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

Nature Protocols volume 16, pages 3382–3418 (2021)Cite this article

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Abstract

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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Fig. 1: Procedure outline.
Fig. 2: Determination of extinction coefficients of cis-WOOH and trans-WOOH.
Fig. 3: Typical chromatogram of 1O2-oxidized l-Trp and characterization of its oxidation products.
Fig. 4: Determination of cis-WOOH and trans-WOOH concentrations.
Fig. 5: Chemical reduction of cis-WOOH and trans-WOOH to cis-WOH and trans-WOH, respectively, by NaBH4.
Fig. 6: Stability of cis-WOOH and trans-WOOH stored as powder and working solutions.
Fig. 7: Characterization of the breakdown products derived from the incubation of cis-WOOH and trans-WOOH in different buffers and media.
Fig. 8: Endogenous formation of 15N2 cis-WOOH from 15N2 l-Trp by porcine arteries pre-treated with 0, 200 or 400 ng/ml recombinant porcine interferon-γ (rpIFNγ).

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All the data relating to this protocol are present in the paper and its associated source files or can be found in the supporting primary research papers.

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Acknowledgements

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.

Author information

Authors and Affiliations

  1. Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia

    Raphael F. Queiroz, Cacang Suarna, Sudhir Shengule, Anita Ayer & Roland Stocker

  2. Department of Natural Sciences, Southwest Bahia State University, Vitoria da Conquista, Brazil

    Raphael F. Queiroz

  3. School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia

    Leo Corcilius, Genevieve E. Sergeant & Richard J. Payne

  4. Starpharma Pty Ltd, Abbotsford, Melbourne, Victoria, Australia

    Sudhir Shengule

  5. Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Brisbane, Queensland, Australia

    Richard J. Payne

  6. The Heart Research Institute, Sydney, New South Wales, Australia

    Anita Ayer & Roland Stocker

  7. Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia

    Anita Ayer

  8. School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia

    Roland Stocker

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  1. Raphael F. Queiroz
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Contributions

A.A. and R.S. designed the study. R.F.Q., C.S. and S.S. carried out the synthesis and purification of l-Trp–derived hydroperoxides. R.F.Q. and C.S. designed and carried out purification of cis- and trans-WOH. R.F.Q. determined extinction coefficients of cis- and trans-WOOH and the corresponding alcohols. R.F.Q. compared hydroperoxide quantification methods. C.S. performed purity testing of all compounds by LC-MS/MS and HPLC-UV. R.F.Q. and C.S. performed stability studies. R.F.Q. carried out and determined the LOD and LOQ of the hydroperoxides and their corresponding alcohols. S.S. and R.J.P. designed, performed and interpreted NMR studies. L.C. and R.J.P. designed, performed and interpreted optical rotation and IR characterization of compounds. G.E.S. and A.A. designed and performed fluorescence studies. All authors interpreted data. R.F.Q., A.A., C.S. and R.S. drafted the manuscript, and all authors critically reviewed the draft manuscript. A.A. and R.S. revised the manuscript, and all authors approved the submitted versions of the manuscript.

Corresponding authors

Correspondence to Anita Ayer or Roland Stocker.

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Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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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

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.

Extended Data Fig. 2 Fluorescence spectra of cis-WOOH, cis-WOH, trans-WOOH and trans-WOH.

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

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