ROS-Responsive Methionine-Containing Amphiphilic Peptides Impart Enzyme-Triggered Phase Transition and Antioxidant Cell Protection.

Reactive oxygen species (ROS) are produced by cellular activities, such as metabolism and immune response, and play important roles in cell signaling and homeostasis. However, overproduced ROS causes irreversible damage to nucleic acids and membrane lipids, supporting genetic mutations and enhancing the effects of aging. Cells defend themselves against ROS using antioxidant systems based on redox-active sulfur and transition metals. Inspired by such biological redox-responsive systems, we developed methionine-containing self-assembling peptides. The Met-containing peptides formed hydrogels that underwent a gel-to-sol phase transition upon oxidation by H2O2, and the sensitivity of the peptides to the oxidant increased as the number of Met residues increased. The peptide containing three Met residues, the largest number of Met residues in our series of designed peptides, showed the highest sensitivity to oxidation and detoxification to protect cells from ROS damage. In addition, this peptide underwent a phase transition in response to H2O2 produced by an oxidizing enzyme. This study demonstrates the design of a supramolecular biomaterial that is responsive to enzymatically generated ROS and can protect cells against oxidative stress.

Rapid Synthesis of Chiral Figure-Eight Macrocycles Using a Preorganized Natural Product-Based Scaffold.

Chiral D2 -symmetric figure-eight shaped macrocycles are promising scaffolds for amplifying the chiroptical properties of π-conjugated systems. By harnessing the inherent and adaptable conformational dynamics of a chiral C2 -symmetric bispyrrolidinoindoline (BPI) manifold, we developed an enantio-divergent modular synthetic platform to rapidly generate a diverse range of chiral macrocycles, spanning from 14- to 66-membered rings, eliminating the need for optical resolution. Notably, a 32-membered figure-eight macrocycle showed excellent circularly polarized luminescence (CPL: |glum |=1.1×10-2 ) complemented by a robust emission quantum yield (Φfl =0.74), to achieve outstanding CPL brightness (BCPL : ϵ×Φfl ×|glum |/2=480). Using quadruple Sonogashira couplings, this versatile synthetic platform enables precise adjustments of the angle, distance, and length among intersecting π-conjugated chromophores. Our synthetic strategy offers a streamlined and systematic approach to significantly enhance BCPL values for a variety of chiral D2 -symmetric figure-eight macrocycles.

Endocytosis-Like Vesicle Fission Mediated by a Membrane-Expanding Molecular Machine Enables Virus Encapsulation for In Vivo Delivery.

Biological membranes are functionalized by membrane-associated protein machinery. Membrane-associated transport processes, such as endocytosis, represent a fundamental and universal function mediated by membrane-deforming protein machines, by which small biomolecules and even micrometer-size substances can be transported via encapsulation into membrane vesicles. Although synthetic molecules that induce dynamic membrane deformation have been reported, a molecular approach enabling membrane transport in which membrane deformation is coupled with substance binding and transport remains critically lacking. Here, we developed an amphiphilic molecular machine containing a photoresponsive diazocine core (AzoMEx) that localizes in a phospholipid membrane. Upon photoirradiation, AzoMEx expands the liposomal membrane to bias vesicles toward outside-in fission in the membrane deformation process. Cargo components, including micrometer-size M13 bacteriophages that interact with AzoMEx, are efficiently incorporated into the vesicles through the outside-in fission. Encapsulated M13 bacteriophages are transiently protected from the external environment and therefore retain biological activity during distribution throughout the body via the blood following administration. This research developed a molecular approach using synthetic molecular machinery for membrane functionalization to transport micrometer-size substances and objects via vesicle encapsulation. The molecular design demonstrated in this study to expand the membrane for deformation and binding to a cargo component can lead to the development of drug delivery materials and chemical tools for controlling cellular activities.

ROS-Triggered Gel-Sol Transition and Kinetics-Controlled Cargo Release by Methionine-Containing Peptides.

The gel-sol transition of self-assembling peptides is a useful switch for environment-dependent drug release. For their applications, kinetics control of the responses is important for matching the velocity of release to the target biological events. Here we demonstrate the chemical control of redox-triggered gel-sol transition kinetics of self-assembling peptides by altering the amino acid sequence. Amphiphilic peptides were developed in which a methionine residue was located in the middle (JigSAP-IMI) or near the N terminus (JigSAP-MII). Both peptides formed hydrogels under physiological conditions-forming ß-sheet-based supramolecular nanofibers. In contrast, the oxidized forms remained in the solution state under identical conditions-adopting α-helix-rich secondary structures. Upon oxidation with H2 O2 , a reactive oxygen species, JigSAP-MII showed a faster gel-to-sol transition and cargo-releasing than JigSAP-IMI, thus indicating that the phase-transition and releasing kinetics of self-assembling peptides can be rationally controlled by the position of the reactive amino acid residue.

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.

Self-assembling Molecular Medicine for the Subacute Phase of Ischemic Stroke.

Ischemic stroke leads to acute neuron death and forms an injured core, triggering delayed cell death at the penumbra. The impaired brain functions after ischemic stroke are hardly recovered because of the limited regenerative properties. However, recent rodent intervention studies manipulating the extracellular environments at the subacute phase shed new light on the regenerative potency of the injured brain. This review introduces the rational design of artificial extracellular matrix (ECM) mimics using supramolecular peptidic scaffolds, which self-assemble via non-covalent bonds and form hydrogels. The facile customizability of the peptide structures allows tuning the hydrogels' physical and biochemical properties, such as charge states, hydrophobicity, cell adhesiveness, stiffness, and stimuli responses. Supramolecular peptidic materials can create safer and more economical drugs than polymer materials and cell transplantation. We also discuss the importance of activating developmental programs for the recovery at the subacute phase of ischemic stroke. Self-assembling molecular medicine mimicking the ECMs and activating developmental programs may stand as a new drug modality of regenerative medicine in various tissues.

Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium.

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) -peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP-PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP-PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP-PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP-PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Invited for the cover of this issue is the group of Takahiro Muraoka at Tokyo University of Agriculture and Technology and collaborators. The image depicts nanofiber formation of an amphiphilic peptide with a central alkylene chain that shows non-cell adhesive properties. Read the full text of the article at 10.1002/chem.202100739.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Amphiphilic peptides bearing terminal alkyl tails form supramolecular nanofibers that are increasingly used as biomaterials with multiple functionalities. Insertion of alkylene chains in peptides can be designed as another type of amphiphilic peptide, yet the influence of the internal alkylene chains on self-assembly and biological properties remains poorly defined. Unlike the terminal alkyl tails, the internal alkylene chains can affect not only the hydrophobicity but also the flexibility and packing of the peptides. Herein, we demonstrate the supramolecular and biological effects of the central alkylene chain length inserted in a peptide. Insertion of the alkylene chain at the center of the peptide allowed for strengthened ß-sheet hydrogen bonds and modulation of the packing order, and consequently the amphiphilic peptide bearing C2 alkylene chain formed a hydrogel with the highest stiffness. Interestingly, the amphiphilic peptides bearing internal alkylene chains longer than C2 showed a diminished cell-adhesive property. This study offers a novel molecular design to tune mechanical and biological properties of peptide materials.

Conjugate of Thiol and Guanidyl Units with Oligoethylene Glycol Linkage for Manipulation of Oxidative Protein Folding.

Oxidative protein folding is a biological process to obtain a native conformation of a protein through disulfide-bond formation between cysteine residues. In a cell, disulfide-catalysts such as protein disulfide isomerase promote the oxidative protein folding. Inspired by the active sites of the disulfide-catalysts, synthetic redox-active thiol compounds have been developed, which have shown significant promotion of the folding processes. In our previous study, coupling effects of a thiol group and guanidyl unit on the folding promotion were reported. Herein, we investigated the influences of a spacer between the thiol group and guanidyl unit. A conjugate between thiol and guanidyl units with a diethylene glycol spacer (GdnDEG-SH) showed lower folding promotion effect compared to the thiol-guanidyl conjugate without the spacer (GdnSH). Lower acidity and a more reductive property of the thiol group of GdnDEG-SH compared to those of GdnSH likely resulted in the reduced efficiency of the folding promotion. Thus, the spacer between the thiol and guanidyl groups is critical for the promotion of oxidative protein folding.

Current Progress in Cross-Linked Peptide Self-Assemblies.

Peptide-based fibrous supramolecular assemblies represent an emerging class of biomaterials that can realize various bioactivities and structures. Recently, a variety of peptide fibers with attractive functions have been designed together with the discovery of many peptide-based self-assembly units. Cross-linking of the peptide fibers is a key strategy to improve the functions of these materials. The cross-linking of peptide fibers forming three-dimensional networks in a dispersion can lead to changes in physical and chemical properties. Hydrogelation is a typical change caused by cross-linking, which makes it applicable to biomaterials such as cell scaffold materials. Cross-linking methods, which have been conventionally developed using water-soluble covalent polymers, are also useful in supramolecular peptide fibers. In the case of peptide fibers, unique cross-linking strategies can be designed by taking advantage of the functions of amino acids. This review focuses on the current progress in the design of cross-linked peptide fibers and their applications.

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.

Formation of Giant and Small Cyclic Complexes from a Flexible Tripeptide Ligand Controlled by Metal Coordination and Hydrogen Bonds.

Formation of giant cyclic complexes by the assembly of small, flexible units is demonstrated by connecting 14 artificial tripeptides (1) with 14 Ni(II) ions. Although tripeptide 1 is very flexible because of the presence of three CH2 groups in the main chain, it formed a tetradecanuclear cyclic complex ([114Ni14]28+) with a large cavity (diameter: ca. 2 nm). In this structure, three tripeptides are coordinated to each Ni(II) center by three different coordination sites in 1, forming a mesh-like structure. Crystal structure analysis and theoretical calculations indicate that the conformation of 1 was controlled by the formation of metal coordination bonds and intramolecular hydrogen bonds. Because of the structural flexibility, the cyclic framework formed both circular and ellipsoidal structures in the crystalline state, depending on the packing structure. In addition, by the conditions of the assembly process, the size of the cavities could be tuned either with a small decrement (dodecanuclear complex [112Ni12]24+) or a large decrement (octanuclear complexes [(1-3H+)4Ni8]4+), in which "shrunk" cavities with a 10-fold difference in diameter (<0.2 nm) were formed by tuning the tripeptide conformation through additional metal coordination to the tripeptide framework. Dynamic light scattering and mass spectrometry studies indicated that the giant cyclic complexes were also present in the solution state.

Glycine Substitution Effects on the Supramolecular Morphology and Rigidity of Cell-Adhesive Amphiphilic Peptides.

Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.

Newly characterized interaction stabilizes DNA structure: oligoethylene glycols stabilize G-quadruplexes CH-π interactions.

Oligoethylene glycols are used as crowding agents in experiments that aim to understand the effects of intracellular environments on DNAs. Moreover, DNAs with covalently attached oligoethylene glycols are used as cargo carriers for drug delivery systems. To investigate how oligoethylene glycols interact with DNAs, we incorporated deoxythymidine modified with oligoethylene glycols of different lengths, such as tetraethylene glycol (TEG), into DNAs that form antiparallel G-quadruplex or hairpin structures such that the modified residues were incorporated into loop regions. Thermodynamic analysis showed that because of enthalpic differences, the modified G-quadruplexes were stable and the hairpin structures were slightly unstable relative to unmodified DNA. The stability of G-quadruplexes increased with increasing length of the ethylene oxides and the number of deoxythymidines modified with ethylene glycols in the G-quadruplex. Nuclear magnetic resonance analyses and molecular dynamics calculations suggest that TEG interacts with bases in the G-quartet and loop via CH-π and lone pair-π interactions, although it was previously assumed that oligoethylene glycols do not directly interact with DNAs. The results suggest that numerous cellular co-solutes likely affect DNA function through these CH-π and lone pair-π interactions.

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.

Design of C2-symmetric alkaloidal chiral amphiphiles and configurational effects on self-assembly.

Alkaloids are a cornerstone in the development of medicinal and synthetic compounds due to their capability of specific recognition of targeted biomacromolecules, and uses in optical resolution and asymmetric reactions. To explore the untapped potential of the rigid and densely functionalized structures of alkaloids with precisely regulated configurations as optically active core scaffolds of self-assembling molecules, here we report the design, syntheses, chiroptical properties and self-assemblies of C2-symmetric alkaloidal amphiphiles with anti/syn stereochemical variations. Bispyrrolidinoindoline (BPI) was chosen as the optically active core scaffold. It was synthetically modified with hydrophobic alkyl chains and hydrophilic tetraethylene glycol tails to provide amphiphilicity. The anti/syn configurational differences in the amphiphiles significantly influenced the chiroptical, dynamic and supramolecular properties. Amphiphiles with anti-configurations responded to a solvent polarity change by altering their conformations, while the conformational changes of the syn-type amphiphiles were largely restricted. Furthermore, the anti-type amphiphile having the highest structural flexibility showed a characteristic split Cotton effect in an organic medium and formed the largest aggregates upon addition of water with a significant change in the circular dichroism (CD) profile, while amphiphiles having conformational restriction by the syn-configuration or a macrocyclic structure showed monomodal CD signals and afforded significantly smaller aggregates upon addition of water. Hence, the C2-symmetric alkaloidal BPI structure is demonstrated to be a useful core scaffold for supramolecular chemistry to design amphiphiles with controllable configurational diversity, which allows for the customization of chiroptical properties, conformational flexibility and self-assembly.

Bio-inspired Multiblock Molecules for Membrane Functionalization.

A multipass transmembrane (MTM) structure is prevalent in membrane proteins for a wide range of functions. Typically, the MTM structure is constructed of bundled multiple α-helices spanning the membrane which are connected by flexible domains. One characteristic feature of MTM proteins is dynamic functions such as stimuli responses and conformational changes. In this review, the development of synthetic molecules forming an MTM structure in membranes is highlighted. The MTM folded structure is developed using an amphiphilic molecular design with a multiblock strategy between rigid hydrophobic components and flexible hydrophilic units. Such synthetic amphiphiles not only form the MTM structure by folding but also self-assemble to construct supramolecular ion channels. An elaborated molecular design of the MTM structure with a ligand-binding pocket allows for ligand-gated regulation of ion transport. Light-triggered membrane deformation for vesicle budding is also demonstrated.

Mechano-Sensitive Synthetic Ion Channels.

Mechanical stress is a ubiquitous stimulus sensed by membrane proteins, but rarely by synthetic molecules. Inspired by mechano-sensitive ion channels found in cell membranes, tension-responsive transmembrane multiblock amphiphiles were developed. In membranes, a single-transmembrane amphiphile responds to both expanding and contracting tensions to weaken and strengthen the stacking of membrane-spanning units, respectively, and ion transportation is triggered by expanding tension to form a supramolecular channel, while little transportation is observed under a tensionless condition. In contrast, a three-transmembrane amphiphile showed little spectroscopic response to tensions, likely due to weaker stacking of membrane-spanning units than in the single-transmembrane amphiphile. Nevertheless, the three-transmembrane amphiphile shows ion transportation by forming a unimolecular channel even under a tensionless condition, and the ion-transporting activity decreased with expanding tension. Interestingly, the estimated operating force of these synthetic systems was comparable to that of the mechano-sensitive proteins. This study opens the door toward new mechano-sensitive molecular devices.

G-Quadruplexes with Tetra(ethylene glycol)-Modified Deoxythymidines are Resistant to Nucleases and Inhibit HIV-1 Reverse Transcriptase.

G-quadruplex formation in virally encoded templates arrests reverse transcription. Methods to stabilize this structure are promising for antiviral approaches. To stabilize G-quadruplex formation, deoxythymidines were modified with tetra(ethylene glycol) (TEG). The TEG-modified G-quadruplexes were stabilized significantly relative to unmodified DNA. In the presence of a TEG-modified oligonucleotide that is capable of forming an intermolecular G-quadruplex with a template containing a hu- man immunodeficiency virus-1 sequence, reverse transcription was inhibited by more than 70 % relative to the reaction in the absence of the TEG-modified oligonucleotide. Moreover, the TEG-modified deoxythymidines protected the DNA oligonucleotide from degradation by various nucleases in human serum. Thus, DNA oligonucleotides modified with TEG have potential in therapeutic applications.