Piperazine-Fused Cyclic Disulfides Unlock High-Performance Bioreductive Probes of Thioredoxins and Bifunctional Reagents for Thiol Redox Biology
Lukas Zeisel, Jan G. Felber, Karoline Scholzen, Carina Schmitt, Alexander J. Wiegand, Leonid Komissarov, Elias S.J. Arnér, Oliver Thorn‐Seshold
Abstract
We report piperazine-fused six-membered-cyclic disulfides as redox substrates that unlock best-in-class bioreduction probes for live cell biology, since their self-immolation after reduction is unprecedentedly rapid. We develop scalable, diastereomerically pure, six-step syntheses that access four key cis - and trans -piperazine-fused cyclic dichalcogenides without chromatography. Fluorogenic redox probes using the disulfide piperazines are activated >100-fold faster than the prior art monoamines, allowing us to deconvolute reduction and cyclization rates during activation. The cis- and trans -fused diastereomers have remarkably different reductant specificities, which we trace back to piperazine boat/chair conformation effects: the c is -fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the t rans - disulfide T-DiThia is activated even by moderate concentrations of monothiols such as GSH. Thus, in cellular applications, cis- disulfide probes selectively report on the reductive activity of the powerful thioredoxin proteins, while t rans -disulfides are rapidly but promiscuously reactive. Finally, we showcase late-stage diversifications of the piperazine-disulfides, promising their broad applicability as redox-cleavable cores for probes and prodrugs that interface powerfully with cellular thiol/disulfide redox biology, for solid phase synthesis and purification, and for stimulus-responsive linkers in bifunctional reagents and antibody-drug conjugates - in addition to their dithiols’ potential as high-performance reducing agents.