Postsynthetic generation of a major acrolein adduct of 2'- deoxyguanosine in oligomeric DNA

Full text of DOI:10.1021/jm980605u

© Copyright 1999 by the American Chemical Society
Volume 42, Number 6
March 25, 1999
Communications to the Editor
P ostsyn th etic Gen er a tion of a Ma jor
Acr olein Ad d u ct of 2′-Deoxygu a n osin e in
Oligom er ic DNA†
Sonia Khullar, Chamakura V. Varaprasad, and
Francis J ohnson*
Departments of Chemistry and Pharmacological Sciences,
State University of New York at Stony Brook,
Stony Brook, New York 11794-3400
Received October 28, 1998
Acrolein, the simplest member of the class of R,â-
unsaturated carbonyl compounds, is a ubiquitous sub-
stance in the environment.2 It arises from a wide range
of sources, from the incomplete combustion of organic
matter to its occurrence as a secondary metabolite of
some anticancer drugs.3 Several research groups have
shown that a number of R,â-unsaturated aldehydes form
adducts with DNA that are mutagenic in bacteria.4 The
adducts5 of acrolein with DNA bases have been char-
acterized,5,6 and of these the cyclic derivative 1 (Figure
1), formed from 2′-deoxyguanosine (dG) residue, is one
of the more dominant. However, little is known about
the precise mutagenic behavior of this adduct at the
level of DNA replication.
F igu r e 1.
For such biological studies, homogeneous segments
of DNA in which adduct 1 is located site-specifically are
required. Unfortunately the base lability of adduct 1
precludes its direct incorporation into DNA by the
standard synthetic protocol whereas the direct treat-
ment of DNA with acrolein leads to attack at multiple
sites.
Because of these problems our group7 and others8
have pioneered the use of the base-stable propano-dG
(3) as a model to study the mutagenic spectrum of these
adducts. The direct treatment of dG with acrolein
results in an initial Michael addition at the C2 amino
group, which is followed by cyclization at N-1 to give
adduct 1. To introduce this lesion into DNA we reasoned
that a viable approach would be to generate the alde-
hyde function in 1a after the synthesis of the DNA was
complete. Although this might be accomplished using
an acetal, the conditions for unmasking this group
would undoubtedly compromise the glycosidic linkages.
F igu r e 2. HPLC spectrum at 260 nm of the hydrolysate after
enzymatic degradation of the 24-mer by phosphodiesterase and
alkaline phosphatase. The peaks represent normal DNA bases
and adduct 1.
Therefore we elected to introduce the N2-dihydroxybutyl
derivative 2 of dG into DNA, as an alkali-stable precur-
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948 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Communications to the Editor
Sch em e 1a
a
Reagents and conditions: (i) amino diol 4, MeOH, CaCl2, 58 °C, 16 h; (ii) Ac2O, Py, rt, 16 h; (iii) HF, Py, rt, 4 h; (iv) DMT-Cl, Py, rt,
1 h; (v) ClP(OCH2CH2CN)NiPr2, NEt3, CH2Cl2, rt, 2 h.
sor to adduct 1. This has the advantages of ease of
protection and a ready fission by periodate to the desired
aldehyde 1a , postsynthetically. The latter could be
expected to cyclize spontaneously to 1 given that this
is thought to be part of the mechanism for the formation
of these adducts (Figure 1).
Thus the fluoro-inosine 5 (Scheme 1), which can be
obtained from 2′-deoxyguanosine in three steps accord-
ing to a literature procedure,9 was treated with the
previously known10 amino diol 4 to obtain11 the dihy-
droxy-dG derivative 6. Acetylation of diol 6 in the
presence of acetic anhydride in pyridine then afforded
the diacetate 7. The cleavage of the silyl ethers in
compound 7 by HF in pyridine resulted in diol 8 which
was smoothly transformed in two steps into the required
DMT-phosphoramidite 10 under standard conditions via
the DMT intermediate 9.
NMR and 13C NMR spectra) indicate that 12 may be a
single isomer.13 Having confirmed that the cyclic adduct
can be formed under these conditions, we directed
attention to the generation of lesion 1 in DNA. This was
accomplished by employing the DMT-phosphoramidite
10 in a standard DNA synthesis protocol14 (Scheme 1).
Normal HPLC methods were used for the purification
of the oligomers. By way of examples, oligomers 5′- CGT
ACX CAT GC-3′ and 5′-GCC ATG CAT GTT XTG CTA
GAT TGC-3′ (where X ) compound 2) were purified
twice by reversed-phase HPLC {eluents: triethylam-
monium acetate (0.1 M, pH 7.1) and CH3CN; a linear
gradient of 16-36% and 0-20% CH3CN over 30 min
was used for DMT-on DNA and DMT-off DNA, respec-
tively}. When these purified oligomers (DMT-off) were
treated with excess of an aqueous solution of sodium
periodate (0.1 M, room temperature) until no starting
material remained (2-6 h, monitored by HPLC), they
afforded single-stranded DNA containing the desired
adduct 1 at position X. These again were purified by
reversed-phase HPLC to give essentially pure material.
Both oligomers were characterized by electrospray mass
spectroscopy (11-mer m/z: obsvd, 3372.5; calcd, 3373.6
To confirm the formation of adduct 1 at the monomer
level, the p-nitrophenethyl group in compound 6 was
removed using DBU in pyridine12 to obtain the diol 11
(Scheme 2). This was then subjected to periodate
cleavage in aqueous MeOH which led to adduct 12,
having a protected sugar moiety. Analytical data (1H

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 949
Sch em e 2a
a
Reagents and conditions: (vi) DBU, MeOH, rt, 16 h; (vii) NaIO4, H2O-MeOH, rt, 2 h.
Sch em e 3a
a
Reagents and conditions: (i) potassium phthalimide (Phth),
DMF, rt, 16 h; (ii) OsO4, N-methylmorpholine N-oxide, THF-H2O,
rt, 16 h; (iii) H2N-NH2, MeOH, rt, 16 h; (iv) acetic acid, NaHCO3.
In conclusion, we have developed a synthetic pathway
that allows the generation in DNA of the alkali-sensitive
acrolein adduct 1. We believe that this methodology has
the general potential to allow the synthesis of DNA
containing other alkali-sensitive lesions. Further work
in this direction is being pursued.
Ack n ow led gm en t. This work was supported in part
by a grant awarded by the National Institutes of Health,
National Cancer Institute (CA799508), and the National
Institute of Environmental Sciences (ES0406812). We
thank Dr. Charles R. Iden and especially Mr. Robert
Rieger for the mass spectra of the nucleosides and DNA
containing the adducts, and we also thank Mrs. Cecilia
Torres for the HPLC analytical data.
F igu r e 3. HPLC spectrum of the hydrolysate as shown in
Figure 2, augmented by the addition of an authentic sample
of adduct 1.
Da) and (24-mer m/z: obsvd, 7428.4; calcd, 7430.8 Da).
The 24-mer was also subjected to enzymatic degradation
by phosphodiesterase and alkaline phosphatase.15 HPLC
of the hydrolysate clearly showed the presence of only
2′-deoxycytidine, 2′-deoxyguanosine, 2′-deoxythymidine,
2′-deoxyadenosine, and the adduct 1selution times
being 7.27, 18.45, 19.13, 24.96, 26.62 min, respectively
(Figure 2). The HPLC peak associated with the acrolein
adduct of dG was identified by the addition of an
authentic sample to the hydrolysate (Figure 3). Work
is now in progress to determine the mutational spec-
trum of adduct 1 in both bacterial and mammalian cell
systems, and to determine the effect of this lesion on
DNA structure using NMR methods.
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and spectral data for compounds 6-12. This material
is available free of charge via the Internet at http://pubs.
acs.org.
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Communications to the Editor
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