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Synthesis of an azido-tagged low affinity ratiometric calcium sensor.

Caldwell ST, Cairns AG, Olson M, Chalmers S, Sandison M, Mullen W, McCarron JG, Hartley RC - Tetrahedron (2015)

Bottom Line: The synthesis of a dual excitation, ratiometric calcium ion sensor with a K d of 90 μM, is described.It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift.The thiohydantoin unit has to be protected with an acetoxymethyl (AM) caging group to allow CuAAC click reaction and incorporation of the KDEL peptide endoplasmic reticulum (ER) retention sequence.

View Article: PubMed Central - PubMed

Affiliation: WestCHEM School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.

ABSTRACT

Changes in high localised concentrations of Ca(2+) ions are fundamental to cell signalling. The synthesis of a dual excitation, ratiometric calcium ion sensor with a K d of 90 μM, is described. It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift. The binding modulating nitro group is introduced to the BAPTA core prior to construction of a benzofuran-2-yl carboxaldehyde by an allylation-oxidation-cyclisation sequence, which is followed by condensation with an azido-tagged thiohydantoin. The thiohydantoin unit has to be protected with an acetoxymethyl (AM) caging group to allow CuAAC click reaction and incorporation of the KDEL peptide endoplasmic reticulum (ER) retention sequence.

No MeSH data available.


Related in: MedlinePlus

Excitation spectra for NitroAzidoFuraRed with free Ca2+ concentrations ranging from 0 μM to 2000 μM. The excitation bandpass was 5 nm and fluorescence emission collected at 630 nm (bandpass 30 nm). The spectra have been corrected for background fluorescence. 5 μM NitroAzido-FuraRed in a 100 mM KCl, 30 mM MOPS, pH 7.2 buffer was incrementally exposed to rising [Ca2+] from 0 to 2 mM. With increasing [Ca2+], fluorescence emission decreases with excitation above 452 nm and increases with excitation below 452 nm (arrows are drawn at 420 nm and 485 nm).
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fig3: Excitation spectra for NitroAzidoFuraRed with free Ca2+ concentrations ranging from 0 μM to 2000 μM. The excitation bandpass was 5 nm and fluorescence emission collected at 630 nm (bandpass 30 nm). The spectra have been corrected for background fluorescence. 5 μM NitroAzido-FuraRed in a 100 mM KCl, 30 mM MOPS, pH 7.2 buffer was incrementally exposed to rising [Ca2+] from 0 to 2 mM. With increasing [Ca2+], fluorescence emission decreases with excitation above 452 nm and increases with excitation below 452 nm (arrows are drawn at 420 nm and 485 nm).

Mentions: Having established that the probe possessed the desired optical properties, we turned our attention to the binding properties. The excitation spectra of NitroAzidoFuraRed were obtained with different concentrations of Ca2+ and showed a good isobestic point at 452 nm (Fig. 3). The Kd of the probe was found to be 90 μM using this titration and the ratio of fluorescence intensities at 630 nm when excited at 420 nm and 485 nm, following the method of Grynkiewitz et al.9


Synthesis of an azido-tagged low affinity ratiometric calcium sensor.

Caldwell ST, Cairns AG, Olson M, Chalmers S, Sandison M, Mullen W, McCarron JG, Hartley RC - Tetrahedron (2015)

Excitation spectra for NitroAzidoFuraRed with free Ca2+ concentrations ranging from 0 μM to 2000 μM. The excitation bandpass was 5 nm and fluorescence emission collected at 630 nm (bandpass 30 nm). The spectra have been corrected for background fluorescence. 5 μM NitroAzido-FuraRed in a 100 mM KCl, 30 mM MOPS, pH 7.2 buffer was incrementally exposed to rising [Ca2+] from 0 to 2 mM. With increasing [Ca2+], fluorescence emission decreases with excitation above 452 nm and increases with excitation below 452 nm (arrows are drawn at 420 nm and 485 nm).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660056&req=5

fig3: Excitation spectra for NitroAzidoFuraRed with free Ca2+ concentrations ranging from 0 μM to 2000 μM. The excitation bandpass was 5 nm and fluorescence emission collected at 630 nm (bandpass 30 nm). The spectra have been corrected for background fluorescence. 5 μM NitroAzido-FuraRed in a 100 mM KCl, 30 mM MOPS, pH 7.2 buffer was incrementally exposed to rising [Ca2+] from 0 to 2 mM. With increasing [Ca2+], fluorescence emission decreases with excitation above 452 nm and increases with excitation below 452 nm (arrows are drawn at 420 nm and 485 nm).
Mentions: Having established that the probe possessed the desired optical properties, we turned our attention to the binding properties. The excitation spectra of NitroAzidoFuraRed were obtained with different concentrations of Ca2+ and showed a good isobestic point at 452 nm (Fig. 3). The Kd of the probe was found to be 90 μM using this titration and the ratio of fluorescence intensities at 630 nm when excited at 420 nm and 485 nm, following the method of Grynkiewitz et al.9

Bottom Line: The synthesis of a dual excitation, ratiometric calcium ion sensor with a K d of 90 μM, is described.It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift.The thiohydantoin unit has to be protected with an acetoxymethyl (AM) caging group to allow CuAAC click reaction and incorporation of the KDEL peptide endoplasmic reticulum (ER) retention sequence.

View Article: PubMed Central - PubMed

Affiliation: WestCHEM School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.

ABSTRACT

Changes in high localised concentrations of Ca(2+) ions are fundamental to cell signalling. The synthesis of a dual excitation, ratiometric calcium ion sensor with a K d of 90 μM, is described. It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift. The binding modulating nitro group is introduced to the BAPTA core prior to construction of a benzofuran-2-yl carboxaldehyde by an allylation-oxidation-cyclisation sequence, which is followed by condensation with an azido-tagged thiohydantoin. The thiohydantoin unit has to be protected with an acetoxymethyl (AM) caging group to allow CuAAC click reaction and incorporation of the KDEL peptide endoplasmic reticulum (ER) retention sequence.

No MeSH data available.


Related in: MedlinePlus