Bicyclic selenenyl sulfides tune stepwise rates of thiol/selenol addition, resolution, and cyclisation, to reveal cellular constraints for bioreductive probes targeting mammalian thioredoxin reductase

The bioreductive activation of dichalcogenide probes by mammalian thiol-type oxidoreductases proceeds via a cascade of consecutive, partly-reversible steps. Stereocontrol elements can exert control over these steps' reaction rates, which was recently exploited in bicyclic reduction substrates that fuse a reducible disulfide ring to a templating nitrogenous ring to reach powerful substrate-controlled kinetic selectivities for, e.g., vicinal dithiol vs monothiol reductants in live cells. We now deploy regio-, diastereo-, template-, and pH-control elements to shape the reactivity of unprecedented bicyclic selenenyl sulfides (SeSP), arriving at probes that selectively address the mammalian selenoenzyme thioredoxin reductase TrxR1. We accessed these densely-functionalised cis- or trans-fused-1,2-thiaselenanes on gram scale over 5 steps, via a regioselective key step that elaborates an unusual, differentially protected 2,2’-biaziridine intermediate through sequential one-pot chalcogen introduction and selenenyl sulfide formation. By profiling all regio- and diastereo-isomeric bicycles for their partly- or fully-reversible reactivity, we showed how effects that slow their reduction steps (addition then resolution) can compensate by vastly accelerating subsequent cyclisation speeds, such that cellular redox processing is effective and TrxR-selective. More broadly, this study shows how multistep cascade probes can leverage conformational effects and internal non-covalent interactions to differentiate step kinetics along their on-target vs off-target reaction pathways, thus achieving reaction-based target selectivity in complex biological settings.