Microtof q 3

Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO, Easton, MD, USA). IR data was collected using a Bruker Alpha Fourier transform infrared spectrophotometer (Bruker, Billerica, MA, USA). NMR spectra were measured at ¹H resonance frequency on a Jeol Eclipse + 400 MHz spectrometer (JEOL, Peabody, MA, USA). Chemical shifts were calibrated internally to the residual signal of deuterated chloroform (CDCl₃ δ_H 7.26, δ_C 77.0). For NMR measurements concentration used was in the range of 3–8 mg of compound (depending on the amount of compound available) dissolved in 600 μL of deuterated chloroform. High-resolution mass spectra were obtained on a Bruker micrOTOF-Q III (Bruker Daltonics, Billerica, MA, USA). HPLC separations were performed using an Agilent 1200 HPLC system equipped with a quaternary pump, a diode array detector (Agilent, Santa Clara, CA, USA) and a normal phase silica gel column (Phenomenex Sphereclone^®, 4.6 mm × 100 mm, 5 µm). Reverse phase solid phase extraction (SPE) separation was carried out using SUPELCO Supelclean™ LC-18 (C-18, octadecyl) solid-phase extraction cartridges (Supelco^® Analytical, Bellefonte, PA, USA).

HRMS was performed with a Bruker Daltonics micrOTOF-Q III (electrospray ionization, ESI) instrument.

N-Glycan derivatives were supplied from GlyTech Inc. (Kyoto, Japan). Boc-miniPEG^TM, Boc-miniPEG-3^TM, and EDC·HCl were obtained from Peptide Institute, Inc. (Osaka, Japan). 5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRA, SE) was obtained from AAT Bioquest (California, USA). 3-(5-Carboxypentyl)-1,1-dimethyl-2-((E)-2-((E)-3((E)-2-(1,1,3-trimethyl-1H-benzo[e]indol-2(3 H)-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-1H-benzo[e]indolium (Cy7.5) chloride was obtained from abcam (Cambridge, UK). All other chemicals and solvents of special grade were obtained from Tokyo Chemical Industry, co., Ltd (Tokyo, Japan) or Wako Pure Chemical Industries, Ltd (Osaka, Japan), and were used without purification. HPLC was performed on Shimadzu liquid chromatograph CBM-20A, LC-20AD, and SPD-20AV (Kyoto, Japan) with an analytical column COSMOSIL ₅C₁₈-AR-300 (4.6 mm ×250 mm, Nacalai Tesque, Inc., Kyoto, Japan) at a flow rate of 1 mL/min, and on JASCO liquid chromatograph LC-NetII/ADC, PU-2089 Plus, and UV-2075 Plus (Tokyo, Japan) with a preparative column COSMOSIL ₅C₁₈-AR-300 (20 mm × 250 mm, Nacalai Tesque, Inc.) at a flow rate of 7 mL/min. Mass spectra were recorded on a Bruker micrOTOF QIII (Rheinstetten, Germany). NMR spectra were recorded on a JEOL AL400 spectrometer (Tokyo, Japan).

The purity and identity of the RNA oligonucleotides was analyzed by anion-exchange HPLC (Dionex DNAPac PA200, 2 × 250 mm, at 60 °C. Solvent A: 25 mM Tris-HCl (pH 8.0), 6 M Urea. Solvent B: 25 mM Tris-HCl (pH 8.0), 6 M Urea, 0.5 M NaClO₄. Gradient: linear, 0–40% solvent B, 4% solvent B per 1 CV), and HR-ESI-MS (Bruker micrOTOF-Q III, negative ion mode, direct injection). An aliquot (200 pmol in 25 µL) was digested by snake venom phosphodiesterase (SVPD, 0.5 U) in the presence of bacterial alkaline phosphatase (BAP, 0.5 U) in 40 mM Tris. pH 7.5, 20 mM MgCl₂, and the resulting mononucleosides were analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry using an RP-18 column (Synergi 4 µm Fusion-RP C18 80 Å, 250 × 2 mm, at 25 °C. aqueous mobile phase A: 5 mM NH₄OAc, pH 5.3. organic mobile phase B: 100% acetonitrile. Gradient: 0–5% B in 15 min, then 5–50% B in 20 min, flow rate 0.2 ml/min) and micrOTOF-Q III with ESI ion source operated in positive ion mode (capillary voltage: 4.5 kV, end plate offset: 500 V, nitrogen nebulizer pressure: 1.4 bar, dry gas flow: 9 l/min). Extracted ion chromatograms and UV absorbance traces at 245 nm confirmed presence of remdesivir.

NMR spectra were measured using a Bruker Avance 400, Bruker, Bremen, Germany (¹H at 400.1 MHz; ¹³C at 100.6 MHz; ²⁹Si {¹H} (INEPT technique) at 79.5 MHz) at 25 °C. ¹H and ¹³C NMR signals of the prepared compounds were assigned to the corresponding atoms using gHSQC, gCOSY, and gHMBC 2D NMR correlation spectra. ¹H and ¹³C chemical shifts (δ/ppm) are given relative to residual solvent signals (δ_H/δ_C: DMSO-d6 2.50/39.52); ²⁹Si spectra were referenced to an external standard hexamethyldisilane (−19.87 ppm). HRMS spectra were measured using a MicroTOF-QIII instrument (Bruker Daltonics, Bruker, Bremen, Germany) with an ESI or APCI ionization source in the positive mode.

Five microliters of HEG (1 mg/mL) was injected into an LC-20AD ultrafast liquid chromatograph (UFLC) (Shimadzu) connected in line with a diode array detector (DAD) (240–800 nm) and a mass spectrometer with electrospray ionization (ESI) and a quadrupole time-of-flight (QTOF) analyzer (120–1200 Da; micrOTOF-Q III, Bruker Daltonics). It was equipped with a C-18 Kinetex column (150 mm × 2.2 mm inner diameter, 2.6 μm) with an oven temperature of 50°C. The mobile phase consisted of deionized water (A) and acetonitrile (B), both containing 0.1% formic acid, with the following gradient: 0–8 min, 3% B; 8–30 min, 3–25% B; and 30–60 min, 25–80% B. The gradient was followed by washing and reconditioning of the column (8 min). The flow rate was 0.3 mL/min.

The analysis of EBSp was performed in a UFLC chromatography Shimadzu Proeminence system coupled to a diode array detector and a mass spectrometer MicrOTOF-Q III (Bruker Daltonics), which is provided with an electrospray ionization source and analyzers quadrupole and time-of-flight. For the analyses, a Kinetex C18 chromatographic column (Phenomenex, 2.6 μm, 100 Å, 150 × 2.1 mm) was used. The flow rate was 0.3 mL.min⁻¹ and an injection volume was 8 μL. The mobile phase was acetonitrile (B) and ultrapure water (A) with 0.1% formic acid (v/v). The chromatographic separation was performed by gradient elution profile (0-2 min -3% B, 2-25 min from 3 to 25% B, 25-40 min from 25 to 80% B, and 40-43 min -80% B) at 50°C. For the MS analyses, nitrogen is applied as nebulization gas (4 bar) and drying gas (9 L.min⁻¹), applying the capillary voltage 3,500 kV. EBSp was analyzed by positive and negative ion mode.