Inhibition of Ice Recrystallization by Nanotube-Forming Cyclic Peptides.

While most native ice-binding proteins are rigid, artificial (macro)molecular ice-binders are usually flexible. Realizing a regular array with precisely positioned ice-binding motifs on synthetic proteins, (macro)molecular ice-binders are thus challenging. Here, we exploit the predictable assembly of cyclic peptides into nanotubes as a starting point to prepare large, rigid ice-binders bearing an ice-binding site that is found in hyperactive ice-binding proteins in insects. First, we designed, synthesized, and purified cyclic octapeptide Lys2CP8 bearing a TaT motif to promote ice binding and investigated their solution assembly and activity using circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, light scattering (LS), cryogenic transmission electron microscopy (cryo-TEM), and ice recrystallization inhibition (IRI) assays. The cyclic peptide Lys2CP8 was synthesized in good yield using Fmoc chemistry and purified by reversed-phase HPLC. Upon dissolution in aqueous solutions, Lys2CP8 was observed to assemble in a pH- and concentration-dependent manner into objects with nanoscopic dimensions. LS revealed the presence of small and large aggregates at pH 3 and 11, held together through a network of intermolecular antiparallel ß-sheets as determined by FTIR and CD spectroscopy. Cryo-TEM revealed the presence of one-dimensional objects at pH 3 and 11. These are mostly well-dispersed at pH 3 but appear to bundle at pH 11. Interestingly, the pH-dependent self-assembly behavior translates into a marked pH dependence of IRI activity. Lys2CP8 is IRI-active at pH 3 while inactive at pH 11 hypothetically because the ice-binding sites are inaccessible at pH 11 due to bundling.

How Subtle Changes Can Make a Difference: Reproducibility in Complex Supramolecular Systems.

The desire to construct complex molecular systems is driven by the need for technological (r)evolution and our intrinsic curiosity to comprehend the origin of life. Supramolecular chemists tackle this challenge by combining covalent and noncovalent reactions leading to multicomponent systems with emerging complexity. However, this synthetic strategy often coincides with difficult preparation protocols and a narrow window of suitable conditions. Here, we report on unsuspected observations of our group that highlight the impact of subtle "irregularities" on supramolecular systems. Based on the effects of pathway complexity, minute amounts of water in organic solvents or small impurities in the supramolecular building block, we discuss potential pitfalls in the study of complex systems. This article is intended to draw attention to often overlooked details and to initiate an open discussion on the importance of reporting experimental details to increase reproducibility in supramolecular chemistry.

Oligodimethylsiloxane-Oligoproline Block Co-Oligomers: the Interplay between Aggregation and Phase Segregation in Bulk and Solution.

Discrete block co-oligomers (BCOs) assemble into highly ordered nanostructures, which adopt a variety of morphologies depending on their environment. Here, we present a series of discrete oligodimethylsiloxane-oligoproline (oDMS-oPro) BCOs with varying oligomer lengths and proline end-groups, and study the nanostructures formed in both bulk and solution. The conjugation of oligoprolines to apolar siloxanes permits a study of the aggregation behavior of oligoproline moieties in a variety of solvents, including a highly apolar solvent like methylcyclohexane. The apolar solvent is more reminiscent of the polarity of the siloxane bulk, which gives insights into the supramolecular interactions that govern both bulk and solution assembly processes of the oligoproline. This extensive structural characterization allows the bridging of the gap between solution and bulk assembly. The interplay between the aggregation of the oligoproline block and the phase segregation induced by the siloxane drives the assembly. This gives rise to disordered, micellar microstructures in apolar solution and crystallization-driven lamellar nanostructures in the bulk. While most di- and triblock co-oligomers adopt predictable morphological features, one of them, oDMS15-oPro6-NH2, exhibits pathway complexity leading to gel formation. The pathway selection in the complex interplay between aggregation and phase segregation gives rise to interesting material properties.

Introducing Hyaluronic Acid into Supramolecular Polymers and Hydrogels.

The use of supramolecular polymers to construct functional biomaterials is gaining more attention due to the tunable dynamic behavior and fibrous structures of supramolecular polymers, which resemble those found in natural systems, such as the extracellular matrix. Nevertheless, to obtain a biomaterial capable of mimicking native systems, complex biomolecules should be incorporated, as they allow one to achieve essential biological processes. In this study, supramolecular polymers based on water-soluble benzene-1,3,5-tricarboxamides (BTAs) were assembled in the presence of hyaluronic acid (HA) both in solution and hydrogel states. The coassembly of BTAs bearing tetra(ethylene glycol) at the periphery (BTA-OEG4) and HA at different ratios showed strong interactions between the two components that led to the formation of short fibers and heterogeneous hydrogels. BTAs were further covalently linked to HA (HA-BTA), resulting in a polymer that was unable to assemble into fibers or form hydrogels due to the high hydrophilicity of HA. However, coassembly of HA-BTA with BTA-OEG4 resulted in the formation of long fibers, similar to those formed by BTA-OEG4 alone, and hydrogels were produced with tunable stiffness ranging from 250 to 700 Pa, which is 10-fold higher than that of hydrogels assembled with only BTA-OEG4. Further coassembly of BTA-OEG4 fibers with other polysaccharides showed that except for dextran, all polysaccharides studied interacted with BTA-OEG4 fibers. The possibility of incorporating polysaccharides into BTA-based materials paves the way for the creation of dynamic complex biomaterials.

Choline-Functionalized Supramolecular Copolymers: Toward Antimicrobial Activity against Streptococcus pneumoniae.

Dynamic binding events are key to arrive at functionality in nature, and these events are often governed by electrostatic or hydrophobic interactions. Synthetic supramolecular polymers are promising candidates to obtain biomaterials that mimic this dynamicity. Here, we created four new functional monomers based on the benzene-1,3,5-tricarboxamide (BTA) motif. Choline or atropine groups were introduced to obtain functional monomers capable of competing with the cell wall of Streptococcus pneumoniae for binding of essential choline-binding proteins (CBPs). Atropine-functionalized monomers BTA-Atr and BTA-Atr3 were too hydrophobic to form homogeneous assemblies, while choline-functionalized monomers BTA-Chol and BTA-Chol3 were unable to form fibers due to charge repulsion. However, copolymerization of BTA-Chol3 with non-functionalized BTA-(OH)3 yielded dynamic fibers, similar to BTA-(OH)3. These copolymers showed an increased affinity toward CBPs compared to free choline due to multivalent effects. BTA-based supramolecular copolymers are therefore a versatile platform to design bioactive and dynamic supramolecular polymers with novel biotechnological properties.

Facilitating functionalization of benzene-1,3,5-tricarboxamides by switching amide connectivity.

Synthetic water-compatible supramolecular polymers based on benzene-1,3,5-tricarboxamides (BTAs) have attracted a lot of interest in recent years, as they are uniquely suited to generate functional multicomponent biomaterials. Their morphologies and intrinsic dynamic behaviour mimic fibrous structures found in nature. Moreover, their modularity allows control of the density of functionalities presented on the surface of the fibres when using functionalized BTA monomers. However, such moieties generally comprise a functionality on only one of three side chains, resulting in lengthy synthetic protocols and limited yields. In this work, we avert the need for desymmetrization of the core by starting from commercially available 5-aminoisophthalic acid. This approach eliminates the statistical reactions and reduces the number of synthetic steps. It also leads to the inversion of the connectivity of one of the amides to the benzene core. By combining spectroscopy, light scattering and cryogenic transmission electron microscopy, we confirm that the inversed amide BTAs (iBTAs) form intermolecular hydrogen bonds and assemble into supramolecular polymers, like previously used symmetrical BTAs, albeit with a slight decrease in water solubility. Solubility problems were overcome by incorporating iBTAs into conventional BTA-based supramolecular polymers. These two-component mixtures formed supramolecular fibres with a morphology and dynamic behaviour similar to BTA-homopolymers. Finally, iBTAs were decorated with a fluorescent dye to demonstrate the synthesis of functional monomers, and to visualize their co-assembly with BTAs. Our results show that functionality can be introduced into supramolecular polymers with monomers that slightly differ in their core structure while maintaining the structure and dynamics of the fibres.

Supramolecular Double Helices from Small C3-Symmetrical Molecules Aggregated in Water.

Supramolecular fibers in water, micrometers long and several nanometers in width, are among the most studied nanostructures for biomedical applications. These supramolecular polymers are formed through a spontaneous self-assembly process of small amphiphilic molecules by specific secondary interactions. Although many compounds do not possess a stereocenter, recent studies suggest the (co)existence of helical structures, albeit in racemic form. Here, we disclose a series of supramolecular (co)polymers based on water-soluble benzene-1,3,5-tricarboxamides (BTAs) that form double helices, fibers that were long thought to be chains of single molecules stacked in one dimension (1D). Detailed cryogenic transmission electron microscopy (cryo-TEM) studies and subsequent three-dimensional-volume reconstructions unveiled helical repeats, ranging from 15 to 30 nm. Most remarkable, the pitch can be tuned through the composition of the copolymers, where two different monomers with the same core but different peripheries are mixed in various ratios. Like in lipid bilayers, the hydrophobic shielding in the aggregates of these disc-shaped molecules is proposed to be best obtained by dimer formation, promoting supramolecular double helices. It is anticipated that many of the supramolecular polymers in water will have a thermodynamic stable structure, such as a double helix, although small structural changes can yield single stacks as well. Hence, it is essential to perform detailed analyses prior to sketching a molecular picture of these 1D fibers.

Exploring the Potential of Benzene-1,3,5-tricarboxamide Supramolecular Polymers as Biomaterials.

The fast dynamics occurring in natural processes increases the difficulty of creating biomaterials capable of mimicking Nature. Within synthetic biomaterials, water-soluble supramolecular polymers show great potential in mimicking the dynamic behavior of these natural processes. In particular, benzene-1,3,5-tricaboxamide (BTA)-based supramolecular polymers have shown to be highly dynamic through the exchange of monomers within and between fibers, but their suitability as biomaterials has not been yet explored. Herein we systematically study the interactions of BTA supramolecular polymers bearing either tetraethylene glycol or mannose units at the periphery with different biological entities. When BTA fibers were incubated with bovine serum albumin (BSA), the protein conformation was only affected by the fibers containing tetraethylene glycol at the periphery (BTA-OEG4). Coarse-grained molecular simulations showed that BSA interacted with BTA-OEG4 fibers rather than with BTA-OEG4 monomers that are present in solution or that may exchange out of the fibers. Microscopy studies revealed that, in the presence of BSA, BTA-OEG4 retained their fiber conformation although their length was slightly shortened. When further incubated with fetal bovine serum (FBS), both long and short fibers were visualized in solution. Nevertheless, in the hydrogel state, the rheological properties were remarkably preserved. Further studies on the cellular compatibility of all the BTA assemblies and mixtures thereof were performed in four different cell lines. A low cytotoxic effect at most concentrations was observed, confirming the suitability of utilizing functional BTA supramolecular polymers as dynamic biomaterials.

Sequence of Polyurethane Ionomers Determinative for Core Structure of Surfactant-Copolymer Complexes.

The core of micelles self-assembled from amphiphiles is hydrophobic and contains little water, whereas complex coacervate core micelles co-assembled from oppositely charged hydrophilic polymers have a hydrophilic core with a high water content. Co-assembly of ionic surfactants with ionic-neutral copolymers yields surfactant-copolymer complexes known to be capable of solubilizing both hydrophilic and hydrophobic cargo within the mixed core composed of a coacervate phase with polyelectrolyte-decorated surfactant micelles. Here we formed such complexes from asymmetric (PUI-A2) and symmetric (PUI-S2), sequence-controlled polyurethane ionomers and poly(N-methyl-2-vinylpyridinium iodide)29-b-poly(ethylene oxide)204 copolymers. The complexes with PUI-S2 were 1.3-fold larger in mass and 1.8-fold larger in radius of gyration than the PUI-A2 complexes. Small-angle X-ray scattering revealed differences in the packing of the similarly sized PUI micelles within the core of the complexes. The PUI-A2 micelles were arranged in a more ordered fashion and were spaced further apart from each other (10 nm vs. 6 nm) than the PUI-S2 micelles. Hence, this work shows that the monomer sequence of amphiphiles can be varied to alter the internal structure of surfactant-copolymer complexes. Since the structure of the micellar core may affect both the cargo loading and release, our findings suggest that these properties may be tuned through control of the monomer sequence of the micellar constituents.

Structure and Dynamics of Supramolecular Polymers: Wait and See.

The introduction of stereogenic centers in supramolecular building blocks is used to unveil subtle changes in supramolecular structure and dynamics over time. Three stereogenic centers based on deuterium atoms were introduced in the side chains of a benzene-1,3,5-tricarboxamide (BTA) resulting in a supramolecular polymer in water that at first glance has a structure and dynamics identical to its achiral counterpart. Using three different techniques, the properties of the double helical polymers are compared after 1 day and 4 weeks. An increase in helical preference is observed over time as well as a decrease in the helical pitch and monomer exchange dynamics. It is proposed that the polymer of the chiral monomer needs time to arrive at its maximal preference in helical bias. These results indicate that the order and tight packing increase over time, while the dynamics of this supramolecular polymer decrease over time, an effect that is typically overlooked but unveiled by the isotopic chirality.

Dilution-induced gel-sol-gel-sol transitions by competitive supramolecular pathways in water.

Fascinating properties are displayed by synthetic multicomponent supramolecular systems that comprise a manifold of competitive interactions, thereby mimicking natural processes. We present the integration of two reentrant phase transitions based on an unexpected dilution-induced assembly process using supramolecular polymers and surfactants. The co-assembly of the water-soluble benzene-1,3,5-tricarboxamide (BTA-EG4) and a surfactant at a specific ratio yielded small-sized aggregates. These interactions were modeled using the competition between self-sorting and co-assembly of both components. The small-sized aggregates were transformed into supramolecular polymer networks by a twofold dilution in water without changing their ratio. Kinetic experiments show the in situ growth of micrometer-long fibers in the dilution process. We were able to create systems that undergo fully reversible hydrogel-solution-hydrogel-solution transitions upon dilution by introducing another orthogonal interaction.

Elucidating dynamic behavior of synthetic supramolecular polymers in water by hydrogen/deuterium exchange mass spectrometry.

A comprehensive understanding of the structure, self-assembly mechanism, and dynamics of one-dimensional supramolecular polymers in water is essential for their application as biomaterials. Although a plethora of techniques are available to study the first two properties, there is a paucity in possibilities to study dynamic exchange of monomers between supramolecular polymers in solution. We recently introduced hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize the dynamic nature of synthetic supramolecular polymers with only a minimal perturbation of the chemical structure. To further expand the application of this powerful technique some essential experimental aspects have been reaffirmed and the technique has been applied to a diverse library of assemblies. HDX-MS is widely applicable if there are exchangeable hydrogen atoms protected from direct contact with the solvent and if the monomer concentration is sufficiently high to ensure the presence of supramolecular polymers during dilution. In addition, we demonstrate that the kinetic behavior as probed by HDX-MS is influenced by the internal order within the supramolecular polymers and by the self-assembly mechanism.

Co-assembly of precision polyurethane ionomers reveals role of and interplay between individual components.

Industrial and household products, such as paints, inks and cosmetics usually consist of mixtures of macromolecules that are disperse in composition, in size and in monomer sequence. Identifying structure-function relationships for these systems is complicated, as particular macromolecular components cannot be investigated individually. For this study, we have addressed this issue, and have synthesized a series of five sequence-defined polyurethanes (PUs): one neutral-hydrophobic, one single-charged hydrophilic, one single-charged hydrophobic and two double-charged amphiphilic PUs (one symmetric and one asymmetric). These novel precision PUs - that were prepared by using stepwise coupling-deprotection synthetic protocols - have a defined composition, size and monomer sequence, where the chosen sequences were inspired by those that are abundantly formed in the production of industrial waterborne PU dispersions. By performing dynamic light scattering experiments (DLS), self-consistent field (SCF) computations and cryogenic transmission electron microscopy (cryo-TEM), we have elucidated the behavior in aqueous solution of the individual precision PUs, as well as of binary and ternary mixtures of the PU sequences. The double-charged PU sequences ('hosts') were sufficiently amphiphilic to yield single-component micellar solutions, whereas the two more hydrophobic sequences did not micellize on their own, and gave precipitates or ill-defined larger aggregates. Both the neutral-hydrophobic PU and the hydrophilic single-charged PU were successfully incorporated in the host micelles as guests, respectively increasing and reducing the micelle radius upon incorporation. SCF computations indicated that double-charged symmetric PUs stretch whilst double-charged asymmetric PUs are expelled from the core to accommodate hydrophobic PU guests within the micelles. For the ternary mixture of the double-charged symmetric and asymmetric hosts and the neutral-hydrophobic guest we have found an improved colloidal stability, as compared to those for binary mixtures of either host and hydrophobic guest. In another ternary mixture of precision PUs, with all three components not capable of forming micelles on their own, we see that the ensemble of molecules produces stable micellar solutions. Taken together, we find that the interplay between PU-molecules in aqueous dispersions promotes the formation of stable micellar hydrocolloids.

Fractal-like R5 assembly promote the condensation of silicic acid into silica particles.

HYPOTHESIS: Despite advances in understanding the R5 (SSKKSGSYSGKSGSKRRIL) peptide-driven bio-silica process, there remains significant discrepancies regarding the physicochemical characterization and the self-assembling mechanistic driving forces of the supramolecular R5 template. This paper investigates the self-assembly of R5 as a function of monovalent (sodium chloride) and multivalent salt (phosphate) to determine if assembly is phosphate ion concentration dependent. Additionally, we hypothesize that the assembled R5 aggregates do not resemble a micelle or unimer structure as proposed in current literature. EXPERIMENTS: R5 peptides were synthesized, and aggregates evaluated for their size, morphology, and association state as a function of salt and ionic strength concentration via dynamic and static light scattering, small angle X-ray and neutron scattering and cryogenic transmission electron microscopy. Furthermore, we compare the proposed R5 template to precipitated silica by scanning electron microscopy. FINDINGS: R5 peptides assemble into large aggregates due to multivalence bridging and the decrease in electrostatic repulsion due to ionic strength. We elucidate the structure of R5 aggregates as mass-fractals composed of small spherical aggregates. Moreover, we discover that phosphate ions not only have a significant role in driving the growth of the R5 scaffold, but additionally in driving the polycondensation of silicic acid during the bio-silification process via electrostatic interactions.

Autonomous mesoscale positioning emerging from myelin filament self-organization and Marangoni flows.

Out-of-equilibrium molecular systems hold great promise as dynamic, reconfigurable matter that executes complex tasks autonomously. However, translating molecular scale dynamics into spatiotemporally controlled phenomena emerging at mesoscopic scale remains a challenge-especially if one aims at a design where the system itself maintains gradients that are required to establish spatial differentiation. Here, we demonstrate how surface tension gradients, facilitated by a linear amphiphile molecule, generate Marangoni flows that coordinate the positioning of amphiphile source and drain droplets floating at air-water interfaces. Importantly, at the same time, this amphiphile leads, via buckling instabilities in lamellar systems of said amphiphile, to the assembly of millimeter long filaments that grow from the source droplets and get absorbed at the drain droplets. Thereby, the Marangoni flows and filament organization together sustain the autonomous positioning of interconnected droplet-filament networks at the mesoscale. Our concepts provide potential for the development of non-equilibrium matter with spatiotemporal programmability.

Insights into the Kinetics of Supramolecular Comonomer Incorporation in Water.

Multicomponent supramolecular polymers are a versatile platform to prepare functional architectures, but a few studies have been devoted to investigate their noncovalent synthesis. Here, we study supramolecular copolymerizations by examining the mechanism and time scales associated with the incorporation of new monomers in benzene-1,3,5-tricarboxamide (BTA)-based supramolecular polymers. The BTA molecules in this study all contain three tetra(ethylene glycol) chains at the periphery for water solubility but differ in their alkyl chains that feature either 10, 12 or 13 methylene units. C10BTA does not form ordered supramolecular assemblies, whereas C12BTA and C13BTA both form high aspect ratio supramolecular polymers. First, we illustrate that C10BTA can mix into the supramolecular polymers based on either C12BTA or C13BTA by comparing the temperature response of the equilibrated mixtures to the temperature response of the individual components in water. Subsequently, we mix C10BTA with the polymers and follow the copolymerization over time with UV spectroscopy and hydrogen/deuterium exchange mass spectrometry experiments. Interestingly, the time scales obtained in both experiments reveal significant differences in the rates of copolymerization. Coarse-grained simulations are used to study the incorporation pathway and kinetics of the C10BTA monomers into the different polymers. The results demonstrate that the kinetic stability of the host supramolecular polymer controls the rate at which new monomers can enter the existing supramolecular polymers.

Impact of the water-compatible periphery on the dynamic and structural properties of benzene-1,3,5-tricarboxamide based amphiphiles.

The consequences of using saccharides versus tetra(ethyleneglycol) chains as water-compatible moieties on the morphology and dynamics of supramolecular polymers in aqueous solutions are investigated. The saccharides form many H-bonds with other saccharides within the polymer and with water, increasing the hydration of the fiber and changing its dynamics.

Dynamic diversity of synthetic supramolecular polymers in water as revealed by hydrogen/deuterium exchange.

Numerous self-assembling molecules have been synthesized aiming at mimicking both the structural and dynamic properties found in living systems. Here we show the application of hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to unravel the nanoscale organization and the structural dynamics of synthetic supramolecular polymers in water. We select benzene-1,3,5-tricarboxamide (BTA) derivatives that self-assemble in H2O to illustrate the strength of this technique for supramolecular polymers. The BTA structure has six exchangeable hydrogen atoms and we follow their exchange as a function of time after diluting the H2O solution with a 100-fold excess of D2O. The kinetic H/D exchange profiles reveal that these supramolecular polymers in water are dynamically diverse; a notion that has previously not been observed using other techniques. In addition, we report that small changes in the molecular structure can be used to control the dynamics of synthetic supramolecular polymers in water.