Photochemical cleavage of duplex DNA by N-benzoyloxy-2-thiopyridone linked to 9-aminoacridine

Article abstract of DOI:10.1039/a702913f

Upon illumination N-aroyloxy-2-thiopyridones induce non-specific single-strand nicks in duplex DNA at micromolar concentrations.

Full text of DOI:10.1039/a702913f

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Photochemical cleavage of duplex DNA by N-benzoyloxy-2-thiopyridone linked
to 9-aminoacridine
Emmanuel A. Theodorakis,* Xin Xiang and Petra Blom
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California
92093-0358, USA
O
Upon illumination N-aroyloxy-2-thiopyridones induce non-
specific single-strand nicks in duplex DNA at micromolar
X
N
H
CO2Me
concentrations.
6 X = Br
7 X = N3
Synthetic reagents that cleave DNA are of great interest as tools
in biochemical sciences. The design of such molecules
constitutes a timely and challenging research topic and has led
to the development of both sequence-specific DNA cleavers1
and DNA footprinting reagents.2
Our studies are directed towards the development of
conceptually new approaches to DNA cleavage, induced by
photoactivation of N-aroyloxy-2-thiopyridone derivatives, such
as 1 (Scheme 1).3,4 To this end, we were guided by the
operational advantages offered by the photoinducible DNA
cleavage using the N-benzoyloxy-2-thiopyridone 4.5 These
i
ii
O
X
N
H
CO2H
8 X = N3
9 X = NH2
iii
iv
H
N
H
N
v
CO2H
5
N
Ar
O
O
N
O
S
10
O
hν
DNA
cleavage
Scheme 2 Reagents and conditions: i, NaN3 (2.0 equiv.), 18-crown-6 (0.05
equiv.) DMF, 25 °C, 3 h, 91%; ii, KOH (2.0 equiv.), THF–H2O (1:1),
25 °C, 24 h, 98%; iii, 10% Pd/C (0.1 equiv.), H2, MeOH, 24 h, 89%; iv,
9-chloroacridine (1.0 equiv.), PhOH, 110 °C, 1 h, 90%; v, 2-mercaptopyr-
idine N-oxide (1.0 equiv.), EDC (1.0 equiv.), DMF, 25 °C, 1 h, 43%
+
Ar
O•
[>350 nm]
S•
N
1
2
3
Scheme 1
molecules could be ideal nucleic acid cleavers since they
possess the following characteristics: a purely organic structure,
facile preparation, prolonged stability in the absence of light,
and well documented radical chemistry.3 The observed nucleic
acid strand scission occurs upon a simple irradiation (l > 350
nm), presumably via the generation of aroyloxyl radicals 2 and
without the need of a metal or external oxidants.
(Scheme 2), followed by saponification of the methyl ester
moiety and reduction of the azido group gave rise to amino acid
9 in 79% overall yield, through intermediates 7 and 8. Coupling
of 9 with 9-chloroacridine followed by esterification with
2-mercaptopyridine N-oxide using 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDC) afforded conjugate 5
in 38% overall yield.
Herein we describe our preliminary data on a novel family of
photoactivated DNA-cleavers represented by 5. The reagent
design is based on linking a DNA photocleaving ligand to the
9-aminoacridine via a polymethylene chain. The aminoacridi-
nyl group could assure high affinity for duplex DNA via
intercalation,6 while the thiopyridone entity could account for
the DNA cleavage. Indeed, upon irradiation (l > 350 nm),
compound 5 produces single strand breaks in duplex DNA with
no intrinsic sequence selectivity.
We further compared the visible light photolysis of 5 and 4 in
the presence of supercoiled circular fX174 DNA (Fig. 1).
Control experiments indicated that both light and the thiopyri-
done derivatives 5 or 4 are necessary for the DNA cleavage
(lanes 1–3). Complete inhibition of the cleavage by glutathione
(lanes 11, 12) further supports the notion that free radicals are
responsible for the DNA damage. Moreover, compound 5
lanes
1
2
3
4
5
6
7
8
9
10 11 12
The synthesis of compound 5 commences with bromide 6,
itself readily available by the condensation of 5-bromovaleroyl
chloride with methyl p-amino benzoate. Azidation of 6,
II
III
I
O
Fig. 1 Concentration-dependent photocleavage of fX174 DNA induced by
4 and 5. The fX174 DNA (50 mm/base pair) was incubated for 1 h at 25 °C
with 4 or 5 (30 mm Tris-HCl, 20 mm NaCl), then subjected to irradiation at
4 °C (lanes 1 and 4–12) with one lamp (GE 300 W) placed at approximately
20 cm from the samples. The results were analysed on 1% agarose gel (Tris-
acetate buffer) stained with ethidium bromide. Lane 1: fX174DNA
(control); lane 2: DNA and 60 mM of 5 (no hn); lane 3: DNA and 1 mm of
4 (no hn) lanes 4–7: DNA and 5 at concentrations of 5: 5, 10, 30 and 60 mm,
respectively; lanes 8–10: DNA and 4 at concentrations of 4: 30, 60 and 1.0
mm, respectively; lane 11: DNA, 60 mm of 5, and 3.0 mm of glutathione;
lane 12: DNA, 1.0 mm of 4 and 3.0 mm of glutathione.
O
N
S
4
5
O
O
H
N
H
N
N
N
S
O
Chem. Commun., 1997
1463

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cleaves DNA at concentrations as low as 5 3 1026 m, while at
6 3 1025 m complete conversion of the form I to form II is
observed. In comparison, derivative 4 produces similar results
at 131023 m concentration. This substantial increase in
efficiency of cleavage is attributed to the intercalating proper-
ties of 9-aminoacridine.6
An autoradiogram illustrating the results obtained upon
irradiation of 5 in the presense of the 5A-32P labelled 93-mer
duplex DNA is shown in Fig. 2. Our data show that both 4 and
5 generate identical DNA ladders upon irradiation and the
photocleavage is indisputably neither base- nor sequence-
specific. Furthermore, comparison of lanes 8–12 indicate that 5
is more efficient than 4 in cleaving DNA and can accurately cut
the duplex at concentrations as low as 5 3 1026 m. This increase
in efficiency of cleavage is attributed to the presence of the
9-aminoacridinyl group. We ruled out the possibility that the
9-aminoacridinyl group enhances cleavage by altering the
conformation of the DNA, since less cleavage was detected
when 9-aminoacridine was added as an external intercalator
(compare lines 12,13). Interestingly, the cleavage is more
enhanced upon subsequent treatment with piperidine at 90 °C
for 30 min without any change in sequence or base specificity
(lines 7,8). Based on the above data we believe that in the case
of 5 the DNA cleavage is performed by the intercalation
complex and is probably mediated by aroyloxyl radicals.
It is evident from the above studies that the N-aroyloxy-
2-thiopyridones 1 can induce non-specific single strand nicks in
duplex DNA in a light-dependent reaction. The efficiency and/
or selectivity of the cleavage could be tuned by the proper
choice of the DNA recognition element. In addition the light
intensity that is responsible for the photoactivation could be
tuned by structurally modifying the thiopyridone core. Thus, the
N-benzoyloxy-2-thiopyridone moiety can be used for the design
of new DNA photocleaving reagents with potential use as
‘photofootprinting reagents’ or as ‘site-directed photonu-
cleases’. Studies across these lines are now under investigation
in our laboratories.
We thank Dr T. Li and Professors Y. Tor and K. C. Nicolaou
of this department for useful discussions and the Donors of The
Petroleum Research Fund, administered by the American
Chemical Society, for partial support of this research.
Footnote
* E-mail: etheodor@ucsd.edu
References
1 A. M. Maxam and W. Gilbert, Methods Enzymol., 1980, 65, 499;
S. A. Kane, H. Sasaki and S. M. Hecht, J. Am. Chem. Soc., 1995, 117,
9107; T. Li, Z. Zeng, V. A. Estevez, K. U. Baldenius, K. C. Nicolaou and
G. F. Joyce, J. Am. Chem. Soc., 1994, 116, 3709; J. G. Muller, P. Zheng,
S. E. Rokita and C. J. Burrows, J. Am. Chem. Soc., 1996, 118, 2320;
M. J. Absalon, J. W. Kozarich and J. Stubbe, Biochemistry, 1995, 34,
2065.
2 R. P. Hertzberg and P. B. Dervan, J. Am. Chem. Soc., 1982, 104, 313;
M. D. Kuwabara and D. S. Sigman, Biochemistry, 1987, 26, 7234;
T. D. Tullius, Nature, 1988, 18, 213; N. Schmid and J.-P. Behr,
Biochemistry, 1991, 30, 4357; P. B. Dervan, Nature, 1992, 359, 87.
3 J. Boivin, E. Crepon and S. Z. Zard, Tetrahedron, 1990, 31, 6869;
D. H. R. Barton, B. Lacher and S. Z. Zard, Tetrahedron, 1987, 43, 4321;
W. Adam, D. Ballmaier, B. Epe, G. N. Grimm and C. R. Saha-Moller,
Angew. Chem., Int. Ed. Engl., 1995, 34, 2156; B. M. Aveline,
I. E. Kochevar and R. W. Redmond, J. Am. Chem. Soc., 1996, 118, 289;
K. M. Hess and T. A. Dix, Anal. Biochem., 1992, 206, 309.
4 For selected reports on light-induced DNA cleaving agents, see
M. M. Becker and J. C. Wang, Nature, 1984, 309, 682; K. Uchida,
A. M. Pyle, T. Morii and J. K. Barton, Nucleic Acids Res., 1989, 17,
10 259; W. Adam, J. Cadet, F. DallAAcqua, B. Epe, D. Ramaiah and
C. R. Saha-Moller, Angew. Chem. Int. Ed. Engl., 1995, 34, 107; I. Saito,
M. Takayama, T. Matsuura, S. Matsugo and S. Kawanishi, J. Am. Chem.
Soc., 1990, 112, 883; A. J. Blacker, J. Jazwinski, J.-M. Lehn and
F. X. Wilhelm, J. Chem. Soc., Chem., Commun., 1986, 1035; J. C. Quada,
M. J. Levy and S. M. Hecht, J. Am. Chem. Soc., 1993, 115, 12 171;
P. A. Wender, S. M. Touami, C. Alayrac and U. C. Philipp, J. Am. Chem.
Soc., 1996, 118, 6522; G. A. Neuhart, C. C. Cheng and H. H. Thorp,
J. Am. Chem. Soc., 1995, 117, 1463.
5 E. A. Theodorakis and K. M. Wilkoxen, Chem. Commun., 1996, 1927.
6 9-Aminoacridine displays strong intercalating properties onto DNA with
no intrinsic sequence selectivity. For selected literature on this topic, see
C. Bailly and J.-P. Henichart, Perspect. Bioconj. Chem., 1991, 112;
A. H. J. Wang, Curr. Opin. Struct. Biol., 1992, 2, 361; P. E. Nielsen,
C. Jeppesen, M. Egholm and O. Buchardt, Biochemistry, 1988, 27, 6338;
A. Lorente, J. F. Espinosa, M. F. Saiz, J.-M. Lehn, W. D. Wilson and
Y. Y. Zhong, Tetrahedron Lett., 1996, 37, 4417; F. C. K. Chiu,
R. T. C. Brownlee, K. Mitchell and D. R. Phillips, Bioorg. Med. Chem.
Lett., 1995, 5, 1689.
Fig. 2 Autoradiogram of a 10% denaturing polyacrylamide gel showing
photocleavage of 5A-32P end-labeled Sal I / Sph I restriction fragment of
pBR322 duplex DNA (93-mer), induced by 4 and 5. DNA was incubated for
1 h at 25 °C with compounds 4 or 5 in buffered solution (30 mm Tris-HCl,
20 mm NaCl) and then irradiated (with one GE 300 W lamp placed 20 cm
from the samples) for another 2 h at 4 °C (lanes 3,4 and 7–13). The resulted
solution was treated with piperidine (1 m) at 90 °C for 30 min, followed by
EtOH precipitation (lanes 8–13). Lane 1: DNA cut by PIe I (14 base pairs)
and EcoN I (28 base pairs); lane 2: DNase footprinting; lane 3: DNA
(control); lane 4: DNA irradiated and piperidine treated without 4 or 5; lane
5: DNA and 200 mm of 4 (no hn); lane 6: DNA and 60 mM of 5 (no hn); lane
7: DNA and 5 mm of 5 (no piperidine treatment); lanes 8–10: DNA and 5 at
concentrations of 5: 10 and 20 mm respectively; lanes 11,12: DNA and 4 at
concentrations of 4: 40 and 60 mm respectively; lane 13: DNA, 4 (60 mm)
and 9-aminoacridine (30 mm).
Received in Corvallis, OR, USA, 28th April 1997; 7/02913F
1464
Chem. Commun., 1997