Unraveling the mechanism of the photodeprotection reaction of 8-bromo- and 8-chloro-7-hydroxyquinoline caged acetates.

Photoremovable protecting groups (PPGs) when conjugated to biological effectors forming "caged compounds" are a powerful means to regulate the action of physiologically active messengers in vivo through 1-photon excitation (1PE) and 2-photon excitation (2PE). Understanding the photodeprotection mechanism is important for their physiological use. We compared the quantum efficiencies and product outcomes in different solvent and pH conditions for the photolysis reactions of (8-chloro-7-hydroxyquinolin-2-yl)methyl acetate (CHQ-OAc) and (8-bromo-7-hydroxyquinolin-2-yl)methyl acetate (BHQ-OAc), representatives of the quinoline class of phototriggers for biological use, and conducted nanosecond time-resolved spectroscopic studies using transient emission (ns-EM), transient absorption (ns-TA), transient resonance Raman (ns-TR(2)), and time-resolved resonance Raman (ns-TR(3)) spectroscopies. The results indicate differences in the photochemical mechanisms and product outcomes, and reveal that the triplet excited state is most likely on the pathway to the product and that dehalogenation competes with release of acetate from BHQ-OAc, but not CHQ-OAc. A high fluorescence quantum yield and a more efficient excited-state proton transfer (ESPT) in CHQ-OAc compared to BHQ-OAc explain the lower quantum efficiency of CHQ-OAc relative to BHQ-OAc.

Synthesis of a caged glutamate for efficient one- and two-photon photorelease on living cells.

We have synthesized a biologically inert, photosensitive derivative of the major excitatory amino acid, L-glutamate (which we call MDNI-glu) that makes more efficient use of incident light than all other caged glutamates. Laser flash photolysis of MDNI-glu in acutely isolated hippocampal brain slices evoked a rapid increase in intracellular Ca2+ concentration in astrocytes.

Light-mediated inhibition of protein synthesis.

The regulation of protein synthesis is vital for a host of cell biological processes, but investigating roles for protein synthesis have been hindered by the inability to selectively interfere with it. To inhibit protein synthesis with spatial and temporal control, we have developed a photo-releasable anisomycin compound, N-([6-bromo-7-hydroxycoumarin-4-yl]methyloxycarbonyl)anisomycin (Bhc-Aniso), that can be removed through exposure to UV light. The area of protein synthesis inhibition can be restricted to a small light-exposed region or, potentially, the volume of two-photon excitation if a pulsed IR laser is the light source. We have tested the compound's effectiveness with an in vitro protein-translation system, CHO cells, HEK293 cells, and neurons. The photo-released anisomycin can inhibit protein synthesis in a spatially restricted manner, which will enable the specific inhibition of protein synthesis in subsets of cells with temporal and spatial precision.

Synthesis and characterization of 4-methoxy-7-nitroindolinyl-D-aspartate, a caged compound for selective activation of glutamate transporters and N-methyl-D-aspartate receptors in brain tissue.

The D-isomer of aspartate is efficiently transported by high-affinity Na(+)/K(+)-dependent glutamate transporters and is an effective ligand of N-methyl-d-aspartate (NMDA) receptors. To facilitate analysis of the regulation of these proteins in their native membranes, we synthesized a photolabile analogue of D-aspartate, 4-methoxy-7-nitroindolinyl-D-aspartate (MNI-D-aspartate). This compound was photolyzed with a quantum efficiency of 0.09 at pH 7.4. Photorelease of d-aspartate in acute hippocampal slices through brief (1 ms) UV laser illumination of MNI-d-aspartate triggered rapidly activating currents in astrocytes that were inhibited by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA), indicating that they resulted from electrogenic uptake of D-aspartate. These transporter currents exhibited a distinct tail component that was approximately 2% of the peak current, which may result from the release of K(+) into the extracellular space during counter transport. MNI-D-aspartate was neither an agonist nor an antagonist of glutamate transporters at concentrations up to 500 muM and was stable in aqueous solution for several days. Glutamate transporter currents were also elicited in Bergmann glial cells and Purkinje neurons of the cerebellum in response to photolysis of MNI-D-aspartate, indicating that this compound can be used for monitoring the occupancy and regulation of glutamate transporters in different brain regions. Photorelease of D-aspartate did not activate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or metabotropic glutamate receptors (mGluRs) in neurons, but resulted in the selective, but transient, activation of NMDA receptors in hippocampal pyramidal neurons; MNI-D-aspartate was not an antagonist of NMDA receptors. These results indicate that MNI-D-aspartate also may be useful for studying the regulation of NMDA receptors at excitatory synapses.

Spontaneous Ca2+ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca2+ pools.

Spontaneous Ca2+ oscillations in vascular smooth muscle cells have been modeled using a single Ca2+ pool. This report describes spontaneous Ca2+ oscillations dependent on two separate Ca2+ sources for the nuclear versus cytoplasmic compartments. Changes in free intracellular Ca2+ were monitored with ratiometric Ca2+- fluorophores using confocal microscopy. On average, spontaneous oscillations developed in 79% of rat aortic smooth muscle cells that were synchronous between the cytoplasm and nucleus. Reduction of extracellular Ca2+ (less than 1 microM)decreased the frequency and amplitude of the cytoplasmic oscillations with 48% of the oscillations asynchronous between the nuclear and cytoplasmic compartments. Similar results were obtained with the Ca2+ channel blockers, nimodipine and diltiazem. Arg-vasopressin (AVP) induced a rapid release of intracellular Ca2+ stores that was greater in the nuclear compartment (4.20 +/- 0.23 ratio units, n = 56) than cytoplasm (2.54 +/- 0.28) in cells that had spontaneously developed prior oscillations. Conversely, cells in the same conditions lacking oscillations had a greater AVP-induced Ca2+ transient in the cytoplasm (4.99 +/- 0.66, n = 17) than in the nucleus (2.67 +/- 0.29). Pre-treatment with Ca2+ channel blockers depressed the AVP responses in both compartments with the cytoplasmic Ca2+ most diminished. Depletion of internal Ca2+ stores prior to AVP exposure blunted the nuclear response, mimicking the response of cells that lacked prior oscillations. Spontaneous oscillating cells had a greater sarcoplasmic reticulum network than cells that did not oscillate. We propose that spontaneous nuclear oscillations rely on perinuclear sarcoplasmic reticulum stores, while the cytoplasmic oscillations rely on Ca2+ influx.

Bhc-diol as a photolabile protecting group for aldehydes and ketones.

[reaction: see text] 6-Bromo-4-(1,2-dihydroxyethyl)-7-hydroxycoumarin (Bhc-diol) can be used under simulated physiological conditions as a photoremovable protecting group for aldehydes and ketones. The single- and two-photon-induced release of benzaldehyde, piperonal, acetophenone, and cyclohexanone is demonstrated.

Brominated hydroxyquinoline as a photolabile protecting group with sensitivity to multiphoton excitation.

The synthesis and photochemistry of a new photolabile protecting group for carboxylic acids based on 8-bromo-7-hydroxyquinoline (BHQ) is described. BHQ possesses a greater single photon quantum efficiency than 4,5-dimethoxy-4-nitrobenzyl ester (DMNB) and 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc), and it has sufficient sensitivity to multiphoton-induced photolysis for use in vivo. Its increased solubility and low fluorescence make it quite useful as a caging group for biological messengers. [structure: see text]