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

Abstract

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.

DNA synthesis technologies to close the gene writing gap

“…97 – 100 ) and Camena Bioscience appears to favour 2-nitrobenzyl 26 as a 3ʹ-PG 101 . Other PGs are attempted for the protection of 3ʹ-OH and the bases of NTPs 102 – 106 , with parallel efforts focusing on PGs for XNA synthesis 90 , 107 . However, TdT must be able to accommodate the protected nucleotides in its active site, which limits the choice of PGs or requires the re-engineering of the enzyme for compatibility with 3ʹ-PG.…”

High-throughput DNA synthesis for data storage

“…11a). 91,119,122,152,179 A variety of reversible terminators are available, such as 3′- O -NH 2 -dNTP (DNA Script and Nuclera), 180–183 3′- O -azidomethyl-dNTP 184,185 (Molecular Assemblies), 3′- O -(2-nitrobenzyl)-dNTP (Camena Bioscience), 186 3′-phosphate 187 (Codexis 188 ) or photo-cleavable nucleotides 189,190 and other protecting groups. 191,192…”

“…11a). 91,119,122,152,179 A variety of reversible terminators are available, such as 3 0 -O-NH 2 -dNTP (DNA Script and Nuclera), [180][181][182][183] 3 0 -O-azidomethyl-dNTP 184,185 (Molecular Assemblies), 3 0 -O-(2nitrobenzyl)-dNTP (Camena Bioscience), 186 3 0 -phosphate 187 (Codexis 188 ) or photo-cleavable nucleotides 189,190 and other protecting groups. 191,192 In 2020, DNA Script launched the world's first desktop DNA printer ''SYNTAX'' based on enzymatic synthesis, enabling automated synthesis, purification and quantification.…”

Boosting the Release of Leaving Group from Blebbistatin Derivative Photocages via Enhancing Intramolecular Charge Transfer and Stabilizing Cationic Intermediate

“…3−7 Photocages are highly prized for their ability to provide spatial and temporal control over the accurate release of various chemicals and bioactive groups when they are excited by light. For example, to control the release of proteins, 8,9 drug molecules, 10,11 biological small molecules, 12 fluorescent dyes, 13,14 and so on. Therefore, photocages can be applied in chemistry, biology, and medicine.…”

“…Photocages or photoactivatable protecting groups (PPGs) are light-sensitive chemical protecting groups that mask substrates (leaving groups, LGs) through a covalent linkage that renders the substrates inert. , Upon light irradiation, the leaving groups will be cleaved by breaking the covalent bond, releasing, and then restoring their function and activity. − Photocages are highly prized for their ability to provide spatial and temporal control over the accurate release of various chemicals and bioactive groups when they are excited by light. For example, to control the release of proteins, , drug molecules, , biological small molecules, fluorescent dyes, , and so on. Therefore, photocages can be applied in chemistry, biology, and medicine.…”