Use of pyridazinediones for tuneable and reversible covalent cysteine modification applied to peptides, proteins and hydrogels.

Reversible cysteine modification has been found to be a useful tool for a plethora of applications such as selective enzymatic inhibition, activity-based protein profiling and/or cargo release from a protein or a material. However, only a limited number of reagents display reliable dynamic/reversible thiol modification and, in most cases, many of these reagents suffer from issues of stability, a lack of modularity and/or poor rate tunability. In this work, we demonstrate the potential of pyridazinediones as novel reversible and tuneable covalent cysteine modifiers. We show that the electrophilicity of pyridazinediones correlates to the rates of the Michael addition and retro-Michael deconjugation reactions, demonstrating that pyridazinediones provide an enticing platform for readily tuneable and reversible thiol addition/release. We explore the regioselectivity of the novel reaction and unveil the reason for the fundamental increased reactivity of aryl bearing pyridazinediones by using DFT calculations and corroborating findings with SCXRD. We also applied this fundamental discovery to making more rapid disulfide rebridging agents in related work. We finally provide the groundwork for potential applications in various areas with exemplification using readily functionalised "clickable" pyridazinediones on clinically relevant cysteine and disulfide conjugated proteins, as well as on a hydrogel material.

Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

High-Throughput Peptide Derivatization toward Supramolecular Diversification in Microtiter Plates.

The evolution of life on earth eventually leads to the emergence of species with increased complexity and diversity. Similarly, evolutionary chemical space exploration in the laboratory is a key step to pursue the structural and functional diversity of supramolecular systems. Here, we present a powerful tool that enables rapid peptide diversification and employ it to expand the chemical space for supramolecular functions. Central to this strategy is the exploitation of palladium-catalyzed Suzuki-Miyaura cross-coupling reactions to direct combinatorial synthesis of peptide arrays in microtiter plates under an open atmosphere. Taking advantage of this in situ library design, our results unambiguously deliver a fertile platform for creating a set of intriguing peptide functions including green fluorescent protein-like peptide emitters with chemically encoded emission colors, hierarchical self-assembly into nano-objects, and macroscopic hydrogels. This work also offers opportunities for quickly surveying the diversified peptide arrays and thereby identifying the structural factors that modulate peptide properties.

In vivo biocompatibility and immunogenicity of metal-phenolic gelation.

In vivo forming hydrogels are of interest for diverse biomedical applications due to their ease-of-use and minimal invasiveness and therefore high translational potential. Supramolecular hydrogels that can be assembled using metal-phenolic coordination of naturally occurring polyphenols and group IV metal ions (e.g. TiIV or ZrIV) provide a versatile and robust platform for engineering such materials. However, the in situ formation and in vivo response to this new class of materials has not yet been reported. Here, we demonstrate that metal-phenolic supramolecular gelation occurs successfully in vivo and we investigate the host response to the material over 14 weeks. The TiIV-tannic acid materials form stable gels that are well-tolerated following subcutaneous injection. Histology reveals a mild foreign body reaction, and titanium biodistribution studies show low accumulation in distal tissues. Compared to poloxamer-based hydrogels (commonly used for in vivo gelation), TiIV-tannic acid materials show a substantially improved in vitro drug release profile for the corticosteroid dexamethasone (from <1 day to >10 days). These results provide essential in vivo characterization for this new class of metal-phenolic hydrogels, and highlight their potential suitability for biomedical applications in areas such as drug delivery and regenerative medicine.

Residue-Specific Solvation-Directed Thermodynamic and Kinetic Control over Peptide Self-Assembly with 1D/2D Structure Selection.

Understanding the self-organization and structural transformations of molecular ensembles is important to explore the complexity of biological systems. Here, we illustrate the crucial role of cosolvents and solvation effects in thermodynamic and kinetic control over peptide association into ultrathin Janus nanosheets, elongated nanobelts, and amyloid-like fibrils. We gained further insight into the solvation-directed self-assembly (SDSA) by investigating residue-specific peptide solvation using molecular dynamics modeling. We proposed the preferential solvation of the aromatic and alkyl domains on the peptide backbone and protofibril surface, which results in volume exclusion effects and restricts the peptide association between hydrophobic walls. We explored the SDSA phenomenon in a library of cosolvents (protic and aprotic), where less polar cosolvents were found to exert a stronger influence on the energetic balance at play during peptide propagation. By tailoring cosolvent polarity, we were able to achieve precise control of the peptide nanostructures with 1D/2D shape selection. We also illustrated the complexity of the SDSA system with pathway-dependent peptide aggregation, where two self-assembly states ( i.e., thermodynamic equilibrium state and kinetically trapped state) from different sample preparation methods were obtained.

Kinetically Controlled Lifetimes in Redox-Responsive Transient Supramolecular Hydrogels.

It remains challenging to program soft materials to show dynamic, tunable time-dependent properties. In this work, we report a strategy to design transient supramolecular hydrogels based on kinetic control of competing reactions. Specifically, the pH-triggered self-assembly of a redox-active supramolecular gelator, N,N'-dibenzoyl-l-cystine (DBC) in the presence of a reducing agent, which acts to disassemble the system. The lifetimes of the transient hydrogels can be tuned simply by pH or reducing agent concentration. We find through kinetic analysis that gel formation hinders the ability of the reducing agent and enables longer transient hydrogel lifetimes than would be predicted. The transient hydrogels undergo clean cycles, with no kinetically trapped aggregates observed. As a result, multiple transient hydrogel cycles are demonstrated and can be predicted. This work contributes to our understanding of designing transient assemblies with tunable temporal control.

Choice of Capping Group in Tripeptide Hydrogels Influences Viability in the Three-Dimensional Cell Culture of Tumor Spheroids.

Two peptide-derived low-molecular-weight gelators bearing different capping groups, 9-fluorenylmethyloxycarbonyl (Fmoc) and phenothiazine, were synthesized and their gel networks were characterized. The variation of the N-terminal capping group affects the viability of these hydrogels as a three-dimensional cell culture for multicellular tumor spheroids. These results indicate that the phenothiazine capping group is a more biocompatible alternative to the widely used Fmoc moiety.