Characterization and Quantification of Tryptophan‐ and Tyrosine‐derived Hydroperoxides^†

Abstract

The reaction of singlet oxygen (1O2) with the amino acids tryptophan and tyrosine, either free or inserted in peptides or proteins, gives rise to hydroperoxides. To understand the impact of these hydroperoxides in complex biological systems, methods allowing their characterization and accurate quantification must be available. In this work, hydroperoxides derived from tryptophan and tyrosine and from peptides containing these amino acids were synthesized by photooxidation, and characterized by high‐resolution mass spectrometry. In addition, experiments were carried out to compare two colorimetric methods commonly used for quantification of peroxides, namely the iodometric and the ferric‐xylenol orange assays. For the tryptophan hydroperoxide, the quantifications obtained by colorimetric methods were then compared to that obtained by NMR. The results showed that for the ferric‐xylenol orange method, the stoichiometry between peroxide and Fe3+ ions varies considerably. On the other hand, for the iodometric assay, the stoichiometry peroxide:I3− ions is always 1:1. However, the kinetics of the reactions of peroxides with I− vary, and the assay must be performed in anaerobic conditions. Thus, the iodometric method is more appropriate for precise quantification of a given peroxide. The characterization and accurate quantification of biological peroxides is key to understand the mechanisms involved in redox processes.

Photosensitized Oxidation of Free and Peptide Tryptophan to N-Formylkynurenine

“…Upon incubation, two of them disappeared and the other one exhibited increased area. Comparison of the spectra of the products (Figure 5) to those reported in the literature 40,57 indicates that the two peaks that disappeared during the incubation are the two isomers of cTrp-OOH and the product that exhibited increased concentration is the cyclic alcohol cTrp-OH (Scheme 1). Besides, the area of the peaks corresponding to NFKyn, in both HPLC-PDA and HPLC-FL chromatograms, increased, as already explained before.…”

“…Therefore, this analysis suggests that while the rate of consumption of Trp via oxidation by 1 O 2 is independent of the substrate used, the reaction pathways to yield NFKyn depend on the nature of the molecule containing Trp. Di Mascio and co-workers have fully characterized Trpderived hydroperoxides 40,57,58 and demonstrated that cTrp-OOH (Scheme 1) is, as a matter of fact, a mix of two isomers (trans-and cis-2-carboxy-3a-hydroperoxy-1,2,3,3a,8,8ahexahydropyrrolo[2,3-b]indole) that can be separated by HPLC. These authors also demonstrated that cTrp-OOH is rather stable in aqueous solution at 25 and 37 °C and is slowly converted into cTrp-OH (also two isomers) and NFKyn (Scheme 1).…”

Antibacterial Mesoporous Silica Granules Containing a Stable N-Halamine Moiety

“…PVA-MBA@SiO 2 granules (1.00 g) were added to 50 mL of deionized water, and then 5.0 mL of 7.5% NaOCl dilute solution was added dropwise under stirring with a pH adjustment to about 7.5 by using 0.50 mol L –1 H 2 SO 4 . After the reaction mixture was stirred for 2 h under room temperature, PVA-MBA-Cl@SiO 2 granules were filtrated out and rinsed repeatedly with deionized water to remove residual hypochlorite ions and then dried under a vacuum at 50 °C for 2 h. The oxidative chlorine content (Cl + %) of PVA-MBA-Cl@SiO 2 granules could be measured by an iodometric method. , The specific steps are as follows. A certain amount of PVA-MBA-Cl@SiO 2 granules were ground into a fine powder and accurately weighed.…”