717 plus autosampler

RP-HPLC analysis was performed using the Waters 625 LC System (encompassing: Waters 625 Pump, Waters 600 Controller, Waters 717plus Autosampler, Waters Degasser and Waters 996 PDA detector) Synergy Max-RP C12 (250 × 4.6 mm, 4u, Phenomenex) column, combined with Synergy Max-RP C12 pre-column according to an adapted procedure [74 (link), 75 (link)]. Separation of nucleosides was conducted in the presence of ‘A’ buffer (0.5 % v/v methanol in 10 mM KH₂PO₄, pH 3.7) and ‘B’ buffer (10 % v/v methanol in 10 mM KH₂PO₄, pH 3.7). The pH of the buffers was adjusted with phosphoric acid. The linear gradient used for separation consisted of 100 % ‘A’ buffer to 100 % of ‘B’ buffer for 10 min, next 100 % of ‘B’ buffer for 10–25 min and then, at the end of 25 min program 100 % ‘A’ buffer was pumped for 5 min. Flow rate was 1 ml per min and column temperature was set at 30 °C. UV-detection was used at the wavelength of 280 nm. The external standard consisted of major DNA (0.5–50 µM) and RNA nucleosides (1.5–150 µM) and 5-methyl-2′-deoxycytidine (5mdC) dissolved in deionized water. Peaks corresponding to 2′-deoxycytidine (dC) and 5mdC had retention time equal to 6.5 and 9.3 min, respectively. The contribution of 5mdC was calculated based on Millennium 32 v. 4.0 software (Waters Corporation, Milford, Massachusetts, USA).

UV and IR spectra were obtained employing Perkin-Elmer Lambda 40 and Perkin-Elmer Spectrum BX instruments (PerkinElmer, Inc., Waltham, USA), respectively. VLC grade (Macherey-Nagel, Polygoprep 60-50C18) was used for vacuum liquid chromatography. All organic solvents were distilled prior to use. HPLC was carried out using a Waters system, controlled by Waters Millenium software, consisting of a 600E pump, a 996 PDA, and a 717 plus autosampler (Waters Corporation, Milford, MA, USA). All NMR spectra were recorded on a Bruker Avance 300 and 500 DRX spectrometers (Bruker Corporation, Rheinstetten, Germany). Spectra were referenced to the residual solvent signals with resonances at δ_H/C 2.04/29.8 (acetone-d₆) and δ_H/C 7.26/77.0 (CDCl₃). ESI mass spectra were obtained on an Applied Biosystems/MDS Sciex API 2000 MS spectrometer (Applera Corporation and MDS Inc., Foster, CA, USA). HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q Time-of-Flight mass spectrometer with ESI source, and UPLC-Synapt G2 HDMS mass spectrometer (Bruker CorporationCompany, Rheinstetten, Germany).

¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer. The chemical shifts reported were referenced to internal tetramethylsilane (0.00 ppm) or to the solvent resonance at the appropriate frequency. The number and weight average molecular weights (M_n and M_w, respectively) of polymers were measured by gel permeation chromatography (GPC) using a Waters HPLC system equipped with a model 1515 isocratic pump, a 717 plus autosampler, and a 2414 refractive index (RI) detector with Waters Styragel columns HT6E and HT2 in series. Empower II GPC software was used for running the GPC instrument and for calculations. Both the Styragel columns and the RI detector were heated and maintained at 40 °C temperature during sample analysis. Chloroform was used as the eluent at a flow rate of 1.0 mL min⁻¹. Sample concentrations of 2 mg mL⁻¹ and injection volumes of 100 µL were used. Polymer molecular weights were determined based on a conventional calibration curve generated by narrow polydispersity polystyrene standards from Aldrich Chemical Co.