Supporting Information Amphiphilic guanidinocalixarenes inhibit lipopolysaccharide (LPS)- and lectin-stimulated Toll-like Receptor 4 (TLR4) signaling Stefania E. Sestito a, Fabio A. Facchini a, Ilaria Morbioli b, Jean-Marc Billod c, Sonsoles Martin-Santamaria c, Alessandro Casnati b, Francesco Sansone b * and Francesco Peri a * - Molecular Modeling, Docking Results - Chemistry: General - Synthesis and compounds characterization - Biology: activity of PHA plant lectin on HEK cells - MTT toxicity test - Activity of compounds 7-9 on HEK cells - References 1
Molecular Modeling-Docking Results Figure S1. Left: superimposition of the best docked pose of compound 2 (orange) and compound 3 (cyan) into human TLR4.MD-2 system (PDB ID 3FXI). Right: detail of the superimposed docked poses (TLR4.MD-2 structure is not displayed). 2
Figure S2. Superimposition of the best docked poses of compounds 2 (yellow), 3 (pink) and 4 (green) into (m)tlr4.md-2 system (PDB ID 2Z65) in the antagonist conformation. Figure S3. Best docked poses for compounds 7, 8 and 9 (green, blue and yellow, respectively) inside TLR4.MD-2 (PDB ID 2Z65). 3
RMSD (Å) t (ns) Figure S4. RMSD along the 90 ns MD simulation of the two TLR4.MD-2/3 complexes starting from the agonist (green) and the antagonist (purple) conformation of MD-2. The RMSD of compound 3 is also displayed: in complex with TLR4.MD-2 in the agonist (red) and the antagonist (cyan) conformation. RMSF (Å) Residue number Figure S5. RMSF along the 90 ns MD simulation of the two TLR4.MD-2/3 complexes in the agonist (red) and the antagonist (cyan) conformation of MD-2. 4
Figure S6. Calculated logp for compounds 1-9. Parameters for nitrogen atom type nj : LogFile: addatomtypes { { "nj" "N" "sp2" } } FRCMOD: MASS nj 14.01 0.530 sp2 N in amino groups (from ff14sb N2) BOND ca-nj 481.0 1.340 JCC,7,(1986),230; ARG,CYT,GUA (from parm10 CA-N2) hn-nj 434.0 1.010 JCC,7,(1986),230; ADE,CYT,GUA,ARG (from parm10 H - N2) 5
ANGLE ca-nj-hn 50.0 120.00 (from parm10 CA-N2-H) nj-ca-nj 70.0 120.00 AA arg (from parm10 N2-CA-N2) ca-nj-ca 50.0 123.20 AA arg (from parm10 CA-N2-CT) hn-nj-hn 35.0 120.00 (from parm10 H -N2-H) ca-ca-nj 70.0 120.00 (from parm10 CA-C -OH) DIHE hn-nj-ca-nj 1 0.000 0.0-4. (H -N2-CA-N2 from ff14sb) hn-nj-ca-nj 1 2.400 180.0 2. --- ca-nj-ca-nj 1 0.000 0.0-4. Arg Lys copied (C8-N2-CA-N2 from ff14sb) ca-nj-ca-nj 1 2.400 180.0 2. --- ca-ca-nj-ca 1 0.065 0.0-4. (CA-C -OH-HO from ff14sb) ca-ca-nj-ca 1 0.883 180.0 2. (CA-C -OH-HO from ff14sb) ca-ca-ca-nj 1 1.1 180.0 2. (CA-CA-C -OH from ff14sb) ca-ca-nj-hn 1 0.065 0.0-4. (CA-C -OH-HO from ff14sb) ca-ca-nj-hn 1 0.883 180.0 2. (CA-C -OH-HO from ff14sb) 6
Chemistry General. The reactions were carried out under a nitrogen atmosphere. TLC were performed using prepared plates of silica gel (Merck 60 F254 on aluminium) and revealed using UV light or staining reagents (H 2 SO 4 (5% in EtOH), ninhydrin (5% in EtOH), basic solution of KMnO 4 (0.75% in H2O), Pancaldi solution (molybdatophosphorus acid and Ce(IV) sulphate in 4% sulphuric acid). 1 H NMR (400 MHz) and 13 C NMR spectra (100 MHz) were recorded on a Bruker AV400 spectrometer using partially deuterated solvents as internal standards. Mass spectra were recorded in Electrospray Ionization (ESI) mode using a SQ Detector, Waters spectrometer. HPLC analyses were performed in Jasco and Waters HPLC systems, using reverse phase C 14 and C 18 columns, water/meoh and water/mecn gradients and UV detection. Bis-Boc-N-triflylguanidine (purity 95.0%) was purchased from Sigma- Aldrich and used as such. All tested compounds were obtained 95% purity according to HPLC analysis. Synthesis and characterization of new compounds 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[4-(bis-Bocguanidine)butoxy]calix[4]arene. To a solution of 5,11,17,23-tetra-tert-butyl- 25,26,27,28-tetrakis-(4-amino)butoxy]calix[4]arene (0.29 g, 0.31 mmol) in dry DCM (5 ml), Et 3 N (0.18 ml, 1.26 mmol) and bis-boc-n-triflylguanidine (0.49 g, 1.26 mmol) were added. The reaction mixture was stirred at rt for two days and monitored by TLC (Hex/EtOAc 3:1). When finished, the solvent was removed under reduced pressure and the crude was triturated in water for 3h and filtered to give the title compound (0.10 g, 0.055 mmol, 17% yield) as a white solid. 1 H-NMR (400 MHz, CDCl 3 ) δ (ppm): 11.53 (s, 4H, BocNH); 8.41 (s, 4H, CH 2 NH); 6.78 (s, 8H, ArH); 4.35 (d, 4H, J = 12.4 Hz, ArCH 2 Ar ax); 3.93 (m, 8H, OCH 2 ); 3.50 (m, 8H, CH 2 NH); 3.14 (d, 4H, J = 12.4 Hz, 7
ArCH 2 Ar eq); 2.02 (m, 8H, OCH 2 CH 2 ), 1.70 (m, 8H, CH 2 CH 2 NH), 1.50 (s, 72H, Boc); 1.09 (s, 36H, t-bu). The spectroscopic data found are in agreement with those reported in literature 1 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis(4-guanidiniumbutoxy)calix[4]arene (2). To a solution of 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetrakis[4-(bis-Bocguanidine)butoxy]calix[4]arene (0.082 g, 0.044 mmol) in 1,4 dioxane (5mL), HCl 37% (0.145 ml, 1.74 mmol) and TES (0.070 ml, 0.44 mmol) were added. The reaction mixture was stirred at room temperature and monitored every hour by ESI-MS (MeOH). After 18 hours the solvent was removed under reduced pressure and the crude was triturated in Et 2 O and then in H 2 O to get compound 2 as a white solid (0.020 g, 0.016 mmol, 36% yield). 1 H-NMR (400 MHz, CDCl 3 ) δ (ppm): 6.84 (s, 8H, ArH); 4.45 (d, 4H, J = 12.4 Hz, ArCH 2 Ar ax); 4.00 (m, 8H, OCH 2 ); 3.46 (m, 8H, CH 2 NH); 3.18 (d, 4H, J = 12.4 Hz, ArCH 2 Ar eq); 2.09 (m, 8H, OCH 2 CH 2 ), 1.80 (m, 8H, CH 2 CH 2 NH), 1.11 (s, 36H, t-bu). The spectroscopic data found are in agreement with those reported in literature. 1 5,11,17,23-Tetrahydroxycarbonyl-25,26,27,28-tetrahexyloxycalix[4]arene. A solution of 5,11,17,23-tetraformyl-25,26,27,28-tetrahexyloxycalix[4]arene 2 (0.20 g, 0.23 mmol) in CHCl 3 /acetone 1:1 (8 ml) was cooled to 0 C. A solution of sulfamic acid (0.27 g, 2.75 mmol) and sodium chlorite (0.21 g, 2.29 mmol) in H 2 O (1 ml) was added. The reaction mixture was stirred at room temperature and monitored with TLC for 4 days. After the reaction was completed the solvent was removed under reduced pressure, 2 N HCl (2 ml) was added and the light yellow solid was filtered and washed with H 2 O. The solid was triturated in MeOH, filtered and washed with cold MeOH to give the title product as a white solid (0.15 g, 0.16 mmol, 68% yield). M.p.: > 300 C. 1 H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.32 (bs, 4H, COOH); 7.32 (s, 8H, ArH); 4.34 (d, 4H, J = 8
13.2 Hz, ArCH 2 Ar ax); 3.91 (m, 8H, J = 7.4 Hz, OCH 2 ); 3.39 (d, 4H, J = 13.2 Hz, ArCH 2 Ar eq); 1.95-1.80 (m, 8H, OCH 2 CH 2 ); 1.45-1.20 (m, 8H, O CH 2 CH 2 (CH 2 ) 3 CH 3 ); 0.95-0.85 (m, 12H, CH 3 ). 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 167.3; 160.3; 134.8; 130.1; 125.1; 75.5; 32.0; 30.5; 30.3; 25.9; 22.8; 14.3. MS (ESI-) m/z: calcd for C 56 H 72 O 12 936.50, found 935.77 [M-H] - 9
Biology HEK-blue cells activation by plant lectin PHA Figure S7. Dose-dependent PHA activation of TLR4 signal in HEK-blue and Null cells. TLR4 HEK Blue and Null cells (control cell line) were stimulated with increasing concentrations of PHA lectin. Data are normalized with LPS (100 ng/ml) and represent the mean of percentage ± SD of at least three independent experiments. Nt= not treated. 10
MTT toxicity test Figure S8. MTT assay of compounds 1-5 in HEK Blue cells. Cells were treated with the same concentrations of compounds used in the other assays; the bars represent the cell viability estimated by using 10 µm of compounds, equivalent to the maximum concentration used previously. Data are normalized with PBS and represent the mean of percentage ± SD of at least 3 independent experiments. 11
Figure S9. Effect on LPS-stimulated HEK-Blue cells activation by calixarenes 7-9. Human TLR4 HEK-Blue were treated with increasing concentrations of compounds and stimulated with LPS (100 ng/ml). The results represent normalized data with positive control (LPS alone) and expressed as the mean of percentage ± SD of at least three independent experiments. References 1. Chen, X.; Dings, R. P.; Nesmelova, I.; Debbert, S.; Haseman, J. R.; Maxwell, J.; Hoye, T. R.; Mayo, K. H. Topomimetics of Amphipathic Beta-sheet and Helix- Forming Bactericidal Peptides Neutralize Lipopolysaccharide Endotoxins. J. Med. Chem. 2006, 49, 7754-7765. 2. Nomura, E.; Hosoda, A.; Takagaki, M.; Mori, H.; Miyake, Y.; Shibakami, M.; Taniguchi, H. Self-organized Honeycomb-Patterned Microporous Polystyrene Thin Films Fabricated by Calix[4]arene Derivatives. Langmuir. 2010, 26, 10266-10270. 12
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