The conjugates of uracil-cyclen Zn(II) complexes: Synthesis, characterization, and their interaction with plasmid DNA

Article abstract of DOI:10.1016/j.bmc.2006.04.048

As an important nucleobase in RNA, uracil was introduced into the side chain of cyclen (1,4,7,10-tetraazacyclododecane) by using phenylene dimethylene group as bridge. The target compounds 5 were obtained in high yields. Subsequent experiments demonstrate

Full text of DOI:10.1016/j.bmc.2006.04.048

Bioorganic & Medicinal Chemistry 14 (2006) 5756–5764
The conjugates of uracil–cyclen Zn(II) complexes: Synthesis,
characterization, and their interaction with plasmid DNA
Chuan-Qin Xia,a Ning Jiang,b Ji Zhang,a Shan-Yong Chen,a
Hong-Hui Lin,b,* Xin-Yu Tan,a Yang Yuea and Xiao-Qi Yua,c,
*
aDepartment of Chemistry, Key Laboratory of Green Chemistry and Technology (Ministry of Education),
Sichuan University, Chengdu 610064, PR China
bCollege of Life Science, Sichuan University, Chengdu 610064, PR China
cState Key Laboratory of Biotherapy, West China Hospital, West China Medical School,
Sichuan University, Chengdu, Sichuan 610041, PR China
Received 10 January 2006; revised 5 April 2006; accepted 6 April 2006
Available online 5 June 2006
Abstract—As an important nucleobase in RNA, uracil was introduced into the side chain of cyclen (1,4,7,10-tetraazacyclododecane)
by using phenylene dimethylene group as bridge. The target compounds 5 were obtained in high yields. Subsequent experiments
demonstrated that the uracil–cyclen conjugates can bind Zn2+ cation rapidly in water, and the catalytic activities of their Zn(II) com-
plexes 6 in DNA cleavage were also studied. The results showed that Zn(II) complexes can catalyze the cleavage of supercoiled DNA
(pUC 19 plasmid DNA) (Form I) to produce nicked DNA (Form II and Form III) with high selectivity. In water solution, complex
6b may form a unique and stable supramolecular structure, which benefits the DNA cleavage process.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
to date are scarce.5 Our goal is to develop novel Zn(II)
complexes prepared from small organic ligands, and
use these complexes to cleave double-strand DNA
effectively. The research areas about synthetic Zn(II)
complexes include following systems: (1) Barton’s
mononuclear Zn(II)-binding peptides tethered to rhodi-
um complex as intercalator.6 (2) Scrimin’s binuclear
Zn(II)-binding peptides’ conjugate with an acridine
intercalator.7 (3) Tecilla’s mononuclear Zn(II)-binding
cis–cis-triaminocyclohexane ligands linked by an
anthraquinone moiety through alkyl spacers with
different lengths.8 (4) Dinuclear macrocyclic polyamine
zinc(II) complexes linked by different spacers, which
was developed in our group.9 All of above complexes
have two functions: the scission part (Zn(II)) and the
recognition part (intercalator).
The cleavage of DNA with high selectivity has become
an invaluable tool in biology, bioorganic chemistry,
and molecular biology. However, the phosphodiester
bonds of DNA are very difficult to be hydrolyzed under
physiological conditions.1 A number of natural enzymes
such as restriction endonucleases and topoisomerases
can efficiently catalyze DNA hydrolysis.2 The special ef-
fects of those enzymes most often depend on their active
sites including metal ions, particularly Zn(II), Mg(II),
and Fe(II), and their ability to act as Lewis acids.3 In re-
cent years, many artificial metallonucleases have been
suggested as substitute for natural enzymes to cleave
DNA. Among the physiologically relevant metal ions,
Zn(II) is probably the best suitable metal ion for artifi-
cial metallonucleases. However, the reactivity of Zn(II)
ion is somewhat lower than that of the other commonly
employed transition-metal ions,4 and this is why the
examples of Zn(II)-based artificial nucleases reported
Cyclen (1,4,7,10-tetraazacyclododecane) has strong
coordination ability toward a wide range of cations,
including transition metal ions and lanthanide ions.10,11
And the complexes derived from this kind of ligands
have been widely used in the fields of DNA recogni-
tion,12,13 DNA cleavage,14 and enzyme mimics.15,16 In
order to increase the DNA cleavage rate at low concen-
tration of metal complex, we introduced nucleobases,
which could be favorable to recognize DNA through
Keywords: 1,4,7,10-Tetraazacyclododecane (cyclen); DNA cleavage;
Synthesis; ESI mass spectrum.
*
Corresponding authors. Fax: +86 28 85415886; e-mail: xqyu@
tfol.com
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.048

C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
5757
Watson–Crick interaction, to the cyclen cycle. In this
2.3. Synthesis of uracil–cyclen conjugates
paper, we designed and synthesized the conjugates of cy-
clen and uracil bridged by rigid spacers, and applied
them to the coordination with Zn(II) to form metal
complexes, which may be used to cleave DNA. These
complexes have some special properties: (1) Uracil, as
a nucleobase, could be favorable to recognize DNA.
(2) More interestingly, the metal ion in the complex
could bind to the nitrogen atom of the imide of uracil
group through an intramolecular or intermolecular
mode, which can form a unique and stable supramolec-
ular complex in aqueous solution. The complex that has
this property displays higher activity in the DNA cleav-
age process than the one without the supramolecular
structure. We also run ESI mass spectra to testify the
special structure.
2.3.1. 1-[40-(Bromomethyl)benzyl]-4,7,10-tris(tert-butoxy-
carbonyl)-1,4,7,10-tetraazacyclododecane (3a). Com-
pound 3 was prepared as previously reported.18 Under
N2 atmosphere, to a solution of 1,4-dibromomethylene
benzene (1.03 g, 4.00 mmol) and Na2CO3 (0.25 g,
2.17 mmol) in 50 mL of dry CH3CN was slowly added
1,4,7-tris(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclod-
odecane 1 (0.50 g, 1.06 mmol) under stirring. The reaction
was stirred at 80 °C for 72 h. The insoluble inorganic salt
was then filtered off and the filtrate was concentrated in
vacuo. The residue was dissolved in CHCl3 and then puri-
fied by silica gel column chromatography (ethyl acetate/
petroleum ether = 1:4) and obtained colorless amorphous
solid (yield, 68.9%). Mp: 69–71 °C. ESI-MS: m/z = 757.5
(M+Na)+.
2. Experimental
2.3.2. 1-[30-(Bromomethyl)benzyl]-4,7,10-tris(tert-butoxy-
carbonyl)-1,4,7,10-tetraazacyclododecane (3b). The syn-
thetic method of 3b was the same as that of 3a except
using 1,3-dibromomethylene benzene as reactant. Color-
less amorphous solid was obtained (yield 73.5%). Elu-
ent: EtOAc/petroleum ether = 1:4. Mp: 83–84 °C. ESI-
MS: m/z = 766.3 (M+Na)+.
2.1. General information
ESI-mass spectra data were recorded on Finnigan
LCQDECA mass spectrometer. HRMS spectral data were
recorded on Bruker Daltonics Bio TOF. 1H NMR spec-
tra were measured on a Varian INOVA-400 spectrome-
ter and chemical shifts in ppm are reported relative to
internal Me4Si (CDCl3) or 3-(trimethylsilyl) propionic-
2,2,3,3-d4 acid sodium salt (D2O). Melting points were
determined by using a micro-melting point apparatus
without any corrections. 1,4,7-Tris(tert-butyloxycarbon-
yl)-1,4,7,10-tetraazacyclododecane (Boc3-cyclen) was
prepared according to the literature.17 All chemicals
and reagents were obtained commercially and used with-
out further purification. Electrophoresis was processed
by using a Biomeans Stack II-Electrophoresis system,
PPSV-010. Bands were visualized by UV light and pho-
tographed followed by the estimation of the intensity of
the DNA bands using a Gel Documentation System,
recorded on an Olympus GRAB-it 2.0 Annotating Im-
age Computer System. Electrophoresis grade agarose
and plasmid DNA (pUC 19) were purchased from
Takara Biotechnology Company.
2.3.3. 1-[40-(100-Uracilmethyl)benzyl]-4,7,10-tris(tert-bu-
toxycarbonyl)-1,4,7,10-tetraazacyclododecane (4a). NaH
(40 mg, 1 mmol) was added to a suspension of uracil
(2.2 mmol, 244.2 mg) and KI (60 mg, catalytic amount)
in dry, degassed DMSO and the mixture was stirred for
0.5 h at 50 °C under N2 atmosphere. A solution of 3a
(2 mmol, 1.37 g) in dry DMSO was added slowly to this
mixture and the reaction mixture was then stirred at
60 °C overnight. Water was added and the solution was
extracted with ethyl acetate. The organic layer was dried
with anhydrous Na2SO4 and then concentrated in vacuo
below 40 °C to give crude product. The resulting crude
product was purified by silica gel chromatography (meth-
anol/chloroform = 1:8) to afford colorless amorphous
1
solid 4a (yield: 80%). Mp: 73–75 °C. H NMR (CDCl3,
400 MHz) d: 1.45–1.47 (m, 27H, OC(CH3)3), 2.63 (s,
4H, CH2-cyclen), 3.27–3.73 (m, 14H, CH2NCH2,cyclen–
CH2Ph), 4.88 (s, 2H, Ph–CH2–uracil), 5.57–5.69 (d, 1H,
H(5) in uracil), 7.12–7.14 (d, H(6)in uracil), 7.14–7.28 (m,
4H, PhH); HRMS (ESI) calcd for C35H54N6O8Na
[M+Na]+: m/z = 709.3895. Found: 709.3872.
2.2. Procedure of DNA cleavage experiments
The preparation of Zn(II) complexes 6 is described as
follows: To the water solutions of the cyclen–uracil con-
jugates 5a and 5b was added 1 equiv of Zn(ClO4)2Æ7H2O
as water solution, and the pH was controlled at 8.0 by
addition of NaOH solution. DNA cleavage experiments
were performed as follows: supercoiled pUC 19 DNA
(10 lL, 0.025 lg/lL) with certain concentration of vita-
min C (Vc, if needed) in 100 mM Tris–HCl buffer was
treated with complexes 6, followed by dilution with
the Tris–HCl buffer to a total volume of 35 lL. The
samples were then incubated at 37 °C and loaded on a
1% agarose gel containing 1.0 lg/lL ethidium bromide.
Electrophoresis was carried out at 40 V for 30 min in
TAE buffer. Bands were visualized by UV light and
photographed followed by the estimation of the intensi-
ty of the DNA bands using a Gel Documentation
System.
2.3.4. 1-[30-(100-Uracilmethyl)benzyl]-4,7,10-tris(tert-butox-
ycarbonyl)-1,4,7,10-tetraazacyclododecane (4b). The syn-
thetic method of 4b was the same as that of 4a except
using 3b as reactant. Colorless amorphous solid was
obtained (yield 78.7%). Mp: 64–66 °C, 1H NMR (CDCl3,
400 MHz) d: 1.42–1.48 (m, 27H, OC(CH3)3), 2.63 (s, 4H,
CH2-cyclen), 3.26–3.72 (m, 14H, CH2NCH2,cyclen–
CH2Ph), 4.88 (s, 2H, Ph–CH2–uracil), 5.68–5.70 (d, 1H,
H(5) in uracil, J = 0.8 Hz), 7.31–7.33 (d, H(6)in uracil),
7.17–7.33 (m, 4H, PhH). HRMS (ESI) calcd for C35H54
N6O8Na [M+Na]+: m/z = 709.3895. Found: 709.3891.
2.3.5. 1-[40-(100-Uracilmethyl)benzyl]-1,4,7,10-tetraazacy-
clododecane tetrahydrobromide (5a). To a solution of 4a
(0.709 g, 1.0 mmol) in dry EtOH (5 mL) at 0 °C, 40%

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C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
aqueous HBr (1 mL) was added slowly. After stirring
overnight at room temperature, the reaction mixture
was concentrated in vacuo below 40 °C to give crude
product. The resulting crude product was recrystallized
from EtOH/40% aqueous HBr to afford 5aÆ4HBr as
2.3.7. 1-[40-(100-Uracilmethyl)benzyl]-1,4,7,10-tetraazacy-
clododecane zinc perchlorate (6a). The hydrobromide
salt of 5a (0.721 g, 1.0 mmol) was dissolved in 5 mL
of water. After adjusting aqueous solution to alkaline
(pH P 8), Zn(ClO4)2Æ6H2O (0.408 g, 1.1 mmol) in
EtOH (5 mL) was added. The mixture was stirred over-
night at room temperature. The solution was gradually
concentrated to give a white powder solid 6a in 88.3%
yield. Mp: >300 °C. 1H NMR (D2O, 400 MHz) d:
2.86–3.12 (m, 16H, NCH2CH2N), 3.73 (cyclen-CH2–
Ph), 5.10 (s, 2H, Ph–CH2–uracil), 5.78–5.83 (d, 1H,
H(5) in uracil, J = 8 Hz), 7.68–7.80 (d, H(6) in uracil,
J = 8 Hz), 7.36–7.42 (m, 4H, Ph). ESI-MS (m/z):
449.0 (MꢀH+).
2.3.8. 1-[30-(100-Uracilmethyl)benzyl]-1,4,7,10-tetraazacy-
clododecane zinc perchlorate (6b). White powder was ob-
tained (yield 88.5%). Mp: >300 °C, 1H NMR (D2O,
400 MHz) d: 2.83–3.20 (m, 16H, NCH2CH2N), 3.69 (cy-
clen–CH2–Ph), 4.82 (s, 2H, Ph–CH2–uracil), 5.61–5.72
(d, 1H, H(5) in uracil, J = 12 Hz), 7.35–7.58 (m, 4H,
Ph), 7.80–7.94 (d, H(6) in uracil, J = 8 Hz). ESI-MS
(m/z): 451.2 (M+H)+.
1
white powder (yield, 78%). Mp: 236–238 °C. H NMR
(D2O, 400 MHz) d: 2.99–3.29 (m, 16H, NCH2CH2N),
3.93 (cyclen–CH2Ph), 5.05 (s, 2H, Ph–CH2–uracil),
5.91–5.93 (d, 1H, H(5) in uracil, J = 8 Hz), 7.80–7.82
(d, H(6) in uracil, J = 8 Hz), 7.43–7.48 (m, 4H, PhH).
HRMS (ESI) calcd for C20H31N6O2 [M+H]+:
m/z = 387.2503. Found: 387.2503.
2.3.6. 1-[30-(100-Uracilmethyl)benzyl]-1,4,7,10-tetraazacy-
clododecane tetrahydrobromide (5b). The synthetic meth-
od of 5b was the same as that of 5a except using 4b as
reactant. White powder was obtained (yield 73.3%).
1
Mp: 190–191 °C. H NMR (D2O, 400 MHz) d: 2.93–
3.27 (m, 16H, NCH2CH2N), 3.89 (cyclen–CH2Ph), 4.88
(s, 2H, Ph–CH2–uracil), 5.67–5.69 (d, 1H, H(5) in uracil,
J = 12 Hz), 7.37–7.53 (m, 4H, PhH), 7.82–7.84 (d, H(6)in
uracil, J = 8 Hz). HRMS (ESI) calcd for C20H31N6O2
[M+H]+: m/z = 387.2503. Found: 387.2516.
R
R
Boc
Boc
Boc
Boc
Boc
H
N
O
O
Br-R-Br
Br
N
N
N
N
N
N
N
N
N
N
N
N
2
uracil
NH
Na2CO3/CH3CN
NaH/DMF
Boc
Boc
Boc
Boc
3
4
1
R
R
N
O
O
H
N
N
O
N
Zn(ClO4)2
NaOH
N
NH
NH
40%HBr
ethanol
NH
NH
+
Zn2
O
HN
N
N
H
H
5
6
6a: R=
6b: R=
Scheme 1. The synthetic route of uracil–cyclen conjugates and their Zn(II) complexes.
12
10
a
R
H
b
N
N
N
N
O
8
Zn2+
NH
N
O
H
H
6
4
2
0
6
R =
a:
R =
b:
0
50
100
150
200
250
300
Eq (OH-)
Figure 1. Typical pH titration curves of (a) 0.286 mM 6a, (b) 0.286 mM 6b. Conditions: 25 °C, I = 0.1 (NaNO3), where Eq (OHꢀ) is the number of
equivalents of base added.

C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
5759
3. Results and discussion
plasmid (Form I) to open-circular form (Form II) and
linear form (Form III). The amounts of the strands were
measured by agarose gel electrophoresis.
3.1. Synthesis of the uracil–cyclen conjugates and their
Zn(II) complexes
First, we choose complex 6a to optimize the conditions
of DNA cleavage. Figure 2A–D shows the effects of
pH value, vitamin C (Vc) concentration, complex
The synthetic routes of target uracil–cyclen compounds
and their Zn(II) complexes are shown in Scheme 1. The
uracil–cyclen conjugate ligands 4a and 4b were synthe-
sized via the reaction of uracil and 1-bromomethylben-
zyl-4,7,10-tris(tert-butyloxycarbonyl)-1,4,7,10-tetraaz-
acyclododecane 3, which were obtained by reacting
1,4,7-tris(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclod-
odecane (Boc3-cyclen) 1 with dibromomethyl (1,4- for a
and 1,3- for b) benzene 2. The Boc-protective groups
were moved by adding 40% HBr aqueous solution. The
cyclen–uracil Zn(II) complexes 6a and 6b were prepared
through the reaction of 5 with zinc perchlorate.19 The
1
2
3
4
5
6
7
A
B
Form II
Form I
Form II
Form I
1
new compounds 4–6 were confirmed by H NMR and
HRMS analysis.
C
D
3.2. Potentiometric studies of 6a and 6b
Form II
Form I
The protonation was determined by potentiometric pH
titration against 0.1 M NaOH with I = 0.10 (NaNO3)
at 25 °C. A typical pH titration curve is shown in
Figure 1, which gave three inflections for complexes 6a
and 6b. The first inflection can be assigned to the depro-
tonation of the three protons at 0 < pH <3, the second
pH is the deprotonation of Zn(II)-bound waters at pH
7.8; the third is the deprotonation of Nꢀ(3) on uracil
in complexes 6a and 6b at pH 9.5 (just as Scheme 2).
Form II
Form I
Figure 2. Agarose gel electrophoresis of cleavage reaction of pUC 19
(DNA) (7 lg/mL) in Tris–HCl buffer (100 mM, pH 8.0) at 37 °C. (A)
By different pH value. Scission condition: [6a] = 5.74 lM, time = 3 h,
[Vc] = 0.05 mM. Lane 1: DNA control; Lanes 2–7: pH 6.0, 6.5, 7.0, 7.5,
8.0, and 9.0. (B) By different Vc concentrations. Scission condition:
[6a] = 5.74 lM, time = 3 h. Lane 1: DNA control; lanes 2–6: [Vc] = 0.7,
0.3, 0.1, 0.05, 0.025 mM. (C) By different concentration of complex 6a.
Scission condition: time = 3 h, [Vc] = 0.05 mM. Lane 1: DNA control;
lanes 2–6: [6a] = 0.82, 2.45, 4.10, 5.74, and 9.02 lM. (D) By different
cleavage time. Scission condition: [6a] = 5.74 lM, [Vc] = 0.05 mM.
Lane 1: DNA control; lanes 2–7: time = 10, 30, 60, 120, and 180 min.
3.3. Interaction between complexes 6 and plasmid DNA
The activities of complexes 6a and 6b with pUC 19
supercoiled DNA were studied. Plasmid pUC 19 DNA
was used as substrate. The DNA cleavage abilities of
Zn(II) complexes were initially studied by monitoring
the conditions of catalytic transforming of supercoiled
R
H
R
R
H
+
N
N
N
O
O
pH=2-3
N
N
H
N
N
O
O
O
H+
NH
NH
NH
H+
N
N
N
H
H
H
H
H
R
H
N
N
N
N
O
N
N
N
O
pH=7.8
Zn2+
Zn2+
NH
N
N
N
O
H
H
H
H
H2O
+
H O
H
2
R
R
H
H
N
N
N
N
N
N
N
O
N
O
pH=9.5
Zn2+
Zn2+
NH
N-
N
N
O
O
H
H
H
H
H2O
R =
a:
b: R =
H2O
Scheme 2. Deprotonation of complexes 6.

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C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
concentration, and cleavage time, respectively. Cleavage
of DNA could be processed under physiological
conditions by using very low complex concentration
(at lM grade, Fig. 2C) in short time (Fig. 2D) associated
with high selectivity (Form III did not appear). Figure 3
shows the quantitation of % plasmid relaxation relative
to plasmid DNA per lane in Figure 2. It is obvious that
Form II increased after DNA cleavage associates with
the increasing of the concentration of Vc or complex.
But interestingly, when we enhanced the complex con-
centration from 5.74 lM to 9.02 lM, the amount of
Form II reduced sharply. This may be due to that Form
I might be more liable to be cleaved to fragment (Form
III) under high concentration of complex 6a. In the
DNA cleavage, Vc was used as reducing agent to prevent
the oxidation caused by oxygen in air. Figure 2B shows
that without the use of Vc, DNA could not be cleaved
smoothly. According to the results, the DNA cleavage
could be processed under the optimized conditions as
below: pH 8.0, [complex] = 4.10 lM, reaction
time = 30 min, [Vc] = 0.05 mM, and using Tris–HCl as
buffer.
Then both of the complexes 6a and 6b were applied to
the DNA cleavage. Figure 4 shows that the Zn(II) ura-
cil–cyclen complexes 6a and 6b as chemical nucleases
can catalyze the cleavage of plasmid DNA (pUC19)
more efficiently than the zinc–cyclen complex (lanes
2–4). The results displayed that cyclen–Zn(II) at the
same concentration (5.74 lM) did not result in any
detectable hydrolysis, but efficient hydrolysis of plasmid
DNA in the presence of 6a and 6b was observed. Elec-
trophoresis and densitometry results indicated that sin-
gle cleavage of the supercoiled form yielded 43.6%,
78.9% nicked form by 6a and 6b, respectively
(Fig. 4b). In contrast, hydrolysis of supercoiled form
in the presence of cyclen–Zn(II) could only give 24%
yield (equal to the DNA control). Compound 6b is bet-
ter catalyst for DNA cleavage than 6a. The data sug-
gested that the structures of the complexes, in
particular the nature of bridging groups between cyclen
and uracil, play an important role in the cleavage reac-
tion. Additionally, 6a and 6b could cleave DNA
(pUC19) into Form II without the use of Vc, the yields
are almost equal to the ones of the experiments using
a
b
50
40
30
20
10
0
50
40
30
20
10
0
2
3
4
5
6
7
2
3
4
5
6
lane
lane
pH effect
Vc concentration Effect
c
d
30
25
20
30
25
20
15
10
5
15
10
5
0
0
2
3
4
5
6
2
3
4
5
6
lane
lane
Effect of concentration of complex 6a
Time Effect
Figure 3. Quantitation of % plasmid relaxation relative to plasmid DNA per lane in Figure 2.

C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
5761
a
1
2
3
4
5
6
7
8
9
10
Form II
Form I
b
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
Lane
Figure 4. (a) Agarose gel electrophoresis of cleavage reaction of pUC 19 DNA (7 lg/mL) in Tris–HCl buffer (100 mM, pH 8.0) at 37 °C for 3 h. Lane
1: DNA control; lane 2: 6b + Vc; lane 3: 6a + Vc; lane 4: cyclen–Zn2+ + Vc; lane 5: Vc; lane 6: 6b + Vc (parallel experiment); lane 7: 6a + Vc (parallel
experiment); lane 8: cyclen–Zn2+ + Vc (parallel experiment); lane 9: only 6b; lane 10: only 6a. (b) Quantitation of % plasmid relaxation relative to
plasmid DNA per lane.
a
1
2
3
4
5
6
Form II
Form I
b
70
60
50
40
30
20
10
0
1
2
3
4
5
6
lane
Figure 5. Effect of concentration of complex 6b on the cleavage reaction of pUC 19 (DNA) (7 lg/mL) in Tris–HCl buffer (100 mM, pH 8.0) at 37 °C
for 3 h. (a) Agarose gel electrophoresis diagram: lane 1: DNA control; lanes 2–6: [6b] = 0.82, 2.45, 4.10, 5.74, and 9.02 lM. (b) Quantitation of %
plasmid relaxation relative to plasmid DNA per lane.

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C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
Vc as reductant (lanes 9 and 10). Our subsequent efforts
focused on the reactivity of complex 6b.
III) within 30 min (lane 6) when we increased the con-
centration of 6b to 9.02 lM. For all experiments using
6b as catalyst, the conversion of Form I was higher than
the reactions under same conditions catalyzed by 6a.
The cleavage of DNA by different concentrations of
complex 6b was also studied (Fig. 5). The amount of
nicked DNA (Form II) observed in agarose gel electro-
phoresis diagram increased associated with the increase
of the concentration of complex 6b in the reaction sys-
tem. Increasing the concentration of 6b in the order of
0.82, 2.45, 4.10, 5.74, 9.02 lM, the nicked DNA (Form
II) was obtained with 9.92%, 26.93%, 39.36%, 44.82%,
58.03%, and 70.34% yields, respectively (Fig. 5b). Like
6a, Form I can be efficiently cleaved to fragment (Form
3.4. Mass study of 6a and 6b
ESI-mass (positive) spectra of 6a and 6b were studied to
analyze the detail structures of the complexes. As shown
above, in the condition of pH 8.0, the cleavage effect of 6
is the highest, so we choose pH 8.0 as ESI-mass experi-
ment condition. Uracil, which has an imide part, can
give lone pair electron on N atom and combine to
a
449.0
×104
6
4
2
999.0
337.1
0
200
400
600
800
1000
c
448.0
b
999.0
×104
451.0
1250
6
4
2
0
1001.0
1000
750
500
250
0
450.0
997.0
1003.0
1000.0
452.0
998.0
1002.0
1004.0
450.5
453.0
451.5
1005.0
448.5
1007.0
452.5
454.0
453.5
447.0
455.0
992.5
995.0
997.5 1000.0 1002.5 1005.0 1007.5 1010.0
101
N
O
O
N
O
O
N
O
O
N
N-
N
H
N
+
N
H
N
+
N
Zn2
Zn2
H
N
N
N
N
-
H
H
ClO4
N
N
Zn2+
H
H
N
N
H
H
B
A
Figure 6. ESI-mass spectra of 6b. (a) Full mass spectra; (b) M+; (c) 2Mþ þ ClO4
.
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C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
5763
N
O
O
N
O
O
N
N
O
O
N
N
N
O
O
N
H
N
+
N
N
H
N
+
N
Zn2
H
N
Zn2 +
N
Zn2
H
N
N
N
H
H
N
N
Zn2 +
H
H
N
N
H
H
N
N
H
H
n
7
Figure 7. Supposed supramolecular structure assembled by 6b.
Figure 8. ESI-mass spectra of 6a. (a) Full mass spectra; (b) M+.
Zn(II). As results, compound A could be formed
through intramolecular recognition mode and com-
pound B could be formed by an intermolecular mode.
Figure 6 gives the ESI-mass spectra of 6b, m/e 449.0
and 999.0 indicate the mass of compounds A and B,
respectively. We supposed that a supramolecular com-
plex like 7 might be formed through the Zn(II)–uracil
interaction (Fig. 7).
clic polyamine Zn(II) complexes of 6a and 6b were also
prepared. The results of DNA cleavage show that these
Zn(II) complexes are able to accelerate the plasmid
DNA cleavage dramatically. The complex with 1,3-
phenylenedimethylene as bridge (6b) is better for DNA
cleavage than the one with 1, 4-phenylenedimethylene
as bridge (6a). In complex 6b, the metal ion in the
complex could bind to the nitrogen atom of the imide
of uracil group intramolecularly or intermolecularly,
which can form a unique reversible and stable supramo-
lecular complex in aqueous solution. The complex that
has this property displays higher activity in the DNA
cleavage process than the one without the supramolecu-
lar structure. ESI mass spectra were studied to testify the
special structure. From the MS of 6b, we can clearly find
the peak belongs to the dimer. As a result, cyclen–uracil
Zn(II) complexes as chemical nucleases are able to accel-
erate the plasmid DNA cleavage at low concentration
within shorter time, and because of the uracil part, some
interesting supramolecular structures might be formed
in water solution.
The existence of dimer or polynuclear complexes may
enhance the DNA cleavage ability. Figure 8 shows
the ESI-mass spectra of 6a. The peak at m/e = 451.3
indicates the mass of M+H+ of 6a. Unlike 6b, the
Zn(II) complex dimer or polymer of 6a cannot be ob-
served in the ESI-mass spectra. This may be due to
that in 6a, the spacer does not have a right angle to
form a supramolecular structure like 7, which might
be the reason for that the cleavage ability of 6b is much
better than that of 6a. The existence of polynuclear
Zn(II) complex in 6b stands a good chance to be favor-
able to cleave DNA.
4. Conclusion
Acknowledgments
In this paper, we first designed and synthesized the con-
jugate ligands of cyclen–uracil, and correlated macrocy-
This work was financially supported by the National
Natural Science Foundation of China (Grant Nos.:

5764
C.-Q. Xia et al. / Bioorg. Med. Chem. 14 (2006) 5756–5764
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20132020, 20372051, and 20471038), Program for New
Century Excellent Talents in University, Specialized
Research Fund for the Doctoral Program of Higher
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