Orthogonal Cysteine–Penicillamine Disulfide Pairing for Directing the Oxidative Folding of Peptides

Precise disulfide pairing in synthetic peptides usually is achieved using orthogonal protecting group strategies or relies on primary sequence manipulation. Orthogonal disulfide pairing technology should be promising for directing the rational folding of multicyclic peptides from the fully reduced peptides. Here, we report a discovery on the orthogonality between heterodisulfide pairing of cysteine (Cys) and penicillamine (Pen) and formation of Cys-Cys/Pen-Pen homodisulfides. The orthogonal Cys-Pen disulfide pairing can be exploited for highly selective production of certain (multi)­cyclic structures (or even a sole structure without isomers) through direct oxidation in air or thiol–disulfide exchanges in redox media. This strategy makes rational folding of multicyclic peptides without protecting groups, sequence manipulation, and complex synthetic reactions a reality, thus providing invaluable assets to peptide communities, and should greatly benefit the development of multicyclic peptide therapeutics and ligands

Aromaticity/Bulkiness of Surface Ligands to Promote the Interaction of Anionic Amphiphilic Gold Nanoparticles with Lipid Bilayers

The presence of large hydrophobic aromatic residues in cell-penetrating peptides or proteins has been demonstrated to be advantageous for their cell penetration. This phenomenon has also been observed when AuNPs were modified with peptides containing aromatic amino acids. However, it is still not clear how the presence of hydrophobic and aromatic groups on the surface of anionic AuNPs affects their interaction with lipid bilayers. Here, we studied the interaction of a range of anionic amphiphilic AuNPs coated by different combinations of hydrophobic and anionic ligands with four different types of synthetic lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic/bulky ligands on the surface of AuNPs into lipid bilayers is sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the interaction of AuNPs with lipid bilayers. Finally, we further correlated the observation on model liposomes with that on cell membranes, demonstrating that AuNPs that are more disruptive to the more negatively charged liposomes are also substantially more disruptive to cell membranes. In addition, our results revealed that certain cellular membrane domains that are more susceptible to disruption caused by hydrophobic interactions with nanoparticle surfaces might determine the threshold of AuNP-mediated cytotoxicity

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

Study on the processes of the thiol-mediated disulfide exchange reactions on the cell surface is not only important to our understanding of extracellular natural bioreduction processes but to the development of novel strategies for the intracellular delivery of synthetic bioactive molecules. However, disulfide-bonded probes have their intrinsic inferiority in exploring the detailed exchange pathway because of the bidirectional reactivity of disulfide bonds toward reactive thiols. In this work, we developed thioether-bonded fluorescent probes that enable us to explore thiol-mediated thioether (and disulfide) exchange reactions on the cell surface through fluorescence recovery and/or cell imaging. We demonstrated that our thioether-bonded probes can be efficiently cleaved through thiol-thioether exchanges with exofacial protein thiols and/or glutathione (GSH) efflux. The exchanges mainly take place on the cell surface, and GSH efflux-mediated exchange reactions can take place without the requirement of pre-exchanges of the probes with cell surface-associated protein thiols. On the basis of our founder methodology, for the first time we demonstrated the interplay of exofacial protein thiols and GSH efflux on the cleavage of external thioether-bonded compounds. Moreover, given that an understanding of the process of GSH efflux and the mechanism on which it relies is crucial to our understanding of the cellular redox homeostasis and the mechanism of multidrug resistance, we expect that our thioether-bonded probes and strategies would greatly benefit the fundamental study of GSH efflux in living cells

Proteolytic Unlocking of Ultrastable Twin-Acylhydrazone Linkers for Lysosomal Acid-Triggered Release of Anticancer Drugs

Targeted prodrugs exploiting cleavable linkers capable of responding to endogenous stimuli have increasingly been explored for cancer therapy. Successful application of these prodrug designs relies on the manipulation of both stability and responsiveness of the cleavable linkers, which, however, are difficult to be finely regulated, particularly for acid-responsive acylhydrazone bonds. Here we developed a new class of peptide-bridged twin-acylhydrazone linkers (PTA linkers) displaying both an ultrahigh stability and a rapid responsivenesshighly stable in neutral and acidic conditions due to the effect of cooperativity between the two acylhydrazone bonds, easily cleavable in acidic conditions after enzymatically triggered unlocking of the two bonds. Moreover, our study shows the design of PTA-linked prodrugs and the proof-of-concept application of the PTA linkers for site-specific release of anticancer drugs into cancer cells

Broad Control of Disulfide Stability through Microenvironmental Effects and Analysis in Complex Redox Environments

Disulfide bonds stabilize the tertiary- and quaternary structure of proteins. In addition, they can be used to engineer redox-sensitive (bio)­materials and drug-delivery systems. Many of these applications require control of the stability of the disulfide bond. It has recently been shown that the charged microenvironment of the disulfide can be used to alter their stability by ∼3 orders of magnitude in a predictable and finely tunable manner at acidic pH. The aim of this work is to extend these findings to physiological pH and to demonstrate the validity of this approach in complex redox milieu. Disulfide microenvironments were manipulated synergistically with steric hindrance herein to control disulfide bond stability over ∼3 orders of magnitude at neutral pH. Control of disulfide stability through microenvironmental effects could also be observed in complex redox buffers (including serum) and in the presence of cells. Such fine and predictable control of disulfide properties is not achievable using other existing approaches. These findings provide easily implementable and general tools for controlling the responsiveness of biomaterials and drug delivery systems toward various local endogenous redox environments

pH-Switchable Fluorescent Probe for Spatially-Confined Visualization of Intracellular Hydrogen Peroxide

Intracellular H₂O₂ plays an important role in regulating a variety of cellular functions. Fluorescent probes that can make response to intracellular levels of H₂O₂ would provide valuable tools for revealing the functions of H₂O₂ in living organisms. However, traditional pH-insensitive probes and lysosome-targetable probes can only provide spatially nonspecific visualization of intracellular H₂O₂ and specific sensing of lysosomal H₂O₂, respectively. In this work, we developed a H₂O₂-responsive and pH-switchable fluorescent probe (<b>HP-L1</b>) which can make response sequentially to intracellular H₂O₂ and lysosomal pH. The fluorescent probe is comprised of a H₂O₂-responsive boronate moiety and a pH-switchable spirobenzopyran fluorophore. When the probe was applied for intracellular H₂O₂ sensing, only fluorescent emission from lysosomes is visible, and the fluorescence from other regions is not able to be obviously detected, which is due to the pH-switchable property of the spirobenzopyran fluorophore. Thus, the developed fluorescence probe enables the spatially confined (i.e., lysosome-specific) visualization of the intracellular H₂O₂. We envisioned that this kind of fluorescent probe (or the proposed sensing strategy) would allow the visualization of the overall levels of intracellular H₂O₂ without interferences of possible fluorescent signals from other sources (e.g., dyes for cellular staining and multiplex analysis)

Biscysteine-Bearing Peptide Probes To Reveal Extracellular Thiol–Disulfide Exchange Reactions Promoting Cellular Uptake

In recent years, delivery systems based on the incorporation of thiols/disulfides have been extensively explored to promote the intracellular delivery of biological cargoes. However, it remains unclear about the detailed processes of thiol–disulfide exchanges taking place on the cell surface and how the exchange reactions promote the cellular uptake of cargoes bearing thiols or disulfide bonds. In this work, we report the rational design of biscysteine motif-containing peptide probes with substantially different ring-closing property and how these peptide probes were employed to explore the thiol–disulfide exchanges on the cell surface. Our results show that extensive thiol–disulfide exchanges between peptides and exofacial protein thiols/disulfides are involved in the cellular uptake of these peptide probes, and importantly glutathione (GSH) exported from the cytosols participates extensively in the exchange reactions. Cysteine−glycine−cysteine (CGC)-containing peptide probes can be more efficiently taken up by cells compared to other probes, and we suggested that the driving force for the superior cellular uptake arises from very likely the unique propensity of the CGC motif in forming doubly bridged disulfide bonds with exofacial proteins. Our probe-based strategy provides firsthand information on the detailed processes of the exchange reactions, which would be of great benefit to the development of delivery systems based on the extracellular thiol–disulfide exchanges for intracellular delivery of biologics

Exploring and Exploiting Dynamic Noncovalent Chemistry for Effective Surface Modification of Nanoscale Metal–Organic Frameworks

Surface properties determine, to a great extent, the biologically relevant functions of various kinds of nanosized materials. Although the modification of the surface of traditional inorganic or polymeric nanoparticles can be routinely achieved through covalent or noncovalent manner or both, the surface modification of nanoscale metal–organic frameworks (nano-MOFs) is extremely challenging because of their rapid degradation in aqueous environments. In this work, we systematically studied the synergistic and dynamic noncovalent interactions between fluorescent probes and iron­(III) carboxylate nano-MOFs (i.e., MIL-101-NH₂ (Fe), one of the most prevalent MOFs used in drug delivery and imaging). We further examined the interplay between the surface binding of fluorescent probes and the degradation of MIL-101-NH₂ (Fe) in aqueous medium. It was demonstrated that the surface binding of probes is not only of high affinity but also dynamic and nonsheddable, even during the degradation, a feature that is essentially different from the covalent conjugation. Subsequently, we developed a unique and straightforward strategy for the surface modification of MIL-101-NH₂ (Fe) with polymer by exploiting the synergy of noncovalent interactions between functionalized copolymers and MIL-101-NH₂ (Fe). We demonstrated that the binding of polymers onto MIL-101-NH₂ (Fe) surface was very effective in aqueous solution and surprisingly nonsheddable during the process of degradation. Surface polymers can creep on the surface of MIL-101-NH₂ (Fe), in a dynamic and real-time manner, to the new sites formed immediately after the degradation. In addition, the stability of MIL-101-NH₂ (Fe) particles in aqueous environments can be improved to some extent by the surface polymer coating. The results presented herein constitute an important innovation for surface engineering of nano-MOFs, which would benefit the application of nano-MOFs as delivery systems in aqueous systems

The Interplay of Disulfide Bonds, α‑Helicity, and Hydrophobic Interactions Leads to Ultrahigh Proteolytic Stability of Peptides

The contribution of noncovalent interactions to the stability of naturally occurring peptides and proteins has been generally acknowledged, though how these can be rationally manipulated to improve the proteolytic stability of synthetic peptides remains to be explored. In this study, a platform to enhance the proteolytic stability of peptides was developed by controllably dimerizing them into α-helical dimers, connected by two disulfide bonds. This platform not only directs peptides toward an α-helical conformation but permits control of the interfacial hydrophobic interactions between the peptides of the dimer. Using two model dimeric systems constructed from the N-terminal α-helix of RNase A and known inhibitors for the E3 ubiquitin ligase MDM2 (and its homologue MDMX), a deeper understanding into the interplay of disulfide bonds, α-helicity, and hydrophobic interactions on enhanced proteolytic stability was sought out. Results reveal that all three parameters play an important role on attaining ultrahigh proteolytic resistance, a concept that can be exploited for the development of future peptide therapeutics. The understanding gained through this study will enable this strategy to be tailored to new peptides because the proposed strategy displays substantial tolerance to sequence permutation. It thus appears promising for conveniently creating prodrugs composed entirely of the therapeutic peptide itself (i.e., in the form of a dimer)