Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds

The l -tryptophan–derived tricyclic hydroperoxide cis -WOOH was recently identified as a novel and biologically important factor for regulating vascular tone and blood pressure under inflammatory conditions and potentially other cellular redox signaling events. cis -WOOH is highly labile and currently not available commercially. In this protocol, we provide procedures for the synthesis, purification, quantification and characterization of cis -WOOH, its epimer trans- WOOH and their respective alcohols ( cis -WOH and trans -WOH). Photo-oxidation of l -tryptophan ( l -Trp) results in a mixture containing cis -WOOH and trans -WOOH, which are separated and purified by semi-preparative HPLC. cis -WOH and trans -WOH are then produced by sodium borohydride reduction and purified by semi-preparative HPLC. Characterization of cis -WOOH and trans -WOOH and the reduced alcohol variants is achieved using HPLC, fluorescence, NMR and liquid chromatography-tandem mass spectrometry. The protocol provides instructions for storage and quantification, as well as ways to test the stability of these hydroperoxides in commonly used buffers and media. Finally, we describe examples of how to monitor the formation of cis -WOOH in biological samples. The protocol ensures reasonable yield (11%) and purity (>99%) of cis -WOOH and control compounds in 5–6 d and outlines conditions under which cis -WOOH is stable for several months. The hydroperoxide l -tryptophan derivative cis -WOOH regulates vascular tone under inflammatory conditions via redox signaling. This protocol describes the preparation of cis -WOOH and related compounds for use as analytical standards and biochemical tools.