Aviii hd 500

Trans-Clovamide was synthesized using methods described by Xie et al. (2013) (link), starting with trans-caffeic acid (Cayman Chemical 70602) and L-DOPA methyl ester (Sigma-Aldrich D1507). The product was dissolved in DMSO-d₆ and analyzed (¹H NMR) on a Bruker AVIII-HD-500. Retention time and absorbance spectrum of the product were compared with commercial clovamide (Cayman Chemical 16138) using HPLC-DAD.

All reactions were performed open to air and without precautions to exclude air/moisture unless specified otherwise. Reagents and solvents were purchased from commercial sources and used without further purification unless specified otherwise. NMR spectra were recorded on Bruker AVIII HD 400, NEO 400, AVIII HD 500 and AVII 500 spectrometers. Chemical shifts (δ) are quoted in parts per million (ppm). ¹H and ¹³C NMR spectra are referenced to residual protons in chloroform-d (δ_H = 7.26, δ_C = 77.16) and acetone-d₆ (δ_H = 2.05, δ_C = 28.95). Peak multiplicities are defined as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Coupling constants (J) are reported to the nearest 0.1 Hz. High-resolution mass spectra (HRMS) were recorded on a Thermo Scientific exactive mass spectrometer (Waters Equity autosampler and pump) for electrospray ionisation (ESI). Flash chromatography refers to normal phase column chromatography on silica gel (Merck Si 60, 0.040–0.063 mm) under a positive pressure of nitrogen.

Choline alkanoate ILs were synthesized via neutralization of the base with the appropriate organic acids, following reported methods [42 (link),43 (link),57 (link)]. Briefly, 0.1 mol organic acid (glacial acetic acid, propionic acid, butyric acid, hexanoic acid, or octanoic acid) was added dropwise into an aqueous solution of choline hydroxide (0.1 mol). The mixture was stirred continuously using a magnetic stirrer (RZR 2051, Heidolph, Schwabach, Germany) for 12 h at room temperature. The obtained ILs were dried for 6 h under vacuum using a rotary evaporator (R-210, Büchi, Flawil, Switzerland), followed by freeze drying (Freezone 2.5, Labconco, Kansas City, MO, USA) for approximately 4 days.
To confirm the identity and purity of the synthesized ILs, 500 MHz NMR spectroscopy (AVIIIHD 500, Bruker, Fällanden, Switzerland) and infrared spectroscopy (Attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectrometer, Alpha, Bruker, Billerica, MA, USA) were used (spectra are included in the Supplementary Materials). Viscosity was measured using a VISCOlab 3000 viscometer (Cambridge Viscosity, Inc., Boston, MA, USA) at 25 °C. The water content of the choline ILs was measured using a Karl Fisher Titrator (C20, Mettler Toledo, Greifensee, Switzerland). The chemical properties of the synthesized ILs are shown in Table 3.

To conduct NMR analysis, samples were prepared by dissolving each sample in 650 μL of DMSO-d₆ (Sigma Aldrich^®) to obtain the concentration of 5 mg/ml then transferred to 5 mm 7″ NMR tubes. J-resolved and proton (¹H NMR) experiments were performed using a Bruker^® AVIII HD 500 (11.7 T) and the spectra were processed using MestReNova x64 software (version 14.1.2).
The data acquisition followed the parameters used in the previous work developed by Sampaio et al. (2016) (link) using 16 scans in the pre-saturation pulse sequence for one-dimensional (1D) proton spectra (¹H NMR) and 32 scans and 64 increments per scan for two-dimensional (2D) ¹H−¹H J-resolved (J-res) NMR spectra; data points widths of 3.56 kHz for F2 (chemical shift axis) and 50 Hz for F1 (spin-spin coupling constant axis). The solvent signal was suppressed by the selective pre-saturation method.

MTT was purchased from Molecular Probes® (Invitrogen™). Fetal bovine serum (FBS) was obtained from Gibco. All absorbance readings were performed using the FilterMax F5 Multi-Mode Microplate Reader spectrofluorimeter (Molecular Devices). Proton (¹H) NMR spectra were recorded at 400 or 500 MHz and carbon (¹³C) NMR spectra at 101 or 126 MHz with ¹H decoupling. Spectra were recorded on Bruker AVIIIHD 400 or Bruker AVIIIHD 500 spectrometers with CDCl₃ as reference. High-resolution mass spectra (HRMS) were recorded by the Departmental Mass Spectrometry Service, University of Oxford on a Thermo Scientific Exactive Mass Spectrometer (using a Waters Equity autosampler and pump) for electrospray ionisation (ESI).