Tagging Live Cells that Express Specific Peptidase Activity with Solid‐State Fluorescence

Prost, Maxime; Canaple, Laurence; Samarut, Jacques; Hasserodt, Jens

doi:10.1002/cbic.201402091

Cited by 29 publications

(26 citation statements)

References 37 publications

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“…As previously reported, the replacement of hydrogen at the R1 position with chlorine-induced bathochromic shifts in HPQ (28,29). Therefore, we tried creating a red-shifted solid-state fluorophore, termed HPQ1, in which chlorine was replaced with a 4-(trifluoromethyl) styrene residue.…”

Section: Resultsmentioning

confidence: 99%

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A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging

Lyu

Huang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Cell membrane–targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro–2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl–4H-chromen–4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

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Section: Resultsmentioning

confidence: 99%

“…2A). The selfimmolative linker in the probe not only minimizes steric hindrance at the reaction sites, but also improves the stability of HYPQG (28,29). Control probes HPQG and HTPQG were also synthesized using the same design strategy.…”

Section: Resultsmentioning

confidence: 99%

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging

Lyu

Huang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…These properties enable the use of high concentrations of substrate to maximize the rate of ruthenium(II)‐photocatalyzed uncaging without compromising the signal with the background of a profluorophore. This new caged QPD complements the prior caged‐QPDs that have been developed as probes for phosphatases, glycosidases, peptidases …”

Section: Resultsmentioning

confidence: 82%

“…This new caged QPD complements the prior caged-QPDst hat have been developeda sp robes for phosphatases, [38,39] glycosidases, [40] peptidases. [41] The attractive properties of this dye (photostability,s patial contrast) have already inspired its use in nucleic acid imaging using an alkaline phosphatase conjugates in fluorescent in situ hybridization (FISH). [42][43] We reasoned that for soluble assays, confining the product of the templated reactiono nab ead would localize the precipitate and enhance its detection.…”

Section: Resultsmentioning

confidence: 99%

Turn On of a Ruthenium(II) Photocatalyst by DNA‐Templated Ligation

Anzola

Winssinger

2018

Chemistry A European J

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Here, the synthesis of a RuII photocatalyst by light‐directed oligonucleotide‐templated ligation reaction is described. The photocatalyst was found to have tremendous potential for signal amplification with >15000 turnovers measured in 9 hours. A templated reaction was used to turn on the activity of this ruthenium(II) photocatalyst in response to a specific DNA sequence. The photocatalysis of the ruthenium(II) complex was harnessed to uncage a new precipitating dye that is highly fluorescent and photostable in the solid state. This reaction was used to discriminate between different DNA analytes based on localization of the precipitate as well as for in cellulo miRNA detection. Finally, a bipyridine ligand functionalized with two different peptide nucleic acid (PNA) sequences was shown to enable template‐mediated ligation (turn on of the ruthenium(II) photocatalysis) and recruitment of substrate for templated photocatalysis.

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“…This three-component approach has been used successfully with more canonical types of peptidases. [18][19][20] Our initial target, 16, was accessed in 9 steps from commercially available materials (see Supporting Information). Notably, the activation of 16 is analogous to precolibactin activation: hydrolysis by ClbP reveals a primary amine, which can undergo a rapid 5-exo-trig cyclization to produce the active species ( Figure 3A).…”

mentioning

confidence: 99%

In Vitro Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe

et al. 2019

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The gut bacterial genotoxin colibactin is linked to the development of colorectal cancer. In the final stages of colibactin's biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual innermembrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, fulllength ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.

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Tagging Live Cells that Express Specific Peptidase Activity with Solid‐State Fluorescence

Cited by 29 publications

References 37 publications

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging

Turn On of a Ruthenium(II) Photocatalyst by DNA‐Templated Ligation

In Vitro Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe

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