60 f254 tlc plate

Merck 60 F254 TLC plates were used in monitoring the reactions.
¹H NMR spectra were recorded with an INOVA 400 instrument with a 5 mm probe. All chemical shifts were quoted relative to deuterated solvent signals (δ in ppm and J in Hz).
HPLC-MS analysis was carried out with an Agilent Technologies HP1100 instrument, equipped with a ZOBRAX-Eclipse XDB-C8 Agilent Technologies column (flow: 0.4 mL/min; mobile phase: CH₃CN/H₂O gradient from 30 to 80% CH₃CN in 8 min and then 80% CH₃CN until 25 min), coupled with an Agilent Technologies MSD1100 single-quadrupole mass spectrometer (full-scan mode from m/z 50 to 2600; scan time of 0.1 s in positive ion mode, ESI spray voltage of 4500 V, nitrogen gas of 35 psi (1 psi = 6894.7 Pa), drying gas flow of 11.5 mL/min, fragmentor voltage of 20 V).

n-Hexane fraction (100 mg) of V. negundo leaves was dissolved in 40 mL of n-hexane and the volume was made up to 50 mL in a volumetric flask. This fraction was used for the TLC (thin layer chromatography) fingerprinting profile. TLC plates consisted of 10 × 10 cm, precoated with silica gel 60 F₂₅₄ TLC plates (E. Merck) (0.2 mm thickness) with aluminum sheet support. The spotting device was a CAMAG Linomat V Automatic Sample Spotter (Camag Muttenz, Switzerland); the syringe was 100 µL (Hamilton). The developing chamber was a CAMAG glass twin trough chamber (20 × 10 cm), densitometer a Camag TLC Scanner 3 linked to winCATS software. The experimental conditions were kept constant where temperature was 25 ± 2°C and relative humidity was 40%. TLC fingerprint was developed by applying 25 µL of n-hexane fraction (100 mg/50 mL) in duplicate along with standards, lupeol, and β-sitosterol with band distance of 12 mm and band size of 8 mm. Plate was developed in a solvent system of toluene : methanol (9.7 : 0.3), dried and observed under UV 254 nm and UV 366 nm. The plate was derivatized with anisaldehyde-sulfuric acid reagent followed by heating at 100°C until the colored band appeared. The R_F value and color of the resolved bands were noted.

All oligonucleotides were synthesized on a MerMade12 (BioAutomation) synthesizer using standard phosphoramidite chemistry^{49 (link)}. FAM-labeled oligonucleotides were synthesized using fluorescein-labeled phosphoramidite. Thin-layer chromatography (TLC) purification of oligonucleotides of length 12 nt was performed on Merck 60 F254 TLC plates with 1-propanol/ammonia/water = 55:35:10 (v/v/v). Oligonucleotides of length 32 nt were purified by polyacrylamide gel electrophoresis. The details of deprotection and purification of oligonucleotides have been described previously^{50 (link)}. Purified oligonucleotides were characterized using MALDI-TOF mass spectrometry.

Oligoribonucleotides for UV-melting experiments, DNA primers for PCR reactions, and 5′-FAM labeled DNA primers for reverse transcription in SHAPE experiments were synthesized on a BioAutomation MerMade12 DNA/RNA synthesizer using β-cyanoethyl phosphoramidite chemistry as described previously [52 (link)], and commercially available phosphoramidites (ChemGenes (Wilmington, MA, USA); GenePharma (Shanghai, China)). The details of deprotection and purification of oligoribonucleotides were also described previously [52 (link)]. Thin-layer chromatography (TLC) purification of the oligonucleotides was carried out on Merck 60 F254 TLC plates with the mixture of 1-propanol/aqueous ammonia/water = 55:35:10 (v/v/v). After synthesis and purification, oligonucleotides were verified using mass spectroscopy (MALDI-ToF).

Total lipids extraction and TLC analysis were performed according to the protocol described by Mu et al.^{23 (link)}. In brief, 10 mg of ENPs were mixed with equal volume of chloroform and methanol and centrifuged at 2000 rpm for 5 min at room temperature. The organic phase obtained was dried under a stream of nitrogen and suspended in chloroform. The extracted lipids were resolved through silica gel 60 F254 TLC plates (Merck) using chloroform/methanol/acetic acid (95:4.5:0.5, by volume) as a solvent system. Plates were dried at room temperature and sprayed with a solution containing 10% copper sulfate and 8% phosphoric acid. Lipid bands were visualized by charring the plates at 120 °C for 10 min.

General: The solvents and reagents utilized in this study were purchased from commercial suppliers and employed without additional purification. The reaction progress was observed by thin-layer chromatography (TLC) using a silica gel 60 F254 TLC plate manufactured by Merck. ¹H and ¹³C NMR spectra were obtained using a Bruker Advance 400 MHz spectrometer, with deuterated dimethylsulfoxide (DMSO) as the solvent. Chemical shifts (δ) are expressed in parts per million (ppm) relative to the internal standard tetramethylsilane (TMS). The abbreviations s, d, t, and m are commonly used to denote singlet, doublet, triplet, and multiplet, respectively. The reported units for coupling constants (J) are hertz (Hz). The acquisition of high-resolution mass spectra (HRMS) was performed using either the Waters Acquity UPLC/Synapt G2 QTOF MS or the JMS 700 mass spectrometer manufactured by Jeol, Japan. The starting material 1 [33 (link)] and key intermediate 2 [34 (link),35 (link)] were prepared adopting the reported procedure.