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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.

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Mentions: With a sensor with desirable properties in hand, we tested the conjugation reaction. A model thiohydantoin derivative 18, prepared from thiohydantoin 16, was used to test the CuAAC with alkyne-tagged phenylalanine derivative 20 (Scheme 3). However, mixtures immediately turned red upon mixing the thiohydantoin with copper(I) ions and the click reaction was ineffective. This is consistent with the reported formation of a thiohydantoin-copper complex.40 The tetracarboxylate BAPTA-based fluorophores are cell-impermeable and are generally caged as acetoxymethyl (AM) esters so that they can cross the plasma membrane.41 The AM esters are hydrolysed rapidly by esterases inside cells to give the active Ca2+ sensors. This strategy has been used for FuraRed,11 which also includes N-acetylmethyl caging of the thiohydantoin group. Since NitroAzidoFuraRed is designed to be used in cells, we investigated whether AM-protection of the model thiohydantoin would allow CuAAC. Happily, conversion of thiohydantoin 18 into the AM derivative 19 was followed by smooth CuAAC to give the triazole adduct 21, a process that occurred without the dramatic colour change observed previously.


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)

© Copyright Policy - CC BY
Related In: Results  -  Collection

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

Mentions: With a sensor with desirable properties in hand, we tested the conjugation reaction. A model thiohydantoin derivative 18, prepared from thiohydantoin 16, was used to test the CuAAC with alkyne-tagged phenylalanine derivative 20 (Scheme 3). However, mixtures immediately turned red upon mixing the thiohydantoin with copper(I) ions and the click reaction was ineffective. This is consistent with the reported formation of a thiohydantoin-copper complex.40 The tetracarboxylate BAPTA-based fluorophores are cell-impermeable and are generally caged as acetoxymethyl (AM) esters so that they can cross the plasma membrane.41 The AM esters are hydrolysed rapidly by esterases inside cells to give the active Ca2+ sensors. This strategy has been used for FuraRed,11 which also includes N-acetylmethyl caging of the thiohydantoin group. Since NitroAzidoFuraRed is designed to be used in cells, we investigated whether AM-protection of the model thiohydantoin would allow CuAAC. Happily, conversion of thiohydantoin 18 into the AM derivative 19 was followed by smooth CuAAC to give the triazole adduct 21, a process that occurred without the dramatic colour change observed previously.

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