Linker-free fluorophore-labeled oligonucleotides:: Synthesis of 3′-fluoresceinylthymidine building blocks and their coupling reactions

Reported here are the syntheses of two thymidine derivatives with an amide-linked 6-carboxyfluorescein residue at their 3 '-position that are suitable for the synthesis of fluorophore-labeled oligonucleotides. The first is a 5 '-phosphoramidite with a pivaloyl-protected carboxyfluorescein residue in the lactone form. It was prepared from 3 '-azido-3 '-deoxythymidine (AZT) in three steps and 73% overall yield and coupled on solid support. The second is an imidazolide of thymidine 5 '-monophosphate that was obtained from AZT in five steps and 48% overall yield. The imidazolide can be coupled to amino-terminated nucleic acids in aqueous solution.