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Enzymatic incorporation of deoxynucleoside 5′triphosphate bearing the photocleavable protecting group is a useful method for the preparation of photocaged oligodeoxynucleotides. Here, we describe the synthesis of new photocaged deoxynucleoside triphosphates N1-(2-nitrobenzyl)-deoxypseudouridine triphosphate (d NB ΨTP) and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine triphosphate (d NPOM ΨTP). We successfully synthesized d NB ΨTP and d NPOM ΨTP and applied them to enzymatic synthesis of photocaged oligonucleotides. In addition, we also synthesized phosphoramidites of N1-(2-nitrobenzyl)-and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine to enable chemical synthesis of photocaged oligonucleotides incorporating them. The photocleavable 2-nitrobenzyl and 6-nitropiperonyloxymethyl in oligonucleotides were cleaved by irradiation at 365 nm for 30 and 10 s, respectively. We also studied the enzymatic incorporation of d NB ΨTP and d NPOM ΨTP using the Klenow fragment exo − . As a result, it was clarified that d NPOM ΨTP could be incorporated to oligonucleotide 193 times more efficiently than d NB ΨTP, as judged by V max /K m . We also performed the incorporation of at least eight d NPOM Ψ residues in a 35-mer oligodeoxynucleotide. It has also been revealed that the oligodeoxynucleotides incorporating photocaged deoxypseudouridine were useful for photocontrol of DNA triplex formation.

Deoxynucleoside 5'-triphosphate was synthesized with 3-oxo-2 H-pyridazin-6-yl (Pz)-a uracil analogue lacking a 2-keto group-as the nucleobase. Theoretical analyses and hybridization experiments indicated that Pz recognizes adenine (A) for formation of a Watson-Crick base pair. Primer extension reactions using nucleoside 5'-triphosphate and the Klenow fragment revealed that the synthetic nucleoside 5'-triphosphate was incorporated into the 3' end of the primer through recognition of A in the template strand. Moreover, the 3'-nucleotide residue harboring Pz as the base was resistant to the 3'-exonuclease activity of Klenow fragment exo+. The primer bearing the Pz base at the 3' end could function in subsequent chain elongation. These properties of Pz were attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond, which was presumed to form an H-bond with the amino acid residue of DNA polymerase for effective recognition of the 3' end of the primer for primer extension. These results provide a basis for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic bond and base-pairing sites for Watson-Crick hydrogen bonding.