A tryptophan-analog host whose interactions with ammonium ions in water are dominated by the hydrophobic effect

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1 Supporting Information for A tryptophan-analog host whose interactions with ammonium ions in water are dominated by the hydrophobic effect Amanda L. Whiting, icole M. eufeld and Fraser Hof* 1. General Information Solvents and reagents were used as obtained from Sigma-Aldrich. Proton ( 1 H) MR and carbon ( 13 C) MR spectra were recorded on a Brüker AC300 (300 MHz) spectrometer or Brüker Avance 500 (500 MHz). Proton ( 1 H) MR spectra for MR titration studies were recorded on a Brüker Avance 360 (360 MHz) spectrometer. Chemical shifts (δ) are given in parts per million (ppm) relative to TMS and referenced to residual protonated solvent (CHCl 3 : δ H 7.26 ppm, δ C ppm) (DH: δ H 4.79 ppm). J values are given in Hz. Abbreviations used are s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Melting points were obtained using a Gallenkamp Melting Point Apparatus and are uncorrected. Low resolution electron impact mass spectra (LR-EIMS) and high resolution electron impact mass spectra (HR-EIMS) were obtained on a double focusing Kratos Concept mass spectrometer. 2. Synthesis 3-(1H-Indol-3-yl)-propionic acid methyl ester (4). Synthesized by the reported procedure. 1 δ H (300 MHz; CDCl 3 ) 2.37 (2H, t, J = 7.7, CH 2 CH 2 C 2 Me), 3.11 (2H, t, J = 7.4, CH 2 CH 2 C 2 Me), 3.68 (3H, s, C 2 Me), 6.98 (1H, s, 2-H), 7.17 (2H, m, indole-h), 7.34 (1H, d, J = 7.8, indole-h), 7.61 (1H, d, J = 7.8, indole-h), 8.00 (1H, s br, H). 3-(1-{3,5-Bis-[3-(2-methoxycarbonyl-ethyl)-indol-1-ylmethyl]-benzyl}-1H-indol-3-yl)-propionic acid methyl ester (6). 60% sodium hydride (0.24 g, 6.1 mmol) and indole 4 (1.2 g, 6.0 mmol) were suspended in anhydrous dimethylformamide (8 ml) and stirred under nitrogen. After 25 min, 1,3,5- tris(bromomethyl)benzene 5 (357 mg, 1.0 mmol) in dimethylformamide (2 ml) was added dropwise and the reaction left to stir for 2 hours. After complete reaction of tris(bromomethyl)benzene, most solvent was removed via vacuum and the reaction quenched with hexanes and tetrahydrofuran. After filtration of solids, column chromatography (15 to 30 % ethyl acetate/hexanes) yielded the product 6 (260 mg, 36%) as an off-white solid. mp o C; δ H (300 MHz; CDCl 3 ) 2.64 (6H, t, J = 7.7, 3 x CH 2 CH 2 C 2 Me), 3.05 (6H, t, J = 7.7, 3 x CH 2 CH 2 C 2 Me), 3.67 (9H, s, 3 x C 2 Me), 5.09 (6H, s, 3 x CH 2 Ph), 6.68 (3H, s, 3 x 2-H), 6.79 (3H, s, 3 x PhH), 7.08 (9H, m, 3 x indole-h), 7.57 (3H, m, 3 x indole-h). δ C (75 MHz; CDCl3) 20.8, 35.0, 49.7, 51.8, 109.8, 114.5, 119.2, 119.3, 122.1, 124.5, 125.6, 128.0, 136.7, 139.2, 174.0; m/z (LR-EIMS) 723 (M +, 8%), 552 (100), 536 (25), 203 (45), and 130 (45); m/z (HR-EIMS) (M +. C 45 H requires ). 3-(1-{3,5-Bis-[3-(2-carboxy-ethyl)-indol-1-ylmethyl]-benzyl}-1H-indol-3-yl)-propionic acid (7). Trisubstituted methyl ester indole host 6 (63 mg, 0.09 mmol) was dissolved in tetrahydrofuran (2 ml) and distilled water (2.5 ml). Excess solid sodium hydroxide (91.5 mg, 2.3 mmol) was added and the reaction was stirred at room temperature for 21 hours under argon. Reaction was diluted with an equal volume of 1 M hydrochloric acid, extracted with ethyl acetate (3 x 30 ml), and dried over magnesium sulfate before concentrating under vacuum. Precipitation from a minimum of tetrahydrofuran with hexanes and sonication gave the triacid 7 (53 mg, 89%) as a white solid. mp o C; δ H (500 MHz;

2 CDCl 3 ) 2.71 (6H, t, J = 7.1, 3 x CH 2 CH 2 C 2 H), 3.06 (6H, t, J = 6.8, 3 x CH 2 CH 2 C 2 H), 5.06 (6H, s, 3 x CH 2 Ph), 6.80 (3H, s, 3 x 2-H), 6.87 (3H, s, 3 x PhH), 7.10 (9H, m, 3 x indole-h), 7.55 (3H, m, 3 x indole-h); δ C (125 MHz; CDCl3) 20.4, 34.6, 50.2, 109.9, 114.0, 119.0, 119.4, 122.1, 125.8, 126.2, 128.2, 136.8, 139.0, 179.5; m/z (LR-EIMS) 681 (M +, 30%), 189 (25), 130 (100), 83 (45); m/z (HR-EIMS) (M +. C 42 H requires ). 3-(1-{3,5-Bis-[3-(2-carboxy-ethyl)-indol-1-ylmethyl]-benzyl}-1H-indol-3-yl)-propionic acid trisodium salt (1). Sodium methoxide in methanol (0.66 ml, M) was added to a suspension of triacid indole host 7 (138 mg, 0.20 mmol) in methanol (30 ml). After 30 minutes, the reaction was filtered to remove suspended solids and the filtrate condensed to dryness. The solid was collected to give trisodium indole host 1 (149 mg, 99%) as an off-white solid. δ H (360 MHz, D 2 ) 2.39 (6H, t, J = 8.0, 3 x CH 2 CH 2 C 2 H), 2.84 (6H, t, J = 8.0, 3 x CH 2 CH 2 C 2 H), 6.44 (3H, s, 2-H), 6.72 (3H, s, PhH), 6.80 (3H, d, J = 8.1, indole-h), 6.93 (3H, t, J = 7.4, indole-h), 7.02 (3H, t, J = 7.4, indole-h), 7.57 (3H, d, J = 7.87, indole-h). Missing (6H, s, 3 x CH 2 Ph) Signal under DH peak. 1-(3,5-Bis-indol-1-ylmethyl-benzyl)-1H-indole (2). Synthesized using a modification of published procedure. 2 60% sodium hydride (0.83 g, 20.8 mmol) and indole (2.56 g, 22.0 mmol) were suspended in anhydrous dimethylformamide (30 ml) and stirred under argon. After 45 min, 1,3,5- tris(bromomethyl)benzene 5 in dimethylformamide (4 ml) was added dropwise and the reaction left to stir 4 hours. After complete reaction of tris(bromomethyl)benzene, most solvent was removed via vacuum and the reaction quenched with hexanes and ethyl acetate. After filtration of solids, column chromatography (1:3 chloroform/hexanes) and precipitation in ethanol with cooling yielded the product (653 mg, 39%) as an off-white solid. δ H (360 MHz, CDCl 3 ) 5.14 (6H, s, 3 x CH 2 Ph), 6.52 (3H, d, J = 3.1, 3 x 3-H), 6.72 (3H, s, 3 x PhH), 7.00 (3H, d, J = 3.1, 3 x 2-H), (9H, m, indole-h), 7.64 (3H, m, indole-h). 3. MR Titrations Binding titrations in D 2 were carried out in D 2 containing a 2 HP 4 /ah 2 P 4 (total phosphate concentration of 50 mm) at ph 8.0 (pd 7.6). Acidity of buffered solutions (pd) in D 2 was measured using a saturated KCl ph electrode and corrected to ph by addition of 0.4 units. 3 Titrations in CDCl 3 were performed in commercial CDCl 3 available from Cambridge Isotope Laboratories. Hosts 1 and 2 were prepared as described in Section 2. All guests were commercially available and used as received. Excel workbooks for curve-fitting MR titration data and MR dilutions were provided for online access by J.M. Sanderson. 4, 5 a) Dilution study. A 15 mm solution of host 1 was prepared in buffered D 2 to create a stock solution. 0.3 ml aliquots of host 1 stock were added sequentially to an MR tube originally containing 0.6 ml of blank buffered D 2. Additions continued until the MR tube contained a total of 1.5 ml. 0.3 ml of the tube solution was then removed to a second MR tube and 0.3 ml additions of stock solution continued. Additions were continued in a third MR tube once the total volume again reached 1.5 ml. The concentrations observed in this study ranged from 15 to 0.4 mm. Representative data and curve fitting are presented in Figure S1. on-linear curve fitting of the dilution data of 1 in buffered D 2 solution gave a K homodimer (400 M -1 ) averaged over eight tracked signals, which was used in further calculations of K assoc in water for 1.

3 (a) (b) Figure S1. (a) Representative dilution data of 1 in phosphate buffered D 2. (b) A representative set of observed data (points) and non-linear fit (line) to a homodimerization isotherm. Dimerization of 2 in CDCl 3 was also investigated. A 50 mm stock solution of 2 in CDCl 3 was titrated in to an MR tube containing 0.5 ml blank CDCl 3 to a total volume of 1.4 ml. Host dimerization of 2 under these conditions was found to be negligible and was not included in further calculations. b) Guest binding. Host solutions (1-2 mm) were prepared fresh in the appropriate solvent. A portion (0.6 ml) was removed to become the receiving solution in the MR tube and a portion was used to make up guest solutions (2-150 mm) in order to ensure that the host concentration remained constant throughout the titration. The maximum concentration of guest solutions to be titrated was determined taking into account both guest solubility and buffer concentration. Guest species which could undergo a proton transfer (e.g. Me 3 H + ) were kept to concentrations well under 50 mm in order not to overwhelm the buffer present. 1 H MR binding titrations were carried out at 295 K on a Brüker Avance 360 spectrometer at 360 MHz. Chemical shift data of multiple signals from each titration were fit to a 1:1 binding isotherm. In the case of 1, the effect of homodimerization on the concentration of free host was taken into account using the standard method as implemented in the fitting spreadsheets made freely available by J. Sanderson, Durham, UK. 4

4 4. 1 H and 13 C MR spectra for synthesized compounds Compound 4 H

5 Compound 6

6 Compound 7 H H H

7 Compound 2

8 5. References 1. M. S. C. Pedras and M. Jha, Bioorg. Med. Chem., 2006, 14, P. Rajakumar and M. Gayatri Swaroop, Tet. Lett., 2006, 47, P. K. Glasoe and F. A. Long, J. Phys. Chem., 1960, 64, J. M. Sanderson, The Centre for Bioactive Chemistry, Department of Chemistry, University Science Laboratories, Durham, UK. 5. H. Adams, C. A. Hunter, K. R. Lawson, J. Perkins, S. E. Spey, C. J. Urch and J. M. Sanderson, Chem. Eur. J., 2001, 7,