Synthesis of Deoxypseudouridine 5′-Triphosphate Bearing the Photoremovable Protecting Group at the N1 Position Capable of Enzymatic Incorporation to DNA

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)Psi TP) and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine triphosphate (d(NPOM)Psi TP). We successfully synthesized d(NB) Psi TP and d(NPOM)Psi TP 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 photo- cleavable 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) Psi TP and d(NPOM)Psi TP using the Klenow fragment exo(-). As a result, it was clarified that d(NPOM)Psi TP could be incorporated to oligonucleotide 193 times more efficiently than d(NB) Psi TP, as judged by V-max/K-m. We also performed the incorporation of at least eight d(NPOM)Psi TP 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.