"Photochemical Surgery" of 1D Metal-Organic Frameworks with a Site-Selective Solubilization/Crystallization Strategy.

One-dimensional (1D) hybrid MOFs are attractive if they consist of different MOF blocks with interconnected channels. However, the precision synthesis of such 1D multiblock MOFs with the desired block lengths and sequences remains a formidable challenge. Herein we propose the "photochemical surgery" method, which combines top-down and bottom-up approaches to enable the site-selective solubilization (removal)/crystallization (reconstruction) of 1D MOFs. We employed photoreactive MOFs, which were prepared by complexing either Cd2+ or Zn2+ with a mixture containing a photochromic bispyridyl ligand (PyDTEopen or PyDTZEopen) and an isophthalate (5-nitroisophthalate (nip2-) or 5-bromoisophthalate (bip2-)). These MOFs were obtained as high-aspect-ratio, needlelike, colorless crystals that bore 1D channels oriented parallel to the long needle axis. When photoreactive DTECdMOFNO2 ([Cd(nip)(PyDTEopen)(H2O)]n), for example, was immobilized at both ends with a metal alloy on a glass substrate and exposed to UV light through a photomask for 60 min in N,N-dimethylformamide/methanol (DMF/MeOH), the unmasked part was removed via solubilization to produce a 50 µm gap. The resulting specimen was immersed for 24 h at 25 °C in DMF/MeOH containing the necessary components for the construction of DTZECdMOFNO2 ([Cd(nip)(PyDTZEopen)(H2O)]n). Eventually, the gap was filled with DTZECdMOFNO2 to produce a triblock hybrid MOF (DTECdMOFNO2-DTZECdMOFNO2-DTECdMOFNO2). The result of a guest diffusion experiment confirmed that the newly formed DTZECdMOFNO2 block shared its 1D channels with the host DTECdMOFNO2 blocks. "Photochemical surgery" can be applied to synthesize 1D hybrid MOFs bearing unconventional sequences and morphologies, e.g., honeycomb- and inverted-honeycomb-patterned hybrids.

Effects of the Magnetic Orientation of M13 Bacteriophage on Phage Display Selection.

Although phage display selection using a library of M13 bacteriophage has become a powerful tool for finding peptides that bind to target materials on demand, a remaining concern of this method is the interference by the M13 main body, which is a huge filament >103  times larger than the displayed peptide, and therefore would nonspecifically adhere to the target or sterically inhibit the binding of the displayed peptide. Meanwhile, filamentous phages are known to be orientable by an external magnetic field. If M13 filaments are magnetically oriented during the library selection, their angular arrangement relative to the target surface would be changed, being expected to control the interference by the M13 main body. This study reports that the magnetic orientation of M13 filaments vertical to the target surface significantly affects the selection. When the target surface was affinitive to the M13 main body, this orientation notably suppressed the nonspecific adhesion. Furthermore, when the target surface was less affinitive to the M13 main body and intrinsically free from the nonspecific adhesion, this orientation drastically changed the population of M13 clones obtained through library selection. The method of using no chemicals but only a physical stimulus is simple, clean, and expected to expand the scope of phage display selection.

ATP-Responsive Nanoparticles Covered with Biomolecular Machine "Chaperonin GroEL".

Herein, we report an ATP-responsive nanoparticle (GroEL NP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroEL NP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroEL NP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable GroEL NP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroEL NP per GroEL was 4.8 and 4.0 times greater than those of precursor cys GroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that GroEL NP could be iteratively extended to double-layered ( GroEL ) 2 ${{^{({\rm GroEL}){_{2}}}}}$ NP.

Reconstitution of microtubule into GTP-responsive nanocapsules.

Nanocapsules that collapse in response to guanosine triphosphate (GTP) have the potential as drug carriers for efficiently curing diseases caused by cancer and RNA viruses because GTP is present at high levels in such diseased cells and tissues. However, known GTP-responsive carriers also respond to adenosine triphosphate (ATP), which is abundant in normal cells as well. Here, we report the elaborate reconstitution of microtubule into a nanocapsule that selectively responds to GTP. When the tubulin monomer from microtubule is incubated at 37 °C with a mixture of GTP (17 mol%) and nonhydrolysable GTP* (83 mol%), a tubulin nanosheet forms. Upon addition of photoreactive molecular glue to the resulting dispersion, the nanosheet is transformed into a nanocapsule. Cell death results when a doxorubicin-containing nanocapsule, after photochemically crosslinked for properly stabilizing its shell, is taken up into cancer cells that overexpress GTP.

Photoreactive Molecular Glue for Enhancing the Efficacy of DNA Aptamers by Temporary-to-Permanent Conjugation with Target Proteins.

We developed a photoreactive molecular glue, BPGlue-N3, which can provide a universal strategy to enhance the efficacy of DNA aptamers by temporary-to-permanent stepwise stabilization of their conjugates with target proteins. As a proof-of-concept study, we applied BPGlue-N3 to the SL1 (DNA aptamer)/c-Met (target protein) conjugate system. BPGlue-N3 can adhere to and temporarily stabilize this aptamer/protein conjugate multivalently using its guanidinium ion (Gu+) pendants that form a salt bridge with oxyanionic moieties (e.g., carboxylate and phosphate) and benzophenone (BP) group that is highly affinitive to DNA duplexes. BPGlue-N3 is designed to carry a dual-mode photoreactivity; upon exposure to UV light, the temporarily stabilized aptamer/protein conjugate reacts with the photoexcited BP unit of adhering BPGlue-N3 and also a nitrene species, possibly generated by the BP-to-N3 energy transfer in BPGlue-N3. We confirmed that SL1, covalently conjugated with c-Met, hampered the binding of hepatocyte growth factor (HGF) onto c-Met, even when the SL1/c-Met conjugate was rinsed prior to the treatment with HGF, and suppressed cell migration caused by HGF-induced c-Met phosphorylation.

Intracellular Photoactivation of Caspase-3 by Molecular Glues for Spatiotemporal Apoptosis Induction.

Caspase-3 (Casp-3) is an enzyme that efficiently induces apoptosis, a form of programmed cell death. We report a dendritic molecular glue PCGlue that enables intracellular delivery of Casp-3 and its photoactivation. PCGlue carrying multiple guanidinium (Gu+) ion pendants via photocleavable linkages can tightly adhere to Casp-3 and deliver it into the cytoplasm mainly by direct penetration through the plasma membrane. Casp-3, whose surface is covered by PCGlue, is unable to interact with its cellular substrates and can therefore not induce apoptosis. However, upon exposure to UV or two-photon near-infrared (NIR) light, PCGlue is cleaved off to liberate Casp-3, triggering the apoptotic signaling cascade. This intracellular photoactivation of Casp-3 allows spatiotemporal induction of apoptosis in irradiated cells.

One-Step Synthesis of an Adaptive Nanographene MOF: Adsorbed Gas-Dependent Geometrical Diversity.

A layered metal-organic framework (MOF) comprising extra-large nanographene sheets, HBCMOF, was successfully synthesized using a dicarboxylic acid derivative of hexa-peri-hexabenzocoronene (HBCLH2), and its structure was characterized by single-crystal X-ray diffraction analysis. The crystal structure shows that 2D layers composed of a dinuclear Zn2+ complex unit and HBCL are located on top of each other through multiple weak interlayer bonds, affording HBCMOF, having three dimensionally connected nanopores with large nanographene surfaces. The HBC-based nanographene sheets are anchored to the MOF framework via two zinc carboxylate linkages and therefore have an axial rotational freedom. The sorption isotherms of gaseous molecules such as carbon dioxide and hydrocarbons (acetylene, propane, propylene, benzene, and cyclohexane) on HBCMOF all displayed a hysteretic profile with reversible structural changes, as observed by in situ powder X-ray diffraction studies.

Molecular Glue that Spatiotemporally Turns on Protein-Protein Interactions.

We developed a dendritic molecular glue PCGlue-NBD that can serve universally to "turn on" protein-protein interactions (PPIs) spatiotemporally. PCGlue-NBD carrying multiple guanidinium ion (Gu+) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PCGlue-NBD on a target protein is photocleaved at butyrate-substituted nitroveratryloxycarbonyl linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PCGlue-NBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PCGlue-NBD/HGF hybrid to light-emitting diode light (365 nm), the PCGlue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The turn-on PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a millimeter-scale resolution by pointwise irradiation with UV light.

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags.

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases. Here, we demonstrate light-triggered nuclear translocation of guests using caged molecular glue (CagedGlue-R) tags, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Guests tagged with CagedGlue-R are taken up into living cells via endocytosis and remain in endosomes. However, upon photoirradiation, CagedGlue-R is converted into uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which facilitates the endosomal escape and subsequent nuclear translocation of the guests. This method is promising for site-selective nuclear-targeting drug delivery, since the tagged guests can migrate into the cytoplasm followed by the cell nucleus only when photoirradiated. CagedGlue-R tags can deliver macromolecular guests such as quantum dots (QDs) as well as small-molecule guests. CagedGlue-R tags can be uncaged with not only UV light but also two-photon near-infrared (NIR) light, which can deeply penetrate into tissue.

Transferrin-Appended Nanocaplet for Transcellular siRNA Delivery into Deep Tissues.

Transferrin (Tf) is known to induce transcytosis, which is a consecutive endocytosis/exocytosis event. We developed a Tf-appended nanocaplet (TfNC⊃siRNA) for the purpose of realizing siRNA delivery into deep tissues and RNA interference (RNAi) subsequently. For obtaining TfNC⊃siRNA, a macromonomer (AzGu) bearing multiple guanidinium (Gu+) ion units, azide (N3) groups, and trityl (Trt)-protected thiol groups in the main chain, side chains, and termini, respectively, was newly designed. Because of a multivalent Gu+-phosphate salt-bridge interaction, AzGu can adhere to siRNA along its strand. When I2 was added to a preincubated mixture of AzGu and siRNA, oxidative polymerization of AzGu took place along the siRNA strand, affording AzNC⊃siRNA, the smallest siRNA-containing reactive nanocaplet so far reported. This conjugate was converted into Glue/BPNC⊃siRNA by the click reaction with a Gu+-appended bioadhesive dendron (Glue) followed by a benzophenone derivative (BP). Then, Tf was covalently immobilized onto Glue/BPNC⊃siRNA by Gu+-mediated adhesion followed by photochemical reaction with BP. With the help of Tf-induced transcytosis, TfNC⊃siRNA permeated deeply into a cancer spheroid, a 3D tissue model, at a depth of up to nearly 70 µm, unprecedentedly.

Nitrobenzoxadiazole-Appended Cell Membrane Modifiers for Efficient Optoporation with Noncoherent Light.

FLNBD-BAMPEG2k, bearing a nitrobenzoxadiazole (NBD) unit and an oleyl terminus conjugated via a poly(ethylene glycol) (PEG) spacer ( Mn = 2,000), was designed to fluorescently label cell membranes by docking its hydrophobic oleyl terminus. During laser scanning microscopy in a minimal essential medium (MEM), human hepatocellular carcinoma Hep3B cells labeled with FLNBD-BAMPEG2k appeared to undergo optoporation at their plasma membrane. We confirmed this unprecedented possibility by a series of cellular uptake experiments using negatively charged and therefore membrane-impermeable quantum dots (QDs; Dh = 4.7 nm). Detailed studies indicated that the photoexcited NBD unit can generate singlet oxygen (1O2), which oxidizes the constituent phospholipids to transiently deteriorate the cell membrane. Reference membrane modifiers FLNBD-Oleyl and FLNBD-BAMPEG8k having shorter or longer hydrophilic spacers between the NBD and oleyl units showed a little or substantially no optoporation. For understanding these results, one must consider the following contradictory factors: (1) The photosensitized 1O2 generation efficiently occurs only when the NBD unit is in aqueous media, and (2) the lifetime of 1O2 in aqueous media is very short (3.0-3.5 µs). As supported experimentally and computationally, the hydrophilic spacer length of FLNBD-BAMPEG2k is optimal for compromising these factors. Further to note, the optoporation using FLNBD-BAMPEG2k is not accompanied by cytotoxicity.

Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells.

We developed dendritic caged molecular glues (CagedGlue-R) as tags for nucleus-targeted drug delivery, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable unit (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Negatively charged CagedGlue-R hardly binds to anionic biomolecules because of their electrostatic repulsion. However, upon exposure of CagedGlue-R to UV light or near-infrared (NIR) light, the BANVOC groups of CagedGlue-R are rapidly detached to yield an uncaged molecular glue (UncagedGlue-R) that carries multiple Gu+ pendants. Because Gu+ forms a salt bridge with PO4-, UncagedGlue-R tightly adheres to anionic biomolecules such as DNA and phospholipids in cell membranes by a multivalent salt-bridge formation. When tagged with CagedGlue-R, guests can be taken up into living cells via endocytosis and hide in endosomes. However, when the CagedGlue-R tag is photochemically uncaged to form UncagedGlue-R, the guests escape from the endosome and migrate into the cytoplasm followed by the cell nucleus. We demonstrated that quantum dots (QDs) tagged with CagedGlue-R can be delivered efficiently to cell nuclei eventually by irradiation with light.

Guanidinium-based "molecular glues" for modulation of biomolecular functions.

Molecular adhesion based on multivalent interactions plays essential roles in various biological processes. Hence, "molecular glues" that can adhere to biomolecules may modulate biomolecular functions and therefore can be applied to therapeutics. This tutorial review describes design strategies for developing adhesive motifs for biomolecules based on multivalent interactions. We highlight a guanidinium ion-based salt-bridge as a key interaction for adhesion to biomolecules and discuss the application of molecular glues for manipulation of biomolecular assemblies, drug delivery systems, and modulation of biomolecular functions.

Swallowing a Surgeon: Toward Clinical Nanorobots.

In this Account, "a step toward clinical nanorobots" is proposed as one of the Holy Grails in chemistry, which could lead to a great leap in the field of biomedicines when accomplished. We review our preliminary contributions to this challenge by engineering chaperonin protein GroEL to generate de novo structures and functions.

Crystalline Nanochannels with Pendant Azobenzene Groups: Steric or Polar Effects on Gas Adsorption and Diffusion?

An azobenzene-containing, zirconium-based metal-organic framework (AzoMOF), upon irradiation with ultraviolet (UV) light at 365 ± 10 nm, underwent trans-to-cis isomerization of its azobenzene pendants to furnish the cis-isomer content of 21% (AzoMOF21%) in 30 min at the photostationary state and underwent backward isomerization into AzoMOF1% upon either irradiation with visible light (420-480 nm) or heating. When the cis-isomer content increased, the diffusion rate and amount of CO2 adsorbed into the nanochannels of AzoMOF decreased considerably. When erythrosine B, a polarity-probing guest, was used, it showed a red shift upon exposure of AzoMOF20%⊃EB to visible light, indicating that the interior environment of AzoMOF turns less polar as the trans-isomer content becomes higher. In sharp contrast, the adsorption profiles of AzoMOF15% and AzoMOF1% for Ar having an analogous kinetic diameter to CO2 but no quadrupole moment and a smaller polarizability were virtually identical to one another. Therefore, it is likely that CO2 experiences a dominant effect of a polar effect rather than a steric effect in the crystalline nanochannels.

Supramolecular Nanotube of Chaperonin GroEL: Length Control for Cellular Uptake Using Single-Ring GroEL Mutant as End-Capper.

How to modulate supramolecular protein nanotubes without sacrificing their thermodynamic stability? This challenging issue emerged with an enhanced reality since our successful development of a protein nanotube of chaperonin GroELMC as a novel ATP-responsive 1D nanocarrier because the nanotube length may potentially affect the cellular uptake efficiency. Herein, we report a molecularly engineered protein end-capper (SRMC) that firmly binds to the nanotube termini since the end-capper originates from GroEL. According to the single-ring mutation of GroEL, we obtained a single-ring version of GroEL bearing cysteine mutations (GroELCys) and modified its 14 apical cysteine residues with merocyanine (MC). Whereas SRMC self-dimerizes upon treatment with Mg(2+), we confirmed that SRMC serves as the efficient end-capper for the Mg(2+)-mediated supramolecular polymerization of GroELMC and allows for modulating the average nanotube length over a wide range from 320 to 40 nm by increasing the feed molar ratio SRMC/GroELMC up to 5.4. We also found that the nanotubes shorter than 100 nm are efficiently taken up into HEP3B cells.

Boronic Acid-Appended Molecular Glues for ATP-Responsive Activity Modulation of Enzymes.

Water-soluble linear polymers GumBAn (m/n = 18/6, 12/12, and 6/18) with multiple guanidinium ion (Gu(+)) and boronic acid (BA) pendants in their side chains were synthesized as ATP-responsive modulators for enzyme activity. GumBAn polymers strongly bind to the phosphate ion (PO4(-)) and 1,2-diol units of ATP via the Gu(+) and BA pendants, respectively. As only the Gu(+) pendants can be used for proteins, GumBAn is able to modulate the activity of enzymes in response to ATP. As a proof-of-concept study, we demonstrated that trypsin (Trp) can be deactivated by hybridization with GumBAn. However, upon addition of ATP, Trp was liberated to retrieve its hydrolytic activity due to a higher preference of GumBAn toward ATP than Trp. This event occurred in a much lower range of [ATP] than reported examples. Under cellular conditions, the hydrolytic activity of Trp was likewise modulated.

Biomolecular robotics for chemomechanically driven guest delivery fuelled by intracellular ATP.

The development of nanocarriers that selectively release guest molecules on sensing a particular biological signal is being actively pursued in nanomedicine for diagnostic and therapeutic purposes. Here we report a protein-based nanocarrier that opens in the presence of intracellular adenosine-5'-triphosphate (ATP). The nanocarrier consists of multiple barrel-shaped chaperonin units assembled through coordination with Mg(2+) into a tubular structure that protects guest molecules against biological degradation. When its surface is functionalized with a boronic acid derivative, the nanocarrier is able to enter cells. The hydrolysis of intracellular ATP into adenosine-5'-diphosphate (ADP) induces conformational changes of the chaperonin units, which in turns generate a mechanical force that leads to the disassembly of the tube and release of the guests. This scission occurs with a sigmoidal dependence on ATP concentration, which means that the nanocarrier can differentiate biological environments in terms of the concentration of ATP for selective guest release. Furthermore, biodistribution tests reveal preferential accumulation of the nanocarriers in a tumour tissue.

Columnarly assembled liquid-crystalline peptidic macrocycles unidirectionally orientable over a large area by an electric field.

Being inspired by naturally occurring peptidic macrocycles, we developed liquid-crystalline (LC) compounds 1 and 2 that are capable of self-assembling into hexagonal columnar mesophases over a wide temperature range that includes room temperature. Their bowl-shaped macrocyclic cores are conformationally robust because of the presence of internal H-bonds, while the columnar assembly is ensured by intermolecular H-bonding interactions involving the exocyclic amide units. When an electric field was applied to their LC films from a direction orthogonal to the film plane, the columns were oriented homeotropically over a large area.

Redox-responsive molecular helices with highly condensed π-clouds.

Helices have long attracted the attention of chemists, both for their inherent chiral structure and their potential for applications such as the separation of chiral compounds or the construction of molecular machines. As a result of steric forces, polymeric o-phenylenes adopt a tight helical conformation in which the densely packed phenylene units create a highly condensed π-cloud. Here, we show an oligomeric o-phenylene that undergoes a redox-responsive dynamic motion. In solution, the helices undergo a rapid inversion. During crystallization, however, a chiral symmetry-breaking phenomenon is observed in which each crystal contains only one enantiomeric form. Crystals of both handedness are obtained, but in a non-racemic mixture. Furthermore, in solution, the dynamic motion of the helical oligomer is dramatically suppressed by one-electron oxidation. X-ray crystallography of both the neutral and oxidized forms indicated that a hole, generated upon oxidation, is shared by the repeating o-phenylene units. This enables conformational locking of the helix, and represents a long-lasting chiroptical memory.