"Self-activating" chemical nuclease: Ferrocenyl cyclen Cu(II) complexes act as efficient DNA cleavage reagents in the absence of reductant

Article abstract of DOI:10.1016/j.ejmech.2008.03.029

The interactions of cyclen Cu(II) complexes functionalized by ferrocenyl group with plasmid DNA indicated that these complexes have high cleavage efficiency via an oxidative mechanism in the absence of any reductant or oxidant.

Full text of DOI:10.1016/j.ejmech.2008.03.029

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1768e1772
http://www.elsevier.com/locate/ejmech
Short communication
‘‘Self-activating’’ chemical nuclease: Ferrocenyl cyclen Cu(II)
complexes act as efficient DNA cleavage reagents in the
absence of reductant
Kun Li a, Li-Hong Zhou a, Ji Zhang a, Shan-Yong Chen a, Zhong-Wei Zhang b,
a,c,
b,**
, Xiao-Qi Yu
*
Jing-Jing Zhang a, Hong-Hui Lin
a Department of Chemistry, Key Laboratory of Green Chemistry and Technology (Ministry of Education),
Sichuan University, No. 29, Wangjiang Road, Chengdu, Sichuan 610064, PR China
b Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences,
Sichuan University, Chengdu, Sichuan 610064, PR China
c State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University,
Chengdu, Sichuan 610041, PR China
Received 6 September 2007; received in revised form 25 December 2007; accepted 21 March 2008
Available online 4 April 2008
Abstract
The interactions of cyclen Cu(II) complexes functionalized by ferrocenyl group with plasmid DNA indicated that these complexes have high
cleavage efficiency via an oxidative mechanism in the absence of any reductant or oxidant.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Chemical nucleases; DNA cleavage; Cyclen; Complexes
1. Introduction
cases, photo-induction was also needed to initiate the cleavage
process. For the above reasons, the in vivo applications of
these reagents were limited [5,6]. Therefore, seeking ‘‘self-
activating’’ chemical nucleases have become a challenge for
chemists, and only few examples were reported [7].
Chemical nucleases, which have potential applications in
the fields of biotechnology and therapeutic reagents [1e3],
have been proved to be efficient tools for DNA cleavage. A
large number of transition metal complexes have been ex-
plored with good to excellent DNA cleavage activities through
either hydrolytic or oxidative pathways [4,5]. However, most
oxidative cleavage reagents that exhibited outstanding DNA
cleavage activity require the addition of external oxidative
(e.g. dihydrogen peroxide, molecular oxygen) or reductive re-
agents (e.g. ascorbic acid, 3-mercaptopropionic acid). In some
Recent efforts in our laboratory focused on the copper
complexes of cyclen derivatives for their excellent DNA cleav-
age activity [8]. For further studies and development of highly
efficient chemical nuclease, we tried to find an electron recep-
tor to transfer electron to DNA strands. Ferrocene, as a charac-
teristic structure of metal atom sandwiched between planar
cyclopentadiene rings, exhibits good redox properties and
has already shown to replace phenyl moieties advantageously
in biologically active compounds [9]. Herein, we introduced
a ferrocenyl group to the cyclen ring and expected that this
kind of new ligands may display better biological activity [10].
Three cyclen Cu(II) complexs containing ferrocene were
designed and synthesized. According to the route shown in
Scheme 1 [8e,11], 3Boc-cyclen reacted with N-Cbz-protected
* Corresponding author. Department of Chemistry, Key Laboratory of Green
Chemistry and Technology (Ministry of Education), Sichuan University, No.
29, Wangjiang Road, Chengdu, Sichuan 610064, PR China. Tel./fax: þ86
28 85415886.
** Corresponding author.
E-mail address: xqyu@tfol.com (X.-Q. Yu).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.029

K. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 1768e1772
1769
O
O
Boc
Boc
Boc
Boc
Boc
NHCbz
NH2
R
HN
N
N
N
N
N
N
N
N
N
N
N
DCC/HOBt
CH2Cl2
Pd/C
MeOH
+
R
R
CbzHN
COOH
1a-c
Boc
Boc
Boc
Boc
2a-c
3a-c
O
H
N
O
H
N
Boc
Boc
N
N
N
4, DCC
THF
TFA
CH2Cl2
NH
N
Fe
O
Fe
R
O
R
N
NHHN
Boc
3CF3COOH
5a-c
6a-c
O
H
N
O
H
H
Cu(NO3)2 6H2O
C2H5OH
N
N
Fe
Cu2+
N
O
R
OH
=
4
N
Fe
H
7a:R=H
7b:R=CH2C6H5
7c:R=CH2OH
Scheme 1. Synthetic route of cycleneferrocene Cu(II) complexes.
amino acids 1 to give 2 by using coupling reagent DCC. Cbz
group was removed by using 10% Pd/C in methanol under hy-
drogen atmosphere. The coupling compounds 5 were obtained
by the reactions of 3 and ferrocenecarboxylic acid 4 in the
presence of DCC in THF. After deprotection by TFA and sub-
sequent metalation, the cycleneferrocene Cu(II) complexes 7
were obtained in moderate yields (45%, 38% and 25% for 7a,
b and c, respectively). All ligands were characterized by IR,
1
2
3
4
5
6
7
A
Form II
100
Form III
Form I
A
Form III
Form II
80
60
40
20
0
B
C
0
1
2
3
4
5
6
7
8
100
80
60
40
20
0
B
C
100
80
60
40
20
0
Form III
Form II
Form II
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
Fig. 1. Agarose gel electrophoresis patterns for the cleavage of pUC19 plasmid DNA (5 mg/mL) by various concentrations of complexes 7aec, and the quantities of
percentage plasmid relaxation (Form II or Form III) relative to plasmid DNA per lane. Reaction was done in an NaH2PO4/Na2HPO4 buffer (100 mM, pH 7.4) at
37 ꢀC for 5 h. (A) Lane 1: DNA control; lane 2e7: DNA þ complex 7a of 16.9, 28.3, 56.6, 106, 170, 424 mM. (B) Lane 1: DNA control; lane 2e6:
DNA þ complex 7b of 14.4, 24.0, 48.0, 90.0, 125 mM. (C) Lane 1: DNA control; lane 2e7: DNA þ complex 7c of 10.7, 17.8, 35.7, 68.0, 107, 267 mM.

1770
K. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 1768e1772
Table 1
Time-dependent cleavage of pUC19 plasmid DNA by complex 7a (170 mM)
and 7b (144 mM)
results indicated that 7a and 7b could catalyze the cleavage of
supercoiled DNA (Form I) to the nicked DNA form (Form II)
in 2 h at the concentration of 170 and 144 mM, respectively. In-
creasing the reaction time led to the increase of Form III.
Meanwhile, unlike 7a and 7b, complex 7c could catalyze the
cleavage of Form I to Form II in 20 min at the concentration of
107 mM. In order to observe the gradual change of the yields in
the DNA cleavage process, we reduced the concentration of
complex 7c to 66.8 mM (Fig. 2). The quantification of decrease
of Form I and increase of Form II allowed evaluating the kinetic
parameters accordingly, as shown in Fig. 2B. Since no Form III
was observed under this reaction condition, it is easy to deduce
that the cleavage reaction followed a pseudo-fist-order kinetic
mechanism with k1 ¼ 7.3 ꢁ 10ꢂ3 minꢂ1 and R2 ¼ 0.98
[1a,12], which was in agreement with the mechanism presented
by other copper complexes [1a,7f].
Complex
Time [h]
Supercoiled [%]
Nicked [%]
Linear [%]
DNA control
12.0
0.5
1.0
2.0
4.0
8.0
12.0
0.5
1.0
2.0
4.0
8.0
12.0
100
33
19
2
0
67
81
90
83
79
57
75
84
87
79
75
68
0
0
7a
7a
7a
7a
7a
7a
7b
7b
7b
7b
7b
7b
0
8
6
11
21
43
2
0
0
23
13
7
3
6
2
19
25
32
0
0
From the above results we might know that complex 7c is
superior to 7a and 7b as catalyst in the DNA cleavage reaction.
This may be due to the hydroxyl group contained in 7c, as this
group could make it easy to form singlet oxygen or metale
oxygen complex [5a,b,13].
To identify the reactive oxygen intermediates, which
might be formed in the DNA cleavage process, experiments
in the presence of a variety of radical scavengers were also
carried out (Fig. 3). DMSO and tert-butyl alcohol were
found to have little effect on DNA cleavage reaction (lanes
4 and 5), which indicated that hydroxyl radicals are not the
active species. However, the DNA cleavage could be
inhibited dramatically when NaI or NaN3 was added to
the system (lanes 2 and 6), and T4 ligase ligation indicated
that the DNA fragments (Form II and Form III) could not be
ligated [14]. These results pointed out that the singlet
oxygen as active species might be involved in the DNA
1H NMR, and HRMS-ESI. The metal complexes 7aec were
characterized by HRMS, IR and element analysis.
The DNA cleavage activities of complexes 7 were studied
under physiological conditions. Firstly, we compared the
catalytic abilities of 7aec for DNA cleavage. The results are
shown in Fig. 1. The cleavage activity was improved signifi-
cantly associated with the increase of the concentration of
complexes 7. Complexes 7a and 7b could convert the super-
coiled DNA (Form I) to nicked DNA (Form II) efficiently at
the concentrations of 106 and 122 mM, respectively. For com-
plex 7c, the yield of Form II increased to nearly 80% when
[7c] was only 35.7 mM, no linear DNA was formed either. Fur-
ther increase of the concentration of 7 led to the appearance of
Form III.
Then we studied the effect of reaction time on the DNA cleav-
age process. The contents of each form are listed in Table 1. The
1
2
3
4
5
6
7
8
A
B100
C
4
80
Form I
Form II
60
40
20
0
2
0
0
100
200 300
Time (min)
400
500
0
100
200 300
Time (min)
400
500
Fig. 2. Effect of reaction time on the cleavage of pUC19 DNA (5 mg/mL) with 7c (66.8 mM) in an Na2HPO4/NaH2PO4 buffer (100 mM, pH 7.4) at 37 ꢀC. (A)
Agarose gel electrophoresis diagrams: lane 1: DNA control; lanes 2e8: complex 7c, 10, 20, 40, 60, 120, 240, 480 min. (B) Quantity of pUC19 plasmid forms.
(C) Linear simulation of pseudo-first-order reaction according to the quantity of supercoiled DNA (Form I).

K. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 1768e1772
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Acknowledgements
This work was financially supported by the National Sci-
ence Foundation of China (nos. 20725206, 20732004 and
20572075), Program for New Century Excellent Talents in
University, Specialized Research Fund for the Doctoral Pro-
gram of Higher Education and Scientific Fund of Sichuan
Province for Outstanding Young Scientist.
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ꢂk1t þ A.
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[15] We examined the Cu(II)eH2O2 system for the control experiment, and
results indicated that part of supercoiled DNA (48%) could be damaged
in higher Cu(II) concentration (0.2 mM) in the presence of H2O2
(143 mM).
[14] Procedure for DNA ligation experiments. After incubation of pUC19
plasmid DNA with complex 7c (0.107 mM) for 6 h at 37 ꢀC, the