Design and synthesis of peptide-polymer conjugates and the characterization of their aggregation
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Peptide-polymer conjugates are a hybrid class of materials that take advantage of natural and synthetic building blocks whose individual properties can be synergistically combined. Their modular design enables the incorporation of chemical functionalities, hierarchal control over assembly, as well as unique chemical, biological, and mechanical properties. These conjugates allow for the design of complex, highly responsive materials with applications in many fields. Within this dissertation, two different peptide-polymer conjugates are discussed. ☐ Peptide-polymer conjugates were synthesized via the copper-catalyzed azide– alkyne cycloaddition (CuAAC) of poly(tert-butyl acrylate) (PtBA) and elastin-like peptides. An azide-functionalized polymer was produced via atom transfer radical polymerization (ATRP) followed by conversion of bromine end groups to azide groups. Subsequent reaction of the polymer with a bis-alkyne-functionalized, elastin-like peptide proceeded with high efficiency, yielding di- and tri-block conjugates, which after deprotection, yielded poly(acrylic acid) (PAA)-based diblock and triblock copolymers. These conjugates were solubilized in dimethyl formamide, and addition of phosphate buffered saline (PBS) induced aggregation. The presence of polydisperse spherical aggregates was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Additionally, a coarse-grained molecular model was designed to reasonably capture inter- and intramolecular interactions for the conjugates and its precursors. This model was used to assess the effect of the different interacting molecular forces on the conformational thermodynamic stability of the copolymers. Our results indicated that the PAA's ability to hydrogen-bond with both itself and the peptide is the main interaction for stabilizing the diblocks and triblocks and driving their self-assembly, while interactions between peptides are suggested to play only a minor role on the conformational and thermodynamic stability of the conjugates. ☐ In the second part of this dissertation, the unfolding thermodynamics of a series of newly designed alanine-rich peptides were modeled and experimentally characterized. A coarse-grained (CG) molecular model was refined to capture thermodynamics and structural changes as a function of temperature for a set of previously published peptide sequences. Circular dichroism spectroscopy (CD) was used to monitor the temperature induced conformational changes and stability of the newly designed sequences. The model predictions were quantitatively or semi-quantitatively accurate in all cases. The simulations and CD results showed that, as expected, in most cases the unfolding of helical peptides is well described by a simply 2-state model, and conformational stability increased with increased length of the helices. A notable exception in a 19-residue helix was when two Ala residues were each replaced with Phe. This stabilized a partly unfolded intermediate state via hydrophobic contacts, and also promoted aggregates at higher peptide concentrations. ☐ Furthermore, the aggregation behavior of these peptides was also characterized by a suite of combination of biophysical techniques. Different light scattering techniques were used to monitor changes in peptide morphology and size distributions as a function of time and temperature. These measurements show large particles immediately upon dissolution in buffer. At room temperature, these particles annealed to reach a mostly monomeric peptide state. At higher temperatures, they grew to form aggregates. Circular Dichroism spectroscopy (CD) was used to monitor the temperature- and time-dependent conformational changes as a function of peptide sequence and incubation time. CD measurements reveal that all the sequences are helical at low temperatures with transitions to coil behavior with increased temperature. Samples incubated at room temperature were able to recover their original helicity. At increased temperature, AQK18 and AQK35, showed some change in conformation, and were not able to recover their original helicity after 72 hours. Furthermore, with increased incubation time of a week, β-sheet conformation was observed in these two samples, while only α-helical conformation loss was observed in AQK27. Transmission Electron Microscopy measurements reveal the formation of fibrils after 72 hours of incubation at 60 °C for all samples, in agreement with the scattering measurements. Additional quenching experiments show that peptide aggregation is stalled upon cooling to room temperature. ☐ Finally, PEG was conjugated to AQK27 and AQK35, using aqueous CuAAC chemistry. These conjugates retained the α-helical conformation of the peptide and underwent reversible unfolding and folding upon heating and cooling. Thermally induced aggregation of the conjugates was characterized by DLS, CD and TEM. Samples incubated at room temperature remained helical after 72 hours and did not show any helical loss. Furthermore, TEM characterization did not reveal the presence of aggregates for these samples. Conjugates incubated at elevated temperature (60 °C) were observed to aggregate after 72 hours, with a shift in autocorrelation function to later times, indicative of the formation of aggregates. In addition, fibrils were observed via TEM characterization. Conformations recorded for AQK35DB and AQK35TB revealed only a small loss in α-helical content as compared to the parent peptide which displayed a mixture of α-helix and b-sheet conformation. This result may indicate that the presence of PEG may sterically hinder the formation of β-sheet aggregation. Qualitatively, there was no apparent difference between the morphologies of the conjugates. This maybe the result of how similar the parent peptides are, and while there are differences in hydrophobicity, they still may behave similarly over longtime scales.
Description
Keywords
Peptides, Polymers, Conjugates, Aggregation