Figure 3. Synthesis of BPA-biotin affinity probes.

Panel A. The BPA-biotin derivative 1 was prepared by Williamson ether synthesis. The 5-hydroxy derivative was converted to 4-(5-iodopentyl)tetrahydro-1H-thieno[3,4-d]imidazol-2(3H)-one using I₂, triphenylphosphine. Selective O-alkylation of BPA with the primary alkyl iodide gave the desired compound 1 as the major product.

Panel B. The BPA-biotin derivative 2 was prepared by sequential Sonogashira coupling and biotinylation. 4-(2-(4-Hydroxyphenyl)propan-2-yl)-2-iodophenol was protected as the bis-TBS ether using standard conditions, and was then coupled with tert-butyl but-3-ynylcarbamate to the alkyne product in excellent yield. The TBS protecting groups were removed with TBAF, and the ^tBoc group was cleaved with TFA to provide the corresponding ammonium salt. Biotinylation with biotin-L₂-NHS in Et₃N/DMF gave the desired compound 2. Both of the biotinylated probes 1 and 2 were purified by silica gel chromatography and structurally characterized by ¹H- and ¹³C-NMR, and HPLC-MS.