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Abstract This protocol describes a method for the incorporation of sensitive functional groups into oligodeoxynucleotides (ODNs). The nucleophile‐sensitive epigeneticN4‐acetyldeoxycytosine (4acC) DNA modification is used as an example, but other sensitive groups can also be incorporated, e.g., alkyl halide, α‐haloamide, alkyl ester, aryl ester, thioester, and chloropurine groups, all of which are unstable under the basic and nucleophilic deprotection and cleavage conditions used in standard ODN synthesis methods. The method uses a 1,3‐dithian‐2‐yl‐methoxycarbonyl (Dmoc) group that carries a methyl group at the carbon of the methoxy moiety (meDmoc) for the protection of exo‐amines of nucleobases. The growing ODN is anchored to a solid support via a Dmoc linker. With these protecting and linking strategies, ODN deprotection and cleavage are achieved without using any strong bases and nucleophiles. Instead, they can be carried out under nearly neutral non‐nucleophilic oxidative conditions. To increase the length of ODNs that can be synthesized using the meDmoc method, the protocol also describes the synthesis of a PEGylated Dmoc (pDmoc) phosphoramidite. With some of the nucleotides being incorporated with pDmoc‐CE phosphoramidite, the growing ODN on the solid support carries PEG moieties and becomes more soluble, thus enabling longer ODN synthesis. The ODN synthesis method described in this protocol is expected to make many sensitive ODNs that are difficult to synthesize accessible to researchers in multiple areas, such as epigenetics, nanopore sequencing, nucleic acid‐protein interactions, antisense drug development, DNA alkylation carcinogenesis, and DNA nanotechnology. © 2024 Wiley Periodicals LLC. Basic Protocol: Sensitive ODN synthesis Support Protocol 1: Synthesis of meDmoc‐CE phosphoramidites Support Protocol 2: Synthesis of a pDmoc‐CE phosphoramidite
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PEGylated Dmoc phosphoramidites for sensitive oligodeoxynucleotide synthesis
Using PEGylated Dmoc (pDmoc) phosphoramidites for oligodeoxynucleotide (ODN) synthesis increases the solubility of ODN on solid support and enables the synthesis of longer sensitive ODNs.
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- PAR ID:
- 10559323
- Publisher / Repository:
- Royal Society of Chemistry
- Date Published:
- Journal Name:
- Organic & Biomolecular Chemistry
- Volume:
- 21
- Issue:
- 45
- ISSN:
- 1477-0520
- Page Range / eLocation ID:
- 9005 to 9010
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D3OB01495A
