Elucidating the Protective Mechanisms of Methimazole against Copper(I)-Mediated Fenton Chemistry Using Online Mass...

Methimazole (MMI), which is primarily known as an antithyroid drug, has received considerable attention for its potent antioxidant properties in a copper(I)-mediated Fenton reaction. However, the antioxidant mechanistic details of MMI, particularly its structural analogy to the natural antioxidant ergothioneine (both of which feature an imidazole-2-thione moiety), remain largely unexplored due to the difficulty of in situ characterizing unstable reaction intermediates. In this study, we combined an in situ reactor-integrated paper-in-tip spray ionization source with mass spectrometry (PTSI-MS) to probe the protection mechanisms of MMI against the Cu^I-catalyzed Fenton reaction online. We present direct MS evidence of the antioxidant mechanisms of MMI (M) in its reduced form (MSH) and oxidized disulfide form (MSSM). The former can directly sequester H₂O₂ (i.e., a H₂O₂ scavenging mechanism). The latter tightly coordinates with Cu^I to form a Cu^I-MSSM complex that stabilizes Cu^I and prevents its oxidation by H₂O₂ and thus the formation of ^•OH (i.e., a metal ion coordination mechanism). The proposed reaction pathway of the Cu^I complexes from MMI disulfide (Cu^I-MSSM, m/z = 289 and 291) to MMI thiosulfonate (Cu^I-MSO₂SM, m/z = 321 and 323) to MMI monosulfide (Cu^I-MSM, m/z 257 and 259) was verified by S-S oxidation, SO₂-S bond cleavage, and extruded sulfur elimination, which confirmed the stable N,N'-bidentate binding of Cu^I to these intermediates, even when attacked by H₂O₂. These findings challenge the traditional radical scavenging mechanism and contribute to our understanding of the relationship between the structure and antioxidant activity of imidazole-2-thiones. Furthermore, the established in situ reactor-integrated PTSI-MS approach provides unique insights into reaction dynamics, offering the advantages of simplicity, speed, and low reaction volume (down to 20 μL).