Xbridge beh c18 obd column

The DMTr-On oligonucleotides were purified by RP-HPLC on an Agilent 1200 equipped with a Waters XBridge BEH C18 OBD column (300 Å, 5 µm, 19 × 250 mm, 4 mL/min, 60 °C). As solvents 400 mM hexafluoroisopropanol (Fluorochem), 16.3 mM Et₃N (Merck), pH 8.3, and MeOH (Fluka) were used with a gradient from 5 to 100% MeOH in 30 min. After separation, the solvent was evaporated in a vacuum concentrator at 4 °C. The DMTr group was removed by incubation of the oligonucleotides in 400 µL 80% aqueous AcOH (Merck) at room temperature for 20 min, followed by evaporating the solvent in a vacuum concentrator at 4 °C. The RNAs were again purified by RP-HPLC under the same conditions as above.

All reactions were performed under an argon atmosphere and anhydrous solvents were used, unless otherwise stated. In flash liquid chromatography purifications, Merck silica gel 60 (0.06–0.2 mm) was used. Final purification of ligands 10, 13 and 14 was carried out in XBridge BEH C18 OBD column, on a Waters 1525EF semi-preparative HPLC system (Milford, MA, USA), equipped with autosampler, diode array detector and fraction collector. NMR spectra were obtained on a Bruker Avance III Ultrashield Plus spectrometer (Billerica, MA, USA), at 500 MHz for ¹H-NMR and 125 MHz for ¹³C-NMR, at 25 °C, chemical shifts relative to tetramethylsilane. MS data were collected on a Bruker Autoflex III Smartbeam MALDI-TOF/TOF instrument (Billerica, MA, USA). UV spectra were recorded on a Jenway 6715 UV-Vis Spectrophotometer (Stone, UK). All DNA sequences used were purchased from Eurogentec (Liège, Belgium) as synthetic oligonucleotides, purified by HPLC and dialysis. Graphs were constructed and data fitting was carried out in OriginPro 8.0 (Northampton, MA, USA).