Designing Molecular Probes To Prolong Intracellular Retention: Application to Nitroxide Spin Probes.

Targeted delivery of molecular probes into cells enables cellular imaging through optical and magnetic modalities. Probe molecules that are well retained by cells can accumulate to higher intracellular concentrations, and thus increase the signal-to-noise ratio of, and widen the temporal window for, imaging. Here we synthesize a paramagnetic spin probe bearing six ionic functional groups and show that it has long intracellular half-life (>12 h) and exceptional biostability in living cells. We demonstrate that judicious incorporation of ionic substituents on probe molecules systematically increases intracellular retention time, and should therefore be beneficial to imaging experiments.

Ncm, a Photolabile Group for Preparation of Caged Molecules: Synthesis and Biological Application.

Ncm, 6-nitrocoumarin-7-ylmethyl, is a photolabile protective group useful for making "caged" molecules. Ncm marries the reliable photochemistry of 2-nitrobenzyl systems with the excellent stability and spectroscopic properties of the coumarin chromophore. From simple, commercially available starting materials, preparation of Ncm and its caged derivatives is both quick and easy. Photorelease of Ncm-caged molecules occurs on the microsecond time scale, with quantum efficiencies of 0.05-0.08. We report the synthesis and physical properties of Ncm and its caged derivatives. The utility of Ncm-caged glutamate for neuronal photostimulation is demonstrated in cultured hippocampal neurons and in brain slice preparations.

Baeyer-Villiger Rearrangement of a Substituted Pyrrole by Oxone.

Pyrroloxyls have been reported to exhibit very narrow EPR spectral lines, essential for in vivo imaging. En route to pyrroloxyls, we observed an unexpected Baeyer-Villiger rearrangement, leading to loss of aromaticity and formation of a 4,5-dihydro-1H-ketopyrrole.

A study on the photophysics of 9-amino-10-cyanoanthracene: probing its dual absorption and emission behavior.

The photophysics of a donor-acceptor substituted chromophore, 9-amino-10-cyanoanthracene (ACAN), has been investigated in polar and nonpolar solvents to understand its intriguing dual absorption and emission behavior. Steady-state and time-resolved fluorescence studies clearly indicate that the short wavelength emission band of ACAN arises from the higher excited singlet state, S2, while the longer wavelength emission band arises from the intramolecular charge transfer (ICT) state, S1. Interestingly, both these states can be populated by direct excitation from the ground state. Temperature dependent studies reveal a pronounced activation controlled nonradiative decay channel for the ICT state of ACAN. It is proposed that this activation controlled nonradiative de-excitation arises because of a large relative displacement and a cross-over of the potential energy (PE) surfaces of ACAN in the ground and the ICT states, as a result of different twist angles of the amino group in these two states. Qualitative PE diagrams have accordingly been presented to correlate and rationalize the observed results. The present study also brings to light the interesting excited state prototropic behavior of ACAN and the consequent modulation of the ICT emission that has not been reported in the literature so far.

Dimethyl 5-acetyl-1-hy-droxy-4-methyl-1H-pyrrole-2,3-di-carboxyl-ate, an oxidation-resistant N-hy-droxy-pyrrole.

The title compound, C11H13NO6, exhibits an intra-molecular O-H⋯O=C hydrogen bond between the N-hydroxyl H atom and carbonyl O atom of the neighboring acetyl group. This finding contradicts a previously published model in which the hydrogen bond was postulated to occur with the neighboring carbomethoxy group. This relatively strong hydrogen bond [O-H⋯O: D = 2.5583 (11) Šand θ = 152°] may underlie the resistance of the title compound to oxidation into the corresponding nitroxide.

Ethyl 2,5-di-tert-butyl-5-eth-oxy-4-oxo-4,5-di-hydro-1H-pyrrole-3-carboxyl-ate.

The title compound, C17H29NO4, contains a chiral center and crystallizes as a racemate. The asymmetric unit consists of two non-equivalent mol-ecules, in which the carbeth-oxy groups have markedly different orientations [C(=O)CC(OEt)=O torsion angles = 59.3 (2) and 156.0 (2)°]. In the crystal, mol-ecules form chains along [101] through N-H⋯O hydrogen bonds.

Characterizing caged molecules through flash photolysis and transient absorption spectroscopy.

Caged molecules are photosensitive molecules with latent biological activity. Upon exposure to light, they are rapidly transformed into bioactive molecules such as neurotransmitters or second messengers. They are thus valuable tools for using light to manipulate biology with exceptional spatial and temporal resolution. Since the temporal performance of the caged molecule depends critically on the rate at which bioactive molecules are generated by light, it is important to characterize the kinetics of the photorelease process. This is accomplished by initiating the photoreaction with a very brief but intense pulse of light (i.e., flash photolysis) and monitoring the course of the ensuing reactions through various means, the most common of which is absorption spectroscopy. Practical guidelines for performing flash photolysis and transient absorption spectroscopy are described in this chapter.

(2)H,(15)N-substituted nitroxides as sensitive probes for electron paramagnetic resonance imaging.

Electron paramagnetic resonance imaging (EPRI) using nitroxides is an emergent imaging method for studying in vivo physiology, including O(2) distribution in various tissues. Such imaging capabilities would allow O(2) mapping in tumors and in different brain regions following hypoxia or drug abuse. We have recently demonstrated that the anion of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (2) can be entrapped in brain tissue to quantitate O(2) concentration in vivo. To increase the sensitivity of O(2) measurement by EPR imaging, we synthesized 3-carboxy-2,2,5,5-tetra((2)H(3))methyl-1-(3,4,4-(2)H(3),1-(15)N)pyrrolidinyloxyl (7). EPR spectroscopic measurements demonstrate that this fully isotopically substituted nitroxide markedly improves signal-to-noise ratio and, therefore, the sensitivity of EPR imaging. The new isotopically substituted nitroxide shows increased sensitivity to changes in O(2) concentration, which will enable more accurate O(2) measurement in tissues using EPRI.

The effect of structure on nitroxide EPR spectral linewidth.

Nitroxides with narrow linewidths are essential for low-frequency EPR spectroscopy and in vivo EPR imaging. In developing a framework for designing narrow-line nitroxides, we sought to understand the unexpectedly narrow line width of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxyl (5). Computational modeling revealed that the carbonyl double bond in the 4-position allows conformational diversity that results in the observed narrowing of the EPR spectral line. In view of this finding, we synthesized two new nitroxides bearing an exocyclic double bond: 4-methoxycarbonylmethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl (7) and 4-acetoxymethoxycarbonylmethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl (9). These nitroxides, like nitroxide 5, exhibited narrow linewidths-consistent with the results of modeling. Nitroxide 8 (4-carboxymethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl), as a prototype, allows for a variety of structural diversity, such as nitroxide 9,that can, for instance, target tissue compartments for in vivo EPR imaging.

Optimization of labile esters for esterase-assisted accumulation of nitroxides into cells: a model for in vivo EPR imaging.

Nitroxide-based electron paramagnetic resonance (EPR) imaging agents are useful quantitative probes of O2 concentration in vivo in real time. Lipophilic, labile alkanoyloxymethyl esters of nitroxides can cross the blood-brain barrier, and after hydrolysis, the corresponding anionic nitroxide is intracellularly entrapped at levels sufficient to permit O2 measurements. The utility of nitroxides as EPR imaging agents depends critically on their ability to accumulate in the brain to high levels. In this study, we systematically investigated the relationship between the structure of the alkanoyl moiety and the ability of the corresponding labile ester to deliver nitroxide intracellularly. We demonstrate, in a cultured cell model, that for nitroxide labile esters with unbranched alkanoyl chains, increasing the chain length improves intracellular loading. Moreover, by studying an isomeric series of labile esters, we conclude that branching of the alkanoyl chain drastically reduces intracellular loading. These structural insights improve our general ability to use labile esters to deliver carboxylates intracellularly, and suggest a strategy for enhancing delivery of nitroxide imaging agents across the blood-brain barrier in a living animal.

Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation.

Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a nonselective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids: biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in approximately 40 micros and approximately 125 micros, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca(2+) indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca(2+)-free extracellular medium, photoreleased vanilloid can still elevate [Ca(2+)](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca(2+) release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in <4 ms at 22 degrees C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision.

Endocannabinoid signaling dynamics probed with optical tools.

Intercellular signaling dynamics critically influence the functional roles that the signals can play. Small lipids are synthesized and released from neurons, acting as intercellular signals in regulating neurotransmitter release, modulating ion channels on target cells, and modifying synaptic plasticity. The repertoire of biological effects of lipids such as endocannabinoids (eCBs) is rapidly expanding, yet lipid signaling dynamics have not been studied. The eCB system constitutes a powerful tool for bioassaying the dynamics of lipid signaling. The eCBs are synthesized in, and released from, postsynaptic somatodendritic domains that are readily accessible to whole-cell patch electrodes. The dramatic effects of these lipid signals are detected electrophysiologically as CB1-dependent alterations in conventional synaptic transmission, which therefore serve as a sensitive reporter of eCB actions. We used electrophysiological recording, photolytic release of caged glutamate and a newly developed caged AEA (anandamide), together with rapid [Ca2+]i measurements, to investigate the dynamics of retrograde eCB signaling between CA1 pyramidal cells and GABAergic synapses in rat hippocampus in vitro. We show that, at 22 degrees C, eCB synthesis and release must occur within 75-190 ms after the initiating stimulus, almost an order of magnitude faster than previously thought. At 37 degrees C, the time could be < 50 ms. Activation of CB1 and downstream processes constitute a significant fraction of the total delay and are identified as major rate-limiting steps in retrograde signaling. Our findings imply that lipid messenger dynamics are comparable with those of metabotropic neurotransmitters and can modulate neuronal interactions on a similarly fast time scale.

Ncm-D-aspartate: a novel caged D-aspartate suitable for activation of glutamate transporters and N-methyl-D-aspartate (NMDA) receptors in brain tissue.

The D-isomer of aspartate is both a substrate for glutamate transporters and an agonist of N-methyl-D-aspartate (NMDA) receptors. To monitor the behavior of these receptors and transporters in intact tissue we synthesized a new photo-labile analogue of D-aspartate, N-[(6-nitrocoumarin-7-yl)methyl]-D-aspartic acid (Ncm-D-aspartate). This compound was photolyzed rapidly (t(1/2)=0.11 micros) by UV light with a quantum efficiency of 0.041 at pH 7.4. In acute hippocampal slices, photolysis of Ncm-D-aspartate by brief (1 ms) exposure to UV light elicited rapidly activating inward currents in astrocytes that were sensitive to inhibition by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA). Neither Ncm-D-aspartate nor the photo-released caging group exhibited agonist or antagonist activity at glutamate transporters, and Ncm-D-aspartate did not induce transporter currents prior to photolysis. Glutamate transporter currents were also elicited in cerebellar Purkinje cells in response to photolysis of Ncm-D-aspartate. Photo-release of D-aspartate from Ncm-D-aspartate did not induce alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor or metabotropic glutamate receptor (mGluR) currents, but triggered robust NMDA receptor currents in neurons; Ncm-D-aspartate and the photolzyed caging group were similarly inert at NMDA receptors. These results indicate that Ncm-D-aspartate can be used to study NMDA receptors at excitatory synapses and interactions between transporters and receptors in brain tissue.

Unique roles of SK and Kv4.2 potassium channels in dendritic integration.

Focal activation of glutamate receptors in distal dendrites of hippocampal pyramidal cells triggers voltage-dependent Ca(2+) channel-mediated plateau potentials that are confined to the stimulated dendrite. We examined the role of dendritic K(+) conductances in determining the amplitude, duration, and spatial compartmentalization of plateau potentials. Manipulations that blocked SK-type Ca(2+)-activated K(+) channels, including apamin and BAPTA dialysis, increased the duration of plateau potentials without affecting their amplitude or compartmentalization. Manipulations that blocked Kv4.2 A-type K(+) channels, including a dominant-negative Kv4.2 construct and 4-aminopyridine, increased the amplitude of plateau potentials by allowing them to recruit neighboring dendrites. Prolongation of plateau potentials or block of Kv4.2 channels at branch points facilitated the ability of dendritic excitation to trigger fast action potentials. SK channels thus underlie repolarization of dendritic plateau potentials, whereas Kv4.2 channels confine these potentials to single dendritic branches, and both act in concert to regulate synaptic integration.