Beilstein J. Org. Chem. 2015, 11, 1553–1560.
C-18 RP column (250 × 4.6 mm, 5 μm; flow rate 1.0 mL min−1; H3&H5 of Ph), 5.42 (s, 8H, N-CH2-Ph), 4.30 (s, 8H, S-CH2-
λ = 260 nm) was used. Reactions were monitored by TLC triazole), 3.97 (s, 8H, CH2-pentaerythritol), 3.79 (t, J = 5.5 Hz,
(Merck, silica gel 60 F254), using short wavelength UV or char- 8H, CH2OH), 2.73 (br t, 8H, S-CH2CH2) ppm; 13C NMR (100
ring with 10% aq H2SO4 for detection (system A: 5% MeOH in MHz, CDCl3) δ 158.9, 144.2, 129.6, 127.2, 121.9, 115.2, 66.4,
CH2Cl2; system B: 10% MeOH in CH2Cl2). Mass spectra were 59.9, 53.7, 44.8, 42.5, 32.8 ppm; ESIMS m/z: [M + H]+ calcd
recorded with a Bruker Daltonics MicrOTOFQ spectrometer for C53H65N12O8S8, 1253.28; found, 1253.27; m/z: [M + Na]+
using ESI mode.
calcd for C53H64N12NaO8S8, 1275.26; found, 1275.26.
Tetrakis-O-(4-azidomethylphenyl)pentaerythritol (1) was Bis(benzotriazol-1-yl) 2-chlorophenyl phosphate: The
prepared as described previously [7].
couplings were carried out essentially as described previously
[8]. A solution of 2-chlorophenyl phosphorodichloridate
Tetrakis-O-{4-[4-(acetylthiomethyl)-1H-1,2,3-triazol-1- (7.6 mmol, 1.88 g) in anhydrous dioxane (5.75 mL) was added
ylmethyl]phenyl}pentaerythritol (2): S-Propargyl thioacetate in one portion to a suspension of 1-hydroxybenzotriazole
(1.73 mL, 15.0 mmol) was added to the solution of compound 1 (15.2 mmol, 2.06 g; dried in vacuo over P2O5 at 55 °C for 3 d)
(1.00 g, 1.51 mmol), sodium ascorbate (30 mg, 0.15 mmol) and and pyridine (15 mmol, 1.2 mL) in anhydrous dioxane (30 mL).
CuI (115 mg, 0.605 mmol) in dry DMAc (5.0 mL) in a Pyrex The reaction mixture was stirred for 2 h, and the precipitate was
tube. The tube was degassed via three freeze–pump–thaw filtered off under anhydrous conditions to give a stock solution
cycles, and the mixture was stirred at 50 °C for 12 h. Water of bis(benzotriazol-1-yl) 2-chlorophenyl phosphate
(10 mL) was added to the reaction mixture and extracted with (0.2 mol L−1) as a clear colorless liquid. The solution
ethyl acetate (20 mL × 3). The combined organic layer was (ρ = 1.057 g L−1) could be stored for several weeks at −20 °C.
washed with saturated NaHCO3, dried with Na2SO4, and the
solvents were evaporated to dryness. The residue was purified General procedure for the coupling of 1-hydroxybenzotria-
by silica gel chromatography (CH2Cl2/MeOH, 97:3 v/v) to give zole-activated phosphotriester building blocks: The coupling
2 (1.2 g, 70%) as a white foam. 1H NMR (400 MHz, CDCl3) δ cycle was analogous to that described previously [8]. 5’-O-
7.35 (s, 4H, H5 of triazole), 7.18 (d, J = 8.6 Hz, 8H, H2&H6 of (4,4’-Dimethoxytrityl)thymidine (0.30 g, 0.54 mmol) was dried
Ph), 6.89 (d, J = 8.6 Hz, 8H, H3&H5 of Ph), 5.38 (s, 8H, by co-evaporation with anhydrous pyridine (3 × 5 mL) and
N-CH2-Ph), 4.32 (s, 8H, AcS-CH2-), 4.13 (s, 8H, CH2- concentrated to a small volume followed by the addition of the
pentaerythritol), 2.31 (s, 12H, -SAc); 13C NMR (125 MHz, stock solution of bis(benzotriazol-1-yl) 2-chlorophenyl phos-
CDCl3) δ 195.2, 158.9, 144.6, 129.7, 127.2, 122.1, 115.1, 66.4, phate in dioxane (0.54 mmol, 0.2 mol L−1, 2.72 mL), giving
53.6, 44.7, 30.4, 23.9 ppm; ESIMS m/z: [M + H]+ calcd for 5’-O-(4,4’-dimethoxytrityl)thymidine 3’-(benzotrizol-1-yl
C53H57N12O8S4, 1117.33, found, 1117.36; m/z: [M + Na]+ 2-chlorophenyl phosphate) 4 in dioxane. The formation of a
calcd for C53H56N12NaO8S4, 1139.31; found, 1139.32.
product with zero mobility on TLC (system B) indicated that
the reaction was complete. In a separate vessel, support 3
Tetrakis-O-{4-[4-(2-hydroxyethyldisulfanylmethyl)-1H- (0.068 mmol, 0.086 g) was dried by co-evaporation with anhy-
1,2,3-triazol-1-ylmethyl]phenyl}pentaerythritol (3). drous pyridine (3 × 5 mL), and then 4 in dioxane and
Degassed butylamine in MeOH (0.21 mL, 1.0 mol L−1, 1-methylimidazole (2 mmol, 0.218 mL) were added under
2.15 mmol) was added to the stirred solution of compound 2 nitrogen. The reaction mixture was stirred for 2 h to obtain the
(600 mg, 0.537 mmol) in degassed MeOH. The mixture was tetravalent nucleoside cluster 7a, transferred to a stoppered
stirred under N2 for 12 h. Degassed MeCN/DCM (5 mL, 1:1 50 mL plastic tube, and MeOH (46 mL) was added. The precip-
v/v) was added and the mixture was neutralized with dry acidic itate formed was kept at −20 °C overnight, isolated by centrifu-
ion exchange resin and filtered off. The solvent was removed by gation, and dried to give 7a (0.20 g, 70%) as a white solid. The
evaporation and the crude product was used in the next reaction precipitate and supernatant were analyzed by HPLC to verify
without purification. 2-(Pyridine-2-yldisulfanyl)ethanol the completeness of precipitation and the identity of the precipi-
(581 mg, 3.11 mmol) was added to the crude compound in tate was verified by ESIMS (Table 1).
degassed MeCN/DCM/MeOH (4 mL, 2:1:1 v/v/v) under N2.
The mixture was stirred for 30 min and the progress was moni- General procedure for detritylation: The detritylation cycle
tored by TLC. Solvents were removed by evaporation and the was analogous to that described previously [8].Tetravalent
product was purified by silica gel chromatography (DCM/ support-bound thymidine monomer 7a (0.048 mmol, 0.20 g)
MeOH, 95:5 v/v) to give 3 (120 mg, 19%) as a thick oil. was dissolved in a mixture of DCM and MeOH (1:1 v/v,
1H NMR (500 MHz, CDCl3) δ 7.40 (s, 4H, H5 of triazole), 7.18 25 mL), and HCl in MeOH (0.115 mL of 1.25 mol L−1 solution)
(d, J = 8.6 Hz, 8H, H2&H6 of Ph), 6.88 (d, J = 8.6 Hz, 8H, was added portion wise. The reaction was monitored by TLC
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