Unit

UNIT 4.55 Non-Nucleoside Phosphoramidites of Xanthene Dyes (FAM, JOE, and TAMRA) for Oligonucleotide Labeling

  1. Maksim V. Kvach1,
  2. Dmitry A. Tsybulsky1,
  3. Vadim V. Shmanai1,
  4. Igor A. Prokhorenko2,
  5. Irina A. Stepanova2,
  6. Vladimir A. Korshun2

Published Online: 1 MAR 2013

DOI: 10.1002/0471142700.nc0455s52

Current Protocols in Nucleic Acid Chemistry

Current Protocols in Nucleic Acid Chemistry

How to Cite

Kvach, M. V., Tsybulsky, D. A., Shmanai, V. V., Prokhorenko, I. A., Stepanova, I. A. and Korshun, V. A. 2013. Non-Nucleoside Phosphoramidites of Xanthene Dyes (FAM, JOE, and TAMRA) for Oligonucleotide Labeling. Current Protocols in Nucleic Acid Chemistry. 52:4.55:4.55.1–4.55.33.

Author Information

  1. 1

    Institute of Physical Organic Chemistry, Minsk, Belarus

  2. 2

    Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

Publication History

  1. Published Online: 1 MAR 2013

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.

Keywords:

  • xanthene fluorophores;
  • fluorescein;
  • JOE;
  • TAMRA;
  • protecting groups;
  • isomer separation;
  • non-nucleoside phosphoramidites;
  • linkers;
  • fluorescence quenching;
  • energy transfer