G1969 85001

H¹ and C¹³ NMR spectra were recorded on a Bruker DRX-400
spectrometer (Bruker, Billerica, MA, USA) at 298 K. ¹H
and ¹³C chemical shifts are reported relative to CHCl₃ (δ_H 7.26 ppm) or CDCl₃ (δ_C 77.16 ppm), DMSO-d₆ (δ_H 2.50 ppm or δ_C 39.52 ppm), and MEOH (δ_H 3.33 ppm) or MeOD (δ_C 49.0 ppm) as an internal
reference. High-resolution mass spectral (HRMS) data were recorded
with an Agilent 1290 binary LC system connected to an Agilent 6230
Accurate-Mass TOF LC/MS (ESI+), calibrated with an Agilent G1969-85001
ES-TOF reference mix containing ammonium trifluoroacetate, purine,
and hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine in 90:10 acetonitrile:water. TLC
was performed on silica gel 60 F₂₅₄ (Merck Millipore) with
detection of UV light. Flash column chromatography [the eluent for
flash chromatography is given between brackets in the Experimental Section] was carried out on silica gel (particle
size, 60 Å; 230–400 mesh; Sigma-Aldrich). Preparative
HPLC was performed using a VP 250/21 Nucleodur C-18, HTEC, 5 μm
column (Macherey-Nagel) on a Gilson 333/334 Prep-Scale system with
a flow rate of 210 mL/min, detection at 210 nm (Gilson 151), and a
CH₃CN (0.005% HCO₂H)/H₂O (0.005%
HCO₂H) eluent system. Compounds 2, 10c, 10e, 10f, 10l, and 10b were purchased from Sigma-Aldrich. Compounds 10d(32 (link)) and 10m(31 ) were synthesized according to previously described literature
procedures.

A Cogent Diamond Hydride Type C column (MicroSolv) was used for normal phase chromatography on 1200 LC system (Agilent Technologies) coupled to an Accurate Mass 6220 TOF (Agilent Technologies) mass spectrometer fitted with an MultiMode ion source. Metabolite extracts were mixed with solvent A 1:1 and separated using mobile phase of solvent gradient A and B: 0–2 min, 85% B; 3–5 min, 80% B; 6–7 min, 75%; 8–9 min, 70% B; 10–11.1 min, 50% B; 11.1–14:10 min 20% B; 14:10-18:10 5% B; 18:10–19 85% B. Solvent A was acetonitrile with 0.2% acetic acid and solvent B was ddH2O with 0.2% acetic acid. Reference mass solution (G1969-85001, Agilent Technologies) was used for continuous mass axis calibration. Analytical amino acids standards (Fluka A9906) was used for retention time match. Ions were identified based on their accurate mass, retention time and spectral information, yielding errors below five ppm. Spectra were analysed using MassHunter Qualitative Analysis B.07.00 and MassHunter Profinder B.08.00 software. Statistical validation of samples/runs were performed using principal component analysis, using Mass Profiler Professional (B.07.01).

The lipidomics data were collected using a standard metabolic profiling method and the same Agilent 6520 QTOF-MS platform [45 (link)]. Briefly each prepared sample (4 μL for positive ESI ionization, 8 μL for negative ESI ionization) was injected onto an Agilent Zorbax 300 SB-C8 column (2.1× 50mm, 1.8-μm), which was heated to 50°C. The flow rate was 0.4 mL/min. Mobile phase A was 5 mM ammonium acetate and 0.1% formic acid in water, and mobile phase B was 5% water in ACN containing 5 mM ammonium acetate and 0.1% formic acid. The mobile phase composition was kept isocratic at 35% B for 1 min, and was increased to 95% B in 19 min; after another 10 min at 95% B, the mobile phase composition was returned to 35% B. The ESI voltage was 3.8 kV. The Q-TOF MS spectrometer was calibrated prior to each batch, and a reference channel infusing the standard reference mixture (G1969-85001, Agilent Technologies) was used during the experiments to ensure mass accuracy. The mass scan range was 100–1600, and the acquisition rate was 1.5 spectra/s. The Q-TOF data were extracted using Agilent MassHunter Qualitative Analysis (version B.07.00) and Mass Profiler Professional (MPP, version B.13.00) software. The absolute intensity threshold for the LC–Q-TOF data extraction was 1000, and the mass accuracy limit was set to 10 ppm.