Bromoacetophenone-based photonucleases: photoinduced cleavage of DNA by 4'-bromoacetophenone-pyrrolecarboxamide conjugates.

Article abstract of DOI:10.1021/ol9903279

[formula: see text] 4'-Bromoacetophenone derivatives which upon excitation can generate monophenyl radicals capable of hydrogen atom abstraction were investigated as photoinducible DNA cleaving agents. Pyrrolecarboxamide-conjugated 4'-bromoacetophenones were synthesized, and their DNA cleaving activities and sequence selectivities were determined.

Full text of DOI:10.1021/ol9903279

ORGANIC
LETTERS
1999
Vol. 1, No. 13
2117-2120
Bromoacetophenone-Based
Photonucleases: Photoinduced
Cleavage of DNA by
4′-Bromoacetophenone−Pyrrolecarboxamide
Conjugates
Paul A. Wender* and Raok Jeon†
Department of Chemistry, Stanford UniVersity, Stanford, California 94305
wenderp@leland.standford.edu
Received October 21, 1999
ABSTRACT
4′-Bromoacetophenone derivatives which upon excitation can generate monophenyl radicals capable of hydrogen atom abstraction were
investigated as photoinducible DNA cleaving agents. Pyrrolecarboxamide-conjugated 4′-bromoacetophenones were synthesized, and their
DNA cleaving activities and sequence selectivities were determined.
The design and synthesis of DNA cleaving agents are goals
of considerable basic and applied significance in chemistry,
biology, and medicine.1 Beginning in 1985, the structures
of a new and now substantial family of highly potent DNA
cleaving agents represented in part by neocarzinostatin,
dynemicin, and calicheamicin began to emerge. Collectively,
these agents share a polyunsaturated nine- or ten-membered
ring core, commonly a cyclic enediyne, which undergoes
cycloaromatization to produce a highly reactive aryl or styryl
diradical, the intermediate which abstracts hydrogen atoms
from DNA initiating its cleavage. The novel structures and
activities of these agents attracted much synthetic interest
from our and other laboratories,2,3 although the complexity
of these agents and even simpler analogues often required
lengthy sequences involving unstable intermediates. As a
consequence, we and others have initiated efforts to identify
simpler, more stable, and synthetically more accessible
precursors to aryl and vinyl radicals, the key intermediates
(2) For recent reviews, see: Enediyne Antibiotics As Antitumor Agents:
Border, D. B., Doyle, T. W., Eds.; Marcel-Dekker: New York, 1995. Maier,
M. E. Synlett 1995, 13-26. Grissom, J. W.; Gunawardena, G. U.; Klingberg,
D.; Huang, D. H. Tetrahedron 1996, 52, 6453-6518. Doyle, T. W.; Kadow,
J. F. Tetrahedron Symposia-In-Print Number 53; Oxford, 1994; Vol. 50,
pp 1311-1538. Wang, K. K. Chem. ReV. 1996, 96, 207. Nicolaou, K. C.;
Dai, W. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1387-1416.
(3) (a) For representative studies from our laboratory, see: Wender, P.
A.; Beckham, S.; O’Leary, J. G. Synthesis 1994, 1278. Wender, P. A.;
Tebbe, M. J. Tetrahedron 1994, 50, 1419. Wender, P. A.; Zercher, C. K.;
Beckham, S.; Haubold, E. J. Org. Chem. 1993, 58, 5867. Wender, P. A.;
Tebbe, M. J. Tetrahedron Lett. 1991, 32, 4863. Wender P. A.; Zercher, C.
K. J. Am. Chem. Soc. 1991, 113, 2311. Wender, P. A.; Harmata, M.; Jeffrey,
D.; Mukai, C.; Suffert, J. Tetrahedron Lett. 1988, 29, 909. (b) Studies from
other laboratories are cited in ref 2 and include contributions from the groups
of Bru¨ckner, Clive, Danishefsky, Doyle, Grierson, Hirama, Isobe, Kadow,
Kende, Krause, Krebs, Magnus, Maier, Myers, Nicolaou, Nuss, Saito,
Schreiber, Semmelhack, Suffert, Takahashi, and Terashima. Recent repre-
sentative examples and further lead references include the following: Myers,
A. G.; Liang, J.; Hammond, M.; Harrington, P. M.; Wu, Y.; Kuo, E. Y. J.
Am. Chem. Soc. 1998, 120, 5319-5320. Clive, D. L. J.; Bo, Y.; Tao, Y.;
Daigneault, S.; Wu, Y.-J.; Meignan, G. J. Am. Chem. Soc. 1998, 120,
10332-10349. Churcher, I.; Hallett, D.; Magnus, P. J. Am. Chem. Soc.
1998, 120, 10350-10358. Suenaga, H.; Nakashima, K.; Mizuno, T.;
Takeuchi, M.; Hamachi, I.; Shinkai, S. J. Chem. Soc., Perkin Trans. 1 1998,
1263-1268.
† Current address: Allergan, Inc., 2525 Dupont Dr, Irvine, CA 92612.
(1) For examples and lead references on nucleic acid cleaving agents,
see: Nucleic Acid Targeted Drug Design; Propst, C. L., Perun, T. J., Eds.;
Dekker: New York, 1992. AdVances in DNA Sequence Specific Agents;
Hurley, L. H., Ed.; JAI Press: London, 1992; Vol. 1, pp 217-243. Armitage,
B. Chem. ReV. 1998, 98, 1171-1200. Gasper, S. M.; Armitage, B.; Shui,
X. Q.; Hu, G. G.; Yu, C. J.; Schuster, G. B.; Williams, L. D. J. Am. Chem.
Soc. 1998, 120, 12402-12409. Ciftan, S. A.; Thorp, H. H. J. Am. Chem.
Soc. 1998, 120, 9995-10000. Kelley, S. O.; Barton, J. K. Science 1999,
283, 375-381. Kino, K.; Saito, I.; Sugiyama, H. J. Am. Chem. Soc. 1998,
120, 7373-7374.
10.1021/ol9903279 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/03/1999

required for DNA cleavage.4,5 Recently, we reported that
simple, commercially available benzotriazoles when com-
bined with a DNA recognition subunit can upon photoacti-
vation cleave DNA in a potent and selective fashion.5 We
have now examined the utility of haloarenes as radical
progenitors for DNA cleavage and report herein that these
readily available compounds upon excitation6 are effective
DNA cleaving agents.
and as a DNA surrogate. In all cases, the debrominated
product, acetophenone, was the major compound produced.
In accord with the intermediacy of an aryl radical, when the
photolysis of 4′-bromoacetophenone was conducted in THF-
d8, the monodeuterated product (Scheme 1)was obtained in
15 min in 57% yield along with unreacted starting material
(22%).
Our studies are based on the observation that excitation
of a haloarene can lead to homolytic cleavage of a carbon-
halogen bond, thereby generating a phenyl radical,7 poten-
tially capable of causing single-stranded lesions to DNA. By
attaching a suitable haloarene to a DNA recognition element,
it was expected that this radical generation could be localized
to specific sites on DNA.8 As a first test of this concept for
DNA cleaving agents, we elected to use 4′-bromoaceto-
phenone derivatives linked to synthetic oligopeptides,
pyrrolecarboximides,9 as shown in Figure 1.
Scheme 1
Having established the utility of 4′-bromoacetophenone
as an aryl radical progenitor, we next sought to attach this
subunit to a series of pyrrolecarboxamide-based DNA minor
groove binders. The pyrrole-linked 4′-bromoacetophenone
conjugates were synthesized as shown in Scheme 2. 3-(4′-
Scheme 2
Figure 1. 4′-Bromoacetophenone-pyrrolecarboxamide conjugates
as photoinducible DNA cleaving agents.
As a reference point for this study, we investigated the
ability of simple haloacetophenones to abstract hydrogen
atoms upon excitation, a required event for DNA cleavage.
The photolytic behavior of 4′-bromoacetophenone is repre-
sentative. The photolyses were performed by using a
medium-pressure mercury arc lamp equipped with a Pyrex
filter under anaerobic conditions. THF was used as solvent
Bromobenzoyl)propanoic acid was prepared by the Friedel-
Crafts succinoylation of bromobenzene and coupled with
pyrrole polyamides 2-4, prepared from N-methylpyrrole
(4) Bregant, T. M.; Groppe, J.; Little, R. D. J. Am. Chem. Soc. 1994,
116, 3635-3636. Jebaratman, D. J.; Kugabalasooriar, S.; Chen, H.; Arya,
D. P. Tetrahedron Lett. 1995, 36, 3123-3126. Griffiths, J.; Murphy, J. A.
J. Chem. Soc., Chem. Commun. 1992, 24-26. Sullivan, R. W.; Coghlan,
V. M.; Munk, S. A.; Reed, M. W.; Moore, H. W. J. Org. Chem. 1994, 59,
2276-2278. Mohler, D. L.; Dain, D. R.; Kerekes, A. D.; Nadler, W. R.;
Scott, T. L. Bioorg. Med. Chem. Lett. 1998, 8, 871-874.
(5) Wender, P. A.; Touami, S. M.; Alayrac, C.; Philipp, U. C. J. Am.
Chem. Soc. 1996, 118, 6522-6523. Touami, S. M.; Poon, C. C.; Wender,
P. A. J. Am. Chem. Soc. 1997, 119, 7611-7612.
(6) (a) Matsumoto, T.; Sakai, Y.; Toyooka, K.; Shibuya, M. Heterocycles
1992, 33, 135-138. (b) Sakai, Y.; Matsumoto, T.; Tanaka, A.; Shibuya,
M. Heterocycles 1993, 36, 565-573. Matsumoto, T.; Utsumi, Y.; sakai,
Y.; Toyooka, K.; Shibuya, M. Heterocycles 1992, 34, 1697-1702. (c)
Quada, J. C., Jr.; Levy, M. J.; Hecht, S. M. J. Am. Chem. Soc. 1993, 115,
12171-12172. (d) Martin, R. F.; Kelly, D. P.; Roberts, M.; Nel, P.; Tursi,
J.; Denison, L.; Rose, M.; Reum, M.; Pardee, M. Int. J. Radiat. Biol. 1994,
66, 517. (e) Chen, T.; Voelk, E.; Platz, M. S.; Goodrich, R. P. Photochem.
Photobiol. 1996, 64, 622.
(7) Hwang, H. J.; El-Sayed, M. A. J. Chem. Phys. 1992, 96, 856-858.
Wagner, P. J.; Sedon, J. Waite, C.; Gudmundsdottir, A. J. Am. Chem. Soc.
1994, 116, 10284-10285. Wagner, P. J.; Sedon, J. H.; Gudmundsdottir,
A. J. Am. Chem. Soc. 1996, 118, 746-754.
(8) For lead references, see: Bouziane, M.; Ketterle, C.; Helissey, P.;
Herfeld, P.; Le Bret, M.; Giorgi-Renault, S.; AuClair, C. Biochemistry 1995,
34, 14051-14058. Boutorine, A. S.; Brault, D.; Takasugi, M.; Delgado,
O. Helene, C. J. Am. Chem. Soc. 1996, 118, 9469-9476. Nakamura, E.;
Tokuyama, H.; Yamago, S.; Shiraki, T.; Sugiura, Y. Bull. Chem. Soc. Jpn.
1996, 69, 2143-2151. Frier, C.; Mouscadet, J. F.; Decout, J. L.; Auclair,
C.; Fontecave, M. Chem. Commun. 1998, 2457-2458. Bailly, C.; Chaires,
J. B. Bioconjugate Chem. 1998, 9, 513-538. Ninomiya, K.; Sugiyama, T.;
Kuroda, R. Nucleic Acids Symp. Ser. 1998, 39, 231-232. Hashimoto, S.;
Nakamura, Y. Chem. Pharm. Bull. 1998, 46, 1941-1943. Herman, D. M.;
Turner, J. M.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1999, 121,
1121-1129.
(9) (a) Carrondo, M. A. A. F. de C. T.; Coll, M.; Aymami, J.; Wang, A.
H.-J.; van der Marel, G. A.; van Boom, J. H.; Rich, A. Biochemistry 1989,
28, 7849-7859. (b) Chen, S.-M.; Leupin, W.; Rance, M.; Chazin, W. J.
Biochemistry 1992, 31, 4406-4413. (c) Edwards, K. J.; Jenkins, T. C.;
Neidle, S. Biochemistry 1992, 31, 7104-7109. (d) Matsumoto, T.; Toyooka,
K.; Nishiwaki, E.; Shibuya, M. Heterocycles 1990, 31, 1629-1633. (e)
Nishiwaki, E.; Lee, H.; Matsumoto, T.; Toyooka, K.: Sakurai, H.; Shibuya,
M. Tetrahedron Lett. 1990, 31, 1299-1302. (f) Parrick, J.; Porssa, M.;
Jenkins, T. C. J. Chem. Soc., Perkin Trans. 1 1993, 2681-2685.
2118
Org. Lett., Vol. 1, No. 13, 1999

according to Shibuya’s method10 to obtain 4′-bromo-
acetophenone-pyrrolecarboxamide conjugates 5-7.
The DNA cleaving activities of compounds 5-7 (Figure
2) were determined by monitoring their effectiveness in
age than 5 over the same concentration range as would be
expected from its stronger association with DNA. In accord
with this finding, the tripyrrole analogue 7 proved to be even
more effective, producing even form III DNA at a concentra-
tion of 30 µM. The 3 µM reaction of distamycin type
analogue 7 gave higher activity than even those observed at
10-fold higher concentrations of the netropsin type analogue
6 with complete disappearance of form I at concentrations
above 20 µM. The DNA cleaving activity of the correspond-
ing desbromoacetophenone analogue of 5 was also tested as
a control. Even at high concentrations (1 mM), this analogue
produced barely detectable DNA cleavage in accord with
the crucial role of the bromide substituent in the cleavage
process.
The inhibitory effects of radical scavengers on the cleavage
process were also examined with TEMPO, a known carbon-
centered radical scavenger, and sodium benzoate, a hydroxyl
radical scavenger. When TEMPO was added to the reaction
mixture, the DNA cleaving activity of compound 7 decreased
as the concentration of TEMPO increased while its activity
was not affected by sodium benzoate as shown in Figure 3.
Figure 2. Light-induced cleavage of DNA by pyrrole-linked 4′-
bromoacetophenones 5, 6, and 7. Supercoiled DNA (φX174RF)
runs at position I, nicked DNA at position II, and linear DNA at
position III. Unless otherwise indicated, all DNA cleavage reactions
were irradiated with Pyrex-filtered light from a 450 W medium-
pressure mercury arc lamp for 30 min at 25 °C. (a) Lane 1-7,
DNA (30 µM/bp) + 5 at concentrations of 3, 10, 20, 30, 50, 100,
and 200 µM, respectively; lane 8, control φX174RF DNA + 5 (200
µM), no hν. (b) Lane 1-7, DNA (30 µM/bp) + 6 at concentrations
of 3, 10, 20, 30, 50, 100, and 200 µM, respectively; lane 8, control
φX174RF DNA + 6 (200 µM), no hν. (c) Lane 1-6, DNA (30
µM/bp) + 7 at concentrations of 3, 10, 20, 30, 50, and 100 µM,
respectively; lane 7, control φX174RF DNA + 7 (200 µM), no
hν.
Figure 3. The effect of radical scavenger on light-induced cleavage
of DNA by peptide-linked 4′-bromoacetophenone 7. Supercoiled
DNA (φX174RF) runs at position I, nicked DNA at position II,
and linear DNA at position III. Unless otherwise indicated, all DNA
cleavage reactions were irradiated with Pyrex-filtered light from a
450 W medium-pressure mercury arc lamp for 30 min at 25 °C.
Lane 1, control φX174RF DNA (30 µM/bp); lanes 2, DNA +
TEMPO (200 mM); lanes 3-5, DNA + 7 (15 µM) + TEMPO at
concentrations of 2, 100, and 200 mM, respectively; lanes 6-8,
DNA + 7 (15 µM) + sodium benzoate at concentrations of 2, 100,
and 200 mM, respectively; lane 9, control DNA + sodium benzoate
(200 mM); lane 10, DNA + 7 (15 µM), no hν.
converting circular supercoiled DNA (form I) to circular
relaxed DNA (form II) and linear DNA (form III). 4′-
Bromoacetophenone and pyrrole polyamide-linked 4′-
bromoactophenones were irradiated at various concentrations
for 30 min in the presence of φX174RFI DNA (30 µM/bp)
in 1:9 DMSO:Tris buffer (20 mM, pH 7.5) under aerobic
conditions. All conjugates tested caused DNA cleavage. The
cleavage efficiency was substrate and concentration depend-
ent. For calibration, 4′-bromoacetophenone itself formed form
III DNA with complete disappearance of form I but only at
high concentrations (10 mM, data not shown).
As expected from design considerations, the efficiency of
DNA cleavage was strongly influenced by the DNA binding
moiety as well as the concentration of the test compound.
Upon irradiation, the monopyrrole linked agent 5 produced
detectable formation of form II DNA in a concentration
dependent fashion over the range of 3-200 µM. The
dipyrrole-linked system 6 produced more pronounced cleav-
The cleavage selectivity of the peptide-linked 4′-bromo-
acetophenone derivatives was determined by sequencing
analyses of the DNA cleavage products obtained when
compounds 5-7 were photolyzed in the presence of a 3′-
32P labeled 517 base pair restriction fragment from pBR322.11
As expected for a cleaving agent bound to a distamycin (7)
or netropsin (6) analogue, the cleavage intensities are the
highest in AT-rich regions of the DNA as shown in Figure
4. A significant cleavage site is marked to the right of the
autoradiogram.
The autoradiogram shown in Figure 4 was quantified by
densitometry, and these data were used to construct histo-
grams for the DNA cleavage observed in the lower regions
(11) Pichersky, E. In DNA Sequencing protocols; Griffin, H. G., Griffin,
A. M., Eds.; Methods in molecular Biology 23; Humana: Totowa, NJ, 1993;
Chapter 31.
(10) Nishiwaki, E.; Tanaka, S.; Lee, H.; Shibuya, M. Heterocycles 1988,
27, 1945-1592.
Org. Lett., Vol. 1, No. 13, 1999
2119

Figure 5. Histograms of DNA cleavage sites by the pyrrole-linked
4′-bromoacetophenone analogues. The relative extent of cleavage
was estimated from the densitometric scans of the lower regions
of the autoradiograms shown in Figure 3, and the height of the bar
represents relative cleavage intensity at the indicated bases: (a)
200 µM of compound 6, (b and c) compound 7 at concentrations
of 15 and 50 µM, respectively.
Figure 4. Autoradiogram of 8% denaturing polyacrylamide gel
showing cleavage of 3′-32P end-labeled 517 base pair restriction
fragment (EcoRI/RsaI) from pBR322 by peptide-linked bromo-
acetophenone 5, 6, and 7. All reactions were irradiated with Pyrex-
filtered light from a 450 W medium-pressure mercury arc lamp
for 30 min at 25 °C. The cleavage site is shown to the right of the
autoradiogram. Lane 1, Maxam-Gilbert G reaction; lane 2, DNA
control; lanes 3-5, DNA + 5 at concentrations of 10, 50, and 200
µM, respectively; lanes 6-8, DNA + 6 at concentrations of 10,
50, and 200 µM, respectively; lanes 9-12, DNA + 7 at concentra-
tions of 5, 15, 50, and 100 µM, respectively; lane 13, DNA + 4′-
bromoacetophenone (500 µM).
sites targeted by the recognition subunit. This work provides
a new class of DNA cleaving agents that are notably easier
to prepare and significantly more stable than enediyne
systems. In addition, the use of light to turn these agents
“on” offers unique external control over the initiation of the
cleavage process relative to bimolecular activations required
with other agents. More generally, given the number of
photochemical and thermal methods to generate phenyl and
vinyl radicals, it is expected that this strategy could be
extended to a variety of other radical progenitor systems.
of the autoradiograms (Figure 5). The histograms show that
compounds 6 and 7 produce cleavage within and adjacent
to sites of multiple contiguous AT base pairs, and the
cleavage pattern remains at high concentration. The cleavage
assay and sequencing of these compounds showed remark-
able correlation between chain lengths and activities with
the highest activity for the distamycin type analogue 7.
In summary, these studies show that 4′-bromoacetophe-
none derivatives upon irradiation can function as DNA
cleaving agents putatively through the generation and reac-
tion of phenyl radicals. Upon conjugation to suitable DNA
recognition elements, these derivatives cleave DNA at the
Acknowledgment. Financial support was provided by the
National Institutes of Health (Grant CA31845) and Korean
Science and Engineering Foundation. We thank Dr. S.
Touami for assistance with the sequencing experiments.
Supporting Information Available: Details of synthesis
and characterization of the listed compounds and experiments
for cleavage of DNA and its assay. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL9903279
2120
Org. Lett., Vol. 1, No. 13, 1999