*2.7. Metabolic Profiling, Compound Isolation, and Chemical Structure Analysis*

The *Streptomyces* broth of TSB, SYP-NaCl, and YD medium cultures (180 mL) was exhaustively extracted using a solvent mixture of 1:1 MeOH:DCM yielding a crude extract (3.89 g). This crude

extract was first separated using SPE on C18 bonded silica gel (Polygoprep C18 (Fisher Scientific, Dublin, Ireland) 12%C, 60 Å, 40–63 μm), eluting with varying solvent mixtures to produce five fractions: H2O (743.62 mg), 1:1 H2O:MeOH (368.6 mg), MeOH (15.4 mg), 1:1 MeOH:DCM (10.9 mg), DCM (8.2 mg). The final three fractions (MeOH, 1:1 MeOH:DCM, DCM, 34.5 mg) were then combined and subject to analytical reverse phase HPLC on a Waters Symmetry (VWR, Dublin, Ireland) C18 5 μm, 4.6 × 250 mm column. The column was eluted with 10% MeCN (0.1% TFA)/90% H2O (0.1% TFA) for 5 min, then a linear gradient to 100% MeCN (0.1% TFA) over 21 min was performed. The column was further eluted with 100% MeCN (0.1% TFA) for 6 min. After the HPLC was complete, a linear gradient back to 10% MeCN (0.1% TFA)/90% H2O (0.1% TFA) over 1 min and then further elution of 10% MeCN (0.1% FA)/90% H2O (0.1% FA) for 4 min was performed. This yielded pure surugamide A (0.8 mg). Surugamide A was characterised using MS and NMR data to confirm the structure for use as an analytical standard.

Surugamide A was quantified in the broth using LC-MS analysis on an Agilent UHR-qTOF 6540 (Agilent Technologies, Cork, Ireland) mass spectrometer. The column used for separation was Waters equity UPLC BEH (Apex Scientific, Kildare, Ireland) C18 1.7 μm 2.1 × 75 mm. The column was eluted with 10% MeCN (0.1% FA)/90% H2O (0.1% FA) for 2 min, then a linear gradient to 100% MeCN (0.1% FA) over 6 min was performed. The column was further eluted with 100% MeCN (0.1% FA) for 4 min. After the UPLC was complete, a linear gradient back to 10% MeCN (0.1% FA)/90% H2O (0.1% FA) over 1 min and then further elution of 10% MeCN (0.1% FA)/90% H2O (0.1% FA) for 3 min was performed before the next run. The MS detection method was positive ion. A calibration curve was produce using the LC-MS method above and injecting the pure surugamide A at seven concentrations (100, 25, 10, 2, 1, 0.2, 0.1 mg/L). Thirty millilitres of each *Streptomyces* strain in broth were extracted using a solvent mixture of 1:1 MeOH:DCM three times to yield a crude extract. These extracts were resuspended in MeOH and filtered through PTFE 0.2 μm filters (Sigma Aldrich, Arklow, Ireland) before being subject to the above LC-MS method.

The surugamide A calibration standards 1–7 and the six extracts were analysed using the Agilent MassHunter Quantification software package. This allowed the quantification of surugamide A in the extracts based on the intensity of peaks in the chromatogram with matching retention time and exact mass.
