Synthesis of protected 2′-O-deoxyribonucleotides on a precipitative soluble support: a useful procedure for the preparation of trimer phosphoramidites

Abstract

Tetrakis-O-(4-azidomethylphenyl)pentaerythritol, derivatized with 5′-O-(4,4′-dimethoxytrityl)-3′-O-{4-[2-(but-3-yn-1-ylamino)-2-oxoethoxy]phenoxyacetyl}thymidine, was used as a soluble support to assemble fully protected 2′-O-deoxyribonucleotide trimers by the phosphotriester chemistry. After the coupling and detritylation steps, the support-bound construct was purified by precipitation in MeOH. The trimers (TAT, AGT, TTA, CAT, GCT), in fully protected form, were released by a treatment with dilute methanolic K₂CO₃ and filtered through a short silica gel column in 65–70% overall yield. Two of the trimers (CAT and GCT), prepared in 0.5 mmol scale, were converted to the corresponding phosphoramidites. The entire procedure for the preparation of trimer phosphoramidites proved straightforward and applicable for the large scale synthesis.

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Introduction

Trimeric phosphoramidite building blocks, coding the amino acids commonly occurring in proteins, have gained popularity as starting materials of combinatorial synthesis of oligonucleotide pools useful for protein mutagenesis^1–3 and generation of antibody libraries.^4–6 Several protocols for the solution phase synthesis of such building blocks by both the phosphoramidite^7–10 and phosphotriester chemistry^11–14 have been published, and even solid-phase preparation of 3′-unprotected trinucleotide phosphotriesters in large scale has been described.¹⁵ An alternative method for their preparation based on utilization of a precipitative soluble support is now described. We have previously shown that short oligodeoxyribonucleotides may be conveniently prepared in hundreds of milligrams scale on a soluble tetrakis-O-[4-(1,2,3-triazol-1-yl)methylphenyl]pentaerythritol support (1 in Fig. 1) that precipitates quantitatively in MeOH.¹⁶ For example, the “outdated” phosphotriester strategy exploiting 3′-(2-chlorophenyl phosphate) building blocks works well on this support.¹⁷ No oxidation step is needed and the coupling cycle, hence, contains only two steps: 5′-deprotection and coupling. In the present study, the same soluble support, derivatized with hydroquinone-O,O′-diacetic acid (the Q-linker)¹⁸ (2 in Fig. 1), allows release of the oligonucleotide in a protected form. Only filtration through a short silica gel column was carried out after the synthesis and the desired 3′-unprotected trinucleotide phosphotriesters (TAT, AGT, TTA, CAT, GCT, 7–12 in Scheme 2) were obtained in 65–70% overall yield. Two of the trimers (CAT and GCT) were converted to the corresponding trimer phosphoramidites (13 and 14). The applicability of 13 and 14 in crude form for the automated phosphoramidite coupling cycle on a solid phase was additionally evaluated.

Fig. 1 Structures of soluble supports.

Results and discussion

Synthesis of the soluble support with the Q-linker (2)

A pentaerythritol-based thymidine cluster (1) has successfully been used as a soluble support for the synthesis of short oligonucleotides in solution.^16,17 The modular structure of the support allows replacement of the linker connecting the 3′-terminal nucleosides. For the synthesis of protected trimers, the Q-linker¹⁸ (hydroquinone-O,O′-diacetic acid) was introduced to the support (2) as described in Scheme 1. 5′-O-(4,4′-Dimethoxytrityl)thymidine (3) was first esterified with hydroquinone-O,O′-diacetic acid in pyridine using N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide (EDC) as an activator and 4-dimethylaminopyridine as a nucleophilic catalyst. A minor amount of symmetrical diester was formed as a byproduct, as reported by Pon and Yu.¹⁸ The free carboxy function of the product (4) was then subjected to amidation with but-3-yn-1-ylamine in DMF using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) as an activator. The alkyne functionalized nucleoside (5) was attached to the tetrakis-O-(4-azidomethylphenyl)pentaerythritol core (6) by 1,3-dipolar cycloaddition using CuI in DMF as a catalyst under oxygen-free conditions.¹⁹ This procedure followed by detritylation gave 2 in 84% overall yield from the tetraazide (6).

Scheme 1 Synthesis of soluble support 2. Conditions: (i) EDC, DMAP, Et₃N, pyridine; (ii) but-3-yn-1-ylamine, HBTU, dioxane; (iii) CuI, DMF, (iv) 10 mmol L⁻¹ HCl in DCM–MeOH (1 : 1, v/v).

Synthesis of protected support-bound deoxyribonucleotide trimers

Five different trimers were assembled on support 2 by applying the HOBt-activation based phosphotriester approach²⁰ as described previously in detail (Scheme 2).¹⁷ The coupling was carried out in dioxane using 1-methylimidazole as a nucleophilic catalyst. After completion of the reaction, MeOH was added to the reaction mixture. The support-bound nucleotides precipitated quantitatively, while all the small molecular reagents remained in solution. 5′-Detritylation was carried out with a mixture of 13 mmol L⁻¹ HCl in DCM–MeOH (1 : 1, v/v), followed by neutralization with pyridine, concentration and precipitation in MeOH. Two coupling cycles gave the desired support bound protected trimers in 70–75% yield.

Scheme 2 Synthesis protected 2′deoxyribonucleotide trimers (7–12) on a soluble support and their conversion to trimer phosphoramidites (13 and 14). Conditions: (i) bis(benzotriazol-1-yl) 2-chlorophenyl phosphate, pyridine, dioxane; (ii) benzotriazolyl phosphotriester of nucleosides, 1-methyl imidazole, dioxane; (iii) 13 mmol L⁻¹ HCl in DCM–MeOH (1 : 1, v/v); (iv) 4.0 mmol L⁻¹ K₂CO₃ in DCM–dioxane–MeOH (3 : 10 : 43, v/v/v); (v) 1-chloro-1-(2-cyanoethoxy)-N,N-diisopropylphosphonamine, Et₃N, DCM.

Selective cleavage of the protected trimers

Virtually efficient and selective cleavage of the trimers, without premature protecting group removal, could be obtained in a mixture of 4 mmol L⁻¹ K₂CO₃ in DCM–dioxane–methanol (3 : 10 : 43, v/v/v, 30 min at r.t.). Even more important for the clean reaction, however, was the quenching step: once the reaction was completed, stoichiometric amount of pyridinium chloride was added. The mixture was then concentrated and eluted through a short silica gel column (desalting and removal of the released branching core) and the product fractions were evaporated to dryness. The overall isolated yields (calculated from 2) for the protected deoxyribonucleotide trimers 7–12 ranged from 65 to 70% (88–99% from the solid supported trimers). RP HPLC-analysis was used to confirm the homogeneity of the products (12 in Fig. 2A, profiles of 7–11 in Fig. S5†) and their authenticity was verified by MS (ESI-TOF) spectroscopy (Table 1). It may be worth of mentioning that the stability of the protecting groups was essential for the success of the approach (alkaline cleavage used). The procedure has been optimized for the trimers, bearing the benzoyl (dC, dA) and isobutyryl (dG) groups on the base moieties and the 2-chlorophenyl group on the phosphate linkages. The 2-chlorophenyl phosphate protection tolerated well also upon the chain elongation and facilitated quantitative precipitation of the products (cf. our previous studies,^16,17 in which the applicability of phosphoramidite and phosphotriester chemistries were evaluated for the liquid phase synthesis of short oligonucleotides).

Fig. 2 (A) RP HPLC profile of 12, (B) RP HPLC profile of crude 2′deoxyoligonucleotide 5′-(CAT)₆T synthesized using 14, (C) ³¹P NMR spectrum (200 MHz, CD₃CN) of crude 14.

Table 1 MS (ESI-TOF) data of 7–12

Protected trimer	Yield^a	Observed monoisotopic mass^b	Calculated monoisotopic mass
a Calculated from the support-bound trimer.b Observed as [(M − 2H)/2]^2−
7	99%	1461.8	1461.3
8	88%	1485.2	1485.3
9	94%	1485.3	1485.3
10	95%	1580.4	1580.4
11	92%	1556.3	1556.4
12	97%	1574.4	1574.4

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Phosphitylation of the protected deoxyribonucleotide trimers

Two of the protected trimers (11 and 12) were synthesized in 0.5 mmol scale and phosphitylated with 1-chloro-1-(2-cyanoethoxy)-N,N-diisopropylphosphanamine. Only aqueous work up with sodium bicarbonate (vs. DCM) was used for the purification of the phosphoramidites (13 and 14, commercially available building blocks^13,21). According to ³¹P NMR spectroscopy (Fig. 2C and S6†), the reaction was nearly quantitative, albeit trace (5%, m/m) of hydrolysed phosphitylation reagent was observed. The applicability of the crude trimer phosphoramidites (13 and 14) in the automated solid phase phosphoramidite coupling cycle was demonstrated. Benzylthiotetrazol as an activator and a 300 s coupling time were used. Couplings were monitored by DMTr-assay that showed 96% and 94% coupling yields for 13 and 14, respectively. 19-mer oligonucleotide [5′-(CAT)₆T-3′] was additionally synthesized on a thymidine-CPG support using six repeated couplings of 14. The oligonucleotide was released from support by concentrated ammonia (first overnight at r.t., then 5 h at 55 °C),¹³ analysed by RP HPLC (Fig. 2B) and the authenticity of the product was verified by MS (ESI-TOF) spectroscopy.

Experimental

General

NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts are given in ppm and referenced relative to the residual solvent signals. RP HPLC conditions: a gradient elution from 50% MeCN in 0.1 mol L⁻¹ Et₃NHOAc to 100% MeCN in 25 min, then continued with MeCN (Fig. 2A); (B) gradient elution from 2.5% MeCN in 0.1 mol L⁻¹ Et₃NHOAc to 50% MeCN in 0.1 mol L⁻¹ Et₃NHOAc in 25 min, then continued with 50% MeCN in 0.1 mol L⁻¹ Et₃NHOAc (Fig. 2B), an analytical C-18 RP column (250 × 4.6 mm, 5 μm) with flow rate 1.0 mL min⁻¹ and detection at λ = 260 nm were used. Mass spectra were recorded with an ESI-Q-TOF MS-spectrometer.

5′-O-(4,4′-Dimethoxytrityl)-3′-O-{4-[2-(but-3-yn-1-ylamino)-2-oxoethoxy]phenoxyacetyl}thymidine (5)

5′-O-(4,4′-Dimethoxytrityl)-3′-O-[4-(carboxymethoxy)phenoxyacetyl] thymidine (4, 8.39 g, 10, 1 mmol), prepared as described previously,¹⁸ was dissolved in a mixture of anhydrous dioxane and pyridine (55 : 45 v/v) and HBTU (5.84 g, 15.1 mmol) and but-3-yn-1-ylamine (1.1 g, 15.1 mmol) were added. The reaction mixture was stirred overnight at ambient temperature, evaporated to an oil, diluted with DCM and washed with water. The organic layers were combined, dried over anhydrous Na₂SO₄ and evaporated to dryness. The residue was purified by silica gel chromatography (MeOH : DCM, 2 : 98, v/v) to give 7.97 g (98%) of 5 as light yellow foam. ¹H NMR (CDCl₃, 500 MHz): δ 8.98 (s, 1H), 7.59 (d, 1H, J = 1.2 Hz), 7.38–7.36 (m, 2H), 7.31–7.22 (m, 7H), 6.97 (b, 1H), 6.86 (m, 4H), 6.83 (m, 4H), 6.38 (dd, 1H, J = 7.8 Hz & 6.9 Hz), 5.55 (m, 1H), 4.64 (d, 1H, J = 16.3 Hz), 4.60 (d, 1H, J = 16.3 Hz), 4.45 (s, 2H), 4.13 (m, 1H), 3.78 (s, 6H), 3.51–3.44 (m, 4H), 2.47–2.43 (m, 4H), 2.00 (t, 1H, J = 2.7 Hz), 1.38 (d, 3H, J = 1.0 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 168.6, 168.5, 163.6, 158.8, 152.7, 152.3, 150.5, 144.1, 135.2, 135.1, 130.1, 130.0, 128.1, 128.0, 127.3, 116.0, 115.9, 2 × 113.4, 111.9, 87.3, 84.2, 83.9, 81.2, 76.5, 70.3, 68.1, 65.9, 63.6, 55.3, 38.6, 37.5, 19.4, 11.7; HRMS (ESI-TOF): m/z calcd for C₄₅H₄₄N₃O₁₁: 802.2976 [M − H]⁻, found 802.3010.

Tetrakis-O-{4-[4-({4-[2-(thymidin-3′-O-yl)-2-oxoethoxy]phenoxyacetamido}ethyl)-1,2,3-triazol-1-ylmethyl]phenyl}pentaerythritol (2)

Tetrakis-O-(4-azidomethylpheny)pentaerytritol (6, 0.48 g, 0.73 mmol)¹⁶ and 5 (3.52 g, 4.4 mmol) were dissolved in DMF (20 mL). The solution was sonicated for 30 minutes followed by bubbling with nitrogen. Solid CuI (0.138 g, 0.70 mmol) and sodium ascorbate (0.144 g, 0.70 mmol) were added and the mixture was stirred for three days at ambient temperature under nitrogen. The reaction mixture was evaporated to dryness, dissolved in DCM and washed with water. The organic layers were combined, dried over Na₂SO₄ and evaporated to dryness. The residue was purified by silica gel chromatography (MeOH : DCM, 5 : 95, v/v) to yield 2.84 g (quant.) of tetrakis-O-{4-[4-({4-[2-(5′-O-DMTr-thymidin-3′-O-yl)-2-oxoethoxy]phenoxyacetamido}ethyl)-1,2,3-triazol-1-ylmethyl]phenyl}pentaerythritol as a light green white foam. The obtained product was dissolved in 10 mmol L⁻¹ HCl in a mixture of MeOH and DCM (1 : 1, v/v). The mixture was stirred for 30 min. At room temperature, neutralized by addition of pyridine and evaporated to dryness. The residue was purified by silica gel chromatography (MeOH : DCM, 1 : 9, v/v) to give light green foam. The foam was dissolved in a mixture of MeOH and DCM (1 : 1, v/v) and washed with excess of aqueous EDTA (removal of Cu²⁺ traces). The organic layer was separated and evaporated to dryness to give 1.63 g (84%) of 2 as a white foam. ¹H NMR (500 MHz, CDCl₃): δ 7.86 (b, 4H), 7.78 (d, 4H, J = 1.0 Hz), 7.44 (s, 4H), 7.14 (d, 8H, J = 8.6 Hz), 6.84 (d, 8H, J = 8.6 Hz), 6.81 (d, 8H, J = 9.5 Hz), 6.78 (d, 8H, J = 9.4 Hz), 6.24 (dd, 4H, J = 6.5 Hz & 6.1 Hz), 5.41 (m, 4H), 5.37 (b, 8H), 4.61 (s, 8H), 4.32 (s, 8H), 4.26 (b, 8H), 4.06 (m, 4H), 3.79 (m, 8H), 3.53 (m, 8H), 2.87 (m, 8H), 2.36–2.26 (m, 8H), 1.86 (d, 12H, J = 0.9 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 169.6, 169.1, 164.9, 158.9, 152.6, 152.4, 150.9, 145.1, 136.3, 129.4, 127.5, 122.0, 2 × 115.7, 115.0, 111.0, 85.0, 84.7, 76.3, 67.7, 66.3, 65.7, 61.7, 53.4, 44.7, 38.3, 37.4, 25.2, 11.8; MS (ESI-TOF): m/z calcd for [(M + 2Cl)/2]²⁻ 1367.45, found 1367.43.

General procedure for the phosphotriester coupling

The protected nucleoside (0.40 mmol, Scheme 2) was dried by coevaporation with dry pyridine (3 × 5 mL) and concentrated to a small volume. Solution of bis(benzotriazol-1-yl) 2-chlorophenyl phosphate in dioxane (0.39 mmol, 0.2 mol L⁻¹, 1.97 mL) was then added to the mixture, which gave the active nucleoside phosphotriester solution.¹⁷ Support 2 (0.025 mmol) was dried by coevaporation with dry pyridine (3 × 5 mL) and one half of the phosphotriester solution (2.0 mmol, 8.0 equiv.) and 1-methylimidazole (1.0 mmol, 0.078 mL) were added under nitrogen. The reaction mixture was stirred for 2 h, transferred to stoppered 50 mL plastic tubes and MeOH (46 mL) was added. The precipitate was isolated by centrifugation and dried under vacuum. The precipitate and supernatant were analyzed by HPLC to verify completeness of the precipitation.

General procedure for the detritylation

The support-bound dimer (0.15 mmol) was dissolved in a mixture of MeOH and DCM (1 : 1, v/v, 100 mL) and 0.125 mol L⁻¹ HCl in methanol (10 mL) was added.¹⁶ The mixture was stirred for 15 min. At ambient temperature, neutralized by addition of pyridine and concentrated to an oil. The oil was dissolved in methanol and DCM (1 : 1, v/v) and the mixture was added to cold methanol. The precipitate formed was isolated (as above) by centrifugation and dried under vacuum. The precipitate and supernatant were analyzed by RP HPLC to verify completeness of the precipitation.

Cleavage of the fully protected deoxyribonucleotide trimers (7–11)

The support-bound trimer (Scheme 2) (0.034 mmol) was dissolved in a 1 : 1 (v/v) mixture of DCM and MeOH (6 mL) and anhydrous dioxane (10 mL) was added. Once the clear solution was obtained, K₂CO₃ in MeOH (5 mmol L⁻¹, 40 mL) was added. The mixture was stirred for 30 min. And pyridinium chloride in MeOH (0.2 mol L⁻¹, 1.0 mL) was added. Volatiles were then removed under reduced pressure. The remained crude was dissolved in a mixture of DCM and MeOH (5 : 95, v/v) and eluted through a short silica gel column (MeOH : DCM, 5 : 95, v/v) to give protected deoxyribonucleotide trimers 7–11 as white foams in 88–99% yield (Table 1) (65–70% overall yield, calculated from 2). The RP HPLC and MS (ESI-TOF) data of 7–11 are shown in Fig. 2A, S5† and Table 1.

Deoxyribonucleotide trimer phosphoramidites 13 and 14

A fully protected deoxyribonucleotide trimer (11 or 12, 0.48 mmol) was dissolved in DCM (5.0 mL). Triethylamine (2.4 mmol) and 1-chloro-1-(2-cyanoethoxy)-N,N-diisopropylphosphonamine (0.58 mmol) were added, the mixture was stirred for 3 h at ambient temperature under nitrogen and poured to cold saturated sodium bicarbonate. The organic layer was separated, dried over anhydrous sodium sulfate, and evaporated to white foam. ³¹P NMR (200 MHz, CDCl₃) of the crude trimer phosphoramidites (14 in Fig. 2C and 13 in Fig. S6†) verified virtually quantitative reaction. The NMR and MS data of the trimers (13 and 14) was consistent with the commercially available building blocks.^13,21 The coupling efficiency of 13 and 14 in the automated DNA/RNA-synthesizer was next studied. 0.1 mol L⁻¹ solutions of the trimer phosphoramidites (13 and 14) in DCM–MeCN (1 : 4, v/v)^13,21 were prepared and loaded to the reagent vessels of a DNA synthesizer. The coupling was carried out on a 1 μmol scale on a thymidine-derived controlled pore glass support (DMTr-T-CPG). Benzylthiotetrazol as an activator with a 300 s coupling time was used. The coupling efficiency was monitored by DMTr-cation assay using a built-in UV-detector that showed 94 and 96% coupling yields for 13 and 14, respectively. 14 was additionally used (using the same coupling cycle) for the assembly of a 19-mer 2′-deoxriboynucleotide (5′-CATCATCATCATCATCATT-3′). The support was treated with concentrated ammonia (first overnight at r.t., then 5 h at 55 °C)¹³ and filtered. The filtrate was evaporated to dryness, dissolved in water and subjected to RP HPLC. RP HPLC profile of the crude product mixture is shown in Fig. 2B. The product peak was isolated and characterized by MS (ESI-TOF): m/z 1891.3 observed for [(M − 3H)/3]³⁻ of the desired product.

Conclusion

A tetrapodal pentaerythritol-based soluble support bearing the Q-linker as a scissile moiety (2) has been prepared and demonstrated to allow gram scale synthesis of fully protected trimeric 2′-O-deoxyribonucleotides 7–12 (overall isolated yield ranged from 65 to 70%). 7–12 may be converted by one step phosphitylation to trimeric 3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite) building blocks (13 and 14 as examples),²¹ bearing 4,4′-dimethoxytrityl group on O-5′,2-chlorophenyl group on phosphodiester linkages and benzoyl (dC, dA) or isobutyryl (dG) group on base moieties. The essential features of the synthesis include removal of small molecular reagents after couplings and a 5′-deprotection step by quantitative precipitation of the soluble support in MeOH and release of the final product in fully protected form by cleavage of the Q-linker under mild basic conditions, followed by simple flash column chromatography and phosphitylation. The entire procedure for the preparation of trimer phosphoramidites was straightforward and applicable for the large scale synthesis.

Acknowledgements

Financial support from the FP7 Marie Curie Actions and from Academy of Finland (No. 251539 and 256214) are gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR and ¹³C NMR data of 2 and 5, ³¹P NMR data of 13 and 14, RP HPLC profiles of 7–11. See DOI: 10.1039/c6ra22316h

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