10348 J. Am. Chem. Soc., Vol. 118, No. 43, 1996
Weinstein et al.
plates (F-254) were from Selecto Scientific. 5′-DMT-riboinosine-2′-
TBDMS-3′-CNEt amidite was purchased from ChemGenes Corp.
Disposable reversed phase C18 columns were purchased from Waters.
Spectroscopy. FAB mass spectra were recorded on a VG Analytical
7070E mass spectrometer operating with a PDP 11/250 data system
and an Ion Tech FAB ion gun working at 8 kV. 3-Nitrobenzyl alcohol
was used as a matrix unless stated otherwise. High-resolution FAB
mass spectra were obtained on a VG ZAB/E spectrometer at the EPSRC
Mass Spectrometry Service Centre (Swansea, U.K.). NMR spectra were
measured on a Bruker AMX400, a Bruker AC200, or a Varian VXR-
300S spectrometer, and chemical shifts are given in parts per million
downfield from an internal standard of tetramethylsilane, unless stated
otherwise. 31P NMR spectra are referenced to 85% phosphoric acid.
Chromatography. Analytical thin-layer chromatography was per-
formed on silica gel coated aluminum plates impregnated with a
fluorescent indicator (254 nm). Nucleosides were visualized either as
black spots by spraying with a solution of 5% (v/v) sulfuric acid and
3% (w/v) phenol in ethanol and charring at 120 °C or by quenched
fluorescence under illumination with short wavelength UV light. Flash
column chromatography was performed using either silica gel 60 (230-
400 mesh) or octadecyl-functionalized silica gel (Aldrich). HPLC was
conducted using a Varian 500 Star liquid chromatograph equipped with
a Varian UV50 detector recording at 260 nm, and analysis performed
on a Nucleosil C18 reversed phase column, using a gradient of 30%
acetonitrile in 50 mM triethylammonium acetate (pH 6.5) over 20 min,
with a flow rate of 1 mL/min.
MHz, CDCl3) δ 8.26 (1H, br s), 8.06 (1H, br s), 7.47-7.19 (12H, m),
6.81 (2H, d, J ) 8.8 Hz), 6.45 (1H, br s), 5.97 (1H, s), 5.29 (1H, br s),
4.45 (1H, m), 4.13 (1H, m), 3.71 (3H, s), 3.6-3.3 (2H, m); 13C NMR
(75 MHz, CDCl3) δ 158.79, 158.12, 148.01, 146.34, 144.11, 143.92,
139.55, 135.14, 130.49, 128.56, 127.93, 127.15, 123.76, 113.24, 91.11,
86.98, 83.44, 79.87, 68.51, 55.12, 29.52; MS m/z (FAB+) 673 (M +
Na+, 6.7), 651 (M + H+, 9.5), 273 (trityl, 100), 137 (hypoxanthine +
2H, 62.8); FAB HRMS found m/z (M + H)+ 651.1119, C30H28N4O5I
requires m/z (M + H)+ 651.1104.
9-[5-O-(Monomethoxytrityl)-3-deoxy-3-thio-3-S,2-O-diacetyl-â-
D-ribofuranosyl]hypoxanthine (6). 9-[5-O-(Monomethoxytrityl)-3-
deoxy-3-iodo-â-D-xylofuranosyl]hypoxanthine (5) (3.85 g, 5.92 mmol)
was dissolved in DMF (40 mL). Potassium thioacetate (2 g, 17.7 mmol,
3 equiv) was added, and the solution stirred for 36-48 h at 40 °C. The
solution was partitioned between CH2Cl2 (100 mL) and saturated
NaHCO3/NaCl (1:1) (100 mL), and the organic phase was washed with
water (3 × 50 mL). The organic phase was dried (MgSO4), filtered,
evaporated in Vacuo, and coevaporated with toluene. The resulting
products were purified by silica gel column chromatography eluting
with MeOH (0-3%) in CH2Cl2. The fractions collected which
contained the products were combined and evaporated in Vacuo to give
6 as a white amorphous solid (1.9 g, 50%): 1Η NMR (200 MHz,
CDCl3) δ 8.19 (1H, br s), 8.12 (1H, br s), 7.43-7.19 (12H, m), 6.82
(2H, d, J ) 8.8 Hz), 6.15 (1H, d, J ) 1.64 Hz), 5.85 (1H, dd, J )
1.64, 5.5 Hz), 4.82 (1H, dd, J ) 5.5, 9.9 Hz), 4.32 (1H, m), 3.77 (3H,
s), 3.45 (2H, m); MS m/z (FAB+) 641 (M + H, 3), 137 (hypoxanthine
+ 2H, 25.4). Anal. Calcd for C34H32O7N4S: C, 63.74; H, 5.03; N,
8.74. Found: C, 63.44; H, 5.01; N, 8.69.
9-[5-O-(Monomethoxytrityl)-3-deoxy-3-S-(5-nitropyridyl-2-disul-
fanyl)-â-D-ribofuranosyl]hypoxanthine (7). The mixed disulfide was
prepared essentially as described.30 To a stirred solution of 5′-O-
(monomethoxytrityl)-3′-S,2′-O-diacetyl-3′-thioinosine (1.28 g, 2 mmol)
dissolved in the minimum amount of methanol was added a solution
of methanolic sodium hydroxide (10 M, 0.6 mL). After stirring for
1-2 h under an atmosphere of nitrogen, the reaction was complete
[TLC, MeOH/CH2Cl2 (9:1) as eluent] and the solution was neutralized
with dilute acetic acid. The solution was partitioned between CH2Cl2
(25 mL) and H2O (25 mL), the CH2Cl2 layer was separated, and 2,2′-
dithiobis(5-nitropyridine) (1.86 g, 6 mmol, 3 equiv) was added. After
stirring for 30 min, saturated aqueous NaHCO3 solution (20 mL) was
added. The organic phase was separated, washed with water (40 mL),
dried (MgSO4), filtered, and evaporated in Vacuo. The crude product
was purified by silica gel column chromatography eluting with a
gradient of MeOH (0-5%) in CH2Cl2. Appropriate fractions were
pooled and evaporated in Vacuo to give a yellow foam (1.1 g, 74%):
1H NMR (200 MHz, CDCl3) δ 9.20 (1H, d, J ) 2.2 Hz,), 8.24 (1H, d,
J ) 9.4 Hz), 8.12 (1H, s), 8.06 (1H, s), 7.57 (1H, d, J ) 9.4 Hz),
7.39-7.14 (12H, m), 6.77 (2H, d, J ) 8.1 Hz), 6.27 (1H, br s), 6.11
(1H, s), 4.72 (1H, m), 4.33 (1H, m), 4.12 (1H, m), 3.77 (3H, s), 3.62(1H,
m), 3.48 (1H, m); MS m/z (FAB+) 711 (M + H+, 3). It was noted
that 7 slowly decomposed to give the symmetrical dinucleoside
disulfide; it was therefore converted to 8 immediately after isolation.
9-[5-O-(Monomethoxytrityl)-3-deoxy-3-S-(5-nitropyridyl-2-disul-
fanyl)-2-O-(tert-fbutyldimethylsilyl)-â-D-ribofuranosyl]hypoxan-
thine (8). Compound 8 was prepared as described.30 The product was
obtained as a pale yellow amorphous solid (88%): 1H NMR (400 MHz,
CDCl3) δ 9.13 (1H, d, J ) 2.2 Hz), 8.24 (1H, dd, J ) 2.6, 9.4 Hz),
8.19 (1H, s), 8.07 (1H, s), 7.67 (1H, d, J ) 9.4 Hz), 7.34-7.20 (12H,
m), 6.75 (2H, d, J ) 8.2 Hz), 6.07 (1H, d, J ) 2.5 Hz), 4.99 (1H, dd,
J ) 2.6, 5.2 Hz), 4.56 (1H, m), 4.05 (1H, dd, J ) 5.4, 7.9 Hz), 3.75
(3H, s, OMe), 3.73 (1H, dd, J ) 2.4, 10.8 Hz), 3.73 (1H, dd, J ) 3.9,
10.8 Hz), 0.94 (9H, s), 0.20 (3H, s), 0.11 (3H, s, Me); MS m/z (FAB+)
825 (M + H+, 3.6), 767 (M - tBu+, 3.4); 13C NMR (100 MHz, CDCl3)
δ 167.36, 159.14, 158.71, 148.45, 145.18, 145.10, 143.83, 143.73,
142.18, 138.78, 134.80, 131.54, 130.40, 128.38, 127.89, 127.18, 125.35,
119.45, 113.14, 90.39, 87.01, 83.65, 77.53, 63.26, 55.21, 53.91, 25.69,
18.13, -4.60, -4.90. Anal. Calcd for C41H44N6O7S2Si: C, 59.69; H,
5.38; N, 10.19. Found: C, 59.59 H, 5.37 N, 10.14.
TLC analysis of enzymatic and chemical reactions was done on PEI
cellulose.86 Solvent systems were (a) 0.9 M NH4OAc (pH 7)/100 mM
DTT, (b) 0.9 M NH4OAc (pH 8)/100 mM DTT, and (c) 0.5 M LiCl/
100 mM DTT. In all cases, resolution was improved by developing
the plate in H2O followed by complete air-drying before applying the
samples.
9-(3-Deoxy-3-iodo-â-D-xylofuranosyl)hypoxanthine (4). Com-
pound 4 was synthesized using modifications of methods reported for
adenosine.87,88 2′,3′-anhydroinosine89 (5.88 g, 23.5 mmol) and sodium
iodide (15.8 g, 105 mmol, 4.5 equiv) were suspended in dry acetonitrile
(120 mL). To this stirred solution was added dropwise boron
trifluoride-etherate (63 mL) at room temperature. After 1 h, the reaction
mixture was quenched with 400 mL of a saturated aqueous NaHCO3
solution, containing NaS2O3 (3 g). The resultant solution was
evaporated in Vacuo to give a white solid. Nucleosidic material was
extracted into methanol (100 mL). The insoluble inorganic salts were
removed by filtration, and the methanolic solution was evaporated in
Vacuo. The crude product was recrystallized from hot water, and a
white solid was collected by filtration. The mother liquor was
concentrated to yield a further crop of material (5.33 g, 60%): 1H NMR
(200 MHz, DMSO-d6) δ 9.05 (1H, br s), 8.38 (1H, s), 8.17 (1H, s),
6.38 (1H, d, J ) 5.5 Hz), 5.86 (1H, d, J ) 4.96 Hz, 5.30 (1H, t, J )
4.94 Hz), 4.99 (1H, dd, J ) 5.48, 6.77 Hz), 4.58 (1H, t, J ) 6.6 Hz),
4.26 (1H, m, H4′), 3.86 (2H, m); 13C NMR (75 MHz, DMSO-d6) δ
156.83, 148.32, 146.23, 138.72, 124.50, 88.32, 81.04, 80.08, 65.88,
28.56; MS m/z (FAB+) 401 (M + Na+, 6.5), 379 (M + H+, 40). FAB
HRMS found m/z (M + Na)+ 400.9725, C10H11N4O4INa requires m/z
(M + Na)+ 400.9722.
9-[5-O-(Monomethoxytrityl)-3-deoxy-3-iodo-â-D-xylofuranosyl]-
hypoxanthine (5). 9-(3-Deoxy-3-iodo-â-D-xylofuranosyl)hypoxan-
thine (5.7 g, 15.1 mmol) was dried by coevaporation with dry pyridine
(3 × 20 mL) and finally suspended in dry pyridine (80 mL).
Monomethoxytrityl chloride (MMTCl) (9.32 g, 30.2 mmol, 2 equiv)
was added, and the reaction was stirred overnight. The solution was
partitioned between CH2Cl2 (250 mL) and saturated NaHCO3/NaCl (1:
1) (250 mL), and the organic phase was washed with water. The
organic phase was dried (Na2SO4), filtered, evaporated in Vacuo, and
then coevaporated with toluene. The resulting residue was purified
by silica gel column chromatography eluting with MeOH (0-6%) in
CH2Cl2 to give 5 as a pale yellow foam (6.4 g, 65%): 1Η NMR (200
(86) Uhlenbeck, O. C. Nature 1987, 328, 596-600.
(87) Russell, A. F.; Greenberg, S.; Moffatt, J. G. J. Am. Chem. Soc. 1973,
95, 4025.
(88) Mengel, R.; Wiedner, H. Chem. Ber. 1976, 109, 1395.
(89) Bhat, V.; Stocker, E.; Ugarkar, B. G. Synth. Commun. 1992, 22,
1481.
2′,3′-Bis(O-tert-butyldimethylsilyl)uridin-5′-yl-H-phosphonate, Tri-
ethylammonium Salt (9). Freshly distilled phosphorus trichloride (1
mL, 11.45 mmol, 5 equiv) and N-methylmorpholine (12.64 mL, 114.5
mmol) were mixed with anhydrous CH2Cl2 (13 mL). To this solution