Practical Method for the Multigram Separation of the 5- and 6-Isomers of Carboxyfluorescein

Abstract

An efficient preparative method for separating 5- and 6-carboxyfluorescein is presented. 6-Carboxyfluorescein dipivalate is isolated as its diisopropylamine salt, which can be converted to the free acid or used directly in coupling reactions. The 5-isomer is isolated from the acidified mother liquor. Isomerically pure carboxyfluoresceins are prepared by hydrolysis of the corresponding dipivalates.

Detection of Epitope-Tagged Proteins in Flow Cytometry: Fluorescence Resonance Energy Transfer-Based Assays on Beads with Femtomole Resolution

“…The temporal order of elution was supplied by Anaspec (San Jose, CA)-the commercial source of the Fmoc-Lys(fluorescein)-OH. This is also consistent with the result that has been reported for the separation by HPLC of 5-and 6-isomers of carboxyfluorescein (19).…”

Facile Large‐Scale Synthesis of 5‐ and 6‐Carboxyfluoresceins: Application for the Preparation of New Fluorescent Dyes

“…Using only resorcinol and pyromellitic dianhydride (12) we obtained the only known derivative described of this type in the literature: Difluorescein (one of the components of FluoroJade B). [8,17,19] This was obtained via dibenzophenone 13 as For further confirmation of the structures of benzophenones 5 and 6 as well as dibenzophenone 13, X-ray structures were obtained ( Figure 3, Figures S1-S3 in the Supporting Information, and X-ray data given in ref. [20] ).…”

“…Carboxyfluoresceins 8 and 7 were purified by simple precipitation without column chromatography. The overall yields starting from trimellitic anhydride (4) and resorcinol were 25 % for 6-carboxyfluorescein (7) and 35 % for 5-carboxyfluorescein (8).…”

“…The maximum wavelengths of absorption (λ max abs ) and fluorescence (λ max em ), pK a values, fluorescence quantum yields (Φ fl ), and photostabilities are summarized in Table 1 for compounds 9-11 and 16-18. For comparison, the photophysical properties of carboxy-JOE (2), carboxy-oregon green (3), and 5-carboxyfluorescein (8) are also included. [15,22] Data for carboxy-SNAFL-1 (1) have not been included due to a large difference in structure compared with fluorescein, and hence incomparable properties.…”

“…[6] Other regioisomeric fluorescein derivatives have also been separated. [7][8][9] To the best of our knowledge, a large-scale synthesis of 100 % regioisomeric pure 5-and 6-carboxyfluorescein without chromatographic purification has never been reported. Carboxyfluoresceins are widely used as fluorescent-labeling reagents by esterification or amidation at the 5-or 6-position.…”