Affinity resins containing enzymatically resistant mRNA cap analogs—a new tool for the analysis of cap-binding proteins

Synthesis of Sepharoses 2–4: (A) Synthesis of m⁷GpCH₂pp-Sepharose: (i) 1. CH₂(POCl₂)₂, (CH₃O)₃PO, 2. triethylamine bicarbonate (TEAB) aq.; (ii) imidazole, 2,2′-dithiodipiridine, Ph₃P, TEA, DMF; (iii) tris(triethylamine) phosphate, ZnCl₂, DMF; (iv) CH₃I, DMSO; (v) 1. tri-n-octylamine, MeOH, 2. DCC, DMF; (vi) p-nitrophenol, TEA, DMF; (vii) Pd/C, H₂, (viii) Sepharose 4B activated witch BrCN, sodium carbonate buffer (pH 9.6). (B) Synthesis of m⁷GpppA-Sepharose and m⁷GpCH₂ppA-Sepharose: (i) 1. POCl₃, (CH₃O)₃PO, 2. TEA/H₂O; (ii) NH₂(CH₂)₆NH₂, H₂O; (iii, v) imidazole, 2,2′-dithiodipiridine, Ph₃P, Et₃N, DMF; (iv) CH₃I, DMSO; (vi) ZnCl₂, DMF; (vii) Sepharose 4B activated witch BrCN, sodium carbonate buffer (pH 9.6).