Emergence of Dynamic Instability by Hybridizing Synthetic Self-Assembled Dipeptide Fibers with Surfactant Micelles.

Supramolecular chemistry currently faces the challenge of controlling nonequilibrium dynamics such as the dynamic instability of microtubules. In this study, we explored the emergence of dynamic instability through the hybridization of peptide-type supramolecular nanofibers with surfactant micelles. Using real-time confocal imaging, we discovered that the addition of micelles to nanofibers induced the simultaneous but asynchronous growth and shrinkage of nanofibers during which the total number of fibers decreased monotonically. This dynamic phenomenon unexpectedly persisted for 6 days and was driven not by chemical reactions but by noncovalent supramolecular interactions between peptide-type nanofibers and surfactant micelles. This study demonstrates a strategy for inducing autonomous supramolecular dynamics, which will open up possibilities for developing soft materials applicable to biomedicine and soft robotics.

Cell-Like Synthetic Supramolecular Soft Materials Realized in Multicomponent, Non-/Out-of-Equilibrium Dynamic Systems.

Living cells are complex, nonequilibrium supramolecular systems capable of independently and/or cooperatively integrating multiple bio-supramolecules to execute intricate physiological functions that cannot be accomplished by individual biomolecules. These biological design strategies offer valuable insights for the development of synthetic supramolecular systems with spatially controlled hierarchical structures, which, importantly, exhibit cell-like responses and functions. The next grand challenge in supramolecular chemistry is to control the organization of multiple types of supramolecules in a single system, thus integrating the functions of these supramolecules in an orthogonal and/or cooperative manner. In this perspective, the recent progress in constructing multicomponent supramolecular soft materials through the hybridization of supramolecules, such as self-assembled nanofibers/gels and coacervates, with other functional molecules, including polymer gels and enzymes is highlighted. Moreover, results show that these materials exhibit bioinspired responses to stimuli, such as bidirectional rheological responses of supramolecular double-network hydrogels, temporal stimulus pattern-dependent responses of synthetic coacervates, and 3D hydrogel patterning in response to reaction-diffusion processes are presented. Autonomous active soft materials with cell-like responses and spatially controlled structures hold promise for diverse applications, including soft robotics with directional motion, point-of-care disease diagnosis, and tissue regeneration.

Visualizing Formation and Dynamics of a Three-Dimensional Sponge-like Network of a Coacervate in Real Time.

Coacervates, which are formed by liquid-liquid phase separation, have been extensively explored as models for synthetic cells and membraneless organelles, so their in-depth structural analysis is crucial. However, both the inner structure dynamics and formation mechanism of coacervates remain elusive. Herein, we demonstrate real-time confocal observation of a three-dimensional sponge-like network in a dipeptide-based coacervate. In situ generation of the dipeptide allowed us to capture the emergence of the sponge-like network via unprecedented membrane folding of vesicle-shaped intermediates. We also visualized dynamic fluctuation of the network, including reversible engagement/disengagement of cross-links and a stochastic network kissing event. Photoinduced transient formation of a multiphase coacervate was achieved with a thermally responsive phase transition. Our findings expand the fundamental understanding of synthetic coacervates and provide opportunities to manipulate their physicochemical properties by engineering the inner network for potential applications in development of artificial cells and life-like material fabrication.

Four distinct network patterns of supramolecular/polymer composite hydrogels controlled by formation kinetics and interfiber interactions.

Synthetic composite hydrogels comprising supramolecular fibers and covalent polymers have attracted considerable attention because their properties are similar to biological connective tissues. However, an in-depth analysis of the network structures has not been performed. In this study, we discovered the composite network can be categorized into four distinct patterns regarding morphology and colocalization of the components using in situ, real-time confocal imaging. Time-lapse imaging of the network formation process reveals that the patterns are governed by two factors, the order of the network formation and the interactions between the two different fibers. Additionally, the imaging studies revealed a unique composite hydrogel undergoing dynamic network remodeling on the scale of a hundred micrometers to more than one millimeter. Such dynamic properties allow for fracture-induced artificial patterning of a network three dimensionally. This study introduces a valuable guideline to the design of hierarchical composite soft materials.

Temporal Stimulus Patterns Drive Differentiation of a Synthetic Dipeptide-Based Coacervate.

The fate of living cells often depends on their processing of temporally modulated information, such as the frequency and duration of various signals. Synthetic stimulus-responsive systems have been intensely studied for >50 years, but it is still challenging for chemists to create artificial systems that can decode dynamically oscillating stimuli and alter the systems' properties/functions because of the lack of sophisticated reaction networks that are comparable with biological signal transduction. Here, we report morphological differentiation of synthetic dipeptide-based coacervates in response to temporally distinct patterns of the light pulse. We designed a simple cationic diphenylalanine peptide derivative to enable the formation of coacervates. The coacervates concentrated an anionic methacrylate monomer and a photoinitiator, which provided a unique reaction environment and facilitated light-triggered radical polymerization─even in air. Pulsed light irradiation at 9.0 Hz (but not at 0.5 Hz) afforded anionic polymers. This dependence on the light pulse patterns is attributable to the competition of reactive radical intermediates between the methacrylate monomer and molecular oxygen. The temporal pulse pattern-dependent polymer formation enabled the coacervates to differentiate in terms of morphology and internal viscosity, with an ultrasensitive switch-like mode. Our achievements will facilitate the rational design of smart supramolecular soft materials and are insightful regarding the synthesis of sophisticated chemical cells.

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue.

Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

Shape-selective one-step synthesis of branched gold nanoparticles on the crystal surface of redox-active PdII-macrocycles.

The synthesis of branched gold nanoparticles (AuNPs) with shape- and size-specific optical properties requires effective control of the particle formation mechanism using appropriate reducing agents and protective agents that prevent particle aggregation in solution. In this context, the heterogeneous synthesis of AuNPs using solid surfaces of graphene oxides and metal-organic frameworks has attracted much attention. These materials are characterized by their ability to immobilize and stabilize the particles grown on the surface without the need for additional protective agents. However, the shape- and size-selective synthesis of AuNPs using solid surfaces remains challenging. Herein, we report the shape-selective one-step synthesis of monodisperse branched AuNPs using a metal-macrocycle framework (MMF), a porous molecular crystal of PdII3-tris(phenylenediamine) macrocycle. Konpeito-Shaped branched AuNPs with uniform size were obtained on the surface of MMF by mixing HAuCl4·4H2O, L-ascorbic acid and MMF microcrystals. Spectroscopic and microscopic observations confirmed that MMF promoted the reduction of gold by its reductive activity as well as acted as a solid support to electrostatically immobilize the pseudo-seed particles for further growth on the crystal surface. In addition, the MMF also served as a substrate for in situ high-speed AFM imaging due to the effective immobilization of AuNPs on the surface, allowing direct visualization of the particle growth. Since the chemical structural features of MMF allow the growth of branched AuNPs via pseudo-seeding, this approach would provide new synthetic methods for obtaining a variety of gold nanostructures.

Phototriggered Spatially Controlled Out-of-Equilibrium Patterns of Peptide Nanofibers in a Self-Sorting Double Network Hydrogel.

Out-of-equilibrium patterns arising from diffusion processes are ubiquitous in nature, although they have not been fully exploited for the design of artificial materials. Here, we describe the formation of phototriggered out-of-equilibrium patterns using photoresponsive peptide-based nanofibers in a self-sorting double network hydrogel. Light irradiation using a photomask followed by thermal incubation induced the spatially controlled condensation of peptide nanofibers. According to confocal images and spectroscopic analyses, metastable nanofibers photodecomposed in the irradiated areas, where thermodynamically stable nanofibers reconstituted and condensed with a supply of monomers from the nonirradiated areas. These supramolecular events were regulated by light and diffusion to facilitate the creation of unique out-of-equilibrium patterns, including two lines from a one-line photomask and a line pattern of a protein immobilized in the hydrogel.

Microscopic Imaging Techniques for Molecular Assemblies: Electron, Atomic Force, and Confocal Microscopies.

Self-assembly is promising for construction of a wide variety of supramolecular assemblies, whose 1D/2D/3D structures are typically relevant to their functions. In-depth understanding of their structure-function relationships is essential for rational design and development of functional molecular assemblies. Microscopic imaging has been used as a powerful tool to elucidate structures of individual molecular assemblies with subnanometer to millimeter resolution, which is complementary to conventional spectroscopic techniques that provide the ensemble structural information. In this review, we highlight the representative examples of visualization of molecular assemblies by use of electron microscopy, atomic force microscopy, confocal microscopy, and super-resolution microscopy. This review comprehensively describes imaging of supramolecular nanofibers/gels, micelles/vesicles, coacervate droplets, polymer assemblies, and protein/DNA assemblies. Advanced imaging techniques that can address key challenges, like evaluation of dynamics of molecular assemblies, multicomponent self-assembly, and self-assembly/disassembly in complex cellular milieu, are also discussed. We believe this review would provide guidelines for deeper structural analyses of molecular assemblies to develop the next-generation materials.

Face-selective adsorption of a prochiral compound on the chiral pore-surface of a metal-macrocycle framework (MMF) directed towards stereoselective reactions.

Molecular adsorption on a surface is a unique way to break the mirror-symmetry of prochiral molecules, and therefore the use of chiral surfaces is an effective strategy for achieving highly selective chiral separation and asymmetric catalytic reactions based on molecular adsorption with high diastereoselectivity. We have previously reported a porous metal-macrocycle framework (MMF) with an enantiomeric pair of chiral pore-surfaces derived from Pd-helical macrocycles as the ingredients of the framework. Aiming at applying the chiral pore-surface of the MMF to asymmetric reactions and chiral separation, herein we propose a strategy to utilize one of the enantiomerically paired pore-surfaces as a homochiral pore-surface with the aid of chiral auxiliaries that can block only one side of the enantiomeric pore-surfaces in a site-selective manner. Single-crystal X-ray diffraction analysis revealed that a chiral auxiliary, (1R)- or (1S)-1-(3-chlorophenyl)ethanol, and a prochiral guest molecule, 2'-hydroxyacetophenone, were cooperatively arranged in each pore unit so that the prochiral guest molecule can face-selectively bind to the homochiral pore-surface.

Control of seed formation allows two distinct self-sorting patterns of supramolecular nanofibers.

Self-sorting double network hydrogels comprising orthogonal supramolecular nanofibers have attracted attention as artificially-regulated multi-component systems. Regulation of network patterns of self-sorted nanofibers is considered as a key for potential applications such as optoelectronics, but still challenging owing to a lack of useful methods to prepare and analyze the network patterns. Herein, we describe the selective construction of two distinct self-sorting network patterns, interpenetrated and parallel, by controlling the kinetics of seed formation with dynamic covalent oxime chemistry. Confocal imaging reveals the interpenetrated self-sorting network was formed upon addition of O-benzylhydroxylamine to a benzaldehyde-tethered peptide-type hydrogelator in the presence of lipid-type nanofibers. We also succeed in construction of a parallel self-sorting network through deceleration of seed formation using a slow oxime exchange reaction. Through careful observation, the formation of peptide-type seeds and nanofibers is shown to predominantly occur on the surface of the lipid-type nanofibers via highly dynamic and thermally-fluctuated processes.

Protein-responsive protein release of supramolecular/polymer hydrogel composite integrating enzyme activation systems.

Non-enzymatic proteins including antibodies function as biomarkers and are used as biopharmaceuticals in several diseases. Protein-responsive soft materials capable of the controlled release of drugs and proteins have potential for use in next-generation diagnosis and therapies. Here, we describe a supramolecular/agarose hydrogel composite that can release a protein in response to a non-enzymatic protein. A non-enzymatic protein-responsive system is developed by hybridization of an enzyme-sensitive supramolecular hydrogel with a protein-triggered enzyme activation set. In situ imaging shows that the supramolecular/agarose hydrogel composite consists of orthogonal domains of supramolecular fibers and agarose, which play distinct roles in protein entrapment and mechanical stiffness, respectively. Integrating the enzyme activation set with the composite allows for controlled release of the embedded RNase in response to an antibody. Such composite hydrogels would be promising as a matrix embedded in a body, which can autonomously release biopharmaceuticals by sensing biomarker proteins.

Force generation by a propagating wave of supramolecular nanofibers.

Dynamic spatiotemporal patterns that arise from out-of-equilibrium biochemical reactions generate forces in living cells. Despite considerable recent efforts, rational design of spatiotemporal patterns in artificial molecular systems remains at an early stage of development. Here, we describe force generation by a propagating wave of supramolecular nanofibers. Inspired by actin dynamics, a reaction network is designed to control the formation and degradation of nanofibers by two chemically orthogonal stimuli. Real-time fluorescent imaging successfully visualizes the propagating wave based on spatiotemporally coupled generation and collapse of nanofibers. Numerical simulation indicates that the concentration gradient of degradation stimulus and the smaller diffusion coefficient of the nanofiber are critical for wave emergence. Moreover, the force (0.005 pN) generated by chemophoresis and/or depletion force of this propagating wave can move nanobeads along the wave direction.

The Power of Confocal Laser Scanning Microscopy in Supramolecular Chemistry: In situ Real-time Imaging of Stimuli-Responsive Multicomponent Supramolecular Hydrogels.

Multicomponent supramolecular hydrogels are promising scaffolds for applications in biosensors and controlled drug release due to their designer stimulus responsiveness. To achieve rational construction of multicomponent supramolecular hydrogel systems, their in-depth structural analysis is essential but still challenging. Confocal laser scanning microscopy (CLSM) has emerged as a powerful tool for structural analysis of multicomponent supramolecular hydrogels. CLSM imaging enables real-time observation of the hydrogels without the need of drying and/or freezing to elucidate their static and dynamic properties. Through multiple, selective fluorescent staining of materials of interest, multiple domains formed in supramolecular hydrogels (e. g. inorganic materials and self-sorting nanofibers) can also be visualized. CLSM and the related microscopic techniques will be indispensable to investigate complex life-inspired supramolecular chemical systems.

pH Nanosensor Using Electronic Spins in Diamond.

Nanoscale measurements provide insight into the nano world. For instance, nanometric spatiotemporal distribution of intracellular pH is regulated by and regulates a variety of biological processes. However, there is no general method to fabricate nanoscale pH sensors. Here, we, to endow pH-sensing functions, tailor the surface properties of a fluorescent nanodiamond (FND) containing nitrogen-vacancy centers (NV centers) by coating the FND with an ionic chemical layer. The longitudinal relaxation time T1 of the electron spins in the NV centers inside a nanodiamond modified by carboxyl groups on the particle surface was found to depend on ambient pH between pH 3 and pH 7, but not between pH 7 and pH 11. Therefore, a single particle of the carboxylated nanodiamond works as a nanometer-sized pH meter within a microscopic image and directly measures the nanometric local pH environment. Moreover, the pH dependence of an FND was changed by coating it with a polycysteine layer, which contains a multitude of thiol groups with higher pKa. The polycysteine-coated nanodiamond obtained a pH dependence between pH 7 and pH 11. The pH dependence of the FND was also observed in heavy water (D2O) buffers. This indicates that the pH dependence is not caused by magnetic noise induced by 1H nuclear spin fluctuations, but by electric noise induced by ion exchanges. Via our method, the sensitive pH range of the nanodiamond pH sensor can potentially be controlled by changing the ionic layer appropriately according to the target biological phenomena.

On-cell coordination chemistry: Chemogenetic activation of membrane-bound glutamate receptors in living cells.

Investigating functions of membrane-bound receptors provides invaluable information about cellular signaling and physiological events. Recently, chemical genetic methods to design chemical switches on the target proteins have intensely been developed for interrogation of the cellular signaling of individual receptor proteins. We recently reported coordination chemistry-based chemogenetics to allosterically activate two types of neurotransmitter receptors, ionotropic and metabotropic glutamate receptors, in living cells. Based on their well-studied structure-activity relationships, we semi-rationally incorporated two His mutations into glutamate receptors near ligand binding pockets as an allosteric site. The engineered glutamate receptors could be allosterically activated upon treatment of Pd(bpy) complex (bpy: 2,2'-bipyridine) through stabilization of the activated conformation in mammalian cells and cultured neurons. Here, we describe the detailed protocol of our approach including the receptor design and activation of the His-engineered receptors and the downstream of the signal transduction cascade in living cells.

Post-assembly Fabrication of a Functional Multicomponent Supramolecular Hydrogel Based on a Self-Sorting Double Network.

Living cells exhibit sophisticated functions because they contain numerous endogenous stimuli-responsive molecular systems that independently and cooperatively act in response to an external circumstance. On the other hand, artificial soft materials containing multiple stimuli-responsive molecular systems are still rare. Herein, we demonstrate a unique multicomponent hydrogel composed of a self-sorting double network prepared through a post-assembly fabrication (PAF) protocol. The PAF protocol allowed the construction of a well-ordered hydrogel with a dual-biomolecule response to two important biomolecules (adenosine triphosphate (ATP) and sarcosine). Such a hydrogel could not be prepared through a one-step mixing protocol. The resultant multicomponent hydrogel responded to ATP and sarcosine through gel-sol transition behavior programmed in an AND logic gate fashion. Finally, we applied the multicomponent hydrogel to the controlled release of an antibody.

Rational synthesis of benzimidazole[3]arenes by CuII-catalyzed post-macrocyclization transformation.

A new series of calix[n]arene analogues, benzimidazole[3]arenes, was rationally synthesized by CuII-catalyzed post-macrocyclization transformation of a tris(o-phenylenediamine) macrocycle, and fully characterized by NMR, MS, and single-crystal X-ray diffraction (XRD) analyses. The resulting syn- and anti-benzimidazole[3]arenes have a bowl-shaped and a warped structure, respectively, in their crystalline states, and both display a dynamic inversion behavior in solution. This modification resulted in strong fluorescence due to the generated benzimidazole moieties. The mechanistic study of the post-macrocyclization transformation demonstrated that the formation of both benzimidazole[3]arenes was catalyzed, via triimine intermediates, by CuII ions in air through oxidation and cyclization of the tris(o-phenylenediamine) macrocycle.

Chemogenetic Approach Using Ni(II) Complex-Agonist Conjugates Allows Selective Activation of Class A G-Protein-Coupled Receptors.

Investigating individual G-protein-coupled receptors (GPCRs) involved in various signaling cascades can unlock a myriad of invaluable physiological findings. One of the promising strategies for addressing the activity of each subtype of receptor is to design chemical turn-on switches on the target receptors. However, valid methods to selectively control class A GPCRs, the largest receptor family encoded in the human genome, remain limited. Here, we describe a novel approach to chemogenetically manipulate activity of engineered class A GPCRs carrying a His4 tag, using metal complex-agonist conjugates (MACs). This manipulation is termed coordination tethering. With the assistance of coordination bonds, MACs showed 10-100-fold lower EC50 values in the engineered receptors, compared with wild-type receptors. Such coordination tethering enabled selective activation of ß2-adrenoceptors and muscarinic acetylcholine receptors, without loss of natural receptor responses, in living mammalian cells, including primary cultured astrocytes. Our generalized, modular chemogenetic approach should facilitate more precise control and deeper understanding of individual GPCR signaling pathways in living systems.

Imaging-Based Study on Control Factors over Self-Sorting of Supramolecular Nanofibers Formed from Peptide- and Lipid-type Hydrogelators.

Multicomponent self-assembly is a fascinating strategy for the construction of smart soft materials. Among them, supramolecular hydrogels comprising self-sorting nanofibers have recently attracted significant attention owing to their rationally incorporated stimulus responsiveness. However, there have been limited investigations of the crucial factors that control the self-sorting phenomena. Here, we describe an imaging-based approach to evaluate the factors that control the formation of self-sorting nanofibers from peptide- and lipid-type hydrogelators. We screened a small library of hydrogelators with distinct chemical properties by direct visualization of their self-assembly behavior by using confocal laser scanning microscopy. Our systematic research identified two important factors that influence the self-sorting behavior of nanofibers: (i) the surface charge of the hydrogelators; and (ii) the hydrophobicity of the side chain on the peptide-type hydrogelators. We determined that the same net/surface charge on the hydrogelators and side chains with a lower hydrophobicity on the peptide-type hydrogelators were preferred. These findings, in combination with the previously reported kinetic factors, were used to design and successfully prepare a three-component orthogonal self-assembly composed of supramolecular nanofibers from peptide- and lipid-type hydrogelators and a cationic organorhodium complex. Our findings would be beneficial for the design of intelligent soft materials based on self-sorting phenomena.