Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution changes.

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed "fixation-driven chemical cross-linking of exogenous ligands (FixEL)," which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules.

Development of a novel PET ligand, [11C]GO289 targeting CK2 expressed in the brain.

Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image ß-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to ß-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration.

Discovery of 2,6-Dihalopurines as Stomata Opening Inhibitors: Implication of an LRX-Mediated H+-ATPase Phosphorylation Pathway.

Stomata are pores in the leaf epidermis of plants and their opening and closing regulate gas exchange and water transpiration. Stomatal movements play key roles in both plant growth and stress responses. In recent years, small molecules regulating stomatal movements have been used as a powerful tool in mechanistic studies, as well as key players for agricultural applications. Therefore, the development of new molecules regulating stomatal movement and the elucidation of their mechanisms have attracted much attention. We herein describe the discovery of 2,6-dihalopurines, AUs, as a new stomatal opening inhibitor, and their mechanistic study. Based on biological assays, AUs may involve in the pathway related with plasma membrane H+-ATPase phosphorylation. In addition, we identified leucine-rich repeat extensin proteins (LRXs), LRX3, LRX4 and LRX5 as well as RALF, as target protein candidates of AUs by affinity based pull down assay and molecular dynamics simulation. The mechanism of stomatal movement related with the LRXs-RALF is an unexplored pathway, and therefore further studies may lead to the discovery of new signaling pathways and regulatory factors in the stomatal movement.

Synthesis, properties, and material hybridization of bare aromatic polymers enabled by dendrimer support.

Aromatic polymers are the first-choice platform for current organic materials due to their distinct optical, electronic, and mechanical properties as well as their biocompatibility. However, bare aromatic polymer backbones tend to strongly aggregate, rendering them essentially insoluble in organic solvent. While the typical solution is to install many solubilizing substituents on the backbones, this often provokes undesired property changes. Herein, we report the synthesis of bare aromatic polymers enabled by a dendrimer support. An initiator arene containing a diterpenoid-based dendrimer undergoes Pd-catalyzed polymerization with monomers bearing no solubilizing substituents to furnish bare aromatic polymers such as polythiophenes and poly(para-phenylene)s. The high solubility of dendrimer-ligated polymers allows not only the unveiling of the properties of unsubstituted π-conjugated backbone, but also mild release of dendrimer-free aromatic polymers and even transfer of aromatic polymers to other materials, such as silica gel and protein, which may accelerate the creation of hybrid materials nowadays challenging to access.

Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock.

CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop" located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

An Isoform-Selective Modulator of Cryptochrome 1 Regulates Circadian Rhythms in Mammals.

Cryptochrome 1 (CRY1) and CRY2 are core regulators of the circadian clock, and the development of isoform-selective modulators is important for the elucidation of their redundant and distinct functions. Here, we report the identification and functional characterization of a small-molecule modulator of the mammalian circadian clock that selectively controls CRY1. Cell-based circadian chemical screening identified a thienopyrimidine derivative KL201 that lengthened the period of circadian rhythms in cells and tissues. Functional assays revealed stabilization of CRY1 but not CRY2 by KL201. A structure-activity relationship study of KL201 derivatives in combination with X-ray crystallography of the CRY1-KL201 complex uncovered critical sites and interactions required for CRY1 regulation. KL201 bound to CRY1 in overlap with FBXL3, a subunit of ubiquitin ligase complex, and the effect of KL201 was blunted by knockdown of FBXL3. KL201 will facilitate isoform-selective regulation of CRY1 to accelerate chronobiology research and therapeutics against clock-related diseases.

Construction of a Fluorescent Screening System of Allosteric Modulators for the GABAA Receptor Using a Turn-On Probe.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABAA receptors (GABAARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABAARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABAARs. We here developed the first turn-on fluorescent imaging probe for GABAARs, which can be used to quantitatively evaluate ligand-receptor interactions under live cell conditions. Using noncovalent labeling of GABAARs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABAAR, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABAARs. These results highlight the power of the use of a turn-on fluorescent probe to screen drugs for complicated membrane proteins that cannot be addressed by conventional methods.

Recognition-driven chemical labeling of endogenous proteins in multi-molecular crowding in live cells.

Endogenous protein labeling is one of the most invaluable methods for studying the bona fide functions of proteins in live cells. However, multi-molecular crowding conditions, such as those that occur in live cells, hamper the highly selective chemical labeling of a protein of interest (POI). We herein describe how the efficient coupling of molecular recognition with a chemical reaction is crucial for selective protein labeling. Recognition-driven protein labeling is carried out by a synthetic labeling reagent containing a protein (recognition) ligand, a reporter tag, and a reactive moiety. The molecular recognition of a POI can be used to greatly enhance the reaction kinetics and protein selectivity, even under live cell conditions. In this review, we also briefly discuss how such selective chemical labeling of an endogenous protein can have a variety of applications at the interface of chemistry and biology.

Affinity-Guided Oxime Chemistry for Selective Protein Acylation in Live Tissue Systems.

Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.

Synthesis of Triarylpyridines in Thiopeptide Antibiotics by Using a C-H Arylation/Ring-Transformation Strategy.

We have described a C-H arylation/ring-transformation strategy for the synthesis of triarylpyridines, which form the core structure of thiopeptide antibiotics. This synthetic method readily gave 2,3,6-triarylpyridines in a regioselective manner by a two-phase approach: C-H arylation (a nickel-catalyzed decarbonylative Suzuki-Miyaura cross-coupling and decarbonylative C-H coupling for the synthesis of 2,4-diaryloxazoles) and ring transformation ([4+2] cycloaddition of 2,4-diaryloxazoles with (hetero)arylacrylic acids). To showcase these methods, we have accomplished the formal synthesis of thiopeptide antibiotics GE2270 s and amythiamicins.

C7-derivatization of C3-alkylindoles including tryptophans and tryptamines.

A versatile strategy for C7-selective boronation of tryptophans, tryptamines, and 3-alkylindoles by way of a single-pot C2/C7-diboronation-C2-protodeboronation sequence is described. The combination of a mild iridium-catalyzed C2/C7-diboronation followed by an in situ palladium-catalyzed C2-protodeboronation allows efficient entry to valuable C7-boroindoles that enable further C7-derivatization. The versatility of the chemistry is highlighted by the gram-scale synthesis of C7-boronated N-Boc-L-tryptophan methyl ester and the rapid synthesis of C7-halo, C7-hydroxy, and C7-aryl tryptophan derivatives.

ß-Selective C-H arylation of pyrroles leading to concise syntheses of lamellarins C and I.

The first general ß-selective C-H arylation of pyrroles has been developed by using a rhodium catalyst. This C-H arylation reaction, which is retrosynthetically straightforward but results in unusual regioselectivity, could result in de novo syntheses of pyrrole-derived natural products and pharmaceuticals. As such, we have successfully synthesized polycyclic marine pyrrole alkaloids, lamellarins C and I, by using this ß-selective arylation of pyrroles with aryl iodides (C-H/C-I coupling) and a new double C-H/C-H coupling as key steps.

Late-Stage C-H Coupling Enables Rapid Identification of HDAC Inhibitors: Synthesis and Evaluation of NCH-31 Analogues.

We previously reported the discovery of NCH-31, a potent histone deacetylase (HDAC) inhibitor. By utilizing our C-H coupling reaction, we rapidly synthesized 16 analogues (IYS-1 through IYS-15 and IYS-Me) of NCH-31 with different aryl groups at the C4-position of 2-aminothiazole core of NCH-31. Subsequent biological testing of these derivatives revealed that 3-fluorophenyl (IYS-10) and 4-fluorophenyl (IYS-15) derivatives act as potent pan-HDAC inhibitor. Additionally, 4-methylphenyl (IYS-1) and 3-fluoro-4-methylphenyl (IYS-14) derivatives acted as HDAC6-insensitive inhibitors. The present work clearly shows the power of the late-stage C-H coupling approach to rapidly identify novel and highly active/selective biofunctional molecules.

Decarbonylative C-H coupling of azoles and aryl esters: unprecedented nickel catalysis and application to the synthesis of muscoride A.

A nickel-catalyzed decarbonylative C-H biaryl coupling of azoles and aryl esters is described. The newly developed catalytic system does not require the use of expensive metal catalysts or silver- or copper-based stoichiometric oxidants. We have successfully applied this new C-H arylation reaction to a convergent formal synthesis of muscoride A.