Synthesis and evaluation of turn-on fluorescent probes for imaging steroid sulfatase activities in cells

Tai, Chih‐Hsuan; Lu, Chun‐Ping; Wu, Shih‐Hsiung; Lo, Lee‐Chiang

doi:10.1039/c4cc01282h

Cited by 22 publications

(21 citation statements)

References 27 publications

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“…25,26 It can be generated by external stimuli under physiological conditions from a stable masked phenol precursor bearing a leaving group (such as a fluorine atom) on the methyl group at the para-or ortho-position. 27 This strategy has been used in inhibitors and prodrugs, [28][29][30] self-immolative linkers in dendrimers, 31 chemical selection of catalytic antibodies, 32,33 activity-based probes for various enzymes, including phosphatase, 34 glycosidases, 22,23,[35][36][37][38][39][40][41] , tyrosine phosphatase, 42 steroid sulfatase, 43 and beta-lactamase, 44 cell imaging 45,46 and probing protein-protein/DNA interactions. 47 Among these applications, the fluorine on either the monofluoromethyl or difluoromethyl group was used as a leaving group to form the active o-quinone methide intermediate via spontaneous elimination of a molecule of HF.…”

Section: Introductionmentioning

confidence: 99%

A Versatile Linker for Probes Targeting Hydrolases via In Situ labeling

Liu

Chen

et al. 2021

Preprint

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Hydrolases are important molecules that are involved in a wide range of biological functions and their activities are tightly regulated in healthy or diseased states. Detecting or imaging the activities of hydrolases, therefore, can reveal underlying molecular mechanisms in the context of cells to organisms, and their correlation with different physiological conditions can therefore be used in diagnosis. Due to the nature of hydrolases, substrate-based probes can be activated in their catalytic cycles, and cleavage of covalent bonds frees reporter moieties. For test-tube type bulk detection, spatial resolution is not a measure of importance, but for cell- or organism-based detection or imaging, spatial resolution is a key factor for probe sensitivity that influences signal-to-background ratio. One strategy to improve spatial resolution of the probes is to form a covalent linkage between the reporter moiety and intracellular proteins upon probe activation by the enzyme. In this work, we developed a generalizable linker chemistry that would allow in situ labeling of various imaging moieties via quinone methide species. To do so, we synthesized probes containing a monofluoromethyl or a difluoromethyl groups for β-galactosidase activation, while using fluorescein as a fluorescent reporter. The labeling efficacy of these two probes was evaluated in vitro. The probe bearing a monofluormethyl group exhibited superior labeling efficiency in imaging β-galactosidase activity in living cells. This study provides a versatile linker for applying quinone methide chemistry in the development of hydrolase-targeting probes involving in situ labeling.

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Section: Introductionmentioning

confidence: 99%

A Versatile Linker for Probes Targeting Hydrolases via In Situ labeling

Liu

Chen

et al. 2021

Preprint

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“…Nevertheless, this could be an advantage for imaging reagents; it may reflect the activities of enzyme more accurately without disturbing the structure of target enzyme. Along this line, QM has been extensively applied as self‐immobilizing imaging reagents for activities of a number of biologically important enzymes, including phosphatases, glycosidases, sulfatases, and β‐lactamases…”

Section: Introductionmentioning

confidence: 99%

“…[8] Nevertheless,t his couldb ea na dvantage for imaging reagents;i tm ay reflect the activities of enzymem ore accurately without disturbing the structure of target enzyme. Along this line, QM has been extensively applieda ss elf-immobilizing imaging reagents for activities of an umber of biologically important enzymes, including phosphatases, [9] glycosidases, [8,10] sulfatases, [11] and b-lactamases. [12] In general,t hese QM-based labelingp robess tructurally consist of four components:e nzymatic recognition moiety,l atent trapping moiety,l inker,a nd reporter ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Highly Efficient Multiple‐Labeling Probes for the Visualization of Enzyme Activities

Song

Chen

et al. 2019

Chemistry A European J

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Quinone methide (QM) as a latent trapping unit has been widely explored in activity‐based self‐immobilizing reagents. However, further application of this strategy has been largely hampered by the limited labeling efficiency to proteins. In this study, a thorough investigation on the labeling efficiency and the structure of QM‐based trapping unit is presented, from which a QM with multiple leaving groups was identified as an optimal trapping unit. An alkaline phosphatase (ALP) immobilizing reagent featured with this multiple‐labeling trapping unit exhibited lower nonspecific binding and, remarkably, a significantly higher labeling efficiency over other immobilizing reagents upon enzymatic activation. The utility of this imaging reagent was further demonstrated with the in vitro and in vivo visualization of the ALP activities. Furthermore, the multiple functional trapping unit may find greater value in the other activity‐based immobilizing probes.

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“…In recent years, substrates including the quinone methide group have been reported to detect cytosol-related enzymatic activity. [13][14][15] Most of these reported substrates were designed to permeate the cell membrane to reach the cytosolic enzymes. However, to effectively detect cell surface antigens on living cells, the substrate should not permeate the cell membrane but should exclusively react with the labeled enzymes on the target antigens.…”

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confidence: 99%

Β-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens

Noguchi¹,

Shimomura²,

Ohuchi³

et al. 2020

Preprint

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The ability to detect cell surface proteins using fluorescent dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter wavelength region. Herein, we report on a new method (CLAMP: quinone methide-based catalyzed signal amplification) in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used β-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, which contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by β-galactosidase labeling of the target cell surface proteins, the resulting quinone methide group-containing product was found to be both cell membrane permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent dye-labeled antibodies.

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Synthesis and evaluation of turn-on fluorescent probes for imaging steroid sulfatase activities in cells

Cited by 22 publications

References 27 publications

A Versatile Linker for Probes Targeting Hydrolases via In Situ labeling

A Versatile Linker for Probes Targeting Hydrolases via In Situ labeling

Highly Efficient Multiple‐Labeling Probes for the Visualization of Enzyme Activities

Β-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens

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