ABSTRACT
The cytoskeleton is essential for spatial and temporal organisation of a wide range of cellular and tissue-level processes, such as proliferation, signalling, cargo transport, migration, morphogenesis, and neuronal development. Cytoskeleton research aims to study these processes by imaging, or by locally manipulating, the dynamics and organisation of cytoskeletal proteins with high spatiotemporal resolution: which matches the capabilities of optical methods. To date, no photoresponsive microtubule-stabilising tool has united all the features needed for a practical high-precision reagent: a low potency and biochemically stable non-illuminated state; then an efficient, rapid, and clean photoresponse that generates a high potency illuminated state; plus good solubility at suitable working concentrations; and efficient synthetic access. We now present CouEpo, a photocaged epothilone microtubule-stabilising reagent that combines these needs. Its potency increases approximately 100-fold upon violet/blue irradiation to reach low-nanomolar values, allowing efficient photocontrol of microtubule dynamics in live cells, and even the generation of cellular asymmetries in microtubule architecture and cell dynamics. CouEpo is thus a high-performance tool compound that can support high-precision research into many microtubule-associated processes, from biophysics to transport, cell motility, and neuronal physiology.
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 cis-fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the trans-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 trans-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.