2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives as fluorescent pigment dyeing substrates and their application for the assay of β-d-galactosidase activities

Otsubo, Tadamune; Minami, Akira; Fujii, Haruna; Taguchi, Risa; Takahashi, Toshifumi; Suzuki, Takashi; Teraoka, Fumiteru; Ikeda, Kiyoshi

doi:10.1016/j.bmcl.2013.01.043

Cited by 30 publications

(25 citation statements)

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“…The fluorescent compound BTP3 has an acid resistance for its fluorescence property, 6) which enabled sialidase reaction under an acidic condition in the present study. BTP3 also has a large stokes shift, which is an excitation of UV (372 nm at maximum wave length) and an emission of green fluorescence (526 nm at maximum wave length).…”

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confidence: 86%

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confidence: 99%

“…The water-soluble non-fluorescent BTP3-Neu5Ac is cleaved to a water-insoluble fluorescent benzothiazolylphenol derivative (BTP3) 6,7) and N-acetylneuraminic acid (Neu5Ac) by sialidase activity. 8) BTP3 locally deposits on sialidase activity-existing sites, resulting in local and fluorescent visualization of sialidase activity in live cells (Fig.…”

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confidence: 99%

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Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses

Takahashi

Takano

Kurebayashi

et al. 2015

Biological & Pharmaceutical Bulletin

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Human parainfluenza virus type 1 (hPIV1) does not form clear plaque by the conventional plaque formation assay because of slightly a cytopathic effects in many cell lines infected with hPIV1, thus making in virus titration, isolation and inhibitor evaluation difficult. We have succeeded in fluorescent histochemical visualization of sialidase activities of influenza A and B viruses, Newcastle disease virus and Sendai virus by using a novel fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). In this study, we applied the BTP3-Neu5Ac assay for rapid detection of hPIV1 and hPIV type 3. The BTP3-Neu5Ac assay could histochemically visualize dot-blotted hPIVs on a membrane and hPIV-infected cells as local fluorescence under UV irradiation. We succeeded in distinct fluorescent visualization of hPIV1-infected cells in only 3 d using the BTP3-Neu5Ac assay. Due to there being no fixation, hPIV1 was isolated directly from fluorescent stained focus cells by the BTP3-Neu5Ac assay. Establishment of a sensitive, easy, and rapid fluorescent focus detection assay for hPIV, hPIV1 in particular will contribute greatly to progress in hPIV studies.Key words human parainfluenza virus (hPIV); sialidase activity; fluorescent visualization; histochemical staining;Human parainfluenza virus type 1 (hPIV1) and human parainfluenza virus type 3 (hPIV3) belong to genus Respirovirus, subfamily Paramyxovirinae, and family Paramyxoviridae. The hPIVs cause flu-like respiratory symptoms, including croup, pneumonia and bronchiolitis, mainly in infants younger than 5 years old and immunocompromised patients. Severe symptoms often lead to hospitalization and occasionally result in sudden death of newborns and infants. hPIVs, the majority of which are hPIV1 and hPIV3, are detected from approximately 20% of viral pneumonia cases in infants younger than 5 years of age.1,2) Although hPIVs are clinically important respiratory viruses for infants and immunocompromised patients, vaccines and medicines against hPIVs have not been developed.Clinical samples and virus culture supernatants include some viruses and mutants. When viruses replicate in the presence of a certain inhibitor, inhibitor-resistant mutant viruses may appear in the culture supernatant. To separate a virus isolate from such samples, the plaque formation assay is frequently used. A virus infects a cell and produces progeny viruses that spread to infect neighboring cells. The population of infected cells shows a cytopathic effect (CPE), for example, cell death or syncytium formation. When virus-cultured cells are overlaid by a medium containing agarose, cell lysis induced by CPE is visualized as a plaque. A virus isolate can be picked up directly from a plaque, which is formed by an isolate. hPIV3 has been reported to form large plaques in human epidermoid cancer (HEp-2) cells, in which hPIV3 shows much syncytium formation.3) On the other hand, hPIV1 does not form plaques by the normal plaq...

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confidence: 86%

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confidence: 99%

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Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses

Takahashi

Takano

Kurebayashi

et al. 2015

Biological & Pharmaceutical Bulletin

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“…16) The new substrate is 2-(benzothiazol-2-yl)-4-bromophenol bonded with N-acetylneuraminic acid (BTP3-Neu5Ac). 2-(Benzothiazol-2-yl)-4-bromophenol (BTP3), which is formed by sialidase reaction of BTP3-Neu5Ac, is a crystalline, insoluble, acid-resistant, and fluorescently stable compound (Ex/ Em=372/526 nm).…”

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“…Therefore, BTP3-Neu5Ac can be used for fluorescent histochemical visualization of sialidase activity. 16,18,19) We developed a new substrate using BTP3 bonded with phosphate (BTP3-Phos) for the purpose of fluorescent histochemical visualization of phosphatase activity (Fig. 1).…”

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Histochemical Imaging of Alkaline Phosphatase Using a Novel Fluorescent Substrate

Takahashi

Otsubo

Ikeda

et al. 2014

Biological & Pharmaceutical Bulletin

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Histochemical visualization of phosphatase is exclusively required for Western immunoblotting and antigen-positive cell staining using an alkaline phosphatase (AP)-labeled secondary antibody. This detection has been performed by several reagents including 5-bromo-4-chloro-3-indolyl-phosphate (X-Phos), nitro blue tetrazolium (NBT), 3-(2'-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-[3H]-quinazolinone (ELF® 97 Phosphate). We previously reported that 2-(benzothiazol-2-yl)-4-bromophenol bonded with N-acetylneuraminic acid (BTP3-Neu5Ac), enabled fluorescent histochemical visualization of sialidase activity. 2-(Benzothiazol-2-yl)-4-bromophenol (BTP3), which is formed from BTP3-Neu5Ac by sialidase reaction, is a crystalline, insoluble and stable fluorogenic compound, deposited at the site of enzyme activity. We developed a BTP3 phosphate ester (BTP3-Phos) for the purpose of fluorescent histochemical visualization of phosphatase activity. BTP3-Phos emitted fluorescence in a manner dependent on the concentration of the AP-labeled antibody. BTP3-Phos also enabled fluorescent histochemical visualization of AP-blotted dots in a manner dependent on the concentration of the AP-labeled antibody. The detection sensitivity of BTP3-Phos was estimated to be greater than that of the conventional method using X-Phos and NBT. Influenza A virus-infected cells were fixed and reacted with anti-influenza A virus antibodies and incubated continuously with an AP-labeled secondary antibody. BTP3-Phos stained the infected cells with distinct green fluorescence. These results indicate that BTP3-Phos can enable fluorescent immunohistochemical staining analysis using an AP-labeled antibody. BTP3-Phos would be beneficial for histochemical staining of AP activity, and may be applicable for multi-color staining or a cell sorter.

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Fluorescent Probes for Diagnostics of β-Galactosidase: From Micro to Macro

Jiang

Tang

et al. 2019

Topics in Medicinal Chemistry

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2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives as fluorescent pigment dyeing substrates and their application for the assay of β-d-galactosidase activities

Cited by 30 publications

References 35 publications

Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses

Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses

Histochemical Imaging of Alkaline Phosphatase Using a Novel Fluorescent Substrate

Fluorescent Probes for Diagnostics of β-Galactosidase: From Micro to Macro

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