Acetylated and Methylated β-Cyclodextrins as Viable Soluble Supports for the Synthesis of Short 2′-Oligodeoxyribo-nucleotides in Solution

Novel soluble supports for oligonucleotide synthesis 11a–c have been prepared by immobilizing a 5′-O-protected 3′-O-(hex-5-ynoyl)thymidine (6 or 7) to peracetylated or permethylated 6-deoxy-6-azido-β-cyclodextrins 10a or 10b by Cu(I)-promoted 1,3-dipolar cycloaddition. The applicability of the supports to oligonucleotide synthesis by the phosphoramidite strategy has been demonstrated by assembling a 3′-TTT-5′ trimer from commercially available 5′-O-(4,4′-dimethoxytrityl)thymidine 3′-phosphoramidite. To simplify the coupling cycle, the 5′-O-(4,4′-dimethoxytrityl) protecting group has been replaced with an acetal that upon acidolytic removal yields volatile products. For this purpose, 5′-O-(1-methoxy-1-methylethyl)-protected 3′-(2-cyanoethyl-N,N-diisopropyl-phosphoramidite)s of thymidine (5a), N4-benzoyl-2′-deoxycytidine (5b) and N6-benzoyl-2′-deoxyadenosine (5c) have been synthesized and utilized in synthesis of a pentameric oligonucleotide 3′-TTCAT-5′ on the permethylated cyclodextrin support 11c.


Introduction
The methods currently applied to preparation of oligonucleotides in the laboratory [1,2] as well as in large-scale production [3][4][5], consist of stepwise addition of nucleoside phosphoramidites to a growing oligomer chain on a solid support. While solid-supported phosphoramidite chemistry OPEN ACCESS undoubtedly is the method of choice for small-scale synthesis of oligonucleotides, solution phase synthesis may well challenge it in cases where multikilogram quantities are needed for clinical phase trials of therapeutic oligonucleotides or as starting materials for the construction of nanomaterials. To ensure efficient coupling on a solid support, the expensive phosphoramidite monomers have to be used in large excess and this requirement tends to become even more stringent in scaling up the synthesis [6]. In addition, the support itself is expensive, comprising one third of the overall raw material cost [4]. For these reasons, several strategies for the assembly of oligonucleotides on soluble supports have been suggested. Most of these are based on use of polyethylene glycol (PEG) as a support. PEG allows coupling in MeCN and may be precipitated by Et 2 O. This protocol has been applied to the synthesis of both oligonucleotides and their phosphoromonothioate analogs by the phosphoramidite [7][8][9][10][11], H-phosphonate [12], and phosphotriester [13,14] strategies. More recently, the 3′-O-(adamantan-1yl)acetyl group [15] and 3′-O-succinyl-tethered 1-ethyl-3-methylimidazolium tetrafluoroborate salt [16] have been used as soluble supports. The (adamantan-1-yl)acetyl group allows extractive work-up of the growing oligonucleotide chain that, besides conventional benzoyl and isobutyryl protections, additionally bears a pivaloyloxymethyl group at N3 of the thymidine residues [15]. The imidazolium tetrafluoroborate tagged oligomers have, in turn, been separated from small molecular reagents by successive precipitations and extractions. As a complementary approach, solid-supported reagents have been exploited to synthesize oligonucleotides in solution [17,18].
We now report on an alternative approach based on the use of fully protected β-cyclodextrin as a soluble support and replacement of the conventional 5′-O-(4,4′-dimethoxytrityl) protecting group with an acetal that upon removal gives only volatile products. The cyclodextrin support is sufficiently small to allow efficient coupling and accurate mass spectrometric analysis, but it still is hydrophobic enough so that the separation of the growing oligonucleotide chain from all the reagents can be rapidly realized by flash chromatography with limited solvent consumption. On using methylated cyclodextrin, the support may be removed by extraction after the ammonolytic release and deprotection of the oligonucleotide.

Preparation of Cyclodextrin-Derived Supports
The soluble supports utilized in the oligonucleotide chain assembly were prepared by immobilizing 5′-protected 3′-O-(hex-5-ynoyl)thymidines 6 or 7 on an azido-functionalized peracetylated or permethylated β-cyclodextrin (Scheme 2). For this purpose, β-cyclodextrin was first monotosylated to 8 [19] and the tosyl group was displaced with azide ion. The azido-functionalized cyclodextrin 9 obtained was then acetylated with excess of acetic anhydride in pyridine, yielding 10a, or methylated to 10b with an equimolar mixture of sodium hydroxide and methyl iodide in DMSO [20,21]. The alkyne-functionalized thymidines, either 6 or 7, were finally conjugated to the azido group by Cu(I) promoted 1,3-dipolar cycloaddition [22,23]. The identity and homogeneity of the products before and after conjugation were checked by ESI-MS and HPLC. The data referring to acetylated and methylated supports 11a and 11c after removal of the 5′-O-protecting group from thymidine is given in Figure 1.

Assembly of Oligonucleotides from 5′-O-(4,4′-Dimethoxytrityl) Building Blocks
The efficiency of phosphoramidite coupling on a cyclodextrin support was first tested by using commercially available 5′-(4,4′-dimethoxytrityl)thymidine 3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite) building blocks for coupling on the peracetylated support 11b. Accordingly, the DMTr group of 11C was removed by 1 h treatment with 3% dichloroacetic acid in DCM, a DCM/HCO 3 − -workup was carried out and the organic phase was evaporated to dryness. Phosphoramidite coupling was then carried out in MeCN (1 h at r.t.), using 1.5 equiv. of the phosphoramidite building block and tetrazole compared to support-bound thymidine. The coupling cycle was completed by conventional I 2 oxidation in aqueous lutidine/THF and subsequent DCM/HSO 3 − -workup. After the second similar coupling step, the overall yield of the support bound fully protected thymidine trimer determined by UV-spectroscopy was 85%, corresponding to 92% coupling yield. The homogeneity of the product was verified by RP-HPLC after ammonolytic release from the support (HPLC trace A in Figure 2). For support A, a linear gradient elution from MeCN 50% at t = 0 to MeCN 100% at t = 20 min. For support B, a linear gradient from MeCN 0% at t = 0 to MeCN 60% at t = 25 min, and then to MeCN 100% at t = 30 min.

Assembly of Oligonucleotides from 5′-O-(1-Methoxy-1-methylethyl) Building Blocks
Although the initial studies described above clearly show that high coupling yield may be achieved on a cyclodextrin support even when using only 50% excess of the monomeric building block, the protocol utilized do not allow synthesis of longer sequences, owing to accumulation of 4,4′-dimethoxytrityl alcohol upon repeated couplings. Removal of this compound from the reaction mixture prior to next coupling is difficult, since the support bearing the protected oligonucleotide and dimethoxytrityl alcohol both are rather hydrophobic and, hence, removal by extraction or flash chromatography is not straightforward. For this reason, it appeared attractive to replace the conventional dimethoxytrityl protection with an acid-labile acetal protection, viz. with 1-methoxy-1-methylethyl A B group, which upon acid-catalyzed transesterification with MeOH was released as volatile dimethyl acetal of acetone. As seen from Figure 3, removal of the 5′-protecting group by HCl in a 2:1 (v/v) mixture of 1,4-dioxane and MeOH was sufficiently faster than depurination of N6-benzoyl-2′-deoxyadenosine to allow virtually quantitative removal of the acetal protection without appreciable depurination. The synthesis of 3′-TTT-5′ was then repeated using 11a as a support and 5a as a building block. The 5′-O-(1-methoxy-1-methylethyl) protection was first removed by 30 min treatment with HCl (0.1 mol·L −1 ) in a 2:1 mixture of dioxane and MeOH. The volatiles were removed under reduced pressure and traces of MeOH were removed by coevaporation with dry pyridine. It is worth noting that traces of pyridine in the product do not harm the subsequent coupling.  Deprotected 11a was then subjected to coupling with 1.5 equiv. of 5a in MeCN under nitrogen, using 4,5-dicyanoimidazole as an activator (6 h at r.t.), and the phosphite triester obtained was oxidized by addition of I 2 in aq. lutidine/THF (30 min at r.t.). Upon DCM/HSO 3 − -workup, lutidine was partly transferred to the organic phase and, hence, the soluble-supported product obtained by evaporation of DCM still contained traces of lutidine. The residue was dissolved in a 2:1 mixture of dioxane and MeOH and the pH of the solution was adjusted between 4 and 5 to remove the 5′-acetal protection and the reaction was allowed to proceed for 1 h. To remove the traces of reagents and the products formed from the unreacted building block upon oxidation, the support was purified by flash chromatography on a short silica gel column before the next coupling. The overall yield after two couplings, determined by UV-spectroscopy, was 87%, corresponding to 93% coupling yield. The trimer released by ammonolysis was homogeneous by RP-HPLC (trace B in Figure 2).
The shortcoming of acetylated cyclodextrin as a support is that it does not withstand ammonolysis, but is converted to hydrophilic products that render the isolation of the oligonucleotide product difficult, necessitating reversed phase HPLC purification. To avoid this, the acetylated cyclodextrin support 10a was replaced with the methylated one, 11c, and the synthesis of 3′-TTT-5′ was repeated. The yield of the synthesis and the homogeneity of the product were similar to those observed for the acetylated support, but now the support could be removed by extraction after the ammonolysis. The HPLC traces of the crude product mixture are shown in Figure 2C.
Finally a hetero-sequence containing 2′-deoxyadenosine and 2′-deoxycytidine in addition to thymidine was assembled on support 11c. Accordingly, the deprotected support bearing the first nuclesoide was dissolved in MeCN and 1.5 equiv. of the monomeric building block in MeCN was added dropwise, followed by 1.5 equiv. of 4,5-dicyanoimidazole in MeCN. The final concentration of 5a in the reaction mixture was 40 mmol L −1 . The reaction was allowed to proceed for 20 h at r.t. after which oxidation with iodine in a 6:1 (v/v) mixture of aq. THF and 2,6-lutidine was carried out (45 min). After aq. NaHSO 3 /DCM work-up, the organic phase was dried over Na 2 SO 4 , filtered and evaporated to dryness under reduced pressure. The residue was dissolved in a 2:1 (v/v) mixture of dioxane and MeOH and HCl in dioxane (0.1 mol·L −1 ) was added to adjust the pH to 4. After 2 h agitation, the mixture was evaporated to dryness and coevaporated with pyridine and MeCN. A rapid column chromatography on a short column was applied to remove the excess of the unreacted building block. Fractions containing the desired product were mixed, evaporated by under reduced pressure and dried under high vacuum. The identity of the support bound dimer was verified by ESI-MS. The same coupling cycle was then repeated by using 5b, 5c and again 5a as building blocks. However, the coupling mixture contained about 20% DCM to ensure complete dissolution of the cyclodextrin-bound starting material. Finally, conventional ammonolysis was carried out to release and deprotect the pentameric oligonucleotide and the support was removed by equilibration between water and DCM. The gravimetrically determined overall yield of the pentamer was 52%, corresponding to 85% average coupling yield. Figure 4 shows the identity and homogeneity of the pentamer.

General
Reactions were monitored by TLC (Merck, Silica gel 60 F 254 ), using short wavelength UV, KMnO 4 staining or burning with 10% aq. H 2 SO 4 for detection. 1D and 2D NMR spectra were recorded on a Bruker Avence 500 MHz or 400 MHz at 25 °C. The chemical shifts are given in ppm. Mass spectra were recorded on a Bruker Daltonics MicrOTOF-Q spectrometer using ESI ionization. RP HPLC was performed on a Thermo ODS Hypersil C18 (250 × 4.6 mm, 5µm) column using UV detection at 260 nm.

3′-O-(tert-Butyldimethylsilyl)thymidine (2a)
. A 1:1:4 (v/v/v) mixture of TFA, water and THF (30 mL) was added dropwise on an ice-bath to compd. 1a dissolved in a minimal volume of THF. After 1.5 h, another portion (30 mL) of the same mixture was added. The progress of desilylation was monitored by TLC and the reaction was quenched after 5.5 h by equilibration between diethyl ether and aq. NaHCO 3 (sat). The organic phase was washed with aq. NaHCO 3 , dried over Na 2 SO 4 and concentrated to white solid foam (81%, 10.38 g). The 1 H-NMR spectrum (500 MHz, CDCl 3 ) was identical to that reported previously [28]. 13 Compd. 1b (1.31 mmol, 0.98 g) was dissolved in DCM (5 mL) containing 3% dichloroacetic acid (150 μL) and MeOH (2 mL) was added. After 2 h stirring at r.t., DCM/aq. NaHCO 3 work-up was carried out and the organic phase was dried over Na 2 SO 4 . The solvent was removed by evaporation, and the residue was purified by a silica gel column chromatography (a stepwise gradient of 1-10% MeOH in DCM) to obtain 2b in 90% yield as white solid foam (1.17 mmol, 0.52 g). The 1 H-and 13 C-NMR spectra were identical with those reported in literature [19].     After stirring for 4.5 h at r.t., the mixture was equilibrated between EtOAc and aq. NaHCO 3 , the organic phase was evaporated to dryness and the residue was purified by silica gel column chromatography using DCM containing 1-5% MeOH as eluent. Yield 91% (111 mg). 1

Chain Assembly from 5′-O-(4,4′-Dimethoxytrityl) Protected Nucleoside Phosphoramidites
Support 11b (0.180 g, 68.2 μmol) was dissolved in DCM containing 3% dichloroacetic acid (5 mL) and stirred for 1 h. The mixture was diluted with DCM (25 mL) and washed with saturated aq. sodium bicarbonate (3 × 10 mL). Combined aqueous phases were back-extracted with DCM (20 mL). The combined organic phases were dried over anhydrous sodium sulfate and evaporated to dryness, giving 0.177 g deprotected material that still contained the DMTr alcohol. The product was subjected to coupling without purification.

Chain Assembly from 5′-O-(1-Methoxy-1-methylethyl) Protected Nucleoside Phosphoramidites
Glassware and stirrer bar were dried in the oven (120 °C). All chemicals and reagents were placed in a box under Nitrogen atmosphere. The solvents were dried over 3 Å molecular sieves.
The deprotected support was dissolved in MeCN (2mL) and the desired 5′-acetal protected building block (5a-c) in MeCN (1.5 equiv. in 1 mL) and 4,5-dicyanoimidazole (1.5 equiv. in 0.52 mL MeCN) were added under nitrogen. The mixture was stirred for 6 h and the oxidation solution (1.1 mL containing 1.5 equiv. of iodine in a 2:4:1 mixture of water, THF and 2,6-lutidine) was added. After 30 min stirring, the mixture was diluted with DCM (20 mL) and washed with an equal volume of aq. NaHSO 3 to remove the traces of iodine. The organic phase was dried over Na 2 SO 4 , filtered and evaporated to dryness under reduced pressure. The residue that still contained traces of 2,6-lutidine, was dissolved in MeOH (2 mL) and a sufficient volume of 0.1 mol L −1 HCl in dioxane was added to neutralize lutidine and adjust pH slightly below 4. After 30 min at r.t., the mixture was evaporated to dryness and the residue was coevaporated with pyridine and MeCN, and subjected to flash chromatographic separation with 1-15% MeOH in DCM to remove the remnants of the first coupling reaction. The subsequent couplings were then carried out in a similar manner, except that upon the two last couplings the support was dissolved in a 4:1 (v/v) mixture of MeCN and DCM instead of MeCN. The identitiy of the product was checked by negative ion ESI-MS after each coupling and deprotection.
On assembling the 3′-TTT-5′ trimer on the acetylated support 11a, the negative ion ESI  Figure 4). The mass of the crude product (after removal of the support by extraction) was 64 mg and the isolated yield (after HPLC purification) 52 mg (37% of theoretical).

Conclusions
A useful method for the preparation of short oligodeoxyribonucleotides in solution has been developed. The essential features of the method include use of 1-methoxy-1-methylethyl group as the 5′-O protecting group and permethylated β-cyclodextrin as a soluble support. Acid-catalyzed methanolysis of this 5′-acetal protection gives easily removable volatile products and hydrophobic cyclodextrin support allows efficient coupling on using only 50% excess of the monomeric building block, rapid flash chromatographic purification after each coupling cycle and extractive removal of the support after ammonolytic release/deprotection of the oligonucleotide.