908 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Shishkina and J ohnson
137.7, 137.6, 128.4, 128.3, 127.9, 127.8, 77.9, 77.2, 73.4, 72.5,
71.5, 71.8, 71.0, 70.9, 69.2, 69.1, 66.9, 66.8, 33.7, 32.8. FAB-
MS: m/z 347.12 (M + H)+. Anal. Calcd for C20H26O5: C, 69.34;
H, 7.56. Found: C, 68.69; H, 7.56.
studies of abasic site-containing DNA-enzyme complexes
would be difficult to prepare using uracil DNA glycosy-
lase. Again, enzymatic reactions in practical terms often
do not run to completion, stopping at 80-90% substrate
conversion. The method described in this paper is novel
and avoids all of the problems described above.
4,6-O-Dib en zyl-1,2,5-O-t r is(ter t-b u t yld im et h ylsilyl)-3-
d eoxyh exitol (7). The dried product 6 (3.2 g, 9 mmol), tert-
butyldimethylsilyl chloride (TBSCl) (5.3 g, 35 mmol), and
imidazole (4.8 g, 70 mmol) were dissolved in anhydrous dim-
ethylformamide (14 mL). The reaction mixture was stirred
under nitrogen for 16 h at 23 °C. Water was then added to the
vigorously stirred reaction mixture, but no precipitate appeared.
The reaction mixture was extracted with ethyl acetate (2 × 200
mL), and the organic layers were combined, then washed with
water, and concentrated in vacuo. The crude reaction product
was purified by silica gel column chromatography (40:60 dichlo-
romethane/hexane) to give 5.6 g of 7 (91%) as an oil. TLC: Rf
0.64 (40:60 dichloromethane/hexane), 0.4 (20:80 chloroform/
hexane), 0.87 (10:90 methanol/dichloromethane). 1H NMR
(CDCl3): δ 7.30 (m, 10H, ArH), 4.52 (d, J ) 2.5 Hz, 4H, CH2-
Ar), 4.02-3.70 (m, 3H, H-1, H-5, H′-1), 3.59-3.44 (m, 4H, H-6,
H-2, H′-6, H-4), 1.75 (m, 2H, H-3), 0.90 (s, 27H, C-CH3), 0.04 (s,
18H, Si-CH3). 13C NMR (CDCl3): δ 137.1, 128.3, 127.5, 78.4,
78.0, 74.3, 73.5, 72.4, 71.2, 71.1, 67.9, 67.8, 36.8, 36.9. Anal.
Calcd for C38H68O5Si3: C, 66.22; H, 9.94. Found: C, 66.45; H,
10.01.
Exp er im en ta l P r oced u r es
All reagents and solvents, unless otherwise specified, were
the best grade commercially available (Aldrich and Fluka) and
were used without further purification. NMR (1H, 13C, and 31P)
spectra were recorded on
a Bruker AC-250 spectrometer.
Chemical shifts are reported in parts per million relative to
tetramethylsilane in the proton spectra and to the deuterated
solvent in the carbon spectra. Mass spectra were recorded on a
Micromass TRIO-2000 instrument in fast atom bombardment
(FAB)1 mode and on a Micromass Quattro spectrometer in
electrospray ionization (ESI) mode. Elemental analyses were
performed by Schwarzkopf Microanalytical Laboratory (Wood-
side, NY). TLC was performed on silica gel sheets (Riendel-
deHae¨n, Sleeze, Germany) containing a fluorescent indicator.
Flash column chromatographic separation was carried out on
60 Å (230-400 mesh) silica gel (TSI Chemical Co., Cambridge,
MA). All experiments dealing with moisture-sensitive com-
pounds were conducted under dry nitrogen.
1,2,5-O-Tr is(ter t-bu tyld im eth ylsilyl)-3-d eoxyh exitol (8).
A reaction mixture containing 7 (4.5 g, 6.5 mmol) and 10 g of
palladium on activated carbon (10% Pd) in 70 mL of methanol
was hydrogenated under pressure (50 psi) for 20 h and then
filtered through Celite. The solvent was removed in vacuo, and
the resulting crude product was purified by silica gel column
chromatography (2:98 methanol/dichloromethane) to give pure
8 (1.9 g, 60%), as a white amorphous solid. TLC: Rf 0.42 (2:98
MeOH/dichloromethane), 0.18 (chloroform). 1H NMR (CDCl3):
δ 3.95-3.78 (m, 2H, H-1), 3.75-3.42 (m, 5H, H-6, H-5, H′-6,
H-2, H-4), 3.16 (bs, 2H, OH), 2.09-1.40 (m, 2H, H-3), 0.92 (s,
27H, C-CH3), 0.09 (s, 12H, Si-CH3), 0.06 (s, 6H, Si-CH3). 13C
NMR (CDCl3): δ 75.1, 73.0, 71.2, 66.5, 64.8, 37.4, 36.8, 25.8,
18.0, -4.5. FAB-MS: m/z 509.3 (M + H)+. Anal. Calcd for
1,3-(R)-O-Diben zyl-2-(S)-h yd r oxy-5-h exen e (5). Butyllith-
ium (52 mL of a 2 M solution in pentane) was added to a stirred
suspension of methyl triphenylphosphonium bromide (36.6 g,
102.5 mmol) in 400 mL of dry toluene under
a nitrogen
atmosphere, and the reaction mixture was further stirred at
room temperature for 2 h. A solution of compound 4 (13.0 g, 41
mmol) prepared according to the method of Vandenriessche et
al. (21) in 50 mL of dry toluene was added slowly to this mixture
using a dropping funnel, and after the addition was complete,
the reaction mixture was heated at 50 °C for 1 h. The reaction
mixture was cooled to room temperature; 100 mL of water was
added, and the reaction mixture was extracted with ethyl
acetate (2 × 300 mL). The combined organic layers were
C
24H56O5Si3: C, 56.64; H, 11.09. Found: C, 57.11; H, 11.02.
successively washed with
a saturated ammonium chloride
6-O-(4,4′-Dim eth oxytr ityl)-1,2,5-O-tr is(ter t-bu tyld im eth -
solution, and then with water, and finally concentrated in vacuo.
The crude reaction mixture was purified by silica gel column
chromatography (20:80 ethyl acetate/hexane) to give 7 g of 5
ylsilyl)-3-d eoxyh exitol (9). Compound 8 (500 mg, 0.98 mmol)
was treated with 4,4′-dimethoxytrityl chloride (DMTCl) (406 mg,
1.2 mmol) in 4 mL of pyridine at room temperature over the
course of 30 min. The pyridine was removed in vacuo, and the
residue was dissolved in dichloromethane (40 mL). This solution
was washed with saturated NaHCO3 (2 × 20 mL) and water.
The organic layer was dried (Na2SO4) and evaporated in a rotary
evaporator. The residue was then purified by silica gel column
chromatography (dichloromethane, containing 0.5% triethy-
lamine) to give pure 9 (796 mg, 98%). TLC: Rf 0.49 (dichlo-
romethane containing 0.5% triethylamine). 1H NMR (CDCl3):
δ 7.47-7.16 (m, 9H, ArH), 6.85-6.78 (m, 4H, ArH), 4.01-3.87
(m, 2H, H-1), 3.78 (s, 6H, O-CH3), 3.63-3.40 (m, 2H, H-5, H-2),
3.23-2.75 (m, 3H, H-6, H-4, H′-6), 1.75-1.49 (m, 2H, H-3), 0.88
(s, 27H, C-CH3), 0.11-0.01 (m, 18H, Si-CH3). 13C NMR
(CDCl3): δ 158.4, 130.1, 128.3, 127.7, 126.7, 113.0, 86.3, 75.8,
71.2, 70.9, 69.3, 67.2, 65.1, 55.2, 36.1, 35.8, 25.9, 18.1, -4.3. ESI-
MS: m/z 809.2 (M - H)-.
6-O-(4,4′-Dim eth oxytr ityl)-4-O-[(N,N-d iisop r op yla m in o)-
(2-cya n oeth oxy)p h osp h in yl]-1,2,5-O-tr is(ter t-bu tyld im eth -
ylsilyl)-3-d eoxyh exitol (10). Compound 9 (150 mg, 0.18 mmol)
was coevaporated with dry toluene (3 × 5 mL), dissolved in
dichloromethane (1.5 mL), and treated with triethylamine (350
µL, 2.5 mmol) and (N,N-diisopropylamino)(2-cyanoethoxy)chlo-
rophosphine (300 µL, 1.26 mmol). The reaction mixture was
stirred at 23 °C for 7 h and then diluted with dichloromethane
(15 mL). The resulting solution was washed with saturated
NaHCO3 (2 × 10 mL), dried (Na2SO4), and then concentrated
in vacuo. The crude product was purified by silica gel column
chromatography (35:65 hexane/dichloromethane containing 0.5%
triethylamine) to obtain the desired product 10 (149 mg, 79%)
1
(54%) as an oil. H NMR (CDCl3): δ 7.38-7.29 (m, 10H, ArH),
6.00-5.83 (m, 1H, H-5), 5.2-5.08 (m, 2H, H-6), 4.62-4.50 (m,
4H, CH2Ar), 3.85 (m, 1H, H-2), 3.66-3.6 (m, 2H, H-1), 3.6-3.5
(m, 1H, H-3), 2.47-2.45 (m, 3H, H-4, H′-4, OH). 13C NMR
(CDCl3): δ 138.2, 137.8, 134.5, 128.3, 127.7, 127.6, 127.5, 117.2,
79.0, 73.4, 72.2, 71.4, 71.0, 34.7. FAB-MS: m/z 313.16 (M + H)+.
4,6-O-Diben zyl-3-d eoxyh exitol (6). To solution of com-
pound 5 (6.4 g, 21.8 mmol) in 14 mL of 6.3% hydrogen peroxide
(25 mmol) in tert-butyl alcohol was added 0.3 mL of a 2.5%
solution of osmium tetroxide (0.03 mmol) in tert-butyl alcohol.
The reaction mixture was stirred for 1 h at room temperature
(by which time the initial orange color had changed to yellow-
green) and then concentrated in vacuo. The resulting residue
was dissolved in ethyl acetate (100 mL) and washed with
aqueous solutions of Na2SO3 (2 × 100 mL), NaHCO3 (2 × 70
mL), and brine (2 × 70 mL). The organic layer was dried (Na2-
SO4) and concentrated in vacuo. The crude reaction mixture was
purified by silica gel column chromatography (10:90 methanol/
dichloromethane) to give 5.2 g of 6 (74%) as a white amorphous
solid. TLC: Rf 0.29 (10:90 methanol/dichloromethane), 0.35
1
(ethyl acetate). H NMR (CDCl3): δ 7.33-7.29 (m, 10H, ArH),
4.53 (s, 4H, CH2Ar), 4.00-3.74 (m, 2H, H-1), 3.74-3.65 (m, 1H,
H-5), 3.65-3.54 (m, 3H, H-2, H-6, H′-6), 3.45-3.39 (q, 1H, H-4),
3.19 (s, 3H, OH), 1.76-1.70 (t, 2H, H-3). 13C NMR (CDCl3): δ
1
Abbreviations: DMT, 4,4′-dimethoxytrityl; dR, deoxyribose; ESI,
electrospray ionization; FAB, fast atom bombardment; F, 1,4-anhydro-
2′-ribitol; TEAAc, triethylammonium acetate; TBS, tert-butyldimeth-
ylsilyl.