Controllable NOdonor (NO-Rosa5)
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Controllable NOdonor (NO-Rosa5)

Price: $300.00
  • Item #: FDV-0032
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Controllable NOdonor (NO-Rosa5)

Cat. No. FDV-0032
Size 0.25 mg
Storage -20 oC

  


Description

NO donor, controlled by visible light
Controllable NO Donor : NO-Rosa5

Nitric oxide (NO), a gaseous free radical, is one of the signaling molecules produced by NO synthases physiologically. NO plays a key role in a wide range of biological processes.
However, NO is extremely unstable in physiological condition, so it is difficult to handle NO molecule in biological experiments. This made all tough to analyze further function of NO in cells. To overcome this point, "NO donors" which are compounds releasing NO molecules in the aqueous buffers have been used in NO research. Although NO donors are valuable tools, NO donors are not controllable spatio-temporally.

Controllable NO donor (original name; NO-Rosa5) is a novel NO donor controllable by visible light. Controllable NOdonor has two moieties, rosamine fluorescent dye as light harvesting group and N-nitrosoaminophenol structure as NO releasing moiety and releases NO molecules triggered by photoinduced electron transfer (PeT). It shows low photo-toxicity, low cytotoxicity, and efficient NO releasing by yellowish green light irradiation.

This product has been commercialized with the support of Nagoya City University.


Product Background

Nitric oxide (NO), a gaseous free radical, is one of the signaling molecules produced by NO synthases physiologically. NO plays a key role in a wide range of biological processes, such as vasodilation, neurotransmission, platelet adhesion, inflammation. NO is extremely unstable in physiological pH and temperature and easily oxidized to nitrite and nitrate. Based on its instability in cells, NO only works very limited space (~100 ?m) from its origin and half time (t1/2) is estimated as only a few seconds. Although its biological importance, it is difficult to handle NO molecule in biological experiments. To investigate the effects of NO on various biological phenomena, synthetic “NO donors”, which are compounds releasing NO molecules, have been used. Many kind of NO donors including organic nitrates/nitrites, NO-coordinated metal ions, N-diazeniumdiolates (also called NONOates) and S-nitrosothiols, have been discovered and contributed to elucidating biological effects of NO molecule. However, these NO donors
generate NO molecules continuously over a period of time and homogenously in the experimental buffer.

It is hard to control NO releasing spatio-temporally and to reproduce physiological role of NO molecules which act as signaling molecule in very limited space (~100 ?m) and period (a few seconds). To overcome this point, recently photocontrollable NO donors, are expected as valuable tools forspatial-temporal control of NO releasing. Although several UV-controllable NO donors have been developed, these compounds are not suitable to biological experiments due to its UV photo-toxicity. Visible light-controllable NO donors are desired for biological applications.

Controllable NOdonor (original name; NO-Rosa5) is a novel yellowish-green light-controllable NO donor originally developed by Drs. Hidehiko Nakagawa and Naoya Ieda, Nagoya City University. Controllable NOdonor has two moieties, rosamine fluorescent dye as light-harvesting group and N-nitrosoaminophenol structure as an NOreleasing moiety, and releases NO molecules triggered by photoinduced electron transfer (PeT). It validated low photo-toxicity, low cellular toxicity, and efficient NO releasing by yellowish-green light irradiation. Ref. 2 reported its biological applications including in cellullo NO-releasing assay and ex vivo vasodilation assay. 


Principal

FDV-0032_fig1.jpg 


Chemical Specs

  • Formulation : C34H33N4O5Cl
  • Molecular weight : 613.11 g/mol as a mono chloride salt
  • Solubility : Soluble in DMSO

Procedure

FDV-0032_fig2.jpg 


Selection guide of compatible fluorophores

Due to Controllable NOdonor releasing NO molecule upon 500-600 nm photo-irradiation, fluorophores, including fluorescent dyes, fluorescent reagent, and fluorescent proteins which are excitable by 500-600 nm wavelength are not available. 
Furthermore, Controllable NOdonor itself shows red fluorescent emission. When customers use any fluorophore with Controllable NOdonor, please refer to following information.
  1. Fluorophores not recommended
    • Excitation with 500-600 nm such as red fluorophores (for example Cy3, TAMRA, RFP protein etc.)
  2. Suitable fluorophores
    • Excitation with <500 nm such as blue fluorophores (for example coumarin dye, BFP protein etc.) or green fluorophores (for example fluorescein, GFP protein etc.)
    • Excitation with >600 nm such as near infrared red or far red fluorophores (for example Cy5, Cy7 etc.)

Application Data

FDV-0032_fig3.jpg 

Fig.1 In cellullo region-specific NO generation with Controllable NOdonor
HEK293T cells were firstly treated with DAF-FM DA (10 uM) for 30 min. After washing, the cells were subsequently treated with 10 uM Controllable NOdonor for 60 min. 
The dish was photoirradiated inside the indicated white circle (r=31 um) using a 543 nm laser of confocal microscopy. 
Left: before irradiation, Right: after irradiation.
Only inside the white circle, NO was detected. (scale bar = 40 um)


FDV-0032_fig4.jpg
Fig.2 Visible light-induced NO releasing in vitro
Quantitative analysis of NO release from Controllable NOdonor with NO electrode. Controllable NOdonor (final 10uM) in 100 mM HEPES buffer was irradiated with a MAX-303 (Asahi Spectra)equipped with a 530-590 nm band-pass filter (light intensity: 70 mW/cm2) . 
Left: Continuous irradiation. Right: Short pulse irradiation.

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FDV-0032_fig8.jpg


Reference Data

  • Visible light-induced NO releasing in vitro
Quantitative analysis of NO release from Controllable NOdonor with NO electrode. Controllable NOdonor (final 10 µM) in 100 mM HEPES buffer was irradiated with a MAX-303 (Asahi Spectra) equipped with a 530-590 nm band-pass filter (light intensity: 70 mW/cm2).
Left: Continuous irradiation. Right: Short pulse irradiation.
FDV-0032_fig9.jpg

  • Cytotoxicity of Controllable NOdonor
HEK293T cells in 96 well plate were treated with 0, 10, 30 and 100 µM of Controllable NOdonor for 48 hours. After incubation, cell viability was assessed by MTT assay. Little toxicity of 10 and 30 µM of Controllable NOdonor was observed. Please note high concentration (100 µM) impaired cell viability.
FDV-0032_fig10.jpg

  • Light-induced NO releasing in cultured cells
HEK293T cells were firstly treated DAF-FM DA (10 µM), a NO detection reagent, for 30 min. After wash cells with PBS two times, the cells were subsequently treated with 10 µM Controllable NOdonor for 30 min. The cells were irradiated with a MAX-303 (Asahi Spectra) equipped with a 530-590 nm band-pass filter (light intensity: 146 mW/cm2) for 15 min. Observed the cells under a confocal fluorescence microscopy (Ex. 500 nm /Em. 515 nm for DAF-FM reagent). (scale bar = 40 µm) 
FDV-0032_fig11.jpg


References

  1. Ieda et al., Sci. Rep., 9, 1430 (2019) Structure-efficiency relationship of photoinduced electron transfer-triggered nitric oxide releasers.
  2. Ieda et al., Chem. Pharm. Bull., 67, 576-579 (2019) , In cellullo and ex vivo availability of yellowish-green-light-controllable NO releaser. 
  3. Okuno et al., Org. Biomol. Chem., 15, 2791-2796 (2017) A yellowish-green-light-controllable nitric oxide donor based on N-nitrosoaminophenol applicable for photocontrolled vasodilation.

 

 

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