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Updated: Dec 24, 2025

Chemical Triphosphorylation of Oligonucleotides
Published on: June 2, 2022
Preparation of DNA containing 5-hydroxymethyl-2'-deoxycytidine modification through phosphoramidites with TBDMS as
1Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA.
Abstract:
This unit describes procedures for preparation of two phosphoramidite building blocks III and IV, both containing a TBDMS as 5-CH(2)OH-protecting group. Phosphoramidites III and IV allow efficient incorporation of 5-hmC into DNA and a "one-step" deprotection procedure to cleanly remove all the protecting groups. A "two-step" deprotection strategy is compatible with ultramild DNA synthesis, which enables the synthesis of 5 hmC-containing DNA with additional modifications. Methods are also presented for their incorporation into oligonucleotides by solid-phase synthesis, subsequent deprotection, and HPLC analysis.
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