Y. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1139–1142
1141
the approximation of the photocleaver to DNA and
facilitated the radicals to damage DNA. However, in
the case of A and B, which photodamaged DNA in elec-
tron transfer mechanism, the high intercalating ability
resulted in the isolation of the photocleaver within the
hydrophobic inner of DNA from oxygen in surrounding
solution might prevent them from undergoing electron
transfer7 to some extent between the amino groups
and oxygen. These two cases implied that the intercala-
tion did not always promote photocleavage and the
influence of intercalating ability of chromophores on
the cleaving ability of photocleavers depended on their
damage mechanisms.
Figure 3. The effect of different additives on the photocleavage of
supercoiled pBR322 DNA (30 ng/lL) in the buffer Tris–HCl (pH 7.5)
under photoirradiation (2300 W/cm2) with a transluminator (366 nm)
for 2 h in the distance of 20 cm at 0 ꢁC; lane 1: DNA and B3 in the
presence of ethanol (1.7 M); lane 2: DNA and B3 in the presence of
superoxide dismutase (SOD, 100 lg/mL); lane 3: DNA and B3 in the
presence of dithiothreitol (DTT, 30 mM); lane 4: DNA and B3 in the
presence of histidine (6mM); lane 5: DNA and B3 at the concentration
of 50 lM; lane 6: DNA alone; lane 7: DNA alone (no hm).
In summary, the present work demonstrated the design
and evaluation of novel and high intercalating photo-
cleavers with five-member thio-heterocyclic fused naph-
thalimides containing aminoalkyl side chains at the
imide. They could damage DNA (supercoiled pBR322)
from form I (closed) to II (nicked) at a concentration
as low as 5 lM and from form I to form III at a concen-
tration of 50 lM. The comparison of these two types of
isomers implied that the influence of intercalating abili-
ties of chromophores on the cleaving abilities of photo-
cleavers depended on their damage mechanisms. The
experimental results suggested that the aminoalkyl side
chain connected with chromophore is also very impor-
tant for photodamage in electron transfer mechanism.
The anti-cancer studies on these photocleavers are also
in progress.
Mechanism experiment was also performed with the
addition of histidine, dithiothreitol, superoxide dismu-
tase and ethanol (Fig. 3). It was found that histidine
(singlet oxygen quencher) ethanol (radical quencher)
had no effect on the cleavage reaction, However, dithio-
threitol (DTT, superoxide anion radical scavenger) re-
tarded the reaction efficiently. It should be pointed out
that superoxide dismutase (SOD, superoxide radical
killer) accelerated the rate of DNA-cleaving reaction,
because the hydrogen peroxide produced by SOD from
superoxide anion radical, could lead to DNA damage in
the presence of UV light or after reduction by the trace
of metal ions. Obviously, superoxide anion radical was
involved in the DNA cleavage. It was supposed by Eri-
ksson and co-workers6 that the reactive superoxide an-
ion radicals formed in the photoirradation had two
pathways. The most common way was reduction of
the excited triplet by an electron donor (e.g., one of
the DNA bases), followed by electron transfer from
the reduced photosensitizer to molecular oxygen. SaitoÕs
research work2d has proved this way. The other way was
a direct ionization of the photosensitizer by radiation,
and electron uptake by molecular oxygen (Ôdirect elec-
tron transferÕ). In our experiment, without using piper-
idine treatment, we could exclude DNA damage by G
oxidation and we found that compounds A4, B4 had
no any cleaving ability in the absence of aminoalkyl side
chain. It implied that the superoxide anion radical was
possibly formed through the electron transfer from
nitrogen in aminoalkyl side chain to oxygen.
Acknowledgements
The National Key Project for Basic Research
(2003CB114400), National Natural Science Foundation
of China, The Science and Technology Foundation of
Shanghai and The Education Commission of Shanghai
partially supported this study.
References and notes
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Compared these two types of isomers (A and B, C and
D), it could be found that the binding abilities of five-
member heterocyclic isomers (binding constants
ꢀ105 MÀ1 for D and B) were much higher than that of
six-member heterocyclic isomers (binding constants
ꢀ103 MÀ1 for C and A). Although the photocleaving
abilities of C and D were parallel to their intercalation
abilities, it could be found that those of A and B were
almost anti-parallel to their intercalation abilities. We
thought that these might be caused by the differences
in DNA damage mechanisms.
The intercalation means that the photocleaver is located
in the hydrophobic inner of duplex DNA. For case of C
and D, the hydroperoxides damaged DNA through rad-
ical mechanism and high intercalating ability promoted