Incorporation of Sensitive Ester and Chloropurine Groups into Oligodeoxynucleotides through Solid‐Phase Synthesis

Abstract

Nucleosides containing ester groups that are sensitive to nucleophiles were incorporated into oligodeoxynucleotides (ODNs) through solid phase chemical synthesis. The sensitive esters are located on a purine nucleobase. They are the esters of ethyl, 2‐methoxyethyl, 4‐methoxyphenyl and phenyl groups, and a thioester. These esters cannot survive the deprotection and cleavage conditions used in known ODN synthesis technologies, which involve strong nucleophiles such as ammonium hydroxide and potassium methoxide (potassium carbonate in anhydrous methanol). To incorporate these sensitive groups into ODNs, the Dmoc (i. e. dimethyl‐1,3‐dithian‐2‐ylmethoxycarbonyl) phosphoramidites and linker were used for solid phase synthesis, which allowed ODN deprotection and cleavage to be carried out under non‐nucleophilic oxidative conditions. Sixteen ODN sequences containing these groups were synthesized and characterized with MALDI MS. In addition, the synthesis and characterization of three ODNs containing a nucleophile sensitive 6‐chloropurine using the same strategy are described.

Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis

“…Under these nearly neutral and non-nucleophilic deprotection and cleavage conditions, a wide range of sensitive groups such as esters, thioesters, activated amides, N-aryl amides, alkyl halides, and α-chloroamides can survive, and therefore, the Dim-Dmoc ODN synthesis technology is well suited for the synthesis of sensitive ODNs. The technology has been demonstrated for the synthesis of ODNs that contain sensitive alkyl esters, phenyl esters, thioesters, alkyl chlorides, and 6-chloropurine (Halami et al, 2018;Lin et al, 2016;Shahsavari et al, 2019a;Shahsavari et al, 2019b). The sensitive ODNs were purified with trityl-on RP HPLC and were characterized with MALDI-TOF MS and enzymatic digestion assay.…”

“…The Dim-Dmoc technology described in this protocol uses Dmoc for the protection of the exo-amino groups of dA, dC, and dG; Dim for the protection of the hydroxyl group of the phosphoramidous acid of all the four nucleoside phosphoramidites; and Dmoc for linking the nascent ODN to solid support ( Fig. 1; Halami et al, 2018;Lin et al, 2016;Shahsavari et al, 2019a;Shahsavari et al, 2019b). Using the technology, the ODN synthesized has the structure represented by 3 (Fig.…”

“…An alternative to avoid aniline evaporation is to use 4-aminobenzyl alcohol. 4-Aminobenzyl alcohol has a higher boiling point (171°C at 11 mm Hg) and is less likely to be evaporated (Halami et al, 2018). Precipitation of ODN by n-BuOH from aqueous solution is usually easy to do but when ODN synthesis yield is low, reducing the volume of solvents is important.…”

“…The new ODN synthesis technology described in this article aims to solve the problem (Halami et al, 2018;Lin et al, 2016;Shahsavari et al, 2019a;Shahsavari et al, 2019b). In this technology, the exo-amino groups of dA, dC, and dG are protected with 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) and the hydroxyl group of the phosphoramidous acid in dA, dC, dG, and dT phosphoramidites, which are later converted to the phosphate diester linkage of the ODN, is protected with 1,3-dithian-2-yl-methyl (Dim).…”

Electrophilic oligodeoxynucleotide synthesis using dM-Dmoc for amino protection

“…To develop a universal technology for the synthesis of ODNs that contain a wide variety of sensitive functionalities, we recently reported the use of 1,3-dithian-2-ylmethoxycabonyl (Dmoc) as protecting groups and linkers for ODN synthesis [40–41]. Due to the low acidity of H-2 (p K a ≈31) in the Dmoc function, these groups and linkers were expected to be stable under ODN synthesis conditions.…”