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

Article abstract of DOI:10.1021/jacs.1c05659

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

Full text of DOI:10.1021/jacs.1c05659

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Article
Controlling Amphiphilic Polymer Folding beyond the Primary
Structure with Protein-Mimetic Di(Phenylalanine)
Jacqueline L. Warren, Peter A. Dykeman-Bermingham, and Abigail S. Knight*
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ABSTRACT: While methods for polymer synthesis have prolif-
erated, their functionality pales in comparison to natural biopol-
ymers−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.
polymer backbone. Helices can be introduced with the BTA-
pendent methacrylate monomer that is incorporated into a
random amphiphilic copolymer through ruthenium-catalyzed
living radical polymerization.6,21 However, the synthetic
versatility of the polymerizable BTA methacrylate is not
currently available for β-sheet-like structures, which have only
been introduced into aqueous assemblies through incorpo-
ration of a peptide block into the polymer backbone.
Oligoleucine has been used as an initiator in nitroxide-
mediated radical polymerization placing a peptide in the
backbone of a polymer and leading to the formation of β-sheet
regions.22,23 These strategies provide a foundation for the
introduction of local structure into polymer scaffolds, but we
lack a full range of tools to create local structure in synthetic
polymers, i.e., moieties that can be easily utilized alongside the
diverse scope of monomers already in the polymer chemist’s
toolbox.
INTRODUCTION
■
Confined to a limited pool of natural amino acid monomers,
proteins have evolved primary structures that yield canonical
structure hierarchy: quaternary structures of multiple polypep-
tide chains, tertiary structure that defines the three-dimen-
sional morphology and global structure, and local rigid regions
dictated by secondary structure. Polymer chemists have far
outpaced natural proteins in increasing the diversity of
monomers; yet, establishing the complex functions of natural
biomacromolecules, and thus the intricate networks of
structure-driving noncovalent interactions, is a remaining
challenge necessary to expand the functionality of next
generation materials.1−3 Each hierarchical level of folding is
required to create subdomains capable of protein-mimetic
functions such as specificity, catalysis, and allostery.
Despite this need for higher-order structure, there are few
examples of synthetic polymer assemblies that demonstrate
these motifs in an aqueous environment.4 Within the single-
chain nanoparticle (SCNP) literature, protein-mimetic collapse
dictating the global morphology has been generated with
hydrogen-bonding using a pendent benzene-1,3,5-tricarbox-
amide (BTA) moiety,5−10 hydrophobic collapse using alkyl
chains,11−16 host−guest interactions,17 metal−ligand interac-
tions,18 and covalent cross-links.19,20 As secondary structure is
driven by backbone hydrogen bonding in proteins, it is
challenging to mimic this local rigidity with an amide-lacking
Received: June 1, 2021
Published: August 10, 2021
https://doi.org/10.1021/jacs.1c05659
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© 2021 American Chemical Society
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Di(phenylalanine), a moiety identified to drive secondary
structure formation in amyloid fibrils, has been implemented
across disciplines (e.g., drug delivery, hydrogel formation, and
metal−organic frameworks) to lead to aggregation in aqueous
environments including driving multichain assembly of hydro-
philic PEG or peptides.24−27 Isolated di- and tripeptides
containing di(phenylalanine) have shown that hydrogen
bonding and aromatic interactions stabilize aggregated
structures as characterized with circular dichroism (CD) and
Fourier transform infrared spectroscopy.28,29 Motivated by
these observations, we hypothesized that di(phenylalanine)
could generate higher-order structure in an amphiphilic
copolymer. Herein we demonstrate the role of hydrophobicity,
aromatic interactions, and hydrogen bonding in the formation
of global structure and novel β-sheet-like local structure
induced through a pendent moiety.
(N-Boc and methyl ester) followed by deprotection of the Boc
and substitution of acryloyl chloride (characterization in
Figures S1−6).
Inspired by protein-mimetic statistical copolymers with
helical domains21 and the natural separation of β-sheet
forming residues in protein sequences, we synthesized random
amphiphilic copolymers with di(phenylalanine) moieties
distributed throughout the polymer scaffold. Dimethylacryla-
mide (DMA) was selected as a hydrophilic monomer as it lacks
a hydrogen bond donor and thus is unlikely to disrupt
hydrogen-bonding between di(phenylalanine) moieties. Due
to the steric bulk of FF and conformational restraints imposed
by its multiple amide bonds, we assessed the ability of the FF
monomer to achieve an approximately statistical distribution
within the copolymer backbone and a Gaussian molecular
weight distribution when copolymerized with DMA. Reversible
addition−fragmentation chain transfer (RAFT) polymeriza-
tion32 performed in DMF yielded polymers with controlled
molecular weight (Mn), low dispersity (Đ), and similar rates of
monomer incorporation (Figures S7−S10 and Tables S1 and
S2).
RESULTS AND DISCUSSION
■
Di(phenylalanine) Monomer (FF) Synthesis and
Copolymerization. Motivated by the unique propensity of
di(phenylalanine) to drive β-sheet formation, we synthesized a
bioinspired monomer with a pendent di(phenylalanine)
moiety (Figure 1a). A di(phenylalanine) acrylamide monomer
was designed (methyl acryloyl di-L-phenylalanine, FF; Figure
1b) to be compatible with common controlled radical
polymerizations, which provide monomer scope versatility,
chain-end fidelity, and control of the polymer molecular weight
and dispersity.30,31 The synthesis was accomplished using an
amide coupling of two orthogonally protected L-phenylalanines
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
(MWPEG) using a PEG standard curve run in both solvent systems
(aqueous and DMF). The peak apex molecular weight (MP) was
determined, and a ratio was calculated. (c) Amphiphilic polymers
compared to FF-DMA. (d) Percent collapse plotted as a function of
wt %.
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 H NMR of the
purified product in DMSO-d6.
1
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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.
Characterization of Polymer Collapse and Assembly.
To investigate the assembled conformation of FF-DMA
copolymers, a series (0 to 40 wt % FF) were synthesized
and their morphologies in neutral aqueous solution (100 mM
phosphate, pH 7) were characterized. To probe the collapse in
an aqueous environment, three methods of assembly were
employed: dissolution, sonication, and a heat−cool cycle
before characterization with dynamic light scattering (DLS)
(Figure 2a and Table S3). For copolymers containing 20 wt %
or less of FF, single-chain nanoparticles (SCNPs) of
comparable sizes were observed for all assemblies. Aggregation
was visible for 30 and 40 wt % FF in the dissolved copolymers;
however, both sonication and a heat−cool cycle led to the
formation of SCNPs. DLS enabled the rapid identification of
single chains and suggested a limited impact of the assembly
strategy on the aggregation state. However, it was challenging
to quantify the anticipated increase in collapse with increasing
hydrophobicity due to the limited instrument accuracy at low
hydrodynamic radii.
To calculate percent collapse that correlated with the
hydrophobic weight percent of the copolymers, we compared
the compact and swollen states of the amphiphilic polymers
using size exclusion chromatography (SEC) in aqueous and
organic (DMF) solvents (Figures 2b and S11), a strategy used
by Sawamoto and co-workers.33 By calibrating with poly-
(ethylene)glycol standards in each solvent, a ratio of the peak
apex molecular weight (MP) in aqueous buffer [MP(aq)] and
DMF [MP(DMF)] is a measure of compactness (Figures S12 and
S13). We converted this value to a percent by multiplying by
100 and plotted this percent compaction as a function of the
FF wt % (Figure 2). Using this strategy, we were able to
observe the anticipated linear relationship, analogous to
previous work with dendrimer collapse,34 and over 50%
collapse at 40 wt % FF (Table S4). Although the more
hydrophobic polymers formed multichain aggregates as
characterized by DLS, the polymers were diluted during SEC
characterization yielding single chains as characterized by
multiangle light scattering (MALS; Figure S14). Additionally,
three separate 20 wt % FF polymers were synthesized and
yielded the same percent collapse values for similar molecular
weights demonstrating reproducible collapse characteristics
(Figure S15). Percent collapse was observed to decrease as the
molecular weight was altered over a range of 7−54 kDa for FF
copolymers. (Figure S16, Table S5). We hypothesize that the
entropic cost of collapse increases with molecular weight,
leading to extended structures as proposed for 25−55 kDa
BTA-containing SCNPs.9
To probe the factors contributing to the collapse of protein-
mimetic FF-DMA, we designed an analogous series of
amphiphilic DMA copolymers with benzyl acrylamide (BAA)
and methyl L-phenylalanine (F) with lower propensity to
hydrogen bond and nonaromatic tert-butyl acrylamide (tBA)
(Figures 2c and S17−S25 and Tables S6 and S7). Both
hydrophobicity and hydrogen bonding are known to
contribute to protein stability,35,36 and di(phenylalanine) is
known to have strong hydrogen-bonding and hydrophobic
interactions.24,37 Thus, either interaction or a combination
could be responsible for the observed collapse.
The three aromatic monomers led to similar collapse
percentages, leading to the hypothesis that the collapse is
primarily driven by hydrophobicity (Figures 2d and S11, S26−
28). To further investigate this, the ratio of collapse to
logP38,39 (estimated by Marvin Sketch) was plotted as a
function of the mole percentage of the hydrophobic monomer
(Figure S29). Linear regressions revealed similar relationships
for FF-, F-, and BAA-DMA and showed lower collapse
percentages than the logP would predict for tBA-DMA (Table
S8). We hypothesize this difference is due to the bulkiness of
tBA close to the backbone, and that the collapse is
predominantly driven by hydrophobicity.
Characterization of Local Polymer Structure. While
collapse is a key component of protein-mimetic polymer
structure, local structure is also critical for complex functions;
this is what motivated the selection of the di(phenylalanine)
moiety. Circular dichroism (CD) spectroscopy and differential
scanning calorimetry (DSC) both revealed more local order in
FF-containing copolymers compared to F-containing copoly-
mers. As anticipated, polymers composed solely of achiral
monomers had no observable CD spectra (Figures 3a and
S30). Unique CD spectra were observed for DMA copolymers
with FF and F in aqueous solution (1 mM phosphate, pH 7).
Although β-sheets commonly display minima, the high
percentage of aromatic amino acids generates noncanonical
spectra. The maxima at 198 and 217 nm have been attributed
to π−π* and n−π* transitions consistent with the aromatic
interactions and hydrogen bonds of di(phenylalanine)
containing β-sheets.25,27,28,40,41 The FF copolymer has both a
narrower peak than the F copolymer at 217 nm and a higher
intensity peak at 198 nm, both indicating a more ordered
structure.42 As the molecular weight was altered over a range
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from 7 to 54 kDa, the spectra maintained their shape for 20 wt
% FF copolymers with DMA (Figure S32). Heating the
aqueous polymer assemblies to 90 °C decreases both maxima,
as is commonly observed for intramolecular interactions
(Figures 3b and S33).43 Characterization of the copolymers
with DSC additionally demonstrates stronger intramolecular
interactions, and thus more ordered local structures for FF-
DMA: the FF-DMA copolymer with 30 wt % FF has a higher
glass transition temperature (Tg) than the DMA homopol-
ymer. Conversely, the F-DMA (30 wt % F) copolymer has a
lower Tg than the DMA homopolymer (Table S9). Although
this characterization is performed in the solid-state, it illustrates
that there are pockets of FF large enough to form
intramolecular interactions that do not form in the
complementary F-DMA copolymers.
Further investigation of polymer internal structure with
thioflavin T (ThT) fluorescence spectroscopy supports the
presence β-sheet-like interactions formed within FF-DMA
(Figures 3c and S34). Emission at 480 nm is commonly
observed for amyloid aggregates due to the restricted rotation
in fibril-containing regions.27,44,45 Copolymers of increasing
weight percentages of FF, F, and BAA demonstrated increasing
fluorescence with increasing percent hydrophobicity. The 30
wt % samples highlight that as the number of cross-links
increases, the FF-DMA is forming more ordered β-sheet-like
structures than copolymers containing F or BAA as illustrated
by the comparatively increased fluorescence.
Positioning F Farther from the Backbone: Alanine-
Phenylalanine Monomer (AF). While the fibrilization of
di(phenylalanine)-containing molecules has been thoroughly
characterized, phenylalanine alone and other phenylalanine-
containing dipeptides have also demonstrated some of the
hallmark characteristics of assembly including fibrilization and
CD spectra similar to di(phenylalanine).24,42,46 To investigate
whether the di(phenylalanine) moiety or simply the presence
of a phenylalanine distanced from the backbone led to the
increased internal structure, an alanine-phenylalanine acryl-
amide was synthesized and characterized (methyl acryloyl L-
alanyl-L-phenylalanine, AF; Figures 4a and S35−S41). Collapse
of AF-DMA copolymers was measured as described for FF-
DMA previously, and trends similar to those of the previous
copolymers with aromatic side chains were observed (Figure
S42 and Table S10). When plotted as a function of mole
percent of the hydrophobic monomer, the ratio of the logP
value to the percent collapse was greater than that observed for
FF and F (Figure S43 and Table S11), suggesting the flexibility
of the alanine linker led to more collapsed structures than
those predicted by the hydrophobicity.
The internal structure of the collapsed AF copolymers
resembles that of the FF copolymers, indicating the AF
copolymers also contain β-sheet-like regions. Fluorescence in
the presence of ThT was compared among polymers with
similar compactness (30 wt % hydrophobic monomer, 1 and 2
in Figure 4a,b) and among those with comparable total
numbers of phenylalanine residues (15 mol % F, 1 and 3 in
Figure 4a,b). Copolymers with equivalent weight percents have
similar numbers of intramolecular physical cross-links, and the
FF-DMA copolymers display more ThT fluorescence. The
polymers with equivalent mole percents have similar numbers
of total phenylalanine residues, and the AF-DMA polymers
display a higher ThT fluorescence likely due to the increased
number of intramolecular physical cross-links as the polymer is
two times more compact. The presence of ordered local
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 and 90 °C.
structure is supported by the CD spectrum that mirrors that of
the FF-DMA copolymers (Figure 4c: both 20 wt %
hydrophobic monomer and Figure S44). However, heating
both samples to 90 °C revealed the AF driven collapse was less
stable as the ellipticity decreases more than that of the FF-
DMA copolymers. This is supported by the minimal difference
in Tg observed for AF-DMA copolymers as compared to
pDMA, as opposed to the increase observed for FF-DMA
(Table S12).
Exploring the Impact of Hydrogen-Bond Donors
within Hydrophilic Monomers. After probing the impact
of systematic variation of the hydrophobic monomer, we
sought to investigate how the presence of hydrogen bond
donors in the hydrophilic monomer would influence the folded
polymer structure. FF-hydrophilic acrylamide amphiphilic
copolymers (20 wt % FF) were synthesized with N-
hydroxyethyl acrylamide (HEAA) and acrylamide (AM)
(Figures 5a, S45, and S46). The amphiphilic copolymers of
comparable molecular weights were characterized via aqueous
SEC alone as acrylamide homopolymers are insoluble in DMF.
As anticipated, due to the difference in hydrophilicity and
additional intramolecular hydrogen bonds, HEAA and AM
containing copolymers were more collapsed than the original
F- and FF-DMA copolymers (Figures 5b and S47−S50; Table
S13). Fluorescence of ThT is also enhanced in the HEAA and
AM copolymers, which suggests the increase in hydrophilicity
leads to a higher propensity for β-sheet-like interactions
(Figures 5c and S51).
CD spectra provide additional insight into the intra-
molecular interactions. Near 218 nm, the narrowest peak is
FF-DMA as compared to FF-HEAA and -AM, suggesting the
DMA copolymer, which lacks additional hydrogen bond
donors, has the most rigid internal structure (Figure 5d).
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continuing to close the gap between synthetic polymers and
biomacromolecules. Additionally, studies with nonrandom
copolymers could reveal control over global structure and
aggregation number including controlling the number of
chains in multichain assemblies and increasing the number of
β-sheet-like interactions. This tunability will facilitate the
development of biocompatible nanomaterials with complex
functions such as selective binding, catalysis, or allostery that
rival and improve upon the capabilities of natural proteins.
ASSOCIATED CONTENT
■
sı
* Supporting Information
Supporting Information containing the experimental details,
synthetic procedures, and supplemental figures and tables
including NMR, SEC, DLS, DSC, and CD characterization is
available free of charge via the Internet at . The Supporting
Information is available free of charge at https://pubs.acs.org/
doi/10.1021/jacs.1c05659.
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
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).
Abigail S. Knight − Department of Chemistry, The University
of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599, United States; orcid.org/0000-0003-
1524-473X; Email: aknight@unc.edu
Authors
Jacqueline L. Warren − Department of Chemistry, The
University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599, United States
Peter A. Dykeman-Bermingham − Department of Chemistry,
The University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599, United States
Copolymers with HEAA and AM no longer have a peak in the
198 nm region. Amide-π interactions have been frequently
observed in proteins and can form stronger interactions than
amide hydrogen bonds;47,48 thus, amide-π interactions may be
disrupting the aromatic interactions observed in FF-DMA.
This suggests that while HEAA and AM copolymers lead to
more collapsed structures, there are fewer interactions between
the di(phenylalanine) moieties in FF-HEAA and -AM
copolymers.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c05659
Notes
The authors declare no competing financial interest.
CONCLUSIONS
■
We have described a series of amphiphilic copolymers with
tunable global and local structure. By comparing the
hydrophobic collapse of amino acid based hydrophobic
monomers (FF, AF, and F) and commercial monomers
(BAA and tBA) we determined that the amount of collapse in
copolymers with DMA is predominantly dependent on the
weight percent of the hydrophobic monomer. However,
internal structure, as characterized by ThT fluorescence and
CD, depends on the composition of the hydrophobic
monomer and the presence or absence of hydrogen-bond
donors in the hydrophilic comonomer. The FF-DMA
copolymer generates the most ordered intramolecular
interactions, while FF-AM leads to the greatest collapse and
ThT fluorescence emission with additional hydrogen bond
donors.
Recent studies have revealed the compaction of SCNPs to
be similar to inherently noncrystalline intrinsically disordered
proteins,49 which comprise close to a third of eukaryotic
proteins.50 This suite of amphiphilic copolymers opens the
door for the development of synthetic single-chain aqueous
assemblies with both helical and β-sheet-like domains,
ACKNOWLEDGMENTS
■
This material is based upon work supported by the U.S.
Department of Energy, Office of Science, Office of Basic
Energy Sciences under Award Number DE-SC0021295. J.L.W.
thanks UNC Department of Chemistry’s E.C Markham
Summer Research Award for a summer undergraduate
fellowship. We acknowledge the Macromolecular Interactions
Facility (SEC-MALS and CD; supported by the National
Cancer Institute of the National Institutes of Health under
award number P30CA016086), the UNC Department of
Chemistry Mass Spectrometry Core Laboratory (HRMS;
supported by the National Cancer Institute of the National
Institutes of Health under award number P30CA016086 and
the National Science Foundation under grant numbers CHE-
0922858 and CHE-1726291), and the Redinbo (ClarioStar
plate reader), Leibfarth (GPC, TGA, and DSC), and Dick
(DLS) laboratories for assistance with instrumentation.
Additionally, the NMR Core Laboratory staff, Drs. Marc ter
Horst and Andrew Camp, for feedback on NMR character-
ization.
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