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Synthesis of a-L-Threofuranosyl Nucleoside Triphosphates Keyong Zou,1 Allen Horhota,3 Biao Yu,2 Jack W. Szostak,1 Larry W.
1Howard Hughes Medical Institute, and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02184, and 2State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai, 200032, China, and 3Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut General Information
Chemicals and reagents were purchased from Sigma/Aldrich unless otherwise noted.
Pyridine and tributylamine were distilled from calcium hydride, DMF from phosphorus pentoxide, and dioxane was dried over sodium. TNA triphosphate syntheses were carried out according to a modified version of the methods developed by Ludwig and Eckstein.1 Advanced intermediates 1 of thymine, 2,6-diaminopurine, and guanine threofuranosyl
nucleosides with 2'-O-DMT and heterocyclic protecting groups were a generous gift of the Eschenmoser laboratory. The protecting groups for the heterocyclic 2,6- diaminopurine and guanine bases were removed prior to phosphorylation, but the 2'-O- DMT protecting group remained in place. Compound 4 was prepared from L-ascorbic
acid and N4-benzoyl cytosine as reported.2 Phosphorylation reactions were monitored by TLC on EMD 60 F253 silica plates run in 1-propanol/NH4OH/H2O (11:7:2) visualized under UV light. Ion exchange chromatography was performed using DEAE Sephadex (Pharmacia) in a Waters 600 HPLC with UV detection eluting with 0-1 M TEAB pH 7.5 followed by reverse phase HPLC purification on a divinyl benzene column eluting with 95% 100 mM TEAA pH 7.0 and a linear gradient from 5% methanol to 100% methanol.
The sodium salt was prepared according to the method described by Hoard and Ott.3 (a-L-Threofuranosyl)thymine-3'-triphosphate, (a-L-threofuranosyl)-2,6-
diaminopurine-3'-triphosphate, and (a-L-threofuranosyl)guanine-3'-triphosphate
(tTTP, tDTP and tGTP, 3)
2'-O-DMT protected threosyl nucleosides were co-evaporated twice from dry pyridine and stored under high vacuum over P2O5. for 1 hr. The nucleosides were dissolved in appropriate solvents and reacted with 2-chlor-4H-1,3,2-benzodiozaphosphorin-4-one in dry dioxane (1 M stock, 1 mol eq.) After 10 minutes tributylammonium pyrophosphate solution in dry DMF (0.5 M stock, 1.5 mol. eq.) and tributylamine (1/3 vol. of pyrophosphate solution) were added and the reaction was allowed to stir for 15 min. I2 solution in pyridine/Water (98:2) (1% stock, 1 mol. eq.) was added to the reaction mixture with 5% sodium sulfite solution (~ 1/3 volume of I2 solution) was added after 10 minutes to quench excess iodine. Solvents were removed under high vacuum. The residue was dissolved in 50% acetic acid for 20 min at 0 oC followed by solvent removal. The residue was then dissolved in water (5 ml) and washed twice with diethyl ether. Ion exchange and reversed-phase HPLC purification followed by conversion to the sodium salts resulted in the threosyl nucleoside 3'-triphosphates in 25-35% yields.
(a-L-Threofuranosyl)thymine-3'-triphosphate
1-{2'-O-[(4',4''-dimethoxytriphenyl)methyl]-a-L-threofuranosyl}thymine (10 mg, 19 mmol) was dissolved in 20 ml pyridine and 60 ml dioxane for the phosphorylation reaction performed as described above. Yield 35%. 31P NMR (161.9 MHz, D2O) d -8.35 (d, Jp,p=19 Hz), -10.60 (d, Jp,p=19 Hz), -21.99 (t, Jp,p=19 Hz). UV (20 mM Tris-HCl pH 7.5) (a-L-Threofuranosyl)-2,6-diaminopurine-3'-triphosphate
N2,N6-Dibenzoyl-9-{2'-O-[(4', 4''-dimetheoxytriphenyl)methyl]-a-L-threofuranosyl}-2,6- diaminopurine was deprotected in 8 M methylamine in ethanol:12 M methylamine in water (1:1) for 3 hr at 50 oC followed by solvent removal and silica gel flash chromatography (0 to 4% methanol in CH2Cl2) yielding 9-{2'-O -[(4', 4''- dimethoxytriphenyl)methyl]-a-L-threofuranosyl}-2,6-diaminopurine in near quantitative yield as confirmed by LRMS. This material (10 mg, 18 mmol) was dissolved in 30 ml pyridine and 120 ml DMF for the phosphorylation reaction. Yield 25%. 31P NMR (202.4 MHz, D2O) d -6.84 (d, Jp,p= 18 Hz), -11.73 (d, Jp,p= 18 Hz), -19.95 (t, Jp,p= 14 Hz). ESI- MS (neg.) 490.9883 (HRMS calc. 490.9886 ). UV (20 mM Tris-HCl pH 7.5) lmax 282 (a-L-Threofuranosyl)guanine-3'-triphosphate
N2-Acetyl-O6-diphenylcarbamoyl)-9-{2'-O-[(4',4''-dimetheoxytriphenyl)methyl]-a-L- threofuranosyl}guanine was deprotected in 8 M methylamine in ethanol:12 M methylamine in water (1:1) for 3 hr at 50 oC followed by solvent removal and silica gel flash chromatography (0 to 4% methanol in CH2Cl2) yielding 9-{2'-O-[(4',4''- dimethoxytriphenyl)methyl]-a-L-threofuranosyl}guanine confirmed by LRMS. This material (10 mg, 18 mmol) was dissolved in 30 ml pyridine and 120 ml DMF for the phosphorylation reaction. Yield 25%. 31P NMR (161.9 MHz, D2O) d -3.08 (d, Jp,p= 18 Hz), -10.40 (d, Jp,p= 18 Hz), -19.07 (t, Jp,p= 18 Hz). UV (20 mM Tris-HCl pH 7.5) lmax N4-Benzoyl-1-{2'-O-acetyl-3'-O-[(4', 4''-dimetheoxytriphenyl)methyl]-a-L-
threofuranosyl}cytosine (5)
N4-Benzoyl-1-{3'-O-[(4',4''-dimetheoxytriphenyl)methyl]-a-L-threofuranosyl}cytosine (4, 1 g, 1.62 mmol), prepared starting from L-ascorboc acid and N4-benzoyl cytosine as
described,2 was co-evaporated twice from dry pyridine (30 ml). Acetic anhydride (275 ml, 2.92 mmol) was added to the solution of 4 in dry pyridine (30 ml). The mixture was
stirred under argon at room temperature for 12 hrs, and then evaporated to dryness after MeOH (2 ml) was added to quench the reaction. The residue was dissolved in CH2Cl2 and washed with sat. aq. NaHCO3 solution, H2O and sat. aq. NaCl solution, dried (Na2SO4) and filtered. The residue after evaporation was purified by silica gel flash chromatography [hexane/CH2Cl2/TEA (1:1:0.0025) to CH2Cl2 /TEA(1:0.0025)] to give 5
TLC (4% MeOH in CH2Cl2): Rf 0.32. 1H-NMR (400 MHz; CDCl3) d 8.82 (bs, 1H), 7.96 (m, 3H), 7.20-7.63 (m, 13H), 6.79-6.84 (m, 4H), 5.93 (bs, 1H), 5.53 (bs, 1H), 4.24 (m, 1H), 3.76-3.78 (2s, 6H), 3.60 (dd, J=14.8, J=7.2, 1H), 3.05 (dd, J=14.8, J=7.6, 1H), 2.08 (s, 3H). 13C NMR (100 MHz; CDCl3) d 168.7, 162.5, 159.0, 145.4, 144.3, 135.8, 135.3, 133.3, 130.1, 130.0, 129.2, 128.3, 128.1, 127.7, 127.5, 113.8, 113.7, 96.6, 91.4, 88.9, 81.3, 76.4, 75.1, 55.6, 21.2 (hexane residues: 31.6, 22.7, 14.1). ESI-MS (pos.) 684.2324 N4-Benzoyl-1-(2'-O-acetyl-a-L-threofuranosyl)cytosine (6)
N4-Benzoyl-1-{2'-O-acetyl-3'-O-[(4',4''-dimetheoxytriphenyl)methyl]-a-L- threofuranosyl}cytosine (5, 500 mg, 0.76 mmol) was treated with 3% TCA in CH2Cl2 (25
ml) at ambient temperature for 3 min. MeOH was added dropwise to quench released DMT cation until the orange color disappeared. After the acid was neutralized by triethylamine (~10 ml), the mixture was immediately evaporated to avoid deacylation by overexposure to the basic solvent in the presence of MeOH. The residue was purified by silica gel flash chromatography (2% MeOH in CH2Cl2). There was still ~ 12% of 5
(tritylated nucleoside) that was subjected to another TCA treatment and column purification. Product fractions were combined to afford 6 (251 mg, 92%) as a white foam.
TLC (4% MeOH in CH2Cl2): Rf 0.23. 1H NMR (400 MHz; DMSO-d6) d 11.26 (bs, 1H), 8.14 (d, J=6.4, 1H), 8.02-7.50 (m, 5H), 7.36 (d, J=6.4, 1H), 5.81 (bs, 1H), 5.72 (bs d,1H), 5.12 (bs, 1H), 4.76 (d, J=9.347, 1H), 4.17 (m, 2H), 2.11 (s, 3H). 13C NMR (100 MHz; DMSO-d6) d 169.7, 168.0, 164.0, 155.0, 146.1, 133.9, 133.3, 129.1, 129.0, 96.4, 91.5, 81.7, 77.2, 73.1, 21.5. ESI-MS (pos.) 382.1015 [M+Na]+ (HSMS calc. 382.1015).
(a-L-Threofuranosyl)cytosine-3'-triphosphate (tCTP, 8)
N4-Benzoyl-1-(2'-O-acetyl-a-L-threofuranosyl)cytosine (6 , 10 mg, 28 m mol) was
dissolved 30 ml dry pyridine and 90 ml dry dioxane for the phosphorylation reaction. The reaction was carried out as described above except for the treatment with acetic acid.
After the excess of iodine was quenched and the solvent was removed under high vacuum, the residue was treated with ammonium hydroxide (28%, 10 ml) at ambient temperature for 3 hours before evaporating to dryness. The residue was dissolved in water (5 ml) and washed twice with CH2Cl2. Ion exchange and reverse phase HPLC purification gave 8 in 30% yield.
31P NMR (161.9 MHz, D2O) d -3.78 (d, Jp,p = 18 Hz), -11.33 (d, Jp,p = 18 Hz), -18.87 (t, Jp,p = 18 Hz). ESI-MS (neg.) [M-H]- 452.0. UV (20 mM Tris-HCl pH 7.5) lmax 271 nm.
References for supporting information:
(1) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635.
(2) Schöning, K.-U., Scholz, P.; Wu X.; Guntha, S.; Delgado, G.; Krishnamurthy, R.; Eschenmoser, A. Helvetica Chimica Acta 2002, 85, 4111-4153.
(3) Hoard, D. E.; Ott, D. G. J. Am. Chem. Soc. 1965, 87, 1785-1788.

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