This Perspective focuses on thiol-mediated uptake, that is, the entry of substrates into cells enabled by oligochalcogenides or mimics, often disulfides, and inhibited by thiol-reactive agents. A short chronology from the initial observations in 1990 until today is followed by a summary of cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs) as privileged scaffolds in thiol-mediated uptake and inhibitors of thiol-mediated uptake as potential antivirals. In the spirit of a Perspective, the main part brings together topics that possibly could help to explain how thiol-mediated uptake really works. Extreme sulfur chemistry mostly related to COCs and their mimics, cyclic disulfides, thiosulfinates/-onates, diselenolanes, benzopolysulfanes, but also arsenics and Michael acceptors, is viewed in the context of acidity, ring tension, exchange cascades, adaptive networks, exchange affinity columns, molecular walkers, ring-opening polymerizations, and templated polymerizations. Micellar pores (or lipid ion channels) are considered, from cell-penetrating peptides and natural antibiotics to voltage sensors, and a concise gallery of membrane proteins, as possible targets of thiol-mediated uptake, is provided, including CLIC1, a thiol-reactive chloride channel; TMEM16F, a Ca-activated scramblase; EGFR, the epithelial growth factor receptor; and protein-disulfide isomerase, known from HIV entry or the transferrin receptor, a top hit in proteomics and recently identified in the cellular entry of SARS-CoV-2.

Oligonucleotide phosphorothioates (OPS) are DNA or RNA mimics where one phosphate oxygen is replaced by a sulfur atom. They have been shown to enter mammalian cells much more efficiently than non‐modified DNA. Thus, solving one of the key challenges with oligonucleotide technology, OPS became very useful in practice, with several FDA‐approved drugs on the market or in late clinical trials. However, the mechanism accounting for this facile cellular uptake is unknown. Here, we show that OPS enter cells by thiol‐mediated uptake. The transient adaptive network produced by dynamic covalent pseudo‐disulfide exchange is characterized in action. Inhibitors with nanomolar efficiency are provided, together with activators that reduce endosomal capture for efficient delivery of OPS into the cytosol, the site of action.

Cyclic oligochalcogenides are emerging as powerful tools to penetrate cells. With disulfide ring tension maximized, selenium chemistry had to be explored next to enhance speed and selectivity of dynamic covalent exchange on the way into the cytosol. We show that diseleno lipoic acid (DiSeL) delivers a variety of relevant substrates. DiSeL‐driven uptake of artificial metalloenzymes enables bioorthogonal fluorophore uncaging within cells. Binding of a bicyclic peptide, phalloidin, to actin fibers evinces targeted delivery to the cytosol. Automated tracking of diffusive compared to directed motility and immobility localizes 79 % of protein‐coated quantum dots (QDs) in the cytosol, with little endosomal capture (0.06 %). These results suggest that diselenolanes might act as molecular walkers along disulfide tracks in locally denatured membrane proteins, surrounded by adaptive micellar membrane defects. Miniscule and versatile, DiSeL tags are also readily available, stable, soluble, and non‐toxic.

To image membrane tension in selected membranes of interest (MOI) inside living systems, the field of mechanobiology requires increasingly elaborated small-molecule chemical tools. We have recently introduced HaloFlipper, i.e., a mechanosensitive flipper probe that can localize in the MOI using HaloTag technology to report local membrane tension changes using fluorescence lifetime imaging microscopy. However, the linker tethering the probe to HaloTag hampers the lateral diffusion of the probe in all the lipid domains of the MOI. For a more global membrane tension measurement in any MOI, we present here a supramolecular chemistry strategy for selective localization and controlled release of flipper into the MOI, using a genetically encoded supramolecular tag. SupraFlippers, functionalized with a desthiobiotin ligand, can selectively accumulate in the organelle having expressed streptavidin. The addition of biotin as a biocompatible external stimulus with a higher affinity for Sav triggers the release of the probe, which spontaneously partitions into the MOI. Freed in the lumen of endoplasmic reticulum (ER), SupraFlippers report the membrane orders along the secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Kinetics of the process are governed by both the probe release and the transport through lipid domains. The concentration of biotin can control the former, while the expression level of a transmembrane protein (Sec12) involved in the stimulation of the vesicular transport from ER to Golgi influences the latter. Finally, the generation of a cell-penetrating and fully functional Sav-flipper complex using cyclic oligochalcogenide (COC) transporters allows us to combine the SupraFlipper strategy and HaloTag technology.

Cytosolic delivery in multicellular 3D spheroids is shown to be an intrinsic advantage of thiol-mediated uptake, which is compatible with proteins and QDs, achieving targeting and controlled release.

Recent progress with chemistry tools to deliver into living cells has seen a shift of attention from counterion-mediated uptake of cell-penetrating peptides (CPPs) and their mimics, particularly the Schmuck cation, toward thiol-mediated uptake with cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs), here exemplified by asparagusic acid. A persistent challenge in this evolution is the simultaneous and quantitative detection of cytosolic delivery and cytotoxicity in a high-throughput format. Here, we show that the combination of the HaloTag-based chloroalkane penetration assay (CAPA) with automated high-content (HC) microscopy can satisfy this need. The automated imaging of thousands of cells per condition in multiwell plates allows us to obtain quantitative data on not only the fluorescence intensity but also on the localization in a very short time. Quantitative and statistically relevant results can be obtained from dose–response curves of the targeted delivery to selected cells and the cytotoxicity in the same experiment, even with poorly optimized cellular systems.

Monomeric cyclic oligochalcogenides (COCs) are emerging as attractive transporters to deliver substrates of interest into the cytosol through thiol-mediated uptake. The objective of this study was to explore COC oligomers. We report a systematic evaluation of monomers, dimers, and trimers of asparagusic, lipoic, and diselenolipoic acid as well as their supramolecular monomers, dimers, trimers, and tetramers. COC dimers were more than twice as active as the monomers on both the covalent and noncovalent levels, whereas COC trimers were not much better than dimers. These trends might suggest that thiol-mediated uptake of COCs is synergistic over both short and long distances, that is, it involves more than two COCs and more than one membrane protein, although other interpretations cannot be excluded at this level of complexity. These results thus provide attractive perspectives for structural evolution as well as imminent use in practice. Moreover, they validate automated HC-CAPA as an invaluable method to collect comprehensive data on cytosolic delivery within a reasonable time at a level of confidence that is otherwise inconceivable.

Oligonucleotide phosphorothioates (OPS) are DNA or RNA mimics where one phosphate oxygen is replaced by a sulfur atom. They have been shown to enter mammalian cells much more efficiently than non‐modified DNA. Thus, solving one of the key challenges with oligonucleotide technology, OPS became very useful in practice, with several FDA‐approved drugs on the market or in late clinical trials. However, the mechanism accounting for this facile cellular uptake is unknown. Here, we show that OPS enter cells by thiol‐mediated uptake. The transient adaptive network produced by dynamic covalent pseudo‐disulfide exchange is characterized in action. Inhibitors with nanomolar efficiency are provided, together with activators that reduce endosomal capture for efficient delivery of OPS into the cytosol, the site of action.