Electrophilic oligodeoxynucleotide synthesis using dM-Dmoc for amino protection

Abstract

Solid-phase synthesis of electrophilic oligodeoxynucleotides (ODNs) was achieved using dimethyl-Dmoc (dM-Dmoc) as amino protecting group. Due to the high steric hindrance of the 2-(propan-2-ylidene)-1,3-dithiane side product from deprotection, the use of excess nucleophilic scavengers such as aniline to prevent Michael addition of the side product to the deprotected ODN during ODN cleavage and deprotection was no longer needed. The improved technology was demonstrated by the synthesis and characterization of five ODNs including three modified ones. The modified ODNs contained the electrophilic groups ethyl ester, α-chloroamide, and thioester. Using the technology, the sensitive groups can be installed at any location within the ODN sequences without using any sequence- or functionality-specific conditions and procedures.

Determination of Optical Density (OD) of Oligodeoxynucleotide from HPLC Peak Area

“…[1] RP HPLC was carried out under typical conditions described elsewhere. [2] UV absorbance was obtained on a Horiba Scientific Duetta Fluorescence and Absorbance Spectrometer at 260 nm in a 1 mL quartz cuvette with a 1 cm light path. Water was used as the blank.…”

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 route for the synthesis is shown in Figure 8. The synthesis has been reported previously (Shahsavari et al, 2019a;Shahsavari et al, 2019b).…”

“…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.…”

“…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).…”