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5' end fluorescent labelling of oligonucleotides with riboflavin-derived phosphitylating reagent
S Mielewczyk1, D Wieckowska, E Krzymańska-Olejnik
1Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
Abstract:
Riboflavin was transformed within six steps into 3-isobutyryl-7,8-dimethyl-10-[2-O-(beta-cyanoethoxy-N,N- diisopropylaminophosphinyl)ethyl]isoalloxazine. This new fluorescent reagent was applied for direct phosphitylation of 5-OH function of protected oligonucleotide assembled on controlled-pore glass support by beta-cyanoethyl phosphoramidite chemistry. As the result of subsequent P(III)----P(V) oxidation and removal of protecting groups with concentrated ammonia, an oligonucleotide 5-labelled with fluorescent flavin moiety could be obtained. Using this procedure 15-mer oligonucleotide of a sequence corresponding to M13 hybridization primer was prepared.