Caging Bioactive Triarylimidazoles: An Approach to Create Visible Light-Activatable Drugs.

Imidazoles are crucial structural components in a variety of small-molecule inhibitors designed to target different kinases in anticancer treatment. However, the effectiveness of such inhibitors is often hampered by nonspecific effects and the development of resistance. Photopharmacology provides a compelling solution by enabling external control over drug activity with spatiotemporal precision. Herein, we introduce a novel strategy for caging bioactive triarylimidazole-based drug molecules. This approach involves introducing a dialkylamino group as a photoremovable group on the carbon atom of the imidazole ring, which intrinsically modulates the core structure from planar imidazole to tetrahedral 2H-imidazole, enabling the caged compound to be selectively uncaged upon visible light exposure. We applied this innovative caging technique to SB431542, a triarylimidazole-based small-molecule inhibitor that targets the pivotal TGF-ß signaling pathway, the dysregulation of which is linked to several human diseases, including cancer. Our results demonstrated the selective inhibition of human breast cancer cell migration in vitro upon light activation, highlighting the potential of our approach to transform triarylimidazole-based drug molecules into visible light-activatable drugs, thereby facilitating spatiotemporal regulation of their pharmacological activity.

Geometry-Induced Oligomerization of Fluorine-Substituted Phenylazothiazole Photoswitches.

Photoswitches are molecules that can absorb light of specific wavelengths and undergo a reversible transformation between their trans and cis isomeric forms. In phenylazo photoswitches, it is common for the less stable cis (Z) isomer to convert back to the more stable trans (E) isomer either through photochemical or thermal means. In this research, we designed new derivatives of phenylazothiazole (PAT) photoswitches, PAT-Fn, which feature fluorine substituents on their phenyl component. These derivatives can reversibly isomerize under visible light exposure with the enrichment of E and Z isomers at photostationary state (PSS). Surprisingly, we observed an unconventional phenomenon when these PAT-Fn (n≧2) photoswitches were in their cis isomeric state in the absence of light. Instead of the anticipated transformation from cis to trans isomer, these compounds converted to an oligomeric compound. Our detailed experimental investigation and theoretical calculations, indicated the crucial role of fluorine substituents and the distinctive geometric arrangement of the cis isomer in driving the unexpected oligomerization process originating from the cis isomeric state.

Phenylazothiazoles as Visible-Light Photoswitches.

A novel class of photoswitches based on a phenylazothiazole scaffold that undergoes reversible isomerization under visible-light irradiation is reported. The photoswitch, which comprises a thiazole heteroaryl segment directly connected to a phenyl azo chromophore, has very different spectral characteristics, such as a redshifted absorption maximum wavelength and well-separated absorption bands of the trans and cis isomers, than conventional azobenzene and other heteroaryl azo compounds. Substituents at the ortho and para positions of the phenyl ring of the photoswitch resulted in a further shift to longer wavelengths up to 525 nm at the absorption maximum with a small thermal stability compensation. These photoswitches showed excellent photostationary distributions of the trans and cis isomers, thermal half-lives of up to 7.2 h, and excellent reductant stability. The X-ray crystal structure analysis revealed that the trans isomers exhibited a planar geometry and the cis isomers exhibited a T-shaped orthogonal geometry. Detailed ab initio calculations further demonstrated the plausible electronic transitions and isomerization energy barriers, which were consistent with the experimental observations. The fundamental design principles elucidated in this study will aid in the development of a wide variety of visible-light photoswitches for photopharmacological applications.

Chaperonin GroEL hydrolyses ortho-nitrophenyl ß-galactoside.

We serendipitously found that chaperonin GroEL can hydrolyze ortho-nitrophenyl ß-galactoside (ONPG), a well-known substrate of the enzyme ß-galactosidase. The ONPG hydrolysis by GroEL follows typical enzyme kinetics. Our experiments and molecular docking studies suggest ONPG binding at the ATP binding site of GroEL.

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.

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists.

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site.

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.

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.

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.

Reductively Cleavable Nanocaplets for siRNA Delivery by Template-Assisted Oxidative Polymerization.

A series of water-soluble telechelic dithiol monomers bearing multiple guanidinium ion (Gu(+)) units in their main chains were synthesized for packaging siRNA by template-assisted oxidative polymerization at their thiol termini. In the presence of siRNA, oxidative polymerization of (TEG)Gu4 affords a uniform-sized (7 ± 2 nm) nanocaplet containing siRNA (P(TEG)Gu4⊃siRNA; P(TEG)Gu4 = polymerized (TEG)Gu4). When this small conjugate is incubated with live cells, cellular uptake occurs, and the nanocaplet undergoes depolymerization in the reductive cytosolic environment to liberate the packaged siRNA. Consequently, gene expression in the live cells is suppressed.

Sub-100 nm carriers by template polymerization for drug delivery applications.

Size-controlled drug delivery systems (DDSs) have gained significant attention in the field of pharmaceutical sciences due to their potential to enhance drug efficacy, minimize side effects, and improve patient compliance. This review provides a concise overview of the preparation method, advancements, and applications of size-controlled drug delivery systems focusing on the sub-100 nm size DDSs. The importance of tailoring the size for achieving therapeutic goals is briefly mentioned. We highlight the concept of "template polymerization", a well-established method in covalent polymerization that offers precise control over molecular weight. We demonstrate the utility of this approach in crafting a monolayer of a polymer around biomolecule templates such as DNA, RNA, and protein, achieving the generation of DDSs with sizes ranging from several tens of nanometers. A few representative examples of small-size DDSs that share a conceptual similarity to "template polymerization" are also discussed. This review concludes by briefly discussing the drug release behaviors and the future prospects of "template polymerization" for the development of innovative size-controlled drug delivery systems, which promise to optimize drug delivery precision, efficacy, and safety.

Induction of molecular chirality by circularly polarized light in cyclic azobenzene with a photoswitchable benzene rotor.

New phototriggered molecular machines based on cyclic azobenzene were synthesized in which a 2,5-dimethoxy, 2,5-dimethyl, 2,5-difluorine or unsubstituted-1,4-dioxybenzene rotating unit and a photoisomerizable 3,3'-dioxyazobenzene moiety are bridged together by fixed bismethylene spacers. Depending upon substitution on the benzene moiety and on the E/Z conformation of the azobenzene unit, these molecules suffer various degrees of restriction on the free rotation of the benzene rotor. The rotation of the substituted benzene rotor within the cyclic azobenzene cavity imparts planar chirality to the molecules. Cyclic azobenzene 1, with methoxy groups at both the 2- and 5-positions of the benzene rotor, was so conformationally restricted that free rotation of the rotor was prevented in both the E and Z isomers and the respective planar chiral enantiomers were resolved. In contrast, compound 2, with 2,5-dimethylbenzene as the rotor, demonstrated the property of a light-controlled molecular brake, whereby rotation of the 2,5-dimethylbenzene moiety is completely stopped in the E isomer (brake ON, rotation OFF), while the rotation is allowed in the Z isomer (brake OFF, rotation ON). The cyclic azobenzene 3, with fluorine substitution on the benzene rotor, was in the brake OFF state regardless of E/Z photoisomerization of the azobenzene moiety. More interestingly, for the first time, we demonstrated the induction of molecular chirality in a simple monocyclic azobenzene by circular-polarized light. The key characteristics of cyclic azobenzene 2, that is, stability of the chiral structure in the E isomer, fast racemization in the Z isomer, and the circular dichroism of enantiomers of both E and Z isomers, resulted in a simple reversible enantio-differentiating photoisomerization directly between the E enantiomers. Upon exposure to r- or l-circularly polarized light at 488 nm, partial enrichment of the (S)- or (R)-enantiomers of 2 was observed.

Dynamic induction of enantiomeric excess from a prochiral azobenzene dimer under circularly polarized light.

The ability to photoinduce enantiomeric excess from the chirality of circularly polarized light (CPL) is pertinent to the study of the origin of homochirality in biomolecules. Such CPL-induced reactions, including both chirality generation and formation of partial enantiomeric imbalance, from nonchiral starting compounds have been known, however, only for the conversion of diarylolefins into chiral helicenes. In this study we synthesized three different prochiral molecules, each featuring a pair of photoisomerizable phenylazo moieties arranged symmetrically upon the phenyl rings of an sp3-hybridized carbon atom (1), the phenyl rings of [2.2]paracyclophane (2), and the ortho positions of a phenyl ring bearing a naphthyl unit (3), and then investigated the possibility of photoinducing enantiomeric excess under CPL. Irradiation of 1-3 with light induced E ↔ Z photoisomerizations of their azobenzene moieties, giving mixtures of their EE, EZ, and ZZ isomers in the photostationary state (PSS). Among these regioisomers, the EZ forms are chiral and existed as racemic mixtures of R and S stereoisomers. Upon CPL irradiation of 3, circular dichroism (CD) revealed enantiomeric enrichment of one of the EZ stereoisomers; furthermore, irradiation with r- or l-CPL gave CD signals opposite in sign, but with equal intensity, in the PSS. In contrast, 1 and 2 did not give any detectable induced CD upon CPL irradiation. These experimental results can be explained by considering the different Kuhn anisotropy factors (g) of the (R)-EZ and (S)-EZ stereoisomers of 1-3, assuming that the origin of the enantiomeric excess is the enantio-differentiating photoisomerization from EZ stereoisomers to nonchiral EE or ZZ regioisomers by r- or l-CPL. In short, we demonstrate the simultaneous induction of chirality and enantiomeric excess from a prochiral azobenzene dimer via a chiral regioisomer formed in situ upon CPL irradiation.