Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
07 2021
07 2021
Historique:
received:
08
09
2020
accepted:
22
03
2021
pubmed:
13
6
2021
medline:
21
7
2021
entrez:
12
6
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34117477
doi: 10.1038/s41596-021-00541-1
pii: 10.1038/s41596-021-00541-1
doi:
Substances chimiques
Superoxides
11062-77-4
Tryptophan
8DUH1N11BX
Hydrogen Peroxide
BBX060AN9V
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3382-3418Références
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