The advances in organic chemistry, catalyst design, and polymerization methodologies have led to the development of a wide range of synthetic polymers with high molecular weight, variable composition and structure, and unique physical states and mechanical properties. Synthetic polymers, produced via step growth, chain growth or ring opening polymerization of small molecule compounds with chemically susceptible groups, consist of long chains of covalently linked monomeric repeat units. [1][2][3][4] With the exception of polypeptides and certain types of nonamide-based polymers, such as poly(isocyanide)s, poly(p-phenylenevinylene), 5 most synthetic polymers exist as random coils or extended chains; they normally do not fold into defined secondary structures seen in natural proteins. Although precise placement of reactive handles or hydrogen bonding units along the polymer backbone or at chain termini has enabled the creation of synthetic polymers with complex molecular architecture, shape, and morphology, [6][7][8][9] these nanoscale molecular structures lack the specificity, complexity, diversity, and flexibility seen in natural proteins.
Synthesized from natural amino acids through transcriptional and translational processes, proteins exhibit well-defined secondary, tertiary, and quaternary structures in aqueous media through concerted non-covalent interactions, including H-bonding, hydrophobic interactions, Van der Waals forces, and ionic interactions. The function of a particular protein is dependent on the peptide sequence as well as their unique higher-order structures. 10 To achieve diverse functions, many natural proteins contain multiple structurally and functionally distinct domains or motifs. For example, the giant muscle protein titin ( 3 MDa) consists of multiple folded immunoglobulin domains arranged in series alternating with unstructured unique sequences. 11,12 The consecutive and reversible unfolding and refolding of the domains are crucial for titin to serve as a modular spring. 13 On the other hand, structural proteins, such as keratins and vimentin, incorporate coiled-coil motifs, i.e. a superhelix of multiple α-helices twisted around each other, to provide mechanical strength to cells through the formation of rigid or semiflexible fibrous networks. 14 The need for advanced materials with tunable properties and adaptive features has motivated researchers to exploit the utility of peptidic building blocks and motifs to create new materials with desired functions. 15 Supramolecular polymerization of cyclic, 16 amphiphilic, and β sheet-forming peptides 17,18 into ordered nanostructures has yielded materials with high molecular weight (200 MDa 17 ), long-range order, and enhanced tunability. Because the assembly is driven by non-covalent interactions, the structures and properties of this type of materials are dependent on pH, temperature and solvent conditions. 19,20 To prevent the polymeric peptide assembly from dissociating, the constituent building blocks must exhibit high affinity for each other, thereby imparting mechanical properties similar to conventional polymers established entirely by covalent bonds. 21 Peptide-based polymers can be synthesized through covalent coupling of pre-assembled peptidic motifs. 22,23 The resultant hybrid materials retain the reversible and dynamic properties of supramolecular motifs, and at the same time are structurally sound and mechanically robust. 24 Side-chain-to-side-chain polymerization of vertically stacked cyclic β-peptide rings via the formation of amide linkages gives rise to nanofibers composed of a single polymer strand that is tougher than natural silk. 25 Alternatively, linkerfree, end-to-end polymerization of antiparallel homotetrameric coiled-coil peptide bundles via thiol-Michael addition leads to the formation of hybrid polymers that are exceptionally rigid with persistence lengths up to 40 µm. 26 As one of the most abundant structural and functional motifs in nature, coiled-coils are essential for mediating strong and specific protein-protein interactions. 10 With a basic heptad repeat, the constituent peptides for coiled-coils can be computationally identified. 27 To expand the range of material properties attainable by one-dimensional nanostructures, an alternative inter-bundle linking chemistry is explored for the preparation of polymeric coiled coils (PCCs).
Herein, using customized, multistranded superhelices as unconventional monomers, we exploited the utility of tetrazine (Tz) ligation with trans-cyclooctene (TCO) for the preparation of semiflexible ultrahigh molecular weight PCCs. This reaction features fast kinetics, high selectivity at low concentrations, and compatibility with biological systems. 28,29 We strategically introduced flexible oligoethylene glycol (OEG3-4) linkers between the peptide backbone and the bioorthogonal handles to afford semiflexible polymer chains. The PCCs were characterized experimentally and computationally to determine the secondary structure, diffusivity, molecular weight, and morphology. When polymerization was conducted at a relatively high bundlemer concentration, interchain entanglement of ultrahigh molecular weight, semiflexible peptidic polymers gave rise to elastic hydrogels that are mechanically robust. 30 Avogadro, 31 and BioRender.com. We have successfully applied tetrazine/TCO ligation, an inverse electron demand Diels-Alder reaction with a second order rate constant k 2 > 10 3 M -1 s -1 , 28,32 to synthesize mechanically robust hydrogel fibers via step-growth polymerization using traditional poly(ethylene glycol)-based monomers. [33][34][35] In this study, computationally designed peptides ("DE" and "DK", Figure 1a) that form stable antiparallel homotetrameric bundles [(DE)4 and (DK)4] 26,36,37 were chosen as supramolecular monomers to produce PCCs via covalent step growth polymerization (Figure 1b). A cysteine residue was appended to the basic sequence at the N-terminus, through which Tz or TCO functionality was introduced using maleimide-OEG4-tetrazine and maleimide-OEG3-TCO, respectively (Figures S1-12). When assembled in H 2 O, the bundlemers contain precisely four identical functional groups evenly distributed on each end.
Homopolymers were prepared using the DE peptide at a Tz/TCO molar ratio of 1/1 and monomer concentrations of 1.0 -4.5 mM. At 2.5 mM, 60% of tetrazine was consumed one minute after mixing, as shown by the attenuation of the absorbance of the tetrazine chromophore (524 nm, Figure 1c).
Tetrazine consumption gradually leveled off after 10 min, and by 25 min, over 95% of tetrazine was converted to cycloadduct (Figure 1d). A single peak at an elution time of 3 min with a molecular mass of 8,249 Da was detected from ultra-performance liquid chromatography (UPLC)-tandem electrospray ionization mass spectrometry (ESI-MS, Figure 1e, Figure S13). During the experiment, all constituent bundles were denatured by acetonitrile, causing the polymer to dissociate into covalently linked dimeric peptides that were readily detectable. Overall, our results confirmed the accessibility of the bioorthogonal reactive groups and the high efficiency of the cycloaddition reaction.
Dilute solution viscometry was employed to indirectly assess the size of poly(DE4) n under native conditions that preserve the secondary structure of the bundles. All measurements were performed in a good solvent (water) under a dilution solution regime with the polymer properly solvated, as evidenced by a logarithmic fit with a slope (concentration exponent) of 1.3 [38][39][40] (Figure 2a), and negative Kraemer constant K K 41 (Table S1). Intrinsic viscosity ([]) was determined by fitting inherent and reduced viscosity to Huggins 42 and Kreamer 43 Equations (Figure S14). As expected, the intrinsic viscosity of poly(DE4) n was significantly higher than that for the monomeric bundlemers (DE-Tz)4 and (DE-TCO)4 (Figure 2b).
Moreover, the intrinsic viscosity increased with the bundlemer concentration used during polymerization.
Because the translational diffusion coefficient of a molecular species is directly related to its molecular weight, we next employed diffusion-ordered spectroscopy (DOSY) to assess poly(DE4) n molecular ( D E -T z ) 4 ( D E -T C O ) 4 P C C 1 .0 m M P C C 2 .5 m M P C C 3 .5 m M 0.00 0.25 0.50 0.75 Intrinsic viscosity (dL/g) 10 -0.5 10 0 10 0.5 10 1 10 -1 10 -0.5 10 0 10 0.5 Concentration (g/dL) Specific viscosity (dL/g) (DE-Tz)4 PCC2.5 mM PCC3.5 mM 1.28 1.28 1.11 (DE-TCO)4 1.14 PCC1.0 mM 1.29 (DE-Tz)4 PCC 1.0 mM PCC 2.5 mM PCC 4.5 mM HDO δ 1 H (ppm) D (m 2 /sec) + [Tz]/[TCO]=1:1 PCC (DE-Tz)4 (DE-TCO)4 a b c d 0 500 1000 1500 2000 2500 -2×10 -7 -1×10 -7 0 Garadient^2 (G/cm) 2 ln(I/I 0 )/[γ 2 δ 2 (Δ-δ/3)*10 4 ] (DE-Tz)4 PCC1.0 mM PCC2.5 mM PCC3.5 mM PCC4.5 mM e ( D E -T z ) 4 P C C 1 .0 m M P C C 2 .5 m M P C C 3 .5 m M P C C 4 .5 m M 1 10 100 1000 10000 Molecular Weight (kDa) ( D E -T z ) 4 P C C 1 .0 m M P C C 2 .5 m M P C C 3 .5 m M P C C 4 .5 m M 0 2 4 6 8 10 Diffusion coefficient (×10 -11 m 2 /s)
weight under native conditions. In this experiment, the NMR signal attenuates as the applied magnetic field gradient strength (g) increases, following the Stejskal-Tanner equation 44 with a linear correlation between the logarithm of signal intensity (I/I 0 ) and g 2 , the slope of which indicates self-diffusion coefficient (D DOSY ) (Figure 2d). The Stejskal-Tanner plot for signals from the aliphatic, aromatic, and amide regions all decayed linearly (Figure S15) for both the monomeric and polymeric species. D DOSY decreased progressively from the monomer to the polymer and PCCs prepared at a higher bundlemer concentration had a lower D DOSY value (Table S2). In Figure 2c and Figure S16, 2D DOSY spectra for monomer (DE-Tz)4 and poly(ED4) n prepared at Tz (or TCO) concentrations of 1, 2.5 and 4.5 mM were overlaid for easy interpretation. In all experiments, the diffusion coefficient of the solvent (D 2 O) was determined to be 1.62×10 -9 m 2 s -1 , with a percent relative standard deviation of 4%, suggesting good viscosity control. 45 The DOSY cross-peaks of (DE-Tz)4 were well-defined in the diffusional dimension while those for poly(DE4) n prepared at higher Tz concentrations exhibited a relatively broader distribution, demonstrating a diversified range of polydispersity. 46 The assembled tetrameric bundles and the OEG linkers mimic the rigid and flexible regions of folded proteins, respectively. Therefore, to estimate poly(DE4) n molecular weight, the average diffusion coefficient was fitted to a standard curve reported by Bodor 47 for folded proteins (Figure 2e, Table S2).
This method is valid because fitting the standard curve with the diffusion coefficient of monomer (DE-Tz)4 yielded 17.7 kDa, very close to the actual molecular weight of the monomer (Table S2). Consistent with the viscometry results, poly(DE4) n prepared at a higher bundlemer concentration had a higher structure with the reactive ends well exposed for continued monomer addition, producing PCCs with exceptionally high molecular weight. 33,[48][49][50] The DE peptide is designed to exhibit an α-helical coiled-coil conformation with D 2 symmetry in water, detectable by circular dichroism (CD) as two minima at 208 and 222 nm. 51 Conjugation of Tz and TCO groups did not affect the ability of the peptide to self-assemble (Figure 3a, Figures S17-18 0.01 0.1 0.001 0.01 0.1 1 10 q (Å -1 ) Intensity (cm -1 ) (DE-Tz)4 poly(DE4) n a 200 220 240 -20 -10 0 10 20 30 40 50 Wavelength (nm) Mean residue ellipticity (deg cm 2 dmol -1 1000 -1 ) 10 o C 20 o C 30 o C 40 o C 50 o C 60 o C 70 o C 80 o C 200 220 240 -20 -10 0 10 20 30 40 50 Wavelength (nm) Mean residue ellipticity (deg cm 2 dmol -1 1000 -1 ) 10 o C 20 o C 30 o C 40 o C 50 o C 60 o C 70 o C 80 o C 20 40 60 80 -25 -20 -15 -10 -5 0 Temperature ( o C) Mean residue ellipticity (deg cm 2 dmol -1 1000 -1 ) (DE-Tz)4 poly(DE4) n (DE-TCO)4 b c d e i ii
appearance of absorbance at 200 nm corresponding to a random coil, and the disappearance of the absorbance at 208 and 222 nm. Importantly, the secondary structure was preserved in the polymer product (Figure 3b). Comparison of the molar residue ellipticity (MRE) at 222 nm (Figure 3c) showed that poly(DE4) n denatures (i.e. denaturation of the helices) at a significantly higher temperature (70-80 C)
than the bundlemers (50-60 C), suggesting stabilization of the helical conformation and strengthening of helix-to-helix association by covalent polymerization. 26,52,53 Examination of the polymerization product, cast on the grid directly from H 2 O, by transmission electron microscopy (TEM) with negative staining showed aggregates composed of semiflexible PCC chains (Figure 3d(i)). Examination under Cryo-TEM conditions also revealed substantional interchain association (Figure S21). Dilution and equilibration of poly(DE4) n in a NaCl solution prior to imaging signficantly reduced polymer aggregation by charge screening, revealing well-dispered single chains that appeared to be semiflexible (Figure 3d(ii)). This appearance is consistent with previously published semiflexible PCC chains prepared using maleimide functionalized bundlemer and tetrathiol pentaerythritol tetrakis(3-mercaptopropionate). 26 However, prior PCCs prepared using short length interbundle coupling chemistry resulted in the formation of stiff molecular fibers with rigid-rod behavior.
Therefore, the physical characteristics of the hybrid polymer is dependent on inter-bundle linker chemistry.
Small angle X-ray scattering (SAXS) was used to determine Kuhn lengths and cross-section radii of poly(DE4) n (Figure 3e and Table S3). The (DE-Tz)4 building block fits reasonably well (reduced chisquared, χ 2 = 1.43) to a cylinder model with a length of 6.7 nm and a radius of 1.2 nm. This radius is consistent with the values for previously published bundlemers, while the length is consistant with a typical bundlemer (l = 3.6 -4.5 nm) 26,36,37,[54][55][56] plus extended OEG linkers at each N-termini. Treating poly(DE4) n as a series of rigid bundlemer segments connected by flexible OEG linkers, we fitted it to a flexible cylinder model in SASview as has been previously done for semiflexible bundlemer rods. 26,56 Additionally, given the relatively long and flexible nature of the OEG linker, with a total of 7 ethylene oxide repeats between adjacent bundlers, it is unlikely for the polymer to have a singular fixed Kuhn length, but instead a range of Kuhn lengths dependent on whether OEG is extended or collapsed. By fitting the Kuhn length with a Gaussian distribution (Figure S22), our analyses revealed that poly(DE4) n (reduced χ 2 = 1.12) had a radius of 1.3 nm and an average Kuhn length of 6.3 nm with a standard deviation of 1.3 nm.
This Kuhn length is slightly smaller than the previously reported 7.1 ± 1 nm for semiflexible bundlemer chains 56 linked with a 4-arm PEG linker but is still indicative of a single bundlemer persistence length.
Separately, a coarse-grain (CG) model was employed to microscopically investigate the conformation of doubly linked bundlemer chains (Figure 4a-b). Based on the rigid rod chain extrapolated from our previous study, 26 attractive interactions among the terminal residues of bundlemers and the intervening chemical linkers are critical in determining the stiffness of the chain. Therefore, both terminus interaction energy, 𝜖 𝑝𝑝 , and the length of the linkers, 𝑁 𝑏𝑒𝑎𝑑𝑠 , were varied to identify the most suitable parameters 0 2 4 6 8 10 0 50 100 150 200 ϵpp Kuhn length b N beads =1 N beads =2 N beads =3 N beads =4 10 0.5 10 1 10 1.5 10 2 10 0.5 10 1 10 1.5 10 2 Chain length N Radius of gyration 0.63 a b d c N=50 N=25 N=100 f e 10 0 10 0.5 10 1 10 1.5 10 2 10 -3 10 -2 10 -1 10 0 Chain length N Diffusion coefficient -1.1 0 10 20 30 40 50 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Kuhn length b Probability (%) N beads =2 N beads =3 N beads =4
that can capture chain properties observed in the experiments. Herein, the Kuhn length, 𝑏, related to the flexibility of the chains, served as a metric for comparison between simulations and experimental results.
As shown in Figure 4c, a competing effect between 𝑁 𝑏𝑒𝑎𝑑𝑠 and 𝜖 𝑝𝑝 w as observed. For chains with 𝑁 𝑏𝑒𝑎𝑑𝑠 = 1, the Kuhn length, 𝑏, continuously increased as the attractive interactions among the adjacent bundlemer termini became larger, ultimately reaching a plateau with increasing 𝜖 𝑝𝑝 , indicating rigid rod formation. In contrast, a sharp transition regime was observed for the chains with a longer linker. As we explored the low 𝜖 𝑝𝑝 range, the Kuhn length for both 𝑁 𝑏𝑒𝑎𝑑𝑠 = 3 and 𝑁 𝑏𝑒𝑎𝑑𝑠 = 4 with 𝜖 𝑝𝑝 = 1.0 were similarly close to the experimentally measured results. Moreover, for 𝑁 𝑏𝑒𝑎𝑑𝑠 = 3 , the Kuhn length distribution plot (Figure 4d) shows the highest probability around 6.0 nm, in good agreement with SAXS analysis of experimentally produced chains in terms of local chain conformation.
We further interrogated the chain conformation of PCCs computationally by varying the number of bundlemer repeat unit N while keeping 𝑁 𝑏𝑒𝑎𝑑𝑠 = 3 and 𝜖 𝑝𝑝 = 1.0 . From the scaling of the radius of gyration (R g ) with N as R g ~N𝜈 , the Flory exponent was determined as 𝜈 = 0.63 , suggesting that the modeled polymer chain was in good solvent with relatively extended conformations (Figure 4e). The calculated ratio 𝑅 𝑒 2 𝑅 𝑔 2 ≈ 6.7, where end-to-end distance 𝑅 𝑒 ≈ 91 for the chain with N = 100 was above the theoretical value 6.3 for flexible chain in a good solvent. 57 Furthermore, the diffusion coefficient D, calculated from the mean square displacement (MSD) (Figure S23) of PCC chains decreased as N increased (Figure 4f), which is consistent with experimental observations (Figure 2e). The scaling of D with N followed 𝐷~𝑁 -1.1 , which indicated a diffusive behavior within the Rouse regimes. 39 Overall, the simulations provide a molecular picture that PCCs consisting of covalently linked coiled-coil bundles behave like semiflexible polymers, even though the constituent bundlemer building blocks are inherently rigid, due to the presence of flexible OEG linkers between neighboring bundles.
When polymerization was conducted at a Tz concentration of 5 mM, a transparent hydrogel with a yellow tint was obtained (Figure 5a-b, Video S1). The distinct pink-red color associated with the tetrazine chromophore disappeared rather rapidly (within minutes) after (DE-TCO)4 and (DE-Tz)4 were mixed.
Characterization by oscillatory rheometry indicated a liquid-to-solid transition at 5 min (Figure S24a), with PCC 5.0 mM PCC 7.5 mM PCC 10.0 mM 0.1 1 10 100 1000 Modulus (Pa) G' G" 0.01 0.1 1 10 0.01 0.1 1 10 100 1000 Frequency (Hz) Modulus (Pa) 20 40 60 80 0.01 0.1 1 10 100 1000 Temperature ( o C) Modulus (Pa) 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 Oscillation strain (%) Modulus (Pa) Fluid state Solid state a c d e f 30 min 12 h, R.T. (DE-TCO)4 Poly(DE4) n hydrogel b (DE-Tz)4 R.T. Bundlemer Solutions 0.01 0.1 1 10 0.01 0.1 1 10 100 1000 Frequency (Hz) Modulus (Pa) G' PCC 10.0 mM G" PCC 10.0 mM G' PCC 7.5 mM G" PCC 7.5 mM G' PCC 5.0 mM G" PCC 5.0 mM
the storage modulus (G') significantly higher than the loss modulus (G"). After 20 min of polymerization, both G' and G" values reached a plateau. The gelation kinetics and hydrogel stiffness are strongly dependent on the Tz concentration in feed (Figure 5c, Figure S24). When polymerization was conducted at a Tz concentration of 10 mM, hydrogels reached a G' of 400 Pa. Although the supramolecular bundlemer building blocks are tetrafunctional, branching and crosslinking via the formation of a stable cycloaddition adduct is not possible owing to the high rigidity of the antiparallel homotetrameric coiledcoils. 26 Once one covalent bond between the bundlemers has formed, a second reaction between the same pair is highly likely due to the proximity of the functional groups. Although the Tz/TCO reaction sites are separated from the ends of the rigid bundlemers by OEG7, these linkages are not sufficiently long to enable branching. As discussed above, owing to the presence of flexible OEG linkers between each bundle, these hybrid polymers exhibited high chain flexibility. Thus, we reason that hydrogel formation is a direct result of physical entanglement of ultrahigh molecular weight PCCs. This hypothesis is supported by our experimental observations; addition of copious amount of water to the as-synthesized hydrogel led to complete dissolution overnight (Figure 5b).
We further analyzed the viscoelastic properties of these hydrogels over a wide range of frequencies (Figure 5d) and strains (Figure S25). The storage modulus remained constant at frequencies of 0.01-10
Hz and oscillation strain of 1%, indicating that the physical entanglement is long-lived. Unlike other entangled hydrogels that fail at very low strains, 23 this type of non-covalent hydrogel exhibits a solid-tofluid transition at 300% strain (Figure 5e) that is attributable to the disentanglement of polymer chains.
Solid-to-fluid transition was also observed with increasing temperature, and the transition occurred at a higher temperature for gels prepared at a higher Tz concentration in feed (Figure 5f). We hypothesized that as the temperature increased, poly(DE4) n became partially denatured via the disassembly of individual bundles, yielding low molecular weight, covalently coupled peptide homodimers (Figure 6a) that are unable to entangle. 58,59 When the peptide solution was subsequently cooled down to 10 C, we did not observe any gel formation (Figure 6b-d).
To further demonstrate the versatility of the bioorthogonal strategy, (DE-Tz)4 was copolymerized with (DK-TCO)4 (Figure 1a, Figures S7-12 However, cycling between 80 and 10 C led to permanent disintegration of the network structure (Figure S27). Cool down to 10 o C Heat up to T m of DE (60 o C) Cool down to 10 o C Heat up to T m of DE (60 o C) 0.01 0.1 1 10 0.01 0.1 1 10 100 1000 Frequency (Hz) Modulus (Pa) 0.01 0.1 1 10 100 0.01 0.1 1 10 100 1000 Oscillation strain (%) Modulus (Pa) 0.01 0.1 1 10 100 0.01 0.1 1 10 100 1000 Oscillation strain (%) Modulus (Pa) 0 100 200 300 0.01 0.1 1 10 100 1000 Time (min) Modulus (Pa) G' Heating G" Heating G' Cooling G" Cooling 0 100 200 300 0.01 0.1 1 10 100 1000 Time (min) Modulus (Pa) G' Heating G" Heating G' Cooling G" Cooling 0.01 0.1 1 10 0.01 0.1 1 10 100 1000 Frequency (Hz) Modulus (Pa) 0.01 0.1 1 10 0.01 0.1 1 10 100 1000 Frequency (Hz) Modulus (Pa) G' w/o heat/cool cycle G" w/o heat/cool cycle G' w/ one heat/cool cycle G" w/ one heat/cool cycle G' w/ ten heat/cool cycles G" w/ ten heat/cool cycles a e b c poly(DE4-DK4) n poly(DE4) n d f g ii i iii ii i iii hydrogels at ambient temperature (i), after heating to 60 C (ii) and then cooling down to 10 C (iii). b,
Storage (G') and loss moduli (G") were plotted as a function of time under temperature cycling. Time sweep experiments were conducted at a frequency of 0.1 Hz and a strain of 0.5%. The temperature was ramped up to 60 C at 5 C/min, held constant for 2 min, then ramped down at the same speed and held constant for 5 min. c, Storage (G') and loss moduli (G") were plotted as a function of frequency at a constant strain of 0.5% for hydrogels initially formed (purple), after the 1st (cyan) and the 10th (orange) heating/cooling cycle. d, Storage (G') and loss moduli (G") were plotted as a function of strain at a constant frequency of 0.1 Hz for hydrogels initially formed (purple), after the 1st (cyan) and the 10th (orange) heating/cooling cycles. e, Proposed molecular structure of poly(DE4) n hydrogels at ambient temperature (i), after heating to 60 C (ii) and then cooling down to 10 C (iii). f, Storage (G') and loss moduli (G") were plotted as a function of time under temperature cycling. Time sweep experiments were conducted at a frequency of 0.1 Hz and a strain of 0.5%. The temperature was ramped up to 60 C at 5 C/min, held constant for 2 min, then ramped down at the same speed and held constant for 5 min. g, Storage (G') and loss moduli (G") were plotted as a function of frequency at a constant strain of 0.5% for hydrogels initially formed (purple), after the 1st (cyan) and the 10th (orange) heating/cooling cycle. h, Storage (G') and loss moduli (G") were plotted as a function of strain at a constant frequency of 0.1 Hz for hydrogels initially formed (purple), after the 1st (cyan) and the 10th (orange) heating/cooling cycles. We speculate that at 60 C, only the DE4 units were denatured; during the cooling cycle, the intact DK4 bundles serve as templates for the association of neighboring DE peptides, leading to the growth of high molecular weight products. However, when the neighboring units are not perfectly aligned, network defects are generated due to the inability of the DE peptides to re-assemble. Repeated heating and cooling results in the accumulation of network defects, compromising the overall hydrogel mechanical properties. This is not the case for the homodimeric hydrogel system. At 60 C, poly(DE4) n dissociates to peptide dimers covalently coupled through a flexible OEG7 linker (Figure 1e, Figure S26). To re-establish the molecular structure of the "homopolymer" at 10 C, four dimeric units must re-assemble in an antiparallel fashion to allow supramolecular polymerization. The flexible OEG7 linker between the two identical peptides permits intramolecular association through back folding, resulting in the formation of a "molecular capper" that distorts the overall reaction stoichiometry and prevents the restoration of the entangled network structure.
In summary, the rapid and highly efficient tetrazine-TCO ligation enabled the synthesis of polymeric coiled-coils with exceptional molecular weight. The long chain hybrid polymer produced at a bundlemer concentration of 4.5 mM consists of up to 200 covalently linked rigid 4-helical peptide bundles. Unlike the stiff, rod-like polymeric coiled-coils reported previously, the hybrid polymers developed here are semiflexible owing to the presence of defined PEG linkers between adjacent bundles. Polymerization at a monomer concentration greater than 4.5 mM resulted in hydrogel formation through the entanglement of the semiflexible polymer chains. The hybrid polymer recapitulated key features of supramolecular polymers, including dynamic and reversible properties, and at the same time, exhibited the robustness and stability of conventional covalently linked polymers. Physical gels established through intermolecular entanglements of the semiflexible polymeric chains behave like covalent networks. While conventional flexible polymers unentangle readily through reptation, semiflexible chains disentangle due to correlated constraint release, which leads to equilibration of internal bending modes before polymers diffuse the full tube length. 60 The bioorthogonality of tetrazine ligation allows the synthesis of new nanometer-scale structures by combining bundlemer building blocks with diverse functionalities and structures. Given the tunability of stiffness and thermodynamic reversibility, the hybrid hydrogel system investigated here may find applications in 3D printing, cell culture, and drug release.
Synthetic procedures, characterization methods, coarse grain simulation, supplemental figures.