1,3-Butadiynyl sulfide-based compact trialkyne platform molecule for sequential assembly of three azides.

A compact trialkyne platform with a silyl-protected 1,3-butadiynyl sulfide moiety and a terminal alkyne group has been developed for sequential regioselective transition metal-catalyzed triazole formation reactions with three azides. This method enabled the facile construction of a low-molecular-weight triazole library and the synthesis of middle-molecular-weight trifunctional probes for protein modification.

Chemical inhibition of phosphatidylcholine biogenesis reveals its role in mitochondrial division.

Phospholipids are major components of biological membranes and play structural and regulatory roles in various biological processes. To determine the biological significance of phospholipids, the use of chemical inhibitors of phospholipid metabolism offers an effective approach; however, the availability of such compounds is limited. In this study, we performed a chemical-genetic screening using yeast and identified small molecules capable of inhibiting phosphatidylcholine (PC) biogenesis, which we designated PC inhibitors 1, 2, 3, and 4 (PCiB-1, 2, 3, and 4). Biochemical analyses indicated that PCiB-2, 3, and 4 inhibited the phosphatidylethanolamine (PE) methyltransferase activity of Cho2, whereas PCiB-1 may inhibit PE transport from mitochondria to the endoplasmic reticulum (ER). Interestingly, we found that PCiB treatment resulted in mitochondrial fragmentation, which was suppressed by expression of a dominant-negative mutant of the mitochondrial division factor Dnm1. These results provide evidence that normal PC biogenesis is important for the regulation of mitochondrial division.

Clickable bisreactive small gold nanoclusters for preparing multifunctionalized nanomaterials: application to photouncaging of an anticancer molecule.

In this study, we successfully synthesized a small-sized gold nanocluster (2 nm) coated with homogeneous tripeptides bearing azido and amino groups that enable facile multifunctionalizations. Using sodium phenoxide to reduce tetrachloroauric(iii) acid in the presence of the cysteine-containing tripeptide, we efficiently prepared the gold nanoclusters without damaging the azido group. We then utilized this clickable bisreactive nanocluster as a versatile platform for synthesizing multifunctionalized gold nanomaterials. The resulting nanoclusters were conjugated with an anticancer compound connected to an indolizine moiety for photoinduced uncaging, a photodynamic therapy agent acting as a photosensitizer for uncaging, and a cyclic RGD peptide. The cytotoxicity of the multifunctionalized gold nanoclusters was demonstrated through red light irradiation of human lung cancer-derived A549 cells treated with the synthesized nanomaterials. The significant cytotoxicity exhibited by the cells underscores the potential utility of this method in advanced cancer therapies.

Palladium-Catalyzed ipso-Borylation of Aryl Halides Promoted by Lewis Acid-Mediated Electrophilic Activation of Aryl(halo)palladium(II) Complex.

Palladium-catalyzed ipso-borylation of aryl halides, well-known as Miyaura borylation, is one of the reliable synthetic methods for organoborons. This reaction involves base-mediated nucleophilic activation of diboron that enables transmetalation of an aryl(halo)palladium(II) intermediate with a diboron. As an alternative, herein, we have established Lewis acid-mediated conditions for borylating (pseudo)haloarenes that require no external base. The electrophilic activation of the aryl(halo)palladium(II) intermediate via dehalogenation with Lewis acidic zinc complexes promotes the borylation.

Synthesis of Multisubstituted Aromatics via 3-Triazenylarynes.

An efficient method for generating 3-triazenylarynes from ortho-iodoaryl triflate-type precursors was developed. The generated arynes reacted with various arynophiles with high regioselectivity because of the triazenyl group. The 3-triazenylaryne precursors functioned as useful intermediates of diverse multisubstituted aromatic compounds through the transformation of the remaining triazenyl group of aryne adducts and triazenyl group-directed ortho-C-H functionalization.

11 C-Cyanation of Aryl Fluorides via Nickel and Lithium Chloride-Mediated C-F Bond Activation.

Aryl fluorides are expected to be useful as radiolabeling precursors due to their chemical stability and ready availability. However, direct radiolabeling via carbon-fluorine (C-F) bond cleavage is a challenging issue due to its significant inertness. Herein, we report a two-phase radiosynthetic method for the ipso-11 C-cyanation of aryl fluorides to obtain [11 C]aryl nitriles via nickel-mediated C-F bond activation. We also established a practical protocol that avoids the use of a glovebox, except for the initial preparation of a nickel/phosphine mixture, rendering the method applicable for general PET centers. This method enabled the efficient synthesis of diverse [11 C]aryl nitriles from the corresponding aryl fluorides, including pharmaceutical drugs. Stoichiometric reactions and theoretical studies indicated a significant promotion effect of lithium chloride on the oxidative addition, affording an aryl(chloro)nickel(II) complex, which serves as a precursor for rapid 11 C-cyanation.

Acute nicotine exposure blocks aromatase in the limbic brain of healthy women: A [11C]cetrozole PET study.

BACKGROUND: Of interest to women's mental health, a wealth of studies suggests sex differences in nicotine addiction and treatment response, but their psychoneuroendocrine underpinnings remain largely unknown. A pathway involving sex steroids could indeed be involved in the behavioural effects of nicotine, as it was found to inhibit aromatase in vitro and in vivo in rodents and non-human primates, respectively. Aromatase regulates the synthesis of oestrogens and, of relevance to addiction, is highly expressed in the limbic brain. METHODS: The present study sought to investigate in vivo aromatase availability in relation to exposure to nicotine in healthy women. Structural magnetic resonance imaging and two [11C]cetrozole positron emission tomography (PET) scans were performed to assess the availability of aromatase before and after administration of nicotine. Gonadal hormones and cotinine levels were measured. Given the region-specific expression of aromatase, a ROI-based approach was employed to assess changes in [11C]cetrozole non-displaceable binding potential. RESULTS: The highest availability of aromatase was found in the right and left thalamus. Upon nicotine exposure, [11C]cetrozole binding in the thalamus was acutely decreased bilaterally (Cohen's d = -0.99). In line, cotinine levels were negatively associated with aromatase availability in the thalamus, although as non-significant trend. CONCLUSIONS: These findings indicate acute blocking of aromatase availability by nicotine in the thalamic area. This suggests a new putative mechanism mediating the effects of nicotine on human behaviour, particularly relevant to sex differences in nicotine addiction.

Synthesis of benzo[b]furans from alkynyl sulfoxides and phenols by the interrupted Pummerer reaction.

The interrupted Pummerer reaction of alkynyl sulfoxides with phenols is disclosed. A wide range of benzo[b]furans were efficiently synthesized through unexplored electrophilic activation of the electron-deficient alkynyl sulfinyl group. Based on the good availability of alkynyl sulfoxides, we successfully prepared various functionalized benzo[b]furans from readily available alkynes, thiosulfonates, and phenols.

Synthesis of Functionalized Dibenzoazacyclooctynes by a Decomplexation Method for Dibenzo-Fused Cyclooctyne-Cobalt Complexes.

A concise route for dibenzoazacyclooctynes (DIBACs) synthesis was developed based on Pictet-Spengler reaction and a novel cobalt decomplexation method established for dibenzo-fused cyclooctyne-cobalt complexes. The method allowed for the facile preparation of functionalized DIBACs, including bisDIBAC, which served as an efficient bisreactive linker for protein modification via the double-click reaction.

Substitution of α-Azido Sulfones with Thiolates to Form α-Azido Sulfides.

Upon treatment of α-azido sulfones with a thiol in the presence of 1,1,3,3-tetramethylguanidine, substitution of the sulfonyl group with a thiolate occurred, resulting in the formation of α-azido sulfides. Based on experimental results and DFT calculations, a reaction mechanism that involves the addition of a thiolate to the azido group and generation of an alkylidene triazene is proposed.

Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate.

Native Oplophorus luciferase (OpLase) and its catalytic 19 kDa protein (wild KAZ) show highest luminescence activity with coelenterazine (CTZ) among CTZ analogs. Mutated wild KAZ with 16 amino acid substitutions (nanoKAZ/nanoLuc) utilizes bis-coelenterazine (bis-CTZ) as the preferred substrate and exhibits over 10-fold higher maximum intensity than CTZ. To understand the substrate selectivity of nanoKAZ between CTZ and bis-CTZ, we prepared the reverse mutants of nanoKAZ by amino acid replacements with the original amino acid residue of wild KAZ. The reverse mutant with L18Q and V27L substitutions (QL-nanoKAZ) exhibited 2.6-fold higher maximum intensity with CTZ than that of nanoKAZ with bis-CTZ. The catalytic properties of QL-nanoKAZ including substrate specificity, luminescence spectrum, luminescence kinetics, luminescence products of CTZ, and luminescence inhibition by deaza-CTZ analogs were characterized and were compared with other CTZ-utilizing luciferases such as Gaussia and Renilla luciferases. Thus, QL-nanoKAZ with CTZ could be used as a potential reporter protein for various luminescence assay systems. Furthermore, the crystal structure of QL-nanoKAZ was determined at 1.70 Å resolution. The reverse mutation at the L18Q and V27L positions of α2-helix in nanoKAZ led to changes in the local structures of the α4-helix and the ß6- and ß7-sheets, and might enhance its binding affinity and oxidation efficiency with CTZ to emit light.

Azido-type-selective triazole formation by iridium-catalyzed cycloaddition with thioalkynes.

The iridium-catalyzed azide-thioalkyne cycloaddition was found to proceed much faster with benzyl azide than with phenyl azide. The high azido-type selectivity was also observed in other combinations of azides with different steric environments. This finding enabled the efficient assembly of three azidophilic molecules to triazido platforms by three sequential triazole-forming reactions.

Clinical evaluation of [18F]pitavastatin for quantitative analysis of hepatobiliary transporter activity.

It is widely accepted that uptake and efflux transporters on clearance organs play crucial roles in drug disposition. Although in vitro transporter assay system can identify the intrinsic properties of the target transporters, it is not so easy to precisely predict in vivo pharmacokinetic parameters from in vitro data. Positron emission tomography (PET) imaging is a useful tool to directly assess the activity of drug transporters in humans. We recently developed a practical synthetic method for fluorine-18-labeled pitavastatin ([18F]PTV) as a PET probe for quantitative evaluation of hepatobiliary transport. In the present study, we conducted clinical PET imaging with [18F]PTV and compared the pharmacokinetic properties of the probe for healthy subjects with or without rifampicin pretreatment. Rifampicin pretreatment significantly suppressed the hepatic maximum concentration and biliary excretion of the probe to 52% and 34% of the control values, respectively. Rifampicin treatment markedly decreased hepatic uptake clearance (21% of the control), and moderately canalicular efflux clearance with regard to hepatic concentration (52% of the control). These results demonstrate that [18F]PTV is a useful probe for clinical investigation of the activities of hepatobiliary uptake/efflux transporters in humans.

Expression and purification of DYRK1A kinase domain in complex with its folding intermediate-selective inhibitor FINDY.

The kinase DYRK1A phosphorylates substrate proteins that are involved in the progression of many diseases. DYRK1A also phosphorylates its own residues on key elements intramolecularly to activate and stabilize itself during the folding process. Once the folding process of DYRK1A has completed, it can no longer catalyzes the intramolecular reaction, suggesting that a transitional intermediate state that catalyzes the autophosphorylation exists. In the previous study, we identified a small molecule, designated as FINDY, that selectively inhibits the folding intermediate of DYRK1A. Although evidence has suggested that FINDY targets the ATP-binding pocket of DYRK1A, it remains elusive as to whether the DYRK1A kinase domain could be purified as a complex with FINDY. In this study, we successfully expressed and purified the kinase domain of DYRK1A in complex with FINDY. The DYRK1A kinase domain was expressed as a fusion protein with a hexahistidine tag and ZZ-domain (His-ZZ-DYRK1A) at 6 °C by using a cold shock induction system in Escherichia coli cells. The cells were incubated with FINDY. The cell pellets were gently extracted on ice and subjected to immobilized-metal affinity chromatography. The amount of FINDY in the elution fraction was measured by UV absorbance specific for FINDY. The eluate contained FINDY with the ratio of FINDY to DYRK1A protein being 0.15 in quadruplicate experiments. Thus, this study demonstrates the direct interaction between the DYRK1A kinase domain and FINDY, paving the way for structural determination of the complex.

Hydride reduction of o-(fluorosilyl)benzodifluorides for subsequent C-F transformations.

An efficient method for sequential C-F transformations of o-hydrosilyl-substituted benzotrifluorides is disclosed. A key to the success is hydride reduction of o-fluorosilyl-substituted difluoromethylenes prepared by a single C-F transformation of o-hydrosilyl-substituted benzotrifluorides. We succeeded in further C-F transformations via hydride abstraction of the resulting o-hydrosilyl group, enabling us to synthesize a wide variety of organofluorine compounds.

Modular synthesis of triazoles from 2-azidoacrylamides having a nucleophilic amino group.

Assembling methods using 2-azidoacrylamides having a nucleophilic amino group are disclosed. Divergent transformations of the amine-type trivalent platform were accomplished with a wide variety of electrophiles to obtain a broad range of 2-azidoacrylamides involving a fluorosulfonyl group-containing trivalent platform. Consecutive click conjugations including triazole formation, thiol-ene-type 1,4-addition, and SuFEx reactions realized the efficient assembly of easily available simple modules.

Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase.

Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase-NADH reaction). CTZ reduced from dCTZ in the FRase-NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase-NADH reaction.

Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+ -Binding Photoprotein Aequorin.

Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca2+ , similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.

Red light-induced conjugation of amines through amide bond formation triggered via photooxidation of 3-acylindolizines.

The development of a conjugation method initiated by irradiation of long-wavelength light (>500 nm) to prepare densely functionalized molecules while avoiding undesired photodegradation has attracted considerable attention. Here we show an amide bond formation method based on the photoreaction of 3-acylindolizines in the presence of amines triggered via red-light irradiation. Photooxidation of 3-acylindolizines using a catalytic amount of a photosensitizer and red light-emitting diodes (660 nm) affords the corresponding conjugated amides in nearly quantitative yields within <5 min. This transformation can be performed in aqueous organic solvents and is applicable to diverse aliphatic amines with various functional groups, including the moieties responsive to short-wavelength light.

Structure-activity relationship for the folding intermediate-selective inhibition of DYRK1A.

DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.