Stepwise Enzymic Oligoribonucleotide Synthesis Including Modified Nucleotides

A method has been developed for the routine synthesis of 2′(3′)-O-monoacyl ribonucleoside 5′-diphosphates for stepwise synthesis of oligoribonucleotides with Escherichia coli polynucleotide phosphorylase. The use of triethyl orthoisovalerate allows the facile preparation of 2′(3′)-O-isovaleryl-UDP, -CDP, -ADP, -GDP, -IDP, -ε-ADP, -ε-CDP, and N ⁶ -isopentenyl-ADP. The synthesis of N ⁶ -isopentenyl-ADP from ADP by N ¹ -alkylation and the Dimroth rearrangement to N ⁶ is reported. The effects of several factors including the nature of the divalent cation, pH, salt concentration, and time on the efficiency of the polynucleotide phosphorylase catalyzed single additions of the 2′(3′)-O-isovaleryl ribonucleoside 5′-diphosphates to an oligoribonucleotide primer are reported. The syntheses of many tetranucleoside triphosphates and two pentanucleoside tetraphosphates in yields of 20-75% are reported. The 2′(3′)-O-isovaleryl derivatives of IDP, ε-ADP, ε-CDP, and N ⁶ -isopentenyl-ADP were all accepted by polynucleotide phosphorylase as substrates for the monoaddition reaction. The extension of the method to include the syntheses of oligoribonucleotides containing modified nucleosides offers a means of studying the roles of these modifications by the use of relatively simple model compounds.