Light-activated tetrazines enable precision live-cell bioorthogonal chemistry

Liu, Luping; Zhang, Dongyang; Johnson, Mai; Devaraj, Neal K.

doi:10.1038/s41557-022-00963-8

“…K. Liu, L. Zhang, D. Johnson, M. Devaraj, N. K. Nat. Chem.202210781085 . This work revealed that photocaged dihydrotetrazines enable light-triggered bioorthogonal reaction with spatiotemporal control.…”

Section: Key Referencesmentioning

confidence: 90%

“…To simplify light-stimulated tetrazine ligation, our group recently developed photocaged dihydrotetrazines for the spatiotemporal control of bioorthogonal reactions with TCO-functionalized probes. 3 These dihydrotetrazine derivatives were readily activated using visible light as a stimulus. Interestingly, we determined that functionalization of dihydrotetrazines with photocleavable protecting groups drastically improved their resilience toward hydrolysis and side reactions with nucleophiles.…”

Section: ■ Development Of Photocaged Lipid Probes For Spatiotemporal ...mentioning

confidence: 99%

“…One potential drawback of prior approaches is the necessity of a photooxidant. To simplify light-stimulated tetrazine ligation, our group recently developed photocaged dihydrotetrazines for the spatiotemporal control of bioorthogonal reactions with TCO-functionalized probes . These dihydrotetrazine derivatives were readily activated using visible light as a stimulus.…”

Section: Development Of Photocaged Lipid Probes For Spatiotemporal Co...mentioning

confidence: 99%

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Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells

Knittel

¹

,

Devaraj

²

2022

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Conspectus The structural boundaries of living cells are composed of numerous membrane-forming lipids. Lipids not only are crucial for the cellular compartmentalization but also are involved in cell signaling as well as energy storage. Abnormal lipid levels have been linked to severe human diseases such as cancer, multiple sclerosis, neurodegenerative diseases, as well as lysosomal storage disorders. Given their biological significance, there is immense interest in studying lipids and their effect on cells. However, limiting factors include the low solubility of lipids, their structural complexity, and the challenge of using genetic techniques to directly manipulate lipid structure. Current methods to study lipids rely mostly on lipidomics, which analyzes the composition of lipid extracts using mass spectrometry. Although, these efforts have successfully catalogued and profiled a great number of lipids in cells, many aspects about their exact functional role and subcellular distribution remain enigmatic. In this Account, we outline how our laboratory developed and applied different bioconjugation strategies to study the role of lipids and lipid modifications in cells. Inspired by our ongoing work on developing lipid bioconjugation strategies to generate artificial cell membranes, we developed a ceramide synthesis method in live cells using a salicylaldehyde ester that readily reacts with sphingosine in form of a traceless ceramide ligation. Our study not only confirmed existing knowledge about the association of ceramides with cell death, but also gave interesting new findings about the structure–function relationship of ceramides in apoptosis. Our initial efforts led us to investigate probes that detect endogenous sphingolipids using live cell imaging. We describe the development of a fluorogenic probe that reacts chemoselectively with sphingosine in living cells, enabling the detection of elevated endogenous levels of this biomarker in human disease. Building on our interest in the fluorescence labeling of lipids, we have also explored the use of bioorthogonal reactions to label chemically synthesized lipid probes. We discuss the development of photocaged dihydrotetrazine lipids, where the initiation of the bioorthogonal reaction can be triggered by visible light, allowing for live cell modification of membranes with spatiotemporal control. Finally, proteins are often post-translationally modified by lipids, which have important effects on protein subcellular localization and function. Controlling lipid modifications with small molecule probes could help reveal the function of lipid post-translational modifications and could potentially inspire novel therapeutic strategies. We describe how our previous studies on synthetic membrane formation inspired us to develop an amphiphilic cysteine derivative that depalmitoylates membrane-bound S-acylated proteins in live cells. Ultimately, we applied this amphiphile mediated depalmitoylation (AMD) in studies investigating the palmitoylation of cancer relevant palmitoylat...

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“…Inducible versions of tetrazine ligation have also been described based on the oxidation of dihydrotetrazine (DHTz) precursors 66,67 Recently, an o-nitrophenyl protected dihydrotetrazine has been used with 405 nm light and without catalysis to uncage tetrazines. 68 The ability to mimic the cellular compartmentalization with nonnatural catalysts would provide a unique tool for regulating cellular processes. Recently, several groups have described photocatalytic proximity labeling as a chemoproteomic tool, [69][70][71][72][73][74][75] including the use of Ir-photocatalysis has been used to promote azide reduction/uncaging in live cells, 72 where the positively charged Ircatalyst was non-specifically localized to the negatively charged environment of the mitochondria.…”

Section: Introductionmentioning

confidence: 99%

Ligand-directed Photocatalysts and Far-red Light Enable Catalytic Bioorthogonal Uncaging inside Live Cells

Rosenberger

¹

,

Xie

²

,

Fang

³

et al. 2022

Preprint

0

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Described are ligand-directed catalysts for live-cell, photocatalytic activation of bioorthogonal chemistry. Catalytic groups are localized via a tethered ligand either to DNA or to tubulin, and red-light (660 nm) photocatalysis is used to initiate a cascade of DHTz-oxidation, intramolecular Diels-Alder reaction, and elimination to release phenolic compounds. Silarhodamine (SiR) dyes, more conventionally used as biological fluorophores, serve as photocatalysts that have high cytocompatibility and produce minimal singlet oxygen. Commercially-available conjugates of Hoechst dye (SiR-H) and Taxol (SiR-T) are used to localize SiR to the nucleus and tubulin, respectively. Computation was used to assist the design of a new class of redox-activated photocage to release either phenol or n-CA4, a microtubule-destabilizing agent. In model studies, uncaging is complete within 5 min using only 2 µM of SiR and 40 µM of the photocage. In situ spectroscopic studies support a mechanism involving rapid intramolecular Diels-Alder reaction and a rate determining elimination step. In cellular studies, this uncaging process is successful at low concentration of both the photocage (25 nM) and the SiR-H dye (500 nM). Uncaging n-CA4 causes microtubule depolymerization and an accompanying reduction in cell area. Control studies demonstrate that SiR-H catalyzes uncaging inside the cell, and not in the extracellular environment. With SiR-T, the same dye serves as photocatalyst and the fluorescent reporter for tubulin depolymerization, and with confocal microscopy it was possible to visualize tubulin depolymerization in real time as the result of photocatalytic uncaging in live cells.

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“…Inducible versions of tetrazine ligation have also been described based on the oxidation of dihydrotetrazine (DHTz) precursors 66,67 Recently, an o-nitrophenyl protected dihydrotetrazine has been used with 405 nm light and without catalysis to uncage tetrazines. 68 The ability to mimic the cellular compartmentalization with nonnatural catalysts would provide a unique tool for regulating cellular processes. Recently, several groups have described photocatalytic proximity labeling as a chemoproteomic tool, [69][70][71][72][73][74][75] including the use of Ir-photocatalysis has been used to promote azide reduction/uncaging in live cells, 72 where the positively charged Ircatalyst was non-specifically localized to the negatively charged environment of the mitochondria.…”

Section: Introductionmentioning

confidence: 99%

Ligand-directed Photocatalysts and Far-red Light Enable Catalytic Bioorthogonal Uncaging inside Live Cells

Rosenberger

¹

,

Xie

²

,

Fang

³

et al. 2022

Preprint

0

View full text Add to dashboard Cite

Described are ligand-directed catalysts for live-cell, photocatalytic activation of bioorthogonal chemistry. Catalytic groups are localized via a tethered ligand either to DNA or to tubulin, and red-light (660 nm) photocatalysis is used to initiate a cascade of DHTz-oxidation, intramolecular Diels-Alder reaction, and elimination to release phenolic compounds. Silarhodamine (SiR) dyes, more conventionally used as biological fluorophores, serve as photocatalysts that have high cytocompatibility and produce minimal singlet oxygen. Commercially-available conjugates of Hoechst dye (SiR-H) and Taxol (SiR-T) are used to localize SiR to the nucleus and tubulin, respectively. Computation was used to assist the design of a new class of redox-activated photocage to release either phenol or n-CA4, a microtubule-destabilizing agent. In model studies, uncaging is complete within 5 min using only 2 µM of SiR and 40 µM of the photocage. In situ spectroscopic studies support a mechanism involving rapid intramolecular Diels-Alder reaction and a rate determining elimination step. In cellular studies, this uncaging process is successful at low concentration of both the photocage (25 nM) and the SiR-H dye (500 nM). Uncaging n-CA4 causes microtubule depolymerization and an accompanying reduction in cell area. Control studies demonstrate that SiR-H catalyzes uncaging inside the cell, and not in the extracellular environment. With SiR-T, the same dye serves as photocatalyst and the fluorescent reporter for tubulin depolymerization, and with confocal microscopy it was possible to visualize tubulin depolymerization in real time as the result of photocatalytic uncaging in live cells.

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Light-activated tetrazines enable precision live-cell bioorthogonal chemistry

Cited by 73 publications

References 45 publications

Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells

Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells

Ligand-directed Photocatalysts and Far-red Light Enable Catalytic Bioorthogonal Uncaging inside Live Cells

Ligand-directed Photocatalysts and Far-red Light Enable Catalytic Bioorthogonal Uncaging inside Live Cells

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