Besides incorporation of acetylated nucleosides into ODNs, the ODN 1g, which contains the 4mcC modification was also synthesized. As shown by its HPLC (Figure ) and CE (Figure ) profiles, as well as MALDI MS (Figure ) and gel electrophoresis analysis (supporting information), this modification is also stable under the Dmoc DNA synthesis, deprotection and cleavage conditions. This finding is predictable because the electron density of the carbonyl carbon of 4mcC should be higher than that of 4acC. Like 2acG and 6acA, 4mcC has not been found in nature, and probably does not exist in nature. However, ODNs containing it may be useful for therapeutic development and other applications. To further confirm that the acetyl and methoxycarbonyl groups in ODNs 1a-g did not fall off from the ODNs during ODN synthesis, deprotection, cleavage, purification and analysis, the ODNs 1a-b and 1e-g were further analyzed with MALDI MS using the ODN 1h, which has the same sequence as 1a-g but does not have any modifications, as an internal standard. As shown in Figure , the mass differences of ODNs 1a-b and 1e-g, which contain one or more modifications, from the internal standard 1h, which are 42.0, 84.8, 41.1, 41.4, and 58.5, respectively, matches the predicted values 42.0, 84.0, 42.0, 42.0, and 58.0. This unambiguously confirms that the Dmoc ODN synthesis method is suitable for the synthesis of sensitive ODNs containing the modifications 4acC, 2acG, 6acA, and 4mcC.