0.14 µm for the best inhibitor); an inhibition level rarely observed with glycosyltransferases. Although the observed "multivalent effects" (i.e., the enhancement of affinity of a ligand when presented in a multimeric fashion) were in general modest, a dramatic effect of the central scaffold on the inhibition level was evidenced: the fullerene and the porphyrin scaffolds being by far superior to the calix- and pillar-arenes. We could also show, by dynamic light scattering analysis, that the best inhibitor had the propensity to form aggregates with the heptosyltransferase. This aggregative property may contribute to the global multivalent enzyme inhibition, but probably do not constitute the main origin of inhibition.

Potent Glycosidase Inhibition with Heterovalent Fullerenes: Unveiling the Binding Modes Triggering Multivalent Inhibition.

Glycosidases are key enzymes in metabolism, pathogenic/antipathogenic mechanisms and normal cellular functions. Recently, a novel approach for glycosidase inhibition that conveys multivalent glycomimetic conjugates has emerged. Many questions regarding the mechanism(s) of multivalent enzyme inhibition remain unanswered. Herein we report the synthesis of a collection of novel homo- and heterovalent glyco(mimetic)-fullerenes purposely conceived for probing the contribution of non-catalytic pockets in glysosidases to the multivalent inhibitory effect. Their affinities towards selected glycosidases were compared with data from homovalent fullerene conjugates. An original competitive glycosidase-lectin binding assay demonstrated that the multivalent derivatives and the substrate compete for low affinity non-glycone binding sites of the enzyme, leading to inhibition by a "recognition and blockage" mechanism. Most notably, this work provides evidence for enzyme inhibition by multivalent glycosystems, which will likely have a strong impact in the glycosciences given the utmost relevance of multivalency in Nature.

Pillar[5]arene-Based Glycoclusters: Synthesis and Multivalent Binding to Pathogenic Bacterial Lectins.

The synthesis of pillar[5]arene-based glycoclusters has been readily achieved by CuAAC conjugations of azido- and alkyne-functionalized precursors. The lectin binding properties of the resulting glycosylated multivalent ligands have been studied by at least two complementary techniques to provide a good understanding. Three lectins were selected from bacterial pathogens based on their potential therapeutic applications as anti-adhesives, namely LecA and LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria. As a general trend, multivalency improved the binding to lectins and a higher affinity can be obtained by increasing to a certain limit the length of the spacer arm between the carbohydrate subunits and the central macrocyclic core.