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

Abstract

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.

Cyclic Dichalcogenides Extend the Reach of Bioreductive Prodrugs to Harness Thiol/Disulfide Oxidoreductases: Applications to seco-Duocarmycins Targeting the Thioredoxin System

“…Cyclic 6-membered disulfide ( SS60 ) or its faster-activated, solubilized analogue ( P-SS60 ) was used as slowly/moderately reduced Trx-selective substrate (minor crosstalk to dithiol Grx is expected). Their unstrained cis- annelated-piperazinyl bicyclic congeners ( P-SS66C , Me-SS66C ) have much faster, though less selective, Trx reductive activation (more crosstalk to dithiol Grx); strained trans- annelated congeners ( Me-SS66T ) are likewise rapidly activated by reductases, but are also moderately labile to monothiols such as GSH, so were expected to produce more unspecifically toxic prodrugs (a more complete view of the cellular selectivity profiles of these dichalcogenides is given in ref ). Separately from these Trx-targeted triggers, we included the specifically TrxR1-activated cyclic 6-membered selenenyl sulfide SeS60 to probe the performance and selectivity possible by targeting the regulator (rather than effector) of the thioredoxin system (Figure ).…”

“… Redox triggers for modular prodrugs. 1st generation Trx-triggers SS60 and solubilized P-SS60 ; 2nd generation solubilized bicyclic Trx-triggers Me-SS66T , Me-SS66C , and P-SS66C ; TrxR-trigger SeS60 . , “Upper limit” control triggers: very labile MC , monothiol-labile SS00 M . “Lower limit” control triggers: hydrolysis benchmarks O56 and P-CC60 , and carbamate stability test OBn .…”

“…The cyclic dichalcogenide redox triggers were previously shown to resist activation by monothiols such as glutathione (GSH) but to allow reduction/cyclization-based release of phenolic fluorophore cargos when treated with disulfide reductase enzymes (e.g., Trx and/or TrxR, Grx, etc. ). − These performance features had not been tested with naphthols, so we used HPLC to test them and show their reduction-based activation sequence (Figure c and Figures S4–S6).…”

“…The biological target space tested by (any) disulfide-based prodrugs has remained limited, as essentially only GSH-labile non-specific linear disulfides and 1,2-dithiolanes , have been examined. Recently, we have developed sets of 6-membered cyclic dichalcogenides with unique reduction-resisting properties as well as reductase selectivity profiles. − Stabilized disulfides in these series were selectively activated by Trxs, with excellent resistance to even thousand-fold higher levels of GSH and monothiols, and bifunctional bicyclic disulfides permitted drastic enhancement of their reductive activation kinetics . Cyclic selenenyl sulfides instead had a selenium-preferring, regioisomer-dependent activation mechanism, making them excellently selective substrates for TrxR1 in live cells. , These bioreductive motifs were validated in fluorogenic probes in acute applications (minutes to hours) in cell culture.…”

Efficient and Scalable Syntheses of 1,2-Thiaselenane-4-amine and 1,2-Thiaselenane-5-amine

“…We focused on cost-and time-efficiency: using cheap commercially available reagents, minimising chromatographic purifications, and reducing the step count. 17,18 The straightforward syntheses we now provide address the growing need for functionalisable selenenyl sulfides in chemical biology and will also fill gaps in organochalcogen chemistry.…”