Synthesis of nucleoside analogues and fluorescent labels for DNA sequencing and other applications in biotechnology

Abstract

Two pyrrolo[2,3-d]pyrimidines and a nucleoside analogue have been prepared. The nucleobases were obtained by spontaneous cyclisation of 4-aminopyrimidyl-acetaldehydes as an acetal. The adenosine analogue has been made by glycosylation of a suitable sugar derivative with the corresponding heterocyclic base. This nucleoside can be converted into fluorescently-labeled chain-terminating substrates for DNA polymerase after subsequent triphosphorylation and coupling to through-bond energy transfer systems. Fluorescein-based donor components to be incorporated into through-bond energy transfer systems have been prepared. The synthesis of 5-ethynylfluorescein-(5-tert-butoxycarbonyl)-pentyl ester has been executed in five steps from 1,3-dihydroxybenzene and phthalic anhydride. The donor fluorescein carboxylate has been alkylated with the tert-butyl ester of 6-bromohexanoic acid to provide a handle for attachment to biomolecules. In the context of regioisomerically pure halofluoresceins, besides the synthesis of pure regioisomers of bromofluorescein derivatives, 5-iodosulfofluorescein and pure regioisomers of 5-nitrofluorescein diacetate, as an intermediate in the synthesis of 5-iodofluoresceins, have been synthesized. New rhodamine-based acceptor components with extended aromatic systems have been prepared from an affordable starting material, tetralin. The attempts to isolate them via repeated recrystallizations and flash chromatography have been unsuccessful. However, these pyrylium cations are expected to fluoresce at longer wavelengths.