2-Mercapto-L-histidine

Ergothioneine (Egt), 2-mercapto-L-histidine betaine (ESH), is a dietary component acting as antioxidant and cytoprotectant. In vitro studies demonstrated that Egt, a powerful scavenger of hydroxyl radicals, superoxide anion, hypochlorous acid and peroxynitrite, protects vascular function against oxidative damages, thus preventing endothelial dysfunction. In order to delve the peculiar oxidative behavior of Egt, firstly identified in cell free-systems, experiments were designed to identify the Egt oxidation products when endothelial cells (EC) benefit of its protection against high-glucose (hGluc). HPLC-ESI-MS/MS analyses revealed a decrease in the intracellular GSH levels and an increase in the ophthalmic acid (OPH) levels during hGluc treatment. Interestingly, in the presence of Egt, the decrease of the GSH levels was lower than in cells treated with hGluc alone, and this effect was paralleled by lower OPH levels. Egt was also effective in reducing the cytotoxicity of H2O2 and paraquat (PQT), an inducer of superoxide anion production, showing a similar time-dependent pattern of GSH and OPH levels, although with peaks occurring at different times. Importantly, Egt oxidation generated not only hercynine (EH) but also the sulfonic acid derivative (ESO3H) whose amounts were dependent on the oxidative stress employed. Furthermore, cell-free experiments confirmed the formation of both EH and ESO3H when Egt was reacted with superoxide anion. In summary, these data, by identifying the EH and ESO3H formation in EC exposed to hGluc, highlight the cellular antioxidant properties of Egt, whose peculiar redox behavior makes it an attractive candidate for the prevention of oxidative stress-associated endothelial dysfunction during hyperglycemia.

Ergothioneine (ESH), the betaine of 2-mercapto-L-histidine, is a water-soluble naturally occurring amino acid with antioxidant properties. ESH accumulates in several human and animal tissues up to millimolar concentration through its high affinity transporter, namely the organic cation transporter 1 (OCTN1). ESH, first isolated from the ergot fungus (Claviceps purpurea), is synthesized only by Actinomycetales and non-yeast-like fungi. Plants absorb ESH via symbiotic associations between their roots and soil fungi, whereas mammals acquire it solely from dietary sources. Numerous evidence demonstrated the antioxidant and cytoprotective effects of ESH, including protection against cardiovascular diseases, chronic inflammatory conditions, ultraviolet radiation damages, and neuronal injuries. Although more than a century after its discovery has gone by, our understanding on the in vivo ESH mechanism is limited and this compound still intrigues researchers. However, recent evidence about differences in chemical redox behavior between ESH and alkylthiols, such as cysteine and glutathione, has opened new perspectives on the role of ESH during oxidative damage. In this short review, we discuss the role of ESH in the complex machinery of the cellular antioxidant defense focusing on the current knowledge on its chemical mechanism of action in the protection against cardiovascular disease.

1. The reactions of 2,2'- and 4,4'-dipyridyl disulphide (2-Py-S-S-2-Py and 4-Py-S-S-4-Py) with l-ergothioneine (2-mercapto-l-histidine betaine), 2-mercaptoimidazole, 1-methyl-2-mercaptoimidazole, thiourea, thioacetamide, 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were investigated spectrophotometrically in the pH range approx. 1-9. 2. These reactions involve two sequential reversible thiol-disulphide interchanges. 3. The reaction of l-ergothioneine with 2-Py-S-S-2-Py and/or with the l-ergothioneine-Py-2-SH mixed disulphide, both of which provide Py-2-SH, is characterized by at least three reactive protonic states. This provides definitive evidence that neutral l-ergothioneine is a reactive nucleophile, particularly towards the highly electrophilic protonated disulphides. 4. A similar situation appears to obtain in the reactions of l-ergothioneine and Py-2-SH with 4-Py-S-S-4-Py and in the reactions of the other 2-mercaptoimidazoles, thiourea and Py-4-SH with 2-Py-S-S-2-Py. The nucleophilic reactivity of Py-4-SH suggests that general base catalysis provided by the disulphide in a cyclic or quasi-cyclic transition state is not necessary to generate nucleophilic reactivity in the other amino-thiones whose geometry could permit such catalysis. 5. The existence of a positive deuterium isotope effect in the l-ergothioneine-2-Py-S-S-2-Py system at pH6-7 provides no evidence for general base catalysis but is in accord with a mechanism involving specific acid catalysis and post-transition-state proton transfer. 6. The pH-dependences of the overall equilibrium positions of the various thiol-disulphide interchanges are described. 7. Reaction of thioacetamide with a stoicheiometric quantity of 2-Py-S-S-2-Py at pH1 provides 2 molecules of Py-2-SH per molecule of thioacetamide and elemental sulphur; these findings can be accounted for by thiol-disulphide interchange to provide a thioacetamide-Py-2-SH mixed disulphide followed by fragmentation to provide CH(3)CN, S and Py-2-SH. 8. Provision of high reactivity in the neutral forms of the members of this series of sulphur nucleophiles by electron donation by the amino group is compared with the well known alpha effect that provides enhanced nucleophilicity in compounds containing an electronegative atom adjacent to the nucleophilic atom. 9. The decrease in the u.v. absorption of l-ergothioneine at 257nm consequent on transformation of its aminothione moiety into an S-alkyl-2-mercaptoimidazole moiety provides a convenient method of following the alkylation of l-ergothioneine by iodoacetamide. 10. The pH dependence of the extinction coefficient of l-ergothioneine at 257nm is described by epsilon(257)={8x10(3)/(1+K(a)/[H(+)]} +6x10(3)m(-1).cm(-1) in which pK(a)=10.8. 11. In the pH range 3-11 the reaction is characterized by two reactive protonic states (X and XH). 12. The X state, reaction of the ionized 2-mercaptoimidazole moiety of the l-ergothioneine dianion with neutral iodoacetamide, is characterized by the second-order rate constant 4.0m(-1).s(-1) (25.0 degrees C, I=0.05). The XH state, characterized by the second-order rate constant 0.03m(-1).s(-1), is interpreted as reaction of the thione form of the neutral 2-mercaptoimidazole moiety of the l-ergothioneine monoanion with neutral iodoacetamide. 13. The XH state of the alkylation reaction does not exhibit a deuterium isotope effect.