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.
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Hydrogen peroxide signaling via its transformation to a stereospecific alkyl hydroperoxide that escapes reductive inactivation
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We thank C. Stanley and G. Maghzal for their assistance with some of the experiments and J. Talib, J. Gebicki, P. Di Mascio and G.E. Ronsein for their advice. This work was funded by Program Grant 1052616 from the National Health and Medical Research Council of Australia (NHMRC) to R.S. R.S. is supported by NHMRC Senior Principal Research Fellowship 1111632. The work also received support from New South Wales Health.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Camilo López-Alarcón and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Stanley, C. P. et al. Nature 566, 548–552 (2019): https://doi.org/10.1038/s41586-019-0947-3
Extended Data Fig. 1 Correlation between absorbance and cis-WOH and trans-WOH concentrations in nanopure H2O containing DTPA (100 µM).
Aliquots of cis-WOOH and trans-WOOH (5 mM; determined by the iodometric method) were incubated with a 20-fold molar excess of NaBH4 (100 mM) for 20 min at room temperature. cis-WOOH was dissolved in HPLC-grade water, whereas trans-WOOH was dissolved initially in DMSO and then diluted to 30% DMSO with nanopure grade H2O. The solutions were then diluted to 10, 25, 50, 100 and 250 µM in nanopure grade H2O before recording their spectra from 400 to 200 nm using a spectrophotometer. Absorbance was plotted against cis- or trans-WOH concentration, and their respective molar extinction coefficients at 295 nm were obtained from the slope as ε = 2.76 ± 0.05 × 103 and ε = 2.23 ± 0.03 × 103 M–1 cm–1. Data are mean ± s.e.m. and are representative of three independent experiments.
a, Excitation and emission spectra of 50 µM cis-WOOH and cis-WOH. b, Excitation and emission spectra of 50 µM trans-WOOH and trans-WOH. Spectra were determined using a Duetta fluorescence and absorbance spectrophotometer (Horiba Scientific). cis-WOOH and cis-WOH were analyzed in milliQ water whereas trans-WOOH and trans-WOH were analyzed in 50:50 (vol:vol) milliQ water/DMSO. All readings were taken at 20 oC.
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Queiroz, R.F., Suarna, C., Corcilius, L. et al. Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds. Nat Protoc 16, 3382–3418 (2021). https://doi.org/10.1038/s41596-021-00541-1