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carboxytetraethylrhodamine N-hydroxysuccinimide esters using
N,N′-disuccinimidyl carbonate (DSC) and DMAP,34 but in our
hands, this gave a mixture of starting material and the di-ester
product (Figure 1).
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Figure 1. Di-ester obtained when treating carboxytetraethylrhodamine
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A. C.; Sánchez-Martín, R. M. Bioorganic
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To achieve 5 or 6-carboxylic acid regioselectivity over the 3-
carboxylic acid, rhodamine must react in its closed lactone form;
however, unlike fluorescein, rhodamine B is in the lactone form
under basic conditions, and in the open form under acidic
conditions.35 Therefore it was reasoned that the active ester of 5
and 6-carboxytetraethylrhodamine would be generated using a
combination of DMAP and DSC with 5 equivalents of
triethylamine to give the desired regioselectivity (Scheme 2).36
Larger quantities of base interfered with the efficiency of the
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In conclusion, methods have been developed for the formation
and separation of the active esters of 5 and 6-isomers of
carboxyfluorescein and carboxyrhodamine B. These methods are
robust and reliable, and make single isomers of these two widely
used fluorophores readily available.
Acknowledgments
Financial support from the MRC and University of Edinburgh is
gratefully acknowledged. Dr Annamaria Lilienkampf is
acknowledged for help with the manuscript preparation.
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32. 5(6)-Carboxyfluorescein diacetate: To a solution of 5(6)-
carboxyfluorescein (15.0 g, 39.9 mmol) in Ac2O (180 mL) was
added pyridine (18 mL, 22.3 mmol) and the reaction was stirred
for 30 min at 110 ºC. The clear solution was concentrated in
vacuo. The crude mixture was dissolved in EtOAc (300 mL) and
washed with aqueous KHSO4 (1M, 2 x 300 mL) and brine (300
mL). The organic layer was dried over Na2SO4, filtered and
concentrated under reduced pressure to give the desired