Rational design of 1,2-dichalcogenides unlocks selective probes for the thioredoxin system

Zeisel, Lukas (2025): Rational design of 1,2-dichalcogenides unlocks selective probes for the thioredoxin system. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Vicinal dithiol oxidoreductases maintain redox homeodynamics by facilitating electron transfer, modulating protein structure and activity, and driving signaling processes. Despite their importance, the Trx/TrxR and Grx/GSH/GR systems lack high-quality turnover probes capable of dynamically monitoring their activity in live cells. This thesis addresses this gap by integrating fundamental knowledge of Trx/GSH system biology & biochemistry with state-of-the-art chemical probe research and basic principles of chalcogenol/dichalcogenide exchange (CDE) chemistry. Dynamic imaging of enzyme activity requires probes that reversibly react with the target protein’s active site. To achieve this for the Trx/GSH systems, the turnover probes developed herein feature an artificial dichalcogenide substrate that is selectively reduced by the target reductase. Upon reduction, the probe undergoes irreversible fragmentation, leading to the accumulation of a fluorescent signal. The rates of the individual steps leading to signal generation govern a probe’s reactivity in biological systems, and achieving selectivity requires the design of artificial substrates whose CDE kinetics favour reduction exclusively by the reductase of interest. This thesis pioneers the rational design of cyclic dichalcogenides for cellular dithiol reductase probes, resulting in practical syntheses of unprecedented cyclic 1,2-dichalcogenides, robust design principles for redox probe development, and the creation of cellular probes for Trx and TrxR. Given the limited synthetic precedent for cyclic 1,2-dichalcogenides, a major focus was the development of efficient synthetic routes to these understudied motifs. Key achievements include regioselective syntheses of 1,2-thiaselenane amines and diastereoselective access to piperazine-fused 1,2-dithianes, 1,2-thiaselenanes and 1,2-diselenanes. Each sequence was designed for scalability & modularity, minimising the need for chromatographic purification. Thioredoxin (Trx) is the most potent dithiol reductase in cells (katt, kfull↑). To achieve selective reduction by Trx, we designed cis-fused bicyclic disulfides that are thermodynamically and kinetically (kretro↑↑) equipped to resist reduction by cellularly abundant monothiols and weaker dithiol reductases (Grxs). Piperazine annellation ensured rapid fluorophore release rates (k’cyc), giving C-DiThias as the first cellular probes that selectively report on thioredoxin activity. Thioredoxin Reductase (TrxR) is a unique selenolthiol reductase, which we targeted by using a 1,2-thiaselenane-4-amine substrate. Incorporation of selenium enabled high katt for TrxR’s selenolate while maintaining rapid reversion rates through intramolecular SN2 at Se (kretro). Kinetic selectivity for TrxR against thiol nucleophiles was reached since this kretro outcompeted the smaller katt for thiolates, and since full reduction from B is uniquely hindered for monothiols via iterative SN2 at the more electrophilic Se. As a result, probe RX1 fully resists >1000-fold challenge with GSH, reacts rapidly with 1/1000 equiv. of TrxR and has excellent TrxR-selective performance in cells, validated by knockout, selenium starvation, knockin, and chemical inhibitors. Overall, this thesis presents a systematic approach to tuning individual probe activation rates through rational molecular design – an area that has received limited attention in the literature thus far. However, the multistep-reactive probe design strategy may prove valuable beyond Trx/GSH system probes: in developing chemical biology reagents or drugs that effectively distuinguish between on-target and off-targets, to achieve unmatched selectivity in complex cellular settings.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: dichalcogenide, selenenyl sulfide, disulfide, thiol/disulfide exchange, reaction rates, thioredoxin reductase, thioredoxin, chemical probes, enzyme kinetics, organochalcogen compounds
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 23. Mai 2025
• 1. Bericht­erstatter:in: Thorn-Seshold, Oliver