Bicyclic Selenenyl Sulfides with Tuned Bioreductive Step Rates Reveal Constraints for Probes Targeting Thioredoxin...

The reductive activation of dichalcogenide probes by thiol-type oxidoreductases proceeds through a cascade of consecutive, partly reversible steps. Stereocontrol elements can modulate the reaction rates of these steps to reach substrate-controlled kinetic selectivity for reductase chemotypes 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 target the mammalian selenoenzyme thioredoxin reductase TrxR1. We accessed these densely functionalised cis- or trans-fused 1,2-thiaselenanes on gram scale over 5 steps by using a regioselective key step that elaborates an unusual, differentially protected 2,2'-bis-aziridine intermediate through sequential one-pot chalcogen introduction and selenenyl sulfide formation. By profiling a set of regio- and diastereoisomeric bicycles for their partly or fully reversible reactivity during reductive activation, we show how effects that slow their reduction steps (addition then resolution) can compensate by vastly accelerating subsequent activation (cyclisation) speeds, such that cellular processing is effective and TrxR-selective. More broadly, this study shows how multistep cascade probes can leverage conformational effects and internal noncovalent interactions to differentiate step kinetics along their on-target versus off-target reaction pathways, thus achieving reaction-based target selectivity in complex biological settings.