Synthesis and characterization of oligodeoxynucleotides containing the major DNA adducts formed by 1,6- and 1,8-dinitropyrene

Article abstract of DOI:10.1021/ol000090c

(Formula presented) An efficient method for the synthesis of oligonucleotides containing a site-specific DNA adduct formed by the carcinogens 1,6- and 1,8-dinitropyrene has been developed. Palladium-catalyzed amination provided a straightforward route for the synthesis of aminonitropyrenes which, following separation, were reduced to the nitrosonitropyrenes. The N-hydroxyaminonitropyrene, generated in situ from each nitrosonitropyrene, was used successfully to introduce the dinitropyrene-derived DNA adduct at a defined site in a oligonucleotide.

Full text of DOI:10.1021/ol000090c

ORGANIC
LETTERS
2000
Vol. 2, No. 13
1871-1874
Synthesis and Characterization of
Oligodeoxynucleotides Containing the
Major DNA Adducts Formed by 1,6- and
1,8-Dinitropyrene
Vandana Purohit and Ashis K. Basu*
Department of Chemistry, UniVersity of Connecticut, Storrs, Connecticut 06269
akbasu@nucleus.chem.uconn.edu
Received April 13, 2000
ABSTRACT
An efficient method for the synthesis of oligonucleotides containing a site-specific DNA adduct formed by the carcinogens 1,6- and 1,8-
dinitropyrene has been developed. Palladium-catalyzed amination provided a straightforward route for the synthesis of aminonitropyrenes
which, following separation, were reduced to the nitrosonitropyrenes. The N-hydroxyaminonitropyrene, generated in situ from each
nitrosonitropyrene, was used successfully to introduce the dinitropyrene-derived DNA adduct at a defined site in an oligonucleotide.
Atmospheric reactions of polycyclic aromatic hydrocarbons
with NO2 or HNO3 generate nitroaromatic compounds of
which nitropyrenes are a major class.1 They are also formed
from incomplete combustions. As a result, nitropyrenes are
ubiquitous in the environment and could arise from sources
as diverse as grilled food and diesel exhaust.1,2 Most of the
nitropyrenes are mutagenic in bacterial3 and mammalian4
cells. They are also tumorigenic in experimental animals.5
The mutagenicity and tumorigenicity of the nitropyrenes are
believed to be mediated through partial nitroreduction by
cellular enzymes followed by formation of covalent adducts
with DNA. The partially reduced derivatives of 1-nitropyrene
(1-NP) and 1,6- and 1,8-dinitropyrene (1,6- and 1,8-DNP)
generate structurally similar adducts at the C8 position of
guanine (Figure 1).6 While 1-NP is the most abundant
nitroaromatic compound in the environment, 1,6- and 1,8-
DNP are the most potent mutagens and carcinogens in this
(2) (a) Ohnishi, Y.; Kinouchi, T.; Manabe, Y.; Tusutsui, H.; Tokiwa,
H.; Otofuji, T. In Short-Term Genetics Bioassays in the EValuation of
Complex EnVironmental Mixtures; Waters, M. D., Shandu, S. S., Lewtas,
J., Claxton, L., Strauss, G., Newnow, S., Eds.; Plenum Press: New York,
1985; pp 195-204. (b) Kinouchi, T.; Tsutsui, H.; Ohnishi, Y. Mutat. Res.
1986, 171, 105.
(3) (a) Mermelstein, R.; Kiriazides, D. K.; Butler, M.; McCoy, E. C.;
Rosenkranz, H. S. Mutat. Res. 1981, 89, 187. (b) Rosenkranz, H. S.;
Mermelstein, R. Mutat. Res. 1983, 114, 217. (c) Fifer, E. K.; Helfich, R.
H.; Djuric, Z.; Howard, P. C.; Beland, F. A. Carcinogenesis 1986, 7, 65.
(1) (a) Pitts, J. N., Jr.; van Cauwenberghe, K. A.; Grosjean, D.; Schmid,
J. P.; Fitz, D. R.; Belser, W. L., Jr.; Knudson, G. B.; Hynds, P. M. Science
1978, 202, 515. (b) Rosenkranz, H. S.; McCoy, E. C.; Sanders, D. R.; Butler,
M.; Kiriazides, D. K.; Mermelstein R. Science 1980, 209, 1039. (c) Tokiwa,
H.; Nakagawa, R.; Morita, K.; Ohnishi, Y. Mutat. Res. 1980, 85, 195.
10.1021/ol000090c CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/27/2000

activated derivatives (or a nitrenium ion) with the N7 of
guanine, which ultimately rearranges to the C8 adduct.8 In
a prior work we were successful in synthesizing adducted
oligonucleotides containing N-(deoxyguanosin-8-yl)-1-ami-
nopyrene (dGAP) by allowing the unmodified oligonucleo-
tides in duplex form to react with N-hydroxy-1-aminopyrene
generated in situ from 1-nitrosopyrene.9 However, the
synthesis of aminonitropyrenes, the precursors of nitroso-
nitropyrenes, is not as straightforward. One published method
of preparation involves nitration of 1-N-acetylaminopyrene
followed by separation and hydrolysis of the products,3c
whereas an alternate approach utilizes Zinin reduction of the
dinitro compounds.6b,10 Unfortunately, both methods gave
very poor yields on our hands. Recent advances in Pd-
catalyzed amination11 provided an alternative route for the
synthesis of the aminonitropyrenes in much better yield as
shown in Scheme 1. Nitration of 1-bromopyrene (2) provided
a mixture of bromonitropyrenes (3a,b,c) that was converted
to a mixture of aminonitropyrenes (6) in two steps. While
nitration only provided three mononitro derivatives in a 2:2:1
ratio as determined by GC-MS, their separation turned out
to be difficult. The mixture of bromonitropyrenes, therefore,
was converted to a mixture of benzophenone imine adducts
by reflux with 1.2 equiv of benzophenone imine in the
presence of Pd(OAc)2, BINAP, and Cs2CO3 in THF for 16
h.11a Acidic cleavage of the mixture of benzophenone imine
adducts was accomplished in high yield (>95%), and the
resulting aminonitropyrenes were separated by chromatog-
raphy on silica gel. The separation of the aminonitropyrenes
was much more efficient than that of either the bromonitro
or benzophenone imine derivatives. With this synthetic
approach, from 2.1 mmol of 1-bromopyrene we were able
Figure 1. a. dG1-AP: N-(deoxyguanosin-8-yl)-1-aminopyrene; x
) y ) H. b. dG1,6-ANP
: N-(deoxyguanosin-8-yl)-1-amino-6-
nitropyrene; x ) NO2, y ) H. c. dG1,8-ANP: N-(deoxyguanosin-
8-yl)-1-amino-8-nitropyrene; x ) H, y ) NO2.
group. The oligodeoxynucleotides containing these adducts
at specific sites are useful probes for exploring the mecha-
nism of mutagenesis and carcinogenesis. Indeed, site-specific
introduction of the DNA adduct formed by 1-NP into an
oligonucleotide allowed a series of structural and biochemical
investigations.7 However, the synthesis of oligonucleotides
containing DNA adducts formed by the DNPs has not been
reported. Herein we describe a convenient method for
synthesis of the reductively activated derivatives of 1,6- and
1,8-DNP, their reaction with an oligonucleotide to form
adducted oligonucleotides, and characterization of the latter.
1,6- and 1,8-DNP undergo a series of reductions in vivo
to form nitroso, N-hydroxy, and amino derivatives. The
ultimate carcinogen is believed to be either the N-hydroxy
compound or an O-esterified derivative formed by an
enzymatic conjugation. The initial DNA adduct formation
is thought to proceed through an SN2 reaction of these
Scheme 1a
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Org. Lett., Vol. 2, No. 13, 2000

to isolate 0.5 mmol each of 1-amino-6-nitropyrene and
1-amino-8-nitropyrene. A major advantage of this synthetic
strategy is that it circumvents the selective reduction problem
of the dinitro compounds.
Each purified isomer of aminonitropyrene was oxidized
to nitrosonitropyrene using m-chloroperoxybenzoic acid. The
nitrosonitropyrene was converted to the N-hydroxy derivative
in situ in the presence of ascorbic acid, which was allowed
to react with the self-complementary decamer, 5′-d(AAAT-
GCATTT), in DMF-H2O (1:9), pH 5.0-5.5 for 16-20 h
at ambient temperature. HPLC analysis of the reaction
mixture showed that the adducted decamer eluted more
slowly than the unmodified oligonucleotide on a reverse-
phase column (Figure 2) and that the typical yields were
Figure 3. The 32P-labeled samples were run on a 84 cm long 20%
polyacrylamide gel for 26 h at 2500 V: lane 1, 5′-AAATGCATTT-
3′; lane 2, 5′-AAATG1,6-ANPCATTT-3′; lane 3, 5′-AAATG1,8-ANP
CATTT-3′.
-
reaction has been shown to cleave DNA at the sites of C8-
or N7-alkylated guanines, similar to the Maxam-Gilbert G
reaction.12 Indeed, polyacrylamide gel electrophoresis of the
piperidine-treated modified decamers (lanes 5 and 7) showed
a band parallel to the Maxam-Gilbert G reaction (lane 3)
of the unmodified decamer (Figure 4). HPLC analysis of
Figure 4. The 32P-labeled samples were run on a 20% polyacyl-
amide gel for 2.5 h at 1800 V: 1, control 10mer; 2, control 10mer
treated with piperidine (no DMS); 3, control 10mer treated with
DMS followed by piperidine; 4, 10mer with 1,8-ANP adduct; 5,
10mer with 1,8-ANP adduct treated with piperidine; 6, 10mer with
1,6-ANP adduct; 7, 10mer with 1,6-ANP adduct treated with
piperidine; 8, 10mer with 1-AP adduct; 9, 10mer with 1-AP adduct
treated with piperidine.
Figure 2. Reverse-phase HPLC chromatograms of oligonucleo-
tides: (A) unreacted 10mer with the absorption spectrum in the
inset; (B) crude reaction mixture of 10mer with N-hydroxy-1-amino-
6-nitropyrene; absorption spectrum of the slower eluting adducted
oligonucleotide is shown in the inset; (C) crude reaction of 10mer
with N-hydroxy-1-amino-8-nitropyrene; absorption spectrum of the
slower eluting adducted oligonucleotide is shown in the inset.
the enzymatically digested adducted decamers revealed the
absence of dG as well as the presence of a late-eluting peak
that cochromatographed with an authentic sample of dG1,6-ANP
(5) (a) Imaida, K.; Lee, M.-S.; Land, S. J.; Wang, C. Y.; King, C. M.
Carcinogenesis 1995, 16, 3027. (b) Smith, B. A.; Manjanatha, M. G.;
Pogribny, I. P.; Mittelstaedt, R. A.; Chen, T.; Fullerton, N. F.; Beland, F.
A.; Helfich, R. H. Mutat. Res. 1997, 379, 61.
10-15% for both the 1,6- and 1,8-isomers. As shown in
Figure 3, the modified decamers have a slower mobility than
the unmodified decamer in polyacrylamide gel electrophore-
sis. The site of adduction was established by treatment of
the modified decamer with piperidine at 90 °C for 1 h. This
(6) (a) Stanton, C. A.; Chow, F. L.; Philips, D. H.; Grover, P. L.; Garner,
R. C.; Martin, C. N. Carcinogenesis 1985, 6, 535. (b) Andrews, P. J.;
Quilliam, M. A.; McCarry, B. E.; Bryant, D. W.; McCalla, D. R.
Carcinogenesis 1986, 7, 105. (c) Djuric, Z.; Fifer, E. K.; Yamazoe, Y.;
Beland, F. A. Carcinogenesis 1988, 9, 357.
(7) (a) Malia, S. A.; Vyas, R. R.; Basu, A. K. Biochemistry 1996, 35,
4568. (b) Gu, Z.; Gorin, A.; Krishnasamy, R.; Hingerty, B. E.; Basu, A.
K.; Broyde, S.; Patel, D. J. Biochemistry 1999, 38, 10843. (c) Nolan, S. J.;
McNulty, J. M.; Krishnasamy, R.; McGregor, W. G.; Basu, A. K.
Biochemistry 1999, 38, 14056.
(4) (a) Cole, J.; Arlett, C. F.; Lowe, J.; Bridges, B. A. Mutat. Res. 1982,
93, 213. (b) Boldt, J.; Mah, M. C.-M.; Wang, Y.-C.; Smith, B. A.; Beland,
F. A.; Maher, V. M.; McCormick, J. J. Carcinogenesis 1991, 12, 119. (c)
Silvers, K. J.; Eddy, E. P.; McCoy, E. C.; Rosenkranz, H. S.; Howard, P.
C. EnViron. Health Perspect. 1994, 102 (Suppl. 6), 195.
(8) (a) Guengerich, F. P.; Mundkowski, R. G.; Voehler, M.; Kadlubar,
F. F. Chem. Res. Toxicol. 1999, 12, 906. (b) Humphreys, W. G.; Kadlubar,
F. F.; Guengerich, F. P. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8278.
Org. Lett., Vol. 2, No. 13, 2000
1873

of 260, which corresponds to the expected value for the mass
of the aminonitropyrene adduct.
In conclusion, we have developed a simple strategy to
synthesize oligonucleotides containing the major DNA
adducts formed by 1,6- and 1,8-DNP. These modified
oligonucleotides will be valuable tools for the investigation
of structural and biological properties of the carcinogen-
DNA adducts.
Acknowledgment. We are grateful to W. McMurray (W.
M. Keck Facility, Yale University) for the ESI-MS analyses
of the adducted oligonucleotides and to J. Soglia and P.
Vouros (the Barnett Institute, Northeastern University) for
the ESI-MS analyses of the adducted nucleoside standards.
The project described was supported by the National
Institutes of Environmental Health Sciences (Grant ES09127),
National Institutes of Health. A.K.B. is a recipient of a
Research Career Development Award (ES00318) from
NIEHS.
Supporting Information Available: Experimental pro-
1
cedures for the synthesis of 6, H NMR of 1,6- and 1,8-
ANP, and the superimposed absorption spectra of ANP,
dGANP, and the adducted decamer. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL000090C
Figure 5. HPLC profile of enzymatically digested oligonucleo-
tides: (A) digest of unmodified 5′-AAATGCATTT; (B) digest of
1,6-ANP-modified 5′-AAATG*CATTT with the inset showing the
absorption spectrum of dG1,6-ANP; (C) digest of 1,8-ANP-modified
5′-AAATG*CATTT with the inset showing the absorption spectrum
(9) Vyas, R. R.; Nolan, S. J.; Basu, A. K. Tetrahedron Lett. 1993, 34,
2247.
(10) Porter, H. K. Org. React. 1973, 20, 455.
(11) (a) Wolfe, J. P.; Ahman, J.; Sadighi, J. P.; Singer, R. A.; Buchwald,
S. L. Tetrahedron Lett. 1997, 38, 6367. (b) Mann, G.; Hartwig, J. F.; Driver,
M. S.; Fernandez-Rivas, C. J. Am. Chem. Soc. 1998, 120, 827. (c) Hartwig,
J. F. Acc. Chem. Res. 1998, 31, 852. (d) Hartwig, J. F. Angew. Chem., Int.
Ed. 1998, 37, 2046. (e) Wolfe, J. P.; Wagaw, S.; Marcoux, J.-F.; Buchwald,
S. L. Acc. Chem. Res. 1998, 31, 805.
(12) (a) Johnson, D. L.; Reid, T. M.; Lee, M.-S.; King, C. M.; Romano,
L. J. Carcinogenesis 1987, 8, 619. (b) Shibutani, S.; Gentles, R. G.; Iden,
C. R.; Johnson, F. J. Am. Chem. Soc. 1990, 112, 5667. (c) Maxam, A.;
Gilbert, W. Methods Enzymol. 1980, 65, 499. (d) Sambrook, J.; Fritsch, E.
F.; T. Maniatis, T. In Molecular Cloning: a laboratory manual, 2nd ed.;
Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989; pp
13.11-13.101.
of dG1,8-ANP
.
and dG1,8-ANP as shown in Figures 5B and 5C, respectively.13
The absorption spectrum of the adducted decamers with the
λ
max at 470 and 480 nm showed a 20-25 nm red shift from
450 and 458 nm, respectively, for dG1,6-ANP and dG1,8-ANP
,
suggesting a stacking interaction of the DNA bases with the
pyrenyl residue. Additional evidence for the structure of the
decamers was obtained by mass spectral analysis with
electrospray ionization. Analysis of the adducted oligonucleo-
tides gave a monoisotopic mass of 3284.63 and 3284.71 for
the dG1,6-ANP- and dG1,8-ANP-containing decamers, respec-
tively, which are in excellent agreement with the theoretical
monoisotopic mass of 3284.62. Comparison of unmodified
(m/z 3024.63) and adducted decamers gave a mass difference
(13) The dGANP standards were isolated by reverse-phase HPLC fol-
lowing adduction of calf thymus DNA and subsequent enzymatic digestion.
The dG adducts were characterized by 1H NMR, which was found to be in
agreement with the reported data.6b,c The absence of the singlet representing
the C8 proton of guanine at ∼7.9 ppm indicated that this position is the
site of adduction. The MS analyses of both standards by ESI gave the correct
mass of m/z 528 as well as fragmentation peaks of 412 and 366,
corresponding to the loss of a sugar and a sugar plus the nitro group,
respectively.
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Org. Lett., Vol. 2, No. 13, 2000