The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2′‐deoxynucleotides with a 2‐amino‐6‐methyl‐8‐oxo‐7,8‐dihydro‐purine (X), a 2‐methyl‐6‐thiopurine (Y) and a 6‐methyl‐4‐pyrimidone (Z) base moiety are described. As demonstrated by Tm measurements, the X and Y bases fail to form a self‐complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W−C recognition (6‐carbonyl and 2‐amino group of purine; 6‐carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases)
