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Synthesis and Application of a New Fluorescein Derivative for Fluorescent Labelling of Oligonucleotides and as a Novel Tool for Non‐radioactive DNA Sequencing
Author(s) -
Holletz Torsten,
Möller Uwe,
Knaf Andrea,
Reinhardt Richard,
Cech Dieter
Publication year - 1993
Publication title -
liebigs annalen der chemie
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.825
H-Index - 155
eISSN - 1099-0690
pISSN - 0170-2041
DOI - 10.1002/jlac.1993199301169
Subject(s) - phosphoramidite , oligonucleotide , chemistry , labelling , fluorescein , dna , nucleoside , nucleic acid , nucleotide , combinatorial chemistry , thymidine , fluorescence , biochemistry , primer (cosmetics) , pyrimidine , organic chemistry , gene , physics , quantum mechanics
The synthesis of 4(5)‐(bromomethyl)fluorescein, which can be used as its 3′,6′‐dibenzoyl derivative 7 for the labelling of nucleosides is described. Pyrimidine nucleosides such as 3′‐and/or5′‐protected 2′‐deoxyuridine and thymidine are labelled by 7 at N 3 of the heterocyclic base. The resulting base‐labelled 3′‐benzoyl‐5′‐dimethoxytrityl nucleosides may, on the one hand, be converted, after cleavage of the protective dimethoxytrityl group, into the 5′‐phosphoramidite 11 by normal phosphorylation; on the other hand, 5′‐triphosphates 13 are prepared by phosphorylation with 2‐chloro‐4 H ‐1,3,2‐benzodioxaphosphorin‐4‐one and pyrophosphate. The phosphoramidites 11 were sucessfully used for the fluorescent labelling of oligonucleotides during automatic DNA synthesis. The oligonucleotides can be used as primer in automatic non‐radioactive DNA sequencing. The 5′‐triphosphates 13 are potential substrates for DNA polymerases or terminal transferase.