Isotope-labeled ergothioneine clarifies the mechanism of reaction with singlet oxygen.

Recently we have uncovered a non-enzymatic multi-step cycle for the regeneration of ergothioneine (ET), after reaction with noxious singlet oxygen (O), by glutathione (GSH). When living cells were loaded with ET labeled with deuterium and N-15 atoms (D5-ET) and exposed to light in the presence of a photosensitizer, no loss of deuterium at position 5 of the imidazole ring was observed, in contradiction to our previous mechanistic proposal. Therefore, it was necessary to reexamine the in vitro products of ET and O by liquid chromatography coupled to high resolution mass spectrometry. Pure O was generated by thermolysis at 37 °C of the endoperoxide DHPNO. The use of D5-ET enabled us to revise and extend the reaction scheme. On the main pathway, O attacks the imidazole ring, and the hydroperoxide intermediates are reduced rapidly by ET or GSH via different mechanisms. The intramolecular water elimination from the 5-hydroperoxide described previously is slower and not a part of the cycle. On another side path, O attacks the sulfur of ET to form a sulfine (S-oxide). The reduction of the sulfine also allows for the complete regeneration of ET. Experiments with methanol instead of water as solvent revealed that, in the absence of GSH, ET was attacked 6 times more frequently at the ring than at the sulfur. In the presence of 1 mM GSH or higher, both side paths were abandoned. ET efficiently captures O with its ring and can then be regenerated to a large extent by GSH, without enzyme involvement.