Alexandre Bruneau

Emilie Gillon

Aurélie Furiga

Etienne Brachet

Mouad Alami

Christine Roques

Annabelle Varrot

Anne Imberty
email: anne.imberty@cermav.cnrs.fr

Samir Messaoudi
email: samir.messaoudi@universite-paris-saclay.fr

Discovery of potent 1,1-diarylthiogalactoside glycomimetic inhibitors of Pseudomonas aeruginosa LecA with antibiofilm properties

Keywords: thiogalactoside, Pseudomonas aeruginosa, antibiofilm activity, structure-activity relationship. Graphical Abstr act Highlights

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen causing severe infections of the respiratory tract particularly in immune-suppressed patients. In addition, P. aeruginosa induces chronic infections in the lungs of patients with cystic fibrosis, damaging the lung epithelium by strains presenting high intrinsic or acquired resistance to antibiotics as loss of susceptibility when under biofilms and resulting in morbidity and mortality for young adults [START_REF] Govan | Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia[END_REF] P. aeruginosa adhesion to epithelial cells involves several proteins able to bind to the glycans at the surface of human tissues and, including two soluble ones: the galactose specific lectin LecA (PA-IL) and the fucose specific lectin LecB (PA-IIL). [START_REF] Gilboa-Garber | Pseudomonas aeruginosa lectins[END_REF][START_REF] Imberty | Structures of the lectins from Pseudomonas aeruginosa: Insights into molecular basis for host glycan recognition[END_REF] Experimental studies on mice and humans demonstrated that monosaccharides and more specifically galactose prevent lung colonization by P. aeruginosa. [START_REF] Bucior | Sugar administration is an effective adjunctive therapy in the treatment of Pseudomonas aeruginosa pneumonia[END_REF][START_REF] Chemani | Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands[END_REF][START_REF] Hauber | Inhalation with fucose and galactose for treatment of Pseudomonas aeruginosa in cystic fibrosis patients[END_REF] Therefore, LecA appears as a target of interest and the development of high affinity glycomimetics as an attractive strategy to treat such infections. [START_REF] Diggle | The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa[END_REF] Furthermore, LecA is also involved in biofilm formation which makes it a relevant target in the context of antibiotic resistance of P. aeruginosa. [START_REF] Diggle | The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa[END_REF] Galactosederivatives targeting this protein were demonstrated to disrupt biofilm formation and restore antibiotic sensitivity. [START_REF] Bergmann | Multivalency effects on Pseudomonas aeruginosa biofilm inhibition and dispersal by glycopeptide dendrimers targeting lectin LecA[END_REF][START_REF] Flockton | Inhibition of Pseudomonas aeruginosa Biofilm Formation with Surface Modified Polymeric Nanoparticles[END_REF][START_REF] Meiers | Directing Drugs to Bugs: Antibiotic-Carbohydrate Conjugates Targeting Biofilm-Associated Lectins of Pseudomonas aeruginosa[END_REF] LecA is a homotetramer with four galactose binding sites, each containing a calcium ion coordinated by both the carbohydrate and the surrounding amino acids (Figure 1A). [START_REF] Cioci | Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa[END_REF] Because of the spatial organization of the four galactose binding sites, the lectin binds with high avidity to natural glycolipids on cell surfaces. [START_REF] Blanchard | Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I (PA-IL)[END_REF] This avidity can be exploited for the design of multivalent glycocompounds that reach high affinity for the lectin. [START_REF] Cecioni | Glycomimetics versus multivalent glycoconjugates for the design of high affinity lectin ligands[END_REF] Although the multivalent galactosides showed a stronger affinity (nanomolar range) against LecA than monovalent analogues, their complex structures and high molecular weight result in lack of druglikeness and poor pharmacokinetic properties that are main drawbacks for reaching clinical trials. Divalent compounds able to cross-link two galactose binding sites on the surface of LecA, were demonstrated to present sufficient avidity to reach efficiency in the nanomolar range (20-80 nM) (Figure 1B). [START_REF] Novoa | Novel LecA ligand identified from glycan array inhibits host cell invasion by Pseudomonas aeruginosa[END_REF][START_REF] Pertici | Optimizing divalent inhibitors of Pseudomonas aeruginosa lectin LecA by using a rigid spacer[END_REF] However, small monovalent ligands such as galactosides bearing an aromatic aglycon are considered to be more drug-like molecules with favorable bioavailability to reach the targeted infected tissues. As highlighted in the Figure 1B, a variety of monovalent aryl-β-O-galactosides were reported to be more active ligands against LecA than galactose.

Libraries were synthetized and tested with net gain in affinity for phenyl (Kd 8.8 µM) and nitrophenyl (PNP-β-Gal; Kd 14 µM). [START_REF] Kadam | A glycopeptide dendrimer inhibitor of the galactosespecific lectin LecA and of Pseudomonas aeruginosa biofilms[END_REF][START_REF] Kadam | CH-pi "T-shape" interaction with histidine explains binding of aromatic galactosides to Pseudomonas aeruginosa lectin LecA[END_REF] The increase in affinity is rationalized by the occurrence of an hydrophobic pocket adjacent to the galactose binding site on LecA surface but also of an unusual T-stacking interaction between the ring of the aglycone and the one of His50. [START_REF] Kadam | CH-pi "T-shape" interaction with histidine explains binding of aromatic galactosides to Pseudomonas aeruginosa lectin LecA[END_REF] The most affine monovalent ligand to date is a phenyl-galactoside extended by the tripeptide Lys-Pro-Leu (GalAG0) from Reymond group (Kd 3 μM). [START_REF] Visini | Structural Insight into Multivalent Galactoside Binding to Pseudomonas aeruginosa Lectin LecA[END_REF] In parallel, Roy et al. reported a novel series of aryl 1thioglycosides, which are more resistant to the metabolic hydrolysis under biological conditions, as a promising and innovative series of glycomimetics with high affinity for LecA. [START_REF] Rodrigue | Aromatic thioglycoside inhibitors against the virulence factor LecA from Pseudomonas aeruginosa[END_REF] Naphtyl thioglycoside was identified as a good candidate with a Kd of 6 μM. In addition, the X-ray crystal analysis of the LecA/thionaphtyl-β-Gal complex revealed that the naphtyl ring plays a pivotal role in the binding pocket by adapting two different conformations in sites A/D and B/C of the tetrameric LecA protein. This difference in the naphthyl group orientation was attributed to changes in both Φ and Ψ torsion angles at the S-glycosidic linkage. Then, the aromatic ring was much closer to the protein surface in protomer B than in protomer A. These advanced achievements created an increased interest to discover new small glycomimetic thioglycosides.

In order to emulate this approach, we sought to explore a new chemical space around the hydrophobic ring with the purpose to identify unique structures with better affinity and antibiofilm activities. In this work, we report the synthesis of a novel series of monovalent and divalent aryl thioglycosides as LecA inhibitors (Figure 2). Their (i) affinity towards LecA, (ii) biofilm inhibition against P. aeruginosa as well as (iii) cytotoxicity against MRC-5 cell line were also presented. Notably, the glycomimetics developed here were potent inhibitors of biofilm formation in stark contrast to the ineffective native carbohydrates.

Results and discussion

Chemistry

We recently reported an efficient Pd-catalyzed method allowing the introduction of glycosyl thiols to various iodo(hetero)aryls [START_REF] Brachet | Stereoselective Palladium-Catalyzed Alkenylation and Alkynylation of Thioglycosides[END_REF] and alkenyl/alkynyl halides. [START_REF] Brachet | Palladium-Catalyzed Cross-Coupling Reaction of Thioglycosides with (Hetero) aryl Halides[END_REF] This method is based on the use of Pd(OAc) 2 as the catalyst, combined with XantPhos as the ligand and Et 3 N as the base in THF or dioxane at 100 °C temperature. Moreover, a variant of this method implying the use of the palladium G3-Xanthphosbiphenyl precatalyst under mild reaction conditions (rt and aqueous solvent) was developed to functionalize challenging substrates with thiosugars [START_REF] Bruneau | Stereoretentive palladium-catalyzed arylation, alkenylation, and alkynylation of 1-thiosugars and thiols using aminobiphenyl palladacycle precatalyst at room temperature[END_REF] including nucleic acids [START_REF] Probst | Efficient Buchwald-Hartwig-Migita Cross-Coupling for DNA Thioglycoconjugation[END_REF] and unprotected long peptides. [START_REF] Montoir | Synthesis of aryl-thioglycopeptides through chemoselective Pd-mediated conjugation[END_REF] With these conditions on hand, we first designed a short series of three phenyl β-S-galactopyranoside 4a-c in the aim to evaluate the substitution of the aromatic ring on the affinity toward LecA. Thus, analogues bearing methoxyl, isopropyl and benzaldehyde groups at the para position of the phenyl ring were prepared in good yields through a sequence of Pd-catalyzed coupling of the iodinated starting materials 1a-c with the tetra-O-acetylated 1-thio-β-D-galactopyranose 2 followed by the Zamplen deprotection of the acetate groups (Scheme 1).

Interestingly, compound 4b which may be considered as a direct aromatic glycomimetic of isopropyl β-D-1thiogalactopyranoside (IPTG) will be a good indicator of the effect of introducing the S-aromatic linkage of IPTG toward the affinity for the lectin (Scheme 1).

Then, in another part of this study, we focused our synthesis on the 1,1 biaryl thiogalactosides 8a-e and 9a (Scheme 1).

We speculated that the presence of two phenyl nucleus linked by Csp 2 carbone bridge could provide strong π-π stacking interactions with residues of the binding pocket and will allow us to understand the role played by hydrophobic aglycones in binding to LecA.

The synthesis of the second series of our thiogalactosides starts from the preparation of the iodinated diphenyl substrates 6a-f through a Pd-catalyzed Barluenga-Valdes cross-coupling between aryl iodides 1a-c and the corresponding tosyl hydrazones 5a-b. [START_REF] Roche | Palladium-Catalyzed One-Pot Reaction of Hydrazones, Dihaloarenes, and Organoboron Reagents: Synthesis and Cytotoxic Activity of 1,1-Diarylethylene Derivatives[END_REF] Then, the coupling of the tetra-O-acetylated 1-thio-β-D-galactopyranose 2 with biaryls 6a-f furnished the desired acetylated galactoside intermediates 7a-f in yields ranging from 63% to 96%. Simple deprotection of the acetate groups by action of sodium methoxide in methanol led to the desired compounds 8a-e and 9a in a quantitative yield (Scheme 1).

Finally, to examine the flexibility versus rigidity of ethylene bridge of 8b, the reduction of this later was performed by hydrogenation with Pd/C. Accordingly, the compound 8f was obtained quantitatively as a mixture of the two diastreosiomers (Scheme 1). Scheme 1. Synthetic route toward thiogalactoside analogues 3a-c, 8a-f and 9a.

Interactions with LecA target 2.2.1. Determination of affinity towards LecA and thermodynamic parameters

The affinity of generated thiogalactosides has been assessed in solution by isothermal titration calorimetry (ITC), a bioanalytical technique for the study of ligand-receptor interactions that generates thermodynamic information on the ligand binding process. Typical thermograms of LecA interacting with the four best ligands are given in Figure 2 and all others are available in Supplementary Information. Titration of LecA by monovalent ligands resulted in exothermic peaks and saturation of 50% of protein occurred with a stoichiometry close to n = 1, indicating the expected binding interaction.

As expected, the divalent compound 9a displays a stoichiometry ligand/protein close to 0.5, indicating that each of the galactose residues is bound to a LecA protomer which is confirmed by aggregation observed in ITC tube.

Table 1 reports the affinity values for all compounds. For monovalent galactosides, the values vary between 1 and 12 µM with higher affinity obtained for compounds 4b, 8a and 8c with Kd lower than 3 μM. This is 30-fold better than the affinity observed for galactose and also stronger than the best affinity reported previously for best thio-galactoside ligands for LecA displayed in Figure 1. [START_REF] Rodrigue | Aromatic thioglycoside inhibitors against the virulence factor LecA from Pseudomonas aeruginosa[END_REF] Interestingly, the affinity of the isopropyl-phenyl-S-galactoside 4b (Kd = 2.45 μM) is 10-time higher than the corresponding non-aromatic IPTG (Kd = 32 μM). [START_REF] Kadam | A glycopeptide dendrimer inhibitor of the galactosespecific lectin LecA and of Pseudomonas aeruginosa biofilms[END_REF] This result demonstrates clearly the gain obtained with S-aryl galactoside ligands compared to S-alkyl analogues. Importantly, the series bearing biaryl aglycones (compounds 8a-f) showed a promising activity particularly with the benzophenone monovalent galactoside 8a with a Kd of 1 μM. To our knowledge, this is the strongest reported affinity for a monovalent galactoside towards LecA. The structure-activity relationship study (SAR) showed that replacing the carbonyl bridge in 8a by a double bond (compound 8b) lead to a lower affinity toward the lectin with a Kd of 6.9 μM. In addition, reducing the double bond into a methyl group (compound 8e) decreased the affinity (Kd = 9 μM). This result suggests a role for the oxygen atom of the carbonyl bridge and indicates that an optimal positioning of the two aromatics is crucial for the binding to LecA.

It is worth noticing that since benzophenone scaffolds are often used as photoactivatable probes, [START_REF] Hassan | Recent Advances in Chemical Biology Using Benzophenones and Diazirines as Radical Precursors[END_REF][START_REF] Murale | Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs)[END_REF][START_REF] Lee | Protein-DNA photo-crosslinking with a genetically encoded benzophenonecontaining amino acid[END_REF][START_REF] Ballard | Investigating γ-secretase protein interactions in live cells using active site-directed clickable dual-photoaffinity probes[END_REF] our hit compound 8a could potentially display photoactivatable properties. It could therefore be envisaged that the irradiation of 8a at 330-365 nm would generate radicals able to react with the protein target by photo-crosslinking.However, during our characterization, particularly during -crystallization experiments and subsequent analysis of crystal structure, we did not observe any photo-crosslinking arising from the alkylation of the benzophenone scaffold to the lectin protein.

The analysis of the thermodynamic contribution demonstrated that the interaction is driven by enthalpy of binding, which is opposed by an entropy barrier, as often observed in protein/glycocompoundinteractions. [START_REF] Dam | Thermodynamic studies of lectin-carbohydrate interactions by isothermal titration calorimetry[END_REF] The compounds that reach the stronger enthalpy, 4b, 8a and 8c, are the ones with higher affinity, indicating that creation of additional contacts around the galactose binding sites is the appropriate strategy to obtain efficient ligands. Compound 8a singles out by its stronger enthalpy and also by a heat integration curve that did not fit perfectly with one site model. Together with baseline noise around saturation of 50% of protein, usually associated with aggregation, this indicates additional interactions, probably with neighboring tetramers in solution (Figure 2). The divalent galactoside 9a displayed an excellent affinity toward LecA with a Kd = 160 nM (Figure 2). In addition, the stoichiometry value of 0.44 indicates that the two thiosugar moieties interact with LecA. Considering the short size of the linker and the precipitation observed during titration, compound 9a does not bind to two adjacent galactose binding sites on one LecA tetramer but rather cross-link two tetramers.

Co-crystal structures of LecA with compounds 8a and 8c

Co-rystallization experiments were conducted with 8a, 8c and 9a ligand with the protein either preincubated with the ligand or deposited on the coverslip with dried ligand on it (dry-soaked method). Co-crystals were obtained for LecA with compounds 8a and 8c only, diffracting to 1.75 and 1.53 Å, respectively. The content of unit cell was different with one lectin tetramer and four ligands for the complex with 8a and one lectin dimer with only one ligand for the complex with 8c (Figure 3 A and B). This difference resulted from the ligand-induced packing. For 8a, stacking contacts are observed between the aryl rings of two ligands in facing tetramer as frequently observed for LecA in complex with aromatic galacto compounds, while for 8c, the distal aryl moiety contacts the binding site of neighbouring LecA, preventing the binding site to be occupied. Such contacts occur only during crystallization and are generally not relevant for analysing the affinity of LecA in solution, although some signs of aggregation were observed during titration of LecA by 8a.

Analysis of contacts established by compound 8c in the binding site reveals the classical contacts for galactose, including calcium coordination by oxygen O3 and O4, direct hydrogen bonds with the protein and water mediated hydrogen bonds for O6 (Figure 3B). The aglycone folds on the protein surface, creating hydrophobic contacts with CH2 of His50 and Pro51 with the proximal and distal benzyl groups, respectively. The rings of His50 and of the ligand are almost perpendicular, corresponding to an unusual type of hydrophobic interaction, described as T-shape and previously observed with other aromatic aglycones for LecA ligands. [START_REF] Kadam | CH-pi "T-shape" interaction with histidine explains binding of aromatic galactosides to Pseudomonas aeruginosa lectin LecA[END_REF] The extensive hydrophobic interactions between the aglycone of 8c and LecA surface provides the structural basis of its excellent affinity (Kd = 3 μM). The compound 8a, which displays the highest affinity for LecA (Kd = 1 μM) compared to the other monovalent ligands, adopts different conformations for its aglycone in the four binding sites, while the galactose always maintains the classical binding mode described above. In protomers A and C, the conformations are very similar, with the two aryl rings adopting an overall shape similar to the one observed for compound 8c thus, creating same hydrophobic contacts with His50 and Pro51 (Figure 3A). In addition, the methoxy group on the proximal benzyl creates hydrophobic contact with Pro38 on the other wall of the binding site. Interestingly, the C=O group between the two benzyl rings establishes a direct hydrogen bond with the side chain of Gln53, that is already interacting with the O6 hydroxyl of the galactose residue. A water bridge is also observed between C=0 and this side chain. These additional hydrogen bonds are in agreement with the more favourable enthalpy of binding measured for compound 8a. The value of ΔH is 50% higher than for homolog 8b that differs only by C=C replacing C=O. The strong enthalpy is partially compensated by increase of entropy barrier but still results in higher affinity. Compound 8a adopts a different conformation in the binding site of chain D and is partially disordered in chain B, resulting in electron density visible only for proximal aryl group (SI, Figure S5 A andC). The resulting extended conformation of the aglycone moiety in chain D displays only limited contact with the protein surface, with methoxy group of first benzyl contacting the CH2 of His50. This illustrates the flexibility of the thio-linkage that allows for different conformations, here, probably induced by the crystal packing.

Since the origin for high affinity of compounds 8a is related to the creation of a hydrogen bond with Gln53, we searched for similar contacts in LecA complexes available in Unilectin3D, the database of crystal structures of lectins. [START_REF] Bonnardel | UniLectin3D, a database of carbohydrate binding proteins with curated information on 3D structures and interacting ligands[END_REF] Synthetic compounds from Winssinger and Titz groups do present a C=O bond linker on the benzyl ring but in para position compared to galactose that is therefore too far from Gln53 for creating a hydrogen bond. [START_REF] Novoa | Novel LecA ligand identified from glycan array inhibits host cell invasion by Pseudomonas aeruginosa[END_REF][START_REF] Siebs | Targeting the Central Pocket of the Pseudomonas aeruginosa lectin LecA[END_REF] On the opposite, the natural ligand, isoGb3 trisaccharide (αGal1-3βGal1-4Glc) demonstrates two hydrogen bonds between the βGal residue and Gln53. [START_REF] Blanchard | Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I (PA-IL)[END_REF] Furthermore, compound 8a adopts a shape very similar to this trisaccharide in the binding site of LecA (Figure 4). The target of LecA in human is proposed to be Gb3, a sphingolipid present on human tissues, and presenting the αGal1-4βGal1-4Glc epitope. No crystal structure is available but a modelling study also proposed that O2

and O3 of the second galactose interact with Gln53 side chain through hydrogen bonds. Values in parentheses correspond to high resolution shell † 5 % of the reflections were set aside for a R free test before initiating any refinement

In vitro cytotoxicity and antibiofilm activity 2.3.a. Antibiofilm activity

Microbial biofilms are becoming increasingly difficult to treat in the medical setting due to their intrinsic resistance to antibiotics. In order to study the potential antibiofilm activity of the most active ligands 8a and 9a, their effect on the inhibition of the biofilm formation was evaluated in a P. aeruginosa assay (Figure 5). Solution with 3 % methanol was considered for control. At first, the effect of monovalent galactoside 8a was investigated with concentrations of 0.08 to 5 mM. Compound 8a showed a dose-response effect on the bacterial biofilm formation with 100 % of inhibition at 5 mM (log reduction > 4) (Figure 5).

The antibiofilm activity with the divalent glycomimetic 9a was also examined. The concentration of 5 mM was fixed first to compare the effect of divalent 9a with the monovalent 8a which showed a strong effect (100 % biofilm inhibition) at this concentration. Surprisingly, at 5 mM, 9a showed a slightly lower reduction of biofilm formation with approximatively 90% inhibition (1 log reduction compared to the 3 % methanol control) (Figure 5). This result is not in agreement with the high affinity observed with 9a (Kd = 0.16 µM) against LecA and at this stage no any obvious correlation between the affinity toward the lectin LecA and the antibiofilm activity could be established. A possible explanation of this result would be a potential aggregating effect displayed by the compound 9a. Several scenari can be envisage for lack of antibiofilm effect of cross-linking compounds, such as the formation of microbial microcolonies that would for the bacteria.

Of note, since the biofilm activity of 9a is less than 8a at 5 mM, the study of its effect at lesser concentrations are not warranted.

Figure 5: Antibiofilm activity of compounds 8a and 9a

b. In vitro cytotoxicity

The toxicity of ligands 8a and 9a against MRC-5 cell line (Human Fetal Lung Fibroblast Cells) was evaluated after a 24 h of contact (Figure 6). In such conditions, no significant loss of cell viability was observed for the higher methanol concentration (3%) and 8a and 9a compounds for concentrations less than or equal to 1.25 mM or 2.5 mM respectively. At the higher concentration tested (5 mM) the cell viability versus control was 7.1% and 5.2% for respectively compounds 8a and 9a. Then diluted 2 fold (2.5 mM) cell viability increased to 41.1% for 8a and 65.1% for 9a to be above 50% for both compounds at the concentration of 1.25mM (61.5% and 91.4% respectively). These results indicate that these two hit compounds display a cytotoxic effect at concentration higher than 1.25 mM. It can be noted that at non-toxic concentration of 1.25 mM (Figure 5), the best compound identified 8a displays approximatively 40% of biofilm inhibition.

Figure 6: % of viability of MRC-5 cells after 24h of contact with compounds 8a and 9a (mean ± SD).

Finally, to exclude any unwanted antibiotic effect on biofilm formation, the MICs (the concentration at which no bacterial growth was visible after 24 h of incubation at 37°C) and MBCs (minimal bactericidal concentration; the concentration at which the number of CFU was reduced by 99.9%) of compounds 8a and 9a against P. aeruginosa PAO1 were recorded as previously described. [START_REF] Trognon | Investigation of Direct and Retro Chromone-2-Carboxamides Based Analogs of Pseudomonas aeruginosa Quorum Sensing Signal as New Anti-Biofilm Agents[END_REF] The MICs and MBCs were higher than 5 mM (the highest concentration tested) for both compounds which demonstrated clearly the absence of classical antibacterial effect (on planktonic cells) for the tested compounds.

Conclusion

In the context of the global threat of antimicrobial resistance, the development of strategies to support or complement antibiotics is crucial. [START_REF] Bassetti | Antimicrobial resistance in the next 30 years, humankind, bugs and drugs: a visionary approach[END_REF] Strategies for developing compounds that would fight infection without or with limited risk inducing resistance include anti-adhesion and anti-biofilm therapy. Glycomimetics that are able to compete with attachment site on tissues and/or biofilm formation have been used to target pathogens. [START_REF] Meiers | Directing Drugs to Bugs: Antibiotic-Carbohydrate Conjugates Targeting Biofilm-Associated Lectins of Pseudomonas aeruginosa[END_REF][START_REF] Blanchard | Secondary sugar binding site identified for LecA lectin from Pseudomonas aeruginosa[END_REF][START_REF] Pertici | Potent divalent inhibitors with rigid glucose click spacers for Pseudomonas aeruginosa lectin LecA[END_REF] Different designs have been proposed, from large multivalent glycodendrimers [START_REF] Bernardi | Multivalent glycoconjugates as antipathogenic agents[END_REF] to non-carbohydrate glycomimetics [START_REF] Kuhaudomlarp | Non-carbohydrate glycomimetics as inhibitors of calcium(II)-binding lectins[END_REF], but the simpler monosaccharide derivatives, as the ones described here, present the advantage of small size, fine specificity through the monosaccharide moiety and reasonable pharmacokinetic and physicochemical properties. Furthermore, the use of thioglycoside linkage insures the metabolic stability.

In summary, we synthetized a series of thio-galactosides compounds directed towards LecA from P. aeruginosa with the aim of increasing the chemical diversity around the aglycone phenyl group, with substituents in meta and ortho positions that were not explored previously. The affinities of a variety of mono-aryl and bi-aryl derivatives were analyzed by ITC as well as a divalent compound. This SAR investigation demonstrated that the bi-aryl derivative with a carbonyl linker on position meta and methoxy on position ortho results in the affinity value that was not previously reached for monovalent ligand of LecA. The crystal structure of the complex rationalized this observation by confirming the establishment of new ligand-protein contacts. It also uncovered the strong similarity with the contact established with natural oligosaccharides in LecA binding site, demonstrating that this hit compound is an excellent mimetic of the natural ligand. This hit compound demonstrated dose-dependent inhibition of biofilm formation, with no antibacterial effect on Solvents and reagents are obtained from commercial suppliers and were used without further purification. Analytical TLC was performed using Merck silica gel F254 (230-400 mesh) plates and analyzed by UV light or by staining upon heating with vanilin solution. For silica gel chromatography, the flash chromatography technique was used, with Merck silica gel 60 (230-400 mesh) and p.a. grade solvents unless otherwise noted. The 1 H NMR and 13 C NMR spectra were recorded in either CDCl 3 , MeOD, or DMSO-d6 on Bruker Avance 300 or 400 spectrometers. The chemical shifts of 1 H and 13 C are reported in ppm relative to the solvent residual peaks. IR spectra were measured on a Perkin Elmer spectrophotometer. High resolution mass spectra (HR-MS) were recorded on a MicroMass LCT Premier Spectrometer. Thiosugars were synthesized as according the following protocols. [START_REF] Floyd | Thiyl Glycosylation of Olefinic Proteins: S-Linked Glycoconjugate Synthesis[END_REF][START_REF] Gamblin | Glyco-SeS: Selenenylsulfide-Mediated Protein Glycoconjugation-A New Strategy in Post-Translational Modification[END_REF] The Xantphos palladium precatalyst third generation was synthesized according to literature protocol. [

Synthesis of the intermediates 7a-g General procedure A for Pd-Catalyzed coupling of thiogalactose with iodoarenes.

A flame-dried resealable Schlenk tube was charged with Pd(OAc) 2 (3 mol%), Xantphos (1.5 mol%), thiogalactose 2 (0.375mmol), aryl halide 6a-f (0.25 mmol), and Et 3 N (0.25 mmol). The Schlenk tube was capped with a rubber septum, evacuated and backfilled with argon; then, dioxane (1.5 mL) was added through the septum. The septum was replaced with a teflon screwcap. The Schlenk tube was sealed, and the mixture was stirred at 100 °C for 1 h. The resulting suspension was cooled to room temperature and filtered through celite eluting with ethyl acetate. The filtrate was concentrated and purification of the residue by silica gel column chromatography gave the desired products.

General procedure B for Pd-G3 XantPhos Catalyzed coupling of thiogalactose with iodoarenes at room temperature.

A round bottom flask is charged with Xantphos PdG 3 precatalyst (1 mol%), thiogalactose 2 (1 equiv.), halide compound (1 equiv.). After Argon flushing, THF (0.25M) is added. Upon stirring the reaction mixture, NEt 3 (1 equiv.) is added to the medium. The reaction mixture was stirred at room temperature under Argon. After completion and solvent evaporation, the residue is purified by silica gel column chromatography, unless otherwise noted, to give the desired product.

(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-{[2-methoxy-5- (3,4,5-trimethoxybenzoyl)phenyl]thio}tetrahydro2H-pyran-3,4,5triyl triacetate 7a [START_REF] Brachet | Palladium-Catalyzed Cross-Coupling Reaction of Thioglycosides with (Hetero) aryl Halides[END_REF] Compound 7a was prepared from the iodophenstatine in 97% yield by using the general method A. White-yellow solid; R f = 0.32 (cyclohexane/ AcOEt, 9:1mp 108-1108C;

(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)thio)tetrahydro-2Hpyran-3,4,5-triyl triacetate 7b

Compound 7b was prepared from the diodide 6b in 80% yield by using the general method A. White solid; R f = 0.22 (Cyclohexane/EtOAc 75/25); 63% after column chromatography (Cyclohexane / EtOAc 75/25); IR (neat): 2970, 2900, 1753, 1647, 1604, 1512, 1368, 1268, 1222, 1084, 1057 cm -1 . m.p.=69.6-71.4°C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.56 (d, J = 2.2 Hz, 1H), 7.22 (dd, J = 8.5, 2.2 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 6.52 (s, 2H), 5.40 (d, J = 3.7 Hz, 2H), 5.33 (s, 1H), 5.27 (t, J = 10.0 Hz, 1H), 5.06 (dd, J = 9.9, 3.4 Hz, 1H), 4.83 (d, J = 10.0 Hz, 1H), 4.06 (dd, J = 6.7, 3.1 Hz, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.80 (s, 6H), 2.10 (s, 3H), 2.05 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H). 13

(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((4-(1-(4-methoxyphenyl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate 7c

Compound 7e was prepared from the diodide 6e in 94% yield by using the general method B . White solid; R f = 0.14 (Cyclohexane/EtOAc 8/2); after column chromatography (Cyclohexane / EtOAc 8/2); IR (neat): 2921, 2849, 1747, 1650, 1603, 1511, 1367, 1210, 1083, 1057 cm -1 . m.p.=51.1-53.0°C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.46 (d, J = 8.4 Hz, 2H), 7.33 -7.20 (m, 4H), 6.86 (d, J = 8.8 Hz, 2H), 5.43 (dd, J = 3.3, 0.8 Hz, 1H), 5.40 (d, J = 1.1 Hz, 1H), 5.36 (d, J = 1.1 Hz, 1H), 5.27 (t, J = 10.0 Hz, 1H), 5.06 (dd, J = 9.9, 3.4 Hz, 1H), 4.74 (d, J = 10.0 Hz, 1H), 4.15 (d, J = 13.4 Hz, 2H), 3.96 (t, J = 6.6 Hz, 1H), 3.82 (s, 3H), 2.13 (s, 3H), 2.10 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H). 13

(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((3-(1-(4-methoxyphenyl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate 7d

Compound 7d was prepared from the diodide 6d in 80% yield by using the general method A. Yellow solid; R f = 0.27 (Cyclohexane/EtOAc 7/3); 75% after column chromatography (Cyclohexane / EtOAc 8/2); IR (neat): 2936, 1744, 1603, 1511, 1368, 1212, 1177, 1083, 1056 cm -1 . m.p.=48.3-50.2°C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.49 (s, 1H), 7.48 -7.41 (m, 1H), 7.34 -7.21 (m, 4H), 6.92 -6.83 (m, 2H), 5.42 (d, J = 1.2 Hz, 2H), 5.38 (d, J = 1.1 Hz, 1H), 5.25 (t, J = 10.0 Hz, 1H), 5.05 (dd, J = 10.0, 3.4 Hz, 1H), 4.73 (d, J = 10.0 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.90 (t, J = 6.2 Hz, 1H), 3.82 (s, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H). 13

(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)ethyl)phenyl)thio)tetrahydro-2Hpyran-3,4,5-triyl triacetate 7g

Compound 7g (reduction of the double bond of 7b) was prepared from the galactoside 7b in a quantitative yield : A round bottom flask was charged with Pd/C (10 weight%) and the acetylated thiogalactoside 7a (1 equiv. 1,5 mmol). MeOH (3mL) was then added. The reaction mixture was stirred at room temperature under hydrogen atmosphere (1atm) for 96h. After completion and solvent evaporation, the residue is purified by silica gel column chromatography to give the desired product. Light brown solid; R f = 0.14 (Cyclohexane/EtOAc 7/3); Quant.; IR (neat): 3391, 2961, 1752, 1590, 1511, 1419, 1369, 1220, 1128, 1084, 1058 cm -1 . m.p.=66.7-67.9°C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.40 (dd, J = 4.6, 2.3 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.41 (s, 2H), 5.43 -5.39 (m, 1H), 5.26 (td, J = 9.9, 7.0 Hz, 1H), 5.08 (dd, J = 3.4, 1.4 Hz, 1H), 4.81 (dd, J = 10.0, 1.6 Hz, 1H), 4.09 (d, J = 6.7 Hz, 2H), 4.06 -3.96 (m, 1H), 3.92 -3.85 (m, 1H), 3.84 (s, 3H), 3.83 -3.78 (m, 9H), 2.10 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H), 1.60 (d, J = 7.2 Hz, 3H). 13

General procedure for the synthesis of unprotected glycosides 8a-f and 9a

To the corresponding acetylated glycoside under argon was added a solution of sodium methoxide [sodium in MeOH (0.25M)]. The reaction mixture was stirred at room temperature during 10 min approx. After control by TLC, the reaction mixture was acidified with Dowex-50®, previously rinsed with MeOH, the resin was taken off by filtration and the solvent evaporated to afford the corresponding unprotected glycoside.

(4-methoxy-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)phenyl)(3,4,5trimethoxyphenyl)methanone 8a

Compound 8a was prepared from the galactoside 7a in quantitative yield. White solid; R f = 0.00 (Cyclohexane/EtOAc 6/4); IR (neat): 3408, 2942, 1751, 1652, 1581, 1414, 1326, 1256, 1229, 1127, 1056 cm -1 . m.p.=89.9-91.7°C; 1 H NMR (300 MHz, Methanol-d 4 ) δ 8.07 (d, J = 2.3 Hz, 1H), 7.73 (dd, J = 8.5, 2.1 Hz, 1H), 7.13 (d, J = 8.7 Hz, 3H), 4.75 (d, J = 9.7 Hz, 1H), 4.00 (s, 3H), 3.96 (d, J = 4.2 Hz, 1H), 3.91 (d, J = 1.6 Hz, 9H), 3.72 (t, J = 9.5 Hz, 1H), 3.64 (t, J = 5.8 Hz, 2H), 3.57 -3.50 (m, 2H). 13

(2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-((2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)thio)tetrahydro-2Hpyran-3,4,5-triol 8b

Compound 8b was prepared from the galactoside 7b in quantitative yield. White solid; R f = 0.00 (Cyclohexane/EtOAc 6/4); IR (neat): 3338, 2989, 1649, 1636, 1536, 1508, 1452, 1415, 1242, 1128, 1056, 1015 cm -1 . m.p.=185.1-186.0°C; 1 H NMR (300 MHz, MeOD) δ 7.55 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 8.5, 2.2 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.59 (s, 2H), 5.44 (d, J = 1.2 Hz, 1H), 5.34 (d, J = 1.2 Hz, 1H), 4.60 (d, J = 9.7 Hz, 1H), 3.90 (d, J = 2.6 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 3H), 3.79 (s, 6H), 3.69 -3.55 (m, 3H), 3.50 -3.41 (m, 2H). 13

Isothermal Titration Calorimetry

LecA was expressed and purified as previously described. [START_REF] Kuhaudomlarp | LecA, a galactose-binding lectin from Pseudomonas aeruginosa[END_REF] All experiments were performed at 25 °C with an ITC200 isothermal titration calorimeter (Microcal-Malvern Panalytical, Orsay, France). The lyophilized LecA protein was dissolved in a buffer composed of 20 mM Tris HCl pH 7.5, 100 mM NaCl and 100µM CaCl2 with 5% DMSO final. All compounds were first dissolved in DMSO then in same buffer for a final concentration of 5% DMSO. The 200 µL sample cell containing LecA (concentrations ranging from 50 to 200 µM) was subjected to injections of ligand solution: 20 to 39 injections of 1 µL or 70 injections of 0.5 µL (0.5 to 2 mM, depending on the ligand) at intervals of 100, 120 or 200s while stirring at 850 rpm. Control experiments were performed by repeating the same protocol, but injecting the ligand into buffer solution. The supplied software Origin 7 or MicroCal PEAQ-ITC was used to fit the experimental data to a theoretical titration curve allowing the determination of affinity (i.e., association constant, Ka), binding enthalpy (∆H), and stoichiometry (n). Values for free energy change (∆G) and entropy contributions (T∆S) were derived from the equation ∆G = ∆H -T∆S = -RT ln Ka (with T = 298.15 K and R = 8.314 J.mol -1 .K -1 .

Crystallization and structure determination of LecA thiogalatosides complexes

The hanging drop vapor diffusion method was used with 2 µL drops containing 50/50 of protein and reservoir solution at 19°C. Crystals of the complex with compound 8c were obtained by cocrystallisation where lyophilized LecA was dissolved in 20 mM Tris/HCl pH 8.0 and 100 mM NaCl to a concentration of 16.9 mg/mL and incubated for 3h at room temperature with 3.8 mM of compound (1 µL of 100 mM compound in 100% DMSO added to 25 µL of protein). Drops were made using the supernatant after centrifugation at 20000 g for 5 minutes since precipitation was observed with a crystallization solution containing 18% PEG3350, 150 mM potassium thiocyanate (KSCN) and 100 mM Tris/HCl pH 8.5. Rod like crystals were transferred in 30% PEG3350 for cryoprotection prior mounting in a cryoloop and flashed freezed in liquid nitrogen. For the complex with compound 8a, the dry soak method was used where 0.2 µL of 25mM compound dissolved in 100% DMSO were deposited on the coverslip prior drying at room temperature for a night. Then, 1 µL of lyophilised LecA dissolved in 20 mM Hepes 7.5 10µM CaCl 2 to 9.8 mg/mL was added and 1 µL of reservoir solution containing 18% PEG3350, 100 mM KSCN and 100 mM sodium acetate pH 4.5. Rod like crystals were obtained and a single rod was transferred in a solution were the PEG concentration was increased to 28% prior mounting and freezing. Data were collected at SOLEIL synchrotron, Saint Aubin, France on Proxima 1 beamline using an Eiger pixel detector (Dectris Ltd). The data were processed using XDS [START_REF] Kabsch | Xds[END_REF] and XDSME. [START_REF] Legrand | XDSME: XDS made easier[END_REF] All further computing was performed using the CCP4 suite. [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF] The structures were solved by molecular replacement using as search model the monomer A coordinates of LecA-Gal complex PDB1OKO in PHASER 2.8 with a search for 2 and 4 copies for LecA-8c and LecA-8a complexes, respectively. [START_REF] Mccoy | Solving structures of protein complexes by molecular replacement with Phaser[END_REF] The model was improved using Buccaneer 1.5 [START_REF] Cowtan | The Buccaneer software for automated model building. 1. Tracing protein chains[END_REF] and ARP/wARP 8.0 [START_REF] Langer | Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7[END_REF] for LecA-8c and LecA-8a complexes, respectively. Restrained maximum likelihood refinement using REFMAC 5.8 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF] was iterated with manual rebuilding in Coot [START_REF] Emsley | Features and development of Coot[END_REF] to refine the structures. Five percent of the observations were set aside to perform cross-validation analysis. Hydrogen atoms were added in their riding positions and used for geometry and structure factor calculations. Ligand library were made using JLigand 2.6 (AceDRG mode) before modelling after inspection of the electron density maps and checked using Privateer. [START_REF] Agirre | Privateer: software for the conformational validation of carbohydrate structures[END_REF] Prior deposition in the Protein Data Bank, the models were validated using both the wwPDB Validation server (http://wwpdb-validation.wwpdb.org) and Molprobity. [START_REF] Williams | MolProbity: More and better reference data for improved all-atom structure validation[END_REF] Coordinates and structure factors have been deposited under PDB accession codes 7Z62 and 7Z62 for LecA in complex with compound 8c and 8a, respectively. Data quality and refinement statistics are described in Table 2. [START_REF] Ballard | Investigating γ-secretase protein interactions in live cells using active site-directed clickable dual-photoaffinity probes[END_REF][START_REF] Rima | Seaweed Extracts: A Promising Source of Antibiofilm Agents with Distinct Mechanisms of Action against Pseudomonas aeruginosa[END_REF][START_REF] Khalilzadeh | Growth inhibition of adherent Pseudomonas aeruginosa by an N-butanoyl-L-homoserine lactone analog[END_REF] P. aeruginosa PAO1 (CIP 104116) was obtained from the Institute Pasteur collection (Paris, France). The strain was frozen and kept at -80°C. The inoculum used in each experiment came from a second subculture on Trypticase soy agar (BioMérieux, Crapone, France) that was incubated under aerobic conditions at 37 °C for 24 h. Bacterial suspensions were freshly prepared in sterile distilled water before each experiment. A low-nutritive medium, named minimum biofilm broth (MBB) was used for the biofilm formation and the evaluation of the antibiofilm activity of extracts to create stressful conditions and subsequently promote biofilm formation and growth of adherent cells rather than planktonic growth. The MBB 10X medium is composed of FeSO 4 , 7 H 2 O (0.005 g/L), Na 2 HPO 4 (12.5 g/L), KH 2 PO 4 (5.0 g/L), (NH 4 ) 2 SO 4 (1.0 g/L), glucose (0.5 g/L) and MgSO 4 , 7 H 2 O (0.2 g/L) Biofilms were developed in 24-well plates (Falcon, TC-treated, polystyrene). The bacterial suspension prepared in MBB (2X) was initially adjusted to 10 8 CFU/mL followed by a serial dilution to 10 -6 with the same medium. One milliliter of the 10 -6 dilution (equivalent to 10 2 CFU/mL) was introduced into each well. In order to test its effect on the biofilm, 1.0 mL of test compound solution prepared at different concentrations in sterile distilled water was added to each well (final concentrations 5, 2.5, 1.25, 0.31 and 0.08 mM). Wells containing 1.0 mL of SDW + 1.0 mL of un-inoculated MBB or 1.0 mL SDW + 1.0 mL inoculated MBB, were considered as sterility and biofilm growth controls, respectively. Plates were then incubated at 37°C. After 2, 4, 6, 20 and 24 h of incubation, the wells were rinsed twice with sterile distilled water and the medium (BBM ± tested product) was replaced. All assays were performed in triplicate.

Antibiofilm activity

After incubation, wells were rinsed twice with 2.0 mL of SDW, and then the attached cells were scraped (for 1 min) with a sterile spatula into 1 mL of SDW. The recovered suspension was diluted by serial dilution (from 10 -1 to 10 -6 ) and each dilution was inoculated by inclusion in TSA agar plates. After 48 h of incubation at 37 ˚C, the numbers of CFU were counted by considering only plates with 15 to 300 CFU. The adhered biomass was then calculated and subjected to logarithmic transformation by Formula (1). The logarithmic reduction and the percentage of biofilm inhibition with respect to the corresponding untreated control were also calculated using Formulas [START_REF] Gilboa-Garber | Pseudomonas aeruginosa lectins[END_REF]

MICs/MBS determination

Determination of the MICs and MBCs of evaluated compounds was performed in Mueller-Hinton broth and Mueller-Hinton agar, respectively, according to EUCAST/CA-SFM guidelines (2020). The range of tested concentrations was from 5,0 mM to 0,0098 mM. The highest used methanol concentration was also tested to confirm no inhibitory of growth nor bactericidal activity.

Cytotoxicity on mammalian cells (MRC-5)

Evaluation of compounds cytotoxicity was performed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) colorimetric assay based on published procedure [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF] with some modifications. The potential cytotoxic activity of the ten most active compounds was assessed against MRC-5 cell lines (Human Fetal Lung Fibroblast Cells: ATCC® CCL-171) purchased from the ATCC® (Manassas, United-States) and cultivated in Modified Eagle Medium (MEM: (Dominique Dutsher, Plaisance-du-Touch, France) in flask. Solutions (2-fold serial dilutions) of selected compounds were prepared in RPMI medium. 96-wells microtiter plates were first filled with 100 µl of a cell suspension prepared in RPMI medium (2 x 10 4 cells/100 µL). After overnight incubation at 37 °C in a humidified 5-6.5% CO 2 incubator, 100 µL of each product dilution were added to achieve final concentrations ranging from 5,0 mM to 0,0098 mM (final volume 200 µL/well). Wells corresponding to the untreated control were supplemented with 100 µL of fresh medium. Phenol was used as positive control for cytotoxicity. The microplate was then incubated for 24h at 37 °C in a humidified 5-6.5% CO 2 incubator. The supernatant was then discarded followed by a rinsing with 100 µL of Dulbeccos's phosphate-buffered saline (D-PBS, Sigma, St. Quentin Fallavier, France). Then 100 µl of a MTT solution prepared in D-PBS at a concentration of 0.5 mg/mL were added in all wells. After 60 min of incubation at 37 °C and in order to solubilize the formed formazan, indicator of cell viability, 100 µL of DMSO were added. After agitation, the OD was measured at 570 nm and the viability percentage was calculated using the following formula. Assays were performed in duplicate with four technical replicates.

𝑉𝑖ability % 𝑥 100

Cytotoxicity was defined for viability percentages below 50%. [START_REF] Pailsson | Pseudomonas aeruginosa uses multiple receptors for adherence to laminin during infection of the respiratory tract and skin wounds[END_REF]

Figure 1 .

 1 Figure 1. (A) Tetrameric structure of LecA (PDB 1OKO) with protein represented as ribbon, calcium as a sphere and galactose ligand as sticks. (B) Selected examples of multivalent and mono-galactoside ligands of LecA.

Figure 2 :

 2 Figure 2: ITC titration of LecA with compound 4b, 8a, 8c and 9a. The plot in the lower panel shows the total heat released as a function of total ligand concentration for the titration shown in the upper panel. The solid line represents the best least-square fit to experimental data using a one site model.

Figure 3 :

 3 Figure 3: Crystal structures of LecA complexed with compounds 8a and 8c. The calcium cataion is presented as a green sphere, the galactoside as sticks, water molecules as red spheres, hydrogen bonds and hydrophobic contacts as blue and magenta dotted lines, respectively. (A) Compound 8a in three different conformations observed in the binding site of chain A, B and D. Binding site of chain C is similar to chain A and is not represented. (B) Compound 8c in binding site A. (C) Superimposition of 8a (two conformations) and 8c in LecA protomer

[ 12 ]Figure 4 :

 124 Figure 4: Superimposition of crystal structures of LecA complexed with compound 8a (chain A -this work) and trisaccharide αGal1-3βGal1-4Glc from PDB 2VXJ (stick colored in cyan).

  the efficient dose, and no toxicity on human cells. It is therefore a very promising hit-compound for therapeutical development, for example as adjuvant to antibiotic actions.

  [a] 24 D : +61.0 (c 1.0, CHCl3); 1 H NMR (300 MHz, CDCl3): d = 8.01 (d, J =2.1 Hz, 1H), 7.71 (dd, J = 8.5, 2.1 Hz, 1H), 6.98 (s, 2H), 6.91 (d, J = 8.6 Hz, 1H), 5.45-5.36 (m, 1H), 5.27 (t, J = 10.0 Hz, 1H), 5.06 (dd, J = 9.9, 3.4 Hz, 1H), 4.88 (d, J = 10.0 Hz, 1H),

  C NMR (75 MHz, CDCl 3 ) δ 170.5(C), 170.4(C), 170.2(C), 169.6(C), 164.5(C), 158.1(C), 153.1(C), 149.2(C), 138.1(C), 137.2(C), 134.4(C), 132.9(CH), 129.5(CH), 120.7(C), 113.1(CH 2 ), 110.6(CH), 105.8(2CH), 85.4(CH), 74.5(CH), 67.5(CH), 67.3(CH), 61.4(CH 2 ), 61.0(CH), 56.3(3CH 3 ), 56.1(CH 3 ), 20.9(CH 3 ), 20.8(3CH 3 ). HR-MS(ESI):m/z calcd for C 32 H 38 O 13 NaS [M+Na] + 685.1931; found 685.1934

  C NMR (75 MHz, CDCl 3 ) δ 170.5(C), 170.3(C), 170.2(C), 169.6(C), 159.6(C), 148.8(C), 141.9(C), 133.6(C), 132.2(2CH), 132.0(C), 129.5(2CH), 128.9(2CH), 113.7(2CH), 113.6(C), 86.8(CH), 74.6(CH), 72.1(CH), 67.4(CH), 67.3(CH), 61.7(CH 2 ), 55.4(CH 3 ), 21.0(CH 3 ), 20.8(2CH 3 ), 20.7(CH 3 ). HR-MS(ESI):m/z calcd for C 29 H 32 O 10 SNa [M+Na] + 595.1614; found 595.1608.

  2(CH), 75.1(CH), 72.5(2CH), 68.5(CH), 68.5(CH), 68.0(CH), 67.9(CH), 62.6(CH 2 ), 62.5(CH 2 ), 20.7(2CH 3 ), 20.7(2CH 3 ), 20.6(2CH 3 ), 20.5(2CH 3 ).HR-MS(ESI):m/z calcd for C 42 H 48 O 18 NaS 2 [M+Na] + 927.2180; found 927.2177 (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((4-(1-(naphthalen-2-yl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate 7f Compound 7f was prepared from the diodide 6f in 80% yield by using the general method A. White solid; R f = 0.16 (Cyclohexane/EtOAc 7/3); 80% after column chromatography (Cyclohexane/EtOAc 7/3); IR (neat): 3059, 1748, 1652, 1591, 1367, 1211, 1153, 1083, 1057, 1041, 1015 cm -1 . m.p.=62.6-64.1°C; 1 H NMR (300 MHz, Chloroform-d) δ 7.86 -7.75 (m, 4H), 7.53 -7.43 (m, 5H), 7.37 -7.31 (m, 2H), 5.61 (d, J = 1.1 Hz, 1H), 5.56 (d, J = 1.1 Hz, 1H), 5.44 (dd, J = 3.4, 1.1 Hz, 1H), 5.29 (t, J = 9.9 Hz, 1H), 5.08 (dd, J = 10.0, 3.4 Hz, 1H), 4.77 (d, J = 10.0 Hz, 1H), 4.24 -4.09 (m, 2H), 4.01 -3.92 (m, 1H), 2.14 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 170.5(C), 170.3(C), 170.2(C), 169.6(C), 149.4(C), 141.5(C), 138.5(C), 133.4(C), 133.1(C), 132.3(C), 132.2(2CH), 129.0(2CH), 128.3(CH), 128.0(CH), 127.8(CH), 127.4(CH), 126.4(CH), 126.4(CH), 126.3(CH), 115.5(CH 2 ), 86.8(CH), 74.6(CH), 72.1(CH), 67.4(CH), 67.4(CH), 61.8(CH 2 ), 21.0(CH 3 ), 20.8(2CH 3 ), 20.7(CH 3 ). HR-MS(ESI):m/z calcd for C 32 H 32 O 9 SNa [M+Na] + 615.1665; found 615.1677.

  C NMR (75 MHz, CDCl 3 ) δ 170.4(C), 170.3(C), 170.2(C), 169.6(C), 157.0(C), 153.3(2C), 142.2(C), 138.9(C), 133.2(CH), 132.2(C), 128.8(CH), 120.2(C), 111.2(CH), 104.9(2CH), 85.5(CH), 74.5(CH), 72.2(CH), 67.6(CH), 67.3(CH), 61.4(CH 2 ), 60.9(CH 3 ), 56.3(2CH 3 ), 56.0(CH 3 ), 44.3(CH), 22.3(CH 3 ), 20.9(CH 3 ), 20.7(3CH 3 ). HR-MS(ESI):m/z calcd for C 32 H 40 O 13 NaS [M+Na] + 687.2087; found 687.2080

  C NMR (75 MHz, MeOD) δ 196.43(C), 161.94(C), 154.29(2C), 134.50(C), 133.16(CH), 131.82(2C), 131.57(CH), 125.86(C), 111.00(CH), 108.77(2CH), 87.99(CH), 80.34(CH), 76.36(CH), 71.20(CH), 70.19(CH), 62.13(CH 2 ), 61.25(CH 3 ), 56.83(2CH 3 ), 56.76(CH 3 ). HR-MS(ESI):m/z calcd for C 23 H 28 O 10 SNa [M+Na] + 519.1301; found 519.1308.

  δ 7.52 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 5.8 Hz, 2H), 7.22 (d, J = 6.4 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 5.35 (dd, J = 7.2, 1.3 Hz, 2H), 4.61 (d, J = 9.6 Hz, 1H), 3.91 (dd, J = 3.4, 1.1 Hz, 1H), 3.81 (s, 3H), 3.78 -3.69 (m, 2H), 3.66 -3.56 (m, 2H), 3.51 (dd, J = 9.2, 3.3 Hz, 1H). 13 C NMR (75 MHz, MeOD) δ 151.3(C), 150.7(C), 141.9(C), 135.6(C), 131.7(2CH), 130.4(2CH), 129.6(2CH), 126.3(C), 114.6(2CH), 113.2(CH 2 ), 90.2(CH), 80.7(CH), 76.4(CH), 71.0(CH), 70.5(CH), 62.7(CH 2 ), 55.7(CH 3

(

  2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-((2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)ethyl)phenyl)thio)tetrahydro-2Hpyran-3,4,5-triol 8eCompound 8e was prepared from the galactoside 7g in quantitative yield. Light brown solid; R f = 0.00 (Cyclohexane/EtOAc 6/4); IR (neat): 3385, 2966, 2938, 1647, 1591, 1490, 1462, 1421, 1332, 1245, 1127, 1063 cm -1 . m.p.=61.3-63.2°C; 1 H NMR (300 MHz, MeOD) δ 7.48 (dd, J = 8.6, 2.2 Hz, 1H), 7.13 (dd, J = 8.5, 2.2 Hz, 1H), 6.91 (dd, J = 8.5, 1.7 Hz, 1H), 6.58 (s, 1H), 6.57 (s, 1H), 4.60 (d, J = 9.7 Hz, 1H), 4.10 (q, J = 6.7 Hz, 1H), 3.95 (d, J = 3.3 Hz, 1H), 3.86 (s, 3H), 3.84 (m, 6H), 3.78 (s, 3H), 3.72 (dd, J = 5.4, 1.2 Hz, 2H), 3.67 (dd, J = 9.4, 5.3 Hz, 1H), 3.60 -3.50 (m, 2H), 1.64 (d, J = 7.2 Hz, 3H).13 C NMR (75 MHz, MeOD) δ 156.9(C), 154.3(C), 144.3(C), 140.6(C), 130.9(CH), 127.6(CH), 124.2(C), 122.2(C), 119.8(C), 111.8(CH), 106.1(2CH), 88.3(CH), 80.6(CH), 76.3(CH), 71.1(CH), 70.4(CH), 62.5(CH 2 ), 61.0(CH 3 ), 56.7(2CH 3 ), 56.5(CH 3 ), 45.5(CH), 22.4(CH 3 ). HR-MS(ESI):m/z calcd for C 24 H 32 O 9 NaS [M+Na] + 519.1665; found 519.1669 (2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-((4-(1-(naphthalen-2-yl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triol 8f Compound 8f was prepared from the galactoside 7f in quantitative yield. White solid; R f = 0.00 (Cyclohexane/EtOAc 6/4); IR (neat): 3359, 1652, 1592, 1347, 1231, 1123, 1057, 1021 cm -1 . m.p.=143.5-144.0°C; 1 H NMR (300 MHz, Methanol-d 4 ) δ 7.93 -7.80 (m, 3H), 7.80 -7.75 (m, 1H), 7.65 -7.56 (m, 2H), 7.49 (td, J = 8.3, 7.7, 2.6 Hz, 3H), 7.33 (d, J = 8.4 Hz, 2H), 5.61 (s, 1H), 5.58 (s, 1H), 4.68 (d, J = 9.6 Hz, 1H), 3.95 (dd, J = 3.3, 1.1 Hz, 1H), 3.87 -3.73 (m, 2H), 3.69 (t, J = 9.5 Hz, 1H), 3.63 (ddd, J = 6.7, 5.1, 1.2 Hz, 1H), 3.56 (dd, J = 9.1, 3.3 Hz, 1H). 13 C NMR (75 MHz, MeOD) δ 151.1(C), 141.5(C), 139.9(C), 135.8(C), 134.7(C), 134.5(C), 131.8(2CH), 129.7(2CH), 129.2(CH), 128.8(CH), 128.6(CH), 128.2(CH), 127.3(CH), 127.2(2CH), 115.2(CH 2 ), 90.1(CH), 80.7(CH), 76.4(CH), 71.0(CH), 70.5(CH), 62.7(CH 2 ). HR-MS(ESI):m/z calcd for C 24 H 24 O 5 SNa [M+Na] + 447.1242; found 427.1237. (2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-((3-(1-(4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2Hpyran-2-yl)thio)phenyl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triol 9a Compound 9a was prepared from the galactoside 7e in quantitative yield. Light yellow solid ; R f = 0.00 (Cyclohexane/EtOAc 6/4); IR (neat): 3366, 2952, 2840, 1750, 1651, 1419, 1370, 1222, 1059, 1015 cm -1 . m.p.=116.1-118.0°C; 1 H NMR (300 MHz, Methanol-d 4 ) δ 7.57 -7.45 (m, 4H), 7.33 -7.19 (m, 3H), 7.18 (d, J = 7.7 Hz, 1H), 5.46 (s, 2H), 4.63 (d, J = 9.5 Hz, 1H), 4.56 (d, J = 9.7 Hz, 1H), 3.92 -3.87 (m, 2H), 3.81 -3.69 (m, 2H), 3.68 -3.54 (m, 5H), 3.49 (td, J = 8.8, 3.1 Hz, 3H).

Table 1 . Microcalorimetry data and thermodynamics contribution for higher affinity compounds binding to LecA. The experiments were realized in duplicate at 298 K unless otherwise stated

 1

	ligand	K D (μM)	n	-G (kJ/mol) H (kJ/mol) TS (kJ/mol)
	4a	11.9 a	1 b	28.1	42.3	-14.2
	4b	2.45 ± 0.4	1 b	32.0	49.9± 0.0	-17.9
	4c	9.2 a	1 b	28.7	41.2	-12.5
	8a	1.02± 0.6	1 b	34.2	54.5 ± 2.7	-20.3
	8b	6.9 ± 0.1	0.98± 0.03	29.4	36.0± 0.7	-6.6
	8c	3.0 ± 0.6	1 b	31.6	46.6 ± 0.1	-15.0
	8d	8.1 ± 0.2	0.97± 0.06	29.1	46.4 ± 0.9	-17.3
	8e	9.0 ± 0.2	1 a	28.8	35.2± 1.0	-6.4
	9a	0.16 ±0.03	0.44± 0.04	38.7	90.2 ± 1.1	-51.5
	a Only one experiment; b ligand concentration adjusted to reach stochiometry of 1(within 10% variation);

Table 2 .

 2 Statistics on Data Collection and Refinement

	Structure	LecA-8c		LecA-8a
	Data collection
	Space Group	P2 1 2 1 2			P2 1 2 1 2 1
	Unit cell a, b, c (Å)	48.12 57.09 83.40		42.26 61.93 180.01
	Beamline	PX1 SOLEIL		PX1 SOLEIL
	Wavelength (Å)	0.97918		0.97856
	Resolution limits (Å)	41.70-1.53 (1.56-1.53)		45.00-1.75 (1.78-1.75)
	Reflections: measured	326966 (16132)		305761 (16876)
	unique	35361 (1733)		48775 (2627)
	Completeness (%)	99.8 (99.4)		100 (99.9)
	R merge (%, within I + /I -)	4.2 (26.4)		6.7 (29.1)
	R meas (%, within I + /I -)	4.7 (29.7)		8.0 (34.7)
	R pim (%, within I + /I -)	2.1 (13.4)		4.3 (18.6)
	Mean I / σI	26.5 (7.3)		16.1 (5.3)
	Multiplicity	9.2 (9.3)		6.3 (6.4)
	CC (1/2)	99.9 (98.7)		99.8 (97.6)
	Refined Statistics
	Resolution (Å)	41.70-1.53		45.00-1.75
	No. reflections / free reflections	33577 / 1749		46191 / 2506
	R-factor / R free (%)	16.5 / 20.6		16.9 / 21.3
	R.m.s.d bond lengths (Å)	0.0155			0.0157
	R.m.s.d bond angles (deg)	1.89			1.77
	R.m.s.d chiral (Å 3 )	0.0965			0.100
	No. of atoms/ Bfac (Å 2 )	A	B	A	B	C	D
	-Protein	942 / 16.5	914 / 20.9	914 / 16.0	907 / 13.8	918 / 9.1	916 / 10.9
	-Calcium	1 / 14.8	1 / 24.6	1 / 15.1	1 / 11.3	1 / 7.6	1 / 9.1
	-Sugar	28 / 21.6		34 / 18.9	23 / 24.0	34 / 15.5	34 /22.0
	-Waters	211 / 29.5	192 / 32.2	201 / 25.7	178 / 24.7	257 / 21.8	225 / 23.3
	Ramachandran Allowed / Favored /Outliers (%)	100 / 97.3 / 0		100 / 97.0 / 0
	PDB Code	7Z62		7Z63

f General procedure for Paladium-Catalyzed coupling of N-Tosylhydrazones with Diodoarenes

  42] 6 and Acetophenone 7 was prepared adopting reported methods spectral data are in agreement with the published ones.[START_REF] Siemann | A review and update of the current status of the vasculature-disabling agent combretastatin-A4 phosphate (CA4P)[END_REF] A round-bottom flask with a condenser under an argon atmosphere was charged with N-tosylhydrazone (1 equiv), Pd(OAc) 2 (4 mol %), SPhos (8 mol %), diodoarene (1.0 equiv, 1 mmol) and LiO-t-Bu (2.4 equiv). Then 7 mL of Dioxane was added via syringe at room temperature. The flask was put into a preheated oil bath and stirred at reflux for 1 h. The resulting suspension was cooled to room temperature and filtered through celite eluting with ethyl acetate. The filtrate was concentrated and purification of the residue by silica gel column chromatography gave the desired products.

	4.2. Synthetic Chemistry
	4.2.1. Synthesis of the intermediates 6a-1 Hz,
	2H), 3.83 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 159.6(C), 148.7(C), 141.5(C), 137.4(2CH), 133.5(C), 130.3(2CH), 129.5(2CH), 113.8(2CH), 113.6(CH 2 ), 93.4(C), 55.5(CH 3 ). HR-MS(APCI):m/z calcd for C 15 H 14 IO [M+H] + 337.0084;
	found 337.0084
	1-iodo-3-(1-(4-iodophenyl)vinyl)benzene 6e
	White solid; R f = 0.62 (Cyclohexane); 45% after column chromatography (Cyclohexane); IR (neat): 3287, 2952, 2925, 1649, 1558, 1414, 1079, 1015 cm -1 . m.p.=64.5-66.1°C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.72 -7.61 (m, 4H), 7.31 -7.19 (m, 1H), 7.12 -6.96 (m, 3H), 5.47 (d, J = 0.6 Hz, 1H), 5.44 (d, J = 0.6 Hz, 1H). 13 C NMR (75 MHz, CDCl 3 ) δ 148.0(C),
	143.3(C), 140.4(C), 137.6(2CH 3 ), 137.2(CH 3 ), 137.1(CH 3 ), 130.1(3CH 3 ), 127.6(CH 3 ), 115.9(CH 2 ), 94.5(C), 93.9(C). HR-MS(APCI):m/z calcd for C 14 H 11 I 2 O [M+H] + 432.8945; found 432.8941
	2-(1-(4-iodophenyl)vinyl)naphthalene 6f
	White solid; R f = 0.47 (Cyclohexane); 51% after column chromatography (Cyclohexane); IR (neat): 3056, 2924, 2853, 1752, 1655, 1557, 1581, 1432, 1368, 1221, 1135, 1111, 1079, 1047, 1006 cm -1 . m.p.=94.8-95.9°C; 1 H NMR (300 MHz,
	Chloroform-d) δ 7.86 -7.78 (m, 3H), 7.75 (dd, J = 1.8, 0.6 Hz, 1H), 7.72 -7.65 (m, 2H), 7.52 -7.43 (m, 3H), 7.19 -
	7.06 (m, 2H), 5.60 (d, J = 1.0 Hz, 1H), 5.54 (d, J = 1.0 Hz, 1H).
	13 C NMR (75 MHz, CDCl 3 ) δ 149.3(C), 141.2(C), 138.4(C), 137.5(2CH), 133.4(C), 133.2(C), 130.4(2CH), 128.3(CH),
	128.0(CH), 127.8(CH), 127.4(CH), 126.4(C), 126.3(2C), 115.4(C), 93.6(C).HR-MS(APCI):m/z calcd for C 18 H 14 I [M+H] + 357.0140; found 357.0147.

1-iodo-3-(1-(4-methoxyphenyl)vinyl)benzene 6d Yellow oil; R f = 0.81 (Cyclohexane/EtOAc 8/2); 52% after column chromatography (Cyclohexane); IR (neat): 3392, 1730, 1608, 1510, 1483, 1283, 1232, 1084 cm -1 . 1 H NMR (300 MHz, CDCl 3 ) δ 7.72 (t, J = 1.7 Hz, 1H), 7.67 (dd, J = 7.8, 1.1 Hz, 1H), 7.34 -7.28 (m, 1H), 7.29 -7.22 (m, 2H), 7.08 (t, J = 7.8 Hz, 1H), 6.93 -6.86 (m, 2H), 5.

43 (d, J = 1.0 Hz, 1H), 5.35 (d, J = 1.0 Hz, 1H), 3.85 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 159.7(C), 148.3(C), 144.3(C), 137.3(CH), 136.7(CH), 133.4(C), 130.0(CH), 129.4(2CH), 127.8(CH), 114.0(CH 2 ), 113.8(2CH), 94.3(C), 55.5(CH 3 ). HR-MS(APCI):m/z calcd for C 15 H 14 IO [M+H] + 337.0084; found 337.0086 1-iodo-4-(1-(4-methoxyphenyl)vinyl)benzene 6c White solid; R f = 0.81 (Cyclohexane/EtOAc 8/2); 37% after column chromatography (Cyclohexane); IR (neat): 2953, 2833, 1753, 1667, 1511, 1483, 1292, 1252, 1180, 1029 cm -1 . m.p.=129.2-130.2°C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.66 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.9 Hz, 2H), 5.37 (dd, J = 16.5, 1.

  CH 2 ), 86.9(CH), 74.5(CH), 72.1(CH), 67.3(CH), 67.3(CH), 61.6(CH), 55.4(CH 3 ), 21.0(CH 3 ), 20.8(2CH 3 ), 20.7(CH 3). HR-MS(ESI):m/z calcd for C 29 H 32 O 10 NaS [M+Na] + 595.1614; found 595.1609 Compound 7e was prepared from the diodide 6e in 96% yield by using the general method B : Light yellow solid; R f = 0.27 (Cyclohexane/EtOAc 5/5); 96% after column chromatography (Cyclohexane / EtOAc 5/5); IR (neat): 2928, 1747, 1637, 1428, 1387, 1368, 1211, 1154, 1083, 1057 cm -1 . m.p.=80.0-81.9°C; 1 H NMR (300 MHz, Acetone) δ 7.59 -7.51 (m, 3H), 7.50 (t, J = 1.7 Hz, 1H), 7.42 -7.31 (m, 3H), 7.26 (ddd, J = 7.7, 1.8, 1.2 Hz, 1H), 5.56 (dd, J = 4.8, 1.0 Hz, 2H), 5.43 (ddd, J = 6.8, 2.5, 1.1 Hz, 2H), 5.26 -5.08 (m, 6H), 4.36 -4.24 (m, 2H), 4.14 (dd, J = 6.3, 1.7 Hz, 2H), 4.10 -4.07 (m, 1H), 4.05 -4.00 (m, 1H), 2.11 (d, J = 7.3 Hz, 6H), 2.03 (d, J = 2.6 Hz, 6H), 1.99 -1.95 (m, 6H), 1.91 (d, J = 3.2 Hz, 6H). 13 C NMR (75 MHz, Acetone) δ 170.7(2C), 170.5(2C), 170.2(2C), 169.9(2C), 149.6(C), 142.8(C), 141.0(C), 134.4(C), 134.3(C), 131.8(2CH), 131.6(2CH), 129.7(CH), 129.4(2CH), 128.2(CH), 115.8(CH 2 ), 86.2(CH), 86.1(CH), 75.

	C NMR (75 MHz, CDCl 3 ) δ 170.5(C), 170.3(C), 170.2(C), 169.6(C), 159.6(C), 148.9(C), 142.8(C), 133.6(C), 132.7(C), 132.0(CH), 131.4(CH), 129.5(2CH), 128.8(CH), 128.2(CH), 113.8((2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((3-(1-(4-(((2R,3S,4R,5R,6S)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)thio)phenyl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate 7e

  C NMR (75 MHz, MeOD) δ 171.8(C), 158.3(C), 154.2(C), 150.8(C), 149.3(C), 139.3(C), 135.8(C), 131.2(CH), 128.6(CH), 124.7(C), 113.5(CH 2 ), 111.4(CH), 106.9(2CH), 88.4(CH), 80.3(CH), 76.3(CH), 71.1(CH), 70.2(CH), 62.2(CH 2 ), 61.2(CH 3 ), 56.6(2CH 3 ), 56.5(CH 3 ). HR-MS(ESI):m/z calcd for C 24 H 30 O 9 NaS [M+Na] + 517.1508; found 517.1504

	(2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-((4-(1-(4-methoxyphenyl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triol 8c
	Compound 8c was prepared from the galactoside 7c in quantitative yield.
	Light yellow solid; R f = 0.00 (Cyclohexane/EtOAc 6/4); IR (neat): 3366, 1650, 1637, 1511, 1466, 1242, 1179, 1089, 1030 cm -1 . m.p.=96.6-98.5°C; 1 H NMR (300 MHz, MeOD)

  ). HR-MS(ESI):m/z calcd for C 21 H 24 O 6 S [M+H] + 427.1191; found 427.1192 J = 5.3, 1.2 Hz, 2H), 4.62 (d, J = 9.6 Hz, 1H), 3.95 (d, J = 2.4 Hz, 1H), 3.85 (s, 3H), 3.72 (d, J = 6.1 Hz, 2H), 3.65 (t, J = 9.4 Hz, 1H), 3.57 (d, J = 6.6 Hz, 1H), 3.53 (dd, J = 9.2, 3.3 Hz, 1H). 13 C NMR (75 MHz, MeOD) δ 159.6(C), 149.3(C), 142.5(C), 134.6(C), 133.5(C), 130.4(CH), 130.0(CH), 128.9(CH), 128.2(CH), 126.6(CH), 113.3(2CH), 112.2(CH 2 ), 88.8(CH), 79.0(CH), 74.9(CH), 69.6(CH), 68.9(CH), 61.0(CH 2 ), 54.3(CH 3 ). HR-MS(ESI):m/z calcd for C 21 H 24 O 6 NaS [M+Na] + 427.1191; found 427.1194

	(2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-((3-(1-(4-methoxyphenyl)vinyl)phenyl)thio)tetrahydro-2H-pyran-3,4,5-triol 8d
	Compound 8d was prepared from the galactoside 7d in quantitative yield.
	Yellow solid; R f = 0.00 (Cyclohexane/EtOAc 6/4); IR (neat): 3366, 2947, 1647, 1607, 1511, 1432, 1415, 1249, 1180, 1084, 1015 cm -1 . m.p.=75.1-75.7°C; 1 H NMR (300 MHz, MeOD) δ 7.63 -7.46 (m, 2H), 7.37 -7.17 (m, 4H), 6.99 -6.89
	(m, 2H), 5.42 (dd,

  13 C NMR (75 MHz, DMSO) δ 148.0(C), 141.2(C), 138.1(C), 135.6(2C), 128.9(3CH), 128.8(2CH), 128.2(2CH), 125.9(CH), 115.1(CH 2 ), 87.6(2CH), 79.2(CH), 79.1(CH), 74.7(2CH), 69.2(2CH), 68.4(CH), 68.3(CH), 60.6(CH 2 ), 60.5(CH 2 ). HR-MS(ESI):m/z calcd for C 26 H 32 O 10 NaS 2 [M+Na] + 591.1335; found 591.1342.

  and (3).

		log of adhered biomass log CFU/mL	log	number of colonies CFU Dilution factor x inoculated volume
		log CFU ml ⁄ reduction log CFU ml ⁄ for control log CFU ml ⁄ for treated biofilm
	IP	%	Adhered cells	CFU mL ⁄ Adhered cells	Adhered cells CFU mL ⁄	CFU mL ⁄	100

Acknowledgments

The authors acknowledge support of this project by the CNRS, University Paris-Saclay, "Agence Nationale de la Recherche" (ANR-15-CE29-0002) and MRES for a doctoral fellowships to A.B and. AI and AV are partially supported bt Glyco@Alps (ANR-15-IDEX-02) and Labex Arcane/CBH-EUR-GS (ANR-17-EURE-0003). We acknowledge SOLEIL for provision of synchrotron radiation facilities (BAG proposal 20170827) and we would like to thank Serena Sirigu and Tatiana Isabet for assistance in using beamline Proxima 1.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/