A highly efficient synthesis of oligodeoxyribonucleotides containing the 2'-deoxyribonolactone lesion [8]

Full text of DOI:10.1021/ja982617a

11810
J. Am. Chem. Soc. 1998, 120, 11810-11811
Scheme 1. Structure of Abasic Sites: A, 2′-Deoxyribose
A Highly Efficient Synthesis of
Oligodeoxyribonucleotides Containing the
2′-Deoxyribonolactone Lesion
(“True” Abasic Site); B, 2′-Deoxyribonolactone; C,
Tetrahydrofuran Analog
Mitsuharu Kotera,* Anne-Gaelle Bourdat, Eric Defrancq, and
Jean Lhomme
LEDSS, Chimie Bioorganique
UMR CNRS 5616, UniVersite´ Joseph Fourier
BP 53, 38041 Grenoble Cedex 9, France
Scheme 2
ReceiVed July 24, 1998
Loss of a base in DNA leaving a 2′-deoxyribonolactone residue
B (Scheme 1) is a common abasic site damage that occurs through
a variety of processes, most of which are interpreted by radical
abstraction of a hydrogen atom at C-1′ of a 2′-deoxyribose unit.1
For example, 2′-deoxyribonolactone formation has been reported
to occur under the action of drugs such as enediyne antibiotics
(e.g., neocarzinostatin)2 and cationic manganese porphyrins3 or
under γ and UV irradiation.4,5 2′-Deoxyribonolactone has also
been suspected to be formed as an intermediate in copper
phenanthroline DNA cleavage.6 The lesion has been reported to
be mutagenic,7 to be resistant to repair nucleases,8 and to lead to
DNA strand scission by â- and δ-elimination processes.6c Indeed
this poor chemical stability of alkaline labile 2′-deoxyribonolac-
tone in DNA has considerably limited our knowledge of the
structural, chemical, and biological consequences of the lesion.
Notably there is no report in the literature of a general method
for preparing oligonucleotides containing the lesion.9
nucleotides containing the 2′-deoxyribonolactone site at any
preselected position in the sequence.
The idea underlying the approach is to mimic the mode of
formation of the lactone in DNA, i.e., to generate a radical at
carbon C-1′ of a deoxyribose residue with total selectivity and
high efficiency in nonaggressive conditions (neutral pH, room
temperature). The biomimetic intramolecular H-abstraction route
has been pioneered by Breslow14 and shown to be quite successful.
The nitro group, when irradiated, is an efficient radical generator
that can be conveniently attached to an aromatic ring.15 The
7-nitroindole nucleoside 2 was thus selected for intramolecular
H-abstraction, on the basis of simple model examination and
molecular modeling calculations, which showed that one of the
oxygen atoms of the nitro group is in close proximity to the H-1′
atom to be abstracted. Nucleoside 2 was prepared by stereose-
lective glycosylation of 2-deoxy-3,5-di-O-p-toluoyl-R-D-erythro-
pentofuranosyl chloride by 7-nitroindole followed by NaOH
treatment (yield 50%). The X-ray structure of the crystalline
nucleoside 2 indicated a 2.4 Å distance between H-1′ and one of
the oxygens of the nitro group.16 The 500 MHz 1H NMR
conformational analysis of the corresponding bis-p-toluoyl deriva-
tive 1 in CDCl3 confirmed the close proximity of those two atoms
in solution. Irradiation17 of 1 in aqueous acetonitrile solution (1:1
by volume, C ) 2 mM) for 1 h yielded the 2′-deoxyribonolactone
derivative 6 along with 7-nitrosoindole 10.18 No trace of side
products was detected in the HPLC chromatogram of the
irradiation mixture.
In the course of programs devoted to synthesis11 and study12
of “true” abasic site A (Scheme 1) and of its recognition by
synthetic molecules,13 we became interested in the examination
of the structurally related 2′-deoxyribonolactone damage. We
report here a general and efficient synthesis of oligodeoxyribo-
* To whom correspondence should be addressed. E-mail: Mitsu.Kotera@
ujf-grenoble.fr. Tel: 33 (0) 4 76 51 48 63. Fax: 33 (0) 4 76 51 43 82.
(1) (a) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor
& Francis Inc.: Philadelphia, PA, 1987. (b) Tronche, C.; Goodman, B. K.;
Greenberg, M. M. Chem. Biol. 1998, 5, 263-271. (c) Goldberg, I. H. Acc.
Chem. Res. 1991, 24, 191-198.
(2) Kappen, L. S.; Goldberg, I. H. Biochemistry 1989, 28, 1027-1032.
(3) (a) Pitie´, M.; Bernadou, J.; Meunier, B. J. Am. Chem. Soc. 1995, 117,
2935-2936. (b) Pratviel, G.; Bernadou, J.; Meunier, B. AdV. Inorg. Chem.
1998, 45, 251-312.
(4) (a) von Sonntag, C.; Schuchmann, H. P. Angew. Chem., Int. Ed. Engl.
1991, 30, 1229-1253. (b) Decarroz, C.; Wagner, J. R.; Cadet, J. Free Radical
Res. Commun. 1987, 2, 295-301.
(5) Urata, H.; Akagi, M. Nucleic Acids Res. 1991, 19, 1773-1778.
(6) (a) Goyne, T. E.; Sigman, D. S. J. Am. Chem. Soc. 1987, 109, 2846-
2848. (b) Zelenko, O.; Gallagher, J.; Xu, Y.; Sigman, D. S. Inorg. Chem.
1998, 37, 2198-2204. (c) Meijler, M. M.; Zelenko, O.; Sigman, D. S. J. Am.
Chem. Soc. 1997, 119, 1135-1136. (d) Chen, T.; Greenberg, M. M. J. Am.
Chem. Soc. 1998, 120, 3815-3816.
The 7-nitroindole nucleoside 2 was then incorporated into
oligonucleotides according to the classical phosphoramidite
technology. Two pentamers were first prepared, containing the
four natural bases with the nitroindole nucleoside (Ni) in the
middle of the sequence flanked either by pyrimidines or by
purines, respectively d(GCNiTA) 3 and d(CANiGT) 4. Coupling
of the Ni nucleoside occurred in satisfactory yield. The oligomers
(7) Povirk, L. F.; Houlgrave, C. W.; Han, Y.-H. J. Biol. Chem. 1988, 263,
19263-19266.
(8) Povirk, L. F.; Goldberg, I. H. Proc. Natl. Acad. Sci. U.S.A. 1985, 82,
3182-3186.
(9) Deoxyoligonucleotides containing a 5′-ABrU site in the middle of a
duplex structure undergo photoreaction to produce a 2′-deoxyribonolactone
residue with release of free adenine.10
(10) Sugiyama, H.; Tsutsumi, Y.; Saito, I. J. Am. Chem. Soc. 1990, 112,
6720-6721.
(14) Breslow, R. Acc. Chem. Res. 1980, 13, 170-177.
(15) (a) Chow, Y. L.; Michon, J.; Michon, P.; Morat, C.; Rassat, A.
Tetrahedron Lett. 1992, 33, 3315-3318. (b) Nakagaki, R.; Mutai, K. Bull.
Chem. Soc. Jpn. 1996, 69, 261-273. (c) Scholl, P. C.; Van De Mark, M. R.
J. Org. Chem. 1973, 38, 2376-2378.
(11) Laayoun, A.; Decout, J.-L.; Defrancq, E.; Lhomme, J. Tetrahedron
Lett. 1994, 28, 4991-4994.
(12) (a) Coppel, Y.; Berthet, N.; Coulombeau, C.; Coulombeau, Ce.; Garcia,
J.; Lhomme, J. Biochemistry 1997, 36, 4817-4830. (b) Fouilloux, L.; Berthet,
N.; Coulombeau, C.; Coulombeau, Ce.; Dheu-Andries, M.-L.; Garcia, J.;
Lhomme, J.; Vatton, P. J. Mol. Struct. 1995, 330, 417-422.
(13) (a) Fkyerat, A.; Demeunynck, M.; Constant, J.-F.; Michon, P.;
Lhomme, J. J. Am. Chem. Soc. 1993, 115, 9952-9959. (b) Berthet, N.;
Boudali, A.; Constant, J.-F.; Decout, J.-L.; Demeunynck, M.; Fkyerat, A.;
Garcia, J.; Laayoun, A.; Michon, P.; Lhomme, J. J. Mol. Recognit. 1994, 7,
99-107. (c) Berthet, N.; Constant, J.-F.; Demeunynck, M.; Michon, P.;
Lhomme, J. J. Med. Chem. 1997, 40, 3346-3352.
(16) Averbuch-Pouchot, M.-T.; Bourdat, A.-G.; Defrancq, E.; Durif, A.;
Kotera, M.; Lhomme, J. Z. Kristallogr. - New Cryst. Struct. 1998, 213, 181-
182.
(17) The photolysis lamp, suspended in a jacketed, water-cooled immersion
well, is a 100-W high-pressure mercury arc Hanovia lamp with Pyrex filter.
Owing to the known character of the ribonolactone moiety, the irradiation
time and concentration as indicated must be strictly controlled to avoid strand
cleavage during irradiation and workup.
10.1021/ja982617a CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/29/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 45, 1998 11811
Figure 3. Irradiation of 5 with Pyrex filter (5 mM sodium phosphate
buffer, pH 6.0, C ) 15 µM). HPLC profiles of 5 (a) before irradiation
and (b) after 30 min of irradiation. 9 is formed along with 7-nitrosoindole
10. HPLC analysis was performed using a Nucleosil C18 column
(Macherey-Nagel) with a detection at 260 and 370 nm; elution was carried
out with a linear gradient of MeOH (5-50%) for 20 min in 5 mM sodium
phosphate buffer (pH 6). The flow rate was 4 mL/min.
observed at 3.14 and 2.71 ppm corresponding to the methylene
protons R to the carbonyl of the deoxyribonolactone moiety. In
the ESMS spectra, the presence of the peaks at m/z 1367.18 and
m/z 683.07, corresponding, respectively, to the calculated mo-
lecular ions [M - H]- and [M - 2H]2-, confirmed the structure
(Figure 1b).
The method was extended to synthesis of longer DNA
fragments. The undecamer 9 was obtained in the same conditions
with identical yields (no secondary peaks visible in the HPLC
spectrum, Figure 3). It was characterized by the ESMS and the
Figure 1. ESMS spectra of the (a) 7-nitroindole-containing oligonu-
cleotide 3 and (b) 2′-deoxyribonolactone-containing oligonucleotide 7.
1
500 MHz H NMR spectra. The undecamer 9 was hybridized
with the complementary strand, and the resulting duplex was
examined for stability. The melting temperature Tm ) 35.6 °C
was determined. This value can be compared to that measured
for the reference unmodified duplex (Tm ) 56.9 °C). This melting
temperature decrease (∆Tm ) 21.3 °C) is indicative of strong
destabilization of the double helix induced by the presence of
the 2′-deoxyribonolactone site. This stability decrease is indeed
quite close to that previously determined for the duplex undecamer
containing the tetrahydrofuran abasic site analog19 C (Scheme 1)
in the same position in the sequence (Tm ) 36.9 °C).13b
As a conclusion the described approach constitutes a highly
efficient route to obtain oligonucleotides containing 2′-deoxyri-
bonolactone. Studies are in progress to determine the influence
of this damage on the structure of the DNA helix and on the
behavior of polymerases and repair enzymes.
Figure 2. 500 MHz 1H NMR spectra of (a) oligonucleotide 3 containing
the nitroindole nucleoside (the asterisks indicate the signals of aromatic
and anomeric protons of the nitroindole nucleoside) and (b) oligonucle-
otide 7 resulting from irradiation of 3 containing the 2′-deoxyribonolactone
residue.
3 and 4 were characterized by their ESMS and 500 MHz 1H NMR
spectra (see spectra of 3 given as an example in Figures 1a and
2a). 3 and 4 were both irradiated in dilute aqueous solution (5
mM sodium phosphate buffer, pH 6.0, C ) 15 µM) at room
temperature.17 After 30 min the nitroindole-containing pentamers
were totally transformed into new oligonucleotides, respectively,
7 and 8, that appeared as single peaks in the HPLC spectrum,
accompanied by the 7-nitrosoindole 10 peak. 7-Nitrosoindole 10
was eliminated by ether extraction. The two pentamers 7 and 8
were isolated and identified by ESMS and 1H NMR spectroscopy.
Supporting Information Available: Experimental procedures for the
preparation of 1 and 2, electrospray mass spectra of oligodeoxynucleotides
4, 5, and 9, and 1H NMR spectra of oligonucleotides 3, 4, and 7 (8 pages,
print/PDF). See any current masthead page for ordering information and
Web access instructions.
JA982617A
1
(18) Compounds 6 and 10 were characterized by their MS and H NMR
spectra. Furthermore, for comparison of the retention time in HPLC, an
authentic sample of 6 was prepared by mCPBA oxidation of 2-deoxy-1-O-
methyl-3,5-bis-p-toluoylribofuranose.
1
The 500 MHz H NMR spectrum of 7 (given for illustration in
(19) As the 2′-deoxyribose site in DNA is unstable and subject to
â-elimination, the chemically stable analogue C is most frequently used in
structural and biological studies. It is indeed a substrate for repair endonu-
cleases.20
(20) Takeshita, M.; Chang, C. N.; Johnson, F.; Will, S.; Grollman, A. P.
J. Biol. Chem. 1987, 262, 10171-10179.
Figure 2b) shows absence of signals between 7.00 and 7.70 ppm
corresponding to indole protons and disappearance of the signal
at 6.25 ppm corresponding to the anomeric proton of the
nitroindole nucleoside in 3. Furthermore, new signals are