PNA/DNA interstrand cross-links from a modified PNA base upon photolysis or oxidative conditions

Article abstract of DOI:10.1016/j.bmcl.2008.08.002

PNA/DNA interstrand cross-links (ICLs) were observed when peptide nucleic acids (PNAs) containing modified thymine derivatives were hybridized with the complementary or one-base mismatched DNA upon photolysis or treatments of oxidative agent. PNA/DNA ICL formation provides a useful method for biological applications such as antisense technologies or PNA chips.

Full text of DOI:10.1016/j.bmcl.2008.08.002

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5054–5057
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
PNA/DNA interstrand cross-links from a modified PNA base upon photolysis
or oxidative conditions
*
Yongtae Kim, In Seok Hong
Department of Chemistry, College of Natural Science, Kongju National University, 182 Shinkwan-dong, Kongju, Chungnam 314-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
PNA/DNA interstrand cross-links (ICLs) were observed when peptide nucleic acids (PNAs) containing
modified thymine derivatives were hybridized with the complementary or one-base mismatched DNA
upon photolysis or treatments of oxidative agent. PNA/DNA ICL formation provides a useful method
for biological applications such as antisense technologies or PNA chips.
Received 4 July 2008
Revised 29 July 2008
Accepted 1 August 2008
Available online 6 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Peptide nucleic acid
Interstrand cross-links
Photolysis
Oxidative condition
Peptide nucleic acids (PNAs)1 have widespread applications in
many areas such as bio-drug for antigene/antisense therapy and
molecular tools for molecular biology and functional genomics.2
Many of theses applications involve the hybridization of PNA olig-
omers to DNA targets to form PNA/DNA duplexes because of the
characteristics of strong PNA/DNA binding modes. If further treat-
ments of these PNA/DNA duplexes such as UV-photolysis or oxi-
dants can produce a covalent linkage between a PNA and a DNA
strand, covalently linked PNA/DNA duplexes could become an inert
molecule permanently under thermodynamic conditions.
Recently, DNA/DNA interstrand cross-links (ICLs) formation
from a modified nucleotide was first reported by the Greenberg
group.3 ICLs formation in duplex DNA after exposure to
c-radioly-
sis is a particularly exciting result because the modified nucleotide
triphosphate could be a useful radiosensitizing agent.4
The Greenberg group showed that a DNA duplex containing a
phenyl selenide derivative (1) produces DNA interstrand cross-
links with the opposing strand dA upon photolysis or oxidative
conditions (Scheme 1). Cross-linking proceeds through the 5-(20-
deoxyuridinyl) methyl radical (2) upon photolysis3 or methide
(3) under oxidative conditions.5 Both reaction conditions produce
a kinetic ICL product believed to be a dA-N1 adduct, which isomer-
izes in solution to the stable dA-N6 adduct (4) by Dimroth
rearrangement.6
Scheme 1. Proposed mechanisms of DNA ICL formation from a modified nucleotide
upon photolysis or oxidative stress.6
modified PNA probe when treated with photolysis or oxidative
stress, they could be a useful tool for PNA biotechnological applica-
tions such as antisense techniques and PNA chips.
A phenyl selenide-modified thymine 1-acetic acid (6) was pre-
pared from thymine 1-acetic acid methyl ester (5) by modifying
the previously reported procedure for thymidine derivative7
(Scheme 2).
Herein, we describe the possibility that ICL formation can be ex-
tended to a PNA/DNA duplex using a PNA probe containing phenyl
selenide-modified thymine (6). If PNA/DNA ICLs are formed using a
First, the photochemical activity of phenyl selenide derivative
(6) was determined under aerobic conditions. After the photolysis
* Corresponding author. Tel.: +82 41 850 8498; fax: +82 41 856 8613.
E-mail address: ishong@kongju.ac.kr (I.S. Hong).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.002

Y. Kim, I. S. Hong / Bioorg. Med. Chem. Lett. 18 (2008) 5054–5057
5055
Scheme 2. Reagents and conditions: (a) NBS, benzene, AIBN; (b) PhSe-SePh, NaBH4,
DMF, 52% (two steps); (c) aq NaOH/Dioxane, 98%.
of 6 at 350 nm, the photolysates were analyzed by HPLC. Consump-
tion of 6 was monitored using an internal standard marker (thymi-
dine, T) by reverse phase HPLC (Fig. 1). Compound 6 (1.0 mM) was
consumed to about 90% within 30 min.
Upon photolysis, the intermediate expected could be a 5-(1-car-
boxymethyl-uracil) methyl radical (7) like the nucleoside analogue
(2). We believe that several stable products that eluted out faster
than the internal standard are derived from the peroxy radical 8,
as proposed previously, and/or the reduction of the hydroperoxide
(Scheme 3).7,8
Next, we tested that oxidative stress can produce a reactive
methide, which is the intermediate for the ICL formation in DNA
under oxidative conditions. We confirmed via NMR experiments
that a reactive methide intermediate (10) is generated under oxi-
dative conditions (NaIO4 treatment).
Phenyl selenide thymine derivative (6) was oxidized into selen-
oxide (9) by NaIO4 in good yield, and allylic selenoxide underwent
[2,3]-sigmatropic rearrangements to produce a methide (10) like
nucleoside analogue 3, as is shown in Figure 2. The production of
methide intermediate was confirmed by the appearance of vinyl
protons (HA and HB in Fig. 2C). Finally, 5-hydroxymethyluracil
1-acetic acid 11 was produced through a reaction with water
during the overnight incubation.
Figure 2. 1H NMR analysis of the reaction of 6 (1 mM) with NaIO4 (2 mM) in
deuterated phosphate buffer (10 mM, pD 7.0). (A) Before NaIO4 addition; 6, (B)
10 min after NaIO4 addition; 9, (C) 2 h after NaIO4 addition; 10, and (D) 16 h after
NaIO4 addition; 11.
The rate constant of the formation of intermediate 10 was
determined by NMR experiments using an internal standard (thy-
midine). The disappearance of the H6 hydrogen of 9 was monitored
in time courses (Fig. 3). The plotting was well fixed to the first-or-
der rate equation. However, phenyl selenoxide derivative
9
(k0 = 5.30 ꢀ 10ꢁ4 sꢁ1
, t1/2 = 21.8 min) rearranged approximately
ten times more slowly than the comparable nucleoside thymidine
derivative (6.1 ꢀ 10ꢁ3 sꢁ1, t1/2 = 1.9 min).9 Hence, we confirmed
that the reactivity of phenyl selenide 6 is similar to that of nucle-
oside analogue 1 upon photolysis or oxidative conditions.
Precursor 6 was independently introduced at the defined site in
PNA oligomer (12) using standard Fmoc-solid phase synthesis
Figure 1. HPLC analysis of the photolysates of 6. (A) Before photolysis, (B) after
photolysis at 350 nm for 30 min, internal standard; thymidine (T).
Figure 3. The rate constant determination of the formation of methide interme-
diate 10.
Scheme 3. Methyl radical intermediate upon the photolysis of 6.

5056
Y. Kim, I. S. Hong / Bioorg. Med. Chem. Lett. 18 (2008) 5054–5057
methods.10 The N-terminal of PNA probe was modified with the
acetyl group (Scheme 4).
DNA oligomers were purchased from Bioneer Inc. DNA13 is a
fully matched 15 mer to the modified PNA (12) and DNA14 is a
mismatched sequence in the base opposite of the precursor site
in PNA (12). DNA15–17 were designed to determine the mis-
matched sequence effects.
In order to determine the ICLs formation between the PNA
probe and DNAs, 50-fluorescein labeled DNAs were hybridized with
a PNA probe under standard hybridization conditions, and then
PNA/DNA duplexes were further treated with photolysis or
NaIO4.11 Denaturing polyacrylamide gel electrophoresis analysis
reveals the formation of products whose migration is severely re-
tarded relatively to unreacted DNA oligomers, indicative of inter-
strand cross-linked products (Fig. 4).
In photolysis, ICL was formed in significant quantities in a fully
matched PNA/DNA13 duplex (Fig. 4A), but the formation of ICLs in
single-base mismatched sequences (PNA/DNA14–17) was less than
10% yield except that of PNA/DNA15. This is consistent with the
previous results from DNA/DNA ICL formation.4 Under oxidative
stress, the extents of ICL formation were dramatically changed. A
fully matched PNA/DNA13 duplex with treatment of NaIO4 did
not produce significant interstrand cross-link. Also, ICL formation
in distal mismatched sequences (DNA16 and 17) was negligible.
However, ICL formation was accelerated in an opposing base or
adjacent base-mismatched sequence PNA/DNA14, PNA/DNA15,
respectively (Fig. 4B).
A characterization of the cross-linked product from the treat-
ment with NaIO4 of PNA/DNA15 was obtained by MALDI-TOF
mass.12 The observed molecular weight (calculated m/z 8666.9;
found m/z 8664.8) is well consistent with that of the reaction be-
tween 6 and the opposing DNA strand (Fig. 5). The observed molec-
ular weight is also consistent with the observation that O2 is
unnecessary for interstrand cross-link formation as in the DNA ICLs
formation.
Figure 5. MALDI-TOF mass of the cross-linked PNA/DNA obtained upon NaIO4
treatment of PNA/DNA15.
PNA/DNA15 treated with NaIO4 (5 mM) (Fig. 6A). Phenyl selenox-
ide derivative 9 (t1/2 = 21.8 min) rearranged approximately 10
times more slowly than the comparable nucleoside thymidine
derivative (t1/2 = 1.9 min).9 Hence, the plotting was carried out at
20 min as an initial background after the treatment of NaIO4. ICL
growth was well fixed first-order kinetics (Fig. 6B). The observed
rate constant for ICL formation (6.9 ꢀ 10ꢁ4 sꢁ1, t1/2 = 16.9 min)
was comparable to that of DNA ICL formation (t1/2 = 28.2 min).
In contrast to the reactivity of the thymidine analogue (1), we
found that the fully matched PNA/DNA13 under oxidative stress
did not produce a significant amount of ICLs as shown in Figure
4B. It can be explained by local duplex stability. The effect of the
phenyl selenide group on duplex thermal stability was analyzed
by measuring the UV-melting temperature (Tm, Table 1). The phe-
nyl selenide group reduced the Tm by 0.8 °C relative to when thy-
In contrast to ICL formations of photolysis, which are produced
from the radical pathway, interstrand cross-links of modified PNA/
DNA under oxidative stress are formed from the methide type
intermediate (10). Further support for this mechanism was ob-
tained by determining the rate constant for ICL formation in
Scheme 4. The sequences of a modified PNA probe, fully matched (DNA13) and
single-base mismatched DNA oligomers (DNA14–17). 50 sites of DNAs were labeled
with fluorescein.
Figure 6. Rate of ICL growth from PNA/DNA15 upon NaIO4 (5 mM, 37 °C)
treatments. (A) Denaturing PAGE analysis, (B) ICL growth follows first-order
kinetics.
Figure 4. Denaturing PAGE analysis of PNA/DNA duplexes. (A) Photolysis (350 nm,
30 min), (B) NaIO4 treatments (5.0 mM, 37 °C for 2 h).

Y. Kim, I. S. Hong / Bioorg. Med. Chem. Lett. 18 (2008) 5054–5057
5057
Table 1
In contrast to photolysis, ICLs formation in NaIO4 treatment
was observed in mismatched DNA sequences in the vicinity of
opposing 6. PNA/DNA ICL formation could be a useful tool for
PNA biotechnological applications such as PNA chips and PCR
clamping.
Comparison of the UV-melting temperature of PNA/DNA duplexes containing
modified 6
PNA
DNAa
Tm (°C)b
18
12
12
12
12
12
13
13
14
15
16
17
63.0 0.0
62.2 0.5
49.4 0.5
49.0 0.1
50.3 0.8
52.8 0.7
Acknowledgments
This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD, Basic Research
Promotion Fund) (KRF-2007-331-C00163) and the new faculty
seed fund of Kongju National University. We thank Dr. Sung Kee
Kim and Dr. Hyunil Lee of Panagene Inc. for the supply of PNA olig-
omers and for technical assistance.
a
Sequences of DNA were the same as those of DNA13–17, but without labeling of
fluorescein.
b
[PNA], [DNA] = 1.0 lM, phosphate buffer (20 mM, pH 7.2).
mine was present. The effect of a mispair opposite 6 was similar to
when one mispair was present in other sites.
References and notes
Recently, the Greenberg group described flanking sequence
effects on cross-linking reactions in a DNA duplex containing a
phenyl selenide-modified 5-methyl-deoxycytidine.9 Almost no
ICL formation in fully matched PNA/DNA13 and distal single-base
mismatched sequences (PNA/DNA16, PNA/DNA17) indicates that
the local duplex stability could be the cause of the sequence effect
on cross-linking because 6 must adopt the syn-conformation in or-
der to react with the opposing DNA strand.
1. (a) Nielsen, P.; Egholm, M.; Berg, R.; Buchardt, O. Science 1991, 254, 1497; (b)
Egholm, M.; Buchardt, O.; Nielsen, P.; Berg, R. J. Am. Chem. Soc. 1992, 114, 1895;
(c) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver,
D. A.; Berg, R.; Kim, S. G.; Norden, B.; Nielsen, P. Nature 1993, 365, 566.
2. Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624.
3. Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 3692.
4. Hong, I. S.; Ding, H.; Greenberg, M. M. J. Am. Chem. Soc. 2006, 128, 2230.
5. Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 10510.
6. Hong, I. S.; Ding, H.; Greenberg, M. M. J. Am. Chem. Soc. 2006, 128, 485.
7. Hong, I. S.; Greenberg, M. M. Org. Lett. 2004, 6, 5011.
The methide type intermediate 10 cannot adopt the syn-confor-
mation in a stable local duplex environment. Hence, no ICL forma-
tions were observed in fully matched and distal single-base
mismatched DNA sequences. However, there was a high yield of
ICL formation in a mispair opposite 6 and adjacent mispair se-
quence due to more flexible local stability. A cross-linking site
opposite of DNA14 strand in the PNA/DNA14 duplex might be a
30-adjacent dA, which was reported by the Greenberg group.9
In summary, PNA/DNA interstrand cross-links were formed from
modified PNA base upon photolysis or oxidative conditions.
8. Romieu, A.; Bellon, S.; Gasparutto, D.; Cadet, J. Org. Lett. 2000, 2, 1085.
9. Peng, X.; Hong, I. S.; Li, H.; Seidman, M. M.; Greenberg, M. M. J. Am. Chem. Soc.
2008, 130, 10299.
10. PNAs were provided from the Panagene Inc. MALDI-TOF-mass: PNA12, calcd
4344.0 (M), found: 4345.1 (M+1); PNA18, calcd 4189.0 (M), found: 4189.8
(M+1).
11. Hybridization conditions: [PNA] = 20 lM, [DNA] = 10 lM, 10 mM, pH 7.2,
phosphate buffer, incubated at 90 °C for 5 min, and then slowly cooled to
room temperature. This is followed by further treatments (5 mM NaIO4 or
350 nm photolysis).
12. Samples for MALDI-TOF mass were pretreated with the C18-Sepak column to
remove salts in reaction solutions.