Bifunctional, piperazine-fused cyclic disulfides for oxidoreductase-activated cellular proagents

We report piperazine-fused six-membered-cyclic dichalcogenides as rapid-response redox sensors for probes, prodrugs, and bifunctional conjugates that interface with cellular thiol/disulfide redox biology. By combining the thermodynamic stability of 1,2-dithianes with unprecedently rapid kinetics of self-immolation after reduction, these motifs are uniquely reliable and flexible reduction-based sensors for live cell applications. We synthesise four cis- and trans-piperazine-fused cyclic disulfides and diselenides by scalable, diastereomerically pure, six-step synthetic routes with just one chromatographic purification. Fluorogenic probes using these redox-active diamines are >100-fold faster activated than the previously known monoamines, which now allowed us to deconvolute the kinetics of the reduction and the cyclisation steps during activation. The diastereomers have remarkably different reductant specificity. In particular, the cis-fused disulfides are only activated by strong vicinal dithiol reductants, while trans-fused disulfides are activated even by monothiols like GSH. Thus, although both disulfides are good substrates for glutaredoxins and thioredoxins in cell-free assays, upon cellular applications the cis-disulfide probes substantially report on oxidoreductase activity in the thioredoxin system (the trans-disulfides remain promiscuously reactive). Finally, we showcase efficient late-stage synthetic diversification of the piperazine-disulfides, promising their broad applicability as cleavable bifunctional cores for redox probes and prodrugs, for solid phase synthesis, and as linkers for antibody-drug conjugates.