Neural and behavioral control in Caenorhabditis elegans by a yellow-light-activatable caged compound.

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 illumination-induced uncaging enables optical control of the behavior of freely moving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.

Towards a Light-mediated Gene Therapy for the Eye using Caged Ethinylestradiol and the Inducible Cre/lox System.

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.

Condensation of Benzyl Carbamate with Glyoxal in Polar Protic and Aprotic Solvents.

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.

Reverse Engineering Caged Compounds: Design Principles for their Application in Biology.

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.

Photochemical Processes of Superbase Generation in Xanthone Carboxylic Salts.

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 .

Optochemical Control of Bacterial Gene Expression: Novel Photocaged Compounds for Different Promoter Systems.

Photocaged compounds are applied for implementing precise, optochemical control of gene expression in bacteria. To broaden the scope of UV-light-responsive inducer molecules, six photocaged carbohydrates were synthesized and photochemically characterized, with the absorption exhibiting a red-shift. Their differing linkage through ether, carbonate, and carbamate bonds revealed that carbonate and carbamate bonds are convenient. Subsequently, those compounds were successfully applied in vivo for controlling gene expression in E. coli via blue light illumination. Furthermore, benzoate-based expression systems were subjected to light control by establishing a novel photocaged salicylic acid derivative. Besides its synthesis and in vitro characterization, we demonstrate the challenging choice of a suitable promoter system for light-controlled gene expression in E. coli. We illustrate various bottlenecks during both photocaged inducer synthesis and in vivo application and possibilities to overcome them. These findings pave the way towards novel caged inducer-dependent systems for wavelength-selective gene expression.

3,3',5,5'-Tetramethoxybenzoin: a forgotten photolabile protecting group.

Although reported several decades ago, 3,3',5,5'-tetramethoxybenzoin esters have not been used as a common photolabile protecting group, contrary to their unsymmetrical 3',5'-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.

Ex Vivo Feedback Control of Neurotransmission Using a Photocaged Adenosine A1 Receptor Agonist.

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.

Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution.

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.

Effect of Photocaged Isopropyl ß-d-1-thiogalactopyranoside Solubility on the Light Responsiveness of LacI-controlled Expression Systems in Different Bacteria.

Photolabile protecting groups play a significant role in controlling biological functions and cellular processes in living cells and tissues, as light offers high spatiotemporal control, is non-invasive as well as easily tuneable. In the recent past, photo-responsive inducer molecules such as 6-nitropiperonyl-caged IPTG (NP-cIPTG) have been used as optochemical tools for Lac repressor-controlled microbial expression systems. To further expand the applicability of the versatile optochemical on-switch, we have investigated whether the modulation of cIPTG water solubility can improve the light responsiveness of appropriate expression systems in bacteria. To this end, we developed two new cIPTG derivatives with different hydrophobicity and demonstrated both an easy applicability for the light-mediated control of gene expression and a simple transferability of this optochemical toolbox to the biotechnologically relevant bacteria Pseudomonas putida and Bacillus subtilis. Notably, the more water-soluble cIPTG derivative proved to be particularly suitable for light-mediated gene expression in these alternative expression hosts.

Optical control of purinergic signaling.

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.

Light-inducible control of cellular proliferation and differentiation by a Hedgehog signaling inhibitor.

The Hedgehog (Hh) signaling pathway is a major regulator of cell differentiation and proliferation. Aberrant activation of the Hh pathway has been implicated in several types of cancer. To understand the Hedgehog pathway and fight against related diseases, it is important to inhibit Hedgehog signaling in a targeted manner. However, no tools are available for the precise inhibition of Hh signaling in a spatiotemporal manner. In this study, we synthesized and evaluated the bioactivity of a light-inducible Hh pathway inhibitor (NVOC-SANT-75). NVOC-SANT-75 inhibits transcription factor Gli1 in NIH3T3 cells and controls proliferation and differentiation of primary cultured mouse cerebellar neurons in a light-irradiation-dependent manner. The light-inducible Hedgehog signaling inhibitors may be a new candidate for light-mediated cancer treatment.

Synthesis and Cellular Labeling of Multifunctional Phosphatidylinositol Bis- and Trisphosphate Derivatives.

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.

Optical Control of Cellular ATP Levels with a Photocaged Adenylate Kinase.

We have developed a new tool for the optical control of cellular ATP concentrations with a photocaged adenylate kinase (Adk). The photocaged Adk is generated by substituting a catalytically essential lysine with a hydroxycoumarin-protected lysine through site-specific unnatural amino acid mutagenesis in both E. coli and mammalian cells. Caging of the critical lysine residue offers complete suppression of Adk's phosphotransferase activity and rapid restoration of its function both in vitro and in vivo upon optical stimulation. Light-activated Adk renders faster rescue of cell growth than chemically inducible expression of wild-type Adk in E. coli as well as rapid ATP depletion in mammalian cells. Thus, caging Adk provides a new tool for direct conditional perturbation of cellular ATP concentrations thereby enabling the investigation of ATP-coupled physiological events in temporally dynamic contexts.

Transformation of COUPY Fluorophores into a Novel Class of Visible-Light-Cleavable Photolabile Protecting Groups.

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.

A Coumarin Triflate Reagent Enables One-Step Synthesis of Photo-Caged Lipid Metabolites for Studying Cell Signaling.

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.

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.

Control of Protein Activity and Gene Expression by Cyclofen-OH Uncaging.

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.

A Click Cage: Organelle-Specific Uncaging of Lipid Messengers.

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.

Optochemical Control of Biological Processes in Cells and Animals.

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.