Chemical synthesis and preliminary structural characterization of a nitrous acid interstrand cross-linked duplex DNA

Full text of DOI:10.1021/ja984426d

J. Am. Chem. Soc. 1999, 121, 5081-5082
5081
duplex DNA, applied to the synthesis of nitrous acid interstrand
cross-linked DNA, and show preliminary structural characteriza-
tion of this DNA by solution NMR spectroscopy. The method
complements that recently reported by Harris and co-workers.15
Chemical Synthesis and Preliminary Structural
Characterization of a Nitrous Acid Interstrand
Cross-Linked Duplex DNA
Exposure of duplex DNA to aqueous nitrous acid is not a viable
route for the preparation of structurally homogeneous interstrand
cross-linked DNA due to concomitant deamination reactions.
Instead, we focused on the synthesis of cross-link lesion 1, found
in small quantities in enzymatic digests of cross-linked DNA,
and its incorporation into DNA by chemical synthesis. Several
unsuccessful attempts were made to prepare 1 by the displacement
of a leaving group in the 2-position of an inosine derivative by
the exocyclic amino group of dG derivatives. However, Pd-
catalyzed reaction of 2 with 3 proceeded to give 4 in good yield
after removal of the silyl groups (Scheme 1).16-18 Hydrogenolysis
of compound 4 removed the benzyl-protecting groups to yield 1,
which was compared to an authentic sample obtained from nitrous
acid interstrand cross-linked DNA.17 However, due to the low
solubility of 1, the benzyl-protecting groups of 4 were removed
after incorporation into DNA.
Two strategies allowed the incorporation of the cross-link lesion
1 into duplex DNA (Scheme 2). In the first, phosphoramidite 6
was synthesized (Scheme 1) and coupled to the growing oligomer
in a standard solid-phase synthesis protocol. Subsequent removal
of the DMT groups by acid treatment and continuation of oligomer
synthesis yielded a three-armed DNA oligomer (Scheme 2A). The
5′-alcohols were capped with acetic anhydride and the allyl
carbonate was removed from the 3′-end with a palladium catalyst
in the presence of butylamine.19,20 The last arm of the cross-linked
DNA was synthesized using inverted phosphoramidites, in which
the 3′-hydroxyl group is protected with a DMT group and the
5′-alcohol has been converted into a phosphoramidite.21 Depro-
tection with aqueous ammonia and purification by denaturing
polyacrylamide gel electrophoresis (DPAGE) gave the cross-
linked oligomer in 10% overall yield.
The second strategy utilized symmetrical phosphoramidite 7
and a more heavily loaded solid support22 (Scheme 2B). Indeed,
we obtained the cross-linked DNA in 10% overall yield after
ammonia deprotection and purification by DPAGE. This approach
is simpler than that illustrated in Scheme 2A; phosphoramidite 7
is synthesized in fewer steps and in significantly higher yield than
6, and less 7 was required. This method was used to prepare
samples for structural characterization.
A method for removing the benzyl-protecting groups in DNA
was needed. Incubation with hydrogen gas in the presence of
palladium on carbon, however, appeared to be inapplicable,
because it has been reported that the double bond in thymidine
can be reduced under these conditions.23 The benzyl groups were
quantitatively removed by transfer hydrogenolysis by means of
palladium black in 35% formamide, 35% ethanol, 10% ethyl
Eric A. Harwood, Snorri Th. Sigurdsson,*
N. B. Fredrik Edfeldt, Brian R. Reid, and Paul B. Hopkins*
Department of Chemistry, UniVersity of Washington
Seattle, Washington 98195-1700
ReceiVed December 28, 1998
Nitrous acid is a mutagenic substance; it converts the exocyclic
amino groups of DNA to carbonyl groups1-6 and forms interstrand
cross-links in duplex DNA.7 There is considerable interest in these
reactions due to the dietary and environmental exposure of humans
to oxides of nitrogen which can initiate these reactions. For
example, sodium nitrite is used extensively in the preparation of
cured meats.8,9 The reactions of nitrous acid with DNA are
believed to proceed by the diazotization of an exocyclic amino
group, followed by displacement by a nucleophile, such as water,
or an amino group of another nucleotide.
Nitrous acid-induced DNA interstrand cross-links form pref-
erentially between two deoxyguanosine (dG) residues at the
sequence [5′-d(CG)]2, forming a cross-link lesion in which the
guanines share a common N2 atom (as in 1).7,10,11 It has been
proposed that this sequence preference is due to the close
proximity of an exocyclic amine of dG on one DNA strand to a
diazonium ion intermediate on the other strand.10,12,13 Molecular
modeling studies suggest that the resulting cross-link lesion can
be accommodated with minimal structural reorganization in
B-form DNA, despite a severe propeller twist of the cross-link
lesion.10,12 A plausible alternative structure for this cross-link
would involve extrusion of the partner dC residues at the cross-
link, as has recently been seen in cisplatin interstrand cross-linked
DNA.14 We desired to obtain a homogeneous sample of nitrous
acid cross-linked DNA to determine experimentally its structure
and to investigate repair of these lesions. We report here two
new methods for the chemical synthesis of interstrand cross-linked
* Corresponding authors.
(1) Strecker, A. Ann. 1861, 118, 151-177.
(2) Kossel, A. Ber. 1885, 85, 1928-1930.
(3) Kossel, A.; Steudel, H. Z. Physiol. Chem. 1903, 37, 377-380.
(4) Schuster, H.; Schramm, G. Z. Naturforschung 1958, 13b, 697-704.
(5) Shapiro, R. J. Am. Chem. Soc. 1964, 86, 2948-2949.
(6) Shapiro, R.; Pohl, S. H. Biochemistry 1968, 7, 448-455.
(7) Shapiro, R.; Dubelman, S.; Feinberg, A. M.; Crain, P. F.; McCloskey,
J. A. J. Am. Chem. Soc. 1977, 99, 302-303.
(15) Tsarouhtsis, D.; Kuchimanchi, S.; DeCorte, B. L.; Harris, C. M.; Harris,
T. M. J. Am. Chem. Soc. 1995, 117, 11013-11014.
(16) Wolfe, J. P.; Wagaw, S.; Buchwald, S. L. J. Am. Chem. Soc. 1996,
118, 7215-7216.
(8) Committee on Nitrite and Alternative Curing Agents in Food. The
Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds; National
Academy Press: Washington, DC, 1981.
(17) Harwood, E. A.; Sigurdsson, S. T.; Hopkins, P. B., manuscript in
preparation.
(9) Handbook of Food AdditiVes, 2nd ed.; Furia, T. E., Ed.; CRC Press:
Cleveland, 1972; pp 150-155.
(18) Preparation of compounds 2-7 is described in the supplementary
(10) Kirchner, J. J.; Hopkins, P. B. J. Am. Chem. Soc. 1991, 113, 4681-
4682.
material.
(19) Hayakawa, Y.; Kato, H.; Uchiyama, M.; Kajino, H.; Noyori, R. J.
Org. Chem. 1986, 51, 2400-2402.
(11) Kirchner, J. J.; Sigurdsson, S. Th.; Hopkins, P. B. J. Am. Chem. Soc.
1992, 114, 4021-4027.
(20) Pirrung, M. C.; Fallon, L.; Lever, D. C.; Shuey, S. W. J. Org. Chem.
1996, 61, 2129-2136.
(12) Kirchner, J. J.; Solomon, M. S.; Hopkins, P. B. In Structure and
Function: Proceedings of the SeVenth ConVersation in Biomolecular Stereo-
dynamics; Sarma, R. H., Sarma, M. H., Eds.; Adenine Press: Albany, 1992;
pp 171-182.
(21) These phosphoramidites can be purchased from Glenn Research, 22825
Davis Drive, Sterling, VA 20164.
(22) These solid supports contain 2.5 mmol of the 3′-base on the same
volume of beads as a normal support for 1 mmol syntheses and can be
purchased from Glenn Research.21
(13) Elcock, A. H.; Lyne, P. D.; Mulholland, A. J.; Nandra, A.; Richards,
W. G. J. Am. Chem. Soc. 1995, 117, 4706-4707.
(14) Huang, H.; Zhu, L.; Reid, B. R.; Drobny, G. P.; Hopkins, P. B. Science
1995, 270, 1842-1845.
(23) Watkins, B. E.; Kiely, J. S.; Rapoport, H. J. Am. Chem. Soc. 1982,
104, 5702-5715.
10.1021/ja984426d CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/14/1999

5082 J. Am. Chem. Soc., Vol. 121, No. 21, 1999
Communications to the Editor
Scheme 1. Preparation of Phosphoramidite Derivatives of
Compound 1 for Synthesis of Nitrous Acid Interstrand
Cross-Linked Duplex DNAa
1
Figure 1. The 750 MHz H NMR spectrum of cross-linked oligomer I
(in 90%H2O/10%D2O, 200 mM NaCl), recorded at 0 °C. The inset shows
the 2D NOESY spectrum (150-ms mixing time) of the imino region. The
intra- and interstrand imino proton connectivities are indicated. Note the
absence of imino protons for the cross-linked guanines, in both the 1D
and 2D spectra.
I as determined by electrospray mass spectrometry was 7278.3
g/mol (calcd 7276.4).
The 1D 1H NMR spectrum of a 0.4 µmol sample of DNA I,25
prepared as illustrated in Scheme 2B, was recorded in water and
the region from 6 to 15 ppm is shown in Figure 1. Due to the
symmetry of the duplex, six imino resonances were expected.
However, inspection of the region between 12 and 14 ppm reveals
only five imino resonances, corresponding to the two AT base
pairs and three CG base pairs. Inspection of cross-peaks in the
imino region of the 2D NOESY spectrum (Figure 1, inset) enables
an “imino-walk” to be made from G1 to G11 to T10 to T4 to
G8. The absence of imino protons at the site of the cross-link
(G7) indicates that this lesion is unlikely to contain base pairing.
The complete assignment of the 1H NMR spectrum and structural
determination are in progress and will be reported in due course.
The method described in Scheme 2B has been used to prepare
several different sequences flanking the lesion site but is restricted
to the preparation of self-complementary sequences. However,
Scheme 2A allows preparation of non-self-complementary cross-
linked duplex DNAs by extending the reverse synthesis to produce
one 3′-overhang which allows ligation to another duplex DNA
of any sequence.
a (a) Tris(dibenzylideneacetone)-dipalladium(0) (Pd2dba3), (R)(+)-2,2′-
bis(diphenylphosphino)-1,1′-binaphthyl (BINAP), sodium tert-butoxide,
toluene, 40%, (b) TBAF, THF, 85%, (c) Pd black, 1,4-cyclohexadiene,
formamide, EtOH, MeOH, EtOAc, 63%, (d) DMT-Cl, pyridine, 79%,
(e) allyl 1-benzotriazolyl carbonate, pyridine, 30%, (f) O-(2-cyanoethyl)
N,N,N′,N′-tetraisopropylphosphoramidite, diisopropylammonium tetra-
zolide, CH2Cl2, 67% (g) DMT-Cl, pyridine, 79%, (h) O-(2-cyanoethyl)
N,N,N′,N′-tetraisopropylphosphoramidite, diisopropylammonium tetra-
zolide, CH2Cl2, 65%.
Scheme 2. Two Strategies for the Synthesis of Nitrous Acid
Interstrand Cross-Linked Duplex DNAa
In summary, we have described a chemical synthesis of a
nitrous acid cross-linked DNA and have shown evidence for the
formation of at least 10 stable base pairs in a 12-nucleotide long
cross-linked duplex. This synthetic strategy should be applicable
for other cross-linked oligonucleotides as long as the cross-link
lesion is stable under the standard conditions of solid-phase
synthesis.
Acknowledgment. We thank the NIH (GM45804 and GM32681) for
financial support and Reliable Pharmaceutical for a generous gift of
deoxyguanosine.
Supporting Information Available: Syntheses of compounds 2-7,
preparation and purification of nitrous acid interstrand cross-linked DNA,
HPLC traces of enzymatic digests of interstrand cross-linked DNA and
co-injections with an authentic sample of compound 1, negative ion mode
electrospray mass spectra of cross-linked DNAs (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
a CPG stands for controlled pore glass.
acetate, and 20% 1,4-cyclohexadiene. HPLC analysis of enzymatic
digests of the phosphodiester backbone revealed cross-link lesion
1 in the expected amount24 along with the appropriate ratio of all
four nucleosides (see Supporting Information). The mass of DNA
JA984426D
(24) Identity verified by co-injection of a sample obtained from nitrous
acid cross-linked DNA.; for quantification, using 16 800 M-1 cm-1 as the
molar extinction coefficient.
(25) This sample was prepared by 12 syntheses using the aforementioned
solid support. The cross-linked product was purified sequentially by DPAGE
and HPLC.