One-pot reaction for the synthesis of m7G5′pppG and m7G5′pppA by using an activatable bifunctional phosphorylating reagent

Solid phase synthesis of 5′-diphosphorylated oligoribonucleotides and their conversion to capped m7Gppp-oligoribonucleotides for uase as primers for influenza A virus RNA polymerasein vitro

“…The 2'-O-methylated m7GpppGmAAUACUCAAG primed transcription to give a transcription product (TP) at -10 times the yield of the products derived from the same primer lacking the 2'-O-methyl group (compare lanes 3 and 6). The transcript primed by m7GpppGmAAUACUCAAG (lane 3) is a doublet, 23 and 24 nt long, previously characterized by partial T1 RNase sequencing, which initiated transcription at the third nucleotide of the added template and was partially elongated at its 3'-end by the addition of a non-templated nucleotide (5). As previously noted (4,5), elongation of primers was not quantitative.…”

“…It should also be possible to automate the reactions described here on the ABI 394 synthesizer, since no precipitates were formed in the reactions. Ultimately, a procedure for the total chemical synthesis of capped oligoribonucleotides is desirable (23). Moreover, the chemical phosphorylation described here should succeed equally well with deoxyribonucleotides, so that other biological applications for the use of 5'-dior triphosphorylated DNA may emerge, e.g.…”

Preparation and Properties of mRNA 5-cap Structure

“…Reaction of 42 with a nucleoside monophosphate in MPD-HMPA solution with AgNO 3 as an activator of the phenylthio group displacement yields an activated nucleoside diphosphate 43 (Scheme 11), which is coupled with a nucleoside monophosphate using CuCl 2 as a catalyst. With this method, m 7 GpppG has been obtained in 55 in 24 hours at room temperature [79]. At a higher temperature, the yield was poorer.…”

“…Activation with chloroquinoylyl reagent 42 provides another method to prepare cap analogs in an organic solvent without the use of nucleoside protecting groups [79]. A 3:1 mixture of 1-methylpyrrolidine (MDP) and HMPA, which was found to dissolve nucleotides, was used as a solvent in coupling reaction.…”

Synthetic mRNA capping

“…Multiple strategies have been reported which mostly rely on the same principle: One of the two nucleotides (typically the monophosphorylated nucleotide) is equipped with a good leaving group while the other one acts as a nucleophile. Different leaving groups have been exploited for the synthesis of cap analogues, comprising phenylthio [ 56 ], 5-chloro-8-quinolyl [ 57 ], morpholidate [ 48 ] and imidazolide moieties [ 58 – 59 ]. Imidazole activation in DMF with ZnCl 2 was first reported by Sekine et al [ 60 ] and is the most often used method for the formation of triphosphates.…”