Synthesis of modified oligodeoxyribonucleotides (ONs) was performed on an DNA synthesizer using 0.2 μmol scale succinyl linked LCAA-CPG (long chain alkyl amine controlled pore glass) columns with a pore size of 500Å. Standard protocols for incorporation of DNA phosphoramidites were used. A ~50-fold molar excess of modified phosphoramidites in anhydrous acetonitrile (at 0.05 M) was used during hand-couplings (performed to conserve material) except with 4Z (~70-fold molar excess in anhydrous CH2Cl2, at 0.07M). Moreover, extended oxidation (45s) and coupling times were used (0.01 M 4,5-dicyanoimidazole as activator, 15 min for monomers V/W/Y, 35 min for monomer Z; 0.01 M 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole [Activator 42], 15 min for monomers Q/S/V). Cleavage from solid support and removal of protecting groups was accomplished upon treatment with 32% aq. ammonia (55 °C, 24 h). Purification of all modified ONs was performed to minimum 80% purity using either of two methods: a) overall synthesis yield >80%: cleavage of DMT using 80% aq. AcOH, followed by precipitation from acetone (−18 °C for 12-16 h) and washing with acetone, or b) overall synthesis yield <80%: purification of ONs by RP-HPLC as described below, followed by detritylation and precipitation as outlined under “a”.
Purification of the crude ONs was performed on a HPLC system equipped with an XTerra MS C18 pre-column (10 μm, 7.8 × 10 mm) and a XTerra MS C18 column (10μm, 7.8 × 150 mm) using the representative gradient protocol depicted in Table S1. The identity of synthesized ONs was established through MALDI-MS/MS analysis (Table S2-S3) recorded in positive ions mode on a Quadrupole Time-Of-Flight Tandem Mass Spectrometer equipped with a MALDI source using anthranilic acid as a matrix, while purity (>80%) was verified by RP-HPLC running in analytical mode.