Tco nhs ester

MagnaBind Amine Derivatized Beads (amine concentration = 12 µmol/mL) from Thermo Scientific, Rockford IL (USA) were also used to assess the binding of the PL effector after functionalization with the TCO-NHS ester (Click Chemistry Tools). For this procedure, 103 µL of magnetic beads (mBeads) were taken from the stock and washed three times with 1 mL of carbonate buffer at pH 8.5 before adding 65.4 µL of TCO previously dissolved in DMF at a concentration of 25.4 mg/mL. The mBeads were left to stir for 30 min protected from light to preserve the transconfiguration of the TCO for the reaction with Tz [19 (link)]. After half an hour, the mBeads were washed three times with carbonate and dissolved in 1.2 mL of carbonate, and 200 µL of 211At-HTzPL and 211At-MeTzPL were added to two reaction vials, each containing 200 µL of TCO-mBeads, corresponding to 10 times excess TCO over Tz, to evaluate the binding. A sample of 50 µL was taken out of the vial and measured on the γ-counter, and later it was washed with PBS and measured again to determine the binding. Unspecific binding to the TCO-mBeads was evaluated by measuring the activity left on the mBeads after reacting with polymer not bearing Tz groups.

For the proof of concept of pretargeting in vivo a model described by Yazdani et al. was applied [20 (link)], based on TCO-modified alendronate for bone targeting. For preparation of TCO–alendronate, briefly, 20 mg sodium alendronate trihydrate (Sigma Aldrich) was dissolved in 400 µL 0.1N sodium-bicarbonate solution pH 8.5. Then 0.6 mL TCO–NHS ester (Click chemistry tools, Scottsdale, AZ, USA), 20 mg/mL in DMSO, were slowly added to the solution and stirred overnight in the dark, followed by freeze drying. As a control the same preparation was prepared, replacing TCO–NHS solution by the same volume of DMSO. For injection in mice the residue was dissolved in water and adjusted to pH 7 (30 mM).