Caging Chromophores Research Articles

Synthesis and characterization of 8-aminoquinoline photocages for biological applications

Inspired by the 8-dimethylaminoquinoline (8-DMAQ) caging chromophore, photocleavable 8-aminoquinoline derivatives were prepared with the aim of furnishing a robust synthetic pathway for novel cage scaffolds. The four-step versatile process allows a facile introduction of various amino-substituents and its overall yield is feasible for scale-up. The novel photocages showed high photolysis efficiency and low fluorescence, comparable to literature reference compounds (as 8-DMAQ-OAc, BHQ-OAc and CyHQ-OAc), demonstrating the utility of the probes for uncaging applications. Several derivatives allow further functionalizations on the side chain, therefore could serve as building blocks for the design of more complex photoresponsive systems.

The yin and yang of intracellular delivery of amphipathic optical probes using n-butyl charge masking.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

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

AbstractWe 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.

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.

Novel Visible Light Photoactivatable Caged Neurotransmitters Based on a N-Methyl Quinolinium Chromophore

The development of novel photolabile protecting groups with practical levels of photolytic efficiency and hydrophilicity can provide smart photochemical tools, such as caged compounds. One of the long-standing problems of most reported photolabile protecting groups is the requirement for one-photon activation, of ultraviolet light (250-400 nm), that is harmful to living cells and has low tissue penetration power. An attractive approach to overcome this would be the use of longer-wavelength light for one-photon activation; advantages would include both lower phototoxicity and higher tissue penetration power than UV irradiation. As part of our research aimed at developing new photochemical tools, we have developed the N-methyl-7-hydroxyquinolinium (N-Me-7-HQm) caging chromophore as a novel photocage, sensitive to visible light. A key to the success of the development of the N-Me-7-HQm photocage was simple N-methylation of the 7-hydroxyquinoline chromophore. This modification allows access to visible light absorbance, facile photoactivation by blue-LED light (458 nm) with high photolytic efficiency, excellent water solubility, and high resistance to spontaneous hydrolysis. The success of the late stage upgrading of a chromophore in the synthetic sequence suggests that further functionalization of the caging chromophore will be possible, and should aid in the rapid generation of structurally diverse libraries of visible light-sensitive photocages.

Chromatically independent, two-color uncaging of glutamate and GABA with one- or two-photon excitation.

Caged compounds enable fast, light-induced, and spatially-defined application of bioactive molecules to cells. Covalent attachment of a caging chromophore to a crucial functionality of a biomolecule renders it inert, while short pulses of light release the caged molecule in its active form. Caged neurotransmitters have been widely used to study diverse neurobiological processes such as receptor distribution, synaptogenesis, transport, and long-term potentiation. Since the neurotransmitters glutamate and gamma-aminobutyric acid (GABA) are the most important, they have been studied extensively using uncaging. However, to be able to probe their interactions on a physiologically relevant timescale, fast and independent application of both neurotransmitters in an arbitrary order is desired. This can be achieved by combining two caging chromophores absorbing non-overlapping and thus orthogonal wavelengths of light, which enables the precise application of two caged molecules to the same preparation in any order, a technique called two-color uncaging. In this chapter, we describe the principles of orthogonal two-color uncaging with one- and two-photon excitation with an emphasis on caged glutamate and GABA. We then give a guide to its practical application and highlight some key studies utilizing this technique.

7-Hydroxy- N-Methylquinolinium Chromophore: A Photolabile Protecting Group for Blue-Light Uncaging.

The development of the N-methyl-7-hydroxymethylquinolinium ( N-Me-7-HQm) caging chromophore as a novel visible-light-sensitive photolabile protecting group is described. N-Me-7-HQm-caged compounds can be photoactivated by blue-light-emitting diode (LED) light (458 nm) with high photolytic efficiency, supporting applications to caging chemistry, and they also have sufficient water solubility and high resistance to spontaneous hydrolysis.

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.

Comparative one- and two-photon uncaging of MNI-glutamate and MNI-kainate on hippocampal CA1 neurons

BackgroundThe light-induced release of neurotransmitters from caging chromophores provides a powerful means to study the underlying receptors in a physiologically relevant context. Surprisingly, most caged neurotransmitters, including the widely used 4-methoxy-7-nitroindolinyl (MNI)-glutamate, show strong antagonism against GABA-A receptors. Kainate has been shown to exhibit a higher efficacy at glutamate receptors compared to glutamate itself. Thus, uncaging of kainate might allow the application of the caged compound at lower, less antagonistic concentrations. New methodsThis study provides a detailed comparison of MNI-glutamate and MNI–kainate uncaging by different modes of one- and two-photon irradiation on hippocampal CA1 pyramidal neurons in acute brain slices. Results/Comparison with existing methodsUnexpectedly, the data revealed that currents in response to MNI-glutamate uncaging were larger compared to MNI-kainate with local one-photon laser uncaging at the soma and two-photon uncaging at the same spines. Furthermore, the direct comparison demonstrates the influence of type of caged agonist and light delivery conditions used for uncaging on the amplitude and kinetic properties of the current response. ConclusionThese findings highlight the importance of experimental design for uncaging experiments and provide a basis for future studies employing one- and two-photon uncaging to understand glutamate-dependent processes. It further provides the first example of two-photon uncaging of kainate at single spines in acute brain slices.

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.

Caged compounds for multichromic optical interrogation of neural systems.

Caged compounds are widely used by neurophysiologists to study many aspects of cellular signaling in glia and neurons. Biologically inert before irradiation, they can be loaded into cells via patch pipette or topically applied in situ to a defined concentration; photolysis releases the caged compound in a very rapid and spatially defined way. As caged compounds are exogenous optical probes, they include not only natural products such neurotransmitters, calcium and IP3 but non-natural products such as fluorophores, drugs and antibodies. In this Technical Spotlight we provide a short introduction to the uncaging technique by discussing the nitroaromatic caging chromophores most widely used in such experiments [e.g. α-carboxy-ortho-nitrobenyl (CNB), dimethoxynitrobenzyl (DMNB), 4-methoxy-7-nitroindolinyl (MNI) and 4-carboxymethoxy-7-nitroindolinyl (CDNI)]. We show that recently developed caging chromophores [rutheniumbipyridial (RuBi) and 7-diethylaminocoumarin (DEAC)450] that are photolyzed with blue light (~ 430-480 nm range) can be combined with traditional nitroaromatic caged compounds to enable two-color optical probing of neuronal function. For example, one-photon uncaging of either RuBi-GABA or DEAC450-GABA with a 473-nm laser is facile, and can block nonlinear currents (dendritic spikes or action potentials) evoked by two-photon uncaging of CDNI-Glu at 720 nm. We also show that two-photon uncaging of DEAC450-Glu and CDNI-GABA at 900 and 720 nm, respectively, can be used to fire and block action potentials. Our experiments illustrate that recently developed chromophores have taken uncaging out of the 'monochrome era', in which it has existed since 1978, so as to enable multichromic interrogation of neuronal function with single-synapse precision.

Wavelength-Selective One- and Two-Photon Uncaging of GABA

We have synthesized photolabile 7-diethylamino coumarin (DEAC) derivatives of γ-aminobutyric acid (GABA). These caged neurotransmitters efficiently release GABA using linear or nonlinear excitation. We used a new DEAC-based caging chromophore that has a vinyl acrylate substituent at the 3-position that shifts the absorption maximum of DEAC to about 450 nm and thus is named "DEAC450". DEAC450-caged GABA is photolyzed with a quantum yield of 0.39 and is highly soluble and stable in physiological buffer. We found that DEAC450-caged GABA is relatively inactive toward two-photon excitation at 720 nm, so when paired with a nitroaromatic caged glutamate that is efficiently excited at such wavelengths, we could photorelease glutamate and GABA around single spine heads on neurons in brain slices with excellent wavelength selectivity using two- and one-photon photolysis, respectively. Furthermore, we found that DEAC450-caged GABA could be effectively released using two-photon excitation at 900 nm with spatial resolution of about 3 μm. Taken together, our experiments show that the DEAC450 caging chromophore holds great promise for the development of new caged compounds that will enable wavelength-selective, two-color interrogation of neuronal signaling with excellent subcellular resolution.

Spectral Evolution of a Photochemical Protecting Group for Orthogonal Two-Color Uncaging with Visible Light

Caged compounds are molecules rendered functionally inert by derivatization with a photochemical protecting group. We describe the design logic behind the development of a diethylaminocoumarin (DEAC) caging chromophore, DEAC450, that absorbs blue light strongly (ε450 = 43,000 M(-1) cm(-1)) and violet light 11-fold more weakly. The absorption minimum is in the wavelength range (340-360 nm) that is traditionally used for photolysis of many widely used nitroaromatic caged compounds (e.g., 4-carboxymethoxy-5,7-dinitroindolinyl(CDNI)-GABA). We used this chromophore to synthesize DEAC450-caged cAMP and found this probe was very stable toward aqueous hydrolysis in the electronic ground state but was photolyzed with a quantum efficiency of 0.78. When DEAC450-cAMP and CDNI-GABA where co-applied to striatal cholinergic interneurons, the caged compounds were photolyzed in an chromatically orthogonal manner using blue and violet light so as to modulate the neuronal firing rate in a bidirectional way.

Optically Selective Two-Photon Uncaging of Glutamate at 900 nm

We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength-selective two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended π-electron moiety at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slices generated postsynaptic responses that were similar to spontaneous postsynaptic excitatory miniature currents, whereas significantly higher energies at 720 nm evoked no currents. Since many nitroaromatic caged compounds are two-photon active at 720 nm, optically selective uncaging of DEAC450-caged biomolecules at 900 nm may allow facile two-color optical interrogation of bimodal signaling pathways in living tissue with high resolution for the first time.

Retraction: Development of Anionically Decorated 2‐(ortho‐Nitrophenyl)‐Propyl‐Caged Neurotransmitters for Photolysis in vitro and in vivo

Three new caged neurotransmitters were synthesized built around the 2-(ortho-nitrophenyl)propyl (NPP) caging chromophore. The NPP-caged L-glutamate (Glu) and γ-aminobutyric acid (GABA) derivatives, which have an extended π-electron system bearing two carboxylates or phosphates were highly soluble (>50 mM) and hydrolytically stable at physiological pH. Uncaging GABA with ultraviolet light blocked network oscillations in layer 1 of the neocortex of a living mouse. Two-photon photolysis of caged Glu at single spine heads evoked changes in membrane voltage that were identical to synaptic stimulations. The implications of solubility complexities for the further development of the NPP scaffold for neurotransmitter uncaging are discussed in the context of other recent developments in this area.

2,5-Dihydroxybenzyl and (1,4-Dihydroxy-2-naphthyl)methyl, Novel Reductively Armed Photocages for the Hydroxyl Moiety

Irradiation of alcohols, phenols, and carboxylic acids "caged" with the 2,5-dihydroxybenzyl group or its naphthalene analogue results in the efficient release of the substrate. The initial byproduct of the photoreaction, 4-hydroxyquinone-2-methide, undergoes rapid tautomerization into methyl p-quinone. The UV spectrum of the latter is different from that of the caging chromophore, thus permitting selective irradiation of the starting material in the presence of photochemical products. These photoremovable protecting groups can be armed in situ by the reduction of photochemically inert p-quinone precursors.

3‐Nitro‐2‐naphthalenemethanol: A Photocleavable Protecting Group for Carboxylic Acids.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

3-Nitro-2-naphthalenemethanol: a photocleavable protecting group for carboxylic acids

Photocleavable protecting groups are important in synthesis and caging. Among many such groups, 2-nitrobenzyl and related groups have been found useful in many applications. However, most of the known 2-nitrobenzyl-based caging chromophores show either low quantum yield or the photolysis wavelength is not suitable for various applications. In this paper, we report 2-nitro-3-naphthalenemethanol (NNM) as an efficient photocleavable protecting group for molecules containing a carboxylic function. NNM possesses photochemical properties better than the 2-nitrobenzyl chromophores as it is photoactivatable at 380 nm in aqueous medium (CH 3CN/H 2O, 3:2 v/v) showing the desired photochemistry. The carboxylic acids are efficiently photoreleased from NNM-based esters in almost quantitative yield.