A synthetic substrate of DNA polymerase deviating from the bases, sugar, and leaving group of canonical deoxynucleoside triphosphates.

The selection of artificial nucleic acids to be used for synthetic biology purposes is based on their structural and biochemical orthogonality to the natural system. We describe the example of a nucleotide mimic that functions as a substrate for polymerases and in which the carbohydrate moiety as well as the base moiety and the leaving group are different from that of the natural building blocks. The nucleotides themselves have two anomeric centers, and different leaving group properties of substituents at both anomeric centers need to be exploited to perform selective glycosylation reactions for their synthesis. In addition, the reversibility of the polymerase reaction at the level of the template has been demonstrated when pyrophosphate functions as leaving group and not with the alternative leaving groups.