Green- to far-red-emitting fluorogenic tetrazine probes – synthetic access and no-wash protein imaging inside living cells

Wieczorek, Achim; Werther, Philipp; Euchner, Jonas

doi:10.1039/c6sc03879d

Cited by 174 publications

(176 citation statements)

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“…To investigate proximity‐directed DA inv with a fluorogenic tetrazine reporter, we envisioned a synthetic strategy based on recent work from our laboratory. In this approach, tetrazinyl benzaldehydes were reacted with diarylethers (or silanes) in Friedel–Crafts reactions to assemble fluorogenic tetrazine (Si)rhodamines which we showed to be suitable for no‐wash protein labeling inside living cells . The design of the fluorophore probe followed the principles: (1) minimal interchromophore distance between the fluorophore and the tetrazine for strong fluorescence quenching and for small probe size, (2) well‐suited fluorophore classes for biological application (3) modular synthetic access under mild conditions.…”

Section: Resultsmentioning

confidence: 99%

“…To be compatible with Lewis acid‐mediated Friedel–Crafts reaction, the carboxylic acid was tert ‐butyl ester‐protected (Scheme II). Tetrazine benzylic alcohol was afforded from hydroxymethyl benzonitrile (39 %) and subsequently oxidized to benzaldehyde 9 using Dess–Martin periodinane (85 %) (Scheme III) …”

Section: Resultsmentioning

confidence: 99%

“…Absorption and emission spectra of Rhod‐Tz were recorded in aqueous phosphate buffered saline (PBS) and the absorption maximum was determined to λ abs =559 nm ( ϵ =4.4×10 4 m −1 cm −1 ), the emission maximum to λ em =582 nm (Figure b). The fluorescence quantum yield Φ f =0.017 is in accordance with previously reported tetrazine quenched rhodamines …”

Section: Resultsmentioning

confidence: 99%

“…Ten equivalents of BCN led to completion of fluorescent product formation after 40 minutes (Figure d). This “turn‐on factor” is slightly improved, compared to recently published fluorogenic tetrazine rhodamines with ultra‐short interchromophore distance (1.6 fold higher) . Furthermore, the water‐soluble dye is highly stable in aqueous buffer at room temperature and no degradation was observed over 12 hours.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, the water‐soluble dye is highly stable in aqueous buffer at room temperature and no degradation was observed over 12 hours. The fluorescence quenching efficiency is in general dependent on the interchromophore distance and a number of short‐distance tetrazine fluorophore conjugates have been reported . The exact quenching mechanism for these fluorogenic tetrazine probes is still under debate, as no thorough physicochemical investigation has been reported yet.…”

Section: Resultsmentioning

confidence: 99%

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A Bifunctional Fluorogenic Rhodamine Probe for Proximity‐Induced Bioorthogonal Chemistry

Werther

Möhler

Wombacher

2017

Chemistry A European J

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Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron demand Diels-Alder reaction (DA ) stands out due to the availability of reactants with very fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable and prone to side reactions. Herein, we evaluate the extent of acceleration of rather unreactive but highly stable dienophiles by DNA-templated proximity. To this end, we developed a modular synthetic route for a novel bifunctional fluorogenic tetrazine rhodamine probe that we used to determine the reaction kinetics of various dienophiles in a fluorescence assay. Under proximity-driven conditions the reaction was found to be several orders of magnitude faster, and we observed almost no background reaction when proximity was not induced. This fundamental study identifies a minimally sized fluorogenic tetrazine dienophile reactant pair that has potential to be generally used for the visualization of biomolecular interactions with temporal and spatial resolution in living systems.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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A Bifunctional Fluorogenic Rhodamine Probe for Proximity‐Induced Bioorthogonal Chemistry

Werther

Möhler

Wombacher

2017

Chemistry A European J

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Labelling Proteins and Biomolecules with Small Fluorescent Sensors

Ghrayche¹,

Graziotto²,

Jeffcoat³

et al. 2022

Molecular Fluorescent Sensors for Cellular Studies

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Dynamic Click Hydrogels for Xeno‐Free Culture of Induced Pluripotent Stem Cells

et al. 2020

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3D cell-laden hydrogels are increasingly used for in vitro and ex vivo cell expansion, controlled stem cell differentiation, and regenerative medicine. [1] Hydrogels can be fabricated by derivatives of purely synthetic polymers (e.g., poly(ethylene glycol) or PEG) and/or naturally derived biomacromolecules (e.g., gelatin, hyaluronic acid, or heparin). [2] Tumor basement membrane derived Matrigel is routinely used as a convenient matrix for 3D cell culture owing to its inherent cell affinity provided by laminin, collagen IV, and residual growth factors. However, Matrigel contains ill-defined and batch-to batch variability of extracellular matrix (ECM) components that may present challenges for studying the contributions of individual matrix cues on cell fate processes. To this end, synthetic PEG-based hydrogels afford a blank slate in which a bottom-up approach can be used to impart precise biochemical and biophysical properties in the cell-laden network. Advances in material chemistry have also enabled the conjugation of a variety of ECM motifs to the otherwise inert PEG-based network, as well as for spatiotemporal manipulation of matrix properties to facilitate the study of cell fate processes within a highly defined and controllable microenvironment. Chemically defined PEG-based hydrogels are particularly advantageous for studying iPSC behaviors in 3D. [3] For example, PEG hydrogels formed by Michael-type additions have proven effective in cell encapsulation owing to the reaction specificity between vinyl sulfone/maleimide (VS/MAL) and thiol (SH) moieties. Hubbell and colleagues utilized VS-SH reactions to determine the role of integrin activation on embryonic stem cell (ESC) self-renewal. [4] Lim et al. later use PEG-based VS-SH hydrogels for maintaining stemness of three human ESC lines. [5] Nonetheless, the reaction rates of Michael-type additions may be difficult to control and vary greatly from seconds to hours, depending on the type of Michael donor-acceptor pairs (e.g., VS-SH ≈ 30-120 min and MAL-SH ≈tens of seconds). [4a] Alternatively, enzymatic crosslinking provides excellent cytocompatibility and tunable gelation kinetics for in situ cell encapsulation. For instance, Xeno-free, chemically defined poly(ethylene glycol) (PEG)-based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene-click chemistries are integrated to form synthetic, dynamically tunable PEG-peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol-norbornene hydrogels crosslinked by multiarm PEG-norbornene (PEG-NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine-norbornene (Tz-NB) click reaction is then employed to dynamically stiffen the cell-laden hydrogels. Fast ...

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Green- to far-red-emitting fluorogenic tetrazine probes – synthetic access and no-wash protein imaging inside living cells

Abstract: Fluorogenic probes for bioorthogonal labeling chemistry are highly beneficial to reduce background signal in fluorescence microscopy imaging.

Cited by 174 publications

References 42 publications

A Bifunctional Fluorogenic Rhodamine Probe for Proximity‐Induced Bioorthogonal Chemistry

A Bifunctional Fluorogenic Rhodamine Probe for Proximity‐Induced Bioorthogonal Chemistry

Labelling Proteins and Biomolecules with Small Fluorescent Sensors

Dynamic Click Hydrogels for Xeno‐Free Culture of Induced Pluripotent Stem Cells

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