Nucleosil 100 7 c18 column

A cell pellet of E. coli BL21(DE3) pGro7 pET28‐Thal containing overexpressed Thal was resuspended in 20 mL of aqueous buffer (50 mM Na₂HPO₄, 30 mM NaBr, pH 7.4) and lysed by French‐Press (3 passages @ 1100 bar cell pressure). Cell lysate was cleared by centrifugation for 30 min (12000×g, 4 °C) and the supernatant was provided in a 100 mL Erlenmeyer flask. To the halogenase lysate, 15–40 μm peptide substrate, PrnF (2.5 U mL⁻¹), PTDH (2 U mL⁻¹), 0.1 mm NAD⁺_, 10 μm FAD, 50 mm sodium phosphite, 30 mm NaBr and 15 mm Na₂HPO₄ (pH 7,4) were added in a total volume of 30–80 mL. Upon incubation for 20 h at 25 °C in a shaking incubator precipitate was removed by centrifugation (10000×g, 30 min, 4 °C), the crude product was freeze dried and purified by preparative RP‐HPLC on a Merck‐Hitachi preparative HPLC system (controller: D7000, pump: L7150, detector: L7420, absorbance monitored at 220 nm) equipped with a Macherey‐Nagel Nucleosil 100–7 C₁₈ column (7 μm, 250×10 mm). A linear gradient from 0→75 % MeCN over 45 min was applied to isolate brominated peptide. Resultant product‐containing fractions were pooled, freeze‐dried and analysed by NMR and HRMS.