To 2-chlorotrityl resin (186 mg, 0.26 mmol) was added DCM (2 ml), 4-[5-(N-fluorenyloxycarbonyl-2-Aminoethyl)-2-hydroxyphenylazo]-benzoic acid (130 mg, 0.26 mmol) and DIEA (175 μl, 1.0 mmol). The mixture was gently shaken overnight at room temperature. Dry methanol (0.2 ml) was added to cap any unreacted resin (10 min). The resin was dried, and the loading efficiency was determined to be 0.85 mmol/g. The resin was swelled with dry DMF (2 ml) for 30 minutes, drained and treated with a 20% solution of piperidine in DMF (2 ml) for 30 minutes. The resin was drained and washed with DMF (5×5 ml) to remove residual traced of piperidine. To the resulting resin was added dry DMF (2 ml), biotin (73.3 mg, 0.30 mmol), PYBOP (156.1 mg, 0.30 mmol) and DIEA (104.5 μl, 0.60 mmol), and the resin was gently shaken at room temperature overnight. The resulting resin was drained, washed with DMF (3×3 ml), DCM (3×3 ml) and hexanes (3×3 ml) and dried in vacuo. Cleavage of the resin was performed using a 10% solution of TFA in DCM (10×2 minutes each). The organic fractions were combined, concentrated in vacuo, and dried via azeotrope with toluene to afford the desired 4-[5-(N-biotinyl-2-Aminoethyl)-2-hydroxyphenylazo]-benzoic acid (2) as an orange solid (65 mg, 50% from the benzoic acid).
