Synthesis of nucleoside 5'-tetraphosphates containing terminal fluorescent labels via activated cyclic trimetaphosphate.

2'-Deoxynucleotide 5'-tetraphosphates in which a fluorescent label is attached to the terminal phosphate are used as key reagents in high-throughput DNA sequencing techniques and in single nucleotide polymorphism typing assays. We demonstrate that this class of compounds can be prepared by reacting fluorophores such as 7-hydroxy-4-methylcoumarin, methylfluorescein, fluorescein and resorufin with an activated form of cyclic trimetaphosphate to give intermediate 11. Reaction of 11 with 2'-deoxynucleoside 5'-monophosphates or a nucleoside 5'-monophosphate gave the target compounds in good yield.