SYNTHESIS AND APPLICATION OF A NEW FLUORESCEIN DERIVATIVE FOR FLUORESCENT LABELING OF OLIGONUCLEOTIDES AND AS A NOVEL TOOL FOR NONRADIOACTIVE DNA-SEQUENCING

The synthesis of 4(5)-(bromomethyl)fluorescein, which can be used as i ts 3',6'-dibenzoyl derivative 7 for the labelling of nucleosides is de scribed. Pyrimidine nucleosides such as 3'-and/or 5'-protected 2'-deox yuridine and thymidine are labelled by 7 at N3 of the heterocyclic bas e. The resulting base-labelled 3'-benzoyl-5'-dimethoxytrityl nucleosid es may, on the one hand, be converted, after cleavage of the protectiv e dimethoxytrityl group, into the 5'-phosphoramidite 11 by normal phos phorylation; on the other hand, 5'-triphosphates 13 are prepared by ph osphorylation with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one and py rophosphate. The phosphoramidites 11 were sucessfully used for the flu orescent labelling of oligonucleotides during automatic DNA synthesis. The oligonucleotides can be used as primer in automatic non-radioacti ve DNA sequencing. The 5'-triphosphates 13 are potential substrates fo r DNA polymerases or terminal transferase.