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 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.

Introduction

The ability to chemically assemble oligodeoxynucleotide (ODN) on a solid support efficiently has made a broad impact in the last few decades on many research areas including molecular biology,^[1–4] chemical biology,^[4–9] synthetic biology,^[9–10] data storage,^[11–13] nanotechnology^[1,5] and medicine.^[1–6] However, the success of ODN synthesis is mainly limited to unmodified ODNs. For modified ODNs, we still face many challenges.^[14–23] In one particular case, due to the use of acyl protecting groups and linkers during solid phase synthesis and their need of strong nucleophiles for deprotection and cleavage, many nucleophile-sensitive groups could not be incorporated into this highly important class of materials. Examples of the sensitive groups include many common ones in organic chemistry such as esters, alkyl halides and epoxides. The availability of ODNs decorated with such groups could benefit many research areas including molecular biology,^[24–26] chemical biology,^[18] and antisense drug development.^[27–28] Recently, we reported a new ODN synthesis technology that can be used to synthesize such modified ODNs.^[16] We used the 1,3-dithiane based Dmoc (i.e. dimethyl-1,3-dithian-2-ylmethoxycarbonyl) phosphoramidites (1a-1c) and linker (2) for the synthesis (Figure 1). With the use of these monomers and linker, after synthesis, the ODN on solid support represented by 3 was obtained. Deprotection and cleavage were then achieved under non-nucleophilic conditions involving using the non-nucleophilic base DBU to remove the 2-cyanoethyl groups and sodium periodate to oxidize the Dmoc function to give 4. Induction of β-elimination of the oxidized Dmoc functions in 4 with aniline, of which the basicity and nucleophilicity are similar as nucleobases, gave the target ODN. Because the entire ODN synthesis, deprotection and cleavage processes did not use any nucleophiles that were appreciably stronger than nucleobases, the new technology is suitable for the synthesis of nucleophile-sensitive ODN analogs. In this paper, we further demonstrate the power of the technology by synthesizing ODNs with nucleobases decorated with sensitive ester groups including the highly sensitive phenyl esters. In addition, the synthesis of ODNs containing a sensitive 6-chloropurine nucleobase is also presented.

Figure 1.

The Dmoc phosphoramidites, linker, and cleavage and deprotection of ODNs assembled with them.

Results and Discussion

To explore the scope of the Dmoc technology for the synthesis of ODNs containing sensitive ester groups, phosphoramidite monomers 5a-5e (Figure 2) were synthesized. Compound 6 was prepared according to reported procedure (Scheme 1).^[29–30] Cross-coupling of 6 with the commercially available 7 using a palladium catalyst gave 8a.^[31] Removing the silyl protecting groups of 8a using TBAF gave 9a. Selective tritylation of the primary hydroxyl group of 9a using DMTr-Cl gave 10a. Converting 10a to the ester-containing phosphoramidite monomer 5a was achieved using the phosphitylation reagent 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite (11) and the activator diisopropylammonium tetrazolide (12).^[32]

Figure 2.

Nucleoside phosphoramidites containing sensitive nucleobases

Scheme 1.

Synthesis of phosphoramidite monomers containing sensitive ester and thioester groups

The other ester-containing phosphoramidite monomers 5b-5e were synthesized from 8a (Scheme 1).^[33] Hydrolysis of 8a under basic conditions gave the nucleoside carboxylic acid 13. Coupling 13 with the alcohols 14b-14d and the thiol 14e gave the esters 8b-8d and the thioester 8e, respectively. Removal of the silyl protecting groups of 8b-8e to give 9b-9e by TBAF was carried out at 0 °C. Performing the reaction at room temperature caused significant hydrolysis of the sensitive ester and thioester groups. The compounds 9b-9e were tritylated to give the DMTr protected nucleosides 10b-10e. Phosphitylation of 10b-10e using reagents 11-12 gave the target ester and thioester containing phosphoramidites 5b-5e.^[32]

With the sensitive phosphoramidite monomers 5a-5e, and our previously reported Dmoc phosphoramidite monomers 1a-1c and Dmoc linker 2,^[16] we had all the tools for assembling ODNs containing the sensitive ester groups. Among the ester groups, the one in 5a is a typical alkyl ester and least reactive under hydrolytic conditions compared with others. We therefore started the study by incorporating this group into ODNs. The solid phase syntheses were carried out under typical ODN synthesis conditions using 2 as the linker and solid support, 1a-1c and the commercial 5’-DMTr 2-cyanoethyl dT phosphoramidite as monomers for incorporating unmodified nucleotides, and 5a for introducing the sensitive nucleoside (Scheme 2). In the coupling step, 0.1 M solutions of 1a-1c, 5a, and dT phosphoramidite were used. Because under our non-nucleophilic ODN deprotection and cleavage conditions, the 5’-acyl groups resulted from capping with an acylation agent will survive, to make the capped failure sequences more hydrophobic and easier to be separated from the full-length sequence using RP HPLC, we used the more hydrophobic phenoxyacetic anhydride instead of acetic anhydride for capping. More details of the synthesis can be found in the experimental section.

Scheme 2.

ODN synthesis, and deprotection and cleavage under non-nucleophilic conditions

The sequences 18a1 and 18a2 (Figure 3) containing the ester group in 5a was synthesized. Before deprotection and cleavage, the structure of the ODNs can be represented as 15 (Scheme 2). Deprotection and cleavage were carried out using a three step procedure under mild non-nucleophilic conditions. First, the 2-cyanoethyl phosphate protecting groups were removed by soaking the CPG (15) in a 10% solution of DBU in acetonitrile at room temperature briefly. After washing away the base, the CPG was suspended in a 0.4 M solution of sodium periodate for about three hours at room temperature to oxidize the disulfide bonds in the Dmoc functions. These treatments converted 15 to 16. After washing away the oxidizing agent, full cleavage and deprotection to give the target ODN 17 was achieved by inducing β-eliminations of the oxidized Dmoc functions by suspending the CPG in a 3% solution of aniline at room temperature. The ODNs (18a1 and 18a2) were then precipitated from the solution with n-butanol. The crude ODNs were analyzed with RP HPLC, and the profile of 18a1 is shown in Figure 4. The major peak was collected and re-inject into RP HPLC to generate the profile of pure ODN. The pure ODN was analyzed with MALDI-TOF MS (Figure 5), and predicted molecular peak was observed. HPLC profiles and MS for 18a2 and values of OD₂₆₀ of the two ODNs are in Supporting Information.

Figure 3.

ODN sequences. M^a-f represent the sensitive modified nucleosides incorporated using phosphoramidites 5a-f, respectively.

Figure 4.

RP HPLC profiles of selected ODNs. Others can be found in Supporting Information. The minor peaks in the profiles of crude ODNs denoted with a and b are from ODNs with the sensitive ester groups hydrolyzed and aminolyzed, respectively.

Figure 5.

MALDI-TOF MS of selected ODNs. Others can be found in Supporting Information.

After successful incorporation of the relatively stable alkyl ester as that in 5a into ODN, we went further to explore the scope of the technology by incorporating more sensitive esters. The ester in 5b has an electron withdrawing methoxy group, which makes the ester more sensitive to hydrolysis. Without the need to adjust the synthesis, deprotection and cleavage conditions used for 18a1 and 18a2, the more sensitive ODNs 18b1-18b4 were prepared conveniently. RP HPLC of the crude ODNs (Supporting Information) indicated that the ester group was completely stable under the non-nucleophilic deprotection and cleavage conditions. With these encouraging results, we took a bold step forward and decided to incorporate the highly nucleophile-sensitive phenyl esters into ODNs. Among the two phenyl esters in 5c and 5d, the former has an electron donating methoxy group on the phenyl ring that makes the ester relatively more stable. Therefore we started by incorporating 5c into ODNs. Under the same synthesis, deprotection and cleavage conditions, we were able to obtain pure ODNs 18c1-18c4, and establish their identity with MALDI MS (for 18c4, see Figures 4 and 5; others are in Supporting Information). However, as indicated in the HPLC profiles of the crude ODNs, significant amount of the ODNs was hydrolyzed and aminolyzed by aniline, which was added during deprotection and cleavage to induce β-elimination of oxidized Dmoc functions and as a scavenger of the elimination alkene product.^[16,34–35] The MALDI MS of the hydrolyzed and aminolyzed products of ODNs 18c1 are provided in Supporting Information. Based on these observations, we were not very optimistic about incorporating the more sensitive electron-neutral phenyl ester in 5d into ODNs. However, we realized that we had not explored the use of conditions even milder than 3% aniline for deprotection and cleavage yet. We therefore went ahead and synthesized the ODNs 18d1-18d3 (Figure 3). Deprotection and cleavage using 3% aniline after sodium periodate oxidation indeed gave more hydrolyzed and aminolyzed products, and the yields of the target ODNs were low. To address the problem, we replaced the 3% aniline with 0.5% 4-aminobenzyl alcohol and carried out the deprotection and cleavage under otherwise identical conditions. The lower nucleophilicity and basicity of 4-aminobenzyl alcohol and lower concentration of the solution were expected to minimize the hydrolysis and aminolysis of the sensitive phenyl ester. Indeed, the results were much better. When the time interval between cleavage and HPLC and MS analyses was minimized, hydrolysis and aminolysis could be avoided as indicated by the analysis data of 18d2 (Figures 4 and 5). If the interval was long, more hydrolysis products were observed, but the yields of target ODNs were still good (Supporting Information).

After demonstrating the suitability of the Dmoc technology for incorporating sensitive esters into ODNs, we made efforts to incorporate the thioester in 5e and the electrophilic 6-chloropurine in 5f into ODNs. For thioester, even though thiols (pKa ~10.5) only have a slightly higher pKa than phenols (pKa ~10), we found that the ODNs 18e1-18e3, which contain a thioester on a nucleobase, were significantly easier to handle than those with a phenyl ester. For deprotection and cleavage, the procedure involving 3% aniline was used. Not much hydrolyzed or aminolyzed products was observed (Supporting Information). The electrophilic 6-chloropurine has been incorporated into ODNs by others before for the synthesis of mutagenic and genotoxic adducts of 1,2,3,4-diepoxybutane and DNA adenine bases.^[33] Special cautions were needed for the synthesis due to the ease of displacement of a chloride by a hydroxide or other nucleophiles through nucleophilic aromatic substitution. For this reason, their synthesis was carried out using the more labile phenoxyacetyl dA and dG phosphoramidites and cleavage and deprotection were achieved with 0.1 M sodium hydroxide at room temperature in three days. Using our technology, synthesis of ODNs containing 6-chloropurine was expected to be simple because 6-chloropurine should be much more stable than phenyl esters. Indeed, ODNs 18f1-18f3 were readily synthesized without any modification of our standard procedure using phosphoramidite 5f, which was prepared according to reported procedure.^[33] No hydrolysis or aminolysis of the ODN products were observed. Their HPLC profiles, MALDI MS and OD₂₆₀ values are in Supporting Information.

Conclusions

In conclusion, we have demonstrated that the Dmoc ODN synthesis technology is capable of synthesizing ODNs that contain nucleobases with sensitive ester and thioester groups. Using a milder condition for β-elimination in the deprotection and cleavage step involving 0.5% 4-aminobenzyl alcohol, we could even incorporate a highly sensitive phenyl ester into ODNs. In addition to esters, we also showed that using the technology, the previously reported synthesis of ODNs containing 6-chloropurine could be achieved more conveniently. Our future endeavors in the area will be to further advance the Dmoc technology and to develop complementary technologies for ODN synthesis with the ultimate goal of being able to make ODNs containing any sensitive functional groups as long as they are compatible with ODN. We expect that the availability of such materials will enable initiation of new projects in many areas including molecular biology, medicine and nanotechnology.