Purification of compounds.

For isolation of compounds 1 to 9, supernatant from 2-L R5A-Cl 6-day cultures was filtrated and then retained by 10-g Sep-Pac Vac C₁₈ cartridge (Waters) conditioned with methanol and milliQ water. The sample held in the cartridge was then fractionated using a collector by elution with an Alliance HPLC preparative chromatographic system (Waters) using as mobile phase solvents methanol and milliQ water, a flow rate of 5 ml/min, and a gradient of 0% to 100% methanol in 55 min. Fractions containing compounds of interest were dried in vacuum in a Rotavapor and resuspended in 2 ml of methanol.

Purification of compounds was carried out by reversed-phase chromatography with the same Alliance HPLC equipment as described above, using a SunFire C₁₈ column (10 μm, 10 by 150 mm; Waters), and isocratic conditions with a flow rate of 5 ml/min and a column temperature of 35°C, using different ratio of solvents A, acetonitrile, and B, milliQ water plus TFA, depending on the compound (acetonitrile 25% for colibrimycin B, 35% for colibrimycins A1 to A3, 40% for colibrimycins C, A4, and A5). TFA was used, ranging between 0.05 and 0.1% depending on the compound (0.05% for colibrimycins A1 to A3 and B, 0.1% for colibrimycins C, A4, and A5).

Peaks containing the desired compounds were collected by a collector setup with the appropriate retention time and lyophilized.

Structural elucidation of each compound was carried out by ESI-time of flight high-resolution mass spectrometry, MS/MS, and NMR spectroscopy. HRMS was employed to determine the molecular formulas based on the experimental accurate masses and the corresponding isotopic distribution. Connectivity of the compounds was established based on one-dimensional (1D) and 2D NMR analyses alongside the fragmentation pattern observed in the MS/MS experiments. HRMS spectra were collected as previously indicated in “HRMS and dereplication analysis,” above. MS/MS of each compound was obtained in the same Bruker maXis high-resolution mass spectrometer by selecting the molecular ion [M+H]⁺ for fragmentation, using N₂ as collision-induced dissociation (CID) gas and the automatically interpolated collision energy provided by the instrument for each parent ion. NMR spectra were recorded in dimethyl sulfoxide (DMSO)-d₆ at 24°C on a Bruker AVANCE III-500 (500 and 125 MHz for ¹H and ¹³C NMR, respectively) equipped with a 1.7-mm TCI MicroCryoProbe, using the residual solvent signal as the internal reference (¹H 2.50 and ¹³C 39.5 for DMSO-d₆). A description of the structural elucidation of the new compounds alongside their HRMS, MS/MS, and NMR spectra is included in Fig. S19 to S100 and Tables S5 to S16 at https://figshare.com/s/526a9777225b6750159c.