Journal articles: 'Photo-uncaging'

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Perdue, Lacey A., Priscilla Do, Andrew Chyong, Emma Costanza, Hye Ryong Kim, Camille David, Anna Kellner, et al. "Chemical modification strategies for photo-induced control of immune cell signaling." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 221.1. http://dx.doi.org/10.4049/jimmunol.204.supp.221.1.

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Abstract Cytokine therapies show great potential to induce cancer immune elimination and have demonstrated therapeutic benefit in a subset of patients. However, controlling the pleotropic effects of these immune signal effectors is a challenge. Here, we present an approach to enable optical control of cytokine activity by caging the activity of these proteins via labeling with wavelength-specific, photo-labile polymers (i.e. photo-caging). We create a system to measure light-induced polymer cleavage (uncaging) through fluorescence dequenching and establish: high wavelength-selectivity, protein activation through tissue mimics, and micrometer-scale uncaging resolution (via photopatterning). We focus on cytokines IL-2, IL-12 and IL-15 and demonstrate ability to reversibly modulate cytokine size and activity. We show photo-caging increases cytokine size by up to 20 fold, resulting in increased serum half-life. Additionally, cytokine activity (via T-cell proliferation) is blunted by 1,000 fold and near fully recovered through light-induced uncaging. Affinity measurements demonstrate photo-caging disrupts the bias of IL-2 towards IL-2Rα expressed on immunosuppressive T-regulatory cells. Further, photo-caging negates the ability of IL-2 to increase antigen specific (OT-1) cell activation, but is restored upon uncaging. In summary, we present the synthesis and therapeutic potential of these optically activated cytokines. In future studies we aim to use cytokine photo-caging as a means to provide new insights into the mechanics of cancer immune elimination and achieve tissue exclusive activation by exploiting spatially and temporally constraining cytokine activation in chip-based organoids and in vivo cancer models.

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Sambath, Karthik, Tinghan Zhao, Zhaoxiong Wan, and Yuanwei Zhang. "Photo-uncaging of BODIPY oxime ester for histone deacetylases induced apoptosis in tumor cells." Chemical Communications 55, no. 94 (2019): 14162–65. http://dx.doi.org/10.1039/c9cc07199g.

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Ichimura, Hideo, Kaushik Parthasarathi, Jens Lindert, and Jahar Bhattacharya. "Lung surfactant secretion by interalveolar Ca2+ signaling." American Journal of Physiology-Lung Cellular and Molecular Physiology 291, no. 4 (October 2006): L596—L601. http://dx.doi.org/10.1152/ajplung.00036.2006.

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Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.

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Wang, Haopei, and Zachary T. Ball. "A photochemical C=C cleavage process: toward access to backbone N-formyl peptides." Beilstein Journal of Organic Chemistry 17 (December 15, 2021): 2932–38. http://dx.doi.org/10.3762/bjoc.17.202.

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Photo-responsive modifications and photo-uncaging concepts are useful for spatiotemporal control of peptides structure and function. While side chain photo-responsive modifications are relatively common, access to photo-responsive modifications of backbone N–H bonds is quite limited. This letter describes a new photocleavage pathway, affording N-formyl amides from vinylogous nitroaryl precursors under physiologically relevant conditions via a formal oxidative C=C cleavage. The N-formyl amide products have unique properties and reactivity, but are difficult or impossible to access by traditional synthetic approaches.

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de Sousa, Aurideia P., Ana C. S. Gondim, Eduardo H. S. Sousa, Mayron A. de Vasconcelos, Edson H. Teixeira, Beatriz Pinheiro Bezerra, Alejandro Pedro Ayala, Patrícia H. R. Martins, Luiz Gonzaga de França Lopes, and Alda K. M. Holanda. "An unusual bidentate methionine ruthenium(II) complex: photo-uncaging and antimicrobial activity." JBIC Journal of Biological Inorganic Chemistry 25, no. 3 (March 14, 2020): 419–28. http://dx.doi.org/10.1007/s00775-020-01772-5.

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Lee, Shin-Rong, Paul J. Adams, and David T. Yue. "Large Ca2+-dependent facilitation of CaV2.1 channels revealed by Ca2+photo-uncaging." Journal of Physiology 593, no. 13 (June 30, 2015): 2753–78. http://dx.doi.org/10.1113/jp270091.

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Rong Lee, Shin, Manu B. Johny, and David T. Yue. "Large Ca2+-Dependent Facilitation of CaV2.1 Channels Induced by Ca2+ Photo-Uncaging." Biophysical Journal 104, no. 2 (January 2013): 461a. http://dx.doi.org/10.1016/j.bpj.2012.11.2549.

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Höglinger, Doris, André Nadler, Per Haberkant, Joanna Kirkpatrick, Martina Schifferer, Frank Stein, Sebastian Hauke, Forbes D. Porter, and Carsten Schultz. "Trifunctional lipid probes for comprehensive studies of single lipid species in living cells." Proceedings of the National Academy of Sciences 114, no. 7 (February 2, 2017): 1566–71. http://dx.doi.org/10.1073/pnas.1611096114.

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Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.

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Leonidova, Anna, Vanessa Pierroz, Riccardo Rubbiani, Yanjun Lan, Anita G. Schmitz, Andres Kaech, Roland K. O. Sigel, Stefano Ferrari, and Gilles Gasser. "Photo-induced uncaging of a specific Re(i) organometallic complex in living cells." Chemical Science 5, no. 10 (June 9, 2014): 4044. http://dx.doi.org/10.1039/c3sc53550a.

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Pickens, Rachael N., Grace L. Judd, and Jessica K. White. "Photo-uncaging a Ru(ii) intercalator via photodecomposition of a bridged Mn(i) photoCORM." Chemical Communications 57, no. 62 (2021): 7713–16. http://dx.doi.org/10.1039/d1cc02371c.

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Carling, Carl-Johan, Jason Olejniczak, Alexandra Foucault-Collet, Guillaume Collet, Mathieu L. Viger, Viet Anh Nguyen Huu, Brendan M. Duggan, and Adah Almutairi. "Efficient red light photo-uncaging of active molecules in water upon assembly into nanoparticles." Chemical Science 7, no. 3 (2016): 2392–98. http://dx.doi.org/10.1039/c5sc03717d.

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Wang, Yan, Shuai Li, Zhenyu Tian, Jiaqi Sun, Shuobin Liang, Bo Zhang, Lu Bai, et al. "Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction." Nucleic Acids Research 47, no. 19 (July 30, 2019): e114-e114. http://dx.doi.org/10.1093/nar/gkz659.

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Abstract Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging–uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.

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Ford, Peter C. "Metal complex strategies for photo-uncaging the small molecule bioregulators nitric oxide and carbon monoxide." Coordination Chemistry Reviews 376 (December 2018): 548–64. http://dx.doi.org/10.1016/j.ccr.2018.07.018.

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Horinouchi, Taeko, Hidehiko Nakagawa, Takayoshi Suzuki, Kiyoshi Fukuhara, and Naoki Miyata. "A novel mitochondria-localizing nitrobenzene derivative as a donor for photo-uncaging of nitric oxide." Bioorganic & Medicinal Chemistry Letters 21, no. 7 (April 2011): 2000–2002. http://dx.doi.org/10.1016/j.bmcl.2011.02.027.

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Dai, Ziwen, and Zhigang Wang. "Photoactivatable Platinum-Based Anticancer Drugs: Mode of Photoactivation and Mechanism of Action." Molecules 25, no. 21 (November 6, 2020): 5167. http://dx.doi.org/10.3390/molecules25215167.

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Platinum-based anticancer drugs are a class of widely used agents in clinical cancer treatment. However, their efficacy was greatly limited by their severe side effects and the arising drug resistance. The selective activation of inert platinum-based drugs in the tumor site by light irradiation is able to reduce side effects, and the novel mechanism of action of photoactivatable platinum drugs might also conquer the resistance. In this review, the recent advances in the design of photoactivatable platinum-based drugs were summarized. The complexes are classified according to their mode of action, including photoreduction, photo-uncaging, and photodissociation. The rationale of drug design, dark stability, photoactivation process, cytotoxicity, and mechanism of action of typical photoactivatable platinum drugs were reviewed. Finally, the challenges and opportunities for designing more potent photoactivatable platinum drugs were discussed.

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Schöneberg, Johannes, Mark Remec Pavlin, Shannon Yan, Maurizio Righini, Il-Hyung Lee, Lars-Anders Carlson, Amir Houshang Bahrami, et al. "ATP-dependent force generation and membrane scission by ESCRT-III and Vps4." Science 362, no. 6421 (December 20, 2018): 1423–28. http://dx.doi.org/10.1126/science.aat1839.

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The endosomal sorting complexes required for transport (ESCRTs) catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated ESCRT-III subunits Snf7, Vps24, and Vps2 and the AAA+ ATPase (adenosine triphosphatase) Vps4 in giant vesicles from which membrane nanotubes reflecting the correct topology of scission could be pulled. Upon ATP release by photo-uncaging, this system generated forces within the nanotubes that led to membrane scission in a manner dependent upon Vps4 catalytic activity and Vps4 coupling to the ESCRT-III proteins. Imaging of scission revealed Snf7 and Vps4 puncta within nanotubes whose presence followed ATP release, correlated with force generation and nanotube constriction, and preceded scission. These observations directly verify long-standing predictions that ATP-hydrolyzing assemblies of ESCRT-III and Vps4 sever membranes.

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Iwamoto, Yuji, Masahito Kodera, and Yutaka Hitomi. "Uncaging a catalytic hydrogen peroxide generator through the photo-induced release of nitric oxide from a {MnNO}6 complex." Chemical Communications 51, no. 46 (2015): 9539–42. http://dx.doi.org/10.1039/c5cc02566d.

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Zheng, Yang, Meichun Gao, Maikel Wijtmans, Henry F. Vischer, and Rob Leurs. "Synthesis and Pharmacological Characterization of New Photocaged Agonists for Histamine H3 and H4 Receptors." Pharmaceuticals 17, no. 4 (April 21, 2024): 536. http://dx.doi.org/10.3390/ph17040536.

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The modulation of biological processes with light-sensitive chemical probes promises precise temporal and spatial control. Yet, the design and synthesis of suitable probes is a challenge for medicinal chemists. This article introduces a photocaging strategy designed to modulate the pharmacology of histamine H3 receptors (H3R) and H4 receptors (H4R). Employing the photoremovable group BODIPY as the caging entity for two agonist scaffolds—immepip and 4-methylhistamine—for H3R and H4R, respectively, we synthesized two BODIPY-caged compounds, 5 (VUF25657) and 6 (VUF25678), demonstrating 10–100-fold reduction in affinity for their respective receptors. Notably, the caged H3R agonist, VUF25657, exhibits approximately a 100-fold reduction in functional activity. The photo-uncaging of VUF25657 at 560 nm resulted in the release of immepip, thereby restoring binding affinity and potency in functional assays. This approach presents a promising method to achieve optical control of H3R receptor pharmacology.

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van Rixel, Vincent H. S., Vadde Ramu, Austin B. Auyeung, Nataliia Beztsinna, David Y. Leger, Lucien N. Lameijer, Stan T. Hilt, et al. "Photo-Uncaging of a Microtubule-Targeted Rigidin Analogue in Hypoxic Cancer Cells and in a Xenograft Mouse Model." Journal of the American Chemical Society 141, no. 46 (October 18, 2019): 18444–54. http://dx.doi.org/10.1021/jacs.9b07225.

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Schuhmacher, Milena, Andreas T. Grasskamp, Pavel Barahtjan, Nicolai Wagner, Benoit Lombardot, Jan S. Schuhmacher, Pia Sala, et al. "Live-cell lipid biochemistry reveals a role of diacylglycerol side-chain composition for cellular lipid dynamics and protein affinities." Proceedings of the National Academy of Sciences 117, no. 14 (March 25, 2020): 7729–38. http://dx.doi.org/10.1073/pnas.1912684117.

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Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol–protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.

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Boucsein, Clemens, Martin Nawrot, Stefan Rotter, Ad Aertsen, and Detlef Heck. "Controlling Synaptic Input Patterns In Vitro by Dynamic Photo Stimulation." Journal of Neurophysiology 94, no. 4 (October 2005): 2948–58. http://dx.doi.org/10.1152/jn.00245.2005.

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Recent experimental and theoretical work indicates that both the intensity and the temporal structure of synaptic activity strongly modulate the integrative properties of single neurons in the intact brain. However, studying these effects experimentally is complicated by the fact that, in experimental systems, network activity is either absent, as in the acute slice preparation, or difficult to monitor and to control, as in in vivo recordings. Here, we present a new implementation of neurotransmitter uncaging in acute brain slices that uses functional projections to generate tightly controlled, spatio-temporally structured synaptic input patterns in individual neurons. For that, a set of presynaptic neurons is activated in a precisely timed sequence through focal photolytic release of caged glutamate with the help of a fast laser scanning system. Integration of synaptic inputs can be studied in postsynaptic neurons that are not directly stimulated with the laser, but receive input from the targeted neurons through intact axonal projections. Our new approach of dynamic photo stimulation employs functional synapses, accounts for their spatial distribution on the dendrites, and thus allows study of the integrative properties of single neurons with physiologically realistic input. Data obtained with our new technique suggest that, not only the neuronal spike generator, but also synaptic transmission and dendritic integration in neocortical pyramidal cells, can be highly reliable.

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Jervis, Peter J., Loic Hilliou, Renato B. Pereira, David M. Pereira, José A. Martins, and Paula M. T. Ferreira. "Evaluation of a Model Photo-Caged Dehydropeptide as a Stimuli-Responsive Supramolecular Hydrogel." Nanomaterials 11, no. 3 (March 11, 2021): 704. http://dx.doi.org/10.3390/nano11030704.

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Short peptides capped on the N-terminus with aromatic groups are often able to form supramolecular hydrogels, via self-assembly, in aqueous media. The rheological properties of these readily tunable hydrogels resemble those of the extracellular matrix (ECM) and therefore have potential for various biological applications, such as tissue engineering, biosensors, 3D bioprinting, drug delivery systems and wound dressings. We herein report a new photo-responsive supramolecular hydrogel based on a “caged” dehydropeptide (CNB-Phe-ΔPhe-OH 2), containing a photo-cleavable carboxy-2-nitrobenzyl (CNB) group. We have characterized this hydrogel using a range of techniques. Irradiation with UV light cleaves the pendant aromatic capping group, to liberate the corresponding uncaged model dehydropeptide (H-Phe-ΔPhe-OH 3), a process which was investigated by 1H NMR and HPLC studies. Crucially, this cleavage of the capping group is accompanied by dissolution of the hydrogel (studied visually and by fluorescence spectroscopy), as the delicate balance of intramolecular interactions within the hydrogel structure is disrupted. Hydrogels which can be disassembled non-invasively with temporal and spatial control have great potential for specialized on-demand drug release systems, wound dressing materials and various topical treatments. Both 2 and 3 were found to be non-cytotoxic to the human keratinocyte cell line, HaCaT. The UV-responsive hydrogel system reported here is complementary to previously reported related UV-responsive systems, which are generally composed of peptides formed from canonical amino acids, which are susceptible to enzymatic proteolysis in vivo. This system is based on a dehydrodipeptide structure which is known to confer proteolytic resistance. We have investigated the ability of the photo-activated system to accelerate the release of the antibiotic, ciprofloxacin, as well as some other small model drug compounds. We have also conducted some initial studies towards skin-related applications. Moreover, this model system could potentially be adapted for on-demand “self-delivery”, through the uncaging of known biologically active dehydrodipeptides.

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Frank, J. G., H. S. Jameson, C. Gorini, and D. Mendelowitz. "Mapping and Identification of GABAergic Neurons in Transgenic Mice Projecting to Cardiac Vagal Neurons in the Nucleus Ambiguus Using Photo-Uncaging." Journal of Neurophysiology 101, no. 4 (April 2009): 1755–60. http://dx.doi.org/10.1152/jn.91134.2008.

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The neural control of heart rate is determined primarily by the activity of preganglionic parasympathetic cardiac vagal neurons (CVNs) originating in the nucleus ambiguus (NA) in the brain stem. GABAergic inputs to CVNs play an essential role in determining the activity of these neurons including a robust inhibition during each inspiratory burst. The origin of GABAergic innervation has yet to be determined however. A transgenic mouse line expressing green florescent protein (GFP) in GABAergic cells was used in conjunction with caged glutamate to identify both clusters and individual GABAergic neurons that evoke inhibitory GABAergic synaptic responses in CVNs. Transverse slices were taken with CVNs patch-clamped in the whole cell configuration. Sections containing both the pre-Botzinger complex as well as the calamus scriptorius were divided into ∼90 quadrants, each 200 × 200 μm and were sequentially photostimulated. Inhibitory post synaptic currents (IPSCs) were recorded in CVNs after a 5-ms photostimulation of 50 μM caged glutamate. The four areas that contained GABAergic cells projecting to CVNs were 200 μm medial, 400 μm medial, 200 μm ventral, and 1,200 μm dorsal and 1,000 μm medial to patched CVNs. Once foci of GABAergic cells projecting to CVNs were determined, photostimulation of individual GABAergic neurons was conducted. The results from this study suggest that GABAergic cells located in four specific areas project to CVNs, and that these cells can be individually identified and stimulated using photouncaging to recruit GABAergic neurotransmission to CVNs.

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Chatterjee, Tanmay, Debjit Roy, Ananya Das, Anup Ghosh, Partha Pratim Bag, and Prasun K. Mandal. "Chemical tweaking of a non-fluorescent GFP chromophore to a highly fluorescent coumarinic fluorophore: application towards photo-uncaging and stem cell imaging." RSC Advances 3, no. 46 (2013): 24021. http://dx.doi.org/10.1039/c3ra44034f.

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Limpitikul, Worawan B., Joseph L. Greenstein, David T. Yue, Ivy E. Dick, and Raimond L. Winslow. "A bilobal model of Ca2+-dependent inactivation to probe the physiology of L-type Ca2+ channels." Journal of General Physiology 150, no. 12 (November 23, 2018): 1688–701. http://dx.doi.org/10.1085/jgp.201812115.

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L-type calcium channels (LTCCs) are critical elements of normal cardiac function, playing a major role in orchestrating cardiac electrical activity and initiating downstream signaling processes. LTCCs thus use feedback mechanisms to precisely control calcium (Ca2+) entry into cells. Of these, Ca2+-dependent inactivation (CDI) is significant because it shapes cardiac action potential duration and is essential for normal cardiac rhythm. This important form of regulation is mediated by a resident Ca2+ sensor, calmodulin (CaM), which is comprised of two lobes that are each capable of responding to spatially distinct Ca2+ sources. Disruption of CaM-mediated CDI leads to severe forms of long-QT syndrome (LQTS) and life-threatening arrhythmias. Thus, a model capable of capturing the nuances of CaM-mediated CDI would facilitate increased understanding of cardiac (patho)physiology. However, one critical barrier to achieving a detailed kinetic model of CDI has been the lack of quantitative data characterizing CDI as a function of Ca2+. This data deficit stems from the experimental challenge of uncoupling the effect of channel gating on Ca2+ entry. To overcome this obstacle, we use photo-uncaging of Ca2+ to deliver a measurable Ca2+ input to CaM/LTCCs, while simultaneously recording CDI. Moreover, we use engineered CaMs with Ca2+ binding restricted to a single lobe, to isolate the kinetic response of each lobe. These high-resolution measurements enable us to build mathematical models for each lobe of CaM, which we use as building blocks for a full-scale bilobal model of CDI. Finally, we use this model to probe the pathogenesis of LQTS associated with mutations in CaM (calmodulinopathies). Each of these models accurately recapitulates the kinetics and steady-state properties of CDI in both physiological and pathological states, thus offering powerful new insights into the mechanistic alterations underlying cardiac arrhythmias.

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Jing, Zhixin, Mark J. McCarron, Michael L. Dustin, and David R. Fooksman. "Germinal center expansion but not plasmablast differentiation is directly proportional to peptide-MHCII density via CD40-CD40L signaling strength." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 102.05. http://dx.doi.org/10.4049/jimmunol.208.supp.102.05.

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Abstract Although cognate peptide-MHCII (pMHCII) is critical for B cell selection in the germinal center (GC), it is unclear how cell intrinsic differences in peptide levels contribute to selection and cell fate decisions. Here, we applied the a-DEC205-OVA (dec) model system, to deliver different levels of OVA peptide to GC B cells in situ in order to interrogate how intermediate and high levels of pMHCII affect selection and cell fate on a per cell basis. We used Ly75+/− (DEC-het) B cells, which expressed ~50% of surface DEC205 protein compared to WT B cells, and presented proportional amount of OVA peptide after dec treatment. Using competitive co-transfers, we found that WT B cells expanded two-fold more than DEC-het B cells. This 2-fold difference in expansion was maintained at a wide range of dec administration. To study T cell recognition in situ, we developed a novel caged ovalbumin peptide, which is readily detected by OT-II T cells upon photo-uncaging, and revealed that initial T cell recognition in the GC leads to increased B-T serial contacts. Differential CD40 signaling, was both necessary and sufficient to mediate 2-fold differences in GC B cell expansion and also promoted GC-like B cell morphology, suggesting CD40 signaling is a rheostat of pMHCII dose. Surprisingly, we found that while plasmablast numbers were increased upon dec stimulation, both WT and DEC-het GC B cells were equally capable of differentiating into plasma cells, suggesting that pMHCII density does not directly control the output or quality of plasma cells generated. Thus, these results delineate distinct roles pMHCII play in expansion vs. differentiation in the GC. Supported by grants from NIH (R01 HL141491-05)

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Korzycka, Karolina A., Philip M. Bennett, Eduardo Jose Cueto-Diaz, Geoffrey Wicks, Mikhail Drobizhev, Mireille Blanchard-Desce, Aleksander Rebane, and Harry L. Anderson. "Two-photon sensitive protecting groups operating via intramolecular electron transfer: uncaging of GABA and tryptophan." Chemical Science 6, no. 4 (2015): 2419–26. http://dx.doi.org/10.1039/c4sc03775h.

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Wan, Zhaoxiong, Shupei Yu, Qi Wang, Karthik Sambath, Roshena Harty, Xiangshan Liu, Hao Chen, Chen Wang, Xuan Liu, and Yuanwei Zhang. "Far-red BODIPY-based oxime esters: photo-uncaging and drug delivery." Journal of Materials Chemistry B, 2023. http://dx.doi.org/10.1039/d3tb01867a.

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Bramham, Jack E., Matja Zalar, and Alexander P. Golovanov. "Controlled release and characterisation of photocaged molecules using in situ LED illumination in solution NMR spectroscopy." Chemical Communications, 2022. http://dx.doi.org/10.1039/d2cc04731d.

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Klausen, Maxime, Victor Dubois, Sébastien Picard, Eduardo Cueto Diaz, Jonathan Daniel, Jean-Baptiste Verlhac, and Mireille Blanchard-Desce. "Click Synthesis of Tweezer Dyads for H‐Bond Assisted Cooperativity in Tandem Uncaging Systems." Chemistry – A European Journal, July 2024. http://dx.doi.org/10.1002/chem.202402076.

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Abstract: “Tandem” uncaging systems, in which a photolabile protecting group (PPG) is sensitized by an energy‐harvesting antenna, may increase the photosensitivity of PPGs by several orders of magnitude for two‐photon (2P) photorelease. Yet, they remain poorly accessible because of arduous multi‐step synthesis. In this work, we design efficient tandem uncaging systems by (i) using a convenient assembly of the building blocks relying on click chemistry, (ii) H‐bonding induced proximity thus facilitating (iii) photoinduced electron transfer (PeT) as a cooperative mechanism. A strong two‐photon absorber electron‐donating quadrupolar antenna and various electron‐accepting PPGs (mDEAC, MNI or MDNI) were clicked stepwise onto a “tweezer‐shaped” pyrido‐2,6‐dicarboxylate platform whose H‐bonding and p‐stacking abilities were exploited to keep the antenna and the PPGs in close proximity. The different electron acceptor ability of the PPGs led to dyads with wildly different behaviors. Whilst the MDNI and MNI dyads showed poor dark stability or no photo‐uncaging ability due to their too high electron accepting character, the mDEAC dyad benefited from optimum redox potentials to promote PeT and slow down charge recombination, resulting in enhanced uncaging quantum yield (Fu=0.38) compared to mDEAC (Fu=0.014). The unique resulted in large 2P photo‐sensitivity in the near‐infrared window (240 GM at 710 nm).

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Denninger, L., H. Brunst, L. J. G. W. van Wilderen, M. Horz, H. M. A. Masood, C. D. McNitt, I. Burghardt, V. V. Popik, and J. Bredenbeck. "Switch the click: Ultrafast photochemistry of photoDIBO-OH tracked by time-resolved IR spectroscopy." Journal of Chemical Physics 160, no. 17 (May 1, 2024). http://dx.doi.org/10.1063/5.0196923.

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Click chemistry refers to selective reactions developed for grafting of bio(macro)molecules in their biological media. Caged click compounds have been employed to spatiotemporally control click reactions. Here, we survey the uncaging of photo-dibenzocyclooctyne-OH (photoDIBO-OH) to its click-chemistry active form DIBO-OH, with particular attention to its conversion timescale and efficiency. Ultraviolet pump–infrared probe experiments reveal a stepwise decarbonylation: first, carbon monoxide (C≡O) is released within 1.8 ps, and then, it converts, within 10 ps, to DIBO-OH. Completion of uncaging is achieved with an efficiency of ∼50%. A successful demonstration of two-photon uncaging of photoDIBO-OH at long wavelength (700 nm) confers enhanced in vivo compatibility and proceeds on the same timescale.

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Kalayci, Kubra, Hendrik Frisch, Christopher Barner-Kowollik, and Vinh X. Truong. "Green Light Enabled Staudinger-Bertozzi Ligation." Chemical Communications, 2022. http://dx.doi.org/10.1039/d2cc00911k.

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We introduce a visible light-induced Staudinger-Bertozzi ligation via photo-uncaging of a triphenylphosphine moiety with a photolabile coumarin derivative. Our action plot study examines the conversion as the function of wavelength,...

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33

Zhang, Yidan, Yixin Zhang, Lili Han, Qiaoling Che, Jiawei Tan, Peng Zou, and Yiyun Chen. "Photo‐Modulation of Gene‐Editing Enzymes CRISPR/Cas9 with Bifunctional Small‐Molecule Ligands." Chinese Journal of Chemistry, September 13, 2023. http://dx.doi.org/10.1002/cjoc.202300452.

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Comprehensive SummaryThe control of protein functions with light is valuable for spatiotemporal probing of biological systems. Current photo‐modulation methods include the light‐induced small‐molecule inhibitors uncaging and chromophore‐assisted light inactivation with reactive oxygen species (ROS). However, constant target protein expression results in inadequate photo‐modulation efficiency, particularly for less potent inhibitors and chromophores. Herein, we report a novel bifunctional small‐molecule ligands strategy to photo‐modulate gene‐editing enzymes CRISPR/Cas9. A coumarin‐derived small‐molecule ligand Bhc‐BRD0539 is developed to uncage the active small‐molecule inhibitor BRD0539 and to generate ROS in the Cas9 proximity upon light irradiation for dual inhibition of Cas9 activity. Our results highlight the synergistic photo‐modulation with bifunctional small‐molecule ligands, which offers a valuable addition to current CRISPR/Cas9 photo‐modulation technologies and may extend to other protein classes.This article is protected by copyright. All rights reserved.

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Morgante, Pierpaolo, Charitha Guruge, Yannick P. Ouedraogo, Nasri Nesnas, and Roberto Peverati. "Competition between cyclization and unusual Norrish type I and type II nitro-acyl migration pathways in the photouncaging of 1-acyl-7-nitroindoline revealed by computations." Scientific Reports 11, no. 1 (January 14, 2021). http://dx.doi.org/10.1038/s41598-020-79701-4.

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AbstractThe 7-nitroindolinyl family of caging chromophores has received much attention in the past two decades. However, its uncaging mechanism is still not clearly understood. In this study, we performed state-of-the-art density functional theory calculations to unravel the photo-uncaging mechanism in its entirety, and we compared the probabilities of all plausible pathways. We found competition between a classical cyclization and an acyl migration pathway, and here we explain the electronic and steric reasons behind such competition. The migration mechanism possesses the characteristics of a combined Norrish type I and a 1,6-nitro-acyl variation of a Norrish type II mechanism, which is reported here for the first time. We also found negligible energetic differences in the uncaging mechanisms of the 4-methoxy-5,7-dinitroindolinyl (MDNI) cages and their mononitro analogues (MNI). We traced the experimentally observed improved quantum yields of MDNI to a higher population of the reactants in the triplet surface. This fact is supported by a more favorable intersystem crossing due to the availability of a higher number of triplet excited states with the correct symmetry in MDNI than in MNI. Our findings may pave the way for improved cage designs that possess higher quantum yields and a more efficient agonist release.

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Höglinger, Doris, Per Haberkant, Auxiliadora Aguilera-Romero, Howard Riezman, Forbes D. Porter, Frances M. Platt, Antony Galione, and Carsten Schultz. "Intracellular sphingosine releases calcium from lysosomes." eLife 4 (November 27, 2015). http://dx.doi.org/10.7554/elife.10616.

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To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

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"Photo-Uncaging of a Microtubule Depolymerizer Reduces Tumor Volume in a Mouse Model." Synfacts 20, no. 03 (February 14, 2024): 0321. http://dx.doi.org/10.1055/s-0043-1773076.

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37

Hudson, Elyse, Christabel Faylinn, Ivonne Rebeca Lopez-Miranda, Josh Milstein, and Andrew Beharry. "Antimicrobial Efficacy of Photocaged β‐lapachone in Bacillus subtilis Biofilms." ChemPhotoChem, June 26, 2024. http://dx.doi.org/10.1002/cptc.202400164.

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With the rise of antibiotic resistance within clinical settings, combating the growth of microbial biofilms presents a unique challenge. Biofilm‐inhabiting bacteria are embedded within a self‐produced, protective matrix, which can reduce the efficacy of treatment. The naturally derived product β‐lapachone is an appealing therapeutic agent that has been reported to inhibit biofilm growth. However, its off‐target toxicity and poor metabolic stability pose a significant hurdle for its application in vivo. Using a photo‐pharmacological approach via a coumarin‐based photocage, the reactivity of β‐lapachone can be tuned so it only becomes active once the photocage is removed. Here we report both the photo‐uncaging efficiency and the effective inhibition concentration of photocaged β‐lapachone within model Bacillus subtilis biofilms. Additionally, the mechanism of action is analyzed with results supporting catalase inhibition. This novel light‐activatable anti‐microbial has potential applications in medical settings to inhibit biofilm growth and provide synergistic treatment with traditional antibiotics.

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Maller, Corina, Eirini Marouda, and Maja Köhn. "Photo‐Claisen Rearrangement in a Coumarin‐Caged Peptide leads to a Surprising Enzyme Hyperactivation." ChemBioChem, August 22, 2024. http://dx.doi.org/10.1002/cbic.202400561.

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Protein phosphatase‐1 (PP1) is a ubiquitous enzyme counteracting hundreds of kinases in cells. PP1 interacts with regulatory proteins via an RVxF peptide motif that binds to a hydrophobic groove on the enzyme. PP1‐disrupting peptides (PDPs) compete with these regulatory proteins, leading to the release of the active PP1 subunit and promoting substrate dephosphorylation. Building on previous strategies employing the ortho‐nitrobenzyl (o‐Nb) group, we introduced coumarin derivatives into a PDP via an ether bond to explore their effects on PP1 activity. Surprisingly, our study revealed that the coumarin‐caged peptides (PDP‐DEACM and PDP‐CM) underwent a photo‐Claisen rearrangement, resulting in an unexpected hyperactivation of PP1. Our findings underscore the importance of linker design in controlling uncaging efficiency and highlight the need for comprehensive in vitro analysis before cellular experiments.

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Schulte, Albert Marten, Lianne M. Smid, Georgios Alachouzos, Wiktor Szymanski, and Ben L. Feringa. "Cation delocalization and photo-isomerization enhance the uncaging quantum yield of a photocleavable protecting group." Chemical Communications, 2024. http://dx.doi.org/10.1039/d3cc05055f.

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40

Zheng, Ying, Zhiwei Ye, Xue Zhang, and Yi Xiao. "Photo-uncaging Triggers on Self-Blinking to Control Single-Molecule Fluorescence Kinetics for Super-resolution Imaging." ACS Nano, June 28, 2024. http://dx.doi.org/10.1021/acsnano.4c03809.

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Otopalik, Adriane G., Alexander C. Sutton, Matthew Banghart, and Eve Marder. "When complex neuronal structures may not matter." eLife 6 (February 6, 2017). http://dx.doi.org/10.7554/elife.23508.

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Much work has explored animal-to-animal variability and compensation in ion channel expression. Yet, little is known regarding the physiological consequences of morphological variability. We quantify animal-to-animal variability in cable lengths (CV = 0.4) and branching patterns in the Gastric Mill (GM) neuron, an identified neuron type with highly-conserved physiological properties in the crustacean stomatogastric ganglion (STG) of Cancer borealis. We examined passive GM electrotonic structure by measuring the amplitudes and apparent reversal potentials (Erevs) of inhibitory responses evoked with focal glutamate photo-uncaging in the presence of TTX. Apparent Erevs were relatively invariant across sites (mean CV ± SD = 0.04 ± 0.01; 7–20 sites in each of 10 neurons), which ranged between 100–800 µm from the somatic recording site. Thus, GM neurons are remarkably electrotonically compact (estimated λ > 1.5 mm). Electrotonically compact structures, in consort with graded transmission, provide an elegant solution to observed morphological variability in the STG.

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Chen, Lujing, Guang Li, Zheng Jiang, and King-Wai Yau. "Unusual phototransduction via cross-motif signaling from G _q to adenylyl cyclase in intrinsically photosensitive retinalganglion cells." Proceedings of the National Academy of Sciences 120, no. 1 (December 29, 2022). http://dx.doi.org/10.1073/pnas.2216599120.

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Nonimage-forming vision in mammals is mediated primarily by melanopsin (OPN4)-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). In mouse M1-ipRGCs, melanopsin predominantly activates, via Gα q,11,14 , phospholipase C-β4 to open transient receptor 6 (TRPC6) and TRPC7 channels. In M2- and M4-ipRGCs, however, a prominent phototransduction mechanism involves the opening of hyperpolarization- and cyclic nucleotide-gated channels via cyclic nucleotide, although the upstream steps remain uncertain. We report here experiments, primarily on M4-ipRGCs, with photo-uncaging of cyclic nucleotides and virally expressed CNGA2 channels to conclude that the second messenger is cyclic adenosine monophosphate (cAMP) – very surprising considering that cyclic guanosine monophosphate (cGMP) is used in almost all cyclic nucleotide-mediated phototransduction mechanisms across the animal kingdom. We further found that the upstream G protein is likewise G q , which via its Gβγ subunits directly activates adenylyl cyclase (AC). Our findings are a demonstration in a native cell of a cross-motif GPCR signaling pathway from G q directly to AC with a specific function.

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Hashimoto, Ryu, Masafumi Minoshima, and Kazuya Kikuchi. "Rational Design of Hydroxylated Thiazole Orange Photocages for Green Light‐Triggered DNA Recombination." ChemBioChem, December 28, 2023. http://dx.doi.org/10.1002/cbic.202300799.

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The precise control of DNA recombination enables the cell‐ or time‐dependent regulation of gene expression in studies of gene function. Caged estrogen receptor ligands combined with a Cre‐ERT2/loxP system are useful tools for light‐triggered DNA recombination. However, the photolysis of most caged compounds requires ultraviolet or blue light, which is toxic and displays low tissue penetration. Although a cyanine‐based photo‐responsive protecting group (PPG) can release estrogen receptor ligands with longer‐wavelength light, its low photolytic efficiency requires long illumination times. We developed a caged estrogen receptor ligand with improved green light‐responsive PPGs. The rational modification of Hydroxylated Thiazole Orange (HTO) photocages using electron‐donating groups (EDGs), such as dimethoxy (DiMeO)‐substituted HTO, resulted in high photolytic efficiency (up to εΦ ≈ 320 M‐1 cm‐1). Theoretical calculations demonstrated that the enhanced photolytic efficiencies were derived from the increased intramolecular charge transfer by EDGs upon excitation. The efficient uncaging of estrogen receptor ligands enabled the control of gene recombination in a ligand‐dependent Cre‐ERT2/loxP system in live cells.

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44

Valera, Jorge S., Álvaro López-Acosta, and Thomas Hermans. "Photoinitiated Transient Self‐Assembly in a Catalytically Driven Chemical Reaction Cycle." Angewandte Chemie International Edition, May 21, 2024. http://dx.doi.org/10.1002/anie.202406931.

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Chemically‐fueled chemical reaction networks (CRNs) are key in controlling dissipative self‐assembly. A key challenge in the field is to store chemical fuel‐precursors or “pre‐fuels” in the system that are catalytically converted into activating or deactivating fuels that drive the CRN. In addition, real‐time control over such catalysis by light is highly desirable, but so far not yet achieved. Here we show a catalytically‐driven CRN that is photoinitiated with 450 nm light, producing activated monomers that go on to perform transient self‐assembly. Monomer activation proceeds via photoredox catalysis, converting the monomer alcohol groups into the corresponding aldehydes that self‐assemble into large supramolecular fibers. Monomer deactivation is achieved by organometallic catalysis that relies on pre‐fuel hydrolysis to release formate (i.e., the deactivating fuel). Additionally, irradiation with 305 nm light accelerates the release of formate by photo‐uncaging the pre‐fuel, leading to a factor ~2 faster deactivation of the monomer. Overall, we show transient self‐assembly upon visible light photoactivation, and tunable life‐times by ultraviolet light.

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45

Valera, Jorge S., Álvaro López-Acosta, and Thomas Hermans. "Photoinitiated Transient Self‐Assembly in a Catalytically Driven Chemical Reaction Cycle." Angewandte Chemie, May 21, 2024. http://dx.doi.org/10.1002/ange.202406931.

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Chemically‐fueled chemical reaction networks (CRNs) are key in controlling dissipative self‐assembly. A key challenge in the field is to store chemical fuel‐precursors or “pre‐fuels” in the system that are catalytically converted into activating or deactivating fuels that drive the CRN. In addition, real‐time control over such catalysis by light is highly desirable, but so far not yet achieved. Here we show a catalytically‐driven CRN that is photoinitiated with 450 nm light, producing activated monomers that go on to perform transient self‐assembly. Monomer activation proceeds via photoredox catalysis, converting the monomer alcohol groups into the corresponding aldehydes that self‐assemble into large supramolecular fibers. Monomer deactivation is achieved by organometallic catalysis that relies on pre‐fuel hydrolysis to release formate (i.e., the deactivating fuel). Additionally, irradiation with 305 nm light accelerates the release of formate by photo‐uncaging the pre‐fuel, leading to a factor ~2 faster deactivation of the monomer. Overall, we show transient self‐assembly upon visible light photoactivation, and tunable life‐times by ultraviolet light.

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Sasikumar, Devika, Yuta Takano, Hanjun Zhao, Reiko Kohara, Morihiko Hamada, Yasuhiro Kobori, and Vasudevanpillai Biju. "Caging and photo-triggered uncaging of singlet oxygen by excited state engineering of electron donor–acceptor-linked molecular sensors." Scientific Reports 12, no. 1 (July 5, 2022). http://dx.doi.org/10.1038/s41598-022-15054-4.

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AbstractSinglet oxygen (1O2), one of the most sought-after species in oxidative chemical reactions and photodynamic cancer therapy, is activated and neutralized in the atmosphere and living cells. It is essential to see "when" and "where" 1O2 is produced and delivered to understand and utilize it. There is an increasing demand for molecular sensor tools to capture, store, and supply 1O2, controlled by light and engineered singlet and triplet states, indicating the 1O2-capturing-releasing state. Here, we demonstrate the outstanding potential of an aminocoumarin-methylanthracene-based electron donor–acceptor molecule (1). Spectroscopic measurements confirm the formation of an endoperoxide (1-O2) which is not strongly fluorescent and remarkably different from previously reported 1O2 sensor molecules. Moreover, the photoexcitation on the dye in 1-O2 triggers fluorescence enhancement by the oxidative rearrangement and a competing 1O2 release. The unique ability of 1 will pave the way for the spatially and temporally controlled utilization of 1O2 in various areas such as chemical reactions and phototherapies.

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Kaye, Esther G., Bijan Mirabi, Ivonne R. Lopez-Miranda, Komadhie C. Dissanayake, Upasana Banerjee, Madelyn Austin, Mark Lautens, Arthur H. Winter, and Andrew A. Beharry. "Photo-Uncaging by C(sp³)–C(sp³) Bond Cleavage Restores β-Lapachone Activity." Journal of the American Chemical Society, June 2, 2023. http://dx.doi.org/10.1021/jacs.3c00398.

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48

Lee, Hyun Kyung, Chelsie E. Conrad, Valentin Magidson, William F. Heinz, Gary Pauly, Ping Yu, Saminathan Ramakrishnan, Jason R. Stagno, and Yun-Xing Wang. "Developing methods to study conformational changes in RNA crystals using a photocaged ligand." Frontiers in Molecular Biosciences 9 (August 16, 2022). http://dx.doi.org/10.3389/fmolb.2022.964595.

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Crystallographic observation of structural changes in real time requires that those changes be uniform both spatially and temporally. A primary challenge with time-resolved ligand-mixing diffraction experiments is asynchrony caused by variable factors, such as efficiency of mixing, rate of diffusion, crystal size, and subsequently, conformational heterogeneity. One method of minimizing such variability is use of a photolabile caged ligand, which can fully saturate the crystal environment (spatially), and whose photoactivation can rapidly (temporally) trigger the reaction in a controlled manner. Our recently published results on a ligand-mixing experiment using time-resolved X-ray crystallography (TRX) with an X-ray free electron laser (XFEL) demonstrated that large conformational changes upon ligand binding resulted in a solid-to-solid phase transition (SSPT), while maintaining Bragg diffraction. Here we investigate this SSPT by polarized video microscopy (PVM) after light-triggered release of a photo-caged adenine (pcADE). In general, the mean transition times and transition widths of the SSPT were less dependent on crystal size than what was observed in previous PVM studies with direct ADE mixing. Instead, the photo-induced transition appears to be heavily influenced by the equilibrium between caged and uncaged ADE due to relatively low sample exposure and uncaging efficiency. Nevertheless, we successfully demonstrate a method for the characterization of phase transitions in RNA crystals that are inducible with a photocaged ligand. The transition data for three crystals of different sizes were then applied to kinetic analysis by fitting to the known four-state model associated with ligand-induced conformational changes, revealing an apparent concentration of uncaged ADE in crystal of 0.43–0.46 mM. These results provide further insight into approaches to study time-resolved ligand-induced conformational changes in crystals, and in particular, highlight the feasibility of triggering phase transitions using a light-inducible system. Developing such approaches may be paramount for the rapidly emerging field of time-resolved crystallography.

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Journal articles on the topic 'Photo-uncaging'

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