Regulating H2S release from self-assembled peptide H2S-donor conjugates using cysteine derivatives.

Self-assembled peptides provide a modular and diverse platform for drug delivery, and innovative delivery methods are needed for delivery of hydrogen sulfide (H2S), an endogenous signaling molecule (gasotransmitter) with significant therapeutic potential. Of the available types of H2S donors, peptide/protein H2S donor conjugates (PHDCs) offer significant versatility. Here we discuss the design, synthesis, and in-depth study of a PHDC containing three covalently linked components: a thiol-triggered H2S donor based on an S-aroylthiooxime (SATO), a GFFF tetrapeptide, and a tetraethylene glycol (TEG) dendron. Conventional transmission electron microscopy showed that the PHDC self-assembled into spherical structures without heat or stirring, but it formed nanofibers with gentle heat (37 °C) and stirring. Circular dichroism (CD) spectroscopy data collected during self-assembly under nanofiber-forming conditions suggested an increase in ß-sheet character and a decrease in organization of the SATO units. Release of H2S from the nanofibers was studied through triggering with various thiols. The release rate and total amount of H2S released over both short (5 h) and long (7 d) time scales varied with the charge state: negatively charged and zwitterionic thiols (e.g., Ac-Cys-OH and H-Cys-OH) triggered release slowly while a neutral thiol (Ac-Cys-OMe) showed ∼10-fold faster release, and a positively charged thiol (H-Cys-OMe) triggered H2S release nearly 50-fold faster than the negatively charged thiols. CD spectroscopy studies monitoring changes in secondary structure over time during H2S release showed similar trends. This study sheds light on the driving forces behind self-assembling nanostructures and offers insights into tuning H2S release through thiol charge state modulation.

Cucurbit[7]uril Macrocyclic Sensors for Optical Fingerprinting: Predicting Protein Structural Changes to Identifying Disease-Specific Amyloid Assemblies.

In a three-dimensional (3D) representation, each protein molecule displays a specific pattern of chemical and topological features, which are altered during its misfolding and aggregation pathway. Generating a recognizable fingerprint from such features could provide an enticing approach not only to identify these biomolecules but also to gain clues regarding their folding state and the occurrence of pathologically lethal misfolded aggregates. We report here a universal strategy to generate a fluorescent fingerprint from biomolecules by employing the pan-selective molecular recognition feature of a cucurbit[7]uril (CB[7]) macrocyclic receptor. We implemented a direct sensing strategy by covalently tethering CB[7] with a library of fluorescent reporters. When CB[7] recognizes the chemical and geometrical features of a biomolecule, it brings the tethered fluorophore into the vicinity, concomitantly reporting the nature of its binding microenvironment through a change in their optical signature. The photophysical properties of the fluorophores allow a multitude of probing modes, while their structural features provide additional binding diversity, generating a distinct fluorescence fingerprint from the biomolecule. We first used this strategy to rapidly discriminate a diverse range of protein analytes. The macrocyclic sensor was then applied to probe conformational changes in the protein structure and identify the formation of oligomeric and fibrillar species from misfolded proteins. Notably, the sensor system allowed us to differentiate between different self-assembled forms of the disease-specific amyloid-ß (Aß) aggregates and segregated them from other generic amyloid structures with a 100% identification accuracy. Ultimately, this sensor system predicted clinically relevant changes by fingerprinting serum samples from a cohort of pregnant women.

Bridging Rigidity and Flexibility: Modulation of Supramolecular Hydrogels by Metal Complexation.

The combination of complementary, noncovalent interactions is a key principle for the design of multistimuli responsive hydrogels. In this work, an amphiphilic peptide, supramacromolecular hydrogelator which combines metal-ligand coordination induced gelation and thermoresponsive toughening is reported. Following a modular approach, the incorporation of the triphenylalanine sequence FFF into a structural (C3 EG ) and a terpyridine-functionalized (C3 Tpy ) C3 -symmetric monomer enables their statistical copolymerization into self-assembled, 1D nanorods in water, as investigated by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). In the presence of a terpyridine functionalized telechelic polyethylene glycol (PEG) cross-linker, complex formation upon addition of different transition metal ions (Fe2+ , Zn2+ , Ni2+ ) induces the formation of soft, reversible hydrogels at a solid weight content of 1 wt% as observed by linear shear rheology. The viscoelastic behavior of Fe2+ and Zn2+ cross-linked hydrogels are basically identical, while the most kinetically inert Ni2+ coordinative bond leads to significantly weaker hydrogels, suggesting that the most dynamic rather than the most thermodynamically stable interaction supports the formation of robust and responsive hydrogel materials.

Evaluation of Charge-Regulated Supramolecular Copolymerization to Tune the Time Scale for Oxidative Disassembly of ß-Sheet Comonomers.

A multistimuli-responsive supramolecular copolymerization is reported. The copolymerization is driven by hydrogen bond encoded ß-sheet-based charge co-assembly into 1D nanorods in water, using glutamic acid or lysine residues in either of the peptide comonomers. The incorporation of methionine as hydrophobic amino acid supports ß-sheet formation, but oxidation of the thioether side-chain to a sulfoxide functional group destabilizes the ß-sheet ordered domains and induces disassembly of the supramolecular polymers. Using H2 O2 as reactive oxygen species, the time scale and kinetics of the oxidative disassembly are probed. Compared to the charge neutral homopolymers, it is found that the oxidative disassembly of the charged ampholytic copolymers is up to two times faster and is operative at neutral pH. The strategy is therefore an important addition to the growing field of amphiphilic polythioether containing (macro)molecular building blocks, particularly in view of tuning their oxidation induced disassembly which tends to be notoriously slow and requires high concentrations of reactive oxygen species or acidic reaction media.

Impact of NDI-Core Substitution on the pH-Responsive Nature of Peptide-Tethered Luminescent Supramolecular Polymers.

The pH-responsive nature of two self-assembled NDI-peptide amphiphile conjugates is reported. The diethoxy substituted NDI showed a pH-dependent assembly behaviour, as expected. In contrast, the isopropylamino- and ethoxy-substituted NDI based supramolecular polymer was stable at acidic and basic aqueous conditions. This finding highlights how subtle changes in the molecular design of π-stacked chromophore-peptide conjugates have a drastic impact on their equilibrium structure and ultimately functional properties.