Next, we wished to test our hypothesis that the selenenylsulfides should interact selectively with mammalian TrxR on the basis of the TrxR selenolthiol. TrxR has two redox-active sites, with the NADPH-driven N-terminal Cys-Cys shuttling electrons to the Cterminal Sec-Cys. As Hondal et al have shown in their paper "No Selenium Required", 32 many compounds that had been assumed to require a selenol for reactivity can be similarly processed by a C-terminal Cys-Cys mutant TrxR U498C , or are better or exclusively processed by the N-terminal dithiol site. We compared probe activation by wildtype TrxR (Sec-Cys) with Cys-Cys mutant rTrxR1 U498C . The mutant no longer reduced either the A-or G-type probes, while its reduction of standard substrates DTNB and juglone was unimpeded (Fig. , Fig. ). This supported that both A-and G-selenenylsulfide probes do indeed require native mammalian TrxR's C-terminal selenolthiol for efficient reduction, matching our designs. With excellent monothiol resistance, and strong processing by selenolthiol TrxR1 rather than its dithiol mutant, it remained an open question whether physiological concentrations of other selenolbearing proteins would also be capable of probe triggering, which is important for enzymatic selectivity. There are only 25 selenoproteins in the human proteome; only a few of these have ubiquitous expression at substantial concentrations; and nearly all of those have monoselenol active sites (not selenolthiols). The highest-concentration ubiquitous selenoproteins are monoselenol glutathione peroxidases GPx1 and GPx4. We challenged RX1 and G1 with GPx1 up to supraphysiological 1 µM concentration, and no probe activation was detected over 6 hours (Fig. ). This was an encouraging step towards the possibility of cyclic selenenylsulfides acting as TrxR-selective probes (see Supporting Note 6).