1100 series lc msd trap

Shunt metabolites (2, 3, 6–9) (S2 Text), dianthones (13) (S2 Text), and modified anthraquinones (structures not shown) (S2 Text) were identified using HPLC-UV-Vis-MS (Agilent, California, US; 1100 series LC/MSD trap). HPLC conditions were as follows: 5 min isocratic gradient of 5% solvent B; 30 min linear gradient from 5 to 95% solvent B; 10 min isocratic gradient of 95% solvent B; 5 min linear gradient from 95–5% solvent B; and 10 min isocratic gradient at 5% B using a C18 2.6 μm 2.1 × 100 mm Accucore LC column (ThermoFisher Scientific) heated to 45°C with a flow rate of 0.25 ml min⁻¹. Injection volume of 20 μl was analysed via electrospray injection MS in negative mode.

The peptides APX, APX-17, and APX-12 were prepared by FMOC solid phase synthesis. Synthesis was performed on rink-amide resin using Fmoc-protected amino acids, HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate), and DIEA (N,N-Diisopropylethylamine) at a 1:1:2 ratio for couplings. Deprotections were performed using a solution of 1:4 v:v: piperidine in DMF (N,N-Dimethylformamide). Cleavage was performed by mixing the resin with a cocktail of TFA (trifluoroacetic acid):TIS (triisopropylsilane):H2O:ethanedithiol (92.5:2.5:2.5:2.5) for ~2 h followed by filtration and peptide precipitation in cold diethyl ether. Remaining peptides were purchased from GenScript (Piscataway, NJ, USA). All peptides were purified by reverse phase high-performance liquid chromatography using a linear gradient of solvent A (H20 with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA). Peptides were separated on an Agilent (5 μm 9.4 × 250 mm) C4 column. Fractions were monitored using UV absorbance at 220 and 280 nm and peptide identity was confirmed using ESI-MS (Agilent 1100 Series LC/MSD Trap, Santa Clara, CA, USA). Purified peptides were lyophilized, reconstituted in an ethanol water mixture and stored at 4 °C. Peptide stock concentration was determined spectrophotometrically, as described previously [31 (link),32 (link)].

IR spectra were recorded on a JASCO FT-IR (4100) spectrometer. High-resolution mass determination was carried out using a Finnigan TSQ quantum ultra mass spectrometer (Thermo Scientific). NMR spectra were recorded at 300 K on Bruker Avance III spectrometers with chloroform-d₁ as solvent and internal standard. The solvent signal was referenced to δ_H 7.24 ppm and δ_C 77.0 ppm, respectively. Analytical HPLC was performed on an Agilent 1100 Series LC/MSD trap. Flash column chromatography was undertaken using silica gel 60 M (230–400 mesh). TLC analyses were performed on silica gel plates (Sil G/UV254 0.20 mm, Macherey-Nagel) using a 9:1 mixture of chloroform and methanol as the eluent. Analytes were detected with vanillin-sulfuric acid spray reagent.