Purification of Mycolactones A/B from M. ulcerans

Mycolactones A/B were purified from M. ulcerans extracts as previously described [6 (link),127 (link)]. Briefly, S4018, an African strain of Mycobacterium ulcerans obtained from a patient in Benin, was grown in Middlebrook 7H10 agar supplemented with oleic albumin dextrose catalase growth supplement. The bacteria were resuspended in chloroform-methanol (2:1, v/v) and cell debris was removed by centrifugation. Folch extraction was performed by adding 0.2 volumes of water. The organic phase was dried and phospholipids were precipitated with ice-cold acetone. The acetone-soluble lipids were loaded onto a thin layer chromatography plate and eluted with chloroform-methanol-water (90:10:1, v/v/v) as the mobile phase. The yellow band with a retention factor of 0.23 was scraped off the plate, filtered, evaporated, resuspended in absolute ethanol and then stored in amber glass tubes in the dark. Its concentration was determined by measuring absorbance (λ_max  =  362 nm, log ε  =  4.29), and its purity (>98%) was evaluated with a Shimadzu Ultra-Fast Liquid Chromatograph (UFLC XR system with a CBM-20A controller, a CTO-10AS Prominence column oven, LC-20AB pumps, an SPD M20A diode array detector (Shimadzu, Japan)) and a reverse C18 column (Zorbax 23 Eclipse XDB-C18, 9.4×250mm, Particle Size: 5 μm (Agilent, USA)).