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The stepwise synthesis of monodisperse polyethylene glycols (PEGs) and their derivatives usually involves using an acid-labile protecting group such as DMTr and coupling the two PEG moieties together under basic Williamson ether formation conditions. Using this approach, each elongation of PEG is achieved in three steps – deprotection, deprotonation and coupling – in two pots. Here, we report a more convenient approach for PEG synthesis featuring the use of a base-labile protecting group such as the phenethyl group. Using this approach, each elongation of PEG can be achieved in two steps – deprotection and coupling – in only one pot. The deprotonation step, and the isolation and purification of the intermediate product after deprotection using existing approaches are no longer needed when the one-pot approach is used. Because the stepwise PEG synthesis usually requires multiple PEG elongation cycles, the new PEG synthesis method is expected to significantly lower PEG synthesis cost. 

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