Synthesis of oligonucleotides containing bulky adducts at guanine N2 via the phosphoramidite of O2-triflate-O6-NPE 2'-deoxyxanthosine

Article abstract of DOI:10.1016/S0040-4039(00)00461-5

Oligodeoxynucleotides bearing bulky adducts at guanine N² have been prepared by a postoligomerization strategy in which oligonucleotides containing a highly reactive O²-trifluoromethanesulfonyl-O⁶-(p- nitrophenethyl) 2'-deoxyxanthosine residue were reacted with (±)-10β-amino- 7,8,9,10-tetrahydro-benzo [a]pyrene-7β,8α,9α-triol and related compounds. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00461-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 3555–3558
Synthesis of oligonucleotides containing bulky adducts at guanine
N2 via the phosphoramidite of O2-triflate-O6-NPE
20-deoxyxanthosine
Monica D. Cooper, Richard P. Hodge, Pamela J. Tamura, Amanda S. Wilkinson,
Constance M. Harris ∗ and Thomas M. Harris ∗
Chemistry Department and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235, USA
Received 22 January 2000; revised 28 February 2000; accepted 3 March 2000
Abstract
Oligodeoxynucleotides bearing bulky adducts at guanine N2 have been prepared by a postoligomerization
strategy in which oligonucleotides containing a highly reactive O2-trifluoromethanesulfonyl-O6-(p-nitrophenethyl)
20-deoxyxanthosine residue were reacted with (±)-10β-amino-7,8,9,10-tetrahydro-benzo[a]pyrene-7β,8α,9α-triol
and related compounds. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: nucleic acids; nucleosides; polycyclic aromatic compounds; purines.
The synthesis of oligonucleotides bearing site- and stereospecific adducts of carcinogens has been an
active field of investigation in recent years. Such adducted oligonucleotides are needed for biological
and structural studies, the goal of which is understanding the basis of chemical carcinogenicity. Several
synthetic strategies have been developed for the preparation of these oligonucleotides. The direct
reaction of oligonucleotides with reactive metabolites such as aflatoxin B1 epoxide1 or benzo[a]pyrene
diol epoxide (BPDE)2 is the simplest and can be highly efficient in cases where there are a very
limited number of target sites in the oligonucleotide, e.g. one or two guanines. In other cases the
desired adduct can be prepared at the nucleoside stage and converted to phosphoramidite to be used in
oligonucleotide synthesis3–5 or a phosphoramidite containing a reactive substituent can be introduced into
the oligonucleotide and the adduct introduced by a postoligomerization reaction.6,7 We have exploited
the third strategy successfully for a number of adducts; e.g. this approach works well for adenine
adducts of BPDE if 6-fluoropurine nucleoside is used as the reactive electrophile in reaction with
aminotriol 1 (prepared by opening of BPDE with ammonia).8 However, progress in the synthesis of
oligonucleotides containing adducts of bay-region diol epoxides at guanine N2 has not moved as fast.
In particular, the problem of preparing NMR quantities of oligonucleotides containing such adducts
in sequences containing multiple guanines has not been fully solved. Initially, we explored the use
of 2-fluoro-20-deoxyinosine (or an O6-protected derivative), but had difficulty achieving reproducible
∗
Corresponding authors. Tel/fax: (615) 322-2649; e-mail: harristm@toxicology.mc.vanderbilt.edu (T. M. Harris)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00461-5
tetl 16741

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results with 1. Reaction at the nucleoside level proceeded satisfactorily but postoligomerization reaction
required more vigorous reaction conditions (80°C, 9 days) than we cared to use. The successful use
of 2-fluoro-20-deoxyinosine in the synthesis of several BPDE-adducted oligonucleotides has recently
been reported, although the yield varied widely with the sequence.4,9 We chose to explore the obvious
approach of using a more reactive leaving group at C-2. Steinbrecher et al. have shown that O6-
protected 20-deoxyxanthosine with a triflate at the 2 position is more reactive than the corresponding
2-fluoro compound and have synthesized a wide variety of N2-substituted guanine derivatives using
this approach.10,11 Although their yield in the reaction of the triflate with the tri-O-acetyl derivative of
aminotriol 1 was very low (5%), we felt that it would be worthwhile to look further at the reactivity
of the triflate. We now report the successful synthesis of the phosphoramidite of O2-triflate-O6-(p-
nitrophenethyl) 20-deoxyxanthosine (2), the use of 2 in the synthesis of oligonucleotides containing
multiple guanines and postoligomerization reactions of these oligonucleotides with hindered aminotriol
1 and related compounds.
The triflate phosphoramidite was prepared from 30,50-O-dibutyryl12 (or the diacetyl analog) 20-
deoxyguanosine (3) (Scheme 1) which was protected at the O6 position by Mitsunobu alkylation with
p-nitrophenethyl alcohol.13–15 Conversion of protected 20-deoxyguanosine 4 to deoxyxanthosine 5 was
initially attempted with pentyl or t-butyl nitrite10 but the aqueous diazotization procedure of Eritja16 was
found to be more reproducible. Protected 5 was converted to 2-triflate 6 with trifluoromethanesulfonyl
chloride and Et3N10 in 70–75% yield. Removal of the acyl groups was accomplished with porcine
pancreatic lipase (PPL)17 to give 7 in 54% yield (75–80%, based on recovered starting material); the
dibutyrate was more satisfactory than the diacetate for this step, being a better substrate for PPL.
Conversion of 7 to phosphoramidite 2 was carried out by the usual methodology.18–21
Scheme 1.
Before continuing with oligonucleotide synthesis a brief NMR study (1H and 19F) was done comparing
the reactivity of aminotriol 1 with triflate 7 and the analogous 2-fluoro compound which clearly showed
that the triflate was more reactive than the 2-fluoro compound under the same experimental conditions
(DMSO, (i-Pr)2NEt, 45°C). After 7 days, 7 had almost completely disappeared (with concomitant forma-

3557
tion of product) whereas the 2-fluoro compound was largely unreacted. The stability of phosphoramidite
2 toward the DNA synthesizer reagents (3% TCA/CH2Cl2, tetrazole, I2/pyridine, t-BuPAC anhydride/1-
Me imidazole) was also examined and appeared to be satisfactory within the time frame for 1-µmol
syntheses of reasonably short oligonucleotides (11–16-mers).
Phosphoramidite 2 was used for the synthesis of a 13-mer, 50-ATCGC-X-CGGCATG-30 (8, Scheme
2). Normal automated synthesis conditions were employed (PerSeptive Biosystems Model 8909 DNA
synthesizer, t-BuPAC amidites) except that a long coupling (15 min) cycle was used for introduction of 2.
Coupling yields of ∼80% were seen for introduction of 2, based on trityl monitoring and recovered failure
sequences. Manual coupling was also explored for this step. It did not give improved yields nor did exten-
ding the machine coupling time to 60 min. The matrix-bound protected oligonucleotide was reacted with
racemic aminotriol 1 in anhydrous DMSO containing (i-Pr)2NEt for 6 days at 45°C. Following deprotec-
tion (concd NH4OH, 60°C, 1 h, then DBU/pyridine, rt, 1–3 days), oligonucleotide 8 was passed through
a Sephadex G-25 column and purified by HPLC on a phenylhexyl column (Phenomenex, Torrance, CA;
0.1 M triethylammonium acetate, pH 7.0/MeOH). The oligonucleotides containing the diastereomeric ad-
ducts were easily separated with the 7S,8R,9S,10R isomer eluting first. The oligomers were characterized
by capillary gel electrophoresis, enzymatic hydrolysis8 and mass spectroscopy (negative-ion MALDI-
TOF, calculated for (M–H)−: 4275.6; found for the (7S,8R,9S,10R) isomer, 4276.0; (7R,8S,9R,10S) iso-
mer, 4275.5). Stereochemistry of adducts was assigned by CD spectroscopy and by HPLC comparison of
adducted nucleosides in enzymatic digests with adducted nucleoside standards of known stereochemistry.
11-Mers, including the G-rich N-ras 12 sequence, 50-GGCAGGTGGTG-30 adducted at the underlined
position with aminotriol 1 or the analogous aminotriols derived from the bay-region (BRBA) and non-
bay-region (nBRBA) anti-diol epoxides of benz[a]anthracene22 were prepared similarly, except that a
first HPLC purification was carried out on NPE-protected product for the BPDE and nBRBA sequences.
Typical yields of purified oligomers ranged from 6 A260 units/diastereomer/1 µmol cassette for nBRBA to
4–5 A260 units/diastereoisomer for the benzo[a]pyrene-adducted oligonucleotides. The major byproducts
seen in the reactions were failure sequences and full-length sequence containing 20-deoxyxanthosine
from hydrolysis of the triflate (which occurred primarily during treatment with NH4OH during workup).
Structural and biological studies are now in progress with these oligonucleotides.
Scheme 2.
Acknowledgements
We wish to acknowledge the generous financial support from the National Institute for Environmental
Health Sciences (ES05355 and ES00267).
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1
21. Phosphoramidite 2: H NMR (CH3CN-d3) δ 8.22 (s, 1H, H8), 8.14 (d, 2H, J=8.7 Hz, PhNO2), 7.54 (d, 2H, J=8.7 Hz,
PhNO2), 7.34–7.30 (m, 2H, DMTr), 7.23–7.16 (m, 7H, DMTr), 6.77–6.69 (m, 4H, DMTr), 6.35 (t, 1H, J=6.8 Hz, H10),
4.84 (t, 2H, J=6.6 Hz, CH2CH2PhNO2), 4.76 (m, 1H, H30), 4.20 (m, 1H, H40), 3.80–3.58 (m, 8H, DMTr, CH(CH3)2),
3.33–3.20 (m, 4H, CH2CH2PhNO2, H50, H500), 2.67–2.60 (m, 1H, H20), 2.53–2.43 (m, 5H, CH2CH2CN, H200), 1.16 (m,
12H, CH(CH3)2). 19F NMR (CH3CN-d3) δ −72.91. 31P NMR (CH3CN-d3) δ 150.19, 150.11. HRMS-FAB+ (NBA) calcd
for C49H53F3N7O12PS (M+H)+ 1052.3241, found 1052.3220. Compound 4: 1H NMR: (CDCl3) δ 8.12 (d, 2H, J=8.7 Hz,
PhNO2), 7.74 (s, 1H, H8), 7.45 (d, 2H, J=8.7 Hz, PhNO2), 6.26 (dd, 1H, J=8.0+6.0, H10), 4.93 (br s, 2H, NH2), 5.39
(dt, 1H, J=6.2+2.4 Hz, H30), 4.69 (t, 2H, J=6.8 Hz, CH2CH2PhNO2), 4.43 (dd, 1H, J=11.5+4.3 Hz, H50), 4.34 (dd, 1H,
J=16.6+4.8 Hz, H500), 4.31 (ddd, 1H, J=6.8+4.6+2.4 Hz, H40), 3.24 (t, 2H, J=7.0 Hz, CH2CH2PhNO2), 2.94 (ddd, 1H,
J=14.2+8.0+6.3 Hz, H20), 2.49 (ddd, 1H, J=14.2+6.0+2.4 Hz, H200), 2.33 (t, 2H, J=7.4 Hz, CH2), 2.29 (t, 2H, J=7.4 Hz,
CH2), 1.68 (p, 2H, J=7.4 Hz, CH2), 1.60 (p, 2H, J=7.4 Hz, CH2), 0.95 (t, 3H, J=7.5 Hz, CH3), 0.90 (t, 3H, J=7.5 Hz, CH3).
HRMS-FAB+ (NBA) calcd for C26H33O8N6 557.2360 (M+H)+, found 557.2344. Compound 5: 1H NMR: (CDCl3) δ 8.20
(d, 2H, J=8.7 Hz, PhNO2), 7.96 (s, 1H, H8), 7.53 (d, 2H, J=8.7 Hz, PhNO2), 6.38 (t, 1H, J=7.0 Hz, H10), 5.33 (p, 1H, J=3.0
Hz, H30), 4.81 (t, 2H, J=6.8 Hz, CH2CH2PhNO2), 4.39 (m, 3H, H40, H50, H500), 3.26 (t, 2H, J=6.8 Hz, CH2CH2PhNO2),
2.65 (m, 2H, H20, H200), 2.34 (m, 4H, 2×CH2), 1.65 (septet, 4H, J=7.3 Hz, 2×CH2), 0.94 (q, 6H, J=7.3 Hz, 2×CH3).
HRMS-FAB+ (NBA) calcd for C26H33O9N5 (M+H)+ 558.2199, found 558.2200. Compound 6: 1H NMR: (CDCl3) δ 8.21
(s, 1H, H8), 8.18 (d, 2H, J=8.7 Hz, PhNO2), 7.49 (d, 2H, J=8.7 Hz, PhNO2), 6.38 (dd, 1H, J=7.7 Hz, 6.0 Hz, H10), 5.39
(dt, 1H, J=6.2+2.5 Hz, H30), 4.85 (t, 2H, J=6.8 Hz, CH2CH2PhNO2), 4.36 (m, 3H, H40, H50, H500), 3.32 (t, 2H, J=6.8 Hz,
CH2CH2PhNO2), 2.83 (ddd, 1H, J=14.2+7.7+6.4 Hz, H20), 2.65 (ddd, 1H, J=14.1+5.9+2.7 Hz, H200), 2.37 (t, 2H, J=7.5
Hz, CH2), 2.30 (t, 2H, J=7.5 Hz, CH2), 1.70 (p, 2H, J=7.5 Hz, CH2), 1.64 (p, 2H, J=7.5 Hz, CH2), 0.98 (t, 3H, J=7.5
Hz, CH3), 0.93 (t, 3H, J=7.5 Hz, CH3). 19F NMR: (CDCl3) δ −73.21 (s). HRMS-FAB+ (NBA) calcd for C27H31O11N5F3S
(M+H)+ 690.1693, found 690.1718. Compound 7: 1H NMR (DMSO-d6) δ 8.71 (s, 1H, H8), 8.17 (d, 2H, J=8.7 Hz, PhNO2),
7.62 (d, 2H, J=8.7 Hz, PhNO2), 6.32 (t, 1H, J=6.5 Hz, H10), 5.37 (d, 1H, J=4.4 Hz, 30-OH, D2O-exch), 4.92 (t, 1H, J=5.4
Hz, 50-OH, D2O-exch), 4.84 (t, 2H, J=6.5 Hz, CH2CH2PhNO2), 4.38 (m, 1H, H30), 3.86 (m, 1H, H40), 3.57–3.46 (m, 2H,
H50, H500), (CH2CH2PhNO2 under H2O), 2.68 (ddd, 1H, J=13.1+6.3+6.3 Hz, H20), 2.35 (ddd, 1H, J=14.1+6.3+3.8 Hz,
H200). 19F NMR (DMSO-d6) δ −72.45 (s). LRMS-FAB+ (glycerol/TFA/DMSO) calcd for C19H19F3N5O9S (M+H)+ 550.4,
found 550.1, 572.1 (M+Na)+, 434.0 (NPE-Xan-OTf+H)+. HRMS-FAB+ (glycerol/TFA/DMSO) calcd for C19H19F3N5O9S
550.0855 (M+H)+, found 550.0867.
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