Synthesis of mono- and dinucleotide photoaffinity probes of ribonucleic acid polymerase

The abortive initiation reaction of RNA polymerase has been used to prepare adenylyl-(3'--5')-uridine 5'-phosphate (pApU) in 74% yield from AMP and UTP. The reactive intermediate p-azidophenyl phosphorimidazolidate has been prepared by starting from p-nitrophenyl phosphate. Reaction of this compound with the terminal phosphates of adenosine 5'-phosphate and adenylyl-(3'--5')-uridine 5'-phosphate gives the corresponding beta-substituted 5'-diphosphates. These products are incorporated into the 5' (leading) end of RNA by RNA polymerase (Escherichia coli) and can be photoactivated at a specific stage of RNA elongation. The dinucleotide photoaffinity label beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate stimulates RNA synthesis more strongly than adenylyl-(3'--5')-uridine.