On-support phosphitylation for diverse oligonucleotide modifications

Fig.1, a. On-support phosphitylation for oligonucleotide functionalisation. General synthetic strategy. b. Diverse functionalised oligonucleotides are accessible, including 3ʹ modifications, charge neutral backbones and internal modifications (bottom).

Oligonucleotides are essential tools in chemical and molecular biology, with applications spanning gene regulation, molecular diagnostics and nucleic acid therapeutics. Their synthesis is dominated by phosphoramidite chemistry, which enables sequence assembly and the incorporation of diverse chemical functionalities [Egli & Manoharan, 2023]. However, introducing new modifications typically requires a dedicated phosphoramidite building block for each functionality. These reagents can be costly and are limited in structural diversity. Their sensitivity to air and moisture further complicates their handling [Krotz et al., 2004]. These limitations create a need for simpler and more flexible approaches to oligonucleotide functionalisation. To overcome these limitations, a direct on-support strategy for oligonucleotide functionalisation was devloped [Dasgupta et al., 2026].

Central to this strategy is the direct phosphitylation of the terminal 5'-hydroxyl group of the immobilised oligonucleotide, generating an on-support phosphoramidite intermediate. Upon activation, this intermediate undergoes in situ coupling with alcohol nucleophiles, followed by oxidation and global deprotection to give the functionalised oligonucleotide. The method is highly chemoselective, enabling the incorporation of diverse bifunctional alcohols (with groups including lipids, fluorophores, azides, amines and thiols) or a nucleoside for oligonucleotide chain extension, typically in high conversion (up to 94%) and without the need for protecting-group manipulation. For example, azido alcohols were coupled to generate 5'-azido-modified oligonucleotides, which enabled cellular fluorescent labelling. Importantly, this strategy is, in principle, agnostic to synthetic directionality and is therefore compatible with oligonucleotide chain extension and modification in either the conventional (3' → 5') or reverse (5' → 3') direction, enabling the installation of diverse functionalities at either terminus. This approach could also be extended to access nuclease-stable phosphotriester backbones. Alkyl phosphorodiamidites were prepared and used to phosphitylate the 5′-hydroxyl group of the immobilised oligonucleotide, followed by coupling with alcohols, oxidation and global deprotection to afford charge-neutral phosphotriesters. A further advantage of this approach is the recovery of unreacted nucleophiles after coupling, enabling the reuse of valuable alcohols and nucleosides in subsequent coupling reactions.

Overall, this work establishes a modular platform that decouples oligonucleotide synthesis from the preparation of specialised phosphoramidite reagents, providing a simpler and more flexible route to chemically diverse oligonucleotides for applications in chemical biology, molecular imaging and nucleic acid therapeutics. Importantly it greatly increases the achievable chemical diversity of modified oligonucleotides, with obvious widespread implications for therapeutics.

Ayan Dasgupta and Tom Brown, September 2026