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Simultaneous covalent and noncovalent hybrid polymerizations

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Science  29 Jan 2016:
Vol. 351, Issue 6272, pp. 497-502
DOI: 10.1126/science.aad4091

Doubling down on polymerization

In biology, structural polymers such as cytoskeletal fibers assemble from covalently polymerized monomers through weaker supramolecular interactions such as hydrogen bonds. Yu et al. report the synthesis of cylindrical fibers when three monomers react, two covalently and one in a supramolecular fashion. When the reaction proceeded stepwise, lower-molecular-weight flat tapes formed instead, which suggests that supramolecular interactions helped to catalyze the covalent polymerization.

Science, this issue p. 497

Abstract

Covalent and supramolecular polymers are two distinct forms of soft matter, composed of long chains of covalently and noncovalently linked structural units, respectively. We report a hybrid system formed by simultaneous covalent and supramolecular polymerizations of monomers. The process yields cylindrical fibers of uniform diameter that contain covalent and supramolecular compartments, a morphology not observed when the two polymers are formed independently. The covalent polymer has a rigid aromatic imine backbone with helicoidal conformation, and its alkylated peptide side chains are structurally identical to the monomer molecules of supramolecular polymers. In the hybrid system, covalent chains grow to higher average molar mass relative to chains formed via the same polymerization in the absence of a supramolecular compartment. The supramolecular compartments can be reversibly removed and re-formed to reconstitute the hybrid structure, suggesting soft materials with novel delivery or repair functions.

Supramolecular soft matter encompasses organic materials in which structural units engage in strong and often complex noncovalent interactions to generate specific properties and functions. Structurally, these materials can be organized nanostructures (1) or supramolecular polymers (2). Supramolecular soft matter has obvious potential to create reversibly dynamic materials, given the finite lifetimes of interunit noncovalent bonds, and development of this area is clearly inspired by biological systems. In cytoskeleton fibers, for example, the monomers are covalent polymers, and it is their reversible noncovalent interactions into a supramolecular polymer that create their dynamic functions in cells (3, 4). Variations in monomer structures (5, 6), covalent templates (7), or catalysts (8) have facilitated great progress toward the design of supramolecular architectures in solution. However, the integration of covalent and supramolecular polymers into hybrid dynamic structures as a source of function has yet to be achieved.

Here we report the synthesis of polymeric systems based on the simultaneous covalent and noncovalent polymerization of structurally matched monomers. We aimed to explore the nature of hybrid structures that might form during this potentially synergistic process. The covalent polymer (C-Polymer) was designed to form by condensation reactions between an aromatic dialdehyde (monomer 1) and an aromatic diamine (monomer 2). These two monomers contained as side chains the amino acid sequence valine–glutamic acid–valine–glutamic acid, connected to the aromatic groups via a dodecyl linkage (Table 1). Monomer 3 of the supramolecular polymer (S-Polymer) (Table 1) is isostructural with the side chains of the C-Polymer and, on the basis of previous results, was expected to form ribbon-shaped supramolecular polymers (9). Consistent with previous work on foldamers (1012), the C-Polymer was designed to have a sixfold helicoidal conformation, in this case promoted in polar media and stabilized by hydrogen bonds among the peptide segments, as well as π-π stacking interactions between aromatic groups.

Table 1 Chemical structures of monomers 1, 2, and 3.

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To synthesize the C-Polymer, we mixed monomers 1 and 2 in a 1:1 molar ratio in aqueous solution at pH 5 to promote the condensation reaction between aldehydes and amines (13). The S-Polymer formed by simply dissolving monomer 3 in water, owing to its strong amphiphilic structure. Cryo–transmission electron microscopy (cryo-TEM) revealed the formation of a heterogeneous collection of one-dimensional (1D) structures in the C-Polymer (Fig. 1A and fig. S9), and the S-Polymer formed the expected ribbon-shaped flat assemblies (Fig. 1B). However, when we mixed solutions of monomers 1, 2, and 3 simultaneously in a molar ratio of 1:1:2 at pH 5, the flat assemblies of the S-Polymer did not form, and we instead observed 1D structures with precisely defined cylindrical shape with uniform diameter as the dominant morphology (in a few uncommon sites, thin ribbonlike defects can be observed) (Fig. 1C and fig. S10). These 1D structures appear well separated, which is possibly the result of the high charge density contributed by the integration of the S-Polymer in the hybrid structure.

Fig. 1 Hybrid CNC polymers.

(A to C) Cryo-TEM images for (A) the covalent polymer (C-Polymer) obtained by mixing monomers 1 and 2 in a 1:1 molar ratio at pH 5 (white arrows point to ribbonlike segments and black arrows to cylindrical ones), (B) the supramolecular polymer (S-Polymer) formed by monomer 3, and (C) the CNC hybrid polymer obtained by simultaneously mixing monomers 1, 2, and 3 in a molar ratio of 1:1:2 at pH 5. (D to G) Molecular graphics illustrations of (D) the covalent polymerization of monomers 1 and 2 [including a magnified representation (E)], (F) the supramolecular polymerization of monomer 3, and (G) the simultaneous covalent and supramolecular polymerizations that yield the hybrid polymer. Phenyl moieties in molecular graphics illustrations in (D), (E), and (G) are shown in yellow. (H) Schematic representation of the CNC hybrid polymer consisting of two distinct covalent (green and yellow) and supramolecular (red) compartments.

We hypothesized that a covalent-noncovalent (CNC) hybrid system was formed by the simultaneous covalent and supramolecular polymerizations. More specifically, we considered that this CNC hybrid integrated distinct covalent and supramolecular compartments as a result of the structural match of their respective monomers (Fig. 1, D to H). In addition, we observed only a homogeneous cylindrical structure, suggesting thorough integration of both polymers. Mechanistically, the preference for helical conformation in the C-Polymer and common structural features in all three monomers could guide directional nucleation and growth of supramolecular compartments to create a cylindrical hybrid structure.

The morphologies of the C-Polymer and the CNC hybrid were also investigated using atomic force microscopy (AFM). In the hybrid samples, AFM experiments revealed the uniform, well-separated fibrils observed with cryo-TEM (fig. S12C), whereas the mixture of monomers 1 and 2 formed bundled fibrous structures (fig. S12B). We attribute the bundling (which was not observed with cryo-TEM) to drying effects as water is removed. This bundling was not observed with AFM when all three monomers (1, 2, and 3) were mixed simultaneously, providing further evidence of the integration of monomer 3 in the hybrid, which should result in highly charged surfaces.

We first used optical spectroscopy to investigate the condensation between monomers 1 and 2 to form the C-Polymer. A 1:1 molar ratio of monomers 1 and 2 in a fresh solution at pH 5, which favors formation of imine bonds for polymerization, yielded a product revealing in its fluorescence spectrum the anticipated excimer emission appearing instantaneously at 430 nm, compared with 358 nm for monomer 1 (Fig. 2A). This shift indicates the existence of strong π-π stacking interactions in the folded backbone of the C-Polymer (14). Immediately upon mixing monomers 1, 2, and 3, we observed substantial quenching of the excimer emission characteristic of the C-Polymer, which is expected with lengthening of the folded backbone (14). This observation and the absence of monomer emission at 358 nm suggest that the covalent polymerization of monomers 1 and 2 within the hybrid was facilitated by the simultaneous polymerizations (Fig. 2A).

Fig. 2 Spectroscopic characterization.

(A) Fluorescence spectra of monomer 1, the C-Polymer, and the hybrid CNC polymer. Fluorescence is measured in units of counts. λ, wavelength. (B) CD spectra of the C-Polymer, the S-Polymer, the hybrid CNC polymer, and the sum of the spectra of the C-Polymer and S-Polymer. (C) Plot of the difference in CD signal intensity at 214 nm, corresponding to the mixture of all three monomers (1, 2, and 3) and that of monomer 3 [Δellipticity = CD intensity (mixture) – CD intensity (monomer 3)], as a function of the added equivalents of monomer 3. All samples were prepared at pH 5.

The typical circular dichroism (CD) signals for β-sheet secondary structure in the peptide side chains were observed in the mixture of monomers 1 and 2 (Fig. 2B), whereas only CD signals corresponding to random coil conformation were observed for the individual monomers (fig. S6). These results indicate that attachment of the peptide to the C-Polymer backbone as a side chain enhanced formation of the β sheets. In turn, these hydrogen bonds can facilitate the growth of the folded backbone by preorganizing monomers. In addition, the absence of a CD signal in the absorption region of the folded backbone (~300 nm) indicates that the chiral centers in peptide segments are too distant or the dodecyl linkers are too flexible to bias the twist sense of the helical backbone (15). When solutions of monomers 1, 2, and 3 were mixed simultaneously, the CD signal for β sheets increased relative to that of the C- or S-Polymer, and the signal intensity was even greater than the sum of both (Fig. 2B). This increase suggests the formation of a highly integrated hybrid structure in which peptide hydrogen bonding is enhanced through synergistic interactions among the three isostructural monomers. The increase in CD intensity depended on the relative concentrations of monomers 1 and 2 versus monomer 3, and the saturation of the signal was observed beyond the addition of two equivalents of monomer 3 (Fig. 2C). We also used cryo-TEM to examine samples resulting from mixtures of monomers 1, 2, and 3 with molar ratios of 1:1:1 and 1:1:4. In both mixtures, we observed a heterogeneous population of structures (fig. S11). Although adding one equivalent of monomer 3 into monomers 1 and 2 gives rise to formation of short cylindrical fibers and ribbons, the mixture containing four equivalents of monomer 3 forms long fibers and ribbons. These results indicate that there is not enough monomer 3 in the first case to form the highly defined structure of the CNC hybrid. However, an excess of monomer 3 in the second case leads to the formation of the CNC hybrid and a ribbon-shaped S-Polymer. On the basis of CD data and cryo-TEM images, we conclude that the supramolecular compartments are formed only by a finite number of monomer 3 molecules per unit length of hybrid structure (Fig. 1G). This is consistent with the well-defined shape and largely uniform diameter of hybrid fibrils.

We tested the possibility of removing the supramolecular compartment from the hybrid CNC polymer and subsequently reconstituting it. We synthesized a fluorescein-labeled version of monomer 3 (fl-3) to quantify this process. Cryo-TEM experiments showed that extraction of monomer 3 from the hybrid by dilution in pH 5 water and dialysis led to the appearance of short fibers (Fig. 3B). Upon addition of fresh monomer 3 to the extracted sample, the long cylindrical morphology of the CNC hybrid was recovered (Fig. 3, A to C), and when the extraction and reconstitution cycle was repeated, identical results were obtained (Fig. 3, D and E). Based on the fluorescence intensity of fl-3 in the polymer solution, 94% of monomer 3 was removed from the hybrid after dilution and dialysis (fig. S17).

Fig. 3 Extraction and reconstitution.

(A to C) Cryo-TEM image of (A) the CNC hybrid polymer, (B) the same material after extraction of the supramolecular compartments by dialysis, and (C) after reconstitution of the hybrid by adding a fresh solution of monomer 3. (D and E) Images corresponding to samples exposed to a second cycle of extraction and reconstitution. (F) Schematic representation of the extraction of supramolecular compartments from CNC hybrid polymers and their reconstitution by adding monomer 3.

To verify covalent polymerization in both the C-Polymer and the CNC hybrid, we used Fourier transform infrared (FTIR) spectroscopy, matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry, and size exclusion chromatography with multiangle light scattering (SEC-MALS). The FTIR measurements provided evidence of imine bond formation in both the C-Polymer and the CNC hybrid (fig. S3), as well as the presence of hydrogen bonds in all samples (fig. S3). MALDI-TOF studies also confirmed the formation of covalent polymer upon mixing monomers 1 and 2 or monomers 1, 2, and 3, as indicated by an appropriate increase in molar mass in both cases (fig. S4). The average molecular weight when 1 and 2 were mixed was determined by SEC-MALS to be on the order of 14 kDa, but a much higher molecular weight of 250 kDa was measured by this technique for the covalent component of the CNC hybrid (fig. S5 and table S1). Based on the average molecular weight measured for the covalent polymer component of the hybrid and the cryo-TEM images, we conclude that cylindrical fibers contain multiple chains condensed by the synergistic secondary interactions among the three structural units. Overall, these results demonstrate the formation of a covalent polymer by mixing monomers 1 and 2 or within the hybrid structure. Furthermore, the results also strongly support the notion that formation of the supramolecular compartment in the hybrid effectively catalyzes covalent polymerization.

We analyzed the covalent component in the CNC hybrid after extraction of the supramolecular compartment using fluorescence and SEC-MALS experiments. Fluorescence spectra of the covalent component after removal of the supramolecular compartment revealed the recovery of quenched excimer emission over time (fig. S18). This result implies that, after removal of monomer 3, the covalent compartment is less stable and dissociates into short covalent chains. In addition, the average molecular weight of the covalent compartment aged for 10 days was determined by SEC-MALS to be ~16 kDa (fig. S18), a decrease of more than one order of magnitude relative to the original covalent component within the hybrid. Both results suggest that the CNC hybrid is more thermodynamically stable than the C-Polymer. Furthermore, CD data as a function of temperature showed that the signals for both the C-Polymer and the CNC hybrid decreased upon heating as a result of thermally induced depolymerization, as indicated by fluorescence results (fig. S7). However, in the CNC hybrid, depolymerization was found to start at a temperature 5°C higher than in the C-Polymer. Again, this finding provides evidence for the stability of the hybrid as a result of the synergistic secondary interactions among its three different structural units. The results also provide mechanistic insight into the CNC hybrid polymerization, strongly suggesting that the synergistic interactions are responsible for the enhanced levels of covalent polymerization in the CNC hybrid structure.

We used small-angle x-ray scattering (SAXS) experiments to further characterize the morphologies of the various supramolecular assemblies in solution. For monomers 2 and 3, the scattering signals showed a –2 slope in the low-q area (q, modulus of the momentum transfer vector), demonstrating the formation of flat structures in solution (Fig. 4A) (16, 17). The C-Polymer exhibited a –1.3 slope, which suggests a heterogeneous mixture of morphologies, consistent with our cryo-TEM observations. In contrast, the CNC hybrid displayed a slope of nearly –1 (Fig. 4A), indicating the formation of highly 1D cylindrical structures without any evidence of the flat structures observed for monomers 2 and 3 (17, 18). Additional geometrical information of the assemblies of the hybrid could be obtained by fitting the scattering curves to a core-shell cylinder model. The diameter for the hybrid was estimated to be 5.9 nm, which is comparable to our observations with cryo-TEM.

Fig. 4 Results of SAXS, cryo-TEM, and MD modeling experiments.

(A) SAXS curves and their corresponding slopes in the linear region obtained from solutions of monomer 2, monomer 3, the C-Polymer, and the hybrid CNC polymer (scattering curves were offset for clarity). The fitting curve for the scattering data of the CNC hybrid is shown in purple. q, modulus of the momentum transfer vector; I, scattered intensity; A.U., arbitrary units. (B) Change in ellipticity at 199 nm as a function of time during formation of the C-Polymer, S-Polymer, and CNC hybrid polymer during simultaneous covalent and supramolecular polymerization (by mixing monomers 1, 2, and 3), as well as the CNC hybrid polymer by adding monomer 3 to a pre-formed C-Polymer. t, time in hours. (C) Plot of the difference in CD signal intensity at 214 nm, corresponding to the mixture of 1 and 2 and that of monomer 3 [Δellipticity = CD intensity (mixture) – CD intensity (monomer 3)], as a function of the added equivalents of monomer 3. The plot shows one curve corresponding to a fresh sample of C-Polymer mixed with 3 and another corresponding to an aged sample. (D and E) Cryo-TEM images of a sample corresponding to a fresh mixture of a pre-formed C-Polymer and monomer 3 (D) and a sample of the same mixture aged for 2 days (E) (both samples contained two equivalents of monomer 3). Black and white arrows in (D) indicate cylindrical fibers and ribbons, respectively. (F) Results from an atomistic MD modeling of the C-Polymer (left) and the CNC hybrid polymer (right) (green, folded aromatic backbone; red, flexible dodecyl linker; blue, the turn and random coil; yellow, β sheet). The white dashed ellipsoids indicate formation of hydrogen bonding between peptides in covalent and supramolecular compartments. (G and H) MM2 MD modeling (see supplementary materials) of the matched structures (monomers 1, 2, and 3) next to the cryo-TEM image of the system prepared by mixing 1, 2, and 3 in a molar ratio of 1:1:2 at pH 5 (G) and, similarly, of the mismatched structures (monomers 1, 2, and 4) with the corresponding cryo-TEM image (H) (red, monomer 3; purple, monomer 4; green, side chains of the covalent compartment; yellow, aromatic units of the covalent compartment).

To gain insight into the mechanism for the formation of the hybrid CNC polymer, we monitored changes in the CD spectrum over time in different types of samples. As shown in Fig. 4B, a mixture of monomers 1 and 2 undergoing covalent polymerization revealed an increasing value of ellipticity that saturates after several hours. The CNC polymer formed by mixing all three monomers simultaneously exhibited a rapid rise in ellipticity, suggesting nucleation and growth of an ordered structure. At the same time, the invariant ellipticity of the S-Polymer formed by monomer 3 indicates that the increase in ellipticity of the CNC hybrid did not arise from independent supramolecular polymerization, but rather from the simultaneous supramolecular and covalent polymerizations. The faster kinetics associated with CNC hybrid formation compared with that of the C-Polymer strongly supports a distinctive mechanism involving simultaneous covalent and supramolecular reactions. These observations could explain why the average molecular weight measured for the covalent compartment of the CNC polymer is so much higher than that of the C-Polymer. In other words, the data are consistent with a synergistic enhancement of C-Polymer formation by supramolecular contacts with monomer 3.

We used CD spectroscopy to follow the interaction between a pre-formed C-Polymer and monomer 3. When different amounts of monomer 3 were added to the pre-formed C-Polymer, we observed only a small initial increase in CD signal intensity in the β-sheet region (Fig. 4C). However, when samples with an excess of monomer 3 aged for 2 days, the CD intensity increased and was comparable to that observed when the hybrid CNC polymers formed through the simultaneous mixing of monomers 1, 2, and 3 (Fig. 4C). Our SEC-MALS results indicate that the average molecular weight of the covalent component in solutions containing two equivalents of monomer 3 increased with time from 21.1 to 190 kDa (table S1). The fact that CD signatures of the ordered hybrid are not observed immediately upon mixing could be explained by diffusional barriers within the system imposed by the pre-formed covalent polymer.

Both SAXS and cryo-TEM experiments supported our interpretation of the CD and molecular weight data. In SAXS experiments, the fresh mixture of pre-formed C-Polymer and monomer 3 revealed a slope of –1.4 in the low-q region, indicating a heterogeneous mixture of morphologies. In marked contrast, we measured a slope of approximately –1 for the aged sample, thus demonstrating the possibility of forming the well-defined cylindrical structure of the CNC hybrid by mixing monomer 3 with pre-formed covalent polymer. In addition, fitting the scattering data to a core-shell cylindrical model yields effectively the same diameter for structures in the aged sample and samples obtained by the simultaneous polymerization of monomers 1, 2, and 3 (fig. S19). Furthermore, cryo-TEM also reveals virtually identical morphologies in these two types of samples (Fig. 4, D and E).

We hypothesize that the structural match of the supramolecular monomer with the side chains of the covalent compartment plays a critical role in the integration of the two compartments and the catalytic effect of the supramolecular polymerization on covalent polycondensation. To test this hypothesis, we used a monomer (4; see supplementary materials) for the S-polymer that would not easily interact noncovalently with the side chains of the C-Polymer. This monomer was also a peptide amphiphile with the same general structural features as monomer 3. However, the structural match in monomer 4 relative to the side chains is lost, both in the length of the hydrophobic region (four additional methylene groups) and its peptide sequence (a different sequence of two amino acids present in monomer 3, valine and glutamic acid, plus two additional glycine residues). Cryo-TEM images of the mismatched system reveal a heterogeneous mixture of morphologies formed by combining monomers 1, 2, and 4 in the molar ratio of 1:1:2 (Fig. 4H and fig. S20). Additionally, in this system we observed only a slight difference in CD intensity after mixing monomers 1, 2, and 4 (fig. S21). These results demonstrate that the new supramolecular monomer does not integrate well with covalent compartments and does not form the distinct hybrid CNC polymer. The average molecular weight of the covalent compartments in the presence of monomer 4 was characterized by SEC-MALS to be ~18 kDa, more than an order magnitude lower than that of the matched system (fig. S21). We conclude from these results that the structural mismatch between monomer 4 and the side chains of the C-Polymer does not promote synergistic interactions responsible for stabilization of the C-Polymer by the S-Polymer, which in turn results in greater growth of the C-Polymer within the CNC hybrid.

In previous work (1922), including our own (2325), systems have been studied in which covalent polymerization is triggered after supramolecular self-assembly of monomers, leading to internally ordered covalent polymers. There is also another system in which an ordered covalent polymer was obtained after polymerization of the monomer in a solvent that does not promote formation of a supramolecular template (26). In our current work, a pathway is described to obtain hybrid polymers in which supramolecular and covalent polymers are integrated. The supramolecular compartment in these systems can be temporarily removed and reconstituted by simply adding its monomer again. Furthermore, we discovered that the supramolecular compartment within the hybrid catalyzes covalent polymerization.

We carried out atomistic molecular dynamics (MD) simulations on the C-Polymer and the hybrid CNC polymer (Fig. 4F). These MD simulations were performed for 24 molecules each of monomers 1 and 2 and 48 molecules of monomer 3 in the presence of water and sodium ions. Details of these simulations can be found in the supplementary materials and in a previous publication (27). The simulations yielded a hybrid CNC structure with a diameter equal to 7 nm, which is reasonably consistent with experimental results (fig. S23). The simulations showed also that β sheets formed among 15 peptide segments within the C-Polymer and 22 peptide segments in the hybrid CNC polymer (fig. S23). Most of the β sheets within the CNC hybrids formed between the supramolecular and covalent compartments (fig. S23 and MD simulations in the supplementary materials). We believe that the integration of the two distinct compartments into the CNC hybrids benefits from these secondary bonds, along with other noncovalent interactions. This integration among isostructural components in all three monomers was an important molecular design criterion.

These polymers self-organize to contain distinct covalent and supramolecular compartments that allow removal and re-formation of the supramolecular component, thus reconstituting the hybrid polymer. These structures could provide functional platforms for novel modes of molecular delivery or repair of structures, as hybrids are disassembled and re-formed by simple addition of small molecules. Our experimental results on these systems also suggest that supramolecular polymerizations can be used to catalyze the formation of covalent macromolecules.

Supplementary Materials

www.sciencemag.org/content/351/6272/497/suppl/DC1

Materials and Methods

Supplementary Text

Schemes S1 to S3

Figs. S1 to S23

Table S1

References (2838)

References and Notes

Acknowledgments: The synthesis and structural characterization of this work was supported by the NSF under award no. DMR-1508731. Experimental work on SAXS was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, under award no. DE-FG02-00ER45810. MD simulations were supported by the Center for Bio-Inspired Energy Science, an Energy Frontier Research Center funded by DOE, Office of Science, Basic Energy Sciences, under award no. DE-SC0000989 (T.Y. and G.C.S.). We thank A. Koltonow for help with AFM measurements and M. Seniw for help with the preparation of graphics. We also acknowledge S. Kewalramani for helpful discussions on the SAXS data. Use of the Advanced Photon Source (APS) was supported by DOE, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. SAXS experiments were performed at the DuPont–Northwestern–Dow Collaborative Access Team (DND-CAT) located at Sector 5 of APS. DND-CAT is supported by E. I. DuPont de Nemours and Co., The Dow Chemical Company, and Northwestern University. We thank the Biological Imaging Facility at Northwestern and the Electron Probe Instrumentation Center facilities of the Northwestern University Atomic and Nanoscale Characterization Experimental Center for the use of TEM. Nuclear magnetic resonance and MS equipment at the Integrated Molecular Structure Education and Research Center was supported by the NSF under grant no. CHE-9871268. We are also grateful to the Peptide Synthesis Core at the Simpson Querrey Institute for BioNanotechnology and Keck Biophysics Facility for instrument use.
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