Synthesis of ¹⁴N- and ¹⁵N-labeled trityl-nitroxide biradicals with strong spin-spin interaction and improved sensitivity to redox status and oxygen

Simultaneous evaluation of redox status and oxygenation in biological systems is of great importance for the understanding of biological functions. Electron paramagnetic resonance (EPR) spectroscopy coupled with the use of the nitroxide radicals have been an indispensable technique for this application but are still limited by low oxygen sensitivity and low EPR resolution in part due to the moderately broad EPR triplet and spin quenching through bioreduction. In this study, we showed that these drawbacks can be overcome through the use of trityl-nitroxide biradicals allowing for the simultaneous measurement of redox status and oxygenation. A new trityl-nitroxide biradical TNN14 composed of a pyrrolidinyl-nitroxide and a trityl and its isotopically labeled ¹⁵N analogue TNN15 were synthesized and characterized. Both biradicals exhibited much stronger spin-spin interaction with J > 400 G compared with that of the previous synthesized trityl-nitroxide biradicals TN1 (∼160 G) and TN2 (∼52 G) with longer linker chain length. The enhanced stability of TNN14 was evaluated using ascorbate as reductant, and the effect of different types of cyclodextrins on its stability in the presence of ascorbate was also investigated. Both biradicals are sensitive to redox status, and their corresponding trityl-hydroxylamines resulting from the reduction of the biradicals by ascorbate share the same oxygen sensitivity. Of note is that the ¹⁵N-labeled TNN15-H with an EPR doublet exhibits improved EPR signal amplitude as compared with that of TNN14-H with an EPR triplet. In addition, cyclic voltammetric studies verify the characteristic electrochemical behaviors of the trityl-nitroxide biradicals.