aDim-aDmoc Protection for ODN Synthesis Allows Deprotection under Non-nucleophilic and Nearly Neutral Conditions

Abstract

Over a hundred non-canonical nucleotides have been found in DNA and RNA. Many of these modified nucleotides are sensitive toward nucleophiles and bases. Because known solid phase DNA and RNA synthesis technologies require strongly nucleophilic and basic conditions for deprotection and cleavage, there is no technology for the synthesis of DNAs and RNAs containing these sensitive nucleotides. The Dim-Dmoc technology has been developed to overcome the challenge. With Dim-Dmoc protection, oligodeoxynucleotide (ODN) deprotection has be achieved with sodium periodate oxidation followed by β-elimination induced by the weak base aniline. Some sensitive groups have been shown to be stable under the deprotection conditions. Besides serving as a base, aniline also serves as a nucleophilic scavenger for the side products of Dim-Dmoc deprotection, which prevents the side products from reacting with the deprotected ODN. For this reason, excess aniline is needed. In this article, we report the use of alkyl Dim (aDim) and alkyl Dmoc (aDmoc) protecting groups for ODN synthesis. With aDim-aDmoc protection, deprotection is demonstrated to be achievable with sodium periodate oxidation followed by the non-nucleophilic base potassium carbonate at pH 8. No scavenger for the side products of deprotection is needed. Over 10 ODNs with length ranging from 10-mer to 23-mer were synthesized, and importantly, the ODNs could be easily purified with RP HPLC. It was further demonstrated that the highly sensitive N4-acetylcytidine nucleoside could survive the oxidative deprotection conditions, and ODNs containing this sensitive nucleotide could be readily synthesized and purified without the need of any special cautions. Work on extending the method for the synthesis of sensitive RNAs such as those containing the biologically important ac4C is in progress.