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Cancer cells change their glucose metabolism towards lactic acid production for various defensive and proliferative advantages. Dichloroacetate (DCA) is an inhibitor of a key enzyme and thus, changes back the OX‐PHOS to glycolysis ratio. We designed and synthesized a singlet oxygen triggered source of dichloroacetate which releases this inhibitor during photodynamic singlet oxygen generation. Synergistic action of DCA and singlet oxygen results in significant enhancement of photocytotoxicity. The results suggest that this approach could offer significant improvement in the therapeutic outcome of PDT. [ABSTRACT FROM AUTHOR]

This work presents the study results on the acid‐catalyzed cascade condensation of p‐toluenesulfonamide with glyoxal in H2SO4 in order to synthesize aza‐ and oxaazaisowurtzitanes—a platform for promising high‐energy‐density compounds—and explore their formation processes. The effects of the ratio of starting reactants, acidity, reaction medium concentration, and temperature on this process were studied in detail. Five novel compounds were derived, including four oxaazaisowurtzitanes comprising one to three aza groups, and one condensation intermediate. The most favorable conditions for the formation of the resultant caged compounds were established and some new regularities of the process were revealed. For instance, the reaction medium concentration was discovered for the first time to influence the generation process of oxaazaisowurtzitanes via direct condensation. The limiting stage of the cage formation process of oxaazaisowurtzitanes was identified. The formation rate of the oxaazaisowurtzitanes was shown to be dependent on their structure. The factor dramatically reducing the yield of the oxaazaisowurtzitanes at elevated synthesis temperature was also revealed. [ABSTRACT FROM AUTHOR]

Increasingly, retinal pathologies are being treated with virus‐mediated gene therapies. To be able to target viral transgene expression specifically to the pathological regions of the retina with light, we established an in vivo photoactivated gene expression paradigm for retinal tissue. Based on the inducible Cre/lox system, we discovered that ethinylestradiol is a suitable alternative to Tamoxifen as ethinylestradiol is more amenable to modification with photosensitive protecting compounds, i.e. "caging." Identification of ethinylestradiol as a ligand for the mutated human estradiol receptor was supported by in silico binding studies showing the reduced binding of caged ethinylestradiol. Caged ethinylestradiol was injected into the eyes of double transgenic GFAP‐CreERT2 mice with a Cre‐dependent tdTomato reporter transgene followed by irradiation with light of 450 nm. Photoactivation significantly increased retinal tdTomato expression compared to controls. We thus demonstrated a first step towards the development of a targeted, light‐mediated gene therapy for the eyes. [ABSTRACT FROM AUTHOR]

The synthesis of substituted 2,4,6,8,10,12-hexaazaisowurtzitane via direct condensation is challenging. The selection of starting ammonia derivatives is very limited. The important step in developing alternative synthetic routes to these compounds is to investigate their formation process in detail. Here, we examined an acid-catalyzed condensation between benzyl carbamate and glyoxal in a ratio of 2:1 in a range of polar protic and aprotic solvents, and discovered a new process occurring during the cascade condensation of glyoxal with ammonia derivatives as well as discovered several processes hindering the formation of caged compounds. More specifically, a cyclic compound, N,N′-bis(carbobenzoxy)-3,6-diamino-1,4-dioxane-2,5-diol, was found to form at the early stage of condensation under low acidity conditions. The formation of this compound is governed by an easier condensation of alcohol groups compared to the amide ones. The condensation intermediates, N,N′-bis(carbobenzoxy)ethan-1,2-diol, N,N′,N″-tris(carbobenzoxy)ethanol, and N,N′,N″,N‴-tetrakis(carbobenzoxy)ethan, were obtained at a higher acidity. A range of solvents were identified: those that react with benzyl carbamate, those that promote the progress of side processes, and those that promote precipitation of condensation intermediates. A few byproducts were isolated and identified. It was found that DMSO exhibits a strong deactivating ability, while CH3CN exhibits a strong activating ability towards the acid-catalyzed condensation process of benzyl carbamate with glyoxal. [ABSTRACT FROM AUTHOR]

Caged compounds, also called phototriggers are formed by a photo-removable protecting group attached to a molecule of interest, hindering its potential interactions or reaction partners. A particular chemical bond is broken when the phototrigger absorbs light of a given wavelength, yielding a non-interacting "cage" and a free interacting molecule. Numerous organic based caged compounds have been devised, and many of them have broad applications, usually in physiology research. The tunability of these phototriggers is scarce, and the common strategy consists in changing the photoremovable group. Conversely, ruthenium-polypyridine caged compounds are built around a Ru center that can accommodate six coordinated molecules or groups including the photo-releasable molecule. The design of the coordination sphere yields many ways to achieve a desired property, or modulate a property, such as hydrophilicity, redox potential, absorption, 2P capabilities, action cross section, etc. In this work we will show how the tuning of quantum yield of photorelease, absorption wavelength and thermal stability is feasible, and discuss the rationale and the limits of the ligand-tuning technique. [ABSTRACT FROM AUTHOR]

Photobase generators are species that allow the photocatalysis of various reactions, such as thiol‐Michael, thiol‐isocyanate, and ring‐opening polymerization reactions. However, existing compounds have complex syntheses and low quantum yields. To overcome these problems, photobase generators based on the photodecarboxylation reaction were developed. We synthesized and studied the photochemistry and photophysics of two xanthone photobase, their carboxylic acid precursors, and their photoproducts to understand the photobase generation mechanism. We determined accurate quantum yields of triplet states and photobase generation. The effect of the intermediate radical preceding the base release was demonstrated. We characterized the photophysics of the photobase by femtosecond spectroscopy and showed that the photodecarboxylation process occurred from the second excited triplet state with a rate constant of 2.2×109 s−1. [ABSTRACT FROM AUTHOR]

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.

Light passes through biological tissue, and so it is used for imaging biological processes in situ. Such observation is part of the very essence of science, but mechanistic understanding requires intervention. For more than 50 years a "second function" for light has emerged; namely, that of photochemical control. Caged compounds are biologically inert signaling molecules that are activated by light. These optical probes enable external instruction of biological processes by stimulation of an individual element in complex signaling cascades in its native environment. Cause and effect are linked directly in spatial, temporal, and frequency domains in a quantitative manner by their use. I provide a guide to the basic properties required to make effective caged compounds for the biological sciences. [ABSTRACT FROM AUTHOR]

The NO dioxygenation reaction catalyzed by heme-containing globin proteins is a crucial aerobic detoxification pathway. Accordingly, the second order reaction of NO with oxymyoglobin and oxyhemoglobin has been the focus of a large number of kinetic and spectroscopic studies. Stopped-flow and rapid-freeze-quench (RFQ) measurements have provided evidence for the formation of a Fe(III)-nitrato complex with millisecond lifetime prior to release of the nitrate product, but the temporal resolution of these techniques is insufficient for the characterization of precursor species. Most mechanistic models assume the formation of an initial Fe(III)-peroxynitrite species prior to homolytic cleavage of the O O bond and recombination of the resulting NO 2 and Fe(IV)=O species. Here we report vibrational spectroscopy measurements for the reaction of oxymyoglobin with a photolabile caged NO donor at cryogenic temperatures. We show that this approach offers efficient formation and trapping of the Fe(III)-nitrato, enzyme-product, complex at 180 K. Resonance Raman spectra of the Fe(III)-nitrato complex trapped via RFQ in the liquid phase and photolabile NO release at cryogenic temperatures are indistinguishable, demonstrating the complementarity of these approaches. Caged NO is released by irradiation <180 K but diffusion into the heme pocket is fully inhibited. Our data provide no evidence for Fe(III)-peroxynitrite of Fe(IV)=O species, supporting low activation energies for the NO to nitrate conversion at the oxymyoglobin reaction site. Photorelease of NO at cryogenic temperatures allows monitoring of the reaction by transmittance FTIR which provides valuable quantitative information and promising prospects for the detection of protein sidechain reorganization events in NO-reacting metalloenzymes. Synopsis The graphic shows the structure of oxymyoglobin, the caged NO compound, and the result of uncaged at 180 K. [Display omitted] • Nitric oxide readily diffuses into the active site of oxymyoglobin at 180 K. • The reaction of NO with oxymyoglobin at 180 K traps a Fe(III)-nitrato complex. • Transmittance FTIR data quantifies trace ferric- and ferrous-nitrosyl contaminants. [ABSTRACT FROM AUTHOR]

The synthesis of substituted 2,4,6,8,10,12-hexaazaisowurtzitane via direct condensation is challenging. The selection of starting ammonia derivatives is very limited. The important step in developing alternative synthetic routes to these compounds is to investigate their formation process in detail. Here, we examined an acid-catalyzed condensation between benzyl carbamate and glyoxal in a ratio of 2:1 in a range of polar protic and aprotic solvents, and discovered a new process occurring during the cascade condensation of glyoxal with ammonia derivatives as well as discovered several processes hindering the formation of caged compounds. More specifically, a cyclic compound, N,N′-bis(carbobenzoxy)-3,6-diamino-1,4-dioxane-2,5-diol, was found to form at the early stage of condensation under low acidity conditions. The formation of this compound is governed by an easier condensation of alcohol groups compared to the amide ones. The condensation intermediates, N,N′-bis(carbobenzoxy)ethan-1,2-diol, N,N′,N″-tris(carbobenzoxy)ethanol, and N,N′,N″,N‴-tetrakis(carbobenzoxy)ethan, were obtained at a higher acidity. A range of solvents were identified: those that react with benzyl carbamate, those that promote the progress of side processes, and those that promote precipitation of condensation intermediates. A few byproducts were isolated and identified. It was found that DMSO exhibits a strong deactivating ability, while CH3CN exhibits a strong activating ability towards the acid-catalyzed condensation process of benzyl carbamate with glyoxal.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.

Physiological responses to the opioid neuropeptide enkephalin often involve both mu and delta opioid receptors. To facilitate quantitative studies into opioid signaling, we previously developed a caged [Leu5]-enkephalin that responds to ultraviolet irradiation, but its residual activity at delta receptors confounds experiments that involve both receptors. To reduce residual activity, we evaluated side-chain, N-terminus, and backbone caging sites and further incorporated the dimethoxy-nitrobenzyl moiety to improve sensitivity to ultraviolet light-emitting diodes (LEDs). Residual activity was characterized using an in vitro functional assay, and the power dependence and kinetics of the uncaging response to 355 nm laser irradiation were assayed using electrophysiological recordings of mu opioid receptor-mediated potassium currents in brain slices of rat locus coeruleus. These experiments identified N-MNVOC-LE as an optimal compound. Using ultraviolet LED illumination to photoactivate N-MNVOC-LE in the CA1 region of hippocampus, we found that enkephalin engages both mu and delta opioid receptors to suppress inhibitory synaptic transmission.

Photocaged inducer molecules, especially photocaged isopropyl-β-d-1-thiogalactopyranoside (cIPTG), are well-established optochemical tools for light-regulated gene expression and have been intensively applied in Escherichia coli and other bacteria including Corynebacterium glutamicum, Pseudomonas putida or Bacillus subtilis. In this study, we aimed to implement a light-mediated on-switch for target gene expression in the facultative anoxygenic phototroph Rhodobacter capsulatus by using different cIPTG variants under both phototrophic and non-phototrophic cultivation conditions. We could demonstrate that especially 6-nitropiperonyl-(NP)-cIPTG can be applied for light-mediated induction of target gene expression in this facultative phototrophic bacterium. Furthermore, we successfully applied the optochemical approach to induce the intrinsic carotenoid biosynthesis to showcase engineering of a cellular function. Photocaged IPTG thus represents a light-responsive tool, which offers various promising properties suitable for future applications in biology and biotechnology including automated multi-factorial control of cellular functions as well as optimization of production processes.

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability. [ABSTRACT FROM AUTHOR]

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca2+ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca2+ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to Go/q or Gs proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca2+ from intracellular stores was much faster than cAMP-mediated Ca2+ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se. [ABSTRACT FROM AUTHOR]

Although reported several decades ago, 3,3ʹ,5,5ʹ-tetramethoxybenzoin esters have not been used as a common photolabile protecting group, contrary to their unsymmetrical 3ʹ,5ʹ-dimethoxybenzoin analogues. While the properties of the latter are superior, their tedious synthesis and chemical instability represent a drawback. In this article, we describe a reliable synthetic access to the symmetrical tetramethoxybenzoin derivatives, and show that their photochemical behaviour remain interesting, in particular chromatically orthogonality with respect to nitroveratryl esters. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The photocaging strategy has been widely used for activating biomolecules and hence regulating the underlying biological processes with high temporal and spatial resolution. Herein, we integrate this strategy into metabolic glycan labeling, which enables optically‐controlled metabolic labeling of cell‐surface sialoglycans with azides. N‐Azidoacetylmannosamine (ManNAz) is caged with a 4,5‐dimethoxy‐2‐nitrobenzyl (DMNB) group at the anomeric hydroxyl group to block its cellular metabolism. After feeding to the cells, irradiation with 365 nm UV light or 405 nm visible light cleaves the photocage and generates ManNAz within the cells. ManNAz is metabolically converted to azido sialic acid, which is then incorporated into cell‐surface sialoglycans. We demonstrated spatial and temporal control of this strategy by specifically labeling sialoglycans on a single cell and tracking the metabolism of ManNAz based on light‐triggered pulse‐chase labeling, respectively. The light‐controlled metabolic glycan labeling strategy should facilitate probing of glycosylation in complex biological systems with high spatiotemporal precision. [ABSTRACT FROM AUTHOR]

There are approximately 50 million people with epilepsy worldwide, even about 25% of whom cannot be effectively controlled by drugs or surgical treatment. A wireless closed-loop system for epilepsy detection and suppression is proposed in this study. The system is composed of an implantable optrode, wireless recording, wireless energy supply, and a control module. The system can monitor brain electrical activity in real time. When seizures are recognized, the optrode will be turned on. The preset photosensitive caged compounds are activated to inhibit the seizure. When seizures are inhibited or end, the optrode is turned off. The method demonstrates a practical wireless closed-loop epilepsy therapy system.

Photoremovable protective groups (PPGs) and related “caged” compounds have been recognized as a powerful tool in an arsenal of life science methods. The present review is focused on recent advances in design of “caged” compounds which function in red or near-infrared region. The naive comparison of photon energy with that of organic bond leads to the illusion that long-wavelength activation is possible only for weak chemical bonds like N-N. However, there are different means to overcome this threshold and shift the uncaging functionality into red or near-infrared regions for general organic bonds. We overview these strategies, including the novel photochemical and photophysical mechanisms used in newly developed PPGs, singlet-oxygen-mediated photolysis, and two-photon absorption. Recent advances in science places the infrared-sensitive PPGs to the same usability level as traditional ones, facilitating in vivo application of caged compounds.

We synthesized the first multifunctionalized phosphoinositide polyphosphate derivatives featuring a photo‐removable protecting group ("cage"), a photo‐crosslinkable diazirine group, and a terminal alkyne group useful for click chemistry. We demonstrate that the lipid derivatives readily enter cells. After photo‐crosslinking, cell fixation and fluorescent tagging via click chemistry, we determined the intracellular location of the lipid derivatives before and after uncaging of the lipids. We find that there is rapid trafficking of PI(3,4)P2 and PI(3,4,5)P3 derivatives to the plasma membrane, opening the intriguing possibility that there is active transport of these lipids involved. We employed the photo‐crosslinking and click chemistry functions to analyze the proteome of PI(3,4,5)P3‐binding proteins. From the latter, we validated by RNAi that the putative lipid binding proteins ATP11A and MPP6 are involved in the transport of PI(3,4,5)P3 to the plasma membrane. [ABSTRACT FROM AUTHOR]

Purinergic signaling plays a pivotal role in physiological processes and pathological conditions. Over the past decades, conventional pharmacological, biochemical, and molecular biology techniques have been utilized to investigate purinergic signaling cascades. However, none of them is capable of spatially and temporally manipulating purinergic signaling cascades. Currently, optical approaches, including optopharmacology and optogenetic, enable controlling purinergic signaling with low invasiveness and high spatiotemporal precision. In this mini-review, we discuss optical approaches for controlling purinergic signaling and their applications in basic and translational science. [ABSTRACT FROM AUTHOR]

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca2+ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca2+ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to Go/q or Gs proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca2+ from intracellular stores was much faster than cAMP-mediated Ca2+ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se.

Caenorhabditis elegans is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist N-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illuminationinduced uncaging enables optical control of the behavior of freelymoving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity. [ABSTRACT FROM AUTHOR]

Although photolabile protecting groups (PPGs) have found widespread applications in several fields of chemistry, biology and materials science, there is a growing interest in expanding the photochemical toolbox to overcome some of the limitations of classical caging groups. In this work, the synthesis of a new class of visible‐light‐sensitive PPGs based on low‐molecular weight COUPY fluorophores with several attractive properties, including long‐wavelength absorption, is reported. Besides being stable to spontaneous hydrolysis in the dark, COUPY‐based PPGs can be efficiently photoactivated with yellow (560 nm) and red light (620 nm) under physiological‐like conditions, thereby offering the possibility of unmasking functional groups from COUPY photocages under irradiation conditions in which other PPGs remain stable. Additionally, COUPY photocages exhibit excellent cellular uptake and accumulate selectively in mitochondria, opening the door to the delivery of caged analogues of biologically active compounds into these organelles. [ABSTRACT FROM AUTHOR]

Photoremovable protecting groups (PPGs) have attracted much attention not only in the field of organic synthesis but also in biology and materials science because of the spatiotemporally controlled release of various functional molecules upon photolysis. In this study, a new PPG, the 2‐(p‐aminophenyl)‐1‐hydroxyinden‐3‐ylmethyl (pAPHi) chromophore, is designed and synthesized for the efficient and fast release of functional molecules with high conversion yields. The photolysis of caged benzoic acid with the pAPHi chromophore in dimethyl sulfoxide released benzoic acid in 96 % yield, with a decomposition quantum yield of 13 %. A high quantum yield of 51 % was observed with the clean formation of benzoic acid (94 % yield) in methanol. Transient absorption spectroscopic analyses clarified the generation of an intermediate carbocation as well as its reactivity. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP) is known as the universal energy source for cellular processes, in addition, ATP also plays an important role in inflammation and cell signaling. Extracellular ATP binds to purinergic receptors and initiates further immune responses. To investigate these processes in‐depth and to understand the complex mechanism of purinergic signaling, chemical tools are necessary. Here we present the synthesis of different photocaged ATP derivatives and the investigation of the light‐induced release of ATP depending on the different synthesized photocleavable protecting groups based on the 2‐nitrobenzyl moiety. Furthermore, we also present the synthesis of a mixed protected ATP‐derivative as an example for a novel class of lipophilically caged nucleoside triphosphates. This new type of compounds is protected with a highly lipophilic non‐toxic bio‐removable acyloxybenzyl group and a photocleavable group. This combination may allow both passive cell uptake and controlled release of ATP by irradiation with non‐harmful UV light. [ABSTRACT FROM AUTHOR]

Caging of pharmacologically active compounds is an interesting approach in biological chemistry and such photoactivatable compounds are also being developed towards novel therapeutic prodrug concepts. In this study we investigated the photolysis of the commonly used 4,5‐dimethoxy‐2‐nitrobenzyl (DMNB) caging moiety attached to the NH‐nitrogen of the pharmacologically relevant N‐phenylpyrimidine‐2‐amine scaffold. Based on this concept, a small set of para‐phenyl substituted derivatives with varying I/M effects (inductive/mesomeric electron‐donating/withdrawing effects) was designed, synthesized and parameters of the photolysis were determined. Hammett constants were related to photokinetic measurements, revealing electron donating effects to be unfavorable for the photolysis reaction. Our findings suggest electronic features of the entire molecule should be taken into account when considering its suitability for the caging concept. The results may also serve as a guideline to make relevant choices for DMNB applications in classical protecting group chemistry. [ABSTRACT FROM AUTHOR]

Photorelease of caged compounds is among the most powerful experimental approaches for studying cellular functions on fast timescales. However, its full potential has yet to be exploited, as the number of caged small molecules available for cell biological studies has been limited by synthetic challenges. Addressing this problem, a straightforward, one‐step procedure for efficiently synthesizing caged compounds was developed. An in situ generated benzylic coumarin triflate reagent was used to specifically functionalize carboxylate and phosphate moieties in the presence of free hydroxy groups, generating various caged lipid metabolites, including a number of GPCR ligands. By combining the photo‐caged ligands with the respective receptors, an easily implementable experimental platform for the optical control and analysis of GPCR‐mediated signal transduction in living cells was developed. Ultimately, the described synthetic strategy allows rapid generation of photo‐caged small molecules and thus greatly facilitates the analysis of their biological roles in live cell microscopy assays. [ABSTRACT FROM AUTHOR]

Free, ionic zinc (Zn 2+ ) modulates neurotransmitter dynamics in the brain. However, the sub-s effects of transient concentration changes of Zn 2+ on neurotransmitter release and uptake are not well understood. To address this lack of knowledge, we have combined the photolysis of the novel caged Zn 2+ compound [Zn(DPAdeCageOMe)] + with fast scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes in live, whole brain preparations from zebrafish ( Danio rerio ). After treating the brain with [Zn(DPAdeCageOMe)] + , Zn 2+ was released by application of light that was gated through a computer-controlled shutter synchronized with the FSCV measurements and delivered through a 1 mm fiber optic cable. We systematically optimized the photocage concentration and light application parameters, including the total duration and light-to-electrical stimulation delay time. While sub-s Zn 2+ application with this method inhibited DA reuptake, assessed by the first-order rate constant ( k ) and half-life ( t 1/2 ), it had no effect on the electrically stimulated DA overflow ([DA] STIM ). Increasing the photocage concentration and light duration progressively inhibited uptake, with maximal effects occurring at 100 μM and 800 ms, respectively. Furthermore, uptake was inhibited 200 ms after Zn 2+ photorelease, but no measurable effect occurred after 800 ms. We expect that application of this method to the zebrafish whole brain and other preparations will help expand the current knowledge of how Zn 2+ affects neurotransmitter release/uptake in select neurological disease states.

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. [ABSTRACT FROM AUTHOR]

Abstract New technologies for spatial and temporal remote control of G protein-coupled receptors (GPCRs) are necessary to unravel the complexity of GPCR signalling in cells, tissues and living organisms. An effective approach, recently developed, consists on the design of light-operated ligands whereby light-dependent GPCR activity regulation can be achieved. In this context, the use of light provides an advantage as it combines safety, easy delivery, high resolution and it does not interfere with most cellular processes. In this review we summarize the most relevant successful achievements in GPCR photopharmacology. These recent findings constitute a significant advance in research studies on the molecular dynamics of receptor activation and their physiological roles in vivo. Moreover, these molecules hold potential toward clinical uses as light-operated drugs, which can overcome some of the problems of conventional pharmacology. Highlights • Photopharmacology consists on the design of light-operated ligands whereby light dependent GPCR activity regulation can be achieved with high spatial and temporal resolution. • Caged compounds and photochromic ligands allow the regulation of endogenous receptors without genetic manipulation. • Photopharmacology has been successfully used in cells and in living organisms. •Ligands irreversibly and reversibly operated by light enable localised therapeutic effects and precise dosing patterns minimizing potential side effects. [ABSTRACT FROM AUTHOR]

Abstract: Lipid messengers exert their function on short time scales at distinct subcellular locations, yet most experimental approaches for perturbing their levels trigger cell‐wide concentration changes. Herein, we report on a coumarin‐based photocaging group that can be modified with organelle‐targeting moieties by click chemistry and thus enables photorelease of lipid messengers in distinct organelles. We show that caged arachidonic acid and sphingosine derivatives can be selectively delivered to mitochondria, the ER, lysosomes, and the plasma membrane. By comparing the cellular calcium transients induced by localized uncaging of arachidonic acid and sphingosine, we show that the precise intracellular localization of the released second messenger is crucial for the signaling outcome. Ultimately, we anticipate that this new class of caged compounds will greatly facilitate the study of cellular processes on the organelle level. [ABSTRACT FROM AUTHOR]

Non-spin-polarized electronic structures and Fermi surface properties of RX 2 Al 20 ( R  =  La, Ce, Yb, Th, U; X  = Ti, V, Cr, Mn) intermetallic compounds were calculated using the full potential all-electron local orbital (FPLO) approach in the framework of the local density approximation (LDA). Trends of the magnetism are discussed in terms of the characteristics of X - 3 d bands with a quantitative analysis of the relationship between band electron filling and crystal electric field splitting. Since coordination icosahedra of X -atoms have small trigonal distortion, crystal electric field splits the fivefold degenerate X -3 d state into low-energy singlet a 1 g and two higher-energy doublets e g . In R Ti 2 Al 20 and R V 2 Al 20 the population of the related 3 d sub-band is not sufficient to cause energetically favorable spin polarization, whereas magnetic instabilities develop in the R Cr 2 Al 20 series. Finally, a manifestation of strong repulsive interactions between itinerant Mn- d electrons become most pronounced in ferromagnetic UMn 2 Al 20 . The influence of non-magnetic R - f states on magnetic and thermodynamic properties is discussed with special emphasis on the role of the f – p and f – d hybridization. For LaTi 2 Al 20 and LaV 2 Al 20 the calculated quantum oscillation frequencies are in accord with experimental reports. [ABSTRACT FROM AUTHOR]

Abstract: Biological processes are naturally regulated with high spatial and temporal control, as is perhaps most evident in metazoan embryogenesis. Chemical tools have been extensively utilized in cell and developmental biology to investigate cellular processes, and conditional control methods have expanded applications of these technologies toward resolving complex biological questions. Light represents an excellent external trigger since it can be controlled with very high spatial and temporal precision. To this end, several optically regulated tools have been developed and applied to living systems. In this review we discuss recent developments of optochemical tools, including small molecules, peptides, proteins, and nucleic acids that can be irreversibly or reversibly controlled through light irradiation, with a focus on applications in cells and animals. [ABSTRACT FROM AUTHOR]

A photolabile ruthenium-based complex, [Ru(bpy)2(4AMP)2](PF6)2, (4AMP=4-(aminomethyl)pyridine) is incorporated into polyurea organo- and hydrogels via the reactive amine moieties on the photocleavable 4AMP ligands. While showing long-term stability in the dark, cleavage of the pyridine-ruthenium bond upon irradiation with visible or near-infrared irradiation (in a two-photon process) leads to rapid de-gelation of the supramolecular gels, thus enabling spatiotemporal micropatterning by photomasking or pulsed NIR-laser irradiation [ABSTRACT FROM AUTHOR]

The title compounds, C17H20O2 (1) and C17H18O2 (2), are allylated caged compounds. In (1), the carbon atoms bearing the allyl groups are far apart [2.9417 (17) Å], hence the expected ring-closing metathesis (RCM) protocol failed to give a ring-closing product. When these carbon atoms are connected by a C—C bond as in (2), the distance between them is much smaller [1.611 (3) Å] and consequently the RCM process was successful. The caged carbon skeleton of (1) can be described as a fusion of four five-membered rings and one six-membered ring. All four five-membered rings exhibit envelope conformations. The structure of compound (2) consists of four five-membered rings, of which two are cyclopentanone rings bonded at the 2, 4 and 5 positions and linked at the 3-carbons by a methylene bridge. It also consists of one four-membered and two six-membered rings. All four five-membered rings adopt envelope conformations. In the crystal of (1), molecules are linked via C—H...O hydrogen bonds, forming sheets lying parallel to (010). In the crystal of (2), molecules are linked via C—H...O hydrogen bonds forming chains along [100].

A systematic study on quinoline-derived light sensitive probes, having third-order rotational symmetry is presented. The electronically linked octupolar structures show considerably improved linear and nonlinear photophysical properties under one- and two-photon irradiation conditions compared to the corresponding monomers. Photolysis of the three acetate derivatives shows strong structure dependency: whereas irradiation of the 6- and 7-aminoquinoline derivatives resulted in fast intramolecular cyclization and only trace amounts of fragmentation products, the 8-aminoquinoline derivative afforded clean and selective photolysis, with a sequential release of their acetate groups ( δu[730]=0.67 GM). [ABSTRACT FROM AUTHOR]

Abstract: To target protein synthesis in defined areas, e. g. neuropiles of small brains or subcellular structures, locally restricted inhibition of protein synthesis is needed and can be realized by caged compounds of protein synthesis inhibitors (PSI). Since organic solvents interfere with protein synthesis themselves, the use of water‐soluble caged PSIs is a prerequisite in studies on protein synthesis. Such compounds are sparsely available. We developed and characterized efficient highly soluble caged compounds of the PSIs anisomycin and emetine masking their biological activity with a {8‐[bis(carboxymethyl)aminomethyl]‐6‐bromo‐7‐hydroxycoumarin‐4‐yl}methoxycarbonyl (BBHCMOC) derivative. The absorption spectra of the resulting BBHCMOC‐caged anisomycin and BBHCMOC‐caged emetine show long‐wavelength maxima and the extinction coefficients are high, allowing uncaging under non‐damaging light conditions. When uncaged, these caged PSIs reliably inhibit protein synthesis in an in vitro translation system and in cell culture. Taken the whole spectrum of properties into account, our BBHCMOC‐caged PSIs are highly qualified for in vivo studies. [ABSTRACT FROM AUTHOR]

We report the synthesis and photochemical properties of a series of dicyanocoumarinylmethyl (DEAdcCM)- and dicyanocoumarinylethyl (DEAdcCE)-based photocages of carboxylic acids and amines with absorption maximum around 500 nm. Photolysis studies with green light have demonstrated that the structure of the coumarin chromophore as well as the nature of the leaving group and the type of bond to be photocleaved (ester or carbamate) have a strong influence on the rate and efficiency of the uncaging process. These experimental observations were also supported by DFT calculations. Such differences in deprotection kinetics have been exploited to sequentially photolyze two dicyanocoumarin-caged model compounds (e.g., benzoic acid and ethylamine), and open the way to increasing the number of functional levels that can be addressed with light in a single system, particularly when combining dicyanocoumarin caging groups with other photocleavable protecting groups, which remain intact under green light irradiation. [ABSTRACT FROM AUTHOR]

The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol-containing molecules and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The control of biological processes plays a central role in life science, especially the tight regulation of gene expression for biotechnological systems. In this context, optogenetic tools have emerged as an important instrument for controlling gene expression by light with high spatiotemporal resolution in a non-invasive way. Here, we present the syntheses and characterization of biofunctional photocaged carbohydrates, on the basis of the biologically most relevant carbohydrates glucose, galactose, rhamnose, and lactose. The single-step cleavage of these compounds allows both, to rapidly activate and temporary or permanently repress the transcription in E. coli after short UV-A light exposure. This study thus presents a versatile toolbox of photocaged carbohydrates for the light-triggered regulation and control of cellular processes useful for synthetic bio(techno)logy applications. [ABSTRACT FROM AUTHOR]

Caged compounds are small organic molecules that can be photoactivated with brief pulses of light. They are widely used to study a great variety of biological processes by physiologists, cell biologists and neuroscientists. Initially made and invented by biologists in the late 1970s, they are now made mostly by chemists, often without any dialogue with the end users, the biologists. The idea for this review is to stimulate interaction between the two communities to further the creative development and application of these powerful optical probes.

A new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment has been developed. By using caged primers in PCR, unnatural sticky-ends of any sequence, which are sufficiently long for ligation-independent cloning (LIC), are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in the desired arrangement in a simple mixture of them, tightly enough to be repaired and ligated in competent cells.

Electrical stimulation has been used for more than 100 years in neuroscientific and biomedical research as a powerful tool for controlled perturbations of neural activity. Despite quickly driving neuronal activity, this technique presents some important limitations, such as the impossibility to activate or deactivate specific neuronal populations within a single stimulation site. This problem can be avoided by pharmacological methods based on the administration of receptor ligands able to cause specific changes in neuronal activity. However, intracerebral injections of neuroactive molecules inherently confound the dynamics of drug diffusion with receptor activation. Caged compounds have been proposed to circumvent this problem, for spatially and temporally controlled release of molecules. Caged compounds consist of a protecting group and a ligand made inactive by the bond between the two parts. By breaking this bond with light of an appropriate wavelength, the ligand recovers its activity within milliseconds. To test these compounds in vivo, we recorded local field potentials (LFPs) from the cerebral cortex of anesthetized female mice (CF1, 60-70 days, 20-30 g) before and after infusion with caged γ-amino-butyric-acid (GABA). After 30 min, we irradiated the cortical surface with pulses of blue light in order to photorelease the caged GABA and measure its effect on global brain activity. Laser pulses significantly and consistently decreased LFP power in four different frequency bands with a precision of few milliseconds (P < 0.000001); however, the inhibitory effects lasted several minutes (P < 0.0043). The technical difficulties and limitations of neurotransmitter photorelease are presented, and perspectives for future in vivo applications of the method are discussed.