Photoinduced cleavage of DNA by bromofluoroacetophenone-pyrrolecarboxamide conjugates

Article abstract of DOI:10.1016/S0960-894X(03)00212-9

Bromofluoroacetophenone derivatives which produce fluorine substituted phenyl radicals that cleave DNA upon excitation were investigated as a novel photonuclease. Pyrrolecarboxamide-conjugated bromofluoroacetophenones; 4′-bromo-2′-fluoroacetophenone and 2′-bromo-4′-fluoroacetophenone were synthesized and their DNA cleaving activities and sequence selectivities were determined. Bromofluoroacetophenone-pyrrolecarboxamide conjugates were found to be effective DNA cleaving agents upon irradiation in concentration dependent manner based on plasma relaxation assay. The DNA cleaving activities of 2′-bromo-4′-fluoroacetophenone derivatives were larger than those of 4′-bromo-2′-fluoroacetophenone derivatives.

Full text of DOI:10.1016/S0960-894X(03)00212-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1763–1766
Photoinduced Cleavage of DNA by
Bromofluoroacetophenone–Pyrrolecarboxamide Conjugates
Paul A. Wendera and Raok Jeonb,*
aDepartment of Chemistry, Stanford University, Stanford, CA 94305, USA
bCollege of Pharmacy, Sookmyung Women’s University, Chungpa-Dong 2-Ka, Yongsan-Ku, Seoul 140-742, South Korea
Received 28 January 2003; accepted 25 February 2003
Abstract—Bromofluoroacetophenone derivatives which produce fluorine substituted phenyl radicals that cleave DNA upon exci-
tation were investigated as a novel photonuclease. Pyrrolecarboxamide-conjugated bromofluoroacetophenones; 40-bromo-20-
fluoroacetophenone and 20-bromo-40-fluoroacetophenone were synthesized and their DNA cleaving activities and sequence selec-
tivities were determined. Bromofluoroacetophenone–pyrrolecarboxamide conjugates were found to be effective DNA cleaving
agents upon irradiation in concentration dependent manner based on plasma relaxation assay. The DNA cleaving activities of
20-bromo-40-fluoroacetophenone derivatives were larger than those of 40-bromo-20-fluoroacetophenone derivatives.
# 2003 Elsevier Science Ltd. All rights reserved.
Development of DNA cleaving agents is an important
subject in chemistry, biology, and medicine.1 Therefore
considerate effort has been made by chemists and bio-
chemists to identify and characterize new molecules
capable of mediating nucleic acids strand scission. Ene-
diynes and other radical progenators are representative
and proven to possess highly potent antitumor activity.2
Especially the design of compounds, which cleave DNA
under photo-irradiation,3 is of great importance not
only from a fundamental biological point of view but
also in a photodynamic therapeutic approach as an
antitumor agent because of their potential ability to
target specific sites. In an effort to design simple photo-
inducible DNA cleaving agents, we recently reported
that commercially available benzotriazoles and halo-
arenes, 40-bromoacetophenones for DNA cleavage
when combined with a DNA recognition subunit can
upon photoactivation cleave DNA in a potent and
selective fashion.4
cleaving agents. Our studies are prompted by a series of
reports that examined the effect of fluorine substitution
of alkyl and aryl radical progenitors on the reactivity of
the radicals generated for hydrogen atom abstraction.
Enhanced reactivities of perfluoroalkyl and fluoroaryl
radicals have proven by several research groups com-
paring with their non-fluorinated counterparts.5 In
addition, as we examined previously, it was expected
that this fluorinated aryl radical generation could be
localized to specific sites on DNA by attaching the
DNA cleaver to DNA recognition element.6 In the
present studies, we elected to use bromofluoro-
acetophenones linked to distamycin type of synthetic
oligopeptide, pyrrolecarboximide7 as shown in Figure 1.
As a preliminary study, we investigated the ability of
two regioisomers of simple bromofluoroacetophenones
In the present paper, we extended our previous studies
on 40-bromoacetophenones to fluoro substituted
bromoacetophenones as radical progenitors for DNA
cleavage and report herein that fluorinated bromoace-
tophenones upon excitation are highly effective DNA
Figure 1. Bromofluoroacetophenone–pyrrolecarboxamide and 40-bromo-
acetophenone–pyrrolecarboxamide conjugates as photoinducible DNA
cleaving agents.
*Corresponding author. Tel.: +82-2-710-9571; fax: +82-2-715-9571;
e-mail: rjeon@sookmyung.ac.kr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00212-9

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P. A. Wender, R. Jeon / Bioorg. Med. Chem. Lett. 13 (2003) 1763–1766
to abstract hydrogen atoms upon excitation, a required
event for DNA cleavage. The photolytic behaviors of 40-
bromo-20-fluoroacetophenone and 20-bromo-40-fluoro-
acetophenone are representative. The photolyses were
performed by using a medium pressure mercury arc
lamp equipped with a Pyrex filter under anaerobic con-
ditions for 15 min. THF-d8 was used as solvent and as a
DNA surrogate. Deuterium incorporation, which is
induced by homolytic cleavage of C–Br bond of methyl
succinyl substituted derivatives 2 was examined. In both
cases, the debrominated products 3 was the major com-
pound produced. In accord with the intermediacy of an
aryl radical, when the photolysis of 40-bromo-20-fluoro-
acetophenone and 20-bromo-40-fluoroacetophenone was
conducted in THF-d8, the monodeuterated 20-fluoro-
acetophenone analogue and 40-fluoroacetophenone were
obtained in 15 min in 26% and 32% yield, respectively
(Scheme 1).
regioisomers were isolated by column chromatography
and then hydrolyzed by treating with barium hydroxide
to succinoic acids 6. Small amount of 2a and 2b were
converted to ortho- or para-fluorophenyl methyl suc-
cinoates by treatment with nBu3SnH to confirm their
geometry as well to get debrominated fluor-
oacetophenone as a standard for photolytic study.
Coupling of succinoic acids 6 with aminopyrrole 7
which was prepared by Shibuya’s method8 gave pyrro-
lecarboxamide-linked bromofluoroacetophenones 1.
The DNA cleaving activities of bromofluoro-
acetophenones were determined as shown in Figures 2
and 3 by monitoring their effectiveness in converting
circular supercoiled DNA (form I) to circular relaxed
DNA (form II) and linear DNA (form III). Simple
bromofluoroacetophenones and pyrrolecarboxamide-
linked bromofluoroactophenones were irradiated at
various concentrations for 30 min in the presence of
fX174RFI DNA (30 mM/bp) in 1:9 DMSO/Tris buffer
(20 mM, pH 7.5) under aerobic conditions. All compounds
tested caused DNA cleavage. The cleavage efficiency was
substrate and concentration dependent. DNA cleaving
activity of 40-bromo-20-fluoroacetophenone was as much
as same with that of non-fluorinated 40-bromoaceto-
phenone. On the other hand, 20-bromo-40-fluoro-
acetophenone showed more effective cleavage of DNA
comparing with 40-bromo-20-fluoroacetophenone in
Having established the utility of bromofluoro-
acetophenone as an aryl radical progenitor, we next
sought to attach this subunit to a distamycin type pyr-
rolecarboxamide-based DNA minor groove binders.
The pyrrolecarboxamide-linked bromofluoroaceto-
phenone conjugates were synthesized as shown in
Scheme 2. Preparation of 3-bromofluorobenzoyl propa-
noic acid 6 was performed by Friedel–Crafts succinoy-
lation of 1-bromo-3-fluorobenzene 4 with succinic
anhydride showing very low yield due to the electron
withdrawing-fluorine
substituent.
Friedel–Crafts
acylation of 1-bromo-3-fluorobenzene by treating
with 3-carbomethoxypropionyl chloride gave bromo-
fluorophenyl methyl succinoates 2 in better yield leading
to two regioisomers, 40-bromo-20-fluoro 2a and
20-bromo-40-fluoro derivatives 2b as 1.5 to 1 ratios. The
Figure 2. Light induced cleavage of DNA by (a) 40-bromo-20-fluoro-
acetophenone; (b) 20-bromo-40-fluoroacetophenone. Supercoiled DNA
(fX174RF) 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 med-
ium pressure mercury arc lamp for 30 min at 25 ꢀC. Lanes 1–4, DNA
(30 mM/bp)+haloacetophenone at concentrations of 0.1, 1, 10, and
100 mM, respectively; lane 5, control fX174RF DNA+haloaceto-
phenone (100 mM), no hn.
Scheme 1. Photolysis of 4-(bromofluorophenyl)-4-oxo-butyric acid
methyl ester. The yields were determined by 1H NMR based on the
internal standard.
Figure 3. Light induced cleavage of DNA by (a) pyrrolecarboxamide-
linked 40-bromo-20-fluoroacetophenone 1a and (b) pyrrolecarbox-
amide-linked 20-bromo-40-fluoroacetophenone 1b. Supercoiled DNA
(fX174RF) 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 med-
ium pressure mercury arc lamp for 30 min at 25 ꢀC. Lane 1, control
fX174RF DNA (30 mM/bp), no hn; lanes 2–6, DNA+1a, 1b at con-
centrations of 3, 7, 15, 20, and 25 mM, respectively; lane 7, DNA+1
(30 mM), no hn.
Scheme 2. Preparation of bromofluroacetophenone–pyrrolecarbox-
amide conjugates: (a) AlCl3, CS2, 80–90 ꢀC, 4 h, 84%; (b) Ba(OH)2,
CH3OH/H2O (2/1), rt, 2 h, 95%; (c) 7, DCC, DMAP, DMF.

P. A. Wender, R. Jeon / Bioorg. Med. Chem. Lett. 13 (2003) 1763–1766
1765
correspondence with the photolytic results of bromo-
fluoroacetophenones in the absence of DNA.
complete disappearance of form I. The 7 mM reaction of
20-bromo-40-fluoroacetophenone derivative gave similar
activity with those observed at 2-fold higher concentra-
tions of 40-bromo-20-fluoroacetophenone derivative. It
was corresponding to the preliminary investigation of
photolytic behavior and cleaving activity of bromo-
fluoroacetophenones.
In the case of pyrrolecarboxamide-linked bromo-
fluoroacetophenones 1 for DNA cleavage, 3 to 25 mM
range of low concentrations were applied. As expected,
the cleavage efficiency of the compounds was remark-
ably increased by attachment of the DNA recognition
element, pyrrolecarboxamide and strongly influenced by
geometry of the fluorine substituent on the phenyl ring
as well as the concentration of the test compound. The
effect of the fluorine substitution itself seemed to be
minor for DNA cleavage but the different geometry of
fluorine substituent led to a significant difference. 20-
Bromo-40-fluoroacetophenone derivative produced
more pronounced cleavage than 40-Bromo-20-fluoro-
acetophenone derivative over the same concentration
range. 20-Bromo-40-fluoroacetophenone derivative pro-
duced form III DNA at a concentration of 15 mM with
The DNA sequence specificities of pyrrolecarboxamide-
linked bromofluoroacetophenones were examined by
DNA sequencing techniques based on electrophoretic
procedures using high resolution denaturing poly-
acrylamide sequencing gels. The 30-32P end labeled 167
base pair restriction enzyme fragment from pBR322 was
used.9 The cleaving sites were interpreted by comparing
with the Maxam-Gilbert G marker which indicates the
G sites of the DNA sequence. Pyrrolecarboxamide-
linked 40-bromoacetophenone 1c was also loaded and
compared it’s sequence selectivity and reactivity with
those of bromofluoroacetophenone derivatives. The
results in Figure 4 show that 20-bromo-40-fluoro-
acetophenone derivative 1b has the highest activity to
DNA in accord with the previous results from agars gel
electrophoresis. As expected for a cleaving agent bound
to pyrrolecarboxamide known as a distamycin or
netropsin analogue, the cleavage intensities are the
highest in AT-rich regions of the DNA as marked on
the autoradiogram.
An autoradiogram (data not shown) obtained was
quantified by densitometry and this data was used to
Figure 4. Autoradiogram of 10% denaturing gel polyacrylamide
gel showing cleavage of 30-32P end-labeled 167 base pair restriction
fragment (EcoRI/RsaI) from pBR322 by pyrrolecarboxamide-linked
40-bromo-20-fluoroacetophenone
1a,
pyrrolecarboxamide-linked
Figure 5. Histograms of DNA cleavage sites by (a) pyrrolecarbox-
amide-linked 40-bromoacetophenone 1c, (b) pyrrolecarboxamide-linked
40-bromo-20-fluoroacetophenone 1a, and (c) pyrrolecarboxamide-linked
20-bromo-40-fluoroacetophenone 1b. The relative extent of cleavage was
estimated from the densitometric scans of the autoradiograms shown in
Figure 4 and the height of the bar represents relative cleavage intensity.
(a–c) 50 mM of compound 1c, 1a and 1b, respectively.
20-bromo-40-fluoroacetophenone 1b, and pyrrolecarboxamide-linked
40-bromoacetophenone 1c. All reactions were irradiated with Pyrex-
filtered light from a 450 W medium pressure mercury arc lamp for 30
min at 25 ꢀC. The cleaving site is shown to the right of the auto-
radiogram. Lanes 1 and 6, Maxam-Gilbert G reaction; lanes 2–4,
DNA+30 mM of 1c, 1a, and 1b, respectively; lane 5, DNA control.

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construct histograms for the DNA cleavage observed.
The histograms shown in Figure 5 clearly show that
40-bromoactophenone derivative and 40-bromo-20-fluoro-
acetophenone derivative produce the similar cleavage
sites and patterns leading to the cleavage within and
adjacent to sites of multiple contiguous AT base pairs.
On the other hand, 20-bomo-40-fluoroacetophenone
derivative seemed to lose the selectivity although the
affinity to AT base sequence still remained at the
applied concentration. It is supposed that the higher
reactivity of the phenyl radical species from the
20-bromo-40-fluoroacetophenone derivative led to the
higher reactivity for the cleavage of DNA with decreas-
ing selectivity. The cleavage assay and sequencing of the
tested compounds showed prominent correlation
between reactivity of the phenyl radical and DNA
cleaving activities with the highest activity of 20-bromo-
40-fluoroacetophenone derivative.
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In summary, 40-bromo-20-fluoroacetophenone and
20-bromo-40-fluoroacetophenone derivatives upon irra-
diation can function as DNA cleaving agents putatively
through the generation and reaction of phenyl radicals.
20-Bromo-40-fluoroacetophenones revealed higher DNA
cleaving activity than 40-bromo-20-fluoroacetophenone
derivatives supposedly induced by the higher reactivity of
the phenyl radical generated from 20-bromo-40-fluoro-
acetophenones. The attachment of a minor groove
binding moiety to bromofluoroacetophenone results in
increased strand scission of DNA mainly in sequence
selective manner. This work provides a new class of
DNA cleaving agents that are easy to prepare and verify.
Acknowledgements
This work was supported by Korea Research Founda-
tion Grant (KRF- 2001-003-F00235) and the National
Institutes of Health (Grant CA31845). We thank Dr. S.
Touami for assistance with the sequencing experiments.
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