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Anti-adhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection Amine M Boukerb, Audric Rousset, Nicolas Galanos, Jean-Baptiste Méar, Marion Thepaut, Teddy Grandjean, Emilie Gillon, Samy Cecioni, Claire Abderrahmen, Karine Faure, David Redelberger, Eric Kipnis, Rodrigue Dessein, Stéphane Havet, Benoit Darblade, Susan E. Matthews, Sophie de Bentzmann, Benoit Guéry, Benoit Cournoyer, Anne Imberty, and Sebastien Vidal J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Nov 2014 Downloaded from http://pubs.acs.org on November 25, 2014

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Journal of Medicinal Chemistry

Vidal, Sebastien; Universite Lyon 1 / CNRS, Institut de Chimie et Biochimie Moleculaires et Supramoleculaires

ACS Paragon Plus Environment


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Anti-adhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection Amine M. Boukerb,† Audric Rousset,‡ Nicolas Galanos,‡,§ Jean-Baptiste Méar,¥ Marion Thépaut,¥ Teddy Grandjean,¥ Emilie Gillon,§ Samy Cecioni,‡,§ Claire Abderrahmen,‡ Karine Faure,¥ David Redelberger,± Eric Kipnis,¥ Rodrigue Dessein,¥ Stéphane Havet, Benoit Darblade Susan E. Matthews,# Sophie de Bentzmann,± Benoit Guéry,¥ Benoit Cournoyer,† Anne Imberty,§ and Sébastien Vidal*‡ † Equipe de recherche « Bactéries Pathogènes Opportunistes et Environnement », UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1 & VetAgro Sup,  Boulevard du  Novembre , F- Villeurbanne, France ‡ Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie Organique  – Glycochimie, UMR CNRS , Université Lyon ,  Boulevard du  Novembre , F- Villeurbanne, France § Centre de Recherche sur les Macromolécules Végétales (CERMAV – CNRS UPR 5301), affiliated with Grenoble Université and ICMG, BP , F- Grenoble, France ¥ Groupe de Recherche Translationelle Relation Hôte-Pathogène Pseudomonas aeruginosa, Faculté de Médecine de Lille UDSL - Université Lille Nord de France, F-59045, Lille Cedex, France ± CNRS Aix Marseille Université, UMR7255, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France  Elicityl SA, 746 Avenue Ambroise Croizat, 38920 Crolles, France # School of Pharmacy, University of East Anglia, Norwich, NR4 7TJ, United Kingdom

ABSTRACT: Pseudomonas aeruginosa lung infections are a major cause of death in cystic fibrosis and hospitalised patients. Treating these infections is becoming difficult due to the emergence of conventional antimicrobial multi-resistance. While monosaccharides have proved beneficial against such bacterial lung infection, the design of several multivalent glycosylated macromolecules has been shown to be also beneficial on biofilm dispersion. In this study, calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized. The characterization of their inhibitory properties ACS Paragon Plus Environment

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on Pseudomonas aeruginosa aggregation, biofilm formation, adhesion on epithelial cells and destruc-

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tion of alveolar tissues were performed. The anti-adhesive properties of the designed glycoclusters were demonstrated through several in vitro bioassays. An in vivo mouse model of lung infection provided an almost complete protection against Pseudomonas aeruginosa with the designed glycoclusters.

INTRODUCTION

Pseudomonas aeruginosa is an opportunistic pathogen involved in a large number of diseases such as septicemia, urinary tract infections, pancreatitis, dermatitis and keratitis mostly in immunocompromised patients.1 Respiratory tract infections are particularly dangerous for patients with chronic lung diseases as well as those under mechanical ventilation (Figure 1). To establish itself in the host, P. aeruginosa has developed a glycostrategy, using auto-produced exopolysaccharides sealing biofilm, elaborating a lipopolysaccharide (LPS) with various oligosaccharide extensions contributing to inflammatory properties and using host carbohydrate recognition for anchoring to mucosa through a battery of carbohydrate binding proteins including soluble lectins or adhesins exposed on pili, flagella or fimbriae.2-4



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Figure 1. Schematic description of adhesion and lung infection by pathogens Two soluble lectins have been identified in P. aeruginosa, LecA (PA-IL) and LecB (PA-IIL) specific to galactose and fucose, respectively.5,6 The crystallographic structures of both lectins have been solved by X-ray diffraction, demonstrating tetrameric arrangement of -sandwich-folded monomers with requirement for calcium in the carbohydrate binding site.7,8 LecA binds to -galactosyl residues present on glycosphingolipids in lung epithelial cell membranes,9,10 while LecB binds to several fucosylated or mannosylated epitopes but display higher affinity for the Lewis a oligosaccharides.8,11 Both lectins possess specific characteristics that allow them to be described as virulence factors. Both lectins have been found on the outer membrane of the bacteria12,13 although no secretion mechanism has been identified and their expression depends on quorum sensing (QS).14,15 Since both lectins bind to human oligosaccharides epitopes recovered on host glycolipids and glycoproteins, they are likely to be involved in bacterial adhesion to cells.16 LecA is involved in the internalization of P. aeruginosa into ACS Paragon Plus Environment

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host cells. It is cytotoxic and able to damage epithelia in lungs and guts.17,18 LecB is not directly cytotoxic itself but has been shown to affect in vitro the ciliary beating frequency of airway epithelial cells.19 Since the binding of lectins to their glycosylated targets can be inhibited by competing monosaccharides, fucose, mannose and galactose have been tested for their ability to limit infection. Indeed, both galactose and fucose could limit the spread of infection in a murine pneumonia model 17 and these monosaccharides are particularly active when used in combination with antibiotics.20 Adhesion of P. aeruginosa to the human respiratory epithelial cell line A549 was also significantly inhibited using human milk oligosaccharides.21 Therefore, a large number of glycoclusters have been synthesized with the aim of competing with carbohydrate-lectin interactions during the infectious process,22-29 using a variety of scaffolds including peptides, modified oligonucleotides, fullerenes, nanoparticles, cyclic oligosaccharides, resorcinarenes, and other scaffolds. The most recent studies achieved affinities for LecA in the nanomolar range based on divalent galactoside compounds.30,31 Small glycoclusters with four galactose residues on calix[4]arene scaffolds32 were among the most efficient architecture against LecA.33,34 We report here the characterization of the binding of both lectins to galactosylated and fucosylated calixarenes, and their inhibitory properties on bacterial aggregation, biofilm formation, adhesion on epithelial cells and alveolar injury. RESULTS AND DISCUSSION Synthesis of monovalent and tetravalent ligands of LecA and LecB Triethyleneglycol was previously identified as a good spacer arm for the connection of the carbohydrate epitope to the multivalent core scaffold. This linker provides flexibility to the designed glycoclusters and more importantly a good solubility in water. The fucosylated ligands targeting LecB were therefore designed using the same linker and an efficient synthesis of the azido-functionalized fucopyranoside derivative 1 was required in order to prepare a large quantity of this starting material for further conjugation to various scaffolds (Scheme 1).



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Fischer glycosylation was used to obtain an excess of the 1,2-cis anomer with an  linkage (Scheme 1). Sulfuric acid was used as the catalyst supported on silica gel. This methodology35,36 provided a rapid access to the -L-fucopyranoside moiety in good yield (52%) although in an inseparable mixture with its -anomer counterpart (α:β, 3:1). The subsequent azidation was performed on the mixture of anomers in good yield (89%) and the final acetylation was achieved in 65% yield when considering only the isolated -L-fucopyranoside 1a.36 Separation was only possible after the acetylation process and using a ternary mixture of solvents differentiating each anomer through their slightly different polarities. Although this protocol was rather cumbersome and long, a large scale (> 5 g) of the desired -Lfucopyranoside 1 could be prepared starting from 15 g of L-fucose in approximately 30% overall yield. The synthesis of a monovalent ligand for LecB was also required for determining of the influence of both the triethyleneglycol linker introduced and multivalency upon binding to the lectin. Cu(I)Catalyzed azide-alkyne cycloaddition37 (CuAAC) of propargyl acetate with the azido-functionalized fucoside 1a afforded the desired triazole derivative 2 and subsequent solvolysis of the acetyl protecting groups provided the monovalent probe 3 (Scheme 1). The galactosylated monovalent probe 4 was previously reported.33 Conjugation of azide 1a and the tetrapropargylated calixarene-based scaffold 5 through CuAAC (Scheme 1) afforded the acetylated glycocluster 6a in high yield (85%, i.e. 96% per reactive center). The deprotection of the acetyl groups was achieved under smooth conditions and all byproducts were evaporated off to afford the hydroxylated glycocluster 7a pure after purification using C18 MPLC purification. The galactosylated tetravalent glycoclusters 6b and 7b were previously reported.33 The mannosylated and glucosylated tetravalent glycoclusters 7c and 7d respectively were synthesized as additional controls according to the same synthetic procedure.


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Scheme 1. Synthesis of the monovalent and tetravalent fucosylated ligands (3 and 7a respectively) of LecB and structure of the corresponding galactosylated ligands (4 and 7b) targeting LecA along with mannosylated and glucosylated ligands 7c and 7d respectively



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Binding studies towards LecA and LecB (ITC studies) Isothermal titration microcalorimetry (ITC) is one of the most powerful bioanalytical techniques for the study of ligand-receptor interactions. Complete thermodynamics can be obtained from such a method and provide a general overview of the binding process involved. While the ITC data for the galactosylated ligands 4 and 7b binding to LecA were previously reported (Table 1),33 the same ITC study for fucosylated ligands 3 and 7a provided an interesting set of data for the better understanding of these mono- or tetravalent lectin-carbohydrate interactions. The dissociation constant (Kd) measured for the monovalent fucosylated ligand 3 was in the nanomolar range and in good agreement with previously reported affinity towards methyl α-L-fucoside38 (FucOMe) thus indicating a limited influence of the triethyleneglycol linker arm introduced (Table 1). The stoichiometry (n) was roughly equal to 1 indicating that a single ligand was bound per binding site of LecB. The tetravalent ligand 7a displayed a nanomolar affinity towards LecB. The n value of 0.32 indicates that at least three carbohydrate epitopes are occupying a fucose binding site of LecB but most probably not from the same tetramer but rather three different tetramers since the distance between the fucose epitopes is too short. In this case, the enthalpy gain is quite remarkable but is partly counterbalanced by the increasing unfavorable entropy term. The relative potency () between the monovalent ligand 3 and the tetravalent fucosylated glycocluster 7a was only 6. This improvement is not very impressive and depicts a “bind and jump” mechanism39 of the LecB lectin around the glycocluster’s structure. The simultaneous interaction of two lectin’s binding site with two fucosides on the same glycocluster, leading to a “chelate” binding mode is not possible based on the distances between each of the protein’s binding sites (40 Å). The ITC data collected previously for the galactosylated ligands highlighted a modest affinity for the methyl β-D-galactoside (GalOMe), methyl -D-galactoside (GalOMe), and the monovalent galactoside 4 while a sub-micromolar affinity was measured for the tetravalent galactosylated glycocluster 7b.33 A chelate aggregative binding mode was hypothesized and later further confirmed by AFM studies.40,41 The mannosylated glycocluster 7c displayed a much higher Kd value in the micromolar range ACS Paragon Plus Environment

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(~50 times lower than for the fucosylated glycocluster 7a) and is in agreement with the poor affinity observed of LecB towards mannosides.38 The glucosylated glycocluster 7d was not recognized at 1.5 mM by LecA nor LecB thus suggesting the absence of non-specific binding properties of such calixarene-based glycoclusters towards LecA and LecB. Table 1. Isothermal titration microcalorimetry (ITC) measurements for binding of ligands FucOMe, 3, 7a, 7c and 7d to LecB and ligands GalOMe,GalOMe, 4, 7b and 7d to LecA.

[a]

[b]

n

Ligand

H°

–TS° –1

–1

G°

Kd –1

(nM)

(kJ.mol )

(kJ.mol )

0.77 ± 0.03

–41.3 ± 1

4.9 ± 1

3

0.90 ± 0.05

–34.3 ± 1.1

–2.9

–37.2

304 ± 31

7a

0.32 ± 0.02

–89.6 ± 2.1

47.8

–41.8

48 ± 0.2

7c

0.22 ± 0.01

–26.7

–5.5

–32.2

2 267 ± 653

FucOMe

[c]

7d

–36.4 ± 0.1

430 ± 10

No binding observed at 1.5 mM

GalOMe

[e]

–39

15

–24 ± 1

70 000

[e]

–40.9 ± 0.3

16.3

–24.5

20 000 ± 300

[e]

–36 ± 1

14 ± 1

–22

150 000 ± 33 000

–104 ± 1

65

–39

176 ± 6

[d]

0.8

GalOMe 

1.0

[g]

1.0

[f]

4

7b

(kJ.mol )

[g]

0.24 ± 0.01

7d

No binding observed at 1.5 mM

[a] Experiments have been duplicated and standard deviations are lower than 10% on stoichiometry and thermodynamic data and lower than 20% on dissociation constants. [b] Binding stoichiometry defined as the number of ligand per monomer of lectin. [c] Data from previous report. previous report.

42

38

[d] Data from

[e] Value fixed during the fitting procedure. [f] Data from previous report.

from previous report.

33


43

[g] Data


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Figure 2. (Top) Raw ITC data obtained by injections of fucosylated glycocluster 7a (150 µM) in LecB solution (50 µM). (Bottom) Corresponding integrated titration curve. Bacterial aggregation properties A series of in vitro aggregation assays were performed to investigate the aggregation properties of the galactosylated and fucosylated ligands towards the PAO1 wild-type strain and its isogenic mutants PAO1ΔlecA and PAO1ΔlecB. The two mutants were used to infer the importance of lectins in glycoside binding specificity. These assays were done at OD600 (optical density at 600 nm) of 1.0. The absence of aggregates in bacterial preparations free of ligands was initially confirmed (Figure 3A and Figure 4δ). Methyl glycosides (FucOMe and GalOMe) ligands in concentrations ranging from 6 to 5000 µM were then studied and, as expected, did not yield any aggregates (Figure 4δ). Tetravalent fucosylated (7a) and galactosylated (7b) glycoclusters in concentrations ranging from 6 to 6600 µM were then tested. Microscopic observations of bacterial-glycoclusters mixtures led to the observation of aggregation phenotypes with filamentous-like (Figure 3B) or spherical (Figure 3C) aggregates. The number of aggregates varied according to the nature and concentration of the tested glycoclusters (Figure 4α and 4β). ACS Paragon Plus Environment

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For the PAO1 strain, only a small number of bacterial aggregates were observed at low (Figure 4-1) and high (Figure 4-3) concentrations of fucosylated and galactosylated glycoclusters. These numbers increased significantly for intermediate concentrations of the fucosylated and galactosylated glycoclusters (Figure 4-2). A study involving a first exposure to galactosylated glycoclusters followed by treatment with fucosylated glycoclusters indicated a greater aggregate forming capability of the fucosylated glycoclusters (Figure 3D). No aggregate was observed for the PAO1∆lecA mutant using the galactosylated glycocluster 7b (Figure 4δ), and significantly reduced numbers were observed for the PAO1∆lecB mutant using the fucosylated glycocluster 7a (Figure 4γ). The aggregation phenotype using the galactosylated glycocluster 7b was therefore LecA-dependent. The involvement of bacterial adhesins other than LecB in the interaction with the fucosylated glycocluster 7a can occur, and LecB-induced interactions were found to greatly increase the number of aggregates. The aggregation assay allowed an investigation of the ability of galactosylated and fucosylated glycoclusters to favour formation of P. aeruginosa aggregates. The most efficient conditions leading to aggregation were those involving intermediate concentrations of glycoclusters (100 to 500 µM). The highest numbers of bacterial aggregates were obtained with the fucosylated tetravalent glycocluster 7a (Figure 4α2, p < 0.01). Lower numbers of aggregates were observed at high concentration of glycocluster (1 000 to 6 600 µM), suggesting a saturation effect of the bacterial cell surface components interacting with such molecules (Figure 4α3 and 43). Much lower numbers of aggregates were also observed at lower concentrations of glycocluster (6 to 33 µM) suggesting a requirement of a critical number of cross-interactions between cells in order to make aggregates of a significant size (Figure 4α1 and 4β1). The best compromise between the number of interacting glycoclusters per cell and a significant formation of aggregates appeared to be obtained at intermediate concentrations around 100 µM (Figure 4α2 and 4β2). This latter concentration of 100 µM was used in a series of additional aggregation assays investigating the specificity of the interactions using monovalent (methyl -D-mannopyranoside – ManOMe and methyl -D-glucopyranoside – GlcOMe) and tetravalent mannosylated (7c) and glucosylated (7d) glyACS Paragon Plus Environment


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coclusters at 100 µM (Figure S18). No aggregate was observed using GlcOMe and the glucosylated glycocluster 7d on the PAO1 strain and its two lectin mutants. For the mannosylated glycocluster 7c, about 70 ± 21 aggregates per 100 µL of PBS cell solution were observed for PAO1 strain, and about 11 ± 4 aggregates for PAO1ΔlecB mutant. This confirms that mannose is a likely ligand for LecB. Other bacterial adhesins could also interact with the mannosylated glycocluster 7c (as previously observed during assay with fucosylated glycoclusters and the PAO1lecB mutant) leading to a residual aggregation effect with the PAO1ΔlecB cells. No aggregate was observed using ManOMe on the PAO1 strain and its two lectin mutants. Fucosylated and galactosylated glycoclusters were further tested in these sets of aggregation assays on the PAO1 strain at 100 µM, and led, respectively, to about 290 ± 40 and 184 ± 34 aggregates per 100 µL of PBS cell solution. These datasets are in agreement with previous observations (Figure 4α2 and 4β2, respectively). About 50 ± 20 aggregates were observed for PAO1ΔlecB mutant using fucosylated glycocluster 7a, while no aggregate was observed for PAO1ΔlecA mutant using galactosylated glycocluster 7b, which is also in agreement with previous datasets (Figure 4γ2 and 4δ2, respectively). A complete set of representative micrographs of the observed aggregates is provided in the supporting information (Figure S18).


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A. Negative control

B. Compound 7a (100 µM)

C. Compound 7b (6 600 µM)

D. Compounds 7a (500 µM) and 7b (660 µM)

Figure 3. Representative micrographs of optical microscopy observations (400×) of bacterial cell aggregates for Pseudomonas aeruginosa PAO1 strain. (A) As a negative control in the absence of methyl glycosides or tetravalent glycoclusters (7a and 7b). (B) With 100 µM final concentration of fucosylated glycocluster (7a). (C) With 6 600 µM final concentration of galactosylated glycocluster (7b). (D) With a mixture of fucosylated and galactosylated glycoclusters (7a and 7b) at 500 and 660 µM final concentrations, respectively. Scale bars: 10 µm.



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Figure 4. Estimated number of aggregates of PAO1, PAO1ΔlecA and PAO1ΔlecB cells (at OD600 of 1.0) in the presence of various concentrations of methyl glycosides or tetravalent glycoclusters (7a and 7b). Three independent slides were analyzed per assay. Numbers of aggregates were estimated for a total volume of 15 µL per slide. Data are expressed per 100 µL of reaction volume. Results are mean±sem. Letter codes indicate statistical test groups used in order to identify conditions favoring the formation of bacterial aggregates. Same letter codes indicate no significant difference between tests (p > 0.05). Different letter codes indicate significant difference between tests (p < 0.05). (δ) Other aggregation assays involving different concentrations of methyl glycosides or tetravalent glycoclusters (7a and 7b) and bacterial strains (PAO1, PAO1∆lecA, and PAO1∆lecB) where no aggregate was observed. (α) Aggregation assays involving PAO1 strain and different concentrations of compound 7a. (β) Aggregation assays involving PAO1 strain and different concentrations of compound 7b. (γ) Aggregation assays involving PAO1∆lecB strain and different con-centrations of compound 7a. The numbered bars present the three groups of low (1), intermediate (2), and high (3) concentrations of ligands.


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Cell adhesion assays The involvement of P. aeruginosa lectins in binding to host cells was further investigated in an in vitro cell culture model using A549 lung epithelial cells. The possibility to interfere with lectin-dependent cell adhesion was investigated using the glycoclusters. A dose-dependent inhibition of adhesion was observed regardless of the compounds assayed. Inhibition of adhesion reached 70% and 90% with galactosylated (7b) and fucosylated (7a) glycoclusters respectively while inhibition only reached 30% and 65% with monovalent GalOMe and FucOMe ligands (Figure 5). The greater effects of fucosylated in comparison to galactosylated compounds, whether monovalent or tetravalent, can be explained by the higher affinities of LecB for fucose (µM) than LecA for galactose (mM). The greater inhibitory effects of glycoclusters 7a and 7b compared to FucOMe and GalOMe may be explained by the higher affinities of tetravalent ligands for LecB and LecA respectively as measured by ITC (vide supra). Fucosylated glycocluster 7a and FucOMe both had an inhibitory effect on adhesion of PAO1lecA strains albeit to a lesser extent than on the wild type PAO1 strain, but not on PAO1lecB. On the opposite, GalOMe and the galactosylated glycocluster 7b lost their effect on PAO1lecA, albeit with remaining activity at high concentrations (250 and 2500 µM) for the latter compound. This suggests that galactosylated glycoclusters may interact with other galactose specific lectins which did not display significant effect n the presence of LecA (PAO1 wild type).



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Figure 5. Bacterial adhesion to A549 cells (3 h infection, MOI 10) in the presence or absence of different carbohydrates at increasing concentrations (μM) after washing off (5 times) excess non-adherent bacteria with PBS. (a) and (b) Inhibition of adhesion of wild-type PAO1 P. aeruginosa. All results are compared to the first column without adhesion inhibitors (PAO1, positive control) (a) fucosylated comACS Paragon Plus Environment

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pounds and (b) galactosylated compounds; (c) and (d) Inhibition of adhesion of PAO1ΔlecA strain. All results are compared to the second column without adhesion inhibitors (PAO1ΔlecA), the first column showing the PAO1 wild-type strain remains as a control (c) fucosylated compounds and (d) galactosylated compounds, (e) and (f) Inhibition of adhesion of PAO1ΔlecB strain. All results are compared to the second column without adhesion inhibitors (PAO1ΔlecB), the first column showing the PAO1 wild-type strain remains as a control (e) fucosylated compounds and (f) galactosylated compounds. All experiments were performed in triplicate. Results are mean±sem. (* p < 0.05); NS: not significant. Additionally, confocal microscopy showed that the bacterial cells are located in close contact to host cell membranes, a localization that is reduced with increasing concentrations of either 7a or 7b (Figure 6), suggesting that glycoclusters are truly interfering with bacterial cell-host interactions.



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Figure 6. Confocal microscopy of murine peritoneal macrophages, nuclei stained with 4’,6’diamidino-2-phenylindole (DAPI), infected by PAO1 strain with a GFP plasmid at MOI 20 alone (rightmost panel) or in the presence of different carbohydrates at either 250 µM or 2500 µM concentrations after washing off (5 times) excess non-adherent bacteria with PBS.

Biofilm studies The potential effects of GalOMe and of the galactosylated glycocluster 7b and of αFucOMe and of the fucosylated glycocluster 7a on LecA- and LecB-dependent biofilms were investigated with concentrations of 0.1 to 5 mM. While GalOMe and methyl FucOMe did not have any significant effect on biofilm formation in the wild-type strain, the tetravalent glycoclusters 7a (Figure 7a) and 7b (Figure 7b) proved beneficial and significantly reduced the PAO1 biofilm formed after 24h at 5 mM, although lower concentration (0.1 mM) did not alter biofilm formation, an effect not observed with the glucosylated glycocluster 7d (Figure S19a). Importantly, these compounds only affected biofilm formation and not bacterial growth since no change in OD600 could be observed (data not shown). To check whether this effect on whole cells was mainly due to lectins, GalOMe and the galactosylated glycocluster 7b on one hand, and FucOMe and the fucosylated glycocluster 7a on the other hand, were tested on lecA and lecB mutants, respectively. This effect was still observed in both mutants at 5 mM of respective glycocluster (7b and 7a), suggesting that tetravalent galactosylated and fucosylated glycoclusters act on other potential targets beside LecA and LecB that remain to be identified (Figure 7c-d). However, this effect was specific since no effect was observed for tetravalent glucosylated (7d) or mannosylated (7c) glycoclusters (Figure S19).



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Figure 7. Quantification of P. aeruginosa biomass in biofilm after 24 h in minimal medium supplemented with vehicle (Veh) or various concentrations of glycoclusters 7a (a) or 7b (b), GalOMe or FucOMe using crystal violet coloration and extraction (* p < 0.01). Strains used were the wild type PAO1 and the isogenic lecA and lecB mutants (c) and (d) respectively.

In vivo lung infection model Glycoclusters 7a and 7b were further evaluated in vivo using a lung infection model was then investigated. The model is designed to assess the effect of antimicrobials and/or adjuvant therapies P. aeruginosa-induced lung injury by measuring the alveolar-capillary permeability of a fluorescent protein (Figure 8). Protein leakage from the serum into the lungs is measured by injecting a tracer (FITCalbumin) and then recovering leaked tracer into the lung through bronchoalveolar lavage 2h later; the proportion of protein leak calculated as the permeability index being correlated to lung injury.44



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Figure 8. In vivo alveolar-capillary barrier permability measurement. While intranasal instillation of saline did not lead to any significant increase in alveolar-capillary permeability, a significant increase up to 4% was caused as expected by P. aeruginosa lung infection (Figure 9). Pre-incubation of bacteria with either αFucOMe (5 mM) or βGalOMe (5 mM) did not significantly affect the injury due to P. aeruginosa. However, both tetravalent glycoclusters 7a and 7b at 1 mM or 5 mM decreased lung permeability by half, to 2%, (Figure 9a). Concomitantly, bacterial load was decreased by more than 3 log CFU in the lung and in the spleen with the tetravalent glycoclusters 6 and 8 (Figure 9b and 9c). While compound 6 led to a decrease of more than 3 log of P. aeruginosa CFU/g in the lung regardless of either 1 mM or 5 mM concentrations, compound 8 led to a decrease of only one log CFU in the lung, and only at the highest concentration (5 mM). Only multivalent glycoclusters displayed protective action against lung infection while the tetravalent carbohydrates were inactive.


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Figure 9. In vivo lung injury as assessed by alveolar-capillary barrier permeability to proteins and bacterial burden as assessed by bacterial load in lung and spleen (** p < 0.001, * p < 0.05). (a) Lung permeability, (b) bacterila load remaining in lungs and (c) bacterial load measured in spleen. ACS Paragon Plus Environment


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CONCLUSION The opportunistic pathogen Pseudomonas aeruginosa is a major cause of pulmonary infections. The increasing multidrug resistance observed for this pathogen compromises antibiotic therapy. While several recent reports have demonstrated the efficacy of monosaccharides against pulmonary infection or bacterial cell adhesion, the design of several multivalent glycosylated macromolecules has proved also beneficial on biofilm dispersion. Calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized through azide-alkyne 1,3-dipolar cycloaddition. Their binding properties towards LecA and LecB, the two majors soluble lectins of Pseudomonas aeruginosa involved in biofilm formation, have then been evaluated by ITC. These glycoclusters induced bacterial clumping in a LecA-dependent and LecB-independent manner. Their anti-biofilm formation properties were also demonstrated only with the multivalent glycoclusters being active while the monosaccharides were not effective. Whether they could participate in biofilm dispersion remains to be shown. Bacterial adhesion to epithelial cells was also inhibited by these glycoclusters as further proof of potential anti-adhesive properties of these molecules. Furthermore, protection against lung injury could be observed in a mouse pulmonary lung infection model. The nanomolar affinities observed by ITC towards the lectins did not translate into nanomolar activities in vivo but rather millimolar concentrations were required. The several competing binding events possible in bacterial, cellular and in vivo assays with other unidentified adhesins, lectins or proteins could explain the higher concentrations required. While previous studies focused on biofilm dispersion45-48 or inhibition of bacterial adhesion to glycosylated surfaces,49 the results presented here (Table 2) represent the most complete set of data in the field of multivalent glycoconjugates as anti-infectious agents against Pseudomonas aeruginosa infection. The tetravalent fucosylated and galactosylated glycoclusters appear to be active both at target level and function level but uncertainties remain concerning the mechanisms of action. Table 2: Summary of biological properties for the calixarene-based glycoclusters Calix-Fuc4 (7a)

Calix-Gal4 (7b)

Calix-Man4 (7c)


Calix-Glc4 (7d)

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Journal of Medicinal Chemistry ITC (Kd)

48 nM (LecB)

176 nM (LecA)

2.3 µM (LecB)

> 1.5 mM (LecA and LecB)

Bacterial aggregation

+++

++

+

-

Biofilm inhibition

+++

+++

-

-

Cell adhesion (A549)

+++

++

Not assayed

Lung infection (mice)

+++

+

Not assayed

The requirement for high concentration (mM) for efficiency in animal models indicates that, as may be expected, the system is more complex in vivo than in vitro. Availability or accessibility of the soluble lectins can be an issue, together with competing with very high affinity natural ligands. Also, the presence of competing unidentified adhesins or other lectins could explain the higher concentrations required, as well as the unexpected action of glycoclusters on biofilm formation in deletion mutants lacking the soluble lectins. Nevertheless, the present results help in confirming the cytotoxic effect of LecA. The lectin effect on alveolar permeability is in agreement with its capacity to cluster glycolipids and to affect cell membrane dynamics.16 LecB appears to be clearly involved in the interaction of P. aeruginosa with its environment, playing a role in both attachment to epithelial cells and formation of bacterial microcolonies. This confirms the ability of inhibiting P. aeruginosa adhesion to epithelial cells using LecB high affinity ligands.21,49 Furthermore, a LecB-related lectin present in Bukholderia cenocepacia has indeed been demonstrated to efficiently bind to heptose residues present in the cell wall of gramnegative bacteria.50 The involvement of LecA and LecB in biofilm is however not clarified: while us and others demonstrated the efficiency of glycoclusters for inhibition or dispersion of biofilm, 45-48 the present data indicate that other receptors, such as fimbriae, are also involved in carbohydrate-based biofilm cohesion. In conclusion, even if not all molecular mechanisms could be elucidated, we could provide a complete panel of target-based and function-based assays of the action of tetravalent glycoclusters on P. aeruginosa lectins. EXPERIMENTAL SECTION ACS Paragon Plus Environment


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Synthesis of glycoclusters General Methods. All reagents for synthesis were commercial and used without further purification. Solvents were purchased dry or methanol was distilled over Mg/I2. All reactions were performed under an Argon atmosphere. Reactions under microwave activation were performed on a Biotage Initiator system. NMR spectra were recorded at 293 K, unless otherwise stated, using a 300 MHz or a 400 MHz Bruker Spectrometer. Shifts are referenced relative to deuterated solvent residual peaks. The following abbreviations are used to explain the observed multiplicities: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet and bs, broad singlet. Assignments were deduced from 2D experiments (HSQC and HMBC). High resolution (HR-ESI-QTOF) mass spectra were recorded using a Bruker MicrOTOF-Q II XL spectrometer. Thin-layer chromatography (TLC) was carried out on aluminum sheets coated with silica gel 60 F254 (Merck). TLC plates were inspected by UV light (λ = 254 nm) and developed by treatment with a mixture of 10% H2SO4 in EtOH/H2O (95:5 v/v) followed by heating. Silica gel column chromatography was performed with silica gel Si 60 (40–63 µm). The glycoclusters tested in bioassays were purified using automated purification systems with medium pressure chromatography on reverse C18 silica gel. Their purity was verified by 1H and 13C NMR techniques indicating ca. 95% purity (data provided in the supporting information). Optical rotation was measured using a Perkin Elmer polarimeter and values are given in 10-1 deg.cm2.g-1.

1-Azido-3,6-dioxaoct-8-yl 2,3,4-tri-O-acetyl--L-fucopyranoside (1a).36 Sulfuric acid (3 mL, 95%) was added slowly and under vigourous stirring to a suspension of silica gel (10 g) in Et 2O (50 mL). After addition, the solvent was evaporated off and the powder dried at 100°C during 4 h. A suspension of L-fucose

(15 g, 90 mmol) and 2-[2-(2-chloroethoxy)ethoxy]ethanol (45 mL, 295 mmol, 3.3 eq.) was

placed in a pre-heated (120°C oil) bath and H2SO4-SiO2 catalyst (300 mg) was added immediately after. The reaction was stirred vigorously at 120°C for 1 h then cooled to r.t. within 15 minutes. The crude mixture was purified by silica gel column chromatography (CH2Cl2/MeOH 9/1) to afford the 1-chloro3,6-dioxaoct-8-yl L-fucopyranoside intermediate (14.75 g, 52%) as an inseparable mixture of anomers ACS Paragon Plus Environment

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(α:β, 3:1) and as a yellow oil. The chlorinated intermediate (5.28 g, 16.8 mmol) was dissolved in DMF (170 mL) with nBu4NI (1.24 g, 3.4 mmol, 0.2 eq.) and sodium azide (5.45 g, 83.9 mmol, 5 eq.). The reaction was stirred at 70°C during 16 h then filtered and purified by silica gel column chromatography (CH2Cl2/MeOH 9/1) to afford the 1-azido-3,6-dioxaoct-8-yl L-fucopyranoside intermediate (4.8 g, 89%) as an inseparable mixture of anomers (α:β, 3:1) and as a yellow oil. The azido-functionalized intermediate was dissolved (5.6 g, 17.5 mmol) in dry pyridine (110 mL) with DMAP (475 mg, 3.9 mmol, 0.2 eq.) then acetic anhydride (70 mL, 736 mmol, 42 eq.) was added at 0°C. The reaction was stirred at r.t. during 16 h. The solution was diluted with EtOAc (500 mL), washed with 1N HCl (5×300 mL), saturated NaCl (2×300 mL), saturated NaHCO3 (3×300 mL), saturated NaCl (2×300 mL). The organic layer was dried (Na2SO4), filtered and evaporated to dryness. The crude mixture was purified by silica gel column chromatography (PE/CHCl3/Acetone 3/1/1) to afford 1a (5.18 g, 65%) as the pure  anomer and as a yellow oil. Data was in agreement with the literature.36

1-(4-Acetoxymethyl-1,2,3-triazol-1-yl)-3,6-dioxaoct-8-yl

2,3,4-tri-O-acetyl--L-fucopyranoside

(2). DIPEA (20 µL, 0.11 mmol, 0.75 eq.) was added to a solution of propargyl acetate (75 µL, 0.75 mmol, 5 eq.), CuI (4 mg, 0.02 mmol, 0.1 eq.) and azido-functionalized fucoside 1a (75 mg, 0.17 mmol) in dry and degassed DMF (2.5 mL) in a Biotage Initiator 2-5 mL vial. The vial was flushed with argon and protected from light (aluminum sheet) and the solution was sonicated for 30 seconds. The vial was sealed with a septum cap and heated at 110°C under microwave irradiation (solvent absorption level: High) during 15 min. After uncapping the vial, the crude mixture was purified by silica gel column chromatography (EtOAc/MeOH 95/5) to afford 2 (73 mg, 80%) as a pale yellow oil. Rf = 0.68 (EtOAc /MeOH 9/1). []20D = – 70.6 (c 0.71 / MeOH). 1H NMR (CD3OD, 400 MHz): δ 8.07 (s, 1H, H-triaz), 5.33 (dd, 1H, J = 10.7 Hz, J = 3.4 Hz, H-3), 5.29-5.24 (m, 1H, H-4), 5.18 (s, 2H, CH2C-triaz), 5.09 (d, 1H, J = 3.7 Hz, H-1), 5.04 (dd, 1H, J = 10.7 Hz, J = 3.7 Hz, H-2), 4.62-4.57 (m, 2H, OCH2CH2N), 4.28 (q, 1H, J = 6.5 Hz, H-5), 3.95-3.87 (m, 2H, CH2CH2N), 3.82-3.56 (m, 8H, OCH2CH2O), 2.15, 2.06, 2.02, 1.95 (4s, 12H, COCH3), 1.11 (d, 3H, J = 6.5 Hz, H-6) ppm. ACS Paragon Plus Environment

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C NMR (CD3OD, 100 MHz): δ


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172.3, 172.3, 172.0, 171.7 (4s, 4C, COCH3), 143.9 (CIV-triaz), 126.6 (CH-triaz), 97.5 (C-1), 72.6 (C-4), 71.6, 71.5, 71.4, 68.5 (3s, 3C, OCH2CH2O), 70.4 (OCH2CH2N), 69.5, 69.4 (2s, 2C, C-2, C-3), 65.7 (C5), 58.3 (OCH2C-triaz), 51.5 (OCH2CH2N), 20.7, 20.7, 20.6, 20.5 (4s, 4C, COCH3), 16.2 (C-6) ppm. HR-ESI-MS: m/z calc. for C23H36N3O12 [M+Na]+ 546.2294, found 546.2283.

1-(4-Hydroxymethyl-1,2,3-triazol-1-yl)-3,6-dioxaoct-8-yl -L-fucopyranoside (3). A solution of compound 2 (73 mg, 0.13 mmol) in MeOH (8 mL), water (2 mL) and triethylamine (2 mL) was stirred at r.t. during 18 h. The solvents were evaporated off then co-evaporated with toluene (3×50 mL) to afford the hydroxylated fucoside 3 (49 mg, 98%) as a pale yellow gum. Rf = 0.06 (EtOAc/MeOH 9/1). []20D = – 68.4 (c 0.43 / MeOH). 1H NMR (CD3OD, 400 MHz): δ 7.98 (s, 1H, H-triaz), 4.79 (d, 1H, J = 2.9 Hz, H-1), 4.68 (s, 2H, OCH2C-triaz), 4.61-4.54 (m, 2H, CH2CH2N), 3.99 (q, 1H, J = 6.6 Hz, H-5), 3.92-3.88 (m, 2H, CH2CH2N), 3.81-3.57 (m, 11H, OCH2CH2O, H-2, H-3, H-4), 1.20 (d, 3H, I = 6.6 Hz, H-6) ppm. 13C NMR (CD3OD, 100 MHz): δ 149.0 (CIV-triaz), 124.9 (CH-triaz), 100.7 (C-1), 73.6 (C4), 71.7 (C-2 or C-3), 71.5, 71.4, 71.4, 68.2 (4s, 4C, OCH2CH2O), 70.5 (CH2CH2N), 70.1 (C-2 or C-3), 67.6 (C-5), 56.5 (OCH2C-triaz), 51.4 (CH2CH2N), 16.7 (C-6) ppm. HR-ESI-MS: m/z calc. for C15H27N3NaO8 [M+Na]+ 400.1690, found 400.1682.

25,26,27,28-Tetra-{1-[1-(2,3,4-tri-O-acetyl--L-fucopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3triazol-4-ylmethyloxy}-p-tert-butyl-calix[4]arene (6a) (1,3-alternate conformation). A solution of alkynylated calix[4]arene 5 (250 mg, 0.3 mmol), CuI (29 mg, 0.15 mmol, 0.5 eq.), azido-functionalized fucoside 1a (838 mg, 1.87 mmol, 6 eq.) and DIPEA (260 µL, 1.5 mmol, 5 eq.) in dry and degassed DMF (5 mL) was introduced in a Biotage Initiator 2-5 mL vial. The vial was flushed with argon and protected from light (aluminum sheet) and the solution was sonicated for 30 seconds. The vial was sealed with a septum cap and heated at 110°C under microwave irradiation (solvent absorption level: High) during 15 min. After uncapping the vial, the crude mixture was purified by silica gel column ACS Paragon Plus Environment

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chromatography (EtOAc/MeOH 95/5) to afford 6a (659 mg, 85%) as a white foam. Rf = 0.73 (EtOAc/MeOH 9/1). []20D = – 67.9 (c 0.76 / MeOH). 1H NMR (CDCl3, 400 MHz): δ 7.26 (s, 4H, Htriaz), 6.87 (s, 8H, H-ar), 5.38-5.32 (m, 4H, H-3), 5.28 (m, 4H, H-4), 5.11 (m, 4H, H-2), 5.09 (m, 4H, H1), 4.52 (m, 16H, OCH2C-triaz, OCH2CH2N), 4.20 (q, 4H, J = 6.5 Hz, H-5), 3.89 (t, 4H, J = 5.2 Hz, CH2OCH2CH2O), 3.76 (m, 4H, ½ FucOCH2), 3.61 (m, 28H, FucOCH2CH2, ½ FucOCH2, CH2OCH2CH2N), 3.47 (s, 8H, Ar-CH2-Ar), 2.15, 2.04, 1.97 (3s, 36H, 3×CH3CO), 1.12 (d, 12H, J = 6.5 Hz, H-6), 1.07 (s, 36H, C(CH3)3) ppm. 13C NMR (CDCl3, 100 MHz): δ 170.8, 170.5, 170.2 (3s, 12C, CH3CO), 153.6 (CIV-ar-O), 144.8, 144.8 (2s, 8C, CIV-triaz, CIV-ar-tBu), 133.8 (CIV-ar-CH2), 127.1 (CHar), 124.0 (CH-triaz), 96.4 (C-1), 71.3 (C-4), 70.7, 70.7, 70.3, 69.6 (4s, 16C, CH2OCH2), 68.3, 68.1 (2s, 8C, C-2, C-3), 67.5 (FucOCH2), 64.5 (C-5), 64.7 (OCH2C-triaz), 50.2 (OCH2CH2N), 38.4 (ar-CH2-ar), 33.9 (C(CH3)3), 31.6 (C(CH3)3), 21.0, 20.9, 20.8 (3s, 12C, CH3CO), 16.0 (C-6) ppm. HR-ESI-MS: m/z calc. for C128H181N12O44 [M+H]+ 2590.2289, found 2590.2198.

25,26,27,28-Tetra-{1-[1-(2,3,4,6-tetra-O-acetyl--D-mannopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3triazol-4-ylmethyloxy}-p-tert-butyl-calix[4]arene (6c) (1,3-alternate conformation). A solution of alkynylated calix[4]arene 5 (1.2 g, 1.49 mmol), CuI (142 mg, 0.74 mmol, 0.5 eq.), azido-functionalized mannoside 1c (3.78 g, 7.47 mmol, 5 eq.) and DIPEA (1.3 mL, 7.47 mmol, 5 eq.) in dry and degassed DMF (20 mL) was introduced in a Biotage Initiator 15-20 mL vial. The vial was flushed with argon and protected from light (aluminum sheet) and the solution was sonicated for 30 seconds. The vial was sealed with a septum cap and heated at 110°C under microwave irradiation (solvent absorption level: High) during 25 min. After uncapping the vial, the crude mixture was diluted with EtOAc (500 mL) and the organic layer washed with aqueous EDTA (0.1 M, 2×150 mL). The organic layer was dried (MgSO4), filtered, evaporated and purified by silica gel column chromatography (EtOAc/MeOH 95/5) to afford 6c (4.12 g, 98%) as a white foam. []20D = + 24.8 (c 1.0 / CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ 7.22 (s, 4H, H-triaz), 6.82 (s, 8H, H-ar), 5.27-5.22 (m, 8H, H-3 H-4), 5.20 (m, 4H, H-2), 4.81 (m, 4H, H-1), 4.53-4.40 (m, 16H, OCH2C-triaz, OCH2CH2N), 4.23 (dd, 4H, J = 3.0, 12.0 Hz, H-6), ACS Paragon Plus Environment


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4.07-3.95 (m, 8H, H-5, H-6’), 3.85 (t, 4H, J = 4.0 Hz, CH2OCH2CH2O), 3.74 (m, 4H, ½ ManOCH2), 3.57 (m, 28H, ManOCH2CH2, ½ ManOCH2, CH2OCH2CH2N), 3.43 (s, 8H, Ar-CH2-Ar), 2.09, 2.04, 1.98, 1.93 (4s, 48H, 4×CH3CO), 1.02 (s, 36H, C(CH3)3) ppm.

13

C NMR (CDCl3, 100 MHz): δ 170.6,

170.0, 169.9, 169.7 (4s, 16C, CH3CO), 153.4 (CIV-ar-O), 144.6, 144.3 (2s, 8C, CIV-triaz, CIV-ar-tBu), 133.5 (CIV-ar-CH2), 126.9 (CH-ar), 123.9 (CH-triaz), 97.6 (C-1), 70.6, 70.5, 70.0, 69.5, 68.4, 67.3 (C-2, C-3, C-4, CH2OCH2, FucOCH2), 69.0 (C-5), 66.1 (OCH2C-triaz), 62.3 (C-6), 49.9 (OCH2CH2N), 38.2 (Ar-CH2-Ar), 33.7 (C(CH3)3), 31.4 (C(CH3)3), 20.9, 20.72, 20.68, 20.65 (4s, 16C, CH3CO) ppm. HRESI-MS: m/z calc. for C136H189N12O52 [M+H]+ 2823.2508, found 2823.2384.

25,26,27,28-Tetra-{1-[1-(2,3,4,6-tetra-O-acetyl--D-glucopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3triazol-4-ylmethyloxy}-p-tert-butyl-calix[4]arene (6d) (1,3-alternate conformation). A solution of alkynylated calix[4]arene 5 (538 mg, 0.67 mmol), CuI (64 mg, 0.33 mmol, 0.5 eq.), azidofunctionalized glucoside 1d (1.69 mg, 3.35 mmol, 5 eq.) and DIPEA (583 µL, 3.35 mmol, 5 eq.) in dry and degassed DMF (10 mL) was introduced in a Biotage Initiator 15-20 mL vials. The vial was flushed with argon and protected from light (aluminum sheet) and the solution was sonicated for 30 seconds. The vial was sealed with a septum cap and heated at 110°C under microwave irradiation (solvent absorption level: High) during 25 min. After uncapping the vial, the crude mixture was diluted with EtOAc (100 mL) and the organic layer washed with aqueous EDTA (0.1 M, 2×100 mL). The organic layer was dried (MgSO4), filtered, evaporated and purified by silica gel column chromatography (EtOAc/MeOH 95/5) to afford 6d (1.35 g, 71%) as a white foam. []20D = – 2.7 (c 1.0 / CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ 7.26 (s, 4H, H-triaz), 6.87 (s, 8H, H-ar), 5.19 (t, 4H, J = 8.0 Hz, H-3), 5.06 (t, 4H, J = 8.0 Hz, H-4), 4.97 (t, 4H, J = 8.0 Hz, H-2), 4.64-4.38 (m, 8H, OCH2C-triaz), 4.56 (d, 4H, J = 8.0 Hz, H-1), 4.25 (dd, 4H, J = 4.0, 12.0 Hz, H-6), 4.13-4.08 (m, 8H, H-6’), 3.96-3.82 (m, 8H, CH2OCH2CH2O), 3.72-3.67 (m, 4H, H-5), 3.66-3.41 (m, 40H, ManOCH2, OCH2CH2N, CH2OCH2CH2N), 3.49 (s, 8H, Ar-CH2-Ar), 2.06, 2.03, 2.01, 1.99 (4s, 48H, 4×CH3CO), 1.05 (s, 36H, C(CH3)3) ppm. 13C NMR (CDCl3, 100 MHz): δ 170.8, 170.3, 169.5, 169.4 (4s, 16C, CH3CO), 154.2 (CIV-ar-O), 144.9, 144.6 (2s, 8C, CIV-triaz, CIVACS Paragon Plus Environment

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ar-tBu), 134.2 (CIV-ar-CH2), 127.4 (CH-ar), 124.2 (CH-triaz), 100.9 (C-1), 72.9 (C-3), 71.9 (C-5), 71.3 (C-2), 70.74, 70.67, 70.3, 69.3 (CH2OCH2, GlcOCH2, OCH2C-triaz), 50.2 (OCH2CH2N), 38.8 (Ar-CH2Ar), 34.2 (C(CH3)3), 31.5 (C(CH3)3), 20.88, 20.83, 20.73, 20.72 (4s, 16C, CH3CO) ppm. HR-ESI-MS: m/z calc. for C136H191N12O52 [M+3H]3+ 941.4218, found 941.4258.

25,26,27,28-Tetra-{1-[1-(-L-fucopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-ylmethyloxy}p-tert-butyl-calix[4]arene (7a) (1,3-alternate conformation). A solution of compound 6a (659 mg, 0.25 mmol) in MeOH (27 mL), water (6 mL) and triethylamine (6 mL) was stirred at r.t. during 18 h. The solvents were evaporated off then co-evaporated with toluene (3×50 mL). C18 MPLC column chromatography (H20 then H2O/MeOH 1:1) afforded the hydroxylated glycocluster 7a (487 mg, 92%) as a white foam. Rf = 0.90 (MeOH/CH2Cl2/H2O 8/2/1). []20D = – 57.5 (c 1.11 / MeOH). 1H NMR (CD3OD, 400 MHz): δ 7.60 (s, 4H, H-triaz), 6.94 (s, 8H, H-ar), 4.77 (d, 4H, J = 3.5 Hz, H-1) 4.64 (s, 8H, OCH2C-triaz), 4.58 (t, 8H, J = 6.8 Hz, OCH2CH2N), 3.97 (d, 4H, J = 9.0 Hz, H-5), 3.91 (m, 8H, OCH2CH2O), 3.80-3.68 (m, 16H, H-2, H-3, OCH2CH2N), 3.65 (m, 28H, H-4, OCH2CH2O, OCH2CH2O), 3.54 (d, 8H, J = 12.1 Hz, Ar-CH2-Ar), 1.18 (d, 12H, J = 8.8 Hz, H-6), 1.09 (s, 36H, C(CH3)3) ppm. 13C NMR (CD3OD, 100 MHz): δ 155.0 (CIV-ar-O), 145.8, 145.4 (2s, 8C, CIV-triaz, CIVar-tBu), 134.7 (CIV-ar-CH2), 128.2 (CH-ar), 126.1 (CH-triaz), 100.7 (C-1), 73.6 (C-4), 71.7 (C-2 or C3), 71.5, 71.5, 71.4, 70.5 (4s, 16C, CH2OCH2), 70.1 (C-2 or C-3), 68.3 (FucOCH2), 67.6 (C-5), 65.4 (OCH2C-triaz), 51.3 (OCH2CH2N), 38.9 (ar-CH2-ar), 34.8 (C(CH3)3), 32.2 (C(CH3)3), 16.8 (C-6) ppm. HR-ESI-MS: m/z calc. for C104H158N12O32 [M+2H]2+ 1043.5547, found 1043.5524.

25,26,27,28-Tetra-{1-[1-(-D-mannopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-ylmethyloxy}p-tert-butyl-calix[4]arene (7c) (1,3-alternate conformation). A solution of compound 6c (4.12 g, 1.45 mmol) in MeOH (20 mL), water (5 mL) and triethylamine (5 mL) was stirred at r.t. during 18 h. The solvents were evaporated off then co-evaporated with toluene (3×50 mL). C18 MPLC column chromatography (H2O then H2O/MeOH 1:1) afforded the hydroxylated glycocluster 7c (1.92 g, 61%) as a white ACS Paragon Plus Environment


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foam. []20D = + 16.2 (c 1.0 / H2O). 1H NMR (D2O, 400 MHz): δ 7.71 (s, 4H, H-triaz), 6.89 (s, 8H, Har), 4.81 (m, 4H, H-1, overlapped with D2O), 4.55 (m, 16H, OCH2C-triaz, OCH2CH2N), 3.90 (m, 4H, H-2), 3.84-3.68 (m, 20H, H-3, H-4, H-5, H-6, H-6’), 3.66-3.52 (m, 40H, CH2OCH2) 3.32 (s, 8H, ArCH2-Ar), 1.02 (s, 36H, C(CH3)3) ppm. 13C NMR (D2O, 100 MHz): δ 153.3 (CIV-ar-O), 144.7, 144.2 (2s, 8C, CIV-triaz, CIV-ar-tBu), 133.3 (CIV-ar-CH2), 126.7 (CH-triaz), 125.4 (CH-ar), 99.9 (C-1), 72.7, 70.5, 69.9, 69.8, 69.6, 69.5, 68.9, 66.6, 66.3, 63.9 (C-2, C-3, C-4, C-5, CH2OCH2, ManOCH2, OCH2C-triaz) 50.3 (OCH2CH2N), 48.8 (Ar-CH2-Ar), 33.4 (C(CH3)3), 31.0 (C(CH3)3) ppm. HR-ESI-MS: m/z calc. for C104H158N12O36 [M+2H]2+ 1075.5445, found 1075.5441.

25,26,27,28-Tetra-{1-[1-(-D-glucopyranosyloxy)-3,6-dioxaoct-8-yl]-1,2,3-triazol-4-ylmethyloxy}-ptert-butyl-calix[4]arene (7d) (1,3-alternate conformation). A solution of compound 6d (1.35 g, 0.48 mmol) in MeOH (20 mL), water (5 mL) and triethylamine (5 mL) was stirred at r.t. during 18 h. The solvents were evaporated off then co-evaporated with toluene (3×50 mL). C18 MPLC column chromatography (H2O then H2O/MeOH 1:1) afforded the hydroxylated glycocluster 7d (687 mg, 67%) as a white foam. []20D = – 16.8 (c 1.0 / H2O). 1H NMR (D2O, 400 MHz): δ 7.77 (s, 4H, H-triaz), 6.89 (s, 8H, H-ar), 4.65-4.51 (m, 19H, OCH2C-triaz, OCH2CH2N), 4.41 (d, 4H, J = 8.0 Hz, H-1), 3.97 (m, 4H, H-5), 3.92 (m, 8H, OCH2CH2O), 3.85 (dd, 4H, J = 2.0, 12.0 Hz, H-6), 3.77-3.59 (m, 32H, OCH2CH2N, CH2OCH2), 3.46 (t, 4H, J = 8.0 Hz, H-3), 3.40-3.35 (m, 4H, H-4), 3.33 (s, 8H, Ar-CH2-Ar), 3.26 (t, 4H, J = 8.0 Hz, H-2), 1.02 (s, 36H, C(CH3)3) ppm. 13C NMR (D2O, 100 MHz): δ 156.1 (CIV-ar-O), 147.1, 146.7 (2s, 8C, CIV-triaz, CIV-ar-tBu), 135.8 (CIV-ar-CH2), 129.7 (CH-ar), 128.2 (CH-triaz), 104.8 (C-1), 78.4 (C-4), 78.2 (C-3), 75.6 (C-2), 72.3, 72.1, 71.4 (CH2OCH2), 71.1 (C-5), 66.6 (GlcOCH2), 63.3 (OCH2C-triaz), 52.4 (OCH2CH2N), 51.3 (Ar-CH2-Ar), 35.9 (C(CH3)3), 33.5 (C(CH3)3) ppm. HR-ESIMS: m/z calc. for C104H158N12O36 [M+2H+Na]3+ 724.6928, found 724.6960.

Biological studies ACS Paragon Plus Environment

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Isothermal Titration Microcalorimetry (ITC). Purified and lyophilized LecB was dissolved in buffer (0.1 M Tris–HCl buffer containing 6 μM CaCl2, pH 7.5) at a concentration of 0.02 mM and degassed. Carbohydrate ligands were dissolved directly into the same buffer at concentration varying from 0.05 to 0.2 mM, degassed, and placed in the injection syringe. Isothermal titration calorimetry was performed with a VP-ITC MicroCalorimeter from MicroCal Incorporated. LecB was placed into the 1.4478-mL sample cell, at 25°C, using 10-μL injections of glycocluster every 300 s. Carbohydrate ligands were also titrated alone against the buffer. Data were fitted with MicroCal Origin 7 software, according to standard procedures. Fitted data yielded the stoichiometry (n), the association constant (Ka) and the enthalpy of binding (ΔH). Other thermodynamic parameters (i.e. changes in free energy, ΔG, and entropy, ΔS) were calculated from the equation ΔG = ΔH – TΔS = – RT lnKa where T is the absolute temperature and R = 8.314 J.mol−1.K−1. Two or three independent titrations were performed for each ligand tested. Bacterial strains. Pseudomonas aeruginosa PAO1 strain is a sequenced, well characterized strain51 used as reference strain. PAO1∆LecA and PAO1∆LecB are in frame deletion mutants deficient respectively for LecA and LecB. P. aeruginosa expressing Green Fluorescent Protein (GFP) were obtained by co-culture transfer of a mini-CTX-GFP from E. coli strain (Kind gift of Dr Ina Atrée). Plasmidcontaining bacteria were selected using tetracycline (200 µg/mL). Bacteria were grown overnight at 37°C in Luria-Bertani broth (LB), under orbital shaking (400 rpm), harvested by centrifugation (2000 g, 5 min) and washed twice with sterile isotonic saline (SIS). Turbidity was then appreciated by measurement of optical density at 600 nm (OD600) (Ultrospec 10 Cell Density Meter, General Electrics, CT, USA), and verified by serial dilution and plating on Bromocresol purple Agar (BCP) (Biomérieux). The following antibiotic concentrations were used. For E. coli: ampicillin (Ap), 50 µg/mL, kanamycin (Km), 15 µg/mL, streptomycin (Sm), 50 µg/mL. For P. aeruginosa: streptomycin (Sm), 2 mg/mL. Construction of P. aeruginosa deletion mutants. PCR was used to generate a 450 bp and a 519 bp DNA fragment upstream of the lecA and lecB genes, respectively, with the (DellecAUp5, DellecAUp3), (DellecBUp5, DellecBUp3) oligonucleotide pairs (See Table S1, supporting information) and a 446 bp and a 649 bp DNA fragment downstream of the lecA and lecB genes, respectively, using the ACS Paragon Plus Environment


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(DellecADn5, DellecADn3), (DellecBDn5, DellecBDn3) oligonucleotide pairs, (See Table S1, supporting information). The resulting DNA fragments were further used as templates for a second overlapping PCR run using a pair of external oligonucleotides (DellecAUp5, DelecADn3), (DellecBUp5, DellecBDn3) respectively, thus leading to a final approximate 1.1 kb DNA fragment that was cloned into the pCR2.1 vector. The resulting DNA fragment bearing appropriate sites, namely BamHI/EcoRV BamHI/ApaI for lecA and lecB, respectively, was further hydrolyzed and cloned into the suicide vector pKNG101. The recombinant plasmid was then mobilized into P. aeruginosa and the deletion mutants were selected on LB plates containing 6% sucrose and appropriate antibiotics as previously described.52 Preparation of cell line. Adherent A549 cells were cultured in cell culture flasks containing Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal calf serum (Dustcher, Vilmorin, France) and 1% Penicillin/Streptomycin. Cells were incubated at 37°C with a 5% (v/v) CO 2 until they formed a confluent monolayer. Cells were then harvested using a 1% (m/v) trypsin/EDTA solution and resuspended in 96 Well cell culture plates containing DMEM (5.104 cells/well) and incubated at 37°C. Once cells have reached confluence (105 cells/well), they were washed with PBS and non specific binding was blocked by incubating cells during one hour in PBS 1% Bovine serum albumin at 37°C. Wells were then washed twice with PBS. Bacteria calibrated at a density of 108 CFU/mL (Multiplicity of infection (MOI) of 10 bacteria for a cell), were incubated with growing concentration of each carbohydrate (0, 25, 250 and 2500 µM of respectively methyl -L-fucoside, methyl -D-galactoside, glycoclusters 7a or 7b) for one hour at room temperature in DMEM without antibiotics. A hundred microliters of each solution was then transferred in the 96 well cell-culture plate. Each condition was performed in quadruplicate. The plate was incubated during 3 hours in a 5% CO2 atmosphere. Non adherent bacteria were removed by washing 5 times with PBS. Cells were lysed by incubation during 30 minutes at 37°C with PBS 1% triton-X100 (v/v). Live CFU were enumerated after a 24 h incubation at 37°C, after serial dilution and plating on BCP agar plates. The percentage of adhesion was calculated as follows:


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Number of CFU × 100 Mean of Number of CFU in the wells without competitor

Immunofluorescence. Cells were infected with GFP expressing bacteria. Cells were washed five times with PBS and fixed for 20 minutes with 4% formalin, and stained with DAPI for 2 minutes. Immunofluorescence images were obtained using a Zeiss LSM 710. Confocal microscopy. Murine peritoneal macrophages were harvested and cultures in IMDM medium over 48h at 37°C with penicillin/streptomycin 100x. 50000 cells were deposited per well in a black 96well plate. Macrophages were then infected with GFP-strains at MOI 20 during 3 hours. Simultaneously, lectin inhibitors were added to the medium at different concentrations (250 µM or 2500 µM). After incubation at 37°C, wells were washed 5 times with PBS. Then, macrophage nuclei were stained with 4’,6’-diamidino-2-phenylindole (DAPI) (1000x). Cells were fixated with 4% formaldehyde. Plates were read with the LSM 710 confocal micropscope a 120x magnification (Carl Zeiss, Switzerland) at 320/456 nm wavelengths (excitation/emission) for DAPI, and 395/504 nm for GFP. In vivo acute lung infection model was induced by intranasal instillation of P. aeruginosa. C57BL6/J mice were lightly anesthetized with inhaled sevoflurane (Forene Abbott), after which 50 µL of the bacterial solution were administered intra-nasally (1.107 CFU of PAO1 strain). Control mice received 50 µL of pyrogen-free isotonic saline solution. All mice were sacrificed at 24 h. In additional experiments, mice received intra-nasally 5 mM or 1 mM of glycoclusters 7a, 7b or 5 mM of GalOMe or FucOMe. Bronchoalveolar Lavage (BAL). Lungs from each experimental group were washed with a total of 1.5 mL of sterile phosphate-buffered saline (PBS). Recovered lavage fluid was centrifuged (200 g for 10 min) and the supernatant was collected.



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Alveolar-capillary barrier permeability was assessed by protein leak from serum into the airway. Mice received intraperitoneally 100 µL of FITC-albumin (Sigma, France) at a concentration of 2 mg/mL, 2 hours before sacrifice. Bronchoalveolar Lavage (100 µL) and serum (100 µL) were each deposited in 96-well plates and fluorescence was read with Mithras 6400 at 504 nm (Berthold technologies, France). The supernatant to serum fluorescence ratio reflecting permeability were then calculated as previously reported.44 Bacterial burden: Mouse lungs and spleens were homogenized in sterile containers with sterile isotonic water. Homogenates were sequentially diluted and cultured on bromocresol purple agar plates for 24 h at 37°C to assess bacterial load. Statistical analysis. Quantitative variables were analyzed by one-way analysis of variance (ANOVA), with a Mann & Whitney post-hoc test when appropriate. Survival was analyzed with a log-rank test. Statistics and graphs were performed with GraphPad Prism 5® software (GraphPad Software Inc., La Jolla, CA, USA). Qualitative variables were analyzed with a χ2 test. p < 0.05 was considered as significant. Results are mean +/- standard error. Bacterial growth. Pseudomonas aeruginosa wild-type strain PAO1 and its two isogenic mutants PAO1∆lecA and PAO1∆lecB were streaked onto a Pseudomonas agar base selective medium (PAB, CM0559 Oxoïd), supplemented with 0.2 g of cetrimide and 0.015 g of nalidixic acid per liter, and grown overnight at 37°C. P. aeruginosa broths were prepared by inoculating cells from a single colony. P. aeruginosa strains were grown separately in Nutrient Broth (NB, Difco, BD France SA) overnight at 37°C and under orbital shaking at 150 rotations per minute (rpm), on a C24 Incubator Shaker (Edison, New Jersey, USA). 1 mL of each pre-culture was used to inoculate 200 mL of NB flasks. Bacterial growth was assessed by measuring the optical density of the broth at 600 nm (OD600) using an Eppendorf visible spectrophotometer (Eppendorf, Biophotometer). Cells were harvested when reaching an OD 600 of 1.0. 10 mL of each culture was centrifuged at 4000 rpm and 22°C for 10 minutes (Eppendorf centrifuge


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5810 R), and cells were then washed two times with a phosphate-buffered saline solution (PBS), and kept in PBS until performing the aggregation assay. Aggregation assay. Methyl glycosides (GalOMe, ManOMe,FucOMe,GlcOMe) and tetravalent (7a-d) ligands were soluble up to 100 mg.mL-1 at 40°C, and were dissolved directly in PBS. These molecules were filtered through 0.2 µm filters. Stock solutions of glucosylated, mannosylated and fucosylated glycoclusters and methyl glycosides were prepared at a final concentration of 25 mM. Stock solution of galactosylated glycocluster 7b was prepared at a final concentration of 33 mM. P. aeruginosa cells and ligands were mixed (using a pipette cone with a large opening) in order to obtain 6, 25, 100, 250, 500, 1000, 2500, and 5000 µM final concentrations of FucOMe and GalOMe, and tetravalent fucosylated ligands, and 8, 33, 132, 330, 660, 1320, 3300, and 6600 µM final concentrations of multivalent galactosylated ligands at an OD600 of 1.0. An additional bacterial aggregation assay was performed using monovalent and tetravalent glucosylated and mannosylated ligands at a final concentration of 100 µM. These reaction mixtures were left at room temperature for 15 minutes. Fifteen microliters of these solutions were then transferred onto a glass slide for microscopic observations. Each condition was performed in triplicate. Microscopic observations were performed at 400 magnification using an optical microscope (Carl Zeiss S.A.S., Zeiss Axioskope), and image analysis was done using an Axiovision software (AxioVs40 V 4.8.2.0, Carl Zeiss MicroImaging). The number of aggregates was estimated by analysis of 40 different fields of the slide. Aggregate status validation was confirmed according to (1) a minimal surface size of aggregate greater than 100 µm2, and (2) the observation of a mass of bacterial cells forming clusters with filamentous of spherical forms. Results were analyzed using the Kruskal-Wallis rank sum test and Wilcoxon-Mann-Whitney rank sum test53 in order to identify conditions favoring the formation of bacterial aggregates.

Biofilm assays. The bacterial macroscopic adhesion assay was performed in 96 microplates. Overnight cultures were diluted at an initial OD600 of 0.1 in M63 medium supplemented with 1 mM MgCl2, 5 ACS Paragon Plus Environment


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mM CaCl2, 0.5% casamino acids, 0.2% glucose in the absence or presence of various concentrations of GalOMe or tetravalent galactosylated glycocluster 7b, under static conditions at 30°C for 24h. Attached bacteria were stained with 1% Crystal Violet for 10 min, washed twice and staining was extracted by treatment with 40% ethanol coupled to sonication of microplates for 30 min at 50°C. OD570 was measured. All quantification assays were made in triplicate. In parallel, OD600 was measured in wells before staining to check that treatment with the monovalent or tetravalent of ligands did not affect bacterial growth.

ASSOCIATED CONTENT Supporting Information. Experimental procedures and NMR spectra for new compounds. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Phone: +0033 472 448 349. E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors thank the Université Claude Bernard Lyon 1 and the CNRS for financial support. Dr F. Albrieux, C. Duchamp and N. Henriques are gratefully acknowledged for mass spectrometry analyses. A.I. acknowledges support from GDR Pseudomonas, Labex ARCANE and COST action BM-1003. S.V. and A.I. are grateful to financial support from the COST Action CM-1102 MultiGlycoNano. Funding for this project was provided by a grant from la Région Rhône-Alpes. This work was funded by the French Fond Unique Interministériel (managed by Oseo and DGCIS), the Conseil Régional RhôneACS Paragon Plus Environment

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Alpes and the Conseil Général des Bouches du Rhône. The authors also thank Lyonbiopôle and Eurobiomed for their support.

ABBREVIATIONS USED BCP: bromocresol purple, CuAAC: Cu(I)-catalyzed azide-alkyne cycloaddition, CFU: colony-forming unit, DAPI: 4’,6’-diamidino-2-phenylindole, DMEM: Dulbecco’s modified Eagle’s medium, EDTA: ethylenediaminetetraacetic acid, FITC: fluorescein isothiocyanate, GFP: green fluorescent protein, HMBC: heteronuclear multiple bond correlation, HSQC: heteronuclear single quantum coherence, IMDM: Iscove’s modified Dulbecco's Medium, ITC: isothermal titration microcalorimetry, LPS: lipopolysaccharide, MOI: multiplicity of infection, MPLC: low pressure liquid chromatography, NMR: nuclear magnetic resonance, OD: optical density, PBS: phosphate buffer saline, QS: quorum sensing, TLC: thin layer chromatography.

REFERENCES (1) de Bentzmann, S.; Plesiat, P. The Pseudomonas aeruginosa opportunistic pathogen and human infections. Environ. Microbiol. 2011, 13, 1655-1665. (2) Imberty, A.; Varrot, A. Microbial recognition of human cell surface glycoconjugates. Curr. Opin. Struct. Biol. 2008, 18, 567-576. (3) Karlsson, K. A. Microbial recognition of target-cell glycoconjugates. Curr. Opin. Struct. Biol. 1995, 622-635. (4) Sharon, N. Carbohydrate-lectin interactions in infectious disease. Adv. Exp. Med. Biol. 1996, 408, 1-8. (5) Gilboa-Garber, N. Pseudomonas aeruginosa lectins. Methods Enzymol. 1982, 83, 378-385. (6) Imberty, A.; Wimmerova, M.; Mitchell, E. P.; Gilboa-Garber, N. Structures of the lectins from Pseudomonas aeruginosa: Insights into molecular basis for host glycan recognition. Microb. Infect. 2004, 6, 222-229. ACS Paragon Plus Environment


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(7) Cioci, G.; Mitchell, E. P.; Gautier, C.; Wimmerova, M.; Sudakevitz, D.; Pérez, S.; Gilboa-Garber, N.; Imberty, A. Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa. FEBS Lett. 2003, 555, 297-301. (8) Mitchell, E.; Houles, C.; Sudakevitz, D.; Wimmerova, M.; Gautier, C.; Pérez, S.; Wu, A. M.; Gilboa-Garber, N.; Imberty, A. Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. Nat. Struct. Biol. 2002, 9, 918-921. (9) Blanchard, B.; Nurisso, A.; Hollville, E.; Tétaud, C.; Wiels, J.; Pokorná, M.; Wimmerová, M.; Varrot, A.; Imberty, A. Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I (PA-IL). J. Mol. Biol. 2008, 383, 837-853. (10) Lanne, B.; Ciopraga, J.; Bergstrom, J.; Motas, C.; Karlsson, K. A. Binding of the galactose-specific Pseudomonas aeruginosa lectin, PA-I, to glycosphingolipids and other glycoconjugates. Glycoconj. J. 1994, 11, 292-298. (11) Perret, S.; Sabin, C.; Dumon, C.; Pokorná, M.; Gautier, C.; Galanina, O.; Ilia, S.; Bovin, N.; Nicaise, M.; Desmadril, M.; Gilboa-Garber, N.; Wimmerova, M.; Mitchell, E. P.; Imberty, A. Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa. Biochem. J. 2005, 389, 325-332. (12) Glick, J.; Garber, N. C. The intracellular localization of Pseudomonas aeruginosa lectins. J. Gen. Microbiol. 1983, 9, 3085-3090. (13) Tielker, D.; Hacker, S.; Loris, R.; Strathmann, M.; Wingender, J.; Wilhelm, S.; Rosenau, F.; Jaeger, K.-E. Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation. Microbiology 2005, 151, 1313-1323. (14) Schuster, M.; Lostroh, C. P.; Ogi, T.; Greenberg, E. P. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol. 2003, 185, 2066-2079.


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Table of Content Graphic


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Scheme 1 172x212mm (300 x 300 DPI)



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Figure 1. Schematic description of adhesion and lung infection by pathogens 238x169mm (96 x 96 DPI)


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Figure 2. (Top) Raw ITC data obtained by injections of fucosylated glycocluster 6 (150 µM) in LecB solution (50 µM). (Bottom) Corresponding integrated titration curve. 201x288mm (150 x 150 DPI)



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Figure 3. Representative micrographs of optical microscopy (400×) observations of bacterial cell ag-gregates for P. aeruginosa PAO1 strain. A. as a negative control in the absence of monovalent (α-L-FucOMe and β-DGalOMe) or multivalent glycoclusters (6 and 8). B. with 100 µM final concentration of fucosylated glycocluster (compound 6). C. with 6 600 µM final concentration of galactosylated gly-cocluster (compound 8). D. with a mixture of fucosylated and galactosylated glycoclusters at 500 and 660 µM final concentrations, respectively. 218x163mm (150 x 150 DPI)


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Figure 8. In vivo alveolar-capillary barrier permability measurement 254x190mm (72 x 72 DPI)


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Antiadhesive Properties of Glycoclusters against ... - ACS Publications

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