A R T I C L E S
Cho et al.
1
3
water, then a 15 min linear gradient to 90% water, 5 min linear gradient
to 80% water, 5 min at 80%, 10 min linear gradient to 20% water, and
butylcarbamoyl)-4-amino-2- C-but-1-ene 10 (58 mg, 65.6%) as a
colorless oil: 1H NMR (CDCl
) δ 5.76 (m, JC-H ) 144 Hz, 1H), 4.45
(br, 1H), 3.01 (m, 2H), 2.24 (m, 2H), 1.45 (s, 9H).
e) N-(tert-Butylcarbamoyl)-3,4-dihydroxy-1-amino-3- C-butane
3
5
min at 20% followed by 5 min linear gradient to initial conditions.
MALDI-TOF mass spectra (negative ion) of modified oligodeoxy-
nucleotides were obtained on a Voyager Elite DE instrument (Perseptive
Biosystems) at the Vanderbilt Mass Spectrometry Resource Facility
using a 3-hydroxypicolinic acid (HPA) matrix containing ammonium
hydrogen citrate (7 mg/mL) to suppress sodium and potassium adducts.
13
(
13
(
11). To a stirred solution of N-(tert-butylcarbamoyl)-4-amino-2- C-
but-1-ene 10 (45 mg, 0.26 mmol), N-methylmorpholine N-oxide (35
mg, 0.286 mmol), THF (0.8 mL), t-BuOH (0.32 mL), and water (0.16
mL) was added osmium tetroxide (10 µL, 0.1 M in benzene, 1.0 µmol).
The mixture was stirred for 12 h, and then a second portion of osmium
tetroxide (6 µL, 0.1 M in benzene, 0.6 µmol) was added, and stirring
continued for an additional 8 h. The reaction was quenched by the
1
13
H and C NMR data for compounds 7-12 were recorded at 300 and
7
5 or 400 MHz and 100 MHz (Bruker Instruments), respectively.
Synthesis of 13C-Labeled γ-OH-PdG Adducted Oligodeoxynucleo-
tide. (a) tert-Butyl N-(2-Hydroxyethyl)carbamic Acid (7). To a stirred
solution of ethanolamine (0.611 g, 10 mmol) and 1 N NaOH (10 mL)
cooled to 0 °C, a solution of di-tert-butyl dicarbonate (2.4 g, 11 mmol)
in methylene chloride (30 mL) was added dropwise. The mixture was
allowed to warm to room temperature and vigorously stirred overnight.
The layers were separated, and the organic phase was successively
addition of 5% aqueous NaHSO (5 mL) and vigorous stirring for 15
3
min and then poured over water. The layers were separated, and the
aqueous phase was extracted with methylene chloride (10 × 3 mL).
The combined organic extracts were dried over Na SO , filtered, and
2
4
concentrated under reduced pressure. Purification by flash chromatog-
raphy on silica gel, eluting with 1-2% methanol in chloroform, gave
1
washed with 0.1 N HCl, 5% NaHCO
MgSO , filtered, and concentrated. Purification by flash chromatography
on silica gel, eluting with 2-3% methanol in chloroform, gave 7 (1.14
3
, and brine, then dried over
11 (37 mg, 69.0%) as an inseparable mixture of diastereomers:
H
4
NMR (CDCl ) δ 4.65-4.50 (br, 1H), 3.70 (m, J
3
C-H
) 135 Hz, 1H),
3.60 (m, 2H), 2.92 (m, 2H), 3.1 (m, 1H), 2.4 (m, 1H), 1.5 (m, 2H),
1.45 (s, 9H).
1
g, 70%): H NMR (CDCl
3
) δ 5.02 (br, 1H), 3.70 (dd, 2H, J ) 3.8, 7.7
Hz), 3.29 (dd, 2H, J ) 3.8, 7.7 Hz), 2.66 (br, 1H), 1.45 (s, 9H).
13
(
f) 4-Amino-2- C-butane-1,2-diol (12). To a solution of 11 (30
1
3
(
b) N-tert-Butyl [2-( C-Cyano)ethyl]carbamic Acid (8). To a
stirred solution of 7 (386 mg, 2.4 mmol) and triethylamine (0.50 mL,
.41 mmol, 3.6 mmol) in anhydrous methylene chloride (5 mL) cooled
to 0 °C was added a solution of methanesulfonyl chloride (310 mg,
.64 mmol) in anhydrous methylene chloride (2 mL) dropwise. The
reaction mixture was stirred at room temperature for 2 h and then
quenched with saturated NaHCO solution. The organic layer was
separated, dried over K CO , filtered, and concentrated under reduced
pressure. The crude mesylate was dissolved in DMSO (10 mL), and
mg, 0.145 mmol) in methylene chloride (1.5 mL) and methanol (0.5
mL) was added clean H-Amberlyst 15 resin (0.3 g).27 The mixture was
9
gently shaken for 14 h, after which time TLC showed that 11 had been
consumed. The resin was removed by filtration and successively washed
with THF and methanol. This amine-bound resin was transferred to a
4 M solution of ammonia in methanol and was gently shaken for 50
min. To this mixture was added methanol (3 × 5 mL) in order to
dissolve all of the deprotected amine. The resin was then removed by
filtration, and the filtrate evaporated under reduced pressure to give
2
3
2
3
13
1
K CN (237 mg, 3.6 mmol) was added in the solution. The solution
12 (14 mg, 91%), which was used without further purification. H NMR
was heated at 40 °C for 15 h then cooled to room temperature. Water
(CD OD) δ 3.73 (m, J
1.6, (m, 1H) 1.5 (m, 1H).
C-H
) 135 Hz, 1H), 3.45 (m, 2H), 2.84 (m, 2H),
3
(
20 mL) was added to the solution, and the mixture was extracted with
ether (3 × 10 mL). The combined organic extracts were washed with
, filtered, and concentrated under
13
Synthesis of γ- C-PdG-Modified Oligodeoxynucleotide 5′-
brine (3 × 10 mL), dried over MgSO
4
6
GCTAGCXAGTCC-3′ (18). The O -trimethylsilylethane-2-fluoro-
inosine-modified oligodeoxynucleotide 16 (100 A260 units) was mixed
reduced pressure. Purification by flash chromatography on silica gel,
34
eluting with 12-15% ethyl acetate in hexanes, gave 8 (288 mg, 70%):
in a plastic test tube with diisopropylethylamine (150 µL), DMSO (500
1
H NMR (CDCl
H).
c) N-(tert-Butylcarbamoyl)-3-amino-1- C-propanal (9). To a
3
) δ 5.01 (br, 1H), 3.40 (m, 2H), 2.60 (m, 2H), 1.45 (s,
13
µL), and 4-amino-2- C-butane-1,2-diol 12 (6 mg). The reaction mixture
9
was stirred at 55 °C for 24 h. The solvents were evaporated under a
vacuum with a centrifugal evaporator, and the residue was dissolved
in 5% acetic acid (500 µL) and stirred for 2 h at room temperature to
1
3
(
stirred solution of 8 (280 mg, 1.64 mmol) in anhydrous methylene
chloride (5 mL) at -78 °C was added diisobutylaluminum hydride (1.0
M in methylene chloride, 4.1 mL) dropwise over 20 min. After the
addition was complete, the reaction mixture was stirred at -78 °C for
6
remove the O -trimethylsilylethane group. The mixture was neutralized
2
with 1 M NaOH and purified by HPLC to give the corresponding N -
13
(
3- C-3,4-dihydroxybutyl)guanine-modified oligodeoxynucleotide 17
10 min and then quenched by the addition of acetone (1 mL), followed
-
(68.7 A260 units - 68%). MALDI-TOF MS: calcd for [M - H]
by saturated aqueous NH Cl (10 mL). The resulting mixture was
4
3
733.7, found 3735.9.
allowed to warm to room temperature and stirred for 40 min. The
mixture was filtered through a pad of Celite, and the filtrate was washed
An aqueous solution of NaIO (500 µL, 20 mM) was added to a
solution of N -(3- C-3,4-dihydroxybutyl)guanine-modified oligode-
oxynucleotide 17 (68.6 A260 units) in 0.05 M, pH 7.0 phosphate buffer,
4
2
13
4
with brine, dried over MgSO , filtered, and evaporated under reduced
pressure. Purification by flash chromatography on silica gel, eluting
(
1
5
2 mL) and the reaction mixture was stirred at room temperature for
1
with 20-25% ethyl acetate in hexanes, gave 9 (86 mg, 30.3%):
NMR (CDCl ) δ 9.81 (d, 1H, JC-H ) 174 Hz), 4.96 (br, 1H), 3.41 (m,
H), 2.71 (m, 2H), 1.45 (s, 9H).
d) N-(tert-Butylcarbamoyl)-4-amino-2- C-but-1-ene (10). To a
stirred suspension of methyltriphenylphosphonium bromide (206 mg,
.576 mmol) in THF (5 mL) was added potassium tert-butoxide in
H
13
0 min. The mixture was purified by HPLC to give 8- C-8-hydroxy-
3
,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one) adducted oligode-
2
oxynucleotide 18. (63.7 A260 units - 93%) MALDI-TOF MS: calcd
1
3
(
-
for [M - H] 3701.6, found 3701.6.
0
(
27) Liu, Y.-S.; Zhao, C.; Bergbreiter, D. E.; Romo, D. J. Org. Chem. 1998,
THF (1 M in THF, 536 µL, 0.536 mmol), and the reaction mixture
was stirred for 30 min. The reaction mixture was cooled to 0 °C, and
a solution of 9 (88 mg, 0.51 mmol) in THF (2 mL) was added. The
reaction mixture was stirred for 1 h at room temperature and then
63, 3471-3473.
(28) Hunter, R.; Richards, P. Org. Biomol. Chem. 2003, 1, 2348-2356.
(
29) Borer, P. N. In Handbook of Biochemistry and Molecular Biology; CRC
Press: Cleveland, OH, 1975.
(30) Piotto, M.; Saudek, V.; Sklenar, V. J. Biomol. NMR 1992, 2, 661-665.
(
31) Mori, S.; Abeygunawardana, C.; Johnson, M.; VanZul, P. C. M. J. Magn.
quenched with saturated aqueous NH
separated. The aqueous phase was extracted with ether (3 × 10 mL).
The combined organic phases were dried over Na SO , filtered, and
4
Cl (10 mL), and the layers
Reson. Series B 1995, 108, 94-98.
(32) Talluri, S.; Wagner, G. J. Magn. Reson. 1996, 112, 200-205.
(33) Markley, J. L.; Bax, A.; Arata, Y.; Hilbers, C. W.; Kaptein, R.; Sykes, B.
D.; Wright, P. E.; W u¨ thrich, K. J. Mol. Biol. 1998, 280, 933-952.
2
4
evaporated under reduced pressure. Purification by flash chromatog-
raphy on silica gel eluting with 3% ethyl acetate in hexanes gave N-(tert-
(
34) Wishart, D. S.; Bigam, C. G.; Yao, J.; Abildgaard, F.; Dyson, H. J.; Oldfield,
E.; Markley, J. L.; Sykes, B. D. J. Biomol. NMR 1995, 6, 135-140.
17688 J. AM. CHEM. SOC.
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VOL. 127, NO. 50, 2005