Synthesis of oligodeoxynucleotides containing a single diastereoisomer of α-(N2-2′-deoxyguanosinyl)-N-desmethyltamoxifen

Article abstract of DOI:10.1016/j.bmcl.2004.05.030

A phosphoramidite chemical synthesis of oligodeoxynucleotides containing a diastereoisomer of (E)-α-(N²-deoxyguanosinyl)-N- desmethyltamoxifen, a major tamoxifen (TAM)-derived DNA adduct in animal and women treated with TAM, was described. The

Full text of DOI:10.1016/j.bmcl.2004.05.030

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4051–4054
Synthesis of oligodeoxynucleotides containing
a single diastereoisomer of
a-(N2-20-deoxyguanosinyl)-N-desmethyltamoxifen
Y. R. Santosh Laxmi, Naomi Suzuki, Sung Yeon Kim 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, USA
Received 21 April 2004; revised 13 May 2004; accepted 14 May 2004
Available online 8 June 2004
Abstract—A phosphoramidite chemical synthesis of oligodeoxynucleotides containing a diastereoisomer of (E)-a-(N2-deoxygua-
nosinyl)-N-desmethyltamoxifen, a major tamoxifen (TAM)-derived DNA adduct in animal and women treated with TAM, was
described. The site-specifically modified oligodeoxynucleotide can be used for mutagenesis, DNA repair, and 3D structural studies
and also as standard for quantitative analysis of TAM-DNA adducts in animal and human.
Ó 2004 Elsevier Ltd. All rights reserved.
Tamoxifen (TAM, 1a; [E-1-[4-{2-(dimethylamino)-eth-
oxy}-phenyl]-1,2-diphenyl]-butene) is widely used in the
endocrine therapy of breast cancer and used as a pro-
phylactic agent for women at high risk of developing
breast cancer.1 However, administration of TAM is
associated with an increased risk of endometrial cancer
in women treated with TAM.2 TAM is metabolized by
phase I enzymes to N-desmethyltamoxifen (N-desTAM,
1c), 4-hydroxytamoxifen, tamoxifen N-oxide, and a-
hydroxytamoxifen (a-OHTAM, 1b).3 N-desTAM is
further metabolized to a-hydroxy-N-desTAM (1d). a-
Hydroxylated TAM metabolites, 1d and 1b, are O-sul-
fonated by rat and human hydroxysteroid sulfotransfe-
rases and react with the exocyclic amino groupof
guanine in DNA via a short-lived carbocation interme-
diate, resulting in the formation of two (E) (fr-1 and fr-
2) and two (Z) (fr-3 and fr-4) diastereoisomers of the a-
with TAM.3 In the present study, we describe a phos-
phoramidite chemical synthesis of (E)-dG-N2-N-
desTAM-modified oligodeoxynucleotides that can be
utilized for exploring biological properties and three-
dimensional structure of dG-N2-N-desTAM adducts.
When following a protocol previously established for
the preparation of dG-N2-TAM-modified oligomer,4 the
synthesis of dG-N2-N-desTAM-modified oligomers was
not successful. Several modifications, including protec-
tion and deprotection of the secondary amino group of
N-desTAM and deletion of the capping step during
automated oligonucleotide synthesis, were required for
preparation of large quantities of (E)-dG-N2-N-
desTAM-modified oligodeoxynucleotide.
The secondary amino groupof ( E)-a-OH-N-desTAM 1d
was protected with
a
benzyloxycarbonyl moiety5
(N2-deoxyguanosyl)-N-desmethyltamoxifen
adducts
(Scheme 1). The protected (E)-a-NH2-N-desTAM 512
was synthesized from the compound 35 using the Mits-
unobu reaction5 followed by hydrolysis.7 The DMT-
derivative of 2-fluoro-(O6-trimethylsilylethyl)-20-deoxy-
inosine 68 was coupled with the (E)-a-NH2-N-desTAM
protected at the aminoethoxy functionality 5 to produce
compound 712 (Scheme 1). Hydrogenation using palla-
dium hydroxide9 (10%) at 50 psi for 48 h successfully
deprotected the benzyloxycarbonyl group to give the
free secondary amino compound 8.12 dG-N2-N-desTAM
phosphoramidite derivative 912 was prepared using
diisopropylammonium tetrazolide.10
(dG-N2-N-desTAM 2b, the structures in Fig. 1) and a-
(N2-deoxyguanosyl)tamoxifen adducts (dG-N2-TAM,
2a), respectively. In fact, (E)-dG-N2-N-desTAM and
(E)-dG-N2-TAM adducts have been detected as major
DNA adducts in rodents, monkey and women treated
Keywords: N-Desmethyltamoxifen; Oligodeoxynucleotide; Phosphor-
amidite; DNA adduct.
* Corresponding author. Tel.: +1-631-444-8018; fax: +1-631-444-3218;
e-mail: shinya@pharm.sunysb.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.030

4052
Y. R. Santosh Laxmi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4051–4054
O
N
HN
R'
OH
N
H
N
N
O
H
H
H
H
H
OH
O
O
R
N
N
R
2
1
a. dG-N2-TAM: R=CH3
b. dG-N2-N-desTAM: R=H
a. TAM: R=CH3; R'=H
α
b. -OHTAM: R=CH3; R'=OH
c. N-desTAM: R=H; R'=H
α
d. -OH-N-desTAM: R=H; R'=OH
Figure 1. Structures of (E)- and (Z)-forms of TAM metabolites and TAM-DNA adducts.
O
OH
OH
N
i
ii
O
O
O
O
O
O
O
O
NH
N
N
1d
3
4
iii
O
Si
O
N
N
HN
N
N
N
ODMT
ODMT
F
N
N
N
O
O
NH2
H
OH
6
OH
O
O
O
iv
O
O
N
N
O
7
5
v
O
N
O
N
N
HN
HN
ODMT
ODMT
N
N
N
O
N
N
O
H
H
vi
OH
O
O
P
CN
O
N(iPr)2
O
8
9
NH
NH
Scheme 1. Synthetic protocol for dG-N2-N-desTAM-modified oligodeoxynucleotides. Reagents and conditions: (i) ClCO2CH2Ph, Et3N, THF, rt,
90%, (ii) phthalimide, TPP, DEAD, rt, 48%, (iii) MeNH2 in EtOH, reflux, 94%; (iv) DMSO, Et3N, 75 °C, 73%; (v) Pd(OH)2 10 mol %, ethylacetate,
Et3N, 50 psi, 78%; (vi) diisopropylammonium tetrazolide, P(N(i-Pr)2)2)OCH2CH2CN, 66%.
The preparation of the dG-N2-N-desTAM-modified
oligodeoxynucleotide using the phosphoramidite pre-
cursor 9, by general method in the DNA synthesizer,
gave more than 90% of oligomer containing an acetyl
group. It was not possible to remove the acetyl group at
the secondary amino groupof the side chain, which was

Y. R. Santosh Laxmi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4051–4054
4053
obtained during the capping process, after the synthesis
of the oligomer in the DNA synthesizer. Therefore, the
capping step was avoided in the synthesizer. The desired
dG-N2-N-desTAM-modified oligodeoxynucleotides was
obtained by HPLC purification.4 This method success-
fully gave the required oligodeoxynucleotide containing
the dG-N2-N-desTAM. The coupling efficiency for
0.25 lmol scale synthesis was >85%, and resulted in
good yields of the oligomers.
synthesis. The sequence of the 15-mer oligomer was
selected from codons 271–275 of P53 mutational hot-
spots. When the (E)-dG-N2-N-desTAM phosphorami-
dite 9 was used, two diastereoisomeric oligomers con-
taining fr-1 or fr-2 of (E)-dG-N2-N-desTAM were
separated by HPLC; the retention times of purified fr-1
and fr-2 of the modified oligomers were 31.9 and
37.6 min, respectively. The molecular weight of dG-N2-
N-desTAM-modified oligomers was measured by LC/
MS/MS in negative ion mode. The spectra of 15-mer
oligomer containing a fr-1 or fr-2 of (E)-dG-N2-N-des-
TAM (50-GAGGTGCXTGTTTGT, where X is dG-N2-
N-desTAM) exhibited an ion at m=z 5025, identifying
The oligodeoxynucleotides (50-GAGGTGCXTGTT-
TGT, where X is dG-N2-N-desTAM as a single diaste-
reoisomer) were prepared by automated chemical
Figure 2. 32P-Postlabeling/HPLC analysis of dG-N2-N-desTAM-modified oligodeoxynucleotide. Standards of a mixture of (E)-forms (fr-1 and fr-2)
and (Z)-forms (a mixture of fr-3 and fr-4) of dG3-N2-N-desTAM (Z)-dG3-N2-TAM were labeled with 32P and subjected on HPLC (A). 15-mer
oligodeoxynucleotides (50 ng, 50-GAGGTGCXTGTTTGT, where X is dG-N2-N-desTAM) containing a single (E)-isoform (fr-1, B; fr-2, C) of dG-
N2-N-desTAM were digested enzymatically, and labeled with 32P. Standards (D) were co-chromatographied with a product from oligomer containing
fr-1 (E) or fr-2 (F) of dG-N2-N-desTAM.

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Y. R. Santosh Laxmi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4051–4054
the molecular mass as 5026 Da. Several (E)-dG-N2-N-
desTAM-modified oligomers having different sequence
context have been successfully synthesized, and these
modified oligomers were also resolved into two diaste-
reoisomeric oligomers by HPLC.
4. Santosh Laxmi, Y. R.; Suzuki, N.; Dasaradhi, L.; John-
son, F.; Shibutani, S. Chem. Res. Toxicol. 2002, 15, 218–
225.
5. Kitagawa, M.; Ravindernath, A.; Suzuki, N.; Rieger, R.;
Terashima, I.; Umemoto, A.; Shibutani, S. Chem. Res.
Toxicol. 2000, 13, 761–769.
6. Mitsunobu, O.; Wada, M.; Sano, T. J. Am. Chem. Soc.
1971, 94, 679–680.
7. Motawia, M. S.; Wengel, J.; Abdel-Megid, A. E. S.;
Pedersen, E. B. Synthesis 1989, 384–387.
8. DeCorte, B. L.; Tsarouhtsis, D.; Kuchimanchi, S.; Coo-
per, M. D.; Horton, P.; Harris, C. M.; Harris, T. M.
Chem. Res. Toxicol. 1996, 9, 630–637.
9. Beaulieu, P. L.; Schiller, P. W. Tetrahedron Lett. 1988, 29,
2019–2022.
10. Mcgee, D. P. C.; Cook, P. N.; Guinosso, C. J. PCT. Int.
Appl., WO 9402501, 1994; p 85.
11. Shibutani, S.; Suzuki, N.; Santosh Laxmi, Y. R.; Schild, L.
J.; Divi, R. L.; Grollman, A. P.; Poirier, M. C. Cancer Res.
2003, 63, 4402–4406.
32P-Postlabeling/HPLC analysis developed recently in
our laboratory11 can also be used to confirm the incor-
poration of dG-N2-N-desTAM into oligomers. As
shown in Figure 2A, standards of (E)- (fr-1 and fr-2) and
(Z)- (a mixture of fr-3 and fr-4) isomers of dG30P-N2-N-
desTAM and dG3-N2-TAM can be resolved in 50 min.
The retention times of a DNA adduct from fr-1 and fr-2
of the modified oligomers were 19.8 min (Fig. 2B) and
20.5 min (Fig. 2C), respectively. When the DNA adduct
from oligomer containing fr-1 (Fig. 2E) or fr-2 (Fig. 2F)
was co-injected with the authentic standards (Fig. 2D),
the products were identified as fr-1 and fr-2 of (E)-dG30p
-
N2-N-desTAM, respectively.
12. Spectral data of new compounds:
1
5: FAB mass: m=z 506 (Mþ). H NMR (CDCl3) d ppm:
1.13 (d, 3H, J ¼ 6:9 Hz, HC–CH3); 3.01 (s, 3H, N(CH3));
3.57–3.61 (m, 2H, N–CH2); 3.90–4.01 (m, 2H, O–CH2);
4.10 (q, 1H, J ¼ 6:96 Hz, NH2HC(CH3)); 6.46–6.55 (m,
2H, H-3,5 of CC6H4O); 6.79–6.85 (m, 2H, H-2,6 of
CC6H4O); 7.20–7.43 (m, 15H, phenyls). 13C NMR
(CDCl3) d ppm: 22.8 (HC–CH3); 35.8 (N(CH3)); 48.7
(NH2HC(CH3)); 47.9 and 48.7 (N–CH2); 65.7 and 66.1
(O–CH2); 66.9 (O–CH2Ph); 113.1, 126.2, 126.6, 127.4,
127.6, 127.7, 127.8, 128.2, 128.3, 129.2, 130.8, 131.0, 134.9,
136.6, 138.4, 138.7, 142.2, 144.1, 156.1, 156.2, 156.4.
7: FAB mass: m=z 1059 (Mþ+1). 1H NMR (CD3OD) d
ppm: 1.39 (m, 3H, HC–CH3); 2.95 (s, 3H, N(CH3)); 3.53–
3.61 (m, 2H, N–CH2); 3.82–3.98 (m, 2H, O–CH2), 5.05–
5.12 (m, 2H, O–CH2–Ph); 5.15–5.24 (m, 1H, N–CH–CH3),
6.39–7.52 (m, 33H, aromatic and C10-H of sugar moiety);
7.84–7.85 (m, 1H, H at C-8 of dG), sugar moiety: 2.40–
2.56 (m, 2H, 20-CH2); 3.20–3.45 (m, 2H, 50CH2–OH); 3.78
(s, 6H, OCH3); 4.15–4.22 (m, 1H, 40-CH); 4.53–4.62 (m,
1H, 30-CH).
8: FAB mass: m=z 925 (Mþ+1). 1H NMR (CD3OD) d
ppm: 1.34–1.36 (m, 3H, HC–CH3); 2.44–2.49 (2s, 3H,
N(CH3)); 2.85–2.98 (m, 2H, N–CH2); 3.81–3.95 (m, 2H,
O–CH2), 5.06–5.21 (m, 1H, N–CH–CH3), 6.36–7.49 (m,
33H, aromatic and C10-H of sugar moiety); 7.84–7.85 (m,
1H, H at C-8 of dG), sugar moiety: 2.62–2.76 (m, 2H, 20-
CH2); 3.32–3.48 (m, 2H, 50CH2–OH); 3.70 (s, 3H, OCH3);
3.73 (s, 3H, OCH3); 4.12–4.21 (m, 1H, 40-CH); 4.48–4.58
(m, 1H, 30-CH).
Thus, phosphoramidite chemical synthesis allows the
preparation of substantial quantities of oligomers con-
taining dG-N2-N-desTAM adduct(s) in virtually any
sequence context. These dG-N2-N-desTAM-modified
oligomers will be used for mutagenesis, DNA repair
studies, 3D NMR structural and crystallographic stud-
ies. Such modified oligomers can also be used as stan-
dards for 32P-postlabeling analysis to quantify dG-N2-
N-desTAM-DNA adducts in animal and human.
Acknowledgements
This research was supported by a Grant ES09418 from
the National Institute of Environmental Health Sci-
ences. We thank Mrs. Cecilia M. Torres for synthesis of
modified oligodeoxynucleotides by automated DNA
synthesizer and Mr. Robert Rieger for mass measure-
ment.
References and notes
1. Jordan, V. C. Br. J. Pharmacol. 1993, 110, 507–517.
2. (a) van Leeuwen, F. E.; Benraadt, J.; Coebergh, J. W. W.;
Kiemeney, L. A. L. M.; Diepenhorst, F. W.; van den Belt-
Dusebout, A. W.; van Tinteren, H. Lancet 1994, 343, 448–
452; (b) 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.; Wormark, N., et al.
J. Natl. Cancer Inst. 1998, 90, 1371–1388.
9: 1H NMR (CD3OD) d ppm: 1.04–1.16 (m, 12H,
2[(CH3)2CH]); 1.32–136 (m, 3H, CHCH3); 2.31–2.39 (m,
2H, C20H); 2.43–2.52 (m, 2H, CH2CN); 2.55–2.61 (m, 3H,
N(CH3)); 2.63–2.76 (m, 2H, NCH2); 3.34–3.54 (m, 4H, P–
O–CH2, 2NCH(CH3)2); 3.57–3.68 (m, 2H, 50CH2OH);
3.70–3.75 (m, 6H, OCH3); 3.86–3.95 (m, 2H, OCH2); 4.12–
4.21 (m, 1H, 40CH of sugar moiety); 4.48–4.58 (m, 1H,
30CH of sugar moiety); 5.11–5.21 (m, 1H, HNCHCH3);
6.33–7.49 (m, 29H, aromatic and 10CH); 7.81–7.85 (m, 1H,
H at C8 of dG). 31P NMR (CD3OD) d ppm: 149.84,
150.27, 150.36.
3. Kim, S. Y.; Suzuki, N.; Santosh Laxmi, Y. R.; Shibutani,
S. Drug Metab. Rev. 2004, 36, 199–218.