Substrate-initiated, self-inactivating, cell-penetrating poly(disulfide)s (siCPDs) are introduced as general transporters for the covalent delivery of unmodified substrates of free choice. With ring-opening disulfide-exchange polymerization, we show that guanidinium-rich siCPDs grow on fluorescent substrates within minutes under the mildest conditions. The most active siCPD transporters reach the cytosol of HeLa cells within 5 min and depolymerize in less than 1 min to release the native substrate. Depolymerized right after use, the best siCPDs are nontoxic under conditions where cell-penetrating peptides (CPPs) are cytotoxic. Intracellular localization (cytosol, nucleoli, endosomes) is independent of the substrate and can be varied on demand, through choice of polymer composition. Insensitivity to endocytosis inhibitors and classical structural variations (hydrophobicity, aromaticity, branching, boronic acids) suggest that the best siCPDs act differently. Supported by experimental evidence, a unique combination of the counterion-mediated translocation of CPPs with the underexplored, thiol-mediated covalent translocation is considered to account for this decisive difference.

The objective of this Feature Article is to reflect on the importance of established and emerging principles of supramolecular organic chemistry to address one of the most persistent problems in life sciences. The main topic is dynamic covalent chemistry on cell surfaces, particularly disulfide exchange for thiol-mediated uptake. Examples of boronate and hydrazone exchange are added for contrast, comparison and completion. Of equal importance are the discussions of proximity effects in polyions and counterion hopping, and more recent highlights on ring tension and ion pair-π interactions. These lessons from supramolecular organic chemistry apply to cell-penetrating peptides, particularly the origin of "arginine magic" and the "pyrenebutyrate trick," and the currently emerging complementary "disulfide magic" with cell-penetrating poly(disulfide)s. They further extend to the voltage gating of neuronal potassium channels, gene transfection, and the delivery of siRNA. The collected examples illustrate that the input from conceptually innovative chemistry is essential to address the true challenges in biology beyond incremental progress and random screening.

Lessons from surface-initiated polymerization are applied to grow cell-penetrating poly(disulfide)s directly on substrates of free choice. Reductive depolymerization after cellular uptake should then release the native substrates and minimize toxicity. In the presence of thiolated substrates, propagators containing a strained disulfide from asparagusic or, preferably, lipoic acid and a guanidinium cation polymerize into poly(disulfide)s in less than 5 min at room temperature at pH 7. Substrate-initiated polymerization of cationic poly(disulfide)s and their depolymerization with dithiothreitol causes the appearance and disappearance of transport activity in fluorogenic vesicles. The same process is further characterized by gel-permeation chromatography and fluorescence resonance energy transfer.Cell-penetrating peptides (CPPs) are short, polycationic peptides or protein domains that are used by viruses to enter cells. 1,2 Their unique ability to transport linked substrates across lipid bilayer membranes has attracted great interest in biomedical applications. Substrates of varying sizes and properties, e.g., small fluorophores to proteins and quantum dots, have been successfully transported into cells using CPPs. The mechanism of cellular uptake is under debate, currently favored are endocytosxis (i.e., macropinocytosis) or passive diffusion across the membrane, depending on conditions. Multiple, moderately hydrophobic cations seem to be all that is needed. Guanidinium cations, as in arginine, are most common, alternatives include ammonium or phosphonium cations. 1 The originally peptidic oligomer backbone has been extensively varied, covering oligocarbamates, β-peptides and several variations of synthetic polymers. 1 Currently, cell-penetrating poly(disulfide)s are emerging as the cell-penetrating molecules of the future because their cytosolic degradation liberates the substrate and eliminates toxicity, one of the key disadvantages associated with CPPs. [3][4][5] However, cell-penetrating poly-(disulfide)s have so far been used mainly in noncovalent polyplexes for gene transfection, and covalent attachment of substrates would be difficult with their preparation methods. We have found recently that poly(disulfide)s can be grown directly on solid substrates by surface-initiated ring-opening disulfide-exchange polymerization. 6 Therefore, we wondered whether the same methodology could be used to Figure 1). Probes or drugs that contain thiol group but cannot penetrate cells without assistance are the ideal substrates, which could serve as an initiator to be appended with a membrane-active poly(disulfide). Thiolated siRNA, for instance, is commercially available. The generality of this approach promises a conceptually innovative solution for a central current challenge, i.e., the noninvasive, nontoxic delivery of unmodified substrates in welldefined, covalent systems rather than complex, noncovalent formulations. In this initial report on the topic, we describe the design, synthesis and evaluation of propag...

This review summarizes the use of orthogonal dynamic covalent bonds to build functional systems. Dynamic covalent bonds are unique because of their dual nature. They can be as labile as non-covalent interactions or as permanent as covalent bonds, depending on conditions. Examples from nature, reaching from the role of disulfides in protein folding to thioester exchange in polyketide biosynthesis, indicate how dynamic covalent bonds are best used in functional systems. Several synthetic functional systems that employ a single type of dynamic covalent bonds have been reported. Considering that most functional systems make simultaneous use of several types of non-covalent interactions together, one would expect the literature to contain many examples in which different types of dynamic covalent bonds are similarly used in tandem. However, the incorporation of orthogonal dynamic covalent bonds into functional systems is a surprisingly rare and recent development. This review summarizes the available material comprehensively, covering a remarkably diverse collection of functions. However, probably more revealing than the specific functions addressed is that the questions asked are consistently quite unusual, very demanding and highly original, focusing on molecular systems that can self-sort, self-heal, adapt, exchange, replicate, transcribe, or even walk and "think" (logic gates). This focus on adventurous chemistry off the beaten track supports the promise that with orthogonal dynamic covalent bonds we can ask questions that otherwise cannot be asked. The broad range of functions and concepts covered should appeal to the supramolecular organic chemist but also to the broader community.