Site-specific generation of deoxyribonolactone lesions in DNA oligonucleotides

Article abstract of DOI:10.1021/ol016255e

(matrix presented) An efficient method for the site-specific generation of 2-deoxyribonolactone oxidative DNA damage lesions from a "photocaged" nucleoside analogue was developed. A nucleoside phosphoramidite bearing a C-1′ nitrobenzyl cyanohydrin was prepared and incorporated into DNA oligonucleotides using automated DNA synthesis. The caged analogue, which was stable in aqueous solution, was converted to the 2-deoxyribonolactone lesion by UV irradiation. DNA containing the caged analogue and the deoxyribonolactone site were characterized by electrospray mass spectrometry (ES-MS).

Full text of DOI:10.1021/ol016255e

ORGANIC
LETTERS
2001
Vol. 3, No. 15
2415-2418
Site-Specific Generation of
Deoxyribonolactone Lesions in DNA
Oligonucleotides
Hamilton J. Lenox, Christopher P. McCoy, and Terry L. Sheppard*
Department of Chemistry, Northwestern UniVersity, 2145 Sheridan Road,
EVanston, Illinois 60208-3113
sheppard@chem.northwestern.edu
Received June 8, 2001
ABSTRACT
An efficient method for the site-specific generation of 2-deoxyribonolactone oxidative DNA damage lesions from a “photocaged” nucleoside
analogue was developed. A nucleoside phosphoramidite bearing a C-1′ nitrobenzyl cyanohydrin was prepared and incorporated into DNA
oligonucleotides using automated DNA synthesis. The caged analogue, which was stable in aqueous solution, was converted to the
2-deoxyribonolactone lesion by UV irradiation. DNA containing the caged analogue and the deoxyribonolactone site were characterized by
electrospray mass spectrometry (ES-MS).
DNA damage by toxic agents compromises the coding
potential and strand integrity of the genetic material.1
Oxidative DNA damage, mediated by free radicals and
reactive oxygen species (ROS), represents a major contributor
to cellular DNA damage.2 Oxidation of DNA may occur at
the nucleobase3 or sugar4 moieties of DNA nucleotides. The
resulting lesions may labilize the otherwise stable DNA
backbone and/or induce mutations during cellular nucleic
acid synthesis. Thus, evolution has developed complex
enzymatic pathways that recognize and repair oxidative DNA
damage.5 An understanding of the chemical mechanisms of
DNA damage and repair is central to uncovering the
biological effects of genomic lesions.
pathways7 produces 2-deoxyribonolactone, or C-1′ oxidized
abasic site, lesions (2) within DNA. Recent studies from the
laboratories of Sigman,8 Greenberg,9 and Chatgilialoglu10
have enumerated diverse chemical mechanisms that produce
(6) For a review, see: Greenberg, M. M. Chem. Res. Toxicol. 1998, 11,
1235-1248.
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1997, 237, 1-5. (b) Harkin, L. A.; Butler, L. M.; Burcham, P. C. Chem.
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Biol. Chem. 1979, 254, 12269-12272. (b) Goyne, T. E.; Sigman, D. S. J.
Am. Chem. Soc. 1987, 109, 2846-2848. (c) Meijler, M. M.; Zelenko, O.;
Sigman, D. S. J. Am. Chem. Soc. 1997, 119, 1135-1136. (d) Zelenko, O.;
Gallagher, J.; Sigman, D. S. Angew. Chem., Int. Ed. Engl. 1997, 36, 2776-
2778.
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1998, 5, 263-271. (b) Tallman, K. A.; Tronche, C.; Yoo, D. J.; Greenberg,
M. M. J. Am. Chem. Soc. 1998, 120, 4903-4909. (c) Chen, T.; Greenberg,
M. M. J. Am. Chem. Soc. 1998, 120, 3815-3816. (d) Greenberg, M. M.;
Yoo, D. J.; Goodman, B. K. Nucleosides Nucleotides 1997, 16, 33-40. (e)
Goodman, B. K.; Greenberg, M. M. J. Org. Chem. 1996, 61, 2-3. (f)
Hwang, J.-T.; Tallman, K. A.; Greenberg, M. M. Nucleic Acids Res. 1999,
27, 3805-3810. (g) Hashimoto, M.; Greenberg, M. M.; Kow, Y. W.;
Hwang, J. T.; Cunningham, R. P. J. Am. Chem. Soc. 2001, 123, 3161-
3162.
Oxidation of the C-1′ position of DNA nucleotides (1) by
exogenous toxic agents6 and endogenous lipid peroxidation
(1) Lindahl, T. Nature 1993, 362, 709-715.
(2) Aust, A. E.; Eveleigh, J. F. Proc. Soc. Exp. Biol. Med. 1999, 222,
246-252.
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(4) (a) Pogozelski, W. K.; Tullius, T. D. Chem. ReV. 1998, 98, 1089-
1107. (b) Povirk, L. F.; Steighner, R. J. Mutat. Res. 1989, 214, 13-22. (c)
Burger, R. M. Chem. ReV. 1998, 98, 1153-1169.
(5) (a) Friedberg, E. C.; Walker, G. C.; Siede, W. DNA Repair and
Mutagenesis; American Society for Microbiology: Washington, DC, 1995.
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Am. Chem. Soc. 1999, 121, 2927-2928. (c) Chatgilialoglu, C.; Ferreri, C.;
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10.1021/ol016255e CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/26/2001

Scheme 1. Deoxyribonolactone Lesion Formation and
Scheme 2. Site-Specific Generation of DNA
Cleavage
Deoxyribonolactone Sites
deoxyribonolactone lesions in DNA. Scheme 1 shows a
generalized pathway for the formation of and subsequent
DNA strand scission at deoxyribonolactone sites. The C-1′
oxidative damage reaction is initiated by hydrogen abstrac-
tion at a deoxyribonucleotide in DNA (1, Scheme 1) to
produce a C-1′ sugar radical. Once formed, the radical species
may undergo one of several possible oxygenation pathways,
which ultimately results in the extrusion of the nucleobase
and the production of the oxidized abasic site, 2. By virtue
of its lactone character, 2 may undergo â-elimination to
produce the R,â-unsaturated lactone DNA strand, 3, and a
5′-phosphorylated DNA product (4). The ene-lactone 3 may
decompose further to produce a 3′-phosphorylated cleavage
product (5) and methylene furanone (6).
Because of the lability of the deoxyribonolactone lesion,
methods for the controlled introduction of oxidized abasic
site lesions into DNA are required. Furthermore, such
methods would provide direct experimental insight into the
chemical properties and biological effects of this oxidative
damage lesion. Existing approaches to deoxyribonolactone
lesions within DNA have relied on pathways analogous to
the natural C-1′ oxidative damage reaction. In particular,
photolysis of C-1′ tert-butyl ketone9,10 or nitroindole11
nucleoside analogues produces C-1′ deoxyribose radicals,
which are efficiently oxygenated to provide oxidized abasic
sites within DNA. These approaches have provided the first
methods for specific deoxyribonolactone generation and have
offered significant insights into the properties of these DNA
damage lesions.9g,11c
photolabile lactone precursor based upon (1) its predicted
compatibility with DNA synthesis and deprotection condi-
tions,12 (2) the expected stability of the caged analogue 8
under aqueous conditions,13 and (3) the known photochem-
istry of o-nitrobenzyl ethers.14 Thus, incorporation of ana-
logue 7 during solid-phase DNA synthesis would produce
the caged-lactone DNA (8). The o-nitrobenzyl ether of 8 then
would be cleaved by UV irradiation to produce the lactone
cyanohydrin, 9, which would be expected to decompose to
the lactone lesion within DNA (2). Herein we describe the
synthesis of caged phosphoramidite 7, its incorporation into
DNA (8), and the efficient photochemical generation and
characterization of 2-deoxyribonolactone lesions (2, Scheme
2) within DNA.
A versatile synthetic route to the caged nucleoside
analogue 7 was developed (Scheme 3). The approach
centered on the construction of a ribosyl donor, cyanobro-
mide 12, which was prepared from the known15 1-chloro-
3,5-di-p-chlorobenzoyl-D-ribofuranose (10). Conversion of
10 into C-1 cyanide (11) was accomplished by reaction with
diethylaluminum cyanide.16 Free-radical bromination of 11
with NBS produced the anomeric mixture of C-1 bromo-
cyanides 12 in 80% yield. The nitrobenzyl group was
installed using silver triflate-promoted glycosylation,17 to
produce 13 in 70% yield as a mixture of C-1′ isomers.
To convert 13 into a form suitable for automated DNA
synthesis, the 3′- and 5′-O-p-chlorobenzoyl protecting groups
were removed with methanolic ammonia18 to produce an
inseparable mixture of the anomeric nucleosides 14. Trans-
formation of 14 to the corresponding mono-methoxytrityl
We now report an efficient method for the site-specific
introduction of 2-deoxyribonolactone lesions in DNA oli-
gonucleotides that is independent of the C-1′ damage
reaction. Our approach, which is outlined in Scheme 2,
involves the generation of lactone sites within DNA from a
stable, photochemically “caged” form of the lesion. The C-1′
cyanohydrin 2-nitrobenzyl ether (7) was selected as a
(12) Ordoukhanian, P.; Taylor, J.-S. J. Am. Chem. Soc. 1995, 117, 9570-
9571.
(13) Olah, G. A.; Arvanaghi, M.; Surya Prakash, G. K. J. Am. Chem.
Soc. 1982, 104, 1628-1631.
(14) Pillai, V. N. Rajasekharan. Synthesis 1980, 1-26.
(15) Eger, K.; Jalalian, M.; Schmidt, M. Tetrahedron 1994, 50, 8371-
8380.
(16) Iyer, R.; Phillips, L.; Egan, W. Synth. Commun. 1991, 20, 2053-
2063.
(17) Buchanan, J. G.; Clelland, A. P. W.; Wightman, R. H.; Johnson,
T.; Rennie, R. A. C. Carbohydr. Res. 1992, 237, 295-301.
(18) Uteza, V.; Chen, G.; Tuoi, J.; Fenet, B.; Grouiller, A. Tetrahedron
1993, 49, 8579-8588.
(11) (a) Kotera, M.; Bourdat, A.; Defrancq, E.; Lhomme, J. J. Am. Chem.
Soc. 1998, 120, 11810-11811. (b) Kotera, M.; Bourdat, A.-G.; Defrancq,
E.; Jourdan, M.; Garcia, J.; Coulombeau, C.; Lhomme, J. Nucleosides
Nucleotides 1999, 18, 1323-1324. (c) Jourdan, M.; Garcia, J.; Defrancq,
E.; Kotera, M.; Lhomme, J. Biochemistry 1999, 38, 3985-3995. (d) Kotera,
M.; Roupioz, Y.; Defranq, E.; Bourdat, A.-G.; Garcia, J.; Coulombeau, C.;
Lhomme, J. Chem. Eur. J. 2000, 6, 4163-4169.
2416
Org. Lett., Vol. 3, No. 15, 2001

completely deprotected the oligonucleotides with minimal
modification of the caged analogue. Reverse-phase HPLC
(RP-HPLC) purification was used to provide oligonucleotides
containing 8 in high synthetic yield and purity.
Scheme 3. Synthesis of Phosphoramidite 7a
Oligonucleotides containing the caged analogue were
characterized by electrospray mass spectrometry (ES-MS)
and base composition analysis. Oligonucleotide 16 was
subjected to ES-MS analysis. As illustrated in Figure 2A
a (a) Et2AlCN, THF, 4 °C to rt, 4 h, 75%; (b) NBS, benzoyl
peroxide, CCl4, 85 °C, 2 h, 80%; (c) 2-nitrobenzyl alcohol, 2,6-
lutidine, AgOTf, CH2Cl2, 3 h, 70%; (d) 4.5 M NH3/MeOH, THF,
40 °C, 3 h, 76%; (e) MMTCl, DMAP, pyridine, 18 h, 80%; (f)
[(i-Pr)2N]2POCH2CH2CN, (i-Pr)2NH2•CHN4, CH2Cl2, 2.5 h, 83%.
(MMT) ethers (15a, 15b) was accomplished in 80% yield
by reaction of 14 with MMTCl in pyridine. Addition of the
hydrophobic MMT group facilitated the chromatographic
separation of the two C-1′ isomers 15a and 15b.19 The MMT
derivatives (15a and 15b) were converted independently into
the corresponding phosphoramidite derivatives (7a or 7b)
using the method of Caruthers.20
Caged phosphoramidite analogues (7) were incorporated
with high efficiency into DNA oligonucleotides with minor
modifications of standard automated DNA synthesis meth-
ods.21 Caged oxidized abasic sites were inserted indepen-
dently into several oligonucleotide sequences, 16, 18, and
19, shown in Figure 1. After DNA synthesis, DNA strands
Figure 2. ES-MS and RP-HPLC characterization of DNA oligo-
nucleotides containing the caged analogue (16) and the oxidized
abasic site (17). (A) ES-MS analysis of RP-HPLC purified 16,
containing the caged lactone analogue (8). (B) RP-HPLC trace of
a crude photolysis reaction of caged-DNA 16 (tR 31.26 min) to
form lactone-containing DNA 17 (tR 29.03 min). (C) ES-MS
analysis of oligonucleotide 17 containing the 2-deoxyribonolactone
lesion (2).
Figure 1. Caged and lactone-containing DNA sequences.
containing 8 were deprotected at room temperature for 14-
16 h with 1.5 M NH3 in methanol. These mild conditions
(19) The stereochemistry of the two cyanohydrin anomers, 15a and 15b,
was assigned by conversion to the corresponding 13a and 13b analogues.
X-ray structural analysis revealed that 13b possessed â-stereochemistry at
the C-1′ position. See Supporting Information.
(20) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.;
Fisher, E. F.; McBride, L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J. Y.
Methods Enzymol. 1987, 154, 287-313.
the observed m/z for 16 was 5188.0, which corresponded
well with the calculated value of 5188.33. In addition,
enzymatic digestion of 16 to its component nucleosides and
quantitative RP-HPLC revealed that 16 contained deoxy-
nucleosides and analogue 14 in the expected ratios.22
Org. Lett., Vol. 3, No. 15, 2001
2417

Oligonucleotides 18 and 19 also gave the expected m/z values
by ES-MS. Thus, solid-phase DNA synthesis using phos-
phoramidite 7 provides an effective route to oligonucleotides
containing the masked lactone analogue 8.
efficiency in double-stranded DNA. For example, photolysis
of 16 in the presence of a complementary strand with dA or
dC paired opposite 8 produced lesion 2 in ∼90% yield in
40 min. Thus, photolytic decaging of lactone analogue 8
offers a clean route to DNA containing the deoxyribonolac-
tone lesion 2.
The synthetic route outlined in Scheme 3 provides a
versatile approach for the construction of other DNA
deoxyribonolactone lesion synthons. For example, a series
of caged lactones with tunable photochemical properties such
as faster deprotection rates or altered absorption maxima
could be synthesized from 12. Other nonphotochemical
caging strategies also could be employed. In addition,
photolabile alkoxycyanohydrins may offer a new protection
strategy for lactones and other carbonyl functional groups.
Several O-protected ester cyanohydrin derivatives have been
reported.23 However, to our knowledge, this report represents
the first demonstration of photolabile 2,2-dialkoxyalkane-
nitriles as masked lactones.
Oligonucleotides containing the caged oxidized abasic site
precursor 8 were converted efficiently by photolysis to
deoxyribonolactone-modified DNA. Irradiation of sequence
16 in buffered aqueous solution provided the oxidized abasic
site 2 within sequence 17 (Figure 1). The time course of the
decaging reaction was analyzed by gel electrophoresis:
photolysis of 16 demonstrated the time-dependent formation
of an alkali labile site over 40 min of irradiation (Supporting
Information). The efficiency of the decaging process is
demonstrated in Figure 2B, which shows a RP-HPLC trace
for the crude reaction mixture resulting from photolysis of
16 for 40 min at 350 nm. Oligonucleotide 16 (retention time
31.26 min) was converted in ∼90% yield to a single new
product that displayed a shorter retention time (29.03 min).
This faster moving material was isolated and subjected to
ES-MS analysis (Figure 2C), which confirmed the identity
of the product as sequence 17 containing the deoxribono-
lactone lesion, 2 (calcd m/z ) 5026.2; found 5026.4). The
high photolytic efficiency for conversion of the masked
analogue 8 to an oxidized abasic site (2) was independent
of the anomeric identity of the starting phosphoramidite (7a
or 7b).
We have outlined an efficient independent synthesis of
the 2-deoxyribonolactone lesion in DNA oligonucleotides,
which is independent of the C-1′ radical DNA damage
pathway. This general strategy will facilitate the further
investigation of the chemical biology of DNA oxidized abasic
site lesions.
Acknowledgment. This work was supported by a Basil
O’Connor Starter Scholar Research Award (#5-FY99-810)
from the March of Dimes Birth Defects Foundation. T.L.S.
is a recipient of the Burroughs Wellcome Fund New
Investigator Award in the Toxicological Sciences. We thank
Dr. Charlotte Stern for assistance with crystallography, and
the University of Illinois, Urbana-Champaign Mass Spec-
trometry Laboratory for MS analysis.
Our approach to deoxyribonolactone-containing DNA has
been generalized to other DNA sequences and to duplex
DNA. Analysis of sequences 18 and 19 demonstrated that
the photolytic decaging of 8 to oxidized abasic sites (2) was
independent of the sequence flanking the caged analogue.
In addition, the decaging process occurs with similar
(21) Phosphoramidite 7a coupled at ∼64% and was extended with an
efficiency of >95%. Analogue 7b coupled at ∼70% and extended at >90%
efficiency. Because DNA contains â-nucleosides, our studies employed
primarily the â-isomer, 7b.
(22) Eadie, J. S.; McBride, L. J.; Efcavitch, J. W.; Hoff, J. B.; Cathcart,
R. Anal. Biochem. 1987, 165, 442-447.
(23) (a) Utimoto, K.; Wakabayashi, Y.; Shishiyama, Y.; Inoue, M.;
Nozaki, H. Tetrahedron Lett. 1981, 22, 4279-4280. (b) Kirchmeyer, S.;
Mertens, A.; Arvanaghi, M.; Olah, G. A. Synthesis 1983, 498-499. (c)
Tomoda, S.; Takeuchi, Y.; Nomura, Y. Chem. Lett. 1982, 1733-1734.
Supporting Information Available: Detailed experi-
mental protocols and spectral data for compounds 7 and 10-
15, X-ray crystal data for 13b. An X-ray crystallographic
file in cif format. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL016255E
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Org. Lett., Vol. 3, No. 15, 2001