We report piperazine-fused six-membered-cyclic dichalcogenides as rapid-response redox substrates that interface with thiol/disulfide redox biology. Combining the stability of 1,2-dithianes with unprecedentedly rapid kinetics of self-immolation after reduction, these motifs are uniquely high-performance reduction-responsive motifs for live cell probes. We develop scalable, diastereomerically pure, six-step synthetic routes with just one chromatographic purification to access four key cis- and trans-piperazine-fused cyclic disulfide and diselenide cores. Fluorogenic redox probes using the disulfide-piperazines are activated >100-fold faster than the previously known monoamines, allowing us to deconvolute the kinetics of the reduction and the cyclisation steps during activation. The cis- and trans-fused diastereomers have remarkably different reductant specificities: the cis disulfides are activated only by strong vicinal dithiol reductants, but the trans-fused disulfides are activated even by moderate concentrations of monothiols such as GSH. Thus, although both disulfides are substrates for redoxins, in cellular applications the cis-disulfide probes were found to selectively report on reductive activity of thioredoxins, while the trans-disulfides are more rapidly but more promiscuously reactive. Finally, we showcase efficient late-stage synthetic diversification of the piperazine-disulfides, promising their broad applicability as robust cleavable cores for redox probes and prodrugs in biology, for solid phase synthesis and purifications, and as stimulus-responsive linkers for bifunctional reagents and antibody-drug conjugates.
Supporting Information PDF - Synthesis, Biochemistry, Cell Biology, Supporting Notes, Analytical Data