Photochemical generation of C4′-oxidized abasic site containing oligodeoxynucleotide and its efficient amine modification

Article abstract of DOI:10.1021/ol060987v

We synthesized oligodeoxynucleotide (ODN, 3), which contains 4′-o-nitrobenzyloxythymidine (4) as a caged precursor of C4′-oxidized abasic site (1). Photoirradiation of 3 at 365 nm followed by amine treatment under neutral conditions afforded the lactam (2

Full text of DOI:10.1021/ol060987v

ORGANIC
LETTERS
2006
Vol. 8, No. 15
3183-3186
Photochemical Generation of
C4 -Oxidized Abasic Site Containing
′
Oligodeoxynucleotide and Its Efficient
Amine Modification
Mariko Aso,*,† Kazuteru Usui,† Mitsuhiro Fukuda,† Yoshie Kakihara,‡
Tsuyoshi Goromaru,‡ and Hiroshi Suemune*,†
Graduate School of Pharmaceutical Sciences, Kyushu UniVersity, Fukuoka,
Japan 812-8582, and Faculty of Pharmacy and Pharmaceutical Sciences,
Fukuyama UniVersity, Fukuyama, Japan 729-0292
aso@phar.kyushu-u.ac.jp; suemune@phar.kyushu-u.ac.jp
Received April 25, 2006
ABSTRACT
We synthesized oligodeoxynucleotide (ODN, 3), which contains 4′-o-nitrobenzyloxythymidine (4) as a caged precursor of C4′-oxidized abasic
site (1). Photoirradiation of 3 at 365 nm followed by amine treatment under neutral conditions afforded the lactam (2) efficiently. Duplexed ODN
3 was converted to 1 faster and more efficiently than single stranded 3, whereas amine treatment of 1 formed from single stranded 3 resulted
in slightly faster lactam formation than with the duplex.
Bleomycin-induced oxidative damage of DNA under limiting
oxygen conditions results in the formation of alkali-labile
C4′-oxidized abasic site (1) (Scheme 1).1 The studies of the
properties and reactivities of 1 are essential because of their
relation to toxicity and the effects on mutagenesis of 1. Under
equilibrium, the 1,4-dihydroxytetrahydrofuran structure of
1 can exist in the open form with the 1,4-dicarbonyl structure.
Reactive aldehydes, especially bifunctional ones, can be toxic
as a result of the reaction of a DNA base and a protein.2 We
found that unsaturated lactam (2a) and a DNA fragment were
formed in the reaction of 1 with amine under mild conditions
(room temperature, pH 7).3 This result indicated the pos-
sibility of modifying amine-containing biomolecules by an
Scheme 1. Reaction of 1 with Amine to Form Lactam
† Kyushu University.
‡ Fukuyama University.
(1) Sugiyama, H.; Kilkuskie, R. E.; Hecht, S. M.; van der Marel, G. A.;
van Boom, J. H. J. Am. Chem. Soc. 1985, 107, 7765.
(2) (a) Nechev, L. V.; Harris, C. M.; Harris, T. M. Chem. Res. Toxicol.
2000, 13, 431. (b) Xu, G.; Liu, Y.; Kansal, M. M.; Sayer, L. M. Chem.
Res. Toxicol. 1999, 12, 855. (c) Chen, L.-J.; Hecht, S, S,; Peterson, L. A.
Chem. Res. Toxicol. 1997, 10, 866.
10.1021/ol060987v CCC: $33.50
© 2006 American Chemical Society
Published on Web 06/24/2006

oligodeoxynucleotide (ODN) containing 1. Lactam formation
was also studied with a C4′-selenated nucleotide, which
generates 1 by treatment with NBS; in this case the lactam
was formed under neutral conditions in good yield.4 How-
ever, quantitative studies on lactam formation from 1 in an
ODN have not been done because formation of 1 in an ODN
by bleomycin is not efficient.5 To study reactivities of 1 in
an ODN, efficient formation of 1 is necessary. We planned
to prepare 1 in an ODN from a caged precursor, which might
be applicable to experiments under physiological conditions.
to form the ODN containing 1 followed by amine treatment,
which results in efficient lactam formation.
Reaction of 4′,5′-unsaturated thymidine (5)8 with m-CPBA
in the presence of o-nitrobenzyl alcohol (10 equiv) gave 7a
and 7b, in 12% and 8%, respectively (Scheme 2). Stereo-
Scheme 2. Synthesis of 4′-o-Nitrobenzyloxythymidine
Derivatives
The lactam formation reaction of 1 does not require extra
reagents such as NaBH4 as in the lysine modification via a
Schiff base. In addition, the structure of the ODN containing
1 should be similar to the unmodified ODN. Based on these
ideas, this lactam formation reaction of 1 may be applicable
to in situ lysine modification and mapping of lysine residues
of DNA binding proteins for their structure-function studies.
Recently, Greenberg et al. reported generation of 1 by
photochemical cleavage of caged sugars, which carry 3,4-
dimethoxy-6-nitrobenzyloxy groups at the C1′ and C4′
positions of 2′-deoxyribose. 5′-O-Silylated O-methyl phos-
phoramidites prepared from the caged sugars were incorpo-
rated into an ODN. They studied chemical stabilities and
biological effects of the ODN containing 1.6 In contrast, we
designed a caged nucleoside that retained the base moiety
at the C1′-position and carried a o-nitrobenzyloxy group at
the C4′-position. We expect that the use of the caged
nucleoside that we have designed will be advantageous for
a site-specific lysine modification, because irradiation of a
caged ODN-protein complex was possible, in which the
interaction of the retained base moiety and the target protein
could be maintained. Thus, we prepared ODN 3 containing
4′-o-nitrobenzyloxythymidine (4). The sequence of the
unmodified parent ODN of 3, 3-nat, contains the binding
site of the DnaA protein that is involved in the initiation of
replication in Escherichia coli. Recently, the X-ray crystal
structure of the complex of DNA binding domain (domain
IV) of DnaA protein and duplex ODN (3-nat) was reported.7
The results of X-ray crystallography suggested that one of
the lysine residues in domain IV (Lys-415) is located near
the phosphate group between C2 and T3. The open form of
1 might react with Lys-415 to modify it if the lysine residue
can come close to carbonyl functions during the molecular
vibrations of the protein conformation in solution. Presently,
we describe the synthesis of 3 and its photochemical reaction
Scheme 3. Photoreaction of 3 Followed by Treatment with
Amine
(3) Aso, M.; Kondo, M.; Suemune, H.; Hecht, S. M. J. Am. Chem. Soc.
1999, 121, 9023.
(4) Aso, M.; Ryuo, K.; Kondo, M.; Suemune, H. Chem. Pharm. Bull.
2000, 48, 1384.
(5) Kozarich et al. quantitated 1 formed from d(CGCGCG) by bleomycins
activated by Fe3+ and H2O2 anaerobically (29%) or by Fe2+ and O2 (22%):
Rabow, L. E.; Stubbe, J.; Kozarich, J. W. J. Am. Chem. Soc. 1990, 112,
3196.
(6) (a) Kim, J.; Gil, J. M.; Greenberg, M. M. Angew. Chem., Int. Ed.
2003, 42, 5882. (b) Greenberg, M. M.; Weledji, Y. N.; Kroeger, K. M.;
Kim, J.; Goodman, M. F. Biochemistry 2004, 43, 2656. (c) Greenberg, M.
M.; Weledji, Y. N.; Kim, J.; Bales, B. C. Biochemistry 2004, 43, 8178. (d)
Kroeger, K. M.; Kim, J.; Goodman, M. F.; Greenberg, M. M. Biochemistry
2004, 43, 13621.
chemistry of 7a was determined by NOESY measurements.
Epoxide 6 could be an intermediate though it was not
isolated.9 Addition of Lewis acid improved the yields of the
(7) Fujikawa, N.; Kurumizaka, H.; Nureki, O.; Terada, T.; Shirouzu, M.;
Katayama, T.; Yokoyama, S. Nucleic Acids Res. 2003, 31 (8), 2077.
3184
Org. Lett., Vol. 8, No. 15, 2006

Figure 2. Time course of (a) formation of 1 from 3 (O) and ds 3
(4) and (b) formation of 2b and (c) 10 from 1 prepared from 3 (O)
and ds 3 (4).
mated DNA synthesizer. After the synthesis of ODN in trityl
on mode, it was purified with HPLC, and the DMTr group
of the ODN was removed under acidic conditions (20%
AcOH, room temperature, 4 h) to give 3 (Supporting
Information). The structure of the obtained ODN was
confirmed by MALDI TOF MS ((m/z) 4107.44 (4109.73,
calcd for 3)). Tm values of the duplex between 3 and the
complementary 13-mer (3-com) were close (38.7 and 46.0
°C in H2O and phosphate buffer) to those of 3-nat (42.4
and 47.7 °C in H2O and phosphate buffer; Supporting
Information).
Figure 1. HPLC analysis. (a) Photoirradiation of 3 (retention time
) 27 min) in H2O containing 100 mM NaCl. 3-nat (retention time
) 20 min) was added as an internal standard. Product A eluted at
18 min. (b) Reaction mixture of the photolyzed 3 with 3-amino-
1,2-propanediol. Products B and C eluted at 9 and 13 min,
respectively. (c) Photoirradiation of ds 3. Complementary strand
of 3 (3-com) eluted at 24 min.
products. To a mixture of 5 and o-nitrobenzyl alcohol (3
equiv) was added m-CPBA at 0 °C. After consumption of
5, 1 equiv of ZnCl2‚OEt2 was added at -78 °C. Diastereo-
mers 7a and 7b were isolated in 32% and 15%, respectively.
Desilylation of 7a afforded C4′-substituted thymidine 4,
which was further converted to phosphoramidite 9 Via 8.
The phosphoramidite 9 was incorporated into the 13-mer
by the conventional phosphoramidite method with an auto-
The photoreaction of 3 was studied with a 10 µM solution
in water or phosphate buffer (10 mM, pH 7) containing 100
mM NaCl and 3-nat (3 µM), which was added to the reaction
mixture as an internal standard.10 Irradiation at 365 nm with
a handy UV lamp led to smooth conversion of 3 to a new
product A in 30 min (Figure 1a). Liberation of thymine was
also confirmed by co-injection of the reaction mixture and
(8) Maag, H.; Rydzewski, R. M.; McRoberts, M. J.; Crawford-Ruth, D.;
Verheyden, J. P. H.; Prisbe, E. J. J. Med. Chem. 1992, 35, 1440.
(9) Tong, W.; Agback, P.; Chattopadhyaya, J. Acta Chem. Scand. 1993,
47, 145.
(10) HPLC analysis of the reaction mixture suggetsed that the peak areas
of 3-nat and 3-com were not changed during reactions.
Org. Lett., Vol. 8, No. 15, 2006
3185

thymine (Supporting Information). The reaction mixture of
photolyzed 3 containing A was subjected to the reaction with
an aqueous solution of 3-amino-1,2-propanediol11 (1000
equiv, pH was adjusted to 7 with AcOH in phosphate buffer).
Heating the reaction mixture at 37 °C resulted in conversion
of A to the new products B and C (Figure 1b). Peaks B and
C comigrated with 10-mer 10 and the synthesized authentic
lactam 2b, respectively. Peak C and 2b were next analyzed
with LC-MS. The mass spectra of C and 2b were identical
with a quasi-molecular ion peak (MH+) at 170 and fragment
peaks at 96 and 138, indicating C was identical with 2b
(Supporting Information). The formation of 10 and 2b
suggested A was the ODN containing 1. The yields of ODN
containing 1, 2b, and 10 were determined on the basis of
comparison of peak areas with that of 3-nat (ODN containing
1; 67% from 3, 2b, and 10; 75% and 61% from ODN
containing 1, respectively; Figure 2a-c).
Next, we studied photoreaction of duplexed 3 (ds 3) under
similar conditions. The formation of the ODN containing 1
occurred faster in duplexed form than it did in the single-
stranded form. The yield of 1 from ds 3 was higher than
that from 3 (82% based on peak area of 3-com10). Treatment
of ODN containing 1 from ds 3 with amine resulted in
slightly slower lactam formation compared to the reaction
of that from 3. However, the efficiencies of lactam formation
were comparable (Figure 2b).
advantageous in the decaging reaction. In regard to the lactam
formation, the duplex containing 1 was more stable than the
single strand, based on HPLC analysis. Thus, the structure
of the ODN affected efficiencies of the photoreaction of 3
and the following lactam formation.
Here, we synthesized ODN (3) containing 4′-o-nitro-
benzyloxythymidine by a conventional phosphoramidite
method. ODN 3 was photolyzed efficiently to give ODN
containing 1. Efficient lactam formation of ODN containing
1 under neutral conditions is a good indication of possibilities
of protein modification.
Acknowledgment. We are grateful for the financial
support from Sapporo Bioscience Foundation.
Supporting Information Available: Experimental pro-
cedures and characterization for compounds 2b, 4, 7a, 7b,
1
8, 9; H NMR spectra of compounds 2b, 4, 7a, 7b, 8, 9,
1-[2-deoxy-3,5-O-methylphosphory-4-o-nitrobenzyloxy-â-D-
ribofuranosyl] thymine (synthesized for determination of the
extinction coefficient of 3) and NOESY spectra of 7a and
7b; HPLC analyses of crude ODN with DMTr group after
cleavage from solid support and crude 3 after treatment under
acidic conditions; MALDI TOF MS spectrum of 3; UV-
melting curves of duplexes of 3 and 3-nat; HPLC traces of
co-injection of the photolyzed 3 and thymine and co-injection
of the reaction mixture of photolyzed 3 and 2b, and 10; LC-
MS spectra of the reaction mixture containing 2b and the
authentic 2b; and time course of formation of 1 and 2b from
3 and ds 3. This material is available free of charge via the
Internet at http://pubs.acs.org.
The o-nitrobenzyl group at the C4′ position of 2′-
deoxyribose might be located in the minor groove of the
duplex. The position of the o-nitrobenzyl group in ds 3 was
(11) Preliminary results showed that the reaction of lysine can give a
mixture of R- and ꢀ-lactams under conditions similar to those in the
formation of 2b. 3-Amino-1,2-propanediol and 2b are soluble in water,
which was appropriate for the reaction and analysis.
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