I have TBDMS (acid labile protecting group) on 2' and 3' of nucleosides and trying to phoshorylate 5' OH. Pocl3 in triethylphosphate generated HCl in situ which deprotected TBDMS protecting group. I am looking for a mild phosphorylation method to install 5' phosphate on A and G.
Chemical synthesis is one way to go. However, nature uses enzymes, and you can phosphorylate the unprotected nucleosides using the right kinase and ATP. Various kinases have their particular substrate preferences, but you can choose a suitable kinase from the list below.
Kinases for ribonucleoside substrates (in your case):
EC 2.7.1.20 - adenosine kinase
EC 2.7.1.73 - inosine kinase
Kinases for 2'-deoxyribonucleoside substrates:
EC 2.7.1.76 - 2'-deoxyadenosine kinase
EC 2.7.1.113 - deoxyguanosine kinase
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