Exactive mass spectrometer

Sim‐H and Sim‐L were prepared as 10 mM solutions in water and DMSO, respectively, and stored at −78°C, and warmed to room temperature immediately prior to injection; 1 μL of each solution was added to 1 mL of water or Tris/HEPES (250 mM HEPES, 80 mM Tris, 10 μM free Ca²⁺, pH 7.2) buffer, respectively, and sampling by LC‐MS was begun immediately. Samples were kept at room temperature for the duration of the analysis. Analyses were performed using a Thermo Exactive mass spectrometer equipped with Waters Acquity liquid chromatography system. Instrument control was performed using Thermo Xcalibur Software. Electrospray source conditions were adjusted to maximize sensitivity; 3 μL of each sample was injected onto the system. The column was a 2.1 × 50 mm (3 μm) ACE equivalence C18. A mixed eluent gradient was employed and is described in Supporting Information Table S3. The four sample types were analysed sequentially; the 5 min analysis time for each sample provided a 20 min sampling frequency for each sample. Mass analysis was performed using MestReNova MS software (2016, V11.0), where the peaks were integrated to determine relative percentages of Sim‐L and Sim‐H over time.

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).