Synthesis and characterization of amino derivatives of persistent trityl radicals as dual function pH and oxygen paramagnetic probes

Triarylmethyl radicals, TAMs, are useful soluble paramagnetic probes for EPR spectroscopic and imaging applications because of their extraordinary stability in living tissues, narrow line width, high analytical resolution at micromolar concentrations and enhanced sensitivity to molecular oxygen. Recently we proposed the concept of dual function pH and oxygen TAM probes based on the incorporation of ionizable groups into the TAM structure (J. Am. Chem. Soc. 2007, 129 (23), 7240-7241). In this paper we report the synthesis of TAM derivatives containing amino groups. The synthesized TAMs combine stability with oxygen and pH sensitivity, in the range of pH from 6.8 to 9.0. To decrease the number of spectral components and improve probe solubility at physiological pH, asymmetric TAM derivatives containing both carboxyl and amino functions were synthesized. The presence of nitrogen and hydrogen atoms in direct proximity to protonatable amino groups resulted in strong pH-induced changes to the corresponding hyperfine splittings, Delta hfs approximately (300-1000) mG, comparable to the values of hfs themselves. Large pH-dependent line shifts of individual spectral components, with narrow linewidths of (160-280) mG, allow for easy discrimination between the pH effect and the observed oxygen-dependent line broadening of about (6 +/- 0.5) mG per % oxygen. The synthesized TAM derivatives represent the first dual function pH and oxygen paramagnetic probes with reasonably valuable properties for biomedical research.     

Reactivity of molecular oxygen with ethoxycarbonyl derivatives of tetrathiatriarylmethyl radicals

Tetrathiatriarylmethyl (TAM) radicals are commonly used as oximetry probes for electron paramagnetic resonance imaging applications. In this study, the electronic properties and the thermodynamic preferences for O2 addition to various TAM-type triarylmethyl (trityl) radicals were theoretically investigated. The radicals' stability in the presence of O2 and biological milieu was also experimentally assessed using EPR spectroscopy. Results show that H substitution on the aromatic ring affects the trityl radical's stability (tricarboxylate salt 1-CO2Na > triester 1-CO2Et > diester 2-CO2Et > monoester 3-CO2Et) and may lead to substitution reactions in cellular systems. We propose that this degradation process involves an arylperoxyl radical that can further decompose to alcohol or quinone products. This study demonstrates how computational chemistry can be used as a tool to rationalize radical stability in the redox environment of biological systems and aid in the future design of more biostable trityl radicals.     

Chiral properties of tetrathiatriarylmethyl spin probes

We report that tetrathiatriarylmethyl (trityl) EPR probes are chiral molecules at room temperature, the two stereoisomers that differ in their helicity being configurationally stable enough to be separated and stored independently.     

Synthesis and characterization of novel indole-containing half-crowns as new emissive metal probes

Two new indole derivatives have been synthesized by a one-pot procedure and their potential as fluorescence probes for metal ions was investigated. The sensor capability of 1 and 2 toward cations such as Ag⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, and Hg²⁻ was studied in dichloromethane solution by absorption, fluorescence emission, and ¹H NMR titrations. Both probes showed selectivity for Ag⁺ and Hg²⁺ ions. The results suggest that these compounds may serve as promising models for future design of novel and more potent sensors.     

Synthesis and characterization of photolabeling probes of miltefosine

Miltefosine is an ether-phospholipid analogue showing remarkable anti-cancer and anti-leishmanial activity thanks to its cell membrane-targeting properties. In order to study the mechanisms responsible for the biological effects of miltefosine using a photolabeling approach, we designed, synthesized and characterized photolabeling probes for studying the effects of miltefosine. In these probes, the photoactivatable tetrafluorophenylazido group is incorporated either at the polar head or in the alkyl chain of miltefosine. The probes showed fast, clear-cut photochemical reactions, which suggests that they are promising tools for use in photolabeling studies.     

Hydrophobic nitroxide radicals as probes to investigate the hydrophobic interaction

The EPR spectrum of 2,2,6,6-tetramethylpiperidine-N-oxide (TEMPO) has been systematically investigated in several solvents and in aqueous solutions of glycerol,t-BuOH, Bu₄NBr,n-HexNH₃Br,n-OctNH₃Br, NaBPh₄, and Ph₄AsCl. Most of the results have been obtained at 25°C, though the temperature dependence of the linewidths has been examined in water and several organic solvents. Data on the spectrum of ditertiarybutylnitroxide in aqueous solutions of glycerol and Bu₄NBr are also presented. The spectrum was simulated to determine W_H, the linewidth after allowance for the unresolved proton hyperfine interaction, in each manifold of the triplet due to the nitrogen hyperfine interaction. The linewidth W_H is analyzed in terms of the reorientational correlation time Äθ and the angular velocity correlation time ÄJ. In most solutions Äθ is determined only by the bulk solution viscosity, except in situations where significant clustering of hydrophobic solute molecules occurs. In pure nonaqueous solvents the temperature dependence results are consistent with ÄJ being determined by the bulk viscosity, while in water or aqueous solutions, a very different behavior is found which is interpreted as a manifestation of clathrate-like hydration of the hydrophobic radical. This interpretation is incorporated in a two-state model developed to account for the W_H data of TEMPO in aqueous solutions of various hydrophobic solutes. The equilibrium parameters derived from the model, for the association of TEMPO with several hydrophobic solutes, support the concept of hydrophobic interactions as pictured from thermodynamic excess functions.     

Synthesis and characterization of glucosamine-bound near-infrared probes for optical imaging

[structure: see text] Two novel near-infrared (NIR) fluorescent probes have been synthesized by linking a carbocyanine fluorophore and glucosamine through different linkers. These probes demonstrated a high quantum yield, low cytotoxicity, reversible pH-dependent fluorescence in the physiological pH range, and a decreased aggregation tendency in aqueous solutions. In vitro NIR optical imaging studies revealed cellular uptake and strong intracellular NIR fluorescence of these two probes in four breast epithelial cell lines.     

Highly stable dendritic trityl radicals as oxygen and pH probe

Novel dendritic trityl radicals (DTR1 and DTR2) with a TAM radical core, PAMAM branching and carboxylate exterior surface exhibit high stability towards oxidoreductants as evidenced by their electrochemical and EPR properties, offering potential application as dual oxygen and pH probe.     

Synthesis and spectral properties of polymethine-cyanine dye-nitroxide radical hybrid compounds for use as fluorescence probes to monitor reducing species and radicals

Various hybrid compounds comprised of two types of nitroxide radicals and either a pentamethine (Cy5) or trimethine cyanine (Cy3) were synthesized. The nitroxide radicals were linked either via an ester-bond to one or two N-alkyl carboxyl-terminated groups of Cy5, or via two amido-bonds (aminocarbonyl or carbonylamino group) to the 5-position of the indolenine moieties of Cy5 and Cy3. Changes in fluorescence and ESR intensities of the hybrid compounds were measured before and after addition of Na ascorbate in PBS (pH 7.0) to reduce the radicals. Among the hybrid compounds synthesized, those that linked the nitroxide radicals via an aminocarbonyl residue at the 5-position of the indolenine moieties on Cy5 and Cy3 exhibited a 1.8- and 5.1-fold increase in fluorescence intensity with the reduction of the nitroxide segment by the addition of Na ascorbate, respectively. In contrast, fluorescence intensity was not enhanced in the other hybrid compounds. Thus, the hybrid compounds which exhibited an increase in fluorescent intensity with radical reduction can be used in the quantitative measurement of reducing species such as Fe(2+) and ascorbic acid, and hydroxyl radicals. Because these hybrid compounds have the advantage of fluorescing at longer wavelengths-661 (Cy5) or 568 (Cy3)nm, respectively, they can be used to measure radical-reducing species or radicals either in solution or in vivo.     

Evaluation of the derivates of phosphorescent Pt-coproporphyrin as intracellular oxygen-sensitive probes

Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH₂ derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH₂ demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0–200 μM O₂). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH₂ represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.     

Design, synthesis, and characterization of novel nanowire structures for photovoltaics and intracellular probes

Semiconductor nanowires (NWs) represent a unique system for exploring phenomena at the nanoscale and are expected to play a critical role in future electronic, optoelectronic, and miniaturized biomedical devices. Modulation of the composition and geometry of nanostructures during growth could encode information or function, and realize novel applications beyond the conventional lithographical limits. This review focuses on the fundamental science aspects of the bottom-up paradigm, which are synthesis and physical property characterization of semiconductor NWs and NW heterostructures, as well as proof-of-concept device concept demonstrations, including solar energy conversion and intracellular probes. A new NW materials synthesis is discussed and, in particular, a new "nanotectonic" approach is introduced that provides iterative control over the NW nucleation and growth for constructing 2D kinked NW superstructures. The use of radial and axial p-type/intrinsic/n-type (p-i-n) silicon NW (Si-NW) building blocks for solar cells and nanoscale power source applications is then discussed. The critical benefits of such structures and recent results are described and critically analyzed, together with some of the diverse challenges and opportunities in the near future. Finally, results are presented on several new directions, which have recently been exploited in interfacing biological systems with NW devices.     

Synthesis and spectroscopic characterization of red-shifted spironaphthoxazine based optical switch probes

Spironaphthoxazine (NISO) is an efficient optical switch probe that has applications in high contrast detection of Förster resonance energy transfer (FRET) using optical lock-in detection (OLID). NISO exists in two distinct states, spiro (SP) and merocyanine (MC), that can be independently controlled by using alternate irradiation with near ultraviolet and visible light. Unfortunately, the SP-state of NISO has an absorption centered at 350 nm, which may lead to phototoxic effects when manipulating the probe within a living cell. To overcome this problem we introduce new, red-shifted amino-substituted NISO probes compared to NISO that undergo an efficient SP to MC transition in response to irradiation by using 405-nm light, which is less damaging to living cells. This study details the synthesis of amino-substituted NISO and their N-hydroxysuccinimide ester and maleimide derivatives and their use in generating covalent attached protein conjugates. This study also presents a characterization of the spectroscopic and optical switching properties of these red-shifted NISO probe in solution.     

Synthesis,characterization, and DNA binding of two copper(II) complexes as DNA fluorescent probes

[Cu(DAPT)₂Cl]Cl·H₂O and [Cu(DBM)(DAPT)Cl] [DAPT = 2,4-diamine-6-(pyrazin-2-yl)-1,3,5-triazine] were synthesized and characterized by IR and UV spectroscopy, elemental analysis, TG–DTA, molar conductivity, and LC–MS. The interaction with calf thymus DNA (ct-DNA) of the two complexes has been studied using UV spectra, fluorescent spectra, cyclic voltammetry, and viscosity measurements. The complexes interact with ct-DNA through classical intercalation. Fluorescence intensity changes of 1 and 2 in the absence and presence of ct-DNA have been investigated for quantitative determination of ct-DNA with the limit of detection of 3.8 and 7.7 ng mL^?1, respectively. From the result, the two complexes are potentially sensitive DNA fluorescent probes.     

Synthesis and fluorescence properties of dimethylaminonaphthalene-deoxyuridine conjugates as polarity-sensitive probes

The design of probes for monitoring various structures and dynamics of DNA and its surroundings is an important step in understanding biological events accompanying interbiomolecular interaction. We have developed novel fluorescent nucleosides in which the uracil base and the fluorophore are tethered by rigid linkers. They show unique absorption and fluorescence emission spectra. Nucleoside 2 is a fluorophore with high CT character and the fluorescence is very sensitive to solvent polarity. Nucleoside 3 shows absorption and emission maxima with longer wavelength due to extension of the DAN-conjugate system. These fluorophore-deoxyuridine conjugates with unique fluorescence properties would work as reporter probes sensitive to the change in microenvironment around specific sites of DNA.     

Synthesis and characterization of oxygen and sulfur bridged aromatic macrocycles

The synthesis of nine 16–18–20–22 membered, oxygen and sulfur bridged aromatic macrocycles is reported, and reaction pathways to macrocycles formation are described. Molecular characterization of the nine compounds has been achieved by their mass spectra. Due to the cyclic nature of the compounds analyzed, the mass spectra are of particular interest. The 16–18-membered macrocycles, containing single oxygen and sulfur bridges, display very strong molecular ion intensities, while the 20–22-membered macrocycles, containing double bridges, show lower stability to electron-impact, and the molecular ion intensities are lower. Some of the salient features of the electron fragmentation processes are briefly discussed in the text.     

Differential pulse polarography and electron spin resonance spectroscopy of nitroxyl free radicals used as ESR-spin probes

Differential pulse polarography (DPP) and electron spin resonance (ESR) were used to study the influence of substituents and of the pH of the medium on DPP peak potentials (electrochemical reduction) resp. kreduction (chemical reduction) of nitroxyl free radicals. The DPP peak potentials can be used to select the appropriate nitroxide spin label for relevant biochemical and biophysical applications.     

Synthesis and characterization of oxygen depleted tert-amine calix[4]arene ligands and study the effect on sigma non-opioid intracellular protein receptor

Structural Chemistry - This study focuses on the biological prospects of oxygen-depleted calix[4]arene ligands on a protein target. Because of their extensive medical relevance, the oxygen-depleted...     

Positrons as probes of Si(100) surface with adsorbed hydrogen and oxygen

Positron annihilation induced Auger spectra from the Si(100) surface exposed to hydrogen and oxygen are analyzed by performing calculations of positron surface states and annihilation characteristics of surface trapped positrons. Positron binding energies and work functions are also computed. It is found that the adsorption of hydrogen and oxygen on the Si(100) surface leads to a displacement of the positron surface state wave function away from the substrate Si atoms. As a result of this displacement, the overlap of the positron wave function with Si core electrons and, consequently, the annihilation probability of Si core electrons reduce, in agreement with experimental data.     

Synthesis and characterization of gallium-based perovskitetype dense membrane with oxygen semipermeability

La0.15Sr0.85Ga0.3Fe0.7O3-δ(LSGFO) and La0.15Sr0.85Co0.3Fe0.7O3-δ(LSCFO) mixed oxygen-ion and electron conducting oxides were synthesized by using a combined EDTA and citrate complexing method, and the corresponding dense membranes were fabricated. The properties of the oxide powders and membranes were characterized with combined SEM, XRD, H2-TPR, O2-TPD techniques, mechanical strength and oxygen permeation measurement. The results showed that LSGFO had much higher thermochemical stability than LSCFO due to the higher valence stability of Ga3+. After the temperature-programmed reduction by 5% H2 in Ar from 20℃to 1020℃, the basic perovskite structure of LSGFO was successfully preserved. LSGFO also favors the oxygen vacancy formation better than LSCFO. Oxygen permeation measurement demonstrated that LSGFO had higher oxygen permeation flux than LSCFO, but they had similar activation energy for oxygen transportation, with a value of 110 and 117 kJ ?mol~(-1), respectively. The difference in oxygen permeation f