Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.

Synthesis and structural analyses of phenylethynyl-substituted tris(2-pyridylmethyl)amines and their copper(ii) complexes.

Three new tris(2-pyridylmethyl)amine-based ligands possessing phenylethynyl units have been prepared using Sonogashira couplings and substitution reactions. Copper(ii) complexes of those tetradentate ligands have also been synthesized. Solid-state structures of the six new compounds have been determined by single-crystal X-ray diffraction analyses. Examination of the molecular structures of the ligands revealed the expected triangular geometries with virtually undeformed carbon-carbon triple bonds. While the tertiary nitrogen of the free ligands seem to be prevented from participation in supramolecular non-covalent interactions by the pyridyl hydrogen at the 3-position, the pyridyl nitrogens play a crucial role in the packing mode of the crystal structure. The nitrogens form weak hydrogen bonds, varied in length between 2.32 and 2.66 Å, with the pyridyl hydrogen of its neighbouring molecule. The [NH-C] contacts enforce one-dimensional columnar assemblies on ligands that organize into wall-like structures, which in turn assemble into three-dimensional structures through CH-π interactions. Structural analyses of Cu(ii) complexes of the ligands revealed propeller-like structures caused by steric crowding of three pyridine ligands. The copper complexes of the ligands having three phenylethynyl substituents showed a remarkably deformed carbon-carbon triple bond enforced by a steric effect of the three phenyl groups. Most significantly, a total of seventy non-covalent interactions, classified into twelve types of hydrogen-involving short contacts, were identified in this study. The phenylethynyl substituent participated in forty-two interactions as a hydrogen bond acceptor, and its role was more distinctive in the crystal structures of the Cu(ii) complexes.

Strong intramolecular charge transfer emission in benzobisoxazole cruciforms: solvatochromic dyes as polarity indicators.

A photophysical study was carried out on a series of nine X-shaped cross-conjugated compounds based on the benzobisoxazole nucleus. These "molecular cruciforms" were terminally substituted with electron-rich 4-(N,N-dimethylamino)phenyl, electron-neutral phenyl or electron-poor 4-pyridyl groups. Intramolecular charge transfer (ICT) transitions in molecular cruciforms were shown to be activated to a different extent by the combinations of these substituents located in different positions along vertical and/or horizontal axes of the molecules, showing strong emission throughout the visible spectrum. The high fluorescence efficiency and bathochromic shifts associated with the increasing polarity of the solvent make these cruciforms interesting candidates for use as fluorescence polarity indicators operating in a broad solvatochromic range.

Qualitative identification of carboxylic acids, boronic acids, and amines using cruciform fluorophores.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms' HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms' frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljanic groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte's structure and - because of cruciforms' charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10(-6) M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Selective and sensitive fluoride detection through alkyne cruciform desilylation.

Desilylation of silylethynyl-substituted benzobisoxazole cruciforms can be achieved using stoichiometric amounts of fluoride, leading to a significant change in their UV-vis absorption and fluorescence. This response is observable at micromolar concentrations of fluoride, and, in the case of a triisopropylsilyl-substituted cruciform fluorophore, extraordinarily selective for fluoride over other small inorganic anions, including hydroxide, acetate, and phosphate.

Benzobisoxazole fluorophore vicariously senses amines, ureas, anions.

A benzobisoxazole-based cruciform fluorophore forms fluorescent complexes with simple boronic acids. Through the changes of their fluorescence emission colours, these complexes can sense and qualitatively distinguish among structurally similar organic nitrogen compounds (amines and ureas) and small organic and inorganic anions. Preliminary results suggest that the intensity of this hybrid sensor's fluorescent response to chloride anions can be quantitatively correlated to chloride concentration.

Benzobisoxazole cruciforms: heterocyclic fluorophores with spatially separated frontier molecular orbitals.

We report the synthesis of nine conjugated cruciform-shaped molecules based on the central benzo[1,2-d:4,5-d']bisoxazole nucleus, at which two conjugated currents intersect at a ~90° angle. Cruciforms' substituents were varied pairwise among the electron-neutral phenyl groups, electron-rich 4-(N,N-dimethylamino)phenyl substituents, and electron-poor pyridines. Hybrid density functional theory calculations revealed that the highest occupied molecular orbitals (HOMOs) are localized (24-99%) in all cruciforms, in contrast to the lowest unoccupied molecular orbitals (LUMOs) which are strongly dependent on the substitution and less localized (6-64%). Localization of frontier molecular orbitals (FMOs) along different axes of these cruciforms makes them promising as sensing platforms, since analyte binding to the cruciform should mandate a change in the HOMO-LUMO gap and the resultant optical properties. This prediction was verified using UV/vis absorption and emission spectroscopy: cruciforms' protonation results in hypsochromic and bathochromic shifts consistent with the preferential stabilization of HOMO and LUMO, respectively. In donor-acceptor-substituted systems, a two-step optical response to protonation was observed, wherein an initial bathochromic shift is followed by a hypsochromic one with continued acidification. X-ray diffraction studies of three selected cruciforms revealed the expected ~90° angle between the cruciform's substituents, and crystal packing patterns dominated by [π···π] stacking and edge-to-face [C-H···π] contacts.

Silent microbleeds and old hematomas in spontaneous cerebral hemorrhages.

OBJECTIVE: The authors studied the risk factors of silent cerebral microbleeds (MBs) and old hematomas (OHs) and their association with concurrent magnetic resonance (MR) imaging findings in the patients of intracerebral hemorrhages (ICHs). METHODS: From April 2002 to June 2007, we retrospectively studied 234 patients of primary hemorrhagic stroke. All patients were evaluated with computed tomography (CT) and 3.0-tesla MR imaging studies within the first week of admission. MBs and OHs were assessed by using T2*-weighted gradient-echo (GRE) MR imaging. The patients were divided into 2 groups, depending on whether or not they had two GRE lesions of chronic hemorrhages. A correlation between MBs and OHs lesions were also statistically tested. Lacunes and white matter and periventricular hyperintensities (WMHs, PVHs) were checked by T1- and T2-weighted spin-echo and fluid attenuated inversion recovery sequences. Variables on the clinical and laboratory data and MR imaging abnormalities were compared between both groups with or without MBs and OHs. RESULTS: MBs were observed in 186 (79.5%) patients and a total of 46 OHs were detected in 45 (19.2%) patients. MBs (39.6%), OHs (80.4%), and ICHs (69.7%) were most commonly located in the ganglionic/thalamic region. Both MBs and OHs groups were more frequently related to chronic hypertension and advanced WMHs and PVHs. The prevalence and number of MBs were more closely associated with OHs groups than non-OH patients. CONCLUSION: This study clearly demonstrated the presence of MBs and OHs and their correlation with hypertension and cerebral white matter microangiopathy in the ICHs patients. Topographic correlation between the three lesions (MBs, OHs, and ICHs) was also noted in the deep thalamo-basal location.