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Article

Synthesis of Sensitive Oligodeoxynucleotides Containing Acylated Cytosine, Adenine, and Guanine Nucleobases

by
Komal Chillar
,
Rohith Awasthy
,
Marina Tanasova
and
Shiyue Fang
*
Department of Chemistry, and Health Research Institute, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA
*
Author to whom correspondence should be addressed.
DNA 2025, 5(2), 25; https://doi.org/10.3390/dna5020025
Submission received: 24 February 2025 / Revised: 9 March 2025 / Accepted: 16 March 2025 / Published: 9 May 2025
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Abstract

:
Background/Objective: Oligodeoxynucleotides (ODNs) containing base-labile modifications such as N4-acetyldeoxycytidine (4acC), N6-acetyladenosine (6acA), N2-acetylguanosine (2acG), and N4-methyoxycarbonyldeoxycytidine (4mcC) are highly challenging to synthesize because standard ODN synthesis methods require deprotection and cleavage under strongly basic and nucleophilic conditions, and there is a lack of ideal alternative methods to solve the problem. The objective of this work is to explore the capability of the recently developed 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) method for the incorporation of multiple 4acC modifications into a single ODN molecule and the feasibility of using the method for the incorporation of the 6acA, 2acG and 4mcC modifications into ODNs. Methods: The sensitive ODNs were synthesized on an automated solid phase synthesizer using the Dmoc group as the linker and the methyl Dmoc (meDmoc) group for the protection of the exo-amino groups of nucleobases. Deprotection and cleavage were achieved under non-nucleophilic and weakly basic conditions. Results: The 4acC, 6acA, 2acG, and 4mcC were all found to be stable under the mild ODN deprotection and cleavage conditions. Up to four 4acC modifications were able to be incorporated into a single 19-mer ODN molecule. ODNs containing the 6acA, 2acG, and 4mcC modifications were also successfully synthesized. The ODNs were characterized using RP HPLC, capillary electrophoresis, gel electrophoresis and MALDI MS. Conclusions: Among the modified nucleotides, 4acC has been found in nature and proven beneficial to DNA duplex stability. A method for the synthesis of ODNs containing multiple 4acC modifications is expected to find applications in biological studies involving 4acC. Although 6acA, 2acG, and 4mcC have not been found in nature, a synthetic route to ODNs containing them is expected to facilitate projects aimed at studying their biophysical properties as well as their potential for antisense, RNAi, CRISPR, and mRNA therapeutic applications.

Graphical Abstract

1. Introduction

Acetylation of the nucleoside cytidine, which gives the N4-acetylcytidine (ac4C, Figure 1) epitranscriptomic modification, has been observed in RNAs, including tRNAs, rRNAs, mRNAs, and various regulatory RNAs. It plays a wide range of roles in biological systems. Errors related to the modification have been found to be associated with many human diseases [1,2,3,4,5]. More recently, acetylation of deoxycytidine, which gives the N4-acetyldeoxycytidine (4acC, Figure 1) epigenetic modification, has also been discovered in the DNA of Arabidopsis, rice, maize, mice, and humans. It is mainly located around transcription start sites and positively correlates with gene expression levels [6,7,8]. Several studies using synthetic oligodeoxynucleotides (ODNs) and oligoribonucleotides (ORNs) have shown that acetylation of cytosine can increase the UV melting temperature of duplex oligonucleotides (ONs) by 1–8 °C [9,10,11]. The knowledge sheds light on the mechanisms by which ac4C plays roles, such as enhancing protein synthesis efficiency in the biological system [12,13,14]. This shows the significance of the synthesis of ONs containing sensitive modifications such as ac4C and 4acC, as well as numerous others [15,16,17,18]. However, standard ON synthesis methods require deprotection and cleavage under strongly basic and nucleophilic conditions, under which ac4C, 4acC, and other sensitive groups are unstable. Although several reported methods may be used for the purpose, they have various limitations, as discussed earlier [19,20,21,22]. For example, some methods can only synthesize ONs that contain thymidine or uridine only and thus do not need nucleobase protection [9,10]. Some require UV irradiation for cleavage, which can damage ON [11]. Some are limited to the synthesis of short ONs [23,24]. Therefore, the development of practically useful methods for sensitive ON synthesis without any sequence limitations and with a broad sensitive group scope is highly significant.
Our research group recently reported base-labile ODN synthesis using the Dmoc function for linking and the meDmoc group for the protection of exo-amino groups of the nucleobases cytosine, adenine, and guanine. Using these protecting and linking strategies, ODN deprotection and cleavage were accomplished under non-nucleophilic and weakly basic conditions [25]. The method enabled us to synthesize ODN sequences containing various sensitive groups, including 4acC. In this paper, we report the synthesis of ODNs containing up to four 4acC modifications (ODNs 1a–d, Table 1), as well as those containing the N2-acetyldeoxyguanosine (2acG, Figure 1, ODN 1e), N6-acetyldeoxyadenosine (6acA, Figure 1, 1f), and N4-methoxycarbonyldeoxycytidine (4mcC, Figure 1, ODN 1g) modifications. Unlike ac4C and 4acC, the 6acA, 2acG, and 4mcC, as well as ac6A, ac2G, and mc4C modifications, have not been found in nature. Our rationale for the synthesis of ODNs containing them includes facilitating projects with aims such as evaluating the potential of the modifications for antisense, RNAi, CRISPR, and mRNA therapeutic applications, wherein the modifications may increase drug cellular stability, improve drug binding affinity and reduce off-target probability [26,27,28,29,30,31,32,33].

2. Materials and Methods

ODN synthesis, deprotection, cleavage, purification, and characterization: All ODNs were synthesized on a MerMade 6 synthesizer on the Dmoc support 2j (Figure 2, 26 µmol/g loading, 20 mg, 0.52 µmol) using phosphoramidite chemistry. Deblocking: DCA (3%, DCM), 90 s × 2. Coupling: Phosphoramidite (Figure 2, 2a–d, 2f–i, and 2e for incorporating dC, dA, dG, T, 4acC, 6acA, 2acG, 4mcC, and the T at 5′-end of ODN, respectively; 0.1 M, ACN), 4,5-dicyanoimidazole (DCI, 0.25 M, ACN), 90 s × 3. Capping: 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphoramidite (0.1 M, ACN), DCI (0.25 M, ACN), 60 s × 3. Oxidation: I2 (0.02 M, THF/pyridine/H2O, 70:20:10, v/v/v), 40 s × 3. The 5′-trityl group was kept to assist RP HPLC. The CPG (3, Scheme 1) was divided into 5 portions (~0.104 µmol ODN each). One portion was subject to deprotection and cleavage (Scheme 1). Deprotect 2-cyanoethyl groups: The suspension of CPG (3, ~0.104 µmol ODN) in the solution of DBU in ACN (1:9, v/v, 1 mL) in a 1.5 mL centrifuge tube was gently shaken at rt for 5 min. The supernatant was removed. The process was repeated two more times. The CPG was washed with ACN (1 mL × 5). This converted 3 to 4. Oxidize meDmoc and Dmoc: The suspension of 4 in the solution of NaIO4 (0.4 M, 1 mL), which has a pH of 4, in a 1.5 mL centrifuge tube was gently shaken at rt for 1.5 h. The supernatant was removed. The process was repeated two times. The CPG was washed with water (1 mL × 5). This converted 4 to 5. Remove oxidized meDmoc groups: The suspension of 5 in the solution of K2CO3 (0.05%, 1 mL), which has a pH of 8, in a 1.5 mL centrifuge tube was gently shaken at rt for 5 h. The supernatant was transferred into a 1.5 mL centrifuge tube using a pipette. The CPG was washed with water (150 μL × 5). The combined supernatant and washes were concentrated to ∼50 μL. To the solution was added nBuOH (450 μL). After mixing by vortex, ODN was precipitated via centrifugation (14.5k rpm, ~14k× g, 15 min). The supernatant was removed, leaving deprotected ODN (6) in the tube. RP HPLC purification: ODN (6) was dissolved in H2O (100 μL). A portion (35 μL) was purified with RP HPLC (see supporting information for HPLC conditions). The fractions of the Tr-on ODN were combined, and volatiles were removed under vacuum. To the ODN was added AcOH (80%, 1 mL). The mixture was shaken gently at rt for 3 h. Volatiles were evaporated. The residue was dissolved in water (100 μL), and a portion (50 μL) was purified with RP HPLC. The purified Tr-off ODN was analyzed with RP HPLC. The ODNs were quantified using a reported method [34] and analyzed with MALDI MS. The purity of the ODNs was further confirmed using capillary gel electrophoresis and PAGE.

3. Results and Discussion

The ODN syntheses were accomplished using phosphoramidites 2a–i and the linker 2j (Figure 2). Among them, 2a–c are standard meDmoc phosphoramidites, and 2j is a standard Dmoc linker. Monomers 2e, 2g–i are known compounds [10,35,36], and they were synthesized in house. The details for the synthesis of 2g–i are provided in the supporting information. Monomers 2d and 2f were purchased from commercial sources. The syntheses were carried out under conditions similar to those using standard phosphoramidite chemistry, except that capping was achieved using 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphoramidite and the nucleotide at the 5′-end was incorporated using phosphoramidite 2e. According to the trityl assay, all the phosphoramidites had similar coupling efficiency as standard commercial phosphoramidites. At the end of syntheses, the ODNs can be represented by 3 (Scheme 1), which contained a Tr group (not shown in 3) introduced by 2e. Deprotection and cleavage were achieved in three steps (Scheme 1). In the first step, the 2-cyanoethyl groups were removed by washing the CPG with 10% DBU in ACN, which converted 3 to 4. In the second step, the Dmoc and meDmoc groups were oxidized with NaIO4, converting 4 to 5. In the third step, the oxidized meDmoc and Dmoc groups were cleaved via β-elimination induced by the weak non-nucleophilic base K2CO3. This gave the fully deprotected ODN with a 5′-Tr group 6 (5′-Tr not shown). All three steps were convenient to operate because, during the first two steps, the ODNs were still on solid support. Excess reagents and side products could be removed simply by washing. For the third step, the solid support and the ODNs in the supernatant were easy to separate, and the quantity of K2CO3 was minute, which did not require to be removed before HPLC purification of the ODN. In addition, all the deprotection and cleavage reactions were carried out at room temperature, which ensured the stability of sensitive groups on the ODNs.
The ODNs were purified by precipitation with nBuOH from water to remove small organic molecules resulting from deprotection. The precipitate was then further purified with RP HPLC, which was made possible with the Tr group at the 5′-end of the ODNs. The typical DMTr group was found unstable in the NaIO4 oxidation step during ODN deprotection and cleavage. The purified Tr-on ODN was then detritylated with 80% AcOH and purified again with RP HPLC. The purity of the ODNs was evaluated by HPLC and capillary electrophoresis (CE, Figure 3), as well as polyacrylamide gel electrophoresis (supporting information). The identity of the ODNs was confirmed with MALDI MS (Figure 3). All the ODNs (1a–h), including those with modifications, once purified with RP HPLC and stored at −20 °C, were found stable for at least one day according to MALDI MS analysis and are predicted to be stable for a much longer time.
Using the Dmoc method, ODNs 1a–g (Table 1) were synthesized. The 19-mers were chosen because some oligonucleotide therapeutics are around this length, and ODNs around this length reliably give sharp peaks in MALDI MS spectra, which is important for determining if the introduced modifications are retained or lost. Among the ODNs, 1a–d contain one to four 4acC modifications, respectively. As shown in Figure 3A–D, the acetylated ODNs showed a major peak in RP HPLC profiles. Unfortunately, besides the major ODN peak, the peak for 1a had a shoulder before the major peak, and in the profiles of 1b–d, a smaller peak appeared at ~10 min. However, we believe that these were caused by non-ODN materials from the HPLC system because CE analysis of the samples gave single sharp peaks (Figure 3H–K). In addition, the samples all gave single sharp peaks in MALDI MS with predicted molecular mass (Figure 3O–R), and gel electrophoresis analysis also gave single bands (supporting information). The MS data indicate that the 4acC modification was stable under the Dmoc ODN synthesis, deprotection, and cleavage conditions. It is well known that 4acC and ac4C are highly sensitive modifications. It is remarkable that the ODN 1d, which contained four 4acC modifications densely packed in a short sequence, could be synthesized. We also made efforts to synthesize an ODN containing five 4acC modifications. However, MALDI MS indicated that the sequence was not stable as the product was found to be contaminated with a small quantity of the sequence containing only four 4acC modifications.
The ODNs 1e–f contain a 2acG and 6acA modification, respectively. As shown in Figure 3E,F, a major peak corresponding to the ODNs was observed, although the peak of 1e had a shoulder after the major peak. Again, we believe that it was caused by our HPLC system because both samples gave a single sharp peak in CE profiles (Figure 3L,M), and MALDI MS gave a single sharp peak with predicted molecular mass (Figure 3S,T). In addition, gel electrophoresis analysis gave single bands (supporting information). The 2acG and 6acA modifications have not been found in nature. However, it is possible that they behave similarly to 4acC and ac4C and may increase ON cellular stability, duplex stability, and mRNA translational efficiency. The success of their incorporation into ODN is expected to facilitate the study of these and other biophysical properties of such ODNs. It is noted that among the ODNs containing the 4acC, 2acG, and 6acA modifications, the ODN containing 2acG is the most labile. The success in synthesizing ODN 1e and purifying and analyzing it indicates that 2acG, like 4acC and 6acA, is stable enough for applications such as antisense, RNAi, CRISPR, and mRNA therapeutic development.
Besides the incorporation of acetylated nucleosides into ODNs, the ODN 1g, which contains the 4mcC modification, was also synthesized. As shown by its HPLC (Figure 3G) and CE (Figure 3N) profiles, as well as MALDI MS (Figure 3U) 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 4, 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, match 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.

4. Conclusions

In summary, using the Dmoc ODN synthesis method, we were able to synthesize sensitive ODNs containing the 6acA, 2acG, and 4mcC, as well as multiple 4acC modifications without any sequence limitations. The 4acC modification is highly sensitive, and therefore, the ability of the Dmoc method to incorporate four of them into one ODN sequence is remarkable. The 6acA, 2acG, and 4mcC modifications have not been found in nature. However, they may behave similarly to 4acC and ac4C in terms of benefits to DNA and RNA duplex stability and may find applications such as antisense, RNAi, CRISPR, and mRNA therapeutics. We expect that the demonstration of their incorporation into ODNs in the present work will facilitate projects aimed at studying their biophysical properties and potential for therapeutic applications.

5. Patent

Sensitive Oligonucleotide Synthesis Using Sulfur-Based Functions as Protecting Groups and Linkers; U.S. Application # 16/946,455; Application Date 23 June 2020; Issuing Date 6 December 2022; Patent Number 11,518,780.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/dna5020025/s1. Experimental details and gel electrophoresis image of ODNs [37,38,39,40,41].

Author Contributions

Conceptualization, S.F.; investigation, K.C., R.A., and M.T.; resources, S.F. and M.T.; writing—original draft preparation, S.F. and K.C.; writing—review and editing, S.F.; supervision, S.F.; project administration, S.F.; funding acquisition, S.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by US NIH, GM109288, and US NSF 1954041.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

Financial support from Robert and Kathleen Lane Endowed Fellowship (K.C.), Michigan Tech Health Research Institute Fellowship (K.C.), Fleming-Skochelak Fellowship (K.C.), and Doctoral Finishing Fellowship (K.C.); assistance from D.W. Seppala (electronics), S. Liu (NMR), Z. Song (MS), and A. Galerneau (MS); and NSF equipment grants (2117318 for NMR, 1048655 and 1531454 for MS); are gratefully acknowledged.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
2acGN2-acetylguanosine
4acCN4-acetyldeoxycytidine
4mcCN4-methyoxycarbonyldeoxycytidine
6acAN6-acetyladenosine
ACNAcetonitrile
CECapillary electrophoresis
CPGControlled pore glass
DBU1,8-Diazabicyclo(5.4.0)undec-7-ene
DCADichloroacetic acid
DCI4,5-Dicyanoimidazole
DCMDichloromethane
Dmoc1,3-dithian-2-yl-methoxycarbonyl
DMTrDimethoxytrityl
meDmoc Methyl Dmoc
ODNOligodeoxynucleotide
ONOligonucleotide
ORNOligoribonucleotide
TrTrityl

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Figure 1. Structure of 4acC, ac4C, 2acG, 6acA, and 4mcC.
Figure 1. Structure of 4acC, ac4C, 2acG, 6acA, and 4mcC.
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Figure 2. Phosphoramidites and linker for sensitive ODN synthesis using the meDmoc method.
Figure 2. Phosphoramidites and linker for sensitive ODN synthesis using the meDmoc method.
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Scheme 1. Deprotection and cleavage of ODNs synthesized using meDmoc phosphoramidites and Dmoc linker. The “b” in 3–6 represents the nucleobase in 2a–j except that in the cases of 2d–e and 2j, there is no meDmoc protected exo-amino group, in the cases of 2f–h, the exo-amine is protected with an acetyl group instead of meDmoc group, and in the case of 2i, the exo-amine is protected with a methoxycarbonyl (mc) group. The “Tr” group at the 5′-end of 3–6, which is introduced with 2e, is not shown. Conditions for deprotection and cleavage of 3: (1) 10% DBU in ACN, rt, 15 min. (2) 0.4 M NaIO4, pH 4, rt, 4.5 h. (3) 0.05% K2CO3, pH 8, rt, 5 h.
Scheme 1. Deprotection and cleavage of ODNs synthesized using meDmoc phosphoramidites and Dmoc linker. The “b” in 3–6 represents the nucleobase in 2a–j except that in the cases of 2d–e and 2j, there is no meDmoc protected exo-amino group, in the cases of 2f–h, the exo-amine is protected with an acetyl group instead of meDmoc group, and in the case of 2i, the exo-amine is protected with a methoxycarbonyl (mc) group. The “Tr” group at the 5′-end of 3–6, which is introduced with 2e, is not shown. Conditions for deprotection and cleavage of 3: (1) 10% DBU in ACN, rt, 15 min. (2) 0.4 M NaIO4, pH 4, rt, 4.5 h. (3) 0.05% K2CO3, pH 8, rt, 5 h.
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Figure 3. RP HPLC, capillary electrophoresis (CE), and MALDI MS of ODNs 1a–g. (A–G) RP HPLC of ODNs 1a–g, respectively. The peak at ~22 min is from the ODN. In some of the profiles, there is a peak at 10 min or a shoulder at the ODN peak. They are from non-ODN materials likely from the HPLC system because the CE, MALDI MS, and gel electrophoresis (see supporting information) analyses all indicate that the ODNs are pure. (H–N) CE profiles of ODNs 1a–g, respectively. (O) MS of ODNs 1a, calcd [M−H]− 5753.0, found 5752.9. (P) MS of ODNs 1b, calcd [M+H]+ 5796.0, found 5797.2. (Q) MS of ODNs 1c, calcd [M+H]+ 5839.0, found 5839.2. (R) MS of ODNs 1d, calcd [M−H]− 5879.0, found 5878.4. (S) MS of ODNs 1e, calcd [M−H]− 5753.0, found 5750.0. (T) MS of ODNs 1f, calcd [M−H]− 5753.0, found 5750.7. (U) MS of ODNs 1g, calcd [M−H]− 5769.0, found 5767.9.
Figure 3. RP HPLC, capillary electrophoresis (CE), and MALDI MS of ODNs 1a–g. (A–G) RP HPLC of ODNs 1a–g, respectively. The peak at ~22 min is from the ODN. In some of the profiles, there is a peak at 10 min or a shoulder at the ODN peak. They are from non-ODN materials likely from the HPLC system because the CE, MALDI MS, and gel electrophoresis (see supporting information) analyses all indicate that the ODNs are pure. (H–N) CE profiles of ODNs 1a–g, respectively. (O) MS of ODNs 1a, calcd [M−H]− 5753.0, found 5752.9. (P) MS of ODNs 1b, calcd [M+H]+ 5796.0, found 5797.2. (Q) MS of ODNs 1c, calcd [M+H]+ 5839.0, found 5839.2. (R) MS of ODNs 1d, calcd [M−H]− 5879.0, found 5878.4. (S) MS of ODNs 1e, calcd [M−H]− 5753.0, found 5750.0. (T) MS of ODNs 1f, calcd [M−H]− 5753.0, found 5750.7. (U) MS of ODNs 1g, calcd [M−H]− 5769.0, found 5767.9.
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Figure 4. MALDI MS of ODNs 1a–b and 1e–g with ODN 1h as an internal standard to indicate that the acyl groups in 4acC and 4mcC were stable under the ODN synthesis, deprotection, and cleavage conditions. (A) ODN 1a, calcd [M−H]− 5753.0, found 5752.8. ODN 1h, calcd [M−H]− 5711.0, found 5710.8. The found mass difference of 42.0 matches 42.0 of the acetyl group (C2H2O). The peak at 5732.3 is from the sodium adduct of 1h. (B) ODN 1b, calcd [M+H]+ 5796.0, found 5796.1. ODN 1h, calcd [M+H]+ 5712.0, found 5711.3. The found mass difference of 84.8 matches 84.0 of the two acetyl groups. The peak at 5752.5 is from the potassium adduct of 1h rather than 1b losing an acetyl group because 1h also gave the potassium adduct peak 5837.1, and in the MS of pure 1b, no corresponding peak was observed. (C) ODN 1e, calcd [M−H]− 5753.0, found 5750.1. ODN 1h, calcd [M−H]− 5711.0, found 5709.0. The found mass difference of 41.1 matches 42.0 of the acetyl group. (D) ODN 1f, calcd [M−H]− 5753.0, found 5751.6. ODN 1h, calcd [M−H]− 5711.0, found 5710.2. The found mass difference of 41.4 matches 42.0 of the acetyl group. (E) ODN 1g, calcd [M−H]− 5769.0, found 5768.0. ODN 1h, calcd [M−H]− 5711.0, found 5709.5. The found mass difference of 58.5 matches 58.0 of the methoxycarbonyl group (C2H2O2). The additional minor peaks are from sodium and potassium adducts.
Figure 4. MALDI MS of ODNs 1a–b and 1e–g with ODN 1h as an internal standard to indicate that the acyl groups in 4acC and 4mcC were stable under the ODN synthesis, deprotection, and cleavage conditions. (A) ODN 1a, calcd [M−H]− 5753.0, found 5752.8. ODN 1h, calcd [M−H]− 5711.0, found 5710.8. The found mass difference of 42.0 matches 42.0 of the acetyl group (C2H2O). The peak at 5732.3 is from the sodium adduct of 1h. (B) ODN 1b, calcd [M+H]+ 5796.0, found 5796.1. ODN 1h, calcd [M+H]+ 5712.0, found 5711.3. The found mass difference of 84.8 matches 84.0 of the two acetyl groups. The peak at 5752.5 is from the potassium adduct of 1h rather than 1b losing an acetyl group because 1h also gave the potassium adduct peak 5837.1, and in the MS of pure 1b, no corresponding peak was observed. (C) ODN 1e, calcd [M−H]− 5753.0, found 5750.1. ODN 1h, calcd [M−H]− 5711.0, found 5709.0. The found mass difference of 41.1 matches 42.0 of the acetyl group. (D) ODN 1f, calcd [M−H]− 5753.0, found 5751.6. ODN 1h, calcd [M−H]− 5711.0, found 5710.2. The found mass difference of 41.4 matches 42.0 of the acetyl group. (E) ODN 1g, calcd [M−H]− 5769.0, found 5768.0. ODN 1h, calcd [M−H]− 5711.0, found 5709.5. The found mass difference of 58.5 matches 58.0 of the methoxycarbonyl group (C2H2O2). The additional minor peaks are from sodium and potassium adducts.
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Table 1. ODN sequences, OD260, and mass data.
Table 1. ODN sequences, OD260, and mass data.
EntryODNSequencesOD260 [a]MALDI MS
CalculatedFound
11a5′-TAGTA4acCTTTATCCAACCTT-3′1.13[M−H]− 5753.05752.9
21b5′-TAGTACTTTAT4acCCAA4acCCTT-3′1.04[M+H]+ 5796.05797.2
31c5′-TAGTA4acCTTTAT4acCCAA4acCCTT-3′0.53[M+H]+ 5839.05839.2
41d5′-TAGTA4acCTTTAT4acCCAA4acC4acCTT-3′0.37[M−H]− 5879.05878.4
51e5′-TA2acGTACTTTATCCAACCTT-3′3.22[M−H]− 5753.05750.0
61f5′-TAGT6acACTTTATCCAACCTT-3′1.07[M−H]− 5753.05750.7
71g5′-TAGTA4mcCTTTATCCAACCTT-3′1.84[M−H]− 5769.05767.9
81h5′-TAGTACTTTATCCAACCTT-3′-[M−H]− 5711.0-
[a] Values were based on 0.52 μmol ODN synthesis.
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Chillar, K.; Awasthy, R.; Tanasova, M.; Fang, S. Synthesis of Sensitive Oligodeoxynucleotides Containing Acylated Cytosine, Adenine, and Guanine Nucleobases. DNA 2025, 5, 25. https://doi.org/10.3390/dna5020025

AMA Style

Chillar K, Awasthy R, Tanasova M, Fang S. Synthesis of Sensitive Oligodeoxynucleotides Containing Acylated Cytosine, Adenine, and Guanine Nucleobases. DNA. 2025; 5(2):25. https://doi.org/10.3390/dna5020025

Chicago/Turabian Style

Chillar, Komal, Rohith Awasthy, Marina Tanasova, and Shiyue Fang. 2025. "Synthesis of Sensitive Oligodeoxynucleotides Containing Acylated Cytosine, Adenine, and Guanine Nucleobases" DNA 5, no. 2: 25. https://doi.org/10.3390/dna5020025

APA Style

Chillar, K., Awasthy, R., Tanasova, M., & Fang, S. (2025). Synthesis of Sensitive Oligodeoxynucleotides Containing Acylated Cytosine, Adenine, and Guanine Nucleobases. DNA, 5(2), 25. https://doi.org/10.3390/dna5020025

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