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

Optical Microscopy in the Nano-World

Scanning near-field optical microscopy (SNOM) is an optical microscopy whose resolution is not bound to the diffraction limit. It provides chemical information based upon spectral, polarization and/or fluorescence contrast images. Details as small as 20 nm can be recognized. Photophysical and photochemical effects can be studied with SNOM on a similar scale. This article reviews a good deal of the experimental and theoretical work on SNOM in Switzerland

Regioselective, efficient and scalable syntheses of 1,2-thiaselenanes

We develop the first regioselective syntheses of 1,2-thiaselenane-4-amine (TSA4) and 1,2-thiaselenane-5-amine (TSA5): redox-active motifs with high value in chemical biology, that until now were hindered by tedious synthesis. We leverage an aziridine intermediate and a kinetically controlled S-acylation for regioselective chalcogen installations. We optimise short, fast sequences with just one or two chromatographic steps that cheaply deliver these motifs on scale for high-throughput inhibitor screening, and provide a robust methodology for assembling other selenenylsulfides

Piperazine-fused cyclic disulfides: high-performance bioreduction-activated cores for bifunctional probes and reagents

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

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 used as the key element in numerous chemical biology probes. Contradictory views of this disulfide populate the literature: some reports describe it as being nonspecifically reduced, others as a highly specific substrate for thioredoxin reductase (TrxR). We here show that 1,2-dithiolane probes are nonspecifically reduced by a broad range of thiol reductants and redox-active proteins, and that their cellular performance is barely affected by TrxR inhibition or knockout. We conclude that inhibitor screenings and "TRFS" probes that have used 1,2-dithiolanes as TrxRselective substrates should be treated with caution, and may need re-evaluation. Understanding 1,2-dithiolanes’ behaviour needs consideration of probe localisation and environmentdependent fluorescence, reduction-independent ring-opening polymerisation, thiol-dependent cellular uptake, and caution when applying thiophilic inhibitors. We present an approach controlling against assay misinterpretation with reducible probes, to ensure that future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research

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

Specialised 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 nonspecific 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 deliver redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.</p

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

Cellular redox networks power a multitude of cellular processes, and are often dysregulated in pathologies including cancer and inflammatory diseases. Quantifying the turnover of the key players in redox homeostasis is crucial for understanding their physiological dynamics and for targeting them in pathologies. However, suitably selective probes for assessing specific redox enzyme activities in cells are lacking. We rationally developed the first chemical probes targeting the mammalian selenoprotein thioredoxin reductase (TrxR) while fully resisting other cellular thiols and oxidoreductases. We used a cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger, oriented to harness the chemistry of TrxR\u27s unique selenolthiol active site, and integrated it into modular probes releasing arbitrary cargos upon reduction. The probes showed remarkable selenocysteine-dependent sensitivity to cytosolic TrxR1, against a panel of oxidoreductases. Lead probe RX1 also had excellent TrxR1-selective performance in cells, as cross-validated by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and TrxRselective chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors. This indicated that tempered SNAr electrophiles may be a more favorable drug class than classically-used electrophiles. The RX1 design is thus a robust, cellularly validated, high-performance modular system for mammalian TrxR1. This sets the stage for in vivo imaging TrxR1 activity in health and disease, and can also drive and reorient TrxR1-inhibitor drug design. The thermodynamic and kinetic considerations behind RX1\u27s selectivity also outline paths towards rationally-designed probes for other key players in redox biology

Marine Omega-3 Phospholipids: Metabolism and Biological Activities

The biological activities of omega-3 fatty acids (n-3 FAs) have been under extensive study for several decades. However, not much attention has been paid to differences of dietary forms, such as triglycerides (TGs) versus ethyl esters or phospholipids (PLs). New innovative marine raw materials, like krill and fish by-products, present n-3 FAs mainly in the PL form. With their increasing availability, new evidence has emerged on n-3 PL biological activities and differences to n-3 TGs. In this review, we describe the recently discovered nutritional properties of n-3 PLs on different parameters of metabolic syndrome and highlight their different metabolic bioavailability in comparison to other dietary forms of n-3 FAs