Difluoro-C4′-oxidized abasic site for efficient amine modification in biological systems

Article abstract of DOI:10.1021/ol302703m

An oligodeoxynucleotide (ODN) containing a 2′,2′-difluorinated analogue of a C4′-oxidized abasic site (C4′-OAS) was designed for the amine modification of biomolecules that interact with nucleic acids. In contrast to the parent C4′-OAS, which yielded amine-modified products accompanied by DNA strand scission, the ODN containing the difluoro C4′-OAS efficiently yielded products carrying ODNs. The amine modification proceeded without additional reagents being required and might be applicable to reactions in biological systems.

Full text of DOI:10.1021/ol302703m

ORGANIC
LETTERS
Difluoro-C40-oxidized Abasic Site
for Efficient Amine Modification
in Biological Systems
2012
Vol. 14, No. 23
5852–5855
Bo Yang, Akiko Jinnouchi, Hiroshi Suemune, and Mariko Aso*
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582,
Japan
aso@phar.kyushu-u.ac.jp
Received September 30, 2012
ABSTRACT
An oligodeoxynucleotide (ODN) containing a 20,20-difluorinated analogue of a C40-oxidized abasic site (C40-OAS) was designed for the amine
modification of biomolecules that interact with nucleic acids. In contrast to the parent C40-OAS, which yielded amine-modified products
accompanied by DNA strand scission, the ODN containing the difluoro C40-OAS efficiently yielded products carrying ODNs. The amine
modification proceeded without additional reagents being required and might be applicable to reactions in biological systems.
DNA that carries a carbonyl group, which can be
synthesized artificially or is formed when DNA is damaged
owing to the actions of various DNA-damaging agents,
Scheme 1. Reaction of 1 with an Amine
can react with nitrogen nucleophiles such as hydrazine and
hydroxylamine under physiological conditions, thereby
leading to the chemical modification of DNA.1ꢀ4 The
reactions of the carbonyl groups in DNA with the amino
groups in DNA-interacting proteins and nucleic acids
can form Schiff bases or carbinolamines, which might
affect the structures and functions of these biomolecules.
Modifications resulting from the formation of irreversible
covalent bonds may be promising, although the required
reducing conditions may not be available in biological
systems.5
The C40-oxidized abasic site (1; C40-OAS) is a type of
oxidatively damaged DNA lesion that is produced by anti-
tumor bleomycins.6 We found that an oligodeoxynucleotide
(ODN) containing 1 reacted with a primary amine under
(1) Dutta, S.; Chowdhury, G.; Gates, K. S. J. Am. Chem. Soc. 2007,
mild conditions that were close to those of biological systems
129, 1852–1853.
and yielded lactam 2 efficiently by eliminating DNA frag-
ments (Scheme 1).7ꢀ9 Since ODN that contains 1 reacts
effectively with amines and its structure is close to that of
(2) Dey, S.; Sheppard, T. L. Org. Lett. 2001, 3, 3983–3986.
(3) Manoharan, M.; Andrade, L. K.; Cook, P. D. Org. Lett. 1999, 1,
311–314.
(4) Angelov, T.; Guaninazzi, A.; Scharer, O. D. Org. Lett. 2009, 11,
661–664.
(5) Sczepanski, J. T.; Wong, R. S.; McKnight, J. N.; Bowman, G. D.;
Greenberg, M. M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 22475–22480.
(6) Sugiyama, H.; Kilkuskie, R. E.; Hecht, S. M.; van der Marel,
G. A.; van Boom, J. H. J. Am. Chem. Soc. 1985, 107, 7765–7767.
(7) Aso, M.; Kondo, M.; Suemune, H.; Hecht, S. M. J. Am. Chem.
Soc. 1999, 121, 9023–9033.
(8) Aso, M.; Usui, K.; Fukuda, M.; Kakihara, Y.; Goromaru, T.;
Suemune, H. Org. Lett. 2006, 8, 3183–3186.
r
10.1021/ol302703m
Published on Web 11/14/2012
2012 American Chemical Society

unmodified natural DNA, it could modify the lysine residues
of DNA-interacting proteins10 andcouldbeusedtomaplysine
residues in DNA-binding proteins as a part of structureꢀ
function investigations. The 1,4-dicarbonyl structure in the
ring-opened form 10, which exists in equilibrium with 1,
might be a key to effective amine modification.11 However,
the ODN containing 1 undergoes β-elimination from the
ring-opened form 10 under basic and heating conditions,
and the resultant cleavage of the binding sequence might
lead to a decrease in the affinity for the target molecules, and
thus a decrease in the modification efficiency.12 Reaction of
the ODN containing 1 with amine-containing molecules
yields molecules bearing lactam but does not result in
conjugation, which is also useful to introduce various func-
tions into DNA13 and to study DNA interacting molecules.
We designed an ODN containing 3, the 2,2-difluorinated
analogue of 1 (Figure 1). The introduction of the difluoro
groups was intended to prevent DNA strand cleavage by
β-elimination. In addition, an increase in the reactivity of the
aldehyde in the ring-opened form 30 was expected owing to
the electron-withdrawing effect of the fluoride. Herein, we
report the synthesis of ODN containing 3 that can carry out
amine modifications to yield ODN products under condi-
tions that are similar to those of biological systems.
The preparation of ODN containing 3 was carried out
by photoirradiating a caged ODN containing 4, which
was similar to generating the ODN containing 1 from its
corresponding caged precursor ODN.9 In this study, ODN
5 was prepared by photoirradiating 6. The synthesis of
caged sugar 4 was carried out using the 4,5-unsaturated
sugar 14 as a key intermediate (Scheme 2). The reaction of
2-deoxy-2,2-difluororibose14 with o-nitrobenzyl bromide
in the presence of DBU resulted in nitrobenzyl ether 9
(30%) and its anomer (40%). After the hydrolysis of
benzoyl groups of 9, 10 was obtained, and its 5-hydroxyl
group was iodinated to yield 11. The attempted conversion
of 11 into 14 by treatment with DBU only led to the
decomposition of 11. On the other hand, the 3-acetylated
12 was successfully converted into 13 by a similar treat-
ment. After hydrolysis of the 3-acetyl group, treatment of
the obtained 14 with m-CPBA in the presence of methanol
resulted in the introduction of a methoxy group at the
4-position, from which 4 was obtained as a major product
along with its C4-epimer. Compound 4 was converted into
phosphoramidite 15, and ODN 6 was synthesized using
the conventional phosphoramidite method by employing
an automated DNA synthesizer: MALDI-TOF MS data:
m/z = 4051 (MW of 6 = 4050). It was anticipated that
the photoirradiation of 6 yielded the ring-opened form 50,
which existed in equilibrium with 5. ODN 5 might be
composed of an equilibrating mixture of isomers at the
anomeric and C4-positions.15
The photoirradiation of 6 was carried out using a 10 μM
solution of 6 in H2O at 365 nm and 0 °C. After 4 h, HPLC
analysis of the reaction mixture revealed the clear conver-
sion of 6 into a new product (13 min; Figure 2). The result
of the MALDI-TOF MS of the photolyzed sample indi-
cated the formation of 5 (m/z = 3904: MW of 5 = 3902).
The yield of 5 was estimated (ca. 80%) on the basis of
a comparison of the area of its peak with that of the peak
of the internal standard. For the amine modification of a
target molecule, 3 must be accessible to the amino group in
the complex. In order to evaluate the reactivity of 3 with a
proximal amine, ODN 5 was subjected to a reaction with
the complementary ODN 7,16 which carried a hexylamine
that could approach 3 in the duplex (Figure 1). ODN 6
(25.4μM in50mMphosphatebuffer; pH valuesof7 and 8)
was hybridizedwithODN 7 (10μM;half the concentration
of 5 after photoirradiation). The decaging of 6 in the
duplex yielded duplex 5:7. The resultant duplex was in-
cubated at 37 °C in a phosphate buffer and was analyzed
Figure 1. ODNs 5 and 6 containing 3 and caged sugar 4 and
amines 7 and 8.
(9) Usui, K.; Aso, M.; Fukuda, M.; Suemune, H. J. Org. Chem. 2008,
8, 241–248.
(10) Jacobs, A. C.; Kreller, C. R.; Greenberg, M. M. Biochemistry
2011, 50, 136–143.
(11) (a) Op de Beeck, M.; Madder, A. J. Am. Chem. Soc. 2011, 133,
796–807. (b) Op de Beeck, M.; Madder, A. J. Am. Chem. Soc. 2012, 134,
10737–10740.
(12) Formation of DNA interstrand cross-link involving cleavage
of the strand containing the C40-OAS was reported by Greenberg.
Fragments (15-mers) formed by cleavage might still have enough affinity
to the complementary 31-mer for cross-link formation. Sczepanski, J. T.;
Jacobs, A. C.; Greenberg, M. M. J. Am. Chem. Soc. 2008, 130, 9646–
9647. Sczepanski, J. T.; Jacobs, A. C.; Majumdar, A.; Greenberg, M. M.
J. Am. Chem. Soc. 2009, 131, 11132–11139.
(14) Chou, T. S.; Heath, P. C.; Patterson, L. E.; Poteet, L. M.; Lakin,
R. E.; Hunt, A. H. Synthesis 1992, 565–570.
(15) The structure of the C40-oxidized abasic site in the duplex was
studied by Stubbe et al.: Chen, J.; Dupradeau, F.-Y.; Case, D. A.;
Turner, C. J.; Stubbe, J. Biochemistry 2007, 46, 3096–3107.
(16) Manoharan, M.; Inamati, G.; Tivel, K. L.; Conklin, B.; Ross,
B. S.; Cook, P. D. Nucleosides Nucleotides 1997, 16, 1141–1143.
(13) Lucas, R.; V.-Climent, E.; G.-Pinto, I.; Avino, A.; Eritja, R.;
Gonzalez, C.; Morales, J. C. Chem. Commun. 2012, 48, 2991–2993.
Org. Lett., Vol. 14, No. 23, 2012
5853

Figure 2. HPLC analysis of the photoreaction of 6. ODNs 6 and
5 were eluted at retention times of 20 and 13 min, respectively.
50-d(ATAC)-30 was added as an internal standard (IS) (retention
time of 11 min).
by HPLC and denaturing polyacrylaminde gel electro-
phoresis (PAGE) (Figure 3a,b; reaction at pH 8). The
formation of two cross-linked products at the expense of
the starting duplex was observed. The major cross-linked
product was eluted at 19 min by HPLC analysis and
corresponded to the slower migrating band seen in the
results of the PAGE analysis. After being isolated, its
structure was judged to be 17 (R-NH2 =7at O-hexylamine;
MW = 7911) from its MALDI-TOF MS spectrum (m/z =
7910). This product might have formed from the five-
membered-ring amine intermediate 18 (MW = 7949) by
the elimination of H2O (MW = 18) and HF (MW = 20;
Scheme 3). The structure of the minor cross-linked product
was also judged to be 19 from its MALDI-TOF MS
spectrum (m/z = 7331, MW of 19 = 7333). The yields of
17 and 19 in the reaction at pH 7, which were 27% and 17%,
respectively, at 24 h, were determined by comparing the
areas of their peaks with that of internal standard. The
yields increased significantly when the reaction was carried
out at pH 8 (55% and 43%, respectively). The cross-link
formation by 3 contrasted with the lactam formation by 1,
although both reactions proceeded without the need for
additional reagents.
Figure 3. (a) HPLC analysis of the reaction of 5 and 7 (50 mM
phosphate buffer; pH 8; 37 °C). 5: retention time of 13 min.
7: retention time of 16 min. 50-d(ATAC)-30 was added as an
internal standard (IS) (retention time of 11 min). The cross-
linked 17 and 19 were eluted at retention times of 19 and 17 min,
respectively. (b) Monitoring of the reaction of 5 and 7 at pH 7
and 8 by denaturing PAGE analysis on a 20% polyacrylamide/
8 M urea gel. (c) UV absorbanceꢀtemperature profile of 17 (red)
and the duplex 5:7 before cross-linking (blue).
only a small amount of 19was formedfrom 17(Supporting
Information).17 These data indicated that the cross-linked
product significantly increased the stability of the duplex
compared to the duplex before the cross-link product had
formed (Tm 42 °C). These data were similar to those
pertaining to the cross-linking between the ODNs containing
an abasic site and an alkyl amine, which was formed by
reductive amination.3 It was assumed that the cross-linked 19
might not have been formed from 17 by the elimination of the
50-ODN fragment. The elimination of the 50-ODN fragment
from 5 was not observed during the course of the reaction.
The reaction of 5 (20 μM in 50 mM phosphate buffer,
pH 8) with excess N-6-aminohexyl dansylsulfonamide 818
(2 mM) was also studied. When the reaction mixture was
incubated at 37 °C and pH 8, the formation of a new
product at the expense of 5 was observed by HPLC
analysis (Supporting Information). The MALDI-TOFF
MS data of the isolated product (m/z = 4182) indicated
that its structure was that of product 20 (R-NH2 = 8; MW
of 20 = 4182), which might have been formed from
the intermediate 18 (R-NH2 = 7; MW = 4220) by the
Cross-link 17 was chemically stable under the reaction
conditions, and conversion of 17 into 19 did not take place
(pH 7 and 8 at 37 °C for 24 h). On heating of a solution of
17 (10 mM phosphate buffer containing 100 mM NaCl;
pH 7) from 20 to 90 °C at 0.5 °C/min, its UV absorbance
profile exhibited a melting transition region at 80 °C
(Figure 3c). Under similar heating conditions (50 mM
phosphate buffer containing 100 mM NaCl; pH 7 and 8),
17 was essentially intact (ca. 97% remained at pH 7), and
(18) Narayanan, R.; Balaram, P. Biochem. Biophys. Res. Commun.
1976, 70, 1122–1128.
(17) At pH 8, 90% of 19 remained (Supporting Information).
5854
Org. Lett., Vol. 14, No. 23, 2012

Scheme 2. Synthesis of the Caged Sugar 4 and the Phosphoramidite 15
elimination of H2O (MW = 18) and HF (MW = 20).
Although the reaction with the proximal amine was much
faster, the reaction proceeded with moderate yield (66%
at 24 h). During the reaction with 8, products that were
lacking the 50-ODN fragment, such as 19, were not ob-
served. This reaction could be useful for synthesizing DNA
conjugates of molecules that contain amines, even if the
conjugates are not stable under reducing conditions.
In summary, we demonstrated effective amine modifica-
tion by a ODN containing 3. After generation of 3 by light,
modification proceeds without the need for additional re-
agents. This method might be applicable to biological sys-
tems. Useful DNA conjugates of amine-containing molecules
can be prepared under mild conditions using this technique.
Scheme 3. Reaction of ODN 5 with an Amine
Supporting Information Available. Experimental pro-
cedures andchracterization of compounds 4 and 9ꢀ15; 1H
NMR spectra of 4 and 9ꢀ15; NOESY spectra of 4 and 9;
13C NMR spectra of 4 and 15; HPLC analyses of the
photoreaction of 6; the reaction of duplex 5:7; heating of
17 to determine Tm; reaction of 5 with 8; MALDI-TOF
MS spectra of 6, 5, 17, 19, and 20.This material is available
free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 23, 2012
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