Controlling reaction pathways to modulate assembly processes
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Abstract
Regulation of the assembly and disassembly of peptide nanostructures is essential to create complex and dynamic systems that exhibit pathway selectivity. This thesis demonstrates the design of synthetically modified peptides that are responsive to stimuli such as light, pH, or redox, thereby controlling their assembly and disassembly behavior. In addition, the concept of stimulus-controlled assembly was investigated in complex environments.
In order to achieve control over the disassembly of nanostructures two short anthracene-containing peptides were designed that also feature a pH sensitive lysine. They assembled into nanosheets or nanoribbons with a distinct secondary structure. These morphologies could further be modulated by pH, leading to different amount of charges on the peptide. Upon irradiation the light-responsive anthracene units performed a bimolecular [4+4] cycloaddition, which disrupted the π-π stacking by distortion of the previously flat aromatic groups and therefore lead to disassembly of the supramolecular architecture. In contrast to monomolecular reactions such as photocleavage, the preorganization of the anthracene units within the supramolecular architecture exerted a notable influence on the kinetics of the bimolecular photodimerization. This highlights the interplay between the supramolecular structure and the molecular chemistry and showcased the controlled disassembly of diverse nanostructures.
The assembly process can also be precisely controlled using light-responsive systems. In conjunction with redox-responsive cysteine, a photolytic reaction cascade was engineered, yielding a disulfide that exhibited pathway selectivity during the assembly process. This was achieved by starting from a caged isopeptide, which released the linear peptide upon photocleavage and subsequently was oxidized by the side products of the cage to a disulfide. The modulation of this process was possible through the introduction of the reducing agent DTT, which affords control over the production and lifetime of the supramolecular monomers. The disulfide displayed short, twisted fibers when directly assembled, yet revealed short and thin fibers when assembled as part of the cascade. By cycling between the redox states, a third morphology of long and thin fibers emerged, whose secondary structure could be tuned by the amount of DTT. This project demonstrated how a combination of light and redox responsiveness enables control over the assembly process with several distinct pathways giving rise to different nanostructures.
A glutathione-responsive isopeptide was then used to control the assembly of nanostructures in a complex environment such as the cell. Elevated levels of glutathione within the cytosol compared to the extracellular matrix could selectively cleave the cage, initiate the reaction cascade and release the linear, assembling peptide. The formation of nanostructures and their effect on MDA-MB-231 breast cancer cells was observed through life-cell imaging and fluorescence spectroscopy, as well as the oxygen consumption of the cell. The supramolecular assembly led to a disruption of mitochondrial integrity and inhibited energy homeostasis. Notably, the observed effects were not confined to two-dimensional cell culture, they could also result in a reduction in tumor size in a three-dimensional tumor spheroid model.
In summary, this thesis presented the modulation of assembly processes by controlling their reaction pathways. This was achieved by incorporating stimuli-responsive groups and tuning of supramolecular interactions. Furthermore, the interplay between molecular chemistry and the supramolecular architecture and vice versa is highlighted. These results should facilitate the discovery of next-generation biomaterials with control over the formation of complex nanostructures.