Transient control of lytic activity via a non-equilibrium chemical reaction system.

The development of artificial non-equilibrium chemical reaction systems has recently attracted considerable attention as a new type of biomimetic. However, due to the lack of bioorthogonality, such reaction systems could not be linked to the regulation of any biological phenomena. Here, we have newly designed a non-equilibrium reaction system based on olefin metathesis to produce the Triton X-mimetic non-ionic amphiphile as a kinetic product. Using phospholipid vesicles encapsulating fluorescent dyes and red blood cells as cell models, we demonstrate that the developed chemical reaction system is applicable for transient control of the resulting lytic activity.

Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection.

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.

Supramolecular Mechanosensitive Potassium Channel Formed by Fluorinated Amphiphilic Cyclophane.

Inspired by mechanosensitive potassium channels found in nature, we developed a fluorinated amphiphilic cyclophane composed of fluorinated rigid aromatic units connected via flexible hydrophilic octa(ethylene glycol) chains. Microscopic and emission spectroscopic studies revealed that the cyclophane could be incorporated into the hydrophobic layer of the lipid bilayer membranes and self-assembled to form a supramolecular transmembrane ion channel. Current recording measurements using cyclophane-containing planer lipid bilayer membranes successfully demonstrated an efficient transmembrane ion transport. We also demonstrated that the ion transport property was sensitive to the mechanical forces applied to the membranes. In addition, ion transport assays using pH-sensitive fluorescence dye revealed that the supramolecular channel possesses potassium ion selectivity. We also performed all-atom hybrid quantum-mechanical/molecular mechanical simulations to assess the channel structures at atomic resolution and the mechanism of selective potassium ion transport. This research demonstrated the first example of a synthetic mechanosensitive potassium channel, which would open a new door to sensing and manipulating biologically important processes and purification of key materials in industries.

Supramolecular Transmembrane Ion Channels Formed by Multiblock Amphiphiles.

Transmembrane proteins located within biological membranes play a crucial role in a variety of important cellular processes, such as energy conversion and signal transduction. Among them, ion channel proteins that can transport specific ions across the biological membranes are particularly important for achieving precise control over those processes. Strikingly, approximately 20% of currently approved drugs are targeted to ion channel proteins within membranes. Thus, synthetic molecules that can mimic the functions of natural ion channel proteins would possess great potential in the sensing and manipulation of biologically important processes, as well as in the purification of key industrial materials.Inspired by the sophisticated structures and functions of natural ion channel proteins, our research group developed a series of multiblock amphiphiles (MAs) composed of a repetitive sequence of flexible hydrophilic oligo(ethylene glycol) chains and rigid hydrophobic oligo(phenylene-ethynylene) units. These MAs can be effectively incorporated into the hydrophobic layer of lipid bilayer membranes and adopt folded conformations, with their hydrophobic units stacked in a face-to-face configuration. Moreover, the folded MAs can self-assemble within the membranes and form supramolecular nanopores that can transport ions across the membranes. In these studies, we focused on the structural flexibility of the MAs and decided to design new molecules able to respond to various external stimuli in order to control their transmembrane ion transport properties. For this purpose, we developed new MAs incorporating sterically bulky groups within their hydrophobic units and demonstrated that their transmembrane ion transport properties could be controlled via mechanical forces applied to the membranes. Moreover, we developed MAs incorporating phosphate ester groups that functioned as ligand-binding sites at the boundary between hydrophilic and hydrophobic units and found that these MAs exhibited transmembrane ion transport properties upon binding with aromatic amine ligands, even within the biological membranes of living cells. We further modified the hydrophobic units of the MAs with fluorine atoms and demonstrated their voltage-responsive transmembrane ion transport properties. These molecular design principles were extended to the development of a transmembrane anion transporter whose transport mechanism was studied by all-atom molecular dynamics simulations.This Account describes the basic principles of the molecular designs of MAs, the characterization of their self-assembled structures within a lipid bilayer, and their transmembrane ion transport properties, including their responsiveness to stimuli. Finally, we discuss future perspectives on the manipulation of biological processes based on the characteristic features of MAs.

Characterization of a novel type of carbonic anhydrase that acts without metal cofactors.

BACKGROUND: Carbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and bicarbonate (HCO3-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, ß, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution. RESULTS: Here, we report that a previously uncharacterized protein group, named "COG4337," constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO2 hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+ß barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO2 metabolism in these organelles. CONCLUSIONS: COG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.

Calcium-induced reversible assembly of phosphorylated amphiphile within lipid bilayer membranes.

Inspired by calcium-induced reversible assembly and disassembly of membrane proteins found in nature, here we developed a phosphorylated amphiphile (PA) that contains an oligo(phenylene-ethynylene) unit as a hydrophobic unit and a phosphate ester group as a hydrophilic calcium-binding unit. We demonstrated that PA can assemble and disassemble in a reversible manner in response to the sequential addition of calcium chloride and ethylene-diaminetetraacetic acid within the lipid bilayer membranes for the first time as a synthetic molecule.

Synthetic Ion Channel Formed by Multiblock Amphiphile with Anisotropic Dual-Stimuli-Responsiveness.

Transmembrane proteins within biological membranes exhibit varieties of important functions that are vital for many cellular activities, and the development of their synthetic mimetics allows for deep understanding in related biological events. Inspired by the structures and functions of natural ion channels that can respond to multiple stimuli in an anisotropic manner, we developed multiblock amphiphile VF in this study. When VF was incorporated into the lipid bilayer membranes, VF formed a supramolecular ion channel whose ion transport property was controllable by the polarity and amplitude of the applied voltage. Microscopic emission spectroscopy revealed that VF changed its molecular conformation in response to the applied voltage. Furthermore, the ion transport property of VF could be reversibly switched by the addition of (R)-propranolol, an aromatic amine known as an antiarrhythmic agent, followed by the addition of ß-cyclodextrin for its removal. The highly regulated orientation of VF allowed for an anisotropic dual-stimuli-responsiveness for the first time as a synthetic ion channel.

Imidazolinium-based Multiblock Amphiphile as Transmembrane Anion Transporter.

Transmembrane anion transport is an important biological process in maintaining cellular functions. Thus, synthetic anion transporters are widely developed for their biological applications. Imidazolinium was introduced as anion recognition site to a multiblock amphiphilic structure that consists of octa(ethylene glycol) and aromatic units. Ion transport assay using halide-sensitive lucigenin and pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) revealed that imidazolinium-based multiblock amphiphile (IMA) transports anions and showed high selectivity for nitrate, which plays crucial roles in many biological events. Temperature-dependent ion transport assay using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) indicated that IMA works as a mobile carrier. 1 H NMR titration experiments indicated that the C2 proton of the imidazolinium ring recognizes anions via a (C-H)+ ⋅⋅⋅X- hydrogen bond. Furthermore, all-atom molecular dynamics simulations revealed a dynamic feature of IMA within the membranes during ion transportation.

Reversible formation of multiple stimuli-responsive polymeric materials through processing control of trifunctional amphiphilic molecules.

A nonionic amphiphile consisting of thermo-responsive tetraethylene glycol chains, redox-responsive thiol groups, and photo-responsive anthracene units was developed. By combining the thermal and redox stimuli in water and bulk to control the assembling processes, two pairs of polymeric materials possessing contrasting responses to thermal treatment and photoirradiation were prepared.

A synthetic ion channel with anisotropic ligand response.

Biological membranes play pivotal roles in the cellular activities. Transmembrane proteins are the central molecules that conduct membrane-mediated biochemical functions such as signal transduction and substance transportation. Not only the molecular functions but also the supramolecular properties of the transmembrane proteins such as self-assembly, delocalization, orientation and signal response are essential for controlling cellular activities. Here we report anisotropic ligand responses of a synthetic multipass transmembrane ion channel. An unsymmetrical molecular structure allows for oriented insertion of the synthetic amphiphile to a bilayer by addition to a pre-formed membrane. Complexation with a ligand prompts ion transportation by forming a supramolecular channel, and removal of the ligand deactivates the transportation function. Biomimetic regulation of the synthetic channel by agonistic and antagonistic ligands is also demonstrated not only in an artificial membrane but also in a biological membrane of a living cell.

Aromatic Fluorination of Multiblock Amphiphile Enhances Its Incorporation into Lipid Bilayer Membranes.

We designed multiblock amphiphiles AmF and AmH, which consist of perfluorinated and non-fluorinated hydrophobic units, respectively. Absorption spectroscopy revealed that both amphiphiles are molecularly dispersed in organic solvent, while they form aggregates under aqueous conditions. Furthermore, we investigated whether AmF and AmH can be incorporated into DOPC lipid bilayer membranes, and found that the maximum concentration of AmF that can be incorporated into DOPC lipid bilayer membranes is 43 times higher than that of AmH.

New Modified Deoxythymine with Dibranched Tetraethylene Glycol Stabilizes G-Quadruplex Structures.

Methods for stabilizing G-quadruplex formation is a promising therapeutic approach for cancer treatment and other biomedical applications because stable G-quadruplexes efficiently inhibit biological reactions. Oligo and polyethylene glycols are promising biocompatible compounds, and we have shown that linear oligoethylene glycols can stabilize G-quadruplexes. Here, we developed a new modified deoxythymine with dibranched or tribranched tetraethylene glycol (TEG) and incorporated these TEG-modified deoxythymines into a loop region that forms an antiparallel G-quadruplex. We analyzed the stability of the modified G-quadruplexes, and the results showed that the tribranched TEG destabilized G-quadruplexes through entropic contributions, likely through steric hindrance. Interestingly, the dibranched TEG modification increased G-quadruplex stability relative to the unmodified DNA structures due to favorable enthalpic contributions. Molecular dynamics calculations suggested that dibranched TEG interacts with the G-quadruplex through hydrogen bonding and CH-π interactions. Moreover, these branched TEG-modified deoxythymine protected the DNA oligonucleotides from degradation by various nucleases in human serum. By taking advantage of the unique interactions between DNA and branched TEG, advanced DNA materials can be developed that affect the regulation of DNA structure.

Thermo-driven self-assembly of a PEG-containing amphiphile in a bilayer membrane.

Self-assembly of lipid molecules in a plasma membrane, namely lipid raft formation, is involved in various dynamic functions of cells. Inspired by the raft formation observed in the cells, here we studied thermally induced self-assembly of a synthetic amphiphile, bola-AkDPA, in a bilayer membrane. The synthetic amphiphile consists of a hydrophobic unit including fluorescent aromatic and aliphatic components and hydrophilic tetraethylene glycol chains attached at both ends of the hydrophobic unit. In a polar solvent, bola-AkDPA formed aggregates to show excimer emission. In a lipid bilayer membrane, bola-AkDPA showed intensified excimer emission upon increase of its concentration or elevation of the temperature; bola-type amphiphiles containing oligoethylene glycol chains likely tend to form self-assemblies in a bilayer membrane triggered by thermal stimuli.

Heat-Triggered Crystallization of Liquid Crystalline Macrocycles Allowing for Conductance Switching through Hysteretic Thermal Phase Transitions.

A polymesomorphic thermal phase-transition of a macrocyclic amphiphile consisting of aromatic groups and oligoethylene glycol (OEG) chains is reported. The macrocyclic amphiphile exists in a highly-ordered liquid crystal (LC) phase at room temperature. Upon heating, this macrocycle shows phase-transition from columnar-lamellar to nematic LC phases followed by crystallization before melting. Spectroscopic studies suggest that the thermally induced crystallization is triggered by a conformational change at the OEG chains. Interestingly, while the macrocycle returns to the columnar-lamellar phase after cooling from the isotropic liquid, it retains the crystallinity after cooling from the thermally-induced crystal. Thanks to this bistability, conductance switching was successfully demonstrated. A different macrocyclic amphiphile also shows an analogous phase-transition behavior, suggesting that this molecular design is universal for developing switchable and memorizable materials, by means of hysteretic phase-transition processes.

Localization of transmembrane multiblock amphiphilic molecules in phase-separated vesicles.

A series of triblock amphiphilic molecules bearing hydrophilic PEG chains at both ends of the long aromatic hydrophobic moieties were obtained serendipitously. The molecules involve linearly connected diarylethyne and diarylbutadiyne units, which show characteristic emissions upon excitation by UV light. These emissions showed red-shifts upon an increase in the solvent polarity, where the shifts are larger for the molecules with longer aromatic moieties. The distribution of these molecules in phase-separated membranes consisting of DOPC/DPPC/cholesterol was studied by fluorescence microscopy. It was found that most compounds, except for that with the longest hydrophobic unit, were selectively distributed in the Ld phase consisting mainly of DOPC. Interestingly, some of them were suggested to encourage delocalization of cholesterol in both the Lo and Ld phases.

Enzymatically cleavable traceless biotin tags for protein PEGylation and purification.

Here we report an example of a protein-PEG conjugate with a biotin tag cleavable by lipase-catalyzed hydrolysis. Very mild cleavage conditions, heterogeneous, easily separable catalysts, and traceless design make this method attractive for the preparation and purification of PEGylated proteins.

Multifarious Polymorphism of a Multiblock Amphiphilic Macrocycle Bearing Thermally Responsive Polyether Segment.

Formation of multiple crystalline phases of a multiblock amphiphilic macrocycle AT2B is demonstrated. AT2B forms a single crystal (Cr-α) by vapor diffusion and shows reversible single-crystal-to-single-crystal transition between two crystalline phases (Cr-α and Cr-ß) by a temperature change, and crystalline AT2B (Cr-ß) melts at 422 K, and the cooling rate from the melt influences the phase of the solid formed. By cooling at 1.0 K min-1, AT2B forms crystalline phases (Cr-γ and Cr-δ), which are different from both Cr-α and Cr-ß. On the other hand, cooling at 2.0 K min-1 results in the formation of an amorphous phase, and a mechanical stress also triggers a crystal-to-amorphous solid transition. Interestingly, the amorphous solid crystallizes to give the fifth crystalline phase (Cr-γ) upon heating before melting. It is suggested that these multiple phase transitions are driven by thermal conformational changes at the tetraethylene glycol chains of AT2B.