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Ergothioneine's Redox Potential And Spectrophotometric Detection Assay: Insights From Redox-Active Dyes
Cao, Chaz T.
Cao, Chaz T.
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Ergothioneine (EGT), a sulfur-containing betaine of 2-thiohistidine, has long been recognized as a unique antioxidant compound of fungal and bacterial origin that accumulates in animal tissues prone to oxidative stress. Despite its biological importance, its redox potential and detection mechanisms remained historically mischaracterized. Canonical values (E°′ = –0.06 V vs SHE) originate from pH 0 oxidation potentials and empirical conversions inconsistent with modern glutathione benchmarks (E°′ = –0.240 V vs SHE). This thesis revisits and redefines EGT’s aqueous redox thermodynamics using complementary electrochemical, spectroscopic, and kinetic assays, and establishes a new, visible-range spectrophotometric method for its detection.Cyclic voltammetry at pH 7 (Ag/AgCl, referenced to Ru(NH₃)₆³⁺/²⁺) established oxidation potentials of Epa (EGT/ESSE) = +0.450 V and Epa (Asc/DHAA) = +0.350 V vs SHE, confirming EGT is a comparatively weak reductant under physiological conditions. Complementary 1H NMR and UV-Vis assays using redox-active probes—oxidized glutathione, DTNB, methylene blue, and cytochrome c—corroborated that EGT exhibits slower reduction kinetics and weaker thermodynamic driving force than ascorbate. Moreover, EGT does not reduce oxidized glutathione, consistent with its more positive formal potential. Together, these orthogonal methods resolve the long‑standing misassignment of EGT’s redox potential. Building on this redox framework, a new colorimetric assay was developed exploiting EGT’s reaction with 2,6‑dichloroindophenol (DCIP). Contrary to classical interpretations, EGT does not reduce DCIP but undergoes a nucleophilic aromatic substitution via a thione‑sulfur addition–elimination pathway to yield mono‑, di‑, and tri‑substituted EGT–DCIP adducts. The adducts exhibit diagnostic absorption maxima at 540 nm and 680 nm, and their formation is accelerated by Group I cations (Li⁺→Cs⁺) in phosphate buffer through cation–phosphate cooperative effects. Mass spectrometry confirmed the structures of the oxidized and reduced adduct species. This mechanism provides a visible‑range, selective, and cost‑effective analytical route to quantify EGT in complex matrices and differentiates it from naturally found thiols and ascorbate. Collectively, these findings redefine EGT as a moderate reductant (Epa ≈ +0.45 V) that engages in soft‑nucleophile substitution chemistry rather than direct electron transfer, and introduce a new cost‑effective, mechanistically validated visible‑range assay for its quantitation. This work reconciles decades of inconsistent data and provides a corrected thermodynamic placement for EGT within biological redox networks. Furthermore, establishing a framework for mechanistic and analytical studies in future biochemical, agricultural, nutritional, and pharmacological investigations of ergothioneine. Thus, enabling accurate evaluation of EGT’s antioxidant capacity in physiological matrices. Keywords: ergothioneine; redox potential; electrochemistry; antioxidant; nucleophilic aromatic substitution; 2,6‑dichloroindophenol; detection assay; spectrophotometry
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2026
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- Embargoed until 2026-12-12