UNIT 4.55 Non‐Nucleoside Phosphoramidites of Xanthene Dyes (FAM, JOE, and TAMRA) for Oligonucleotide Labeling. Issue 1 (March 2013)

• Record Type: Journal Article
• Authors: Kvach, Maksim V.; Tsybulsky, Dmitry A.; Shmanai, Vadim V.; Prokhorenko, Igor A.; Stepanova, Irina A.; Korshun, Vladimir A.
• Is Part Of: Current protocols in nucleic acid chemistry. Volume 52:Issue 1(2013)
• Page Start: 4.55.1
• Page End: 4.55.33
• Publication Date: 2013-03
• Subjects: xanthene fluorophores -- fluorescein -- JOE -- TAMRA -- protecting groups -- isomer separation -- non‐nucleoside phosphoramidites -- linkers -- fluorescence quenching -- energy transfer; Nucleic acids -- Laboratory manuals; Nucleic Acids -- chemical synthesis; Nucleic acids; Laboratory Manuals; Electronic books; Laboratory manuals; 572.8028
• DOI: 10.1002/0471142700.nc0455s52
• Languages: English
• ISSNs: 1934-9270

Abstract

Abstract: This unit describes the preparation of 5‐ and 6‐carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4′, 5′‐dichloro‐2′, 7′‐dimethoxy‐fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non‐nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy‐FAM and carboxy‐JOE as pentafluorophenyl esters. Acylation of 3‐dimethylaminophenol with 1, 2, 4‐benzenetricarboxylic anhydride gave a mixture of 4‐dimethylamino‐2‐hydroxy‐2′, 4′(5′)‐dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5‐ or 6‐carboxytetramethylrhodamines. The dyes were converted into 6‐aminohexanol‐ (JOE), 4‐ trans ‐aminocyclohexanol‐ (FAM and JOE), and hydroxyprolinol‐based (TAMRA) phosphoramidite reagents. Curr. Protoc. Nucleic Acid Chem. 52:4.55.1‐4.55.33. © 2013 by John Wiley & Sons, Inc.