Luna 5 m c18

UV spectra were measured with a Thermo Ultimate 3000 DAD. IR spectra were measured with a PerkinElmer Spectrum One FTIR spectrometer. All NMR experiments were run on a Varian Unity spectrometer (500 MHz for ¹H), a Varian UNITY INOVA spectrometer (600 MHz for ¹H) equipped with a 5 mm triple resonance (HCN) cold probe, or a Bruker spectrometer (800 MHz for ¹H) outfitted with a 5 mm triple resonance (HCN) inverse cold probe. Residual solvent shifts for DMSO-d₆ or CD₃OD were defined as δ_H 2.50 or 3.31, respectively, for proton spectra and δ_C 39.52 or 49.00 for carbon spectra in accordance with reference spectra. Accurate mass measurements for molecular formula determinations were obtained on a Thermo Velos Pro Orbitrap ESI-FTMS. Prefractionation HPLC was performed using a Phenomenex Gemini-NX 5 µm C₁₈ (50 × 21.1 mm) column. Semipreparative HPLC fractions were generated using a Phenomenex Luna 5 µm C₁₈ (250 × 10 mm) column. Analytical LC-MS analysis was performed on samples at a concentration of approximately 0.5–10 mg/mL, using a reversed-phase 150 × 4.60 mm 5 µm C₁₈ Phenomenex Luna column in conjunction with a 4.0 × 3.0 mm C₁₈ (octadecyl) guard column and cartridge (holder part number: KJ0-4282; cartridge part number: AJ0-4287, Phenomenex, Inc.).

An overview of the general method is shown in Figure S14. A 20 L amount of fermentation broth from strain M125SB302Ax, grown in medium M4 (Table S1), as stated above, was extracted with EtOAc, which was dried by rotary evaporation. A portion of the organic extract (192 mg of 319.7 mg total) was prefractionated by HPLC using methods reported previously.^{5 (link)} Analytical HPLC fractions were generated using a Phenomenex Luna 5 µm C18 (150 × 4.60 mm) column. Prefraction F3 (2.8 mg) was subjected to analytical HPLC using MeCN/H₂O (with 0.1% formic acid) at 1 mL/min, UV absorbance detection at 310 nm, and the following step gradient: 10% MeCN for 3.0 min, linear gradient to 82.5% MeCN over 17 min, linear gradient to 100% MeCN over 0.10 min, hold at 100% MeCN for 15 min. Scale-up isolation yields (see Figure S9) were 0.4 mg of F3H1 (t_R = 5.2 min), 0.2 mg of F3H2 (t_R = 7.4 min), 0.4 mg of F3H3 (t_R = 11.7 min), 0.9 mg of F3H4 (1-methyl-4-methylthio-β-carboline (1)) (t_R = 14.8 min), 0.1 mg of F3H5 (t_R = 15.1 to 20.0 min), and 0.6 mg of F3H6 (t_R = 20.0 to 35 min).
1-Methyl-4-methylthio-β-carboline (1): off-white film; UV (MeCN/H₂O) λ_max 256, 294, 379 nm (Figure S12); IR (film) ν_max 3152, 2924, 1620, 1455, 1389, 1324, 739 cm⁻¹ (Figure S13); ¹³C and ¹H NMR data, Table 1; ESITOFMS m/z [M + H]⁺ 229.0787 (calcd for C₁₃H₁₃N₂S, 229.0799).