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Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl−channel. The most common mutation results in a deletion of phenylalanine at position 508 (ΔF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of ΔF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores ΔF508-CFTR-mediated Cl−transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of ΔF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.

In the reaction centers of photosynthetic organisms, chlorophyll triplet states are sometimes formed by recombination of charge-separated intermediates. These triplets are excellent sensitizers for singlet oxygen formation. Carotenoid polyenes can provide photoprotection from singlet oxygen generation by rapidly quenching chlorophyll triplet states via triplet-triplet energy transfer. Because in bacteria the reaction center carotenoid is not located adjacent to the bacteriochlorophyll special pair, which is the origin of the charge separation, it has been postulated that quenching may occur via a triplet relay involving an intermediate chlorophyll monomer. We now report the synthesis and spectroscopic study of a covalently linked carotenoid (C)-porphyrin (P)-pyropheophorbide (Ppd) triad molecule which mimics this triplet relay. The pyropheophorbide singlet-state C-P-[sup 1]Ppd (generated by direct excitation or energy transfer from the attached porphyrin) undergoes intersystem crossing to the triplet C-P-[sup 3]Ppd. In oxygen-free solutions, this triplet decays to [sup 3]C-p-Ppd through a triplet-transfer relay involving an intermediate C-[sup 3]P-Ppd species. In aerated solutions, quenching of C-P-[sup 3]Ppd by the attached carotenoid competes with singlet oxygen sensitization and thus provides a degree of photoprotection. In a similar traid containing a zinc porphyrin moiety, triplet transfer is slow due to the higher energy of the C-[sup 3]P[sub Zn]-Ppd intermediate, more » and photoprotection via the relay is nonexistent. The triplet relay ceases to function at low temperatures in both the natural and biomimetic cases due to the endergonicity of the first step. 37 refs., 6 figs., 1 tab. « less

A variety of molecular triads and dyads consisting of covalently linked carotenoid (C) andor porphyrin (P) moieties have been prepared and studied with transient absorption and time-resolved fluorescence techniques. Diporphyrins of the type P A -P B and C-P A -P B triads demonstrate interporphyrin singlet-singlet energy transfer with rate constants ranging from 8.1×10 8 to 2.3×10 10 s −1 . Interporphyrin photoinitiated electron transfer is observed in molecules possessing sufficient thermodynamic driving force to produce P A •+ -P B •− and C-P A •+ -P B •− charge-separated states. The electron-transfer rate constant increases with increasing reaction free energy change for the molecules studied, and rate constants up to 3.5×10 9 s −1 and quantum yields up to 0.68 were measured. The carotenodiporphyrin triad systems undergo a subsequent electron-transfer step to give final C •+ -P A -P B •− states

Excitation of carotenoid-porphyrin-quinone (C-P-Q) triad molecules initiates a two-step electron transfer to yield a final charge separated state of the form C·+-P-Q·-.

Triplet state electron spin resonance (ESR) spectroscopy has been used to study triplet-triplet energy transfer in B800–850 light-harvesting complexes and carotenoporphyrin molecules. The B800–850 complexes were isolated from the photosynthetic bacteria Rhodobacter sphaeroides GA, aerobically and anaerobically grown Rb. sphaeroides wild type and Rhodopseudomonas acidophila 7750. Free-base and zinc-substituted carotenoporphyrins featuring various linkage structures and different orientations of the pigments were studied. The carotenoporphyrins which contain a short bridging link display ESR spectra which resemble those of the B800–850 complexes. The results indicate a close spatial proximity between the bacteriochlorophyll and carotenoid pigments in the B800–850 complexes which leads to efficient triplet-triplet energy transfer. Computer simulations of the observed ESR spectral line-shapes yielded values for the zero-field splittings which can be understood in terms of the varying extents of π-electron conjugation in the carotenoids. The rate constants for population and decay of the observed triplet states were also obtained from the computer simulations. All of the carotenoid triplet states exhibit similar ESR spectral lineshapes and are characterized by the spin polarization pattern eae aea . The molecular basis for the spectral uniformity may be explained by triplet energy transfer according to the exchange mechanism and conformational changes of the carotenoid which lead to triplet spin relaxation.

Carotenoporphyrin-quinone triad molecules have previously been found to successfully mimic many aspects of photosynthetic electron and energy transfer. These molecules generate long-lived, energetic charge-separated states via a biomimetic multistep electron transfer scheme. In many of these molecules, the overall quantum yield for charge separation has been limited by an unfavorable partitioning of an intermediate charge-separated state between charge recombination and further reaction to yield the desired species. One strategy for overcoming this limitation is based on the fact that, in general, electron transfer rate constants do not depend linearly on reaction free energy change. In the triads, raising the energy of the intermediate charge-separated state would be expected to speed up the desired electron transfer (“normal” Marcus behavior), but slow down charge recombination (which formally lies in the “inverted” region). Laser flash photolysis and transient fluorescence measurements on two new triad molecules reveal enhanced quantum yields of long-lived charge-separated states which are consistent with this expectation.

Carotenoid-porphyrin-quinone triad molecules undergo a photodriven two-step electron-transfer reaction which results in the generation of a high-energy charge-separated state with a lifetime on the microsecond time scale at ambient temperatures in fluid solution. These systems mimic the initial charge separation steps of photosynthesis. A series of these tripartite molecules which differ systematically in the nature of the linkages joining the porphyrin to the quinone and carotenoid moieties has been synthesized in order to investigate the effect of structure on the yield and lifetime of the charge-separated state. The time-averaged solution conformations of these molecules have been determined from porphyrin ring current induced shifts in the /sup 1/H NMR resonances of the carotenoid and quinone moieties. Studies of the triads and related molecules in dichloromethane solution using time-correlated single photon counting fluorescence lifetime techniques have yielded the rate constant for the first of the photoinitiated electron-transfer steps as a function of the linkage joining the porphyrin and the quinone. The rate constants range from 1.5 x 10/sup 8/ to 9.7 x 10/sup 9/ s/sup -1/. For most members of the series, the results are consistent with an exponential dependence of the electron-transfer rate on the experimentally determined donor-acceptor separation, with themore » exponential factor ..cap alpha.. = 0.6 A/sup -1/.« less

The role of nitric oxide (NO) in the induction of longterm depression (LTD) in the cerebellum was explored using a new, organic, membrane-impermeant form of caged NO. NO photolytically released inside Purkinje neurons mimicked parallel fiber (PF) activity in synergizing with brief postsynaptic depolarization to induce LTD. Such LTD required a delay of

Background : Fluorescent indicators that show alterations in excitation and/or emission spectra in response to changes in [Ca 2+ ] are widely used for quantitative cytosolic [Ca 2+ ] measurements. There are several reports of changes in apparent [Ca 2+ ] due only to illumination, however. These results have been attributed either to photodamage to the cells or to photodegradation of the indicator. Light-induced alteration in the behavior of the dye or cells would severely hamper the interpretation of experimental data. We examined this phenomenon in indo-1 loaded cells using confocal laser scanning microscopy. Results: Illumination of indo-1 loaded GH3 cells leads to a decrease in apparent basal [Ca 2+ ] and decreased peaks after depolarization with KCI. When cells were double loaded with indo-1 and fluo-3, the effect of UV illumination was noticed only with the former dye. UV irradiation of indo-1 in simple buffers caused overall photobleaching and conversion to a fluorescent but Ca 2+ -insensitive species. The latter effect cannot be canceled by ratiometric calibration and is due to loss of carboxymethyl groups from the anilino nitrogens. This photodegradation was inhibited by extracellular administration of 10 to 100 μM Trolox, a water-soluble vitamin E analog. Conclusions: Photodegradation processes like that observed for indo-1 are likely to be possible for all cation indicators that contain bis(carboxymethyl)anilino moieties, which include essentially all fluorescent indicators for Ca 2+ and Mg 2+ currently in biological use. If unrecognized, this photochemical dealkylation leads to an underestimation of the analyte concentrations depending on the intensity and duration of illumination. The problem can be avoided by including cell-permeant antioxidants such as Trolox in the bathing solution. The ultimate solution would be to redesign the indicators to minimize photodegradation in the absence of antioxidants.

A four-component molecular device, which consists of two covalently linked porphyrins, one bearing a carotenoid polyene and the other a napthoquinone electron-acceptor, has been prepared to attempt to explain the electron-transfer strategy of photosynthesis. This tetrad was found to demonstrate singlet and triplet energy transfer behavior, which mimics photosynthetic antenna function by chlorophylls and carotenoids and carotenoid photoprotection from singlet oxygen damage. 27 refs., 1 fig.

A digital software‐controlled back‐off system for probe lamp dc signal nullification has been designed and incorporated into a laser flash photolysis spectrometer. This method takes advantage of modern high‐speed operational amplifiers already employed in the detection circuitry and the dedicated small computer that runs the spectrometer. The determination of I0, necessary for absorbance calculations, is inherent in the method.

Photosynthesis converts light to chemical potential energy in the form of long-lived charge separation across a bilayer membrane. Recombination of the charge-separated state is prevented by the large distance between the final electron donor and acceptor. Because the quantum yield of a single long-range electron transfer across the bilayer would be vanishingly small, reaction centers employ a series of electron-transfer steps, each of which occurs over a short distance with high yield. Synthetic carotenoid-porphyrin-quinone triad molecules and other molecular systems which begin to model this approach have recently been reported. In photosynthetic bacteria, the charge separation sequence includes electron donation from a bacteriopheophytin to a quinone which then transfers an electron to a second quinone. An elegant molecule that models such electron transfer has been prepared, although it did not reproduce the long-lived charge separation characteristic of the natural system. The authors now report the synthesis of a tetrachromophoric molecule 1 consisting of a porphyrin (P) covalently linked to both a carotenoid polyene (C) and a rigid diquinone moiety (Q/sub A/-Q/sub B/), excitation of which produces a long-lived (460 ns in dichloromethane, 4 ..mu..s in acetonitrile charge-separated state C/sup .+/-P-Q/sub A/-Q/sub B//sup 0 -/ with a quantum yield of 0.23more » at ambient temperatures and 0.50 at 240 K.« less

In natural photosynthesis membranes, chlorophyll molecules serve as the site of the initial photodriven charge separation. In addition, they play a role in subsequent electron-transfer steps, accept singlet excitation energy from carotenoid antenna molecules, and transfer triplet energy to carotenoid acceptors (thereby preventing sensitized singlet oxygen production and subsequent photodamage to the organism). The authors report herein the synthesis and study of chlorophyll-based carotenopyropheophorbide-quinone triad molecules which mimic all of these natural processes.

Excitation of carotenoid-porphyrin-quinone (C-P-Q) triad molecules initiates a two-step electron transfer to yield a final charge separated state of the form C·+-P-Q·-.

Photosynthesis converts light to chemical potential energy in the form of long-lived charge separation across a bilayer membrane. Recombination of the charge-separated state is prevented by the large distance between the final electron donor and acceptor. Because the quantum yield of a single long-range electron transfer across the bilayer would be vanishingly small, reaction centers employ a series of electron-transfer steps, each of which occurs over a short distance with high yield. Synthetic carotenoid-porphyrin-quinone triad molecules and other molecular systems which begin to model this approach have recently been reported. In photosynthetic bacteria, the charge separation sequence includes electron donation from a bacteriopheophytin to a quinone which then transfers an electron to a second quinone. An elegant molecule that models such electron transfer has been prepared, although it did not reproduce the long-lived charge separation characteristic of the natural system. The authors now report the synthesis of a tetrachromophoric molecule 1 consisting of a porphyrin (P) covalently linked to both a carotenoid polyene (C) and a rigid diquinone moiety (Q/sub A/-Q/sub B/), excitation of which produces a long-lived (460 ns in dichloromethane, 4 ..mu..s in acetonitrile charge-separated state C/sup .+/-P-Q/sub A/-Q/sub B//sup 0 -/ with a quantum yield of 0.23more » at ambient temperatures and 0.50 at 240 K.« less