Piperazine-Fused Cyclic Disulfides Unlock High-Performance Bioreductive Probes of Thioredoxins and Bifunctional Reagents for Thiol Redox Biology.

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.

Correction: Synthesis of highly substituted fluorenones via metal-free TBHP-promoted oxidative cyclization of 2-(aminomethyl)biphenyls. Application to the total synthesis of nobilone.

[This corrects the article DOI: 10.3762/bjoc.17.181.].

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

Small-molecule prodrug approaches that can activate cancer therapeutics selectively in tumors are urgently needed. Here, we developed the first antitumor prodrugs designed for activation by thiol-manifold oxidoreductases, targeting the thioredoxin (Trx) system. The Trx system is a critical cellular redox axis that is tightly linked to dysregulated redox/metabolic states in cancer, yet it cannot be addressed by current bioreductive prodrugs, which mainly cluster around oxidized nitrogen species. We instead harnessed Trx/TrxR-specific artificial dichalcogenides to gate the bioactivity of 10 "off-to-on" reduction-activated duocarmycin prodrugs. The prodrugs were tested for cell-free and cellular reductase-dependent activity in 177 cell lines, establishing broad trends for redox-based cellular bioactivity of the dichalcogenides. They were well tolerated in vivo in mice, indicating low systemic release of their duocarmycin cargo, and in vivo anti-tumor efficacy trials in mouse models of breast and pancreatic cancer gave promising indications of effective tumoral drug release, presumably by in situ bioreductive activation. This work therefore presents a chemically novel class of bioreductive prodrugs against a previously unaddressed reductase chemotype, validates its ability to access in vivo-compatible small-molecule prodrugs even of potently cumulative toxins, and so introduces carefully tuned dichalcogenides as a platform strategy for specific bioreduction-based release.

Selective cellular probes for mammalian thioredoxin reductase TrxR1: rational design of RX1, a modular 1,2-thiaselenane redox probe.

Quantifying the activity of key cellular redox players is crucial for understanding physiological homeostasis, and for targeting their perturbed states in pathologies including cancer and inflammatory diseases. However, cellularly-selective probes for oxidoreductase turnover are sorely lacking. We rationally developed the first probes that selectively target the mammalian selenoprotein thioredoxin reductase (TrxR), using a cyclic selenenylsulfide oriented to harness TrxR's unique selenolthiol chemistry while resisting the cellular monothiol background. Lead probe RX1 had excellent TrxR1-selective performance in cells, cross-validated by knockout, selenium starvation, knock-in, and chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors, which indicated that tempered SNAr electrophiles may be more selective TrxR drugs than the classical electrophiles used hitherto. The RX1 design thus sets the stage for in vivo imaging of the activity of this key oxidoreductase in health and disease, and can also drive TrxR1-inhibitor drug design.

Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically.

The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as "TRFS" probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes' complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.

Synthesis of highly substituted fluorenones via metal-free TBHP-promoted oxidative cyclization of 2-(aminomethyl)biphenyls. Application to the total synthesis of nobilone.

Highly substituted fluorenones are readily prepared in mostly fair to good yields via metal- and additive-free TBHP-promoted cross-dehydrogenative coupling (CDC) of readily accessible N-methyl-2-(aminomethyl)biphenyls and 2-(aminomethyl)biphenyls. This methodology is compatible with numerous functional groups (methoxy, cyano, nitro, chloro, and SEM and TBS-protective groups for phenols) and was further utilized in the first total synthesis of the natural product nobilone.

Selective, Modular Probes for Thioredoxins Enabled by Rational Tuning of a Unique Disulfide Structure Motif.

Specialized cellular networks of oxidoreductases coordinate the dithiol/disulfide-exchange reactions that control metabolism, protein regulation, and redox homeostasis. For probes to be selective for redox enzymes and effector proteins (nM to µM concentrations), they must also be able to resist non-specific triggering by the ca. 50 mM background of non-catalytic cellular monothiols. However, no such selective reduction-sensing systems have yet been established. Here, we used rational structural design to independently vary thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual disulfide reduction trigger units designed for stability to monothiols. We integrated the motifs into modular series of fluorogenic probes that release and activate an arbitrary chemical cargo upon reduction, and compared their performance to that of the literature-known disulfides. The probes were comprehensively screened for biological stability and selectivity against a range of redox effector proteins and enzymes. This design process delivered the first disulfide probes with excellent stability to monothiols yet high selectivity for the key redox-active protein effector, thioredoxin. We anticipate that further applications of these novel disulfide triggers will deliver unique probes targeting cellular thioredoxins. We also anticipate that further tuning following this design paradigm will enable redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.

1-AminoTriazole Transition-Metal Complexes as Laser-Ignitable and Lead-Free Primary Explosives.

This unique complex study describes two isomeric aminotriazoles as auspicious nitrogen-rich ligands for energetic coordination compounds (ECCs) to replace the commonly used highly poisonous and environmentally harmful lead-based primary explosives. The triazoles were obtained by easily scalable and convenient synthetic routes starting solely from commercially available starting materials. 1-Amino-1,2,3-triazole (1, 1-ATRI) and, for the first time, 1-amino-1,2,4-triazole (2, 1A-1,2,4-TRI) were employed as ligands to form highly energetic transition-metal(II) complexes. The desired characteristics could be altered successively by using various nonpoisonous metal(II) centers (Cu2+ , Mn2+ , Fe2+ , and Zn2+ ) and anions (Cl- , NO3 - , ClO3 - , ClO4 - , picrate, styphnate, 2,4,6-trinitro-3-hydroxyphenolate, and 2,4,6-trinitro-3,5-dihydroxyphenolate). The 14 synthesized coordination compounds were characterized comprehensively by XRD, IR and UV/Vis spectroscopy, elemental analysis, and differential thermal and thermogravimetric analyses. Ball-drop impact, electrostatic discharge (ESD), and mechanical (impact and friction) sensitivities were determined according to BAM standard methods. In addition to laser ignition experiments, selected ECCs were evaluated in classical secondary explosive initiation tests (detonators filled with pentaerythritol tetranitrate (nitropenta)), which revealed their enormous potential and proved them to be very attractive for future applications in explosives.