A clickable oxysterol photolabel retains NMDA receptor activity and accumulates in neurons

Oxysterol analogs that modulate NMDA receptor function are candidates for therapeutic development to treat neuropsychiatric disorders. However, the cellular actions of these compounds are still unclear. For instance, how these compounds are compartmentalized or trafficked in neurons is unknown. In this study, we utilized a chemical biology approach combining photolabeling and click chemistry. We introduce a biologically active oxysterol analog that contains: (1) a diazirine group, allowing for the permanent labeling of cellular targets, and (2) an alkyne group, allowing for subsequent in situ visualization using Cu2+ catalyzed cycloaddition of an azide-conjugated fluorophore. The physiological properties of this analog at NMDA receptors resemble those of other oxysterols, including occlusion with other oxysterol-like compounds. Fluorescent imaging reveals that the analog accumulates diffusely in the cytoplasm of neurons through an energy-independent mechanism. Overall, this work introduces a novel chemical biology approach to investigate oxysterol actions and introduces a tool useful for further cell biological and biochemical studies of oxysterols.</p

Spectroscopic Analyses of Excited-State Phenomena in Organized Media: Proton Transfer and Photoisomerization and Measurement of Modified Low Density Lipoproteins Using Rapid-Mixing Multidimensional Stopped-Flow Spectroscopic Methods.

The research presented in this dissertation generally involves aspects of molecular spectroscopy and contains two areas of focus. In the first section, the influences of organized media on the excited-state phenomena of intramolecular proton transfer (ESIPT) and photoisomerization have been investigated. Specifically, the photochemical properties of the novel ESIPT molecule 10-hydroxybenzo(h) quinoline (HBQ), as well as 2-(2\sp\prime-hydroxyphenyl)benzazoles (HBAs), have been examined in the presence of cyclodextrins (CDs) and micelles using absorbance, steady-state, and time-resolved emission spectroscopies. Additionally, the influence of cyclodextrins upon the photoisomerization of trans-stilbene (TS) has been studied. Results from spectroscopic studies of HBQ and HBAs in organized media suggest reduced rates of radiationless transition of the excited tautomer states of these molecules. HBQ exhibits enhanced tautomer emission in both cyclodextrins and micelles through the formation of 1:1 complexes. Moreover, interaction of HBAs with CDs (e.g., $\beta$- and methylated $\beta$-CD\rm\sb{x}) enhance both the ground and excited electronic state (S\sb0 and S\sb1, respectively) intermolecular proton transfer (PT) of two of the HBAs (2-(2\sp\prime-hydroxyphenyl) benzimidazole and -benzothiazole), while enhancing the S\sb0 state intermolecular PT and reducing the S\sb1 state PT rate of 2-(2\sp\prime-hydroxyphenyl)-benzoxazole. Data obtained from induced circular dichroism and time-resolved emission studies are used to further suggest differences in the structural orientation of these molecules in the S\sb0 and S\sb1 states, and the existence of zwitterionic tautomer species of HBAs, respectively. In the case of trans-stilbene, a dual fluorescence is observed for the molecule in ternary aqueous solutions of $\gamma$-cyclodextrin ($\gamma$-CD\rm\sb{x}). The ternary component (cyclohexane or toluene) plays an active role in increasing the excimer emission of the molecule through the formation of ternary complexes that restrict the photoisomerization of trans-stilbene in the cyclodextrin cavity. The results from time-resolved emission and anisotropy studies further support the formation of extended linear aggregates of trans-stilbene:$\gamma$-CD\rm\sb{x} with these components. The second section of this dissertation concerns the development of analytical assays for modified low-density lipoproteins (LDLs) using multidimensional stopped-flow (SF) spectrophotometric methods. Specifically, SF assays for lipid peroxides have been developed using triiodide and indigo carmine dye as chromophores. The data obtained are used to assess the suitability of these assays for modified LDLs

Lipid-membrane-incorporated hydrophobic photochromic molecules prepared by the exchange method using cyclodextrins

It was found that the exchange method for the preparation of lipid-membrane-incorporated guest molecules was applicable not only to fullerenes but also to other hydrophobic molecules such as azobenzene and stilbene. Advantages of this method are that the long-term stability of lipid-membrane-incorporated azobenzene solution and the maximum ratio of [stilbene]/[lipid] were higher than those prepared by the classical method, which we call the ‘premixing method’. Photoisomerisations of these photochromic guest molecules in the lipid membranes maintained the morphology of liposomes.This file includes Electronic Supplementary Information.This work was supported by JSPS KAKENHI a Grant-in-Aid for Scientific Research (B) (Grant No. 25288037), a Grant-in-Aid for Challenging Exploratory Research (Grant Nos. 24655128 and 25650053) and a Grant-in-Aid for Young Scientists (A) (Grant No. 24681028)

Porphyrin‐uptake in liposomes and living cells using an exchange method with cyclodextrin

The water‐solubilisation of porphyrin derivatives is very important for biological applications. Although liposomal drug carriers for porphyrin derivatives have shown significant promise in the field of medicinal chemistry (e.g., as sensitisers for photodynamic therapy), it is currently not possible to prepare lipid‐membrane‐incorporated tetraphenylporphyrin (TPP) with a high concentration of TPP using conventional methods. In this study, we have succeeded in preparing lipid-membrane‐incorporated TPP and zinc(II) tetraphenylporphyrin (ZnTPP) from the corresponding TPP or ZnTPP•cyclodextrin complex using the exchange method in lipid‐membranes composed of liposomes. Furthermore, the exchange method allowed for the incorporation of TPP or ZnTPP into the plasma membranes of HeLa cells. However, it was not possible to prepare lipid‐membrane‐incorporated porphyrin derivatives with polar and hydrophilic groups in the meso positions using this exchange reaction.Electronic supplementary information (ESI) available: Experimental procedures, 1H NMR spectra, DLS measurements, cryo-TEM images, phase contrast and fluorescence images. See DOI: 10.1039/c5ra24985This work was supported by JSPS KAKENHI a Grant‐in‐Aid for Scientific Research (B) (Grant No. 25288037) and a Grant‐in‐Aid for Young Scientists (A) (Grant No. 24681028)

Photosensitizer, pH sensing and optical limiting properties of BODIPY dyes

A series of BODIPY dyes have been successfully synthesised and structurally characterised to examine the effect of halogenation at the 2,6-positions and the introduction of styryl and vinylene groups at the 3,5-positions. The photophysical properties were studied, to assess the effect of the enhancement of the rate of intersystem crossing through halogenation on the fluorescence properties and the generation of reactive oxygen species. This is important in the assessment of the suitability of applying these molecules as photosensitizer dyes for photodynamic therapy and photodynamic antimicrobial chemotherapy. Upon bromination, the dyes showed moderately high singlet oxygen quantum yields. The inclusion of BODIPY dyes into cyclodextrins was explored since it makes them water soluble and hence suitable for biomedical applications, but no singlet oxygen was detected in aqueous media for the inclusion complexes. In order to red-shift the main spectral band of the BODIPY dyes into the therapeutic window, styryl groups were introduced at the 3,5-positions via a modified Knoevenagel condensation reaction. Since the main spectral band lies well above 532 nm, the second harmonic of the Nd:YAG laser, there is relatively weak absorbance at this wavelength. The 3,5-distyryl and 3,5-divinylene BODIPY dyes were assessed for their potential utility for application in nonlinear optics (NLO), and they demonstrated typical nonlinear absorption behaviour characterised by reverse saturable absorption (RSA) in z-scan measurements. Furthermore, the dyes possess excellent optical limiting parameters, such as their third-order suspectibility and hyperpolarizability values, in a wide range of solvents. One dye containing dimethylamino moieties on styryl groups attached at the 3,5-positions was assessed for potential application as an on/off fluorescence sensor. The dye proved to be successful, since intramolecular charge transfer in the S1 state was eliminated in the presence of acid and this results in a fluorescence “turn on” effect. This process was found to be reversible with the addition of a base

Use of gadolinium in 13C NMR studies

The addition of the paramagnetic reagents gadolinium(18- crown-6) nitrate, gadolinium(dicyclohexano-18-crown-6) nitrate, and gadolinium nitrate pentahydrate to each of the substrates ethylbenzene, and thymidine has been studied in an attempt to obtain quantitative 13C spectra. These reagents reduce the nuclear Overhauser effect and shorten the spin-lattice relaxation times of the carbons of these substrates

Mass transfer of cadmium ions in a hollow-fiber module by pertraction

The facilitated transport of Cd(II) ions through a hollow-fiber-supported liquid membrane with bis-2-ethylhexyl phosphoric acid as carrier was studied. The mass transfer rate, expressed as permeability P, was measured as a function of mean aqueous solution velocity and carrier concentration. Characteristic ion permeabilities of 10-26×10-7m/s were measured at feed velocities between 1×10-2-19×10-2 m/s at stripping velocities between 0.22×10-2-7×10-2 m/s with constant feed flow. The measured permeabilities were compared to generally accepted mass transfer correlations. The predicted permeabilities adequately fit the experimental data, indicating that the rate limiting step in the transport of the ion was the diffusion through both aqueous films, feed and stripping, whereas the organic resistance of the membrane was negligible. Furthermore, the proposed model allowed the prediction of the permeability of cadmium for different experimental conditions, which is useful to perform experiments to reduce metal levels in water or other effluents.Fil: Marchese, Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Física Aplicada "Dr. Jorge Andrés Zgrablich". Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto de Física Aplicada "Dr. Jorge Andrés Zgrablich"; Argentina. Universidad Nacional de San Luis. Facultad de Ciencias Fisico Matematicas y Naturales. Departamento de Fisica. Laboratorio de Ciencias de Superficies y Medios Porosos; ArgentinaFil: Campderrós, Mercedes Edith. Universidad Nacional de San Luis. Facultad de Ciencias Fisico Matematicas y Naturales. Departamento de Fisica. Laboratorio de Ciencias de Superficies y Medios Porosos; Argentin

Reversible Supramolecular System of Porphyrin Exchange between Inclusion in Cyclodextrin and Intercalation in DNA by Change in pH

Under the coexistence of cyclodextrin and DNA in water, 5,10,15,20-tetra(4-pyridyl)porphyrin interacted with the cavities of two cyclodextrin molecules in a solution at an around neutral pH and intercalated into DNA under acidic conditions. The supramolecular phenomena occurred completely and reversibly owing to the change in pH.This work was supported by a JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (Grant No. JP16H04133) and a Grant-in-Aid for Challenging Exploratory Research (Grant No. JP16K13982)