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.

Glycofullerene-AuNPs as multivalent ligands of DC-SIGN and bacterial lectin FimH: tuning nanoparticle size and ligand density.

Glycoclusters have been extensively investigated for their inhibition of multivalent carbohydrate-protein interactions, which is often the first step for bacterial and viral pathogens to selectively bind their host cells. Glycoclusters may thus prevent infections by blocking the microbe attachment onto the host cell surface. The potency of multivalent carbohydrate-protein interactions is largely derived from the spatial arrangement of the ligand and the nature and flexibility of the linker. The size of the glycocluster may also have a dramatic impact on the multivalent effect. The main objective of this study is to provide a systematic comparison of gold nanoparticles of three representative sizes and ligand densities at their surface. Therefore, AuNPs with diameters of 20, 60, and 100 nm were coupled either to a monomeric D-mannoside or a decameric glycofullerene. Lectin DC-SIGN and lectin FimH were selected as representative models of viral and bacterial infections, respectively. We also report the synthesis of a hetero-cluster built from 20 nm AuNPs and a mannose-derived glycofullerene and monomeric fucosides. All final glycoAuNPs were evaluated as ligands of DC-SIGN- and FimH using the GlycoDiag LectProfile technology. This investigation revealed that the 20 nm AuNPs bearing glycofullerenes with short linker are the most potent binders of both DC-SIGN and FimH. Moreover, the hetero-glycoAuNPs showed an enhanced selectivity and inhibitory ability towards DC-SIGN. Hemagglutination inhibition assays using uropathogenic E. coli corroborated the in vitro assays. Overall, these results showed smaller glycofullerene-AuNPs (20 nm) exhibited the best potential as anti-adhesive materials for a variety of bacterial and viral pathogens.

Evaluation of Azido 3-Deoxy-d-manno-oct-2-ulosonic Acid (Kdo) Analogues for Click Chemistry-Mediated Metabolic Labeling of Myxococcus xanthus DZ2 Lipopolysaccharide.

Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus-a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence of distinct azido (-N3) analogues of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N3-Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy-d-arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N3-Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide-alkyne cycloaddition (SPAAC) "click" chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N3-Kdo and 7-N3-Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N3-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N3-Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N3-Kdo is indeed a substrate of the enzyme, 7-N3-Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N3-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N3-Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.

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.

Synthesis of Spirocyclic Cyclopropyl Glycosyl-1-phosphate Analogues.

A general methodology allowing the preparation of phosphonylated 1-spirocyclopropyl analogues of glycosyl-1-phosphates is reported. The scope of this reaction has been assessed using various exo-glycals easily obtained from the corresponding pyranoses and furanoses. The cyclopropanation was found to be stereospecific, and the cis/trans selectivity only depends on the E/Z configuration of the starting exo-glycal. The four possible isomers of spirocyclopropyl ribose-1-phosphonate could thus be prepared in a controlled manner, protected and deprotected.

Multivalent 9-O-Acetylated-sialic acid glycoclusters as potent inhibitors for SARS-CoV-2 infection.

The recent emergence of highly transmissible SARS-CoV-2 variants illustrates the urgent need to better understand the molecular details of the virus binding to its host cell and to develop anti-viral strategies. While many studies focused on the role of the angiotensin-converting enzyme 2 receptor in the infection, others suggest the important role of cell attachment factors such as glycans. Here, we use atomic force microscopy to study these early binding events with the focus on the role of sialic acids (SA). We show that SARS-CoV-2 binds specifically to 9-O-acetylated-SA with a moderate affinity, supporting its role as an attachment factor during virus landing to cell host surfaces. For therapeutic purposes and based on this finding, we have designed novel blocking molecules with various topologies and carrying a controlled number of SA residues, enhancing affinity through a multivalent effect. Inhibition assays show that the AcSA-derived glycoclusters are potent inhibitors of cell binding and infectivity, offering new perspectives in the treatment of SARS-CoV-2 infection.

Pillar[5]arene-Based Polycationic Glyco[2]rotaxanes Designed as Pseudomonas aeruginosa Antibiofilm Agents.

Pseudomonas aeruginosa (P.A.) is a human pathogen belonging to the top priorities for the discovery of new therapeutic solutions. Its propensity to generate biofilms strongly complicates the treatments required to cure P.A. infections. Herein, we describe the synthesis of a series of novel rotaxanes composed of a central galactosylated pillar[5]arene, a tetrafucosylated dendron, and a tetraguanidinium subunit. Besides the high affinity of the final glycorotaxanes for the two P.A. lectins LecA and LecB, potent inhibition levels of biofilm growth were evidenced, showing that their three subunits work synergistically. An antibiofilm assay using a double ΔlecAΔlecB mutant compared to the wild type demonstrated that the antibiofilm activity of the best glycorotaxane is lectin-mediated. Such antibiofilm potency had rarely been reached in the literature. Importantly, none of the final rotaxanes was bactericidal, showing that their antibiofilm activity does not depend on bacteria killing, which is a rare feature for antibiofilm agents.

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.

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.

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.

A General Strategy to Synthesize ADP-7-Azido-heptose and ADP-Azido-mannoses and Their Heptosyltransferase Binding Properties.

The multistep synthesis of a novel ADP-7-azido-7-deoxy-l-glycero-ß-d-manno-heptopyranoside 2a and several analogues as heptosyltransferase ligands is described. The synthesis of the key intermediate heptoside-1-ß-phosphate 3a involved a ß-stereoselective phosphorylation of lactol 4 employing diallyl chlorophosphate as a phosphorylating reagent. Five deprotected nucleotide sugars were generated by this synthetic sequence and evaluated as heptosyltransferase substrates (KM, kcat).

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 functionalized copillar[4+1]arenes and rotaxane as heteromultivalent scaffolds.

In this study, novel copillar[4+1]arenes were used as central heteromultivalent scaffolds via orthogonal couplings with a series of biologically relevant molecules such as carbohydrates, α-amino acids, biotin and phenylboronic acid. Further modifications by introducing maleimides or cyclooctyne groups provided molecular probes adapted to copper-free click chemistry. An octa-azidated fluorescent rotaxane bearing two distinct ligands was also generated in a fully controlled manner.

Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor.

Study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. The novel coronavirus SARS-CoV-2 entry into host cells is mediated by its spike glycoprotein (S-glycoprotein), and the angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which the S-glycoprotein binds to the ACE2 receptor. We demonstrate, both on model surfaces and on living cells, that the receptor binding domain (RBD) serves as the binding interface within the S-glycoprotein with the ACE2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. Altogether, these results provide a picture of the established interaction on living cells. Finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the SARS-CoV-2 infection.

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.

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.

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.