A new efficient method for chemical 5'-phosphorylation of synthetic ol
igonucleotides is described. Accordingly, 2-cyanoethyl dimethoxytrityl
oxy)-2,2-di(ethoxycarbonyl)propyl-1 N,N-diisopropyl phosphoramidite (1
) was introduced as the 5'-terminal building block during the normal c
hain assembly. Conventional ammonolysis gave rise to an oligomer prote
cted at 5'-phosphate with a dimethoxytritylated tether. At this step,
the oligonucleotide may be easily separated from truncated impurities
by RP HPLC. Successive detritylation and brief treatment with aqueous
ammonia gave the oligonucleotide 5'-monophosphate. Alternatively, the
yield of the last coupling may be quantified by detritylation of the o
ligonucleotide still anchored to the solid support. Usual deprotection
then leads directly to the 5'-phosphorylated oligomer.