Alternative Synthesis of α-l-Threofuranosyl Guanosine 3′-triphosphate

Abstract

Artificial genetic polymers (XNAs) have attracted considerable attention due to their unique physicochemical properties that include enhanced chemical and biological stability. Unfortunately, some of the most interesting XNAs are constructed from monomers that are not readily available and must be prepared by chemical synthesis. The need to generate building-block materials for these systems warrants careful optimization, as syntheses of XNA monomers can easily exceed ten chemical steps. Here, we evaluate the synthesis of α-l-threofuranosyl guanosine 3′-triphosphate (tGTP), a key substrate in the enzymatic synthesis of α-l-threofuranosyl nucleic acids. Previously, tGTP was prepared by a Vorbrüggen glycosylation reaction from N-acetyl-O-(diphenylcarbamoyl)guanine and a suitably protected threose sugar. However, the preparation of the protected nucleobase was a laborious process that merited further evaluation. We now describe an alternative approach that is easier to perform and does not compromise the overall yield or regioselectivity.

Key words

threose nucleic acids - artificial genetic polymers - medicinal chemistry - threofuranosyl guanine triphosphate - phosphorylation - glycosylation

• References and Notes

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• 17 Intermediate 6 N,O-Bis(trimethylsilyl)acetamide (15 mL, 61.4 mmol, 2.3 equiv) was added to a suspension of 6-chloropurine-2-amine (5.0 g, 29.5 mmol, 1.1 equiv) in anhyd MeCN (70 mL), and the mixture was stirred for 1 h at 60 °C then cooled to 24 °C. A solution of furanose derivative 5 (13.5 g, 26.8 mmol, 1.0 equiv) in anhyd MeCN (70 mL) was added dropwise to the mixture, followed by TMSOTf (13.3 mL, 73.6 mmol, 2.75 equiv), and the resultant mixture was heated for 2.5 h at 60 °C. After removal of MeCN, the syrup was diluted with EtOAc (200 mL) and washed with sat. aq NaHCO₃ and brine, then dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by column chromatography [silica gel, EtOAc–hexane (5–30%)] to give a white solid; yield: 10.9 g (66.3%). ¹H NMR (400 MHz, DMSO-d ₆): δ = 8.30 (s, 1 H), 7.84 (d, J = 7.5 Hz, 2 H), 7.64 (t, J = 7.4 Hz, 1 H), 7.54 (d, J = 6.9 Hz, 2 H), 7.51–7.32 (m, 8 H), 7.27 (t, J = 7.3 Hz, 2 H), 6.93 (s, 2 H), 6.11 (s, 1 H), 5.88 (s, 1 H), 4.61 (s, 1 H), 4.21 (s, 1 H), 4.14 (s, 1 H), 0.91 (s, 9 H). ¹³C NMR (101 MHz, DMSO-d ₆): δ = 164.97, 160.35, 153.92, 150.18, 141.21, 135.68, 134.43, 132.55, 132.44, 130.71, 129.99, 129.26, 128.87, 128.49, 124.12, 87.49, 81.69, 75.69, 74.90, 39.98, 26.97, 19.02. HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₃₂H₃₂ClN₅NaO₄Si: 636.1810; found: 636.1786. Intermediate 7 DMF-DMA (4.4 mL, 33.4 mmol, 5.0 equiv) was added dropwise to a stirred solution of 6 (4.1 g, 6.68 mmol, 1.0 equiv) and DMAP (0.2 g, 1.64 mmol, 0.25 equiv) in anhyd pyridine (15 mL), and the mixture was stirred for 20 h at RT under N₂. The solvent was evaporated under reduced pressure, and the residue was extracted with EtOAc (2 × 100 mL) and washed with H₂O and brine (2 × 100 mL). The combined organic layer was dried (Na₂SO₄) and evaporated under reduced pressure. The residue was purified by column chromatography [silica gel, EtOAc–hexane (5–30% containing 2% Et₃N)] to give a white solid; yield: 3.1 g (69.5%). ¹H NMR (400 MHz, CDCl₃): δ = 8.77 (s, 1 H), 8.48 (s, 1 H), 7.92 (d, J = 7.9 Hz, 2 H), 7.59 (m, 5 H), 7.35 (m, 8H), 6.36 (s, 1 H), 5.89 (s, 1 H), 4.50 (s, 1 H), 4.13 (dd, J = 17.5, 8.8 Hz, 2 H), 3.21 (s, 3 H), 3.18 (s, 3 H), 1.03 (s, 9 H). ¹³C NMR (101 MHz, CD₃OD): δ = 164.55, 162.38, 159.87, 152.13, 149.92, 142.07, 135.43, 135.22, 133.47, 131.93, 129.94, 129.87, 129.35, 128.33, 127.69, 127.55, 126.95, 89.31, 80.58, 76.34, 75.79, 40.39, 34.19, 25.80, 18.26. HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₃₅H₃₈ClN₆O₄Si: 669.2412; found: 669.2413. Intermediate 8
  7 (3.1 g, 4.63 mmol, 1.0 equiv) and NaNO₂ (3.6 g, 52.2 mmol, 11.3 equiv) were dissolved in DMSO (25 mL), and the mixture was stirred mixture at 75 °C for 22 h before H₂O was added to give a white precipitate. EtOAc (300 mL) was added to the precipitate, and the mixture was washed with H₂O and brine (3 × 100 mL). The combined organic layers were dried (Na₂SO₄) and concentrated to give an orange oil that was purified by column chromatography [silica gel, MeOH–CH₂Cl₂ (1–5%)] to give a light-yellow solid; yield: 2.93 g (97%). ¹H NMR (400 MHz, CD₃OD): δ = 8.75 (s, 1 H), 8.01 (s, 1 H), 7.89 (d, J = 7.9 Hz, 2 H), 7.64–7.16 (m, 13 H), 6.26 (s, 1 H), 6.01 (s, 1 H), 4.52 (s, 1 H), 4.41–4.27 (m, 2 H), 3.11 (s, 3 H), 3.09 (s, 3 H), 0.87 (s, 9 H). ¹³C NMR (101 MHz, DMSO-d ₆): δ = 164.9, 158.79, 158.14, 157.85, 149.77, 136.67, 135.69, 135.57, 134.37, 134.37, 132.64, 132.56, 130.69, 130.57, 129.97, 129.25, 128.99, 128.50, 128.31, 120.59, 88.09, 81.51, 75.92, 74.85, 63.07, 52.50, 45.83, 35.03, 26.90, 19.08. HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₃₅H₃₉N₆O₅Si: 651.2751; found: 651.2753. Intermediate 9 A 1 M solution of TBAF in THF (13 mL, 13 mmol, 3.0 equiv) was added dropwise to a cold (0–5 °C; ice bath) solution of 8 (2.75 g, 4.23 mmol, 1.0 equiv) in THF (35 mL), and the mixture was stirred at 0 °C for 2 h. The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc. The organic layer was washed with H₂O and brine, then dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by column chromatography [silica gel, MeOH–CH₂Cl₂ (1–2% containing 2% Et₃N)] to give a white solid; yield: 1.00 g (57.3%). ¹H NMR (400 MHz, DMSO-d ₆) 11.33 (s, 1H), δ = 8.61 (s, 1 H), 7.99 (d, J = 7.8 Hz, 2 H), 7.94 (s, 1 H), 7.67 (t, J = 7.4 Hz, 1 H), 7.53 (t, J = 7.72 Hz, 2 H), 6.06 (s, 1 H), 5.77 (s, 1 H), 4.52 (s, 1 H), 4.26–4.11 (m, 2 H), 3.04 (s, 3 H), 2.98 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 165.40, 158.43, 157.6, 157.04, 149.11, 138.16, 133.88, 129.82, 128.92, 128.65, 120.73, 89.77, 83.30, 76.05, 74.66, 45.68, 41.54, 35.18. HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₉H₂₁N₆O₅: 413.1573; found: 413.1573.
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