Chemical tuning of photoswitchable azobenzenes: a photopharmacological case study using nicotinic transmission.

We have developed photochromic probes for the nicotinic acetylcholine receptor that exploit the unique chemical properties of the tetrafluoroazobenzene (4FAB) scaffold. Ultraviolet light switching and rapid thermal relaxation of the metastable cis configuration are the main drawbacks associated with standard AB-based switches. We designed our photoprobes to take advantage of the excellent thermodynamic stability of the cis-4FAB configuration (thermal half-life > 12 days at 37 °C in physiological buffer) and cis-trans photostationary states above 84%. Furthermore, the well-separated n-π* absorption bands of trans- and cis-4FAB allow facile photoswitching with visible light in two optical channels. A convergent 11-step synthetic approach allowed the installation of a trimethylammonium (TA) head onto the 4FAB scaffold, by means of an alkyl spacer, to afford a free diffusible 4FABTA probe. TAs are known to agonize nicotinic receptors, so 4FABTA was tested on mouse brain slices and enabled reversible receptor activation with cycles of violet and green light. Due to the very long-lived metastable cis configuration, 4FAB in vivo use could be of great promise for long term biological studies. Further chemical functionalization of this 4FAB probe with a maleimide functionality allowed clean cross-linking with glutathione. However, attempts to conjugate with a cysteine on a genetically modified nicotinic acetylcholine receptor did not afford the expected light-responsive channel. Our data indicate that the 4FAB photoswitch can be derivatized bifunctionally for genetically-targeted photopharmacology whilst preserving all the favorable photophysical properties of the parent 4FAB scaffold, however, the tetrafluoro motif can significantly perturb pharmacophore-protein interactions. In contrast, we found that the freely diffusible 4FABTA probe could be pre-set with green light into an OFF state that was biologically inert, irradiation with violet light effectively "uncaged" agonist activity, but in a photoreversible manner. Since the neurotransmitter acetylcholine has fully saturated heteroatom valences, our photoswitchable 4FABTA probe could be useful for physiological studies of this neurotransmitter.

Dendrimer Conjugation Enables Multiphoton Chemical Neurophysiology Studies with an Extended π-Electron Caging Chromophore.

We have developed a caged neurotransmitter using an extended π-electron chromophore for efficient multiphoton uncaging on living neurons. Widely studied in a chemical context, such chromophores are inherently bioincompatible due to their highly lipophilic character. Attachment of two polycarboxylate dendrimers, a method we call "cloaking", to a bisstyrylthiophene (or BIST) core effectively transformed the chromophore into a water-soluble optical probe, whilst maintaining the high two-photon absorption of over 500 GM. Importantly, the cloaked caged compound was biologically inert at the high concentrations required for multiphoton chemical physiology. Thus, in contrast to non-cloaked BIST compounds, the BIST-caged neurotransmitter can be safely delivered onto neurons in acutely isolated brain slices, thereby enabling high-resolution two-photon uncaging without any side effects. We expect that our cloaking method will enable the development of new classes of cell-compatible photolabile probes using a wide variety of extended π-electron caging chromophores.

Optical probing of acetylcholine receptors on neurons in the medial habenula with a novel caged nicotine drug analogue.

KEY POINTS: A new caged nicotinic acetylcholine receptor (nAChR) agonist was developed, ABT594, which is photolysed by one- and two-photon excitation. The caged compound is photolysed with a quantum yield of 0.20. One-photon uncaging of ABT594 elicited large currents and Ca2+ transients at the soma and dendrites of medial habenula (MHb) neurons of mouse brain slices. Unexpectedly, uncaging of ABT594 also revealed highly Ca2+ -permeable nAChRs on axons of MHb neurons. ABSTRACT: Photochemical release of neurotransmitters has been instrumental in the study of their underlying receptors, with acetylcholine being the exception due to its inaccessibility to photochemical protection. We caged a nicotinic acetylcholine receptor (nAChR) agonist, ABT594, via its secondary amine functionality. Effective photolysis could be carried out using either one- or two-photon excitation. Brief flashes (0.5-3.0 ms) of 410 nm light evoked large currents and Ca2+ transients on cell bodies and dendrites of medial habenula (MHb) neurons. Unexpectedly, photorelease of ABT594 also revealed nAChR-mediated Ca2+ signals along the axons of MHb neurons.

Thermodynamically Stable, Photoreversible Pharmacology in Neurons with One- and Two-Photon Excitation.

Photoswitchable bioprobes enable bidirectional control of cell physiology with different wavelengths of light. Many current photoswitches use cytotoxic UV light and are limited by the need for constant illumination owing to thermal relaxation in the dark. Now a photoswitchable tetrafluoroazobenzene(4FAB)-based ion channel antagonist has been developed that can be efficiently isomerized in two separate optical channels with visible light. Importantly, the metastable cis configuration showed very high stability in the dark over the course of days at room temperature. In neurons, the 4FAB antagonist reversibly blocks voltage-gated ion channels with violet and green light. Furthermore, photoswitching could also be achieved with two-photon excitation yielding high spatial resolution. 4FAB probes have the potential to enable long-term biological studies where both ON and OFF states can be maintained in the absence of irradiation.

Coumarin-diene photoswitches for rapid and efficient isomerization with visible light.

We have developed 7-diethylaminocoumarin-based chromophores that photoisomerize with visible light. These photoswitches possess many desirable attributes, including large extinction coefficients (18 600-59 100 M-1 cm-1), high quantum yields (0.45-0.50) and resistance to photofatigue. Additionally, time-resolved spectroscopy indicates that both isomerization reactions are complete in less than 1 ns.

Two-color, one-photon uncaging of glutamate and GABA.

Neuronal cells receive a variety of excitatory and inhibitory signals which they process to generate an output signal. In order to study the interaction between excitatory and inhibitory receptors with exogenously applied transmitters in the same preparation, two caging chromophores attached to glutamate and GABA were developed that were selectively photolyzed by different wavelengths of light. This technique has the advantage that the biologically inactive caged compound can be applied at equilibrium prior to the near instantaneous release of the transmitters. This method therefore mimics the kinetics of endogenously released transmitters that is otherwise not possible in brain slice preparations. Repeated photolysis with either of the two wavelengths resulted in GABA- or glutamate-induced activation of both ionotropic and metabotropic receptors to evoke reproducible currents. With these compounds, the interaction between inhibitory and excitatory receptors was examined using whole field photolysis.

Cloaked Caged Compounds: Chemical Probes for Two-Photon Optoneurobiology.

Caged neurotransmitters, in combination with focused light beams, enable precise interrogation of neuronal function, even at the level of single synapses. However, most caged transmitters are, surprisingly, severe antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors. By conjugation of a large, neutral dendrimer to a caged GABA probe we introduce a "cloaking" technology that effectively reduces such antagonism to very low levels. Such cloaked caged compounds will enable the study of the signaling of the inhibitory neurotransmitter GABA in its natural state using two-photon uncaging microscopy for the first time.

Development of Anionically Decorated Caged Neurotransmitters: In Vitro Comparison of 7-Nitroindolinyl- and 2-(p-Phenyl-o-nitrophenyl)propyl-Based Photochemical Probes.

Neurotransmitter uncaging, especially that of glutamate, has been used to study synaptic function for over 30 years. One limitation of caged glutamate probes is the blockade of γ-aminobutyric acid (GABA)-A receptor function. This problem comes to the fore when the probes are applied at the high concentrations required for effective two-photon photolysis. To mitigate such problems one could improve the photochemical properties of caging chromophores and/or remove receptor blockade. We show that addition of a dicarboxylate unit to the widely used 4-methoxy-7-nitroindolinyl-Glu (MNI-Glu) system reduced the off-target effects by about 50-70 %. When the same strategy was applied to an electron-rich 2-(p-Phenyl-o-nitrophenyl)propyl (PNPP) caging group, the pharmacological improvements were not as significant as in the MNI case. Finally, we used very extensive biological testing of the PNPP-caged Glu (more than 250 uncaging currents at single dendritic spines) to show that nitro-biphenyl caging chromophores have two-photon uncaging efficacies similar to that of MNI-Glu.

C-H functionalization of cyclic amines: redox-annulations with α,ß-unsaturated carbonyl compounds.

Cyclic amines such as pyrrolidine and 1,2,3,4-tetrahydroisoquinoline undergo redox-annulations with α,ß-unsaturated aldehydes and ketones. Carboxylic acid promoted generation of a conjugated azomethine ylide is followed by 6π-electrocylization, and, in some cases, tautomerization. The resulting ring-fused pyrrolines are readily oxidized to the corresponding pyrroles or reduced to pyrrolidines.

Redox-neutral α-sulfenylation of secondary amines: ring-fused N,S-acetals.

Secondary amines react with thiosalicylaldehydes in the presence of catalytic amounts of acetic acid to generate ring-fused N,S-acetals in redox-neutral fashion. A broad range of amines undergo α-sulfenylation, including challenging substrates such morpholine, thiomorpholine, and piperazines. Computational studies employing density functional theory indicate that acetic acid reduces the energy barriers of two separate steps, both of which involve proton transfer.

Redox-neutral α-oxygenation of amines: reaction development and elucidation of the mechanism.

Cyclic secondary amines and 2-hydroxybenzaldehydes or related ketones react to furnish benzo[e][1,3]oxazine structures in generally good yields. This overall redox-neutral amine α-C-H functionalization features a combined reductive N-alkylation/oxidative α-functionalization and is catalyzed by acetic acid. In contrast to previous reports, no external oxidants or metal catalysts are required. Reactions performed under modified conditions lead to an apparent reductive amination and the formation of o-hydroxybenzylamines in a process that involves the oxidation of a second equivalent of amine. A detailed computational study employing density functional theory compares different mechanistic pathways and is used to explain the observed experimental findings. Furthermore, these results also reveal the origin of the catalytic efficiency of acetic acid in these transformations.

Facile Access to Ring-Fused Aminals via Direct α-Amination of Secondary Amines with ortho-Aminobenzaldehydes. Synthesis of Vasicine, Deoxyvasicine, Deoxyvasicinone, Mackinazolinone and Ruteacarpine.

Secondary amines undergo redox-neutral reactions with aminobenzaldehydes under conventional and microwave heating to furnish polycyclic aminals via amine α-amination/N-alkylation. This unique α-functionalization reaction proceeds without the involvement of transition metals or other additives. The resulting aminal products are precursors for various quinazolinone alkaloids and their analogues.

Selective copper(II) acetate and potassium iodide catalyzed oxidation of aminals to dihydroquinazoline and quinazolinone alkaloids.

Copper(II) acetate/acetic acid/O2 and potassium iodide/tert-butylhydroperoxide systems are shown to affect the selective oxidation of ring-fused aminals to dihydroquinazolines and quinazolinones, respectively. These methods enable the facile preparation of a number of quinazoline alkaloid natural products and their analogues.

Metal-free α-amination of secondary amines: computational and experimental evidence for azaquinone methide and azomethine ylide intermediates.

We have performed a combined computational and experimental study to elucidate the mechanism of a metal-free α-amination of secondary amines. Calculations predicted azaquinone methides and azomethine ylides as the reactive intermediates and showed that iminium ions are unlikely to participate in these transformations. These results were confirmed by experimental deuterium-labeling studies and the successful trapping of the postulated azomethine ylide and azaquinone methide intermediates. In addition, computed barrier heights for the rate-limiting step correlate qualitatively with experimental findings.

The decarboxylative Strecker reaction.

α-Amino acids react with aldehydes in the presence of a cyanide source to form α-amino nitriles in what can be considered a decarboxylative variant of the classical Strecker reaction. This unprecedented transformation does not require the use of a metal catalyst and provides facile access to valuable α-amino nitriles that are inaccessible by traditional Strecker chemistry.