2.8. Synthesis of TMP Coupled to Agarose Beads

The 4′-methoxy group of TMP was cleaved as previously described [31]. TMP (10.0 g, 34.5 mmol) was dissolved in 48% hydrobromic acid (120 mL) which had been preheated to 95 °C and stirred at 95–100 °C for 20 min. The heat bath was then removed and sodium hydroxide (50% aq., 30 mL) was added dropwise. Stirring was then stopped and the reaction mixture was allowed to cool to room temperature over 2 h, yielding a white precipitate. Precipitated crystals were filtered, washed with ice-cold water and allowed to mostly dry in Buchner funnel. They were then dissolved in boiling water (150 mL) and neutralized to pH 7 by slow addition of aq. ammonium hydroxide (30% w/w). This solution was then placed at 4 °C overnight, resulting in crystal precipitation. The precipitate was then filtered, rinsed with ice-cold water and dried in vacuo, yielding 1 as beige crystals (4.99 g, 53%). ¹H NMR (300 MHz, CD₃OD) δ ppm: 7.21 (s, 1H), 6.54 (s, 2H), 3.82 (s, 6H), 3.63 (s, 2H).

5-Iodovalerate (2) was prepared as previously described [32], with slight modification. Sodium iodide (6.16 g, 41.1 mmol) was dissolved in dry acetone (26 mL) and brought to reflux. Ethyl 5-bromovalerate (5.0 mL, 31.6 mmol) was then added and the reaction was stirred at reflux for 3 h. Additional sodium iodide (2.37 g, 15.8 mmol) was then added and the solution was stirred at reflux for 1 h. The reaction was cooled and 13 mL ether was added. The solution was filtered and solvent evaporated in vacuo. The residue was dissolved in 40 mL ether and then washed twice each with 12–13 mL of 1% NaOH (aq), ddH₂O and brine. The organic fraction was then dried over sodium sulfate and the solvent removed in vacuo, yielding a pale-yellow oil (3.76 g, 93%). ¹H NMR (300 MHz, CDCl₃) δ ppm: 4.01 (q, 2H), 3.08 (t, 2H), 2.22 (t, 2H, 1.75 (m, 2H), 1.64 (m, 2H), 1.15 (t, 3H).

To generate the 4′-substituted phenol 4, 1 (2.00 g, 7.24 mmol), 2 (2.04 g, 7.97 mmol) and cesium carbonate (4.72 g, 14.48 mmol) were dissolved in dimethylformamide (DMF) (180 mL) and stirred at 70 °C for 6 h. Solvent was then removed in vacuo. The crude product was directly purified by flash silica gel chromatography (DCM→10% MeOH/DCM), yielding a brown solid (1.55 g, 53%). 3 (1.55 g, 3.82 mmol) was then dissolved in MeOH (7.6 mL) and NaOH (5 M, 2.30 mL, 11.5 mmol) was added. The reaction was stirred at room temperature for 1 h. The solvent was then removed, water (26.0 mL) was added and acidified to pH 4 with 1 M HCl. The precipitate was filtered, rinsed with ice-cold water and dried in vacuo, yielding a light brown solid (0.94 g, 66%). ¹H NMR (300 MHz, DMSO-d₆) δ ppm: 7.48 (s, 1H), 6.53 (s, 2H), 6.34 (s, 2H), 5.95 (s, 2H), 3.76 (t, 2H), 3.70 (s, 6H), 3.52 (s, 2H), 2.24 (t, 2H), 1.61 (m, 4H).

To prepare the immobilized TMP ligand 5, 4 (8.0 mg, 21 µmol) and 1,1′-carbonyldiimidazole (4.4 mg, 27 µmol) were dissolved in DMF (1 mL) and mixed at 50 °C, 1000 rpm for 30 min in an Eppendorf Thermomixer (Eppendorf, Hauppauge, NY, USA). CarboxyLink^TM Coupling Gel (Thermo Fisher, Rockford, IL, USA) (1 mL) was washed with DMF (1 mL × 3), then combined with the activated acid and mixed overnight at 50 °C, 1000 rpm. The beads were then centrifuged and solvent removed. Coupling efficiency was monitored by UV absorbance of solvent before and after reaction and determined to be 50–60%. The beads were washed with DMF (1 mL × 3) and N-acetoxysuccinimide (1 mL of 1 M solution in DMF) was added to the beads and mixed for 2 h at room temperature to protect residual free amines. The beads were then washed with DMF (1 mL × 3) and PBS (1 mL × 3) and resuspended in 0.05% NaN₃ in PBS (500 µL) to make a 50% slurry. To prepare N-acetoxysuccinimide, N-hydroxysuccinimide (0.50 g, 4.35 mmol) and acetic anhydride (1.24 mL, 13.1 mmol) were combined and stirred overnight at room temperature. The solvent was removed. Crystals were filtered and rinsed with hexanes and dried in vacuo, yielding white crystals (0.637 g, 93%). ¹H NMR (CDCl₃, 300 MHz) δ ppm: 2.82 (s, 4H), 2.32 (s, 3H).