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.

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.

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.

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.

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.

HaloTag-based conjugation of proteins to barcoding-oligonucleotides.

Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Synthesis of Azidoanilines by the Buchwald-Hartwig Amination.

We report a Buchwald-Hartwig amination compatible with azido functionality. Treatment of azidoaryl iodides and amines with fourth-generation Buchwald precatalyst coordinated by CPhos and sodium tert-butoxide in 1,4-dioxane at 50 °C afforded the corresponding azidoanilines while leaving the azido groups intact. The method showed a broad substrate scope and was applicable to the synthesis of diazido compounds as photoaffinity probe candidates of pharmaceutical amines and multiazido platform molecules.

Direct 3-Acylation of Indolizines by Carboxylic Acids for the Practical Synthesis of Red Light-Releasable Caged Carboxylic Acids.

To enhance the practicality of photouncaging system using 3-acyl-2-methoxyindolizines, direct acylation of indolizines with carboxylic acids was developed using condensation reagents, generally used for peptide coupling. This method allowed for caging a broad range of carboxylic acids with indolizines. The method enabled a facile synthesis of water-soluble caged bioactive carboxylic acids having an intramolecular photosensitizer. The efficient release of carboxylic acids from the synthesized caged compounds upon red light irradiation was confirmed in neutral buffered solutions.

Convergent Synthesis of Fluoroalkenes Using a Dual-Reactive Unit.

Fluoroalkenes have shown importance as a metabolically stable isostere of amide compounds. To expedite the synthesis of diverse fluoroalkenes, we have developed a dual-reactive C2-unit, (Z)-1-boryl-1-fluoro-2-tosyloxyethene, containing nucleophilic and electrophilic moieties. Consecutive palladium-catalyzed cross-coupling reactions of this unit with aryl bromides and aryl boronic acids allow for the convergent synthesis of diverse trans-1,2-diaryl-substituted fluoroethenes in a chemoselective and stereoretentive manner.

Generation of Alkyl Radical through Direct Excitation of Boracene-Based Alkylborate.

The generation of tertiary, secondary, and primary alkyl radicals has been achieved by the direct visible-light excitation of a boracene-based alkylborate. This system is based on the photophysical properties of the organoboron molecule. The protocol is applicable to decyanoalkylation, Giese addition, and nickel-catalyzed carbon-carbon bond formations such as alkyl-aryl cross-coupling or vicinal alkylarylation of alkenes, enabling the introduction of various C(sp3) fragments to organic molecules.

A novel yellow fluorescent protein of recombinant apoPholasin with dehydrocoelenterazine.

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λmax = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H2O2 system, but not in sodium hypochlorite.

Quantification of aromatase binding in the female human brain using [11 C]cetrozole positron emission tomography.

Aromatase, the enzyme that in the brain converts testosterone and androstenedione to estradiol and estrone, respectively, is a putative key factor in psychoneuroendocrinology. In vivo assessment of aromatase was performed to evaluate tracer kinetic models and optimal scan duration, for quantitative analysis of the aromatase positron emission tomography (PET) ligand [11 C]cetrozole. Anatomical magnetic resonance and 90-min dynamic [11 C]cetrozole PET-CT scans were performed on healthy women. Volume of interest (VOI)-based analyses with a plasma-input function were performed using the single-tissue and two-tissue (2TCM) reversible compartment models and plasma-input Logan analysis. Additionally, the simplified reference tissue model (SRTM), Logan reference tissue model (LRTM), and standardized uptake volume ratio model, with cerebellum as reference region, were evaluated. Parametric images were generated and regionally averaged voxel values were compared with VOI-based analyses of the reference tissue models. The optimal reference model was used for evaluation of a decreased scan duration. Differences between the plasma-input- and reference tissue-based methods and comparisons between scan durations were assessed by linear regression. The [11 C]cetrozole time-activity curves were best described by the 2TCM. SRTM nondisplaceable binding potential (BPND ), with cerebellum as reference region, can be used to estimate [11 C]cetrozole binding and generated robust and quantitatively accurate results for a reduced scan duration of 60 min. Receptor parametric mapping, a basis function implementation of SRTM, as well as LRTM, produced quantitatively accurate parametric images, showing BPND at the voxel level. As PET tracer, [11 C]cetrozole can be employed for relatively short brain scans to measure aromatase binding using a reference tissue-based approach.

Synthesis of Diverse Aromatic Ketones through C-F Cleavage of Trifluoromethyl Group.

An efficient synthetic method of aromatic ketones through C-F cleavage of trifluoromethyl group is disclosed. The high functional group tolerance of the transformation and the remarkable stability of trifluoromethyl group in various reactions enabled multi-substituted aromatic ketone synthesis in an efficient route involving useful transformations such as ortho-lithiation, aryne chemistry, and cross-couplings.

Functionalization of a Single C-F Bond of Trifluoromethylarenes Assisted by an ortho-Silyl Group Using a Trityl-Based All-in-One Reagent with Ytterbium Triflate Catalyst.

Catalytic thiolation and azidation of a single C-F bond of trifluoromethylarenes were achieved assisted by an ortho-silyl group with all-in-one reagents to generate a trityl cation and nucleophiles. The reactions catalyzed by ytterbium triflate efficiently afforded a wide variety of difluoromethylenes avoiding the further C-F bond cleavage, by virtue of the mild conditions without the generation of a Brønsted acid.

Practical Synthesis of [18F]Pitavastatin and Evaluation of Hepatobiliary Transport Activity in Rats by Positron Emission Tomography.

We developed a practical synthetic method for fluorine-18 (18F)-labeled pitavastatin ([18F]PTV) as a positron emission tomography (PET) tracer to assess hepatobiliary transporter activity and conducted a PET scan as a preclinical study for proof-of-concept in rats. This method is a one-pot synthesis involving aromatic 18F-fluorination of an arylboronic acid ester followed by deprotection under acidic conditions, which can be reproduced in general clinical sites equipped with a standard radiolabeling system due to the simplified procedure. PET imaging confirmed that intravenously administered [18F]PTV was rapidly accumulated in the liver and gradually transferred into the intestinal lumen through the bile duct. Radiometabolite analysis showed that [18F]PTV was metabolically stable, and 80% of the injected dose was detected as the unchanged form in both blood and bile. We applied integration plot analysis to assess tissue uptake clearance (CLuptake, liver and CLuptake, kidney) and canalicular efflux clearance (CLint, bile), and examined the effects of inhibitors on membrane transport. Treatment with rifampicin, an organic anion transporting polypeptide inhibitor, significantly reduced CLuptake, liver and CLuptake, kidney to 44% and 64% of control, respectively. In contrast, Ko143, a breast cancer resistance protein inhibitor, did not affect CLuptake, liver but significantly reduced CLint, bile to 39% of control without change in [18F]PTV blood concentration. In addition, we found decreased CLuptake, liver and increased CLint, bile in Eisai hyperbilirubinemic rats in response to altered expression levels of transporters. We expect that [18F]PTV can be translated into clinical application, as our synthetic method does not need special apparatus in the radiolabeling system and PET scan with [18F]PTV can quantitatively evaluate transporter activity in vivo.

Expression, purification, and characterization of recombinant apoPholasin.

Pholasin is a reactive oxygen-sensitive photoprotein that consists of an apoprotein (apoPholasin) and an unknown chromophore. The preferred human codon-optimized apoPholasin gene was transiently expressed in mammalian cells and apoPholasin was detected using an anti-recombinant apoPholasin antibody. For the first time, we found that apoPholasin secreted into the culture medium could catalyze the oxidation of coelenterazine (CTZ, a luciferin) to produce continuous luminescence. The fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) was successfully expressed as a soluble form in bacterial cells using the cold induction system. The purified GST-apoPholasin also had luminescence activity with CTZ, showing the bioluminescence emission peak at 461 nm, and the resultant product showed purple blue fluorescence under 365 nm light. Unexpectedly, the main oxidation product of CTZ was identified as coelenteramine (CTM), not coelenteramide (CTMD).

Consecutive Aryne Generation Strategy for the Synthesis of 1,3-Diarylpyrazoles.

An efficient method for the synthesis of diverse 1,3-diarylpyrazoles via consecutive aryne generation has been developed. The bisaryne precursors bearing o-iodo- and o-silylaryl triflate moieties were prepared by sequential Suzuki-Miyaura and Chan-Lam-Evans cross-coupling reactions. The selective generation of the first aryne triggered by a silylmethyl Grignard reagent followed by the second aryne generation mediated by a fluoride ion allowed for the synthesis of diverse multisubstituted 1,3-diarylpyrazoles in a modular synthetic manner with various arynophiles.

Identification of a novel oxidation product from yellow fluorophore in the complex of apoPholasin and dehydrocoelenterazine.

The complex of the recombinant fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) with non-fluorescent dehydrocoelenterazine (dCTZ) (GST-apoPholasin/dCTZ complex) shows yellow fluorescence at 539 nm by excitation at 430 nm. The GST-apoPholasin/dCTZ complex with a fluorophore (dCTZ*) has considerably weak luminescence activity, converting slowly to a blue fluorescence protein with the emission peak at 430 nm. The main oxidation products from dCTZ* for blue fluorescence were identified as coelenteramine (CTM) and an unreported pyrazine derivative, 3-benzyl-5-(4-hydroxyphenyl)pyrazin-2(1H)-one (CTO) that was confirmed by chemical synthesis.

Boracene-based alkylborate enabled Ni/Ir hybrid catalysis.

Boracene-based alkylborate enabled visible light-mediated metallaphotoredox catalysis. The directly excited borate was easily oxidatively quenched by an excited Ir photoredox catalyst. Ni/Ir hybrid catalysis afforded the products under significantly low irradiance.