Controlling Amphiphilic Polymer Folding beyond the Primary Structure with Protein-Mimetic Di(Phenylalanine)

Abstract

While methods for polymer synthesis have proliferated, their functionality pales in comparison to natural biopolymers-strategies are limited for building the intricate network of noncovalent interactions necessary to elicit complex, protein-like functions. Using a bioinspired di(phenylalanine) acrylamide (FF) monomer, we explored the impact of various noncovalent interactions in generating ordered assembled structures. Amphiphilic copolymers were synthesized that exhibit β-sheet-like local structure upon collapsing into single-chain assemblies in aqueous environments. Systematic analysis of a series of amphiphilic copolymers illustrated that the global collapse is primarily driven by hydrophobic forces. Hydrogen-bonding and aromatic interactions stabilize local structure, as β-sheet-like interactions were identified via circular dichroism and thioflavin T fluorescence. Similar analysis of phenylalanine (F) and alanine-phenylalanine acrylamide (AF) copolymers found that distancing the aromatic residue from the polymer backbone is sufficient to induce β-sheet-like local structure akin to the FF copolymers; however, the interactions between AF subunits are less stable than those formed by FF. Further, hydrogen-bond donating hydrophilic monomers disrupt internal structure formed by FF within collapsed assemblies. Collectively, these results illuminate design principles for the facile incorporation of multiple facets of protein-mimetic, higher-order structure within folded synthetic polymers.

Figure 1.

Di(phenylalanine) acrylamide monomer. a) Schematic illustrating the hydrogen-bonding and aromatic interactions leading to β-sheet-like local structure in amphiphilic copolymers. b) Schematic of the FF monomer 3-step synthesis and resulting ¹H NMR of the purified product in DMSO-d₆.

Figure 2.

Assembled structure of FF-DMA copolymers. a) DLS of FF-DMA copolymers at various wt%s when dissolved, sonicated for 1 h, or heated for 30 min and slowly cooled to r.t. in phosphate buffer (100 mM, pH 7) at a polymer concentration of 10 mg/mL. Asterisks (*) represent multichain aggregation where the hydrodynamic diameter was measured as >100 nm. Error bars represent standard deviations (n = 3). b) Calculation of percent collapse by converting retention time (min) into apparent molecular weight of PEG (MW_PEG) using a PEG standard curve run in both solvent systems (aqueous and DMF). The peak apex molecular weight (M_P) was determined, and a ratio was calculated. c) Amphiphilic polymers compared to FF-DMA. d) Percent collapse plotted as a function of wt%.

Figure 3.

Secondary structure of FF-DMA copolymers. a) CD spectra of 20 wt% F-, BAA- and FF-DMA polymers at 20 °C. b) Temperature dependence of CD spectra for 20 wt% FF-DMA polymer taken at 20 - 90 °C. c) ThT fluorescence of F-, BAA-, and FF-DMA at varying wt%s. Inset shows the fluorescence spectra of 30 wt% F-, BAA-, and FF-DMA polymers compared to pDMA. Error bars represent standard deviation (n = 3-5). All characterization was performed in 1 mM phosphate buffer at pH 7.

Figure 4.

Positioning of F, a comparison of AF and FF. a) Structure of AF and percent collapse of AF- and FF-DMA as a function wt% hydrophobic monomer. b) ThT fluorescence of AF- and FF-DMA compared at equivalent wt%s (1 and 2) and mol%s (1 and 3) of F units. Error bars represent standard deviation (n = 4-5). c) Temperature dependence of intramolecular interactions of 20 wt% FF- and AF-DMA polymers taken at 20 °C and 90 °C.

Figure 5.

Evaluating the impact of hydrogen-bond donors within hydrophilic monomers. a) Copolymer structure of FF-DMA, -HEAA, and -AM. b) Comparison of SEC of 20 wt% FF-DMA, -HEAA, and -AM. Comparison of c) ThT fluorescence and d) CD spectra of 20 wt% FF-DMA, -HEAA, and -AM. Error bars represent standard deviation (n = 3-5).