Preparation of oligodeoxynucleotides containing a diastereoisomer of α-(N2-2′-deoxyguanosinyl)tamoxifen by phosphoramidite chemical synthesis

Article abstract of DOI:10.1021/tx0101494

Women treated with an antiestrogen tamoxifen (TAM) for endocrine therapy or prevention of breast cancer show an increased risk of developing endometrial cancer. TAM-DNA adducts have been detected in the liver of rodents treated with TAM and in the endometrium of women taking TAM. The major TAM adducts have been identified as diastereoisomers of trans- and cis -forms of α-(N²-deoxyguanosinyl)tamoxifen (dG-N²-TAM) and α-(N²-deoxyguanosinyl)-N-desmethyltamoxifen. In the study presented here, we prepared oligodeoxynucleotides containing a diastereoisomer of dG-N²-TAM by phosphoramidite chemical synthesis. Initially, the trans- and cis-forms of α-aminotamoxifen (α-NH₂-TAM) were synthesized from α-hydroxytamoxifen using the Mitsunobu reaction followed by hydrolysis. Thereafter by coupling the trans- and cis-form of α-NH₂-TAM with the DMT-derivative of 2-fluoro-(O⁶-2-(trimethylsilyl)ethyl)-2′-deoxyinosine, the trans- and cis-forms of DMT-dG-N²-TAM, respectively, were prepared in high yield and used in the preparation of the phosphoramidite precursors. Large quantities of oligodeoxynucleotides containing a trans- or a cis-form of dG-N²-TAM were prepared efficiently by automated DNA synthesizer. The incorporation of dG-N²-TAM adduct into the oligodeoxynucleotides was confirmed using ³²P-postlabeling/polyacrylamide gel electrophoresis analysis. These site-specifically modified oligodeoxynucleotides will be used for exploring biological properties and three-dimensional structure of TAM - DNA adducts.

Full text of DOI:10.1021/tx0101494

218
Chem. Res. Toxicol. 2002, 15, 218-225
P r ep a r a tion of Oligod eoxyn u cleotid es Con ta in in g a
Dia ster eoisom er of r-(N2-2′-Deoxygu a n osin yl)ta m oxifen
by P h osp h or a m id ite Ch em ica l Syn th esis
Y. R. Santosh Laxmi, Naomi Suzuki, Lakkaraju Dasaradhi,
Francis J ohnson, and Shinya Shibutani*
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New
York at Stony Brook, Stony Brook, New York 11794-8651
Received September 11, 2001
Women treated with an antiestrogen tamoxifen (TAM) for endocrine therapy or prevention
of breast cancer show an increased risk of developing endometrial cancer. TAM-DNA adducts
have been detected in the liver of rodents treated with TAM and in the endometrium of women
taking TAM. The major TAM adducts have been identified as diastereoisomers of trans- and
cis-forms of R-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM) and R-(N2-deoxyguanosinyl)-N-
desmethyltamoxifen. In the study presented here, we prepared oligodeoxynucleotides containing
a diastereoisomer of dG-N2-TAM by phosphoramidite chemical synthesis. Initially, the trans-
and cis-forms of R-aminotamoxifen (R-NH2-TAM) were synthesized from R-hydroxytamoxifen
using the Mitsunobu reaction followed by hydrolysis. Thereafter by coupling the trans- and
cis-form of R-NH2-TAM with the DMT-derivative of 2-fluoro-(O6-2-(trimethylsilyl)ethyl)-2′-
deoxyinosine, the trans- and cis-forms of DMT-dG-N2-TAM, respectively, were prepared in high
yield and used in the preparation of the phosphoramidite precursors. Large quantities of
oligodeoxynucleotides containing a trans- or a cis-form of dG-N2-TAM were prepared efficiently
by automated DNA synthesizer. The incorporation of dG-N2-TAM adduct into the oligodeoxy-
nucleotides was confirmed using 32P-postlabeling/polyacrylamide gel electrophoresis analysis.
These site-specifically modified oligodeoxynucleotides will be used for exploring biological
properties and three-dimensional structure of TAM-DNA adducts.
two cis (fr-3 and fr-4) diastereoisomers of the R-(N2-
deoxyguanosinyl)tamoxifen (dG-N2-TAM, the structures
in Figure 1) adducts (20, 21). In fact, dG-N2-TAM and
R-(N2-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N2-
N-desTAM) adducts have been detected as major DNA
adducts in the liver of rodents treated with TAM (22-
24). dG-N2-TAM adducts have also been detected in the
endometrium of certain women taking TAM (25, 26). dG-
N2-TAM adducts are miscoding and mutagenic lesions,
generating primarily G f T transversions in mammalian
cells (27, 28). This adduct can be removed from DNA by
nucleotide excision repair enzymes (29). TAM-DNA ad-
ducts, if not readily repaired, may contribute to the
initiation of endometrial cancer.
dG-N2-TAM-modified oligodeoxynucleotides were pre-
pared previously in our laboratory by allowing an oli-
godeoxynucleotide containing a single dG to react with
R-acetoxytamoxifen or TAM R-sulfate (27, 28). However,
such postsynthetic methods do not allow the preparation
of oligodeoxynucleotides having a variety of sequence
contexts nor can they be used to synthesize large quanti-
ties of such oligomers. To resolve these difficulties we
synthesized the DMT-phosphoramidite derivative of dG-
N2-TAM and performed a synthesis of dG-N2-TAM-
modified oligodeoxynucleotides using an automated DNA
synthesizer. Using this technique, dG-N2-TAM adducts
can be inserted into any sequence such as known
mutational hot-spots, and used for mutagenesis, DNA
repair, and 3D NMR structural studies.
In tr od u ction
Tamoxifen (TAM;1 [E-1-[4-{2-(dimethylamino)-ethoxy}-
phenyl]-1,2-diphenyl]-butene) is widely used in the en-
docrine therapy of breast cancer (1). This drug was
recently approved as a prophylactic agent for women at
high risk of developing this disease. Unfortunately, an
increased incidence of endometrial cancer has been
observed in breast cancer patients treated with TAM (2-
7) and in women undergoing chemoprevention (8). TAM
is a potent hepatocarcinogen in rats (9-11) and is listed
as a human carcinogen by the International Agency of
Research on Cancer (12).
TAM is metabolized in the liver of rodents and humans
to R-hydroxytamoxifen (R-OHTAM), N-desmethyltamox-
ifen (N-desTAM), tamoxifen N-oxide (TAM N-oxide), and
4-hydroxytamoxifen (4-OHTAM) (13-15). Some N-des-
TAM is further metabolized to R-hydroxy-N-desmethyl-
tamoxifen (R-OH-N-desTAM) (14, 16). We found that
R-OHTAM is sulfonated by rat and human hydroxy-
steroid sulfotransferases (HST) (17, 18), after which they
react with the exocyclic amino group of guanine in DNA
via a short-lived carbocation intermediate (19). This
results in the formation of two trans (fr-1 and fr-2) and
* To whom correspondence should be addressed. Phone: (631) 444-
8018. Fax: (631) 444-3218. E-mail: shinya@pharm.sunysb.edu.
1
Abbreviations: dG, 2′-deoxyguanosine; dG3′P, 2′-deoxyguanosine
3′-monophosphate; TAM, tamoxifen; R-OHTAM, R-hydroxytamoxifen;
R-NH2-TAM, R-aminotamoxifen; dG-N2-TAM, R-(N2-deoxyguanosinyl)-
tamoxifen; PAGE, polyacrylamide gel electrophoresis; HPLC, high-
performance liquid chromatography.
10.1021/tx0101494 CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/29/2002

Synthesis of dG-N2-TAM-Modified Oligomers
Chem. Res. Toxicol., Vol. 15, No. 2, 2002 219
F igu r e 1. Structure of diastereoisomers of trans- and cis-dG-N2-TAM.
en-2-ol (2b) (0.56 g, 1.44 mmol) was allowed to react with
phthalimide (0.22 g, 1.49 mmol), triphenylphosphine (0.39 g,
1.49 mmol), and diethylazodicarboxylate (0.26 g, 1.49 mmol) in
20 mL of THF. This gave the desired product 2-[2-{(Z)-4-(4-(2-
dimethylaminoethoxy)phenyl)-3,4-diphenyl-but-3-enyl}]isoindole-
1,3-dione (3b), (0.4 g 53%), Rf ) 0.14 (eluent, ether:Et3N, 9:1).
FAB mass: m/z 517 (M+ + 1). 1H NMR (CDCl3) δ ppm: 1.61 (d,
3H, J ) 7.5 Hz, HC-CH3); 2.34 (s, 6H, N(CH3)2); 2.71 60 (t,
2H, J ) 5.7 Hz, N-CH2); 4.01 (dt, 2H, J ) 6.0, 2.1 Hz, O-CH2);
5.46 (q, 1H, J ) 7.5 Hz, H-C(CH3)); 6.82-7.70 (m, 18H,
aromatic). 13C NMR (CDCl3) δ ppm: 18.21 (CH-CH3), 45.82
(N(CH3)2), 50.54 (CH-CH3), 58.20 (N-CH2), 65.79 (O-CH2),
114.46, 122.58, 125.82, 126.52, 127.20, 127.39, 128.33, 128.52,
129.61, 129.70, 130.60, 131.68, 131.93, 132.09, 133.48, 133.94,
138.84, 139.15, 141.49, 142.43, 157.68, 167.96 (CdO).
Exp er im en ta l P r oced u r es
Ca u tion : TAM and its derivatives are genotoxic and should
be handled with proper care. Waste materials must be discarded
according to appropriate safety procedures.
Ch em ica ls. Organic chemicals used for synthesis were
supplied by Aldrich Chemical (Milwaukee, WI) and Fisher
Scientific (Pittsburgh, PA). Whenever necessary, solvents were
purified by standard procedures (30). Unless specified silica gel
TLC plates were used. [γ-32P]ATP (specific activity, 6000 Ci/
mmol) was obtained from Amersham Corp (Arlington Heights,
IL). Potato apyrase and alkaline phosphatase (type III) were
purchased from Sigma (St. Louis, MO) and nuclease P1 was
from Boehringer Mannheim (Indianapolis, IN). T4 polynucle-
otide kinase was purchased from Stratagene (La J olla, CA).
Syn th esis of Ch em ica l Ma ter ia ls. The trans- and cis-forms
of R-hydroxytamoxifen (R-OHTAM) were synthesized by the
established protocol (31). Standard stereoisomers of trans- and
cis-dG3′p-N2-TAM for 32P-postlabeling analysis have been pre-
pared previously (18). NMR spectroscopy was performed on
Bruker AC 250 and Varian Gemini 300 MHz NMR instruments.
FAB mass and the ESI negative ion mode were recorded on
Quatro LC-micromass (Manchester) instrument. HPLC analyses
and purifications were performed on a Waters 990 instrument,
equipped with a photodiode array detector.
P r ep a r a tion of d G-N2-TAM-Mod ified Oligod eoxyn u cle-
otid es by Ch em ica l Syn th esis. 2-[2-{(E)-4-(4-(2-Dimethylami-
noethoxy)phenyl)-3,4-diphenyl-but-3-enyl}]isoindole-1,3-dione (3a ).
trans-(E)-4-[4-(2-Dimethylamino-ethoxy)-phenyl]-3,4-diphenyl-
but-3-en-2-ol (2a ) was prepared by the procedure reported by
Foster et al. (31). To a solution of 2a (1.80 g, 4.65 mmol) in THF
(60 mL) were added triphenylphosphine (1.25 g, 4.77 mmol) and
phthalimide (0.71 g, 4.8 mmol) under an inert atmosphere, and
stirring of the solution was continued at 0 °C for 5 min. Then
diethylazodicarboxylate (0.84 g, 4.8 mmol) was added, and the
mixture was stirred at 0 °C for 30 min. The reaction mixture
was allowed to come to room temperature and stirred overnight.
The solvent was evaporated and the residue was purified by
flash chromatography using silica gel and as the eluent, ether:
Et3N, 9:1. Compound 3a was obtained as pale yellow solids (1.14
g, 48%). Rf ) 0.29 (eluent, ether:Et3N, 9:1). The product was
[2-{(E)-4-(4-(2-Dimethylaminoethoxy)phenyl)-3,4-diphenyl but-
3-enyl}]amine (4a ). A 33% solution of methylamine in absolute
ethanol (36.4 mL) was added to a stirred solution of 3a (1.13 g,
2.2 mmol) in 99.9% ethanol (18.3 mL) at room temperature.
After 5 min, the reaction mixture was refluxed for 2.5 h. The
mixture was then cooled to room temperature and the solvent
was evaporated under vacuum. The residue was subjected flash
chromatography on basic alumina, using as the eluent CH2Cl2:
MeOH, 9:1. The title compound 4a was obtained as a pale yellow
solid (0.53 g, 63%), Rf ) 0.42 on alumina TLC (eluent, CH2Cl2:
1
MeOH, 9:1). FAB mass: m/z 387 (M+ + 1). H NMR (CDCl3) δ
ppm: 1.09 (d, 3H, J ) 6.6 Hz, HC-CH3); 2.27 (s, 6H, N(CH3)2);
2.62 (t, 2H, J ) 5.7 Hz, N-CH2); 3.89 (t, 2H, J ) 5.7 Hz,
O-CH2); 4.05 (q, 1H, J ) 6.6 Hz, NH2HC(CH3)); 6.53 (d, 2H, J
) 6.6 Hz, H-3,5 of CC6H4O); 6.80 (d, 2H, J ) 6.6 Hz, H-2,6 of
CC6H4O); 7.16-7.39 (m, 10H, phenyls). 13C NMR (CDCl3) δ
ppm: 22.41 (HC-CH3); 45.34 (N(CH3)2); 48.28 (NH2HC(CH3));
57.70 (N-CH2); 65.12 (O-CH2); 112.82, 125.81, 126.17, 127.05,
127.75, 128.79, 130.45, 130.59, 134.24, 138.09, 138.46, 141.91,
143.59, 156.26.
[2-{(Z)-4-(4-(2-Dimethylaminoethoxy)phenyl)-3,4-diphenyl but-
3-enyl}]amine (4b). The procedure is similar to that used for
the trans-isomer (4a ). Compound 3b (0.53 g, 1.02 mmol) in 8.5
mL of ethanol and 17 mL of a 33% solution of methylamine in
ethanol gave 4b as a pale orange colored solid (0.3 g, 76%). Rf
) 0.25 (eluent, CH2Cl2:MeOH, 9:1). FAB mass: m/z 387 (M+
+
1). 1H NMR (CDCl3) δ ppm: 1.11 (d, 3H, J ) 6.6 Hz, HC-CH3);
2.36 (s, 6H, N(CH3)2); 2.75 (t, 2H, J ) 5.7 Hz, N-CH2); 4.07-
4.17 (m, 3H, O-CH2 and NH2HC(CH3)); 6.89-7.20 (m, 14H,
aromatic). 13C NMR (CDCl3) δ ppm: 22.90 (HC-CH3); 45.85
(N(CH3)2); 48.58 (NH2HC(CH3)); 58.23 (N-CH2); 65.88 (O-CH2);
114.23, 125.66, 126.22, 127.15, 127.35, 129.87, 130.33, 130.92,
134.50, 138.41, 139.40, 142.5, 144.8, 157.5.
characterized as 2-[2-{(E)-4-(4-(2-dimethylaminoethoxy)phenyl)-
3,4-diphenyl-but-3-enyl}]isoindole-1,3-dione (3a ). FAB mass:
1
m/z 517 (M+ + 1). H NMR (CDCl3) δ ppm :1.56 (d, 3H, J ) 7.2
Hz, HC-CH3); 2.26 (s, 6H, N(CH3)2); 2.60 (t, 2H, J ) 5.6 Hz,
N-CH2); 3.88 (t, 2H, J ) 5.8 Hz, O-CH2); 5.41 (q, 1H, J ) 7.5
Hz, H-C(CH3)); 6.51 (d, 2H, J ) 9 Hz, H-3,5 of CC6H4O); 6.83
(d, 2H, J ) 9 Hz, H-2,6 of CC6H4O); 7.05-7.71 (m, 14H, aromatic
protons). 13C NMR (CDCl3) δ ppm: 17.66 (CH-CH3), 45.05
(N(CH3)2), 49.69 (CH-CH3), 57.36 (N-CH2), 64.88 (O-CH2),
112.67, 121.84, 125.90, 126.07, 127.69, 127.76, 127.88, 127.98,
130.02, 130.22, 131.17, 131.32, 132.68, 132.84, 133.88, 137.74,
138.43, 140.63, 141.18, 156.18, 166.99 (CdO).
2-[2-{(Z)-4-(4-(2-Dimethylaminoethoxy)phenyl)-3,4-diphenyl-
but-3-enyl}]isoindole-1,3-dione (3b). The procedure employed
was similar to that used for the trans-isomer (3a ), in which cis-
(Z)-4-[4-(2-dimethylamino-ethoxy)-phenyl]-3,4-diphenyl-but-3-
N2-[2-{(E)-4-(4-(2-Dimethylaminoethoxy)phenyl)-3,4-diphen-
yl but-3-enyl}]-2′-deoxyguano-sine (6a ). Compounds 4a (84 mg,
0.22 mmol) and 5 (81 mg, 0.22 mmol) were dissolved in dry
DMSO (5 mL), and the reaction mixture was stirred under an
inert atmosphere at 75 °C for 3 days [until the disappearance
of starting material was evident by TLC (eluent, CH2Cl2:MeOH,
7:3)]. The solvent was removed under high vacuum and 0.1%
of acetic acid solution (2 mL) was added, and the mixture stirred

220 Chem. Res. Toxicol., Vol. 15, No. 2, 2002
Santosh Laxmi et al.
at room temperature for 2 h. The solvent was removed, and the
dry residue was purified by flash chromatography on silica gel
using as the eluent CH2Cl2:MeOH:Et3N, 9:0.9:0.1. The title
compound 6a was obtained as a pale yellow solid (50 mg, 38%)
Rf ) 0.20 (TLC eluent CH2Cl2:MeOH, 7:3). FAB mass: m/z 637
(M+ + 1). 1H NMR (CD3OD) δ ppm: 1.36 (d, 3H, J ) 7.2 Hz,
HC-CH3); 2.30 (s, 6H, N(CH3)2); 2.70 (t, 2H, J ) 5.3 Hz,
N-CH2); 3.92 (t, 2H, J ) 5.6 Hz, O-CH2), 5.09-5.17 (m, 1H,
N-CH-CH3), 6.55 and 6.65 (d, 2H, J ) 9 Hz, H-3,5 of CC6H4O);
6.82 and 6.83 (d, 2H, J ) 9.3 Hz, H-2,6 of CC6H4O); 7.09-7.51
(m, 10H, phenyls); 8.08, 8.06 (2s, 1H, H at C-8 of dG). Sugar
moiety: 2.33-2.63 (m, 2H, 2′-CH2); 3.69-3.81 (m, 2H, 5′CH2-
OH); 4.02-4.08 (m, 1H, 4′-CH); 4.50-4.56 (m, 1H, 3′-CH), 6.53-
6.41 (m, 1H, 1′-CH). 13C NMR (CD3OD) δ ppm: 21.49 (CHCH3),
41.66 and 42.45 (C2′ of sugar moiety), 45.76 (N(CH3)2); 50.44
and 50.65 (NHCHCH3), 59.05 (N-CH2), 63.31 and 63.37 (C5′
of sugar moiety), 66.22 and 66.26 (O-CH2), 72.55 and 72.68 (C3′
of sugar moiety), 84.57 and 84.69 (C1′ of sugar moiety), 89.32
and 89.45 (C4′ of sugar moiety), 114.56 and 114.59, 117.56 and
117.57, 127.84, 128.30, 129.00, 129.72, 130.53 and 130.56,
131.95 and 132.03, 132.36 and 132.42, 132.47 and 132.52, 136.60
and 136.94, 137.34 and 137.59, 140.31 and 140.43, 141.75 and
141.89, 142.05 and 142.31, 143.65 and 143.72, 152.90 and
152.97, 153.14 and 153.18, 157.93 and 157.99.
were dissolved in dry DMSO (5 mL) and triethylamine (0.046
mL, 0.36 mmol) was added. Reaction and workup were con-
ducted as before to give the title compound 8b. Both of the
diastereoisomers could be separated and isolated by flash
chromatography on silica gel using CH2Cl2:CH3OH:Et3N, 95:4:
1, as the eluent. Fraction 1 (71 mg) Rf ) 0.39, and fraction 2
(67 mg) Rf ) 0.29 (eluent CH2Cl2:MeOH:Et3N, 95:4:1). Overall
yield ) 47%.
1
Fraction 8b-1. FAB mass: m/z 939 (M+ + 1). H NMR (CD3-
OD) δ ppm: 1.36 (d, 3H, J ) 6.9 Hz, HC-CH3); 2.26 (s, 6H,
N(CH3)2); 2.69 (t, 2H, J ) 5.4 Hz, N-CH2); 4.06 (t, 2H, J ) 5.4
Hz, O-CH2), 5.26 (q,1H, J ) 6.9 Hz, N-CH-CH3), 6.76-7.41
(m, 28H, aromatic); 7.85 (s, 1H, H at C-8 of dG). Sugar moiety:
2.50-2.58 (m, 2H, 2′-CH2); 3.69 (broad base singlet, 8H, OCH3
and 5′CH2-OH); 4.12-4.21 (m, 1H, 4′-CH); 4.51-4.61 (m, 1H,
3′-CH); 6.40 (t, 1H, J ) 6.5 Hz, 1′CH).
Fraction 8b-2: FAB mass: m/z 939 (M+ + 1). 1H NMR (CD3-
OD) δ ppm: 1.34 (d, 3H, J ) 6.9 Hz, HC-CH3); 2.30 (s, 6H,
N(CH3)2); 2.73 (t, 2H, J ) 5.1 Hz, N-CH2); 4.09 (t, 2H, J ) 5.1
Hz, O-CH2), 5.17 (q, 1H, J ) 6.9 Hz, N-CH-CH3), 6.72-7.41
(m, 28H, aromatic); 7.83 (s, 1H, H at C-8 of dG). Sugar moiety:
2.28-2.54 (m, 2H, 2′-CH2); 3.68 (singlet, 6H, OCH3); 3.58-3.74
(m, 2H. 5′CH2-OH); 4.14-4.21 (m, 1H, 4′-CH); 4.47-4.58 (m,
1H, 3′-CH); 6.41 (t, 1H, J ) 6.5 Hz, 1′CH).
N2-[2-{(Z)-4-(4-(2-Dimethylaminoethoxy)phenyl)-3,4-diphen-
yl but-3-enyl}]-2′-deoxyguano-sine (6b). The procedure was
similar to that used for the trans-isomer (6a ). Compounds 4b
(100 mg, 0.26 mmol) and 5 (100 mg, 0.27 mmol) in 5 mL of dry
DMSO after reaction were treated with 0.1% AcOH (5 mL) and
gave the title compound 6b as a pale yellow solid (63 mg, 38%),
Rf ) 0.15 (eluent CH2Cl2:MeOH, 7:3). FAB mass: m/z 637 (M+
5′-O-(4,4′-Dimethoxytrityl)-N2-[2-{(E)-4-(4-(2-dimethylamino-
ethoxy)phenyl)-3,4-diphenyl but-3-enyl}]-2′-deoxyguanosine 3′-
O-(2-cyanoethoxy, N,N-diisopropyl) phosphoramidite (10a ). Com-
pound 8a (0.20 g, 0.21 mmol) was dried by azeotropic treatment
with anhydrous pyridine (2 × 10 mL) and placed under vacuum
for 3 h. Anhydrous 1-H-tetrazole (17 mg, 0.25 mmol) was added
to a flame dried flask and kept under a nitrogen atmosphere. A
solution of 8a in anhydrous CH2Cl2 (4 mL) was injected into
the reaction flask followed by 2-cyanoethyl N,N,N,N-tetraiso-
propyl phosphoramidite (100 mg, 0.31 mmol). The reaction
mixture was stirred at room temperature for 3 h and then added
to a saturated solution of sodium bicarbonate (27 mL) and
extracted with CH2CL2 (5 × 30 mL). The organic layer was
separated and dried (MgSO4) then evaporated. The residue was
purified by flash column chromatography using silica gel (CH2-
Cl2:MeOH:Et3N, 95:4:1) to give 10a as a pale yellow solid (225
mg, 95%) Rf ) 0.61 (eluent, CH2Cl2:CH3OH:Et3N, 95:4:1). FAB
1
+ 1). H NMR (CD3OD) δ ppm: 1.38 (d, 3H, J ) 7.2 Hz, HC-
CH3); 2.35 (s, 6H, N(CH3)2); 2.78 (t, 2H, J ) 5.5 Hz, N-CH2);
4.01-4.09 (m, 2H, O-CH2), 5.19 (q, 1H, J ) 7.1 Hz, N-CH-
CH3), 6.89-7.37 (m, 14H, phenyls); 8.08, 8.05 (2s, 1H, H at C-8
of dG). Sugar moiety: 2.44-2.61 (m, 2H, 2′-CH2); 4.06 (m, 2H,
5′CH2-OH); 4.13 (m, 1H, 4′-CH); 4.52 (m, 1H, 3′-CH), 6.41 (t,
1H, J ) 6.9 Hz, 1′-CH). 13C NMR (CD3OD) δ ppm: 21.42
(CHCH3), 41.42 and 42.45 (C2′ of sugar moiety), 47.16 (N(CH3)2);
50.25 and 50.44 (NHCHCH3), 59.25 and 59.30 (N-CH2), 63.32
and 63.40 (C5′ of sugar moiety), 66.69 and 66.74 (O-CH2), 72.59
and 72.73 (C3′ of sugar moiety), 84.56 and 84.75 (C1′ of sugar
moiety), 89.35 and 89.47 (C4′ of sugar moiety), 115.65, 117.41
and 117.62, 126.93, 127.70, 128.37 and 128.40, 128.80, 130.98
and 131.12, 131.60 and 131.66, 131.89 and 131.94, 135.82,
137.26, 137.58, 140.06, 140.22, 141.90, 142.10, 142.33, 142.60,
144.10, 152.80, 152.92, 153.10, 153.14,157.35.
1
mass: m/z 1156 (M+ + H2O). H NMR (CD3OD) δ ppm: 0.99-
1.22 (m, 12H, 2[(CH3)2CH]); 1.34 (d, 3H, J ) 6.6 Hz, CHCH3);
2.18, 2.23 (2s, 6H, N(CH3)2); 2.34-2.79 (m, 6H, 2′CH2 of sugar
moiety, NCH2, CH2CN); 3.31-3.68 (m, 6H, 5′CH2 of sugar
moiety, P-O-CH2, 2NCH(CH3)2); 3.69 (s, 3H, OCH3); 3.72 (s,
3H. OCH3); 3.89 (t, 2H, J ) 5.3 Hz, OCH2); 4.22-4.36(m, 1H,
4′CH of sugar moiety); 4.65-4.76 (m, 1H, 3′CH of sugar moiety);
5.05-5.19 (m,1H, HNCHCH3); 6.38-7.50 (m, 29H, aromatic and
1′CH); 7.88-7.91 (m, 1H, H at C8 of dG). 31P NMR (CD3OD) δ
ppm: 154.66, 154.09, 153.88, 153.84.
5′-O-(4,4′-Dimethoxytrityl)-N2-[2-{(E)-4-(4-(2-dimethylamino-
ethoxy)phenyl)-3,4-diphenyl-but-3-enyl}]-2′-deoxyguanosine (8a ).
Compounds 4a (100 mg, 0.26 mmol) and 9 (175 mg, 0.26 mmol)
were dissolved in anhydrous DMSO (4 mL), and triethylamine
(0.036 mL, 0.29 mmol) was added, and the reaction mixture was
stirred at 75 °C for 5 days (i.e., until the disappearance of the
starting materials as indicated by TLC (eluent, CH2Cl2:CH3-
OH:Et3N, 95:4:1). The solvent was removed under vacuum and
the residue was purified by flash chromatography on silica gel
using CH2Cl2:CH3OH:Et3N, 95:4:1, as eluent. The product 8a
was obtained as a pale yellow solid (164 mg, 67%), Rf ) 0.39
(eluent CH2Cl2:CH3OH:Et3N, 95:4:1). FAB mass: m/z 939 (M+
5′-O-(4,4′-Dimethoxytrityl)-N2-[2-{(Z)-4-(4-(2-dimethylamino-
ethoxy)phenyl)-3,4-diphenyl but-3-enyl}]-2′-deoxyguanosine 3′-
O-(2-Cyanoethoxy,N,N-diisopropyl) Phosphoramidite (10b). The
title compound was prepared by the procedure similar to that
used for the trans-isomer 10a . The phosphoramidite’s derivative
of both the diastereoisomers was prepared separately. 8b-1 (71
mg, 0.08 mmol) in 1.5 mL of CH2Cl2 was added to 1-H-tetrazole
(6.1 mg, 0.09 mmol) and 2-cyanoethyl N,N,N,N-tetraisopropyl
phosphoramidite (36.6 mg, 0.09 mmol) to give 10b-1 (60 mg,
1
+ 1). H NMR (CD3OD) δ ppm: 1.37 (d, 3H, J ) 7.1 Hz, HC-
CH3); 2.21, 2.26 (2s, 6H, N(CH3)2); 2.57 (t, 2H, J ) 5.7 Hz,
N-CH2); 3.91 (t, 2H, J ) 5.4 Hz, O-CH2), 5.04-5.21 (m,1H,
N-CH-CH3), 6.36-7.51 (m, 29H, aromatic and C1′-H of sugar
moiety); 7.85 (s, 1H, H at C-8 of dG). Sugar moiety: 2.32-2.54
(m, 1H, 2′-CH2); 2.81-2.98 (m, 1H, 2′′CH2); 3.27-3.24 (m, 1H,
5′CH2-OH); 3.51-3.59 (m, 1H, 5′′CH2-OH); 3.71 (s, 3H, OCH3);
3.75 (s, 3H, OCH3); 4.14-4.18 (m, 1H, 4′-CH); 4.49-4.56 (m,
1H, 3′-CH).
1
70%) Rf ) 0.35 (eluent, CH2Cl2:CH3OH:Et3N, 95:4:1). H NMR
(CD3OD) δ ppm: 0.99-1.14 (m, 12H, 2[(CH3)2CH]); 1.38 (d, 3H,
J ) 6.9 Hz, CHCH3); 2.27 (2s, 6H, N(CH3)2); 2.27-2.37 (m, 6H,
2′CH2 of sugar moiety, NCH2, CH2CN); 3.31-3.57 (m, 4H,
P-O-CH2, 2NCH(CH3)2); 3.73 (broad base singlet, 8H, 2OCH3,
5′CH2 of sugar moiety); 4.08 (t, 2H, J ) 5.2 Hz, OCH2); 4.23-
4.39 (m, 1H, 4′CH of sugar moiety); 4.63-4.79 (m, 1H, 3′CH of
sugar moiety); 5.15-5.25 (m, 1H, HNCHCH3); 6.35-6.51 (m,
1H, 1′CH of sugar moiety); 6.79-7.48 (m, 28H, aromatic); 7.922,
7.91 (2s, 1H, C8-H of dG). 31P NMR (CD3OD) δ ppm: 153.77,
153.86. FAB mass: m/z 1156 (M+ + H2O). 8b-2 (67 mg, 0.07
mmol) in 1.5 mL of CH2Cl2, was added to 1-H-tetrazole (5.8 mg,
5′-O-(4,4′-Dimethoxytrityl)-N2-[2-{(E)-4-(4-(2-dimethylamino-
ethoxy)phenyl)-3,4-diphenyl-but-3-enyl}]-2′-deoxyguanosine (8b).
The procedure was similar to that used for the trans-isomer 8a .
Compounds 4b (126 mg, 0.33 mmol) and 9 (200 mg, 0.33 mmol)

Synthesis of dG-N2-TAM-Modified Oligomers
Chem. Res. Toxicol., Vol. 15, No. 2, 2002 221
0.08 mmol), 2-cyanoethyl N,N,N,N-tetraisopropylphosphora-
midite (34.3 mg, 0.08 mmol) to give 10b-2 (50 mg, 61%) Rf )
0.31 (eluent, CH2Cl2:CH3OH:Et3N, 95:4:1). FAB mass: m/z 1156
(M+ + H2O). 1H NMR (CD3OD) δ ppm: 0.99-1.21 (m, 12H,
2[(CH3)2CH]); 1.34(d, 3H, J ) 4.8 Hz, CHCH3); 2.32 (s, 6H,
N(CH3)2); 2.39 (t, 1H, J ) 5.7 Hz, 1H of CH2CN); 2.52-2.94 (m,
5H, 2′CH2 of sugar moiety, NCH2, 1H of CH2CN); 3.28-3.35
(m, 2H, P-O-CH2); 3.53-3.73 (m, 4H, 2NCH(CH3)2, 5′CH2 of
sugar moiety); 3.70 (s, 6H, 2OCH3,); 4.10 (t, 2H, J ) 5.2 Hz,
OCH2); 4.18-4.32 (m, 1H, 4′CH of sugar moiety); 4.72-4.85 (m,
1H, 3′CH of sugar moiety); 5.12-5.21 (m,1H, HNCHCH3); 6.39-
6.52 (m, 1H, 1′CH of sugar moiety); 6.72-7.41(m, 28H, aro-
matic); 7.89 (s, 1H, C8-H of dG). 31P NMR (CD3OD) δ ppm:
154.39, 153.60.
Syn th esis of Oligod eoxyn u cleotid es. dG-N2-TAM-modi-
fied oligodeoxynucleotides were synthesized on an Applied
Biosystems DNA synthesizer at the 0.25, 1.0, or 10 µmol, as
described previously (32). Upon completion of the synthesis, the
beads from the cassette were incubated at 55 °C for 16 h in
concentrated ammonium hydroxide. The supernatant liquid was
decanted and the residual beads were washed twice with water
(2 × 500 µL). The combined aqueous fractions were lyophilized
and dissolved in 0.5 mL of 0.1 M TEAA buffer at pH 6.8, filtered,
and subjected to HPLC for isolation of the DMT-protected
oligodeoxynucleotides. HPLC separation was performed on
PRP-1 column (0.46 × 25 cm) at room temperature using a
linear gradient of (A) 0.1 M TEAA, pH 6.8, containing 16-40%
of acetonitrile over a period of 30 min, with a flow rate of 1.0
mL/min. The fractions containing the oligomer were collected
and lyophilized. To remove the DMT protection, these samples
were treated with 80% acetic acid (1 mL) for 30 min, neutralized
with 0.1 M TEAA buffer pH 6.8 (2 mL), and lyophilized. The
DMT-deprotected oligodeoxynucleotides were purified on µBonda-
pak C18 (0.78 × 30 cm, Waters) using 0.05 M TEAA, pH 7.0,
containing 10-20% acetonitrile, eluted over 60 min at a flow
rate 2.0 mL/min.
F igu r e 2. HPLC separation of trans- and cis-dG-N2-TAM.
trans-isoforms (A) or cis-isoforms (B) of dG-N2-TAM were
isolated on a µBondapak C18 (0.39 × 30 cm), using a linear
gradient of 0.05 M triethylammonium acetate (pH 7.0), contain-
ing 10 to 70% acetonitrile with an elution time of 45 min and a
flow rate of 1.0 mL/min.
32P -P ost la b elin g/P olya cr yla m id e Gel E lect r op h or esis
An a lysis. An unmodified or dG-N2-TAM-modified oligodeoxy-
nucleotide (250 pg) was digested at 37 °C for 2 h in 30 µL of 17
mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2,
using micrococcal nuclease (15 units) and spleen phosphodi-
esterase (0.15 units). The reaction mixture was incubated at
37 °C for another 1 h with nuclease P1 (1.0 unit). After the
incubation, 100 µL of water was added. The reaction samples
were extracted twice with 200 µL of butanol. The butanol
fractions were combined, back-extracted with 50 µL of distilled
water, evaporated to dryness, and then used for 32P-postlabeling
analysis. Approximately 95% of TAM adducts were recovered
by butanol extraction. The concentration of the oligodeoxynucle-
otide was estimated as 30 µg ) O.D.260nm 1.0.
The DNA digests were incubated at 37 °C for 30 min with 10
µCi of [γ-32P]ATP (6000 Ci/mmol) and 1 µL of 3′-phosphatase-
free T4 polynucleotide kinase (10 units/µL) (33) and then
incubated with apyrase (50 milliunits) for another 30 min, as
described previously (18). A part of the 32P-labeled sample was
electrophoresed for 5 h on a nondenaturing 30% polyacrylamide
gel (35 × 42 × 0.04 cm) with 1400-1600 V/20-40 mA. A 30%
polyacrylamide gel was prepared from 40% polyacrylamide
solution (60 mL), 10× TBE buffer, pH 7.0 (10 mL), distilled
water (10 mL), 10% ammonium persulfate (0.6 mL), and
TEMED (35 µL). A 10× TBE buffer (pH 7.0) was prepared from
1 M tris-base, 2.24 M boric acid, and 25.5 mM EDTA. Standard
stereoisomers of trans- and cis-forms of dG3′p-N2-TAM (18) were
also labeled with 32P and subjected to PAGE. The position of
32P-labeled adducts was established by a â-phosphorimager
analysis (Molecular Dynamics Inc.).
of 0.05 M triethylammonium acetate, pH 7.0, and 35% aceto-
nitrile were also used for the purification. HPLC analysis was
performed on a Waters 990 HPLC instrument, equipped with a
photodiode array detector.
Resu lts a n d Discu ssion
P r ep a r a tion of tr a n s- a n d cis-d G-N2-TAM. (E)-R-
NH2-TAM (4a ) and (Z)-R-NH2-TAM (4b) were synthe-
sized from (E)-R-OHTAM (2a ) and (Z)-R-OHTAM (2b),
respectively, using the Mitsunobu reaction (34) followed
by hydrolysis (35), as shown in Scheme 1. The detailed
synthetic conditions, and purification of products are
described in the Materials and Methods. 2-Fluoro-(O6-
trimethylsilylethyl)-2′-deoxyinosine (5) was coupled with
(E)-R-NH2-TAM (4a ) or (Z)-R-NH2-TAM (4b) in anhy-
drous DMSO. The products were treated with a low
concentration of acetic acid to remove the O6-silyl group
and then purified by column chromatography to give the
trans- (6a ) or cis-forms (6b) of dG-N2-TAM in ap-
proximately 38% yield. When the trans-form of dG-N2-
TAM (6a ) was subjected to HPLC, two diastereoisomers
(fr-1 and fr-2) were detected at 30.1 and 30.4 min (Figure
2A) although the separation was not well as reported
previously (20). With the cis-form of dG-N2-TAM (6b), two
diastereoisomers (fr-3 and fr-4) were detected at 34.2 and
1
35.0 min (Figure 2B). The H NMR, FAB mass and the
UV spectra of 6a and 6b were consistent with those of
the products obtained by reacting dG with TAM R-sulfate
or R-acetoxytamoxifen (20). Since the yield of dG-N2-TAM
was much higher than that obtained from the postsyn-
thetic method (20), 13C NMR spectra of 6a and 6b were
determined and described in the Materials and Methods.
Isola tion of d G-N2-TAM. To isolate dG-N2-TAM, the pooled
butanol extracts were subjected to liquid chromatography on a
reverse-phase µBondapak C18 column (0.78 × 30 cm, Waters),
eluted over 45 min at a flow rate of 2.0 mL/min with a linear
gradient of 0.05 M triethylammonium acetate, pH 7.0, contain-
ing 10 to 70% acetonitrile. Isocratic HPLC conditions using 65%

222 Chem. Res. Toxicol., Vol. 15, No. 2, 2002
Santosh Laxmi et al.
Sch em e 1. Syn th etic P r otocol for d G-N2-TAM-Mod ified Oligod eoxyn u cleotid es
a
The synthetic steps are described only for the trans-isomers (a ), but the same procedure was employed for the cis-isomers (b).
P r ep a r a tion of d G-N2-TAM-Mod ified Oligod eoxy-
reason may be due to the steric interference of the bulky
TAM moiety on the N2-position of the base hindering the
formation of the DMT derivative. Therefore, the DMT-
derivative of 2-fluoro-(O6-trimethylsilylethyl)-2′-deoxy-
inosine (9) was prepared according to a reported method
(36) and coupled separately with the trans- and cis-forms
of R-NH2-TAM (4a and 4b) (Scheme 1). This procedure
successfully accomplished the synthesis of the required
DMT derivatives (8a and 8b).
n u cleotid es by P h osp h or a m id ite Au tom a ted Syn -
th esis. To incorporate the dG-N2-TAM adduct into an
oligodeoxynucleotide by automated DNA synthesis, the
preparation of the DMT-phosphoramidite derivative is
required. On the basis of the efficient production of dG-
N2-TAM by allowing 2-fluoro-(O6-trimethylsilylethyl)-2′-
deoxyinosine (5) to react with R-NH2-TAM, we attempted
to synthesize the DMT derivatives of both trans- and cis-
dG-N2-TAM using DMTrCl (Scheme 1). However, this
reaction failed to give the 5′-O-tritylated product. The
The phosphoramidite derivative of trans-dG-N2-TAM
(10a ) was prepared from 8a (Scheme 1). Since DMT

Synthesis of dG-N2-TAM-Modified Oligomers
Chem. Res. Toxicol., Vol. 15, No. 2, 2002 223
8b-2, fr-4) on the TLC plate, the phosphoramidite deriva-
tives of cis-dG-N2-TAM (10b-1 and 10b-2) were prepared
from each of the separated DMT derivatives, 8b-1 and
8b-2. Using the phosphoramidite precursors (10a , 10b-
1, and 10b-2), several oligodeoxynucleotides containing
a dG-N2-TAM were prepared by solid-phase synthesis
and purified by HPLC as described in the Materials and
Methods. The coupling efficiency for 0.25-10 µmol scale
synthesis was >98% and resulted in good yields of the
DNA oligomers.
For example, the oligodeoxynucleotides 5′GAGGTGCX-
TGTTTGT, (where X is dG-N2-TAM as a single diaste-
reoisomer) were prepared by automated chemical syn-
thesis. The sequence of the 15-mer oligomer was selected
from codons 271-275 of P53 mutational hot-spots. When
the trans-form of dG-N2-TAM phosphoramidite (10a ) was
used, two oligomers containing fr-1 or fr-2 of trans-dG-
N2-TAM were separated by HPLC at 32.7 and 36.8 min,
respectively (Figure 3B). Oligodeoxynucleotides contain-
ing fr-3 or fr-4 of cis-dG-N2-TAM were prepared sepa-
rately from the DMT-cis-dG-N2-TAM phosphoramidite
(10b-1 or 10b-2). The retention times of the oligomers
containing fr-3 or fr-4 were 41.0 and 43.8 min, respec-
tively (Figure 3, panels C and D). These modified oligo-
mers can be separated from the corresponding unmodi-
fied oligomer (Figure 3A). The UV spectra of the trans-
and cis-dG-N2-TAM-modified oligomers were similar to
that of the corresponding unmodified oligomer (data not
shown). Since monomeric dG-N2-TAM shows weak UV
absorbance above 300 nm as noted previously (20), dG-
N2-TAM-modified oligomers also have weak UV absor-
bance in this region. Several other 15-mer, 19-mer, and
24-mer trans-dG-N2-TAM-modified oligomers were syn-
thesized, and again these modified oligomers were re-
solved into the two diastereoisomeric oligomers by HPLC
(data not shown). The molecular weight of dG-N2-TAM-
modified oligomers was measured by FAB mass spec-
troscopy in negative ion mode. For example, the spectra
F igu r e 3. HPLC separation of 15-mer oligomers containing a
single trans- or cis-dG-N2-TAM. A mixture of 15-mer oligode-
oxynucleotides (5′GAG GTG CXT GTT TGT, where X is dG-N2-
TAM) containing fr-1 and fr-2 of trans-dG-N2-TAM (B) or 15-
mer containing fr-3 (C) or fr-4 (D) of the cis-dG-N2-TAM was
isolated by HPLC. Separation of the unmodified 15-mer is shown
in panel A. These oligomers were isolated on a µBondapak C18
,
using a linear gradient of 0.05 M triethylammonium acetate (pH
7.0), containing 10 to 30% acetonitrile with an elution time of
60 min and a flow rate of 1.0 mL/min.
derivative of cis-dG-N2-TAM (8b) (but not the trans-form)
was resolved into the two diastereoisomers (8b-1, fr-3 and
F igu r e 4. LC/MS/MS analysis of dG-N2-TAM-modified oligodeoxynucleotides. A 15-mer oligodeoxynucleotide (5′GAG GTG CXT
GTT TGT, where X is dG-N2-TAM, 5040 Da) containing a trans-isoform (fr-2, A) or a cis-isoform (fr-4, B) of dG-N2-TAM. The instrument
was operated in negative ion mode using electrospray ionization, as described in Materials and Methods.

224 Chem. Res. Toxicol., Vol. 15, No. 2, 2002
Santosh Laxmi et al.
taining dG-N2-TAM lesion(s) in virtually any sequence
context. These dG-N2-TAM-modified oligomers will be
used for mutagenesis, DNA repair, and 3D NMR struc-
tural studies. In addition, such modified oligomers can
be used as standards for 32P-postlabeling analysis to
quantify TAM-DNA adducts in animal and human DNA
samples.
Ack n ow led gm en t. This research was supported by
Grant ES09418 (to S.S.) from the National Institute of
Environmental Health Sciences. We thank Mrs. Cecilia
M. Torres for synthesis of modified oligodeoxynucleotides
by automated DNA synthesizer and Mr. Robert Rieger
for FAB mass measurements.
Refer en ces
(1) J ordan, V. C. (1993) A current view of tamoxifen for the treatment
and prevention of breast cancer. Br. J . Pharmacol. 110, 507-
517.
(2) Killackey, M., Hakes, T. B., and Pierce, V. K. (1985) Endometrial
adenocarcinoma in breast cancer patients receiving antiestrogens.
Cancer Treat. Rep. 69, 237-238.
(3) Seoud, M. A.-F., J ohnson, J ., and Weed, J . C. (1993) Gynecologic
tumors in tamoxifen-treated women with breast cancer. Obstet.
Gynecol. 82, 165-169.
(4) Fischer, B., Costantino, J . P., Redmond, C. K., Fisher, E. R.,
Wickerham, D. L., and Cronin, W. M. (1994) Endometrial cancer
in tamoxifen-treated breast cancer patients: Findings from the
National Surgical Adjuvant Breast and Bowel Project (NSABP)
B-14. J . Natl. Cancer Inst. 86, 527-537.
(5) van Leeuwen, F. E., Benraadt, J ., Coebergh, J . W. W., Kiemeney,
L. A. L. M., Diepenhorst, F. W., van den Belt-Dusebout, A. W.,
and van Tinteren, H. (1994) Risk of endometrial cancer after
tamoxifen treatment of breast cancer. Lancet 343, 448-452.
(6) Clarke, M., Cillins, R., Davies, C., Godwin, J ., Gray, R., and Peto,
R. (1998) Tamoxifen for early breast cancer: An overview of the
randomized trials. Lancet 351, 1451-1467.
(7) Bernstein, L., Deapen, D., Cerhan, J . R., Schwartz, S. M., Liff, J ,
McGann-Maloney, E., Perlman, J . A., and Ford, L. (1999) Tamox-
ifen therapy for breast cancer and endometrial cancer risk. J .
Natl. Cancer Inst. 91, 1654-1662.
(8) Fischer, B., Costantino, J . P., Wickerham, L., Redmond, C. K.,
Kavanah, M., Cronin, W. M., Botel, V., Robidoux, A., Dimitrov,
N., Atkins, J ., Daly, M., Wieand, S., Tan-Chiu, E., Ford, L., and
Wormark, N., et al. (1998) Tamoxifen for prevention of breast
cancer: report of the National Surgical Adjuvant Breast and
Bowel Project P-1 Study. J . Natl. Cancer Inst. 90, 1371-1388.
(9) Hard, G. C., Iatropoulos, M. J ., J ordan, K., Radi, L., Kaltenberg,
O. P., Imondi, A. R., and Williams, G. M. (1993) Major difference
in the hepatocarcinogenicity and DNA adduct forming ability
between toremifene and tamoxifen in female Crl:CD(BR) rats.
Cancer Res. 53, 4534-4541.
(10) Williams, G. M., Iatropoulos, M. J ., Djordjevic, M. V., and
Kaltenberg, O. P. (1993) The triphenylethylene drug tamoxifen
is a strong liver carcinogen in the rat. Carcinogenesis 14, 315-
317.
(11) Greaves, P., Goonetilleke, R., Nunn, G., Topham, J ., and Orton,
T. (1993) Two-year carcinogenicity study of tanoxifen in Alderley
Park Wister-derived rats. Cancer Res. 53, 3919-3924.
(12) Some pharmaceutical drugs. IARC Monographs on the evaluation
of carcinogenic risks to humans (1996) Vol 66, Lyon, IARC.
(13) Phillips, D. H., Carmichael, P. L., Hewer, A., Cole, K. J ., and Poon,
G. K. (1994) R-Hydroxytamoxifen, a metabolite of tamoxifen with
exceptionally high DNA-binding activity in rat hepatocytes.
Cancer Res. 54, 5518-5522.
(14) J arman, M., Poon, G. K., Rowlands, G., Grimshaw, R. M., Horton,
M. N., Potter, G. A., and McCague, R. (1995) The deuterium
isotope effect for the R-hydroxylation of tamoxifen by rat liver
microsomes accounts for the reduced genotoxicity of [D5-ethyl]-
tamoxifen. Carcinogenesis 16, 683-688.
(15) Poon, G. K., Walter, B., Lonning, P. E., Horton, M. N., and
McCague, R. (1995) Identification of tamoxifen metabolites in
human Hep G2 cell line, human liver homogenate, and patients
on long-term therapy for breast cancer. Drug Metab. Dispos. 23,
377-382.
F igu r e 5. 32P-Postlabeling analysis of dG-N2-TAM-modified
oligodeoxynucleotides. 15-mer oligodeoxynucleotides (250 pg, 5′
GAGGTGCXTGTTTGT, where X is dG or dG-N2-TAM) contain-
ing a single dG or single diastereoisomer of trans-isoforms (fr-1
and fr-2) or cis-isoforms (fr-3 and fr-4) of dG-N2-TAM were
digested enzymatically, and labeled with 32P as described in
Material and Methods. 32P-labeled samples were subjected for
5 h to a nondenaturing 30% polyacrylamide gel (35 × 42 × 0.04
cm). Standard of trans- and cis-forms of dG3′p-N2-TAM were also
labeled with 32P and subjected on PAGE. The position of 32P-
labeled adducts was established using a â-phosporimager.
-
of 15-mer oligomers containing a trans (fr-2)- or cis
(fr-4)-dG-N2-TAM (5′GAGGTGCXTGTTTGT, where X is
dG-N2-TAM) exhibited an ion at m/z 5039, identifying the
molecular mass as 5040 Da (Figure 4, panels A and B).
To confirm the incorporation of the adduct into the
oligomer, the modified oligomer is generally digested
enzymatically and the resulting adducted nucleoside is
isolated by HPLC and identified using a standard (27).
Instead of this approach, we demonstrate that 32P-
postlabeling/polyacrylamide gel electrophoresis (32P-post-
labeling/PAGE) developed recently in our laboratory can
be used to confirm the incorporation of dG-N2-TAM
adducts into oligomers. When dG-N2-TAM-modified 15-
mer oligodeoxynucleotides were analyzed (Figure 5), the
trans-dG-N2-TAM adducts migrated slower than the cis
adducts. The migration of each adduct was consistent
with that of standard trans-diastereoisomers (fr-1 and
fr-2) or cis-diastereoisomers (a mixture of fr-3 and fr-4).
The two trans-diastereoisomers were resolved on the gel
while the two cis-diastereoisomers were not. No adducts
were observed in the unmodified oligomer (Figure 5).
Thus, the oligomers referred as fr-1, fr-2, fr-3, and fr-4
were confirmed to contain the expected diastereoisomer
of dG-N2-TAM adduct.
Thus, phosphoramidite chemical synthesis permits the
preparation of substantial quantities of oligomers con-
(16) Mani, C., Gelboin, H. V., Park, S. S., Pearce, R., Parkinson, A.,
and Kupfer, D. (1993) Metabolism of the antimammary cancer

Synthesis of dG-N2-TAM-Modified Oligomers
Chem. Res. Toxicol., Vol. 15, No. 2, 2002 225
antiestrogenic agent tamoxifen. I. Cytochrome P-450-catalyzed
N-demethylation and 4-hydroxylation. Drug. Metab. Dispos. 21,
645-656.
(26) Shibutani, S., Ravindernath, A., Suzuki, N., Terashima, I.,
Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (2000)
Identification of tamoxifen-DNA adducts in the endometrium of
women treated with tamoxifen. Carcinogenesis 21, 1461-1467.
(27) Shibutani, S., and Dasaradhi, L. (1997) Miscoding potential of
tamoxifen-derived DNA adducts: R-(N2-deoxyguanosinyl)tamox-
ifen. Biochemistry 36, 13010-13017.
(28) Terashima, I., Suzuki, N., and Shibutani, S. (1999) Mutagenic
potential of R-(N2-deoxyguanosinyl)tamoxifen lesions, the major
DNA adducts detected in endometrial tissues of patients treated
with tamoxifen. Cancer Res. 59, 2091-2095.
(29) Shibutani, S., Reardon, J . T., Suzuki, N., and Sancar, A. (2000)
Excision of tamoxifen-DNA adducts by the human nucleotide
excision repair system. Cancer Res. 60, 2607-2610.
(30) Perrin, D. D., and Armarego, W. L. F. (1988) Purification of
Laboratory Chemicals, 3rd ed., pp 1-391, Pergamon, Oxford,
England.
(31) Foster, A. B., J arman, M., Leung, O.-T., McCague, R., Leclercq,
G., and Devleeschouwer, N. (1985) Hydroxy derivatives of tamox-
ifen. J . Med. Chem. 28, 1491-1497.
(32) Bodepudi, V., Shibutani, S., and J ohnson, F. (1992) Synthesis of
2′-deoxy-7,8-dihydro-8-oxoguanosine and 2′-deoxy-7,8-dihydro-8-
oxoadenosine and their incorporation into oligomeric DNA. Chem.
Res. Toxicol. 5, 608-617.
(33) Maniatis, T., Fritsch, E. F., and Sambrook, J . (1982) Molecular
Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Plainview, NY.
(34) Mitsunobo, O., Wada, M. and Sano, T. (1971) Stereospecific and
stereoselective reactions I. Preparation of amines from alcohols.
J . Am. Chem. Soc. 94, 679-680.
(17) Shibutani, S., Shaw, P. M., Suzuki, N., Dasaradhi, L., Duffel, M.
W., and Terashima, I. (1998) Sulfation of R-hydroxytamoxifen
catalyzed by human hydroxysteroid sulfotransferase results in
tamoxifen-DNA adducts. Carcinogenesis 19, 2007-2011.
(18) Shibutani, S., Dasaradhi, L., Terashima, I., Banoglu, E., and
Duffel, M. W. (1998) R-Hydroxytamoxifen is
a substrate of
hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen
DNA adducts. Cancer Res. 58, 647-653.
(19) Sanchez, C., Shibutani, S., Dasaradhi, L., Bolton, J . L., Fan, P.
F., and MaClelland, R. A. (1998) Lifetime and reactivity of the
ultimate tamoxifen carcinogen: the tamoxifen carbocation. J . Am.
Chem. Soc. 120, 13513-13514.
(20) Dasaradhi, L., and Shibutani, S. (1997) Identification of tamox-
ifen-DNA adducts formed by R-sulfate tamoxifen and R-acetoxy-
tamoxifen. Chem. Res. Toxicol. 10, 189-196.
(21) Osborne, M. R., Hewer, A., Hardcastle, I. R., Carmichael, P. L.,
and Phillips, D. H. (1996) Identification of the major tamoxifen-
deoxyguanosine adduct formed in the liver DNA of rats treated
with tamoxifen. Cancer Res. 56, 66-71.
(22) Rajaniemi, H., Rasanen, I., Koivisto, P., Peltonen, K., and
Hemminki, K. (1999) Identification of the major tamoxifen-DNA
adducts in rats liver by mass spectroscopy. Carcinogenesis 20,
305-309.
(23) Umemoto, A., Monden, Y., Suwa, M., Kanno, Y., Suzuki, M., Lin,
C.-X., Ueyama, Y., Momen, M. A., Ravindernath, A., Shibutani,
S., and Komaki, K. (2000) Identification of hepatic tamoxifen-
DNA adducts in mice: R-(N2-deoxyguanosinyl)tamoxifen and
R-(N2-deoxyguanosinyl)tamoxifen N-oxide. Carcinogenesis 21,
1737-1744.
(24) Umemoto, A., Komaki, K., Monden, Y., Suwa, M., Kanno, Y.,
Kitagawa, M., Suzuki, M., Lin, C.-X., Ueyama, Y., Momen, M.
A., Ravindernath, A., and Shibutani, S. (2001) Identification and
quantification of tamoxifen-DNA adducts in the liver of rats and
mice. Chem. Res. Toxicol. 14, 1006-1013.
(35) Motawia, M. S., Wengel, J ., Abdel-Megid, A. E. S., and Pedersen,
E. B. (1989) A convenient route to 3′-amino-3′-deoxythymidine.
Synthesis 384-387.
(36) DeCorte, B. L., Tsarouhtsis, D., Kuchimanchi, S., Cooper, M. D.,
Horton, P., Harris, C. M., and Harris, T. M. (1996) Improved
strategies for postoligomerization synthesis of oligodeoxynucleo-
tides bearing structurally defined adducts at the N2 position of
deoxyguanosine. Chem. Res. Toxicol. 9, 630-637.
(25) Shibutani, S., Suzuki, N., Terashima, I., Sugarman, S. M.,
Grollman, A. P., and Pearl, M. L. (1999) Tamoxifen-DNA adducts
detected in the endometrium of women treated with tamoxifen.
Chem. Res. Toxicol. 12, 646-653.
TX0101494