Disulfide-Linked/Peptide-Incorporated Macrocycles: Unique Redox-Responsiveness and Application for Intracellular Cargo-Delivery

Abstract

The use of disulfide-linked macrocycles for biomedicine applications was not explored, which is, at least partially, due to the lack of a straightforward strategy to incorporate a cell-surface binding ligand into the macrocycles for facilitating cellular uptake. Herein, we discover that twin disulfides consisting of a flexible peptide and a structurally rigid organic molecule can be transformed into stable and trimeric macrocycles upon triggering with thiols. The disulfide linked/peptide incorporated macrocycles are substantially more stable in weakly reducing environments compared to their non-cyclic analogues due to the cooperativity of the three interlinked disulfide bonds. We further demonstrated that the trimeric macrocycles can be a promising scaffold for the development of intracellular cargodelivery systems by incorporating a cell-penetrating peptide into the macrocycles.Disulfide-linked macrocycles are a category of cyclic compounds containing two or more interlinked disulfide bonds, which are at present mainly produced through dynamic combinatorial chemistry. [1] Despite extensive progress in this field and the presence of multiple disulfides within the macrocycles, the response of the macrocycles to redox-stimulation has not been systematically studied. [2] Furthermore, the utility of the disulfide-linked macrocycles for the development of delivery systems for drugs or imaging agents is also unexplored, though disulfide-linked macrocycles are expected to exhibit unique redox-responsiveness. [2a, 3] This is, at least partially, due to the lack of a straightforward strategy to incorporate a cell-surface binding unit into the macrocycles to facilitate cellular uptake.Here, we discovered, for the first time, that a new type of twin-disulfide systems comprised of two building blocks with different structural rigidity can be efficiently transformed into stable and trimeric disulfide-linked macrocycles when triggering with external thiols (Figure 1). The formation of the trimeric macrocycle requires a flexible peptide and a structurally rigid organic molecule that both contain two cysteine residues; hence the use of the peptide dithiol motif facilitates the incorporation of functional peptide sequences (e. g., cell-penetrating peptide (CPP) [4] ) into the trimeric macrocycle, which can conveniently convert the macrocycle into an intracellular cargodelivery system. The disulfide-linked and peptide-incorporated macrocycles exhibit an unconventional redox-responsiveness due to the cooperativity of the three interlinked disulfide bonds; as a consequence, they are substantially more stable in weakly reducing environments as compared to their non-cyclic trimeric analogues. We further demonstrated the potential of exploiting CPP-incorporated trimeric macrocycles for intracellular cargo-delivery.We first synthesized a new type of twin disulfides into which were incorporated both the flexible and rigid dithiol building blocks (Figure 1). The rigid dithiol compound comes from previous reports, [1a, b, 5] that is 1,...

Thioether-Bonded Fluorescent Probes for Deciphering Thiol-Mediated Exchange Reactions on the Cell Surface

“…This result implies that the cleavage of the thioether-bonded probes by thiol-mediated exchanges mainly takes place on the cell surface and, accordingly, exofacial protein thiols are very likely the primary biothiol species getting involved in the thiol–thioether exchange reactions. We further examined if incubating the cells with 5,5′-dithiobis­(2-nitrobenzoic acid) (DTNB), a cell membrane impermeable thiol oxidant, ,, which can be used to reversibly block the cell surface thiols, can influence the cleavage of the probes. Though the fluorescence recovery of Hcy-STat-F is obviously inhibited by the temporary block of exofacial thiols through DTNB coincubation (Figure b), it does not remarkably influence the fluorescence intensity of cells incubated with Hcy-STat (Figure S7), that is, the formation of Hcy-GSH .…”

“…Given that all cells spontaneously export GSH through diffusion and certain cells (e.g., cancer cells) accelerate the efflux through overexpression of plasma membrane-associated GSH transporters, thiol–disulfide exchanges on the surface of cells should be much more complex than ever known when taking into account of the GSH efflux. ,, In the past the processes of thiol–disulfide exchanges on the cell surface have usually been explored using disulfide-bonded fluorescent probes. − Though these probes enable the determination of the overall kinetics of disulfide reduction, the detailed pathway of thiol–disulfide exchanges involving the interplay of exofacial protein thiols and GSH efflux is largely hidden behind the overall reactions. We argue that disulfide-bonded probes have their intrinsic inferiority in deciphering the detailed exchange pathway because of the bidirectional reactivity of disulfide bonds toward reactive thiols (Figure b), − a feature that can result in the formation of either mixed disulfides or free thiols in each thiol–disulfide exchange step, which creates chaos and confusion for exploration of the pathway. To address this, dynamic thioether-bonded (−C–S−) analogues might be exploited for the exploration, − which should be more suited to the mission as their reactivity toward thiols is unidirectional (relative to disulfide bonds; Figure b).…”