Synthesis and Application of a New Fluorescein Derivative for Fluorescent Labelling of Oligonucleotides and as a Novel Tool for Non-radioactive DNA Sequencing

✍ Scribed by Holletz, Torsten ;Möller, Uwe ;Knaf, Andrea ;Reinhardt, Richard ;Cech, Dieter

• Publisher: John Wiley and Sons
• Year: 1993
• Tongue: English
• Weight: 579 KB
• Volume: 1993
• Category: Article
• ISSN: 0947-3440
• DOI: 10.1002/jlac.1993199301169

✦ Synopsis

Abstract

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.