Improved Synthesis of Phosphoramidite-Protected N6-Methyladenosine via BOP-Mediated SNAr Reaction

N6-methyladenosine(m6A) is the most abundant modification in mRNA. Studies on proteins that introduce and bind m6A require the efficient synthesis of oligonucleotides containing m6A. We report an improved five-step synthesis of the m6A phosphoramidite starting from inosine, utilising a 1-H-benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP)-mediated SNAr reaction in the key step. The route manifests a substantial increase in overall yield compared to reported routes, and is useful for the synthesis of phosphoramidites of other adenosine derivatives, such as ethanoadenosine, an RNA analogue of the DNA adduct formed by the important anticancer drug Carmustine.

Here we describe a five-step synthesis of the m 6 A phosphoramidite from inosine employing BOP-mediated S N Ar reaction. The lack of use of POCl 3 or methyliodide enables a two to three-fold improvement in the m 6 A phosphoramidite yield compared to reported methods (Scheme 2) [4][5][6][7]. BOP-or PyBOP-mediated S N Ar reaction is also useful for the synthesis of m 6 A analogues [10,11], e.g., methylethanoadenosine and ethylethanoadenosine.

Synthesis of m 6 A Analogues
To test the generality of the SNAr reaction, we explored the synthesis of m 6 A analogues (11a-g, Scheme 5). Thus, treatment of inosine with BOP/DBU in DMF (40 min) was followed by addition of an amine and reaction overnight to give the desired products in generally good non-optimised yields.

Synthesis of m 6 A Analogues
To test the generality of the S N Ar reaction, we explored the synthesis of m 6 A analogues (11a-g, Scheme 5). Thus, treatment of inosine with BOP/DBU in DMF (40 min) was followed by addition of an amine and reaction overnight to give the desired products in generally good non-optimised yields.

Discussion
Overall, we have developed a modular and efficient five-step synthesis of phosphoramidite-protected m 6 A starting from inosine, which avoids the use of POCl 3 /the Vilsmeier reagent (Scheme 1A). The key step comprises the efficient (90% yield) BOPmediated S N Ar reaction of methylamine with a readily prepared protected inosine derivative, Although, there are reports of using BOP-mediated S N Ar to prepare ribonucleoside analogues [10,[14][15][16][17], use of the reaction to prepare phosphoramidite-protected materials suitable for oligonucleotide synthesis has been limited [18,19]. Investigations on the mechanism of BOP-mediated S N Ar reactions are reported [11]. Thus, the oxide formed by base-mediated deprotonation of the hydroxyl group on the aromatic inosine ring reacts with the electrophilic phosphorus of BOP to form an (acyloxy)phosphonium intermediate (I, Figure 1), which undergoes S N Ar reaction with the oxybenzotriazole to form an intermediate (II, Figure 1) which is subsequently replaced by a nucleophile in a second S N Ar reaction to form the desired product (III, Figure 1). The method is also suited for the synthesis of phosphoramidites of other purines/pyrimidines modified at the C-6/C-4 positions, respectively, as shown by the preparation of N 1 ,N 6 -ethanoadenosine and its analogues using BOP-mediated S N Ar (Scheme 6); these reactions resulted in moderate (22%) to very good (90%) non-optimised yields ( (11b, Scheme 5) (in the case of N 6 -ethyladenosine, the yield was low due to a non-optimal work-up procedure), showing the generality of the reaction. The method also works with alcohol nucleophiles, though since MeOH is a poor nucleophile, after the formation of the proposed oxybenzotriazol intermediate (II, Figure 1), the crude mixture was evaporated and redissolved in excess methanol in the presence of a base (DBU) to give the desired O 6 -methylinosine (11d) product (72%) (Scheme 5). The absence of protection of the hydroxyl groups of inosine does not substantially impact on  . 13(a-c), although, more polar solvent is required to dissolve unprotected inosine.
The modified nucleosides described here will have use in ongoing investigations to probe the selectivity and inhibition of RNA modifying enzymes, including m 6 A demethylases and m 6 A reader proteins. The BOP-mediated S N Ar reaction may also have other applications, e.g., in improving the reported route to N 1 ,N 6 -ethanodeoxyadenosine phosphoramidite [20] or because N 1 ,N 6 -ethanoadenosine can be oxidised to N 1 ,N 6 -ethenoadenosine using MnO 2 , to give a fluorescent analogue of adenosine [21].

General Experimental Considerations
Reagents for synthesis were from Sigma-Aldrich, Alfa Aesar, Cambridge Biotech, Fischer Scientific, or Link Technology, unless otherwise stated. Anhydrous solvents used in reactions were either analytical grade, as obtained commercially (Alfa Aesar), or were freshly distilled. HPLC grade solvents were employed for work-up and chromatography. For the chromatographic purification of phosphoramidites, solvents were dried over P 2 O 5 prior to use. Reactions involving moisture-sensitive reagents were carried out under an argon atmosphere; glassware was oven dried and cooled under nitrogen before use. Reagents were used as supplied (analytical or HPLC grade) without prior purification. Anhydrous MgSO 4 was used as a drying agent.
Thin layer chromatography was performed using aluminium plates coated with 60 F254 silica. Plates were visualised using UV light (254 nm), or 1% (m/v) aq. KMnO 4 stain. Flash column chromatography was performed using Kieselgel 60 silica in a glass column, or on a Biotage SP4 flash column chromatography platform. Retention factors (R f ) are quoted to a precision of 0.05.
Deuterated solvents were from Sigma and Apollo Scientific Ltd. 1 H-NMR and 13 C-NMR spectra were recorded using Bruker AVIII400, AVII500, AVIII600 and AVIII700 NMR spectrometers (Bruker, Banner Lane, UK). Fields were locked by external referencing to the relevant residual deuterium resonance. Chemical shifts (δ) are reported in ppm; coupling constants (J) are recorded in Hz to the nearest 0.5 Hz; when peak multiplicities are reported, the following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broadened, dd = doublet of doublets, dt = doublet of triplets, td = triplet of doublets. Spectra were recorded at room temperature unless otherwise stated. 1 H and 13 C-NMR spectra of compounds are available in the online Supplementary Materials.
Low-resolution mass spectra (m/z) and high-resolution mass spectra (HRMS) were recorded using an LCT Premier XE (Waters, Elstree, UK) or a microTOF machine (Bruker, Banner Lane, UK).
Melting points were recorded on a Gallenkamp Hot Stage apparatus (Gallenkamp, Loughborough, UK). IR spectra were recorded using a Bruker Tensor 27 FT-IR spectrometer (Bruker, Banner Lane, UK)as thin films. Selected characteristic peaks are reported in cm −1 .

Experimental Details for Synthesis
General Procedure A: Synthesis of N-Alkyladenosine DBU (1.5 mmol) was added dropwise to a stirred solution of inosine (1 mmol) and BOP (1.2 mmol) in DMF; the mixture was then heated at 40 • C. After the consumption of starting material (approximately 40 min, as assessed by TLC), the reaction was cooled to room temperature and the appropriate amine (5 mmol) was added dropwise and the reaction was stirred overnight. The crude product mixture was concentrated under reduced pressure, then diluted with ethyl acetate and was washed with water (3 × 10 mL). The organic layer was dried (anhydrous MgSO 4 ) and concentrated under vacuum. The resulted solid was recrystallised twice from iso-propanol.  (2). The desired compound was prepared according to a modified version of the reported procedure [22]. To a stirred suspension of inosine (2.12 g, 8 mmol) in 40 mL anhydrous DMF at 0 • C, di-t-butylsilyl ditrifluoromethanesulfonate (3.0 mL, 8.8 mmol) was added dropwise under an N 2 atmosphere. After consumption of starting material (30 min, as assessed by TLC), the reaction was quenched immediately with imidazole (2.7 g, 40 mmol) at 0 • C. After 5 min, the reaction was warmed to room temperature. t-Butyldimethylsilyl chloride (1.5 g, 9.6 mmol) was then added portionwise and the reaction was refluxed at 60 • C for 12 h. The suspension was then cooled to room temperature, water was added, and the precipitate was collected by suction filtration. The filtrate was discarded, and the white precipitate was washed with cold methanol. The methanol layer was evaporated under reduced pressure and the product was crystallised from CH 2 Cl 2 to give a white solid  (4). The desired compound was prepared according to the reported procedure [4]. To a stirred solution of 3 ,5 -O-Bis(tertbutylsilyl)-2 -O-(tert-butyldimethylsilyl)-N 6 -methyladenosine (3; 240 mg, 0.45 mmol) in 4 mL of CH 2 Cl 2 at −15 • C, a cooled solution of (HF) x ·pyridine (0.06 mL, 2.3 mmol) in 365 µL pyridine was added. The reaction temperature was maintained at -15 • C and stirred for 12 h. The reaction was diluted with CH 2 Cl 2 , then washed first with sat. aq. NaHCO 3 solution, then with water (3 × 10 mL). The organic layer was dried (anhydrous MgSO 4 ) and concentrated under reduced pressure. The residue was purified by column chromatography   [4].

-O-(4,4 -Dimethoxytrityl)−2 -O-dimethyl(tert-butyl)silyl-N6-methyladenosine (5).
The desired compound was prepared according to the reported procedure [4]. To a stirred solution of 2 -O-dimethyl(tert-butyl)silyl-N6-methyladenosine (4) (2.6 g, 6.6 mmol) in 4 mL anhydrous pyridine at 0 • C, DMTrCl (2.7 g, 8.0 mmol) was added portionwise at regular intervals for 12 h. The reaction was quenched by addition of an excess of anhydrous methanol (0.5 mL) at room temperature. After 1 h, the solution was concentrated under vacuum. The crude solid was first dissolved and fractioned between aqueous NaHCO 3 and ethyl acetate; the organic layer was then washed with water (3 × 10 mL). The organic layer was dried (MgSO 4 ) and concentrated under vacuum. The residue was purified by column chromatography (9:1 to 3:2 cyclohexane/ethyl acetate) resulted in a green oil  (12). To a stirred solution of inosine (3.75 g, 13.24 mmol) and imidazole (3.6 g, 53.0 mmol) in anhydrous DMF in a 50 mL round bottom flask, TBDMSCl (6.6 g, 43.7 mmol) was added portionwise. The reaction was heated at 60 • C for 12 h. The suspension was cooled to room temperature, water was added and the precipitate was collected by suction filtration. The filtrate was discarded, and the white precipitate was washed with cold methanol. The methanol layer was evaporated under vacuum; the product was crystallised as a white solid from CH 2 C1 2 (7.8 g, 94%). TLC R f 0.6 (1:9 MeOH/CH 2 Cl 2 ); 1 6 ,N 6 -methyl(2-hydroxyethyl) adenosine (13b; 0.5 g, 0.75 mmol) and Et 3 N (0.54 mL, 3.75 mmol) in 30 mL of anhydrous DMF in a 50 mL round bottom flask, methyltriphenoxyphosphonium iodide (0.85 g, 1.9 mmol) was added and the mixture was stirred at room temperature for 1 h. Anhydrous methanol was added and the crude product mixture was concentrated under reduced pressure, then diluted with ethyl acetate and washed with NaHCO 3 and water (3 × 10 mL). The organic layer was dried (anhydrous MgSO 4 ), then concentrated under reduced pressure. The residue was purified by alumina column chromatography (99:1 to 90:10 CH 3 Cl/MeOH) which resulted in an oil. 1

Conclusions
Overall, we have developed an improved the synthesis of the m 6 A phosphoramidite starting from inosine. Following alcohol group protection, a BOP-mediated S N Ar reaction was employed to introduce the desired N 6 -methylamino group in a suitably protected form to be efficiently converted to the phosphoramidite for incorporation into oligonucleotides. The BOP-mediated S N Ar reaction can be employed to prepare other N 6 -alylamino substituted adenosine derivatives, including ethanoadenosine, an RNA analogue of the DNA adduct formed by the important anticancer drug Carmustine.
Supplementary Materials: The following are available online. 1 H and 13 C-NMR spectra of compounds are available.