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Preparation of a disulfide-linked precipitative soluble support for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks

Summary The preparation of a disulfide-tethered precipitative soluble support and its use for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks is described. To obtain the building blocks, N-acyl protected 2´-deoxy-5´-O-(4,4´-dimethoxytrityl)ribonucleosides were phosphorylated with bis(benzotriazol-1-yl) 2-chlorophenyl phosphate. The “outdated” phosphotriester strategy, based on coupling of PV building blocks in conjunction with quantitative precipitation of the oligodeoxyribonucleotide with MeOH is applied. Subsequent release of the resulting phosphate and base-protected oligodeoxyribonucleotide trimer 3’-pTpdCBzpdGibu-5’ as its 3’-(2-chlorophenyl phosphate) was achieved by reductive cleavage of the disulfide bond.


Introduction
Synthetic nucleic acids have been used to regulate gene expression through different mechanisms of action, such as antisense oligonucleotides [1,2], ribozymes [3], interfering RNAs (siRNA) [4,5] and immunostimulatory CpG [6] based therapeutics. At the same time, the interest in detailed understanding of the factors that govern the interaction of nucleic acids with small molecular entities and other biopolymers has increased. In particular, for NMR spectroscopic studies of such interactions, oligonucleotides are often required in quantities that are inconvenient to prepare by laboratory scale solid-phase synthesis. We have previously reported that short oligonucleotides may be conveniently prepared in hundreds of milligrams scale on a soluble tetrakis-O- [4-(1,2,3-triazol-1-yl)methylphenyl]pentaerythritol support that precipitates quantitatively from MeOH [7][8][9][10]. For example, the "outdated" phosphotriester strategy [11][12][13][14], exploiting 3´-(2-chlorophenyl phosphate) building blocks works well on this support [8]. No oxidation step is needed and the coupling cycle, hence, contains only two steps: 5´-deprotection and coupling. To prepare long sequences, it may, however, be necessary to apply convergent solution-phase coupling of oligomeric 3´-(2-chlorophenyl phosphate) building blocks [15]. For the preparation of such building blocks, a 2-hydroxyethyldisulfanyl functionalized support may in principle be used [16][17][18][19][20][21]. After completion of the chain assembly on the hydroxy function, the disulfide linkage may be reductively cleaved and the phosphate bound 2-mercaptoethyl group is removed. Accordingly, the oligomer expectedly is released in a fully protected form. We now report on the synthesis of such a soluble support, 3, and show that it allows efficient coupling by the 1-hydroxybenzotriazole promoted phosphotriester coupling [11,22,23] and efficient purification of the support bound product by precipitation from MeOH after each deprotection and coupling step.
A trimeric oligodeoxyribonucleotide containing three different 2'-deoxyribonucleosides was assembled on support 3 as depicted in Scheme 3. The couplings were carried out essentially as described previously [8]. Accordingly, the dried support was treated under nitrogen with the thymidine derived building block 4 (2 equiv per support-bound OH), in dioxane in the presence of 1-methylimidazole. The coupling was completed in 12 h, and the excess of 4 and the coupling reagents were removed by precipitating the support with MeOH to obtain 7a. According to TLC analysis, the precipitation was quantitative. Detritylation of 7a with HCl in a 1:1 mixture of MeOH and DCM, followed by neutralization with pyridine, concentration to oil and precipitation from MeOH, afforded 7b.
HPLC-analysis verified the completeness of precipitation ( Figure 1).
Building blocks 5 and 6 were then coupled similarly to obtain 9 (Scheme 3). The identity and homogeneity of the product were verified by ESIMS and HPLC after each coupling and 5´-depro-  tection step. The MS data are given in Table 1. Figure 2 and Figure 3 show as an illustrative example the HPLC traces for 8a, 8b and 9 precipitated from MeOH and the filtrate of precipitation. As seen, the precipitation is virtually quantitative.
The trimer prepared was then released from the support by cleaving the disulfide linkage by TCEP reduction. The phosphate bound 2-mercaptoethyl group was removed spontaneously giving the oligonucleotide trimer expectedly as a in fully Table 1: ESIMS characterization of the support-bound nucleotides indicated in Scheme 3.
General procedure for detritylation: The detritylation cycle was analogous to that described previously [8].Tetravalent support-bound thymidine monomer 7a (0.048 mmol, 0.20 g) was dissolved in a mixture of DCM and MeOH (1:1 v/v, 25 mL), and HCl in MeOH (0.115 mL of 1.25 mol L −1 solution) was added portion wise. The reaction was monitored by TLC (system A). Once completed, the reaction mixture was neutralized with pyridine (1 mL), and the liquid was concentrated. The resulting oil was dissolved in DCM/MeOH (1:1, 3 mL), and MeOH was added (40 mL). The precipitate formed was kept at -20 °C overnight, collected by centrifugation and dried to give the product 7b (0.120 g, 85%) as a white solid. The precipitate and supernatant were analyzed by HPLC (Figure 1) and the precipitate by ESIMS (Table 1).

Cleavage from tetravalent soluble support:
To the solution of support-bound trimer 9 (0.0061 mmol, 0.045 g) in MeOH (1 mL), triethylamine (1.44 mmol, 0.2 mL) and tris(2carboxyethyl)phosphine (0.027 mmol, 0.008 g), stirred for 3 h, and then volatiles were removed under reduced pressure. The residue was stirred with CH 3 CN (3 mL) for 15 min and the precipitated support was removed by filtration, the filtrate was concentrated and dried in vacuo to give a yellow oil. The oily residue was purified by semi-preparative HPLC to afford the phosphate protected trimer oligodeoxyribonucleotide 10 (20 mg, 57%