Dynamic Covalent Michael Acceptors to Penetrate Cells: Thiol‐Mediated Uptake with Tetrel‐Centered Exchange Cascades, Assisted by Halogen‐Bonding Switches

Abstract

Chalcogen-centered cascade exchange chemistry is increasingly understood to account for thiol-mediated uptake, that is, the ability of reversibly thiol-reactive agents to penetrate cells. Here, reversible Michael acceptors are shown to enable and inhibit thiolmediated uptake, including the cytosolic delivery of proteins. Dynamic cyano-cinnamate dimers rival the best chalcogen-centered inhibitors. Patterns generated in inhibition heatmaps reveal contributions from halogen-bonding switches that occur independent from the thyroid transporter MCT8. The uniqueness of these patterns supports that the entry of tetrel-centered exchangers into cells differs from chalcogen-centered systems. These results expand the chemical space of thiol-mediated uptake and support the existence of a universal exchange network to bring matter into cells, abiding to be decoded for drug delivery and drug discovery in the broadest sense.

Tetrel‐Centered Exchange Cascades to Decouple Inhibition and Induction of Thiol‐Mediated Uptake: Introducing Cell‐Penetrating Thiolactones, Focus on Reversible Michael Acceptor Dimers

“…This inhibition was three times weaker than the dimeric Michael acceptor 1 and also weaker than the locked thioacetal control 8 with 9±1 μ m . This result thus confirmed that exchange with the central disulfide bridge is negligible for activity and implied that the halogen‐bond switches identified with Michael acceptor monomers [26] also operate on the dimer level ( Figure 2 ,a ). Namely, para trifluoromethyls have a Hammett σ p =0.54 well beyond ketone and aldehyde acceptors with σ p =0.42, not to speak of iodines with σ p =0.18 [86] .…”

“…The activity of tetrel‐centered CAXs as inhibitors of thiol‐mediated uptake was assessed with fluorescently labeled epidithiodiketopiperazine 9 as a reporter ( Figure 4). This natural product derived CAX provides one of the most active and brightest TMU probes, [79] which has been used extensively to characterize new TMU inhibitors [18,22,26] . Inhibition efficiencies were assessed using automated high‐content high‐throughput imaging microscopy [18,22,26] .…”

“…Tetrel‐centered CAXs such as 1 contain highly reactive Michael acceptors to exchange with cellular thiolates ( I – II , Figure 2 ,a ) [26] . The extra cyano acceptor sufficiently acidifies the central hydrogen to achieve reversibility, that is dynamic covalent Michael addition ( II ) [38–51] .…”

“…For drug discovery perspectives and mechanistic investigations such as target identification, CAXs that can act as inhibitors without simultaneously penetrating cells could be particularly valuable. The reactivity of tetrel‐centered CAXs appeared well suited for this purpose [26] . The cell‐penetrating power of the original chalcogen‐centered CAXs, most popular with strained 1,2‐dithiolanes A , implied that thiol‐mediated uptake operates with dynamic covalent cascade exchange along disulfide arrays ( Figure 1 ,a ) [1,27] .…”

“…Unlike these classical, chalcogen‐centered CAXs, the recently introduced tetrel‐centered CAXs, i. e ., dynamic covalent Michael acceptors, should exchange exclusively with thiol/ate arrays ( Figure 1 ,b ) [26] . While monomeric reversible Michael acceptors exhibited moderate abilities to promote and inhibit thiol‐mediated uptake, dimeric Michael acceptors B were identified as particularly strong inhibitors.…”