Abstract
Base-modified adenosine-5'-triphosphate (ATP) analogues are highly sought after as building blocks for mRNAs and non-coding RNAs, for genetic code expansion or as inhibitors. Current synthetic strategies lack efficient and robust 5'-triphosphorylation of adenosine derivatives or rely on costly phosphorylation reagents. Here, we combine the efficient organic synthesis of base-modified AMP analogues with enzymatic phosphorylation by a promiscuous polyphosphate kinase 2 class III from an unclassified Erysipelotrichaceae bacterium (EbPPK2) to generate a panel of C2-, N(6)-, or C8-modified ATP analogues. These can be incorporated into RNA using template independent poly(A) polymerase. C2-halogenated ATP analogues were incorporated best, with incorporations of 300 to >1000 nucleotides forming hypermodified poly(A) tails.