A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid-based imaging technology

In the development of orthogonal extra base pairs for expanding the genetic alphabet, we created novel, unnatural base pairs between fluorophore and quencher nucleobase analogues. We found that the nucleobase analogue, 2-nitropyrrole (denoted by Pn), and its 4-substitutions, such as 2-nitro-4-propynylpyrrole (Px) and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (NH(2)-hx-Px), act as fluorescence quenchers. The Pn and Px bases specifically pair with their pairing partner, 7-(2,2'-bithien-5-yl)imidazo[4,5-b]pyridine (Dss), which is strongly fluorescent. Thus, these unnatural Dss-Pn and Dss-Px base pairs function as reporter-quencher base pairs, and are complementarily incorporated into DNA by polymerase reactions as a third base pair in combination with the natural A-T and G-C pairs. Due to the static contact quenching, the Pn and Px quencher bases significantly decreased the fluorescence intensity of Dss by the unnatural base pairings in DNA duplexes. In addition, the Dss-Px pair exhibited high efficiency and selectivity in PCR amplification. Thus, this new unnatural base pair system would be suitable for detection methods of target nucleic acid sequences, and here we demonstrated the applications of the Dss-Pn and Dss-Px pairs as molecular beacons and in real-time PCR. The genetic alphabet expansion system with the replicable, unnatural fluorophore-quencher base pair will be a useful tool for sensing and diagnostic applications, as well as an imaging tool for basic research.     

Proton‐Coupled Electron Transfer Induced by Near‐Infrared Light

A proton‐coupled electron transfer reaction induced by near‐infrared light (>710 nm) has been achieved using a dye that shows intense NIR absorption property and electron/proton‐accepting abilities. The developed system generated long‐lived radical species and showed high reversibility and robustness. Mechanistic investigations suggested that the rate‐determining step of the reaction involves the proton transfer process.     

Photo‐Cross‐Linked Small‐Molecule Microarrays as Chemical Genomic Tools for Dissecting Protein–Ligand Interactions

We have developed a unique photo‐cross‐linking approach for immobilizing a variety of small molecules in a functional‐group‐independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on‐array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo‐cross‐linked microarrays of about 2000 natural products and drugs were constructed. This photo‐cross‐linked microarray format was found to be useful not merely for ligand screening but also to study the structure–activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo‐cross‐linking process.     

Pin1 and Par14 peptidyl prolyl isomerase inhibitors block cell proliferation

Disruption of the parvulin family peptidyl prolyl isomerase (PPIase) Pin1 gene delays reentry into the cell cycle when quiescent primary mouse embryo fibroblasts are stimulated with serum. Since Pin1 regulates cell cycle progression, a Pin1 inhibitor would be expected to block cell proliferation. To identify such inhibitors, we screened a chemical compound library for molecules that inhibited human Pin1 PPIase activity in vitro. We found a set of compounds that inhibited Pin1 PPIase activity in vitro with low microM IC50s and inhibited the growth of several cancer lines. Among the inhibitors, PiB, diethyl-1,3,6,8-tetrahydro-1,3,6,8-tetraoxobenzo[lmn] phenanthroline-2,7-diacetate ethyl 1,3,6,8-tetrahydro-1,3,6,8-tetraoxo-benzo[lmn] phenanthroline-(2H,7H)-diacetate, had the least nonspecific toxicity. These results suggest that Pin1 inhibitors could be used as a novel type of anticancer drug that acts by blocking cell cycle progression.     

Sensitivity and specificity of the Sanita-kun Aerobic Count: internal validation and independent laboratory study

The Sanita-kun Aerobic Count consists of a transparent cover film, an adhesive sheet, a layer of nonwoven fabric, and a water-soluble compound film, including a culture medium formula for detection of aerobic microorganisms. The Sanita-kun sheet was validated for 14 food categories in an internal study and an independent study was conducted on ground beef and hot dogs. Both studies showed no significant difference in performance between 5 or 8 replicates of the Sanita-kun sheets and AOAC Method 966.23, excluding some lots of foods. The correlation coefficient to plate count agar in the internal accuracy study was 0.99. The average relative standard deviation for repeatability of total foods was 0.26 and 0.19, respectively, excluding < 10 average counts. The ruggedness study, which examined the influence of incubation temperature and period, recommended incubation of the Sanita-kun sheet at 32.5 +/- 2.5 degrees C for 46 +/- 2 h. Comparison of 3 lots of Sanita-kun sheets showed no decrease of performance in the older lot. The shelf-life of the sheet is at least 14 months. The Sanita-kun Aerobic Counts has been granted AOAC Performance Tested Method status.     

Tuning of redox potentials by introducing a cyclometalated bond to bis-tridentate ruthenium(II) complexes bearing bis(N-methylbenzimidazolyl)benzene or -pyridine ligands

A series of asymmetrical bis-tridentate cyclometalated complexes including [Ru(Mebib)(Mebip)](+), [Ru(Mebip)(dpb)](+), [Ru(Mebip)(Medpb)](+), and [Ru(Mebib)(tpy)](+) and two bis-tridentate noncyclometalated complexes [Ru(Mebip)(2)](2+) and [Ru(Mebip)(tpy)](2+) were prepared and characterized, where Mebib is bis(N-methylbenzimidazolyl)benzene, Mebip is bis(N-methylbenzimidazolyl)pyridine, dpb is 1,3-di-2-pyridylbenzene, Medpb is 4,6-dimethyl-1,3-di-2-pyridylbenzene, and tpy is 2,2':6',2″-terpyridine. The solid-state structure of [Ru(Mebip)(Medpb)](+) is studied by X-ray crystallographic analysis. The electrochemical and spectroscopic properties of these ruthenium complexes were studied and compared with those of known complexes [Ru(tpy)(dpb)](+) and [Ru(tpy)(2)](2+). The change of the supporting ligands and coordination environment allows progressive modulation of the metal-associated redox potentials (Ru(II/III)) from +0.26 to +1.32 V vs Ag/AgCl. The introduction of a ruthenium cyclometalated bond in these complexes results in a significant negative potential shift. The Ru(II/III) potentials of these complexes were analyzed on the basis of Lever's electrochemical parameters (E(L)). Density functional theory (DFT) and time-dependent DFT calculations were carried out to elucidate the electronic structures and spectroscopic spectra of complexes with Mebib or Mebip ligands.     

Preparation and properties of fluorine-containing polyarylates from tetrafluoroisophthaloyl chloride and bisphenols

Fluorine-containing polyarylates having inherent viscosities of 0.2–0.8 dL/g were prepared from tetrafluoroisophthaloyl chloride and various bisphenols by low temperature solution polycondensation in chloroform with triethylamine or by two-phase polycondensation in a dichloromethane-water or nitrobenzene-water system with benzyltriethylammonium chloride as the phase transfer catalyst. These polyarylates were amorphous and were readily soluble in various solvents, including chloroform and N-methyl-2-pyrrolidone. The glass transition temperature of the polymer derived from 2,2-bis(4-hydroxyphenyl) propane was 150°C. These polyarylates started to lose weight around 350°C in an air or nitrogen atmosphere.