Abstract
Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside monophosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).
Highlights
Photocleavable protecting groups[1] are gaining growing attention due to their applications in organic synthesis, chemical biology and many other areas
There were some reports on selective modification of hydroxymethyl groups in 5-hydroxymethyl-2′-deoxyuridine,[25] for Scheme 1 Synthesis of photocaged 2’-deoxyuridine monophosphates and triphosphates (i) ROH, 2,2,6,6-tetramethylpiperidine, silver triflate, CH3CN (ii) NaHCO3, MeOH (iii) Et3N.3HF, THF (iv) POCl3, PO(OMe)[3], (NHBu3)2H2P2O7 (v) POCl3, PO(OMe)[3, 2] M TEAB. *Prepared according to the literature.[22]
The extended modified oligonucleotide (ON) products were confirmed by the MALDI-TOF analysis showing masses corresponding to Uhm-containing ONs (Fig. S13 in the Electronic supplementary information (ESI)†) because the laser irradiation using in MALDI cleaves the photoremovable groups
Summary
Photocleavable protecting groups[1] are gaining growing attention due to their applications in organic synthesis, chemical biology and many other areas. It is desirable to develop alternative photocaging groups for 5hmU which would be compatible with the synthesis of modified dNTPs, polymerase incorporation and would be cleavable by visible light.
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