Synthesis and DNA cleavage activities of mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes which linked with uracil

Article abstract of DOI:10.1016/j.bmc.2006.05.049

Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid (PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and ¹

Full text of DOI:10.1016/j.bmc.2006.05.049

Bioorganic & Medicinal Chemistry 14 (2006) 6745–6751
Synthesis and DNA cleavage activities of mononuclear
macrocyclic polyamine zinc(II), copper(II), cobalt(II)
complexes which linked with uracil
Xiao-Yan Wang,a Ji Zhang,a Kun Li,a Ning Jiang,b Shan-Yong Chen,a
Hong-Hui Lin,b,* Yu Huang,a Li-Jian Maa 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 14 April 2006; revised 25 May 2006; accepted 25 May 2006
Available online 22 June 2006
Abstract—Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid
(PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and 1H
NMR spectroscopy. The catalytic activities on DNA cleavage of these mononuclear complexes with different central metals were sub-
sequently studied, which showed that copper complex was better catalyst in the DNA cleavage process than zinc and cobalt complexes.
The effects of reaction time, concentration of complexes were also investigated. The results indicated that the copper(II) complexes
could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selec-
tively nicked DNA (Form II, no Form III produced) with high yields. The mechanism of the cleavage process was also studied.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
can produce both cationic and anionic complexes. The
most widely studied ligand is 1,4,7,10-tetraazacyclod-
odecane (cyclen), which has strong coordination ability
toward a wide range of cations. Many cyclen complexes
as chemical nucleases have been used in DNA recogni-
tion and cleavage.14–22 So far the metal complex of
cyclen is one kind of the most effective synthetic cata-
lysts for DNA cleavage. We have synthesized several
metal compounds containing cyclen unit and studied
their catalytic abilities on DNA cleavage.23–26 More
recently, we reported the first synthesis of chiral peptide
nucleic acid (PNA) monomer–cyclen conjugates.27
There are many reports using the oligoPNA as recogni-
tion motif.28,29 However, to the best of our knowledge,
PNA monomer has not been reported in the literature.
In these PNA monomer–cyclen conjugates, uridine
could recognize adenine in the strand of DNA, and
the cyclen moiety has high affinity with DNA. Hence
we considered that these conjugates could be good cata-
lysts in DNA cleavage. This prompted us to do more
in-depth studies on the cleavage of DNA catalyzed by
these kinds of complexes. Considering the p–p stacking
effect will be helpful, we introduced phenyl group in the
DNA is naturally stable polymer with an estimated half-
life spontaneous hydrolysis of ꢀ130,000 years under
physiological conditions.1 The main hindrance in
DNA hydrolysis is the large negative charge that inhib-
its the attack of nucleophile to the DNA backbone.
Studies on DNA hydrolysis agents are stimulated by
the understanding of their potential application in bio-
technology and as therapeutic agents.2 Many groups
reported different complexes,3–13 which could accelerate
the cleavage of DNA.8 However, the catalytic efficiency
of currently available chemical nucleases cannot be men-
tioned in the same breath as that of natural enzyme.
Recently, many polyamine macrocyclic ligands were
studied because of their special properties. These ligands
Keywords: 1,4,7,10-Tetraazacyclododecane (cyclen); Macrocyclic poly-
amine; Copper complexes; pUC19 DNA; Cleavage of DNA.
*
Corresponding authors. Tel./fax: +86 28 8541 5886; e-mail:
xqyu@tfol.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.049

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X.-Y. Wang et al. / Bioorg. Med. Chem. 14 (2006) 6745–6751
side chain.30 In this paper, we describe the synthesis and
characterization of noval PNA–cyclen metal complexes
and their applications in the DNA cleavage. The result
shows that the complexes can catalyze the DNA cleav-
age efficiently.
Cu, Co complexes 7a, 7b, and 7c in 68–76% yield as
white, blue, and brown solid, respectively. These
complexes were characterized by NMR, ESI-MS, and
HRMS.
2.2. Cleavage of plasmid DNA
2. Results and discussion
The cleavage activities of complexes 7a–c with pUC 19
supercoiled DNA were studied. The cleavage of the
supercoiled plasmid DNA (Form I) was processed under
physiological conditions to produce open-circular form
(Form II) selectively. The amounts of strand scission
were assessed by agarose gel electrophoresis.
2.1. Preparation of the PNA–cyclen metal complexes
Scheme 1 shows the synthetic route of novel PNA–
cyclen metal complexes 7. N-tert-Butoxycarbonyl-
thyminyl-cysteine 2 was prepared from 5-hydroxymeth-
yluracil according to the literature procedure.27
Compound 2 was used in a peptide coupling reaction
with L-phenylalanine methyl ester, best results were
achieved by using N-methyl morphine (NMM) and
i-C4H9OCOCl as acyl reagent. Deprotection of the
methyl ester by NaOH and subsequent acidification
gives 4. Then the tri-Boc-protected cyclen was
introduced by using i-C4H9OCOCl as acyl reagent to
form 5. The hydrobromide salts of compound 6 were
obtained by deprotection of 5 with 47.5% HBr in EtOH
at low temperature. The salt was converted to the free
based by adjusting pH to 8–9. The free ligand was
allowed to react with Zn(ClO4)2Æ6H2O, Cu(NO3)2, or
Co(NO3)2 in EtOH overnight to give the target Zn,
First, we compared the cleavage abilities of the PNA–
cyclen metal complexes with different central metals.
Figure 1 shows the results. It is obvious that Cu(II) com-
plex (7b, lane 3) catalyzed the cleavage of plasmid DNA
(pUC 19) much more efficiently than the Zn(II) (7a, lane
2) and Co(II) complex (7c, lane 4) under physiological
conditions. Electrophoresis and densitometry indicated
that single cleavage of the supercoiled form yielded
23%, 87%, and 31% nicked form, respectively, by 7a–c
(Fig. 1B). It is obvious that copper may provide addi-
tional and critical elements for the catalytic process.
Although Zn2+, Cu2+, and Co2+ have almost the
same charge density (2.7, 2.8, and 2.8 Aꢁ1), Cu2+ is a
˚
stronger Lewis acid based on their respective ionization
H
H
N
H
N
O
N
O
O
NH
NH
NH
O
S
O
O
(Boc) O
2
S
S
HClH N
COOCH
2
3
96%
COOCH
NH
3
OH
NMM, i-C H OCOCl
4
9
OH
BocHN
C
C
HClH N
2
90%
C
O
BocHN
O
O
3
1
2
H
N
H
O
O
N
NH
NH
O
1) NaOH
2) HCl
O
Boc
N
S
S
3Boc-Cyclen
i-C H OCOCl
O
C
98%
COOH
NH
NH
4
9
N
N
Boc
BocHN
BocHN
C
O
C
65%
N
O
Boc
4
5
H
N
H
N
O
O
NH
NH
.
Zn(ClO ) 6H O
4 2
2
O
S
O
or Cu(NO )
3 2
H
S
H
N
47.5%HBr
77%
or Co(NO )
O
3 2
O
C
H
N
H
N
N
C
pH=8~9
C
O
N
H2N
N
H
H2N
C
N
N
H
N
H
O
N
H
7a
=Zn2+
=Cu2+
=Co2+
¡¤4HBr
7b
7c
7
6
Scheme 1. Synthetic route of novel PNA–cyclen metal complexes 7.

X.-Y. Wang et al. / Bioorg. Med. Chem. 14 (2006) 6745–6751
6747
B
80
A
1
2
3
4
60
40
20
0
Form II
Form I
1
2
3
4
Figure 1. Effect of different complexes 7a–c (0.143 mM) on the cleavage reactions of pUC 19 DNA (7 lg/mL) in a NaH2PO4/Na2HPO4 buffer
(100 mM, pH 7.4) at 37 ꢁC for 48 h. (A) Agarose gel electrophoresis diagram: lane 1, DNA control; lane 2, 7a; lane 3, 7b; lane 4, 7c. (B) Quantitation
of % plasmid relaxation relative to plasmid DNA per lane.
potentials (ICu2+ = 20.29 eV, IZn2+ = 17.96 eV, and ICo2+
=
II. An increase in the intensity of Form II was observed
associated with the increase of reaction time. 69%, 90%
nicked form (Form II) was observed when the reaction
time is 12 and 48 h, respectively (Fig. 2B).
17.06 eV).31 The Lewis acidity of Cu2+ might activate
phosphodiester bond toward nucleophilic attack via
charge neutralization, and, at the same time, might sig-
nificantly lower the pKa of coordinated water mole-
cule.32–34 Our subsequent efforts focused on the
reactivity of complex 7b under physiological conditions.
The cleavage of DNA by different concentrations of
complex 7b was also studied (Fig. 3). The amount of
nicked DNA (Form II) observed in agarose gel electro-
phoresis diagram increased in accord with the change
trend of the concentration of complex 7b in the reaction
A solution containing plasmid DNA was incubated in a
0.5 mL tube with catalyst 7b at 37 ꢁC, pH 7.0, as shown
in Figure 2, the disappearance of supercoiled Form I
was accompanied by appearance of open-circular Form
A
1
2
3
4
5
6
7
8
Form II
Form I
B
100
80
60
40
20
0
1
2
3
4
5
6
7
8
Figure 3. Effect of concentration of complex 7b on the cleavage
reactions of pUC 19 DNA (7 lg/mL) in a NaH2PO4/Na2HPO4 buffer
(100 mM, pH 7.0) at 37 ꢁC for 24 h. (A) Agarose gel electrophoresis
diagram: lane 1, DNA control 24 h; lane 2, DNA control 0 h; lane 3,
[7b] = 14.3 lM; lane 4, [7b] = 28.6 lM; lane 5, [7b] = 0.072 mM; lane 6,
[7b] = 0.143 mM; lane 7, [7b] = 0.286 mM; lane 8, [7b] = 0.572 mM. (B)
Quantitation of % plasmid relaxation relative to plasmid DNA per
lane.
Figure 2. Effect of reaction time on the cleavage reaction of pUC 19
DNA (7 lg/mL) with complex 7b (0.143 mM) in NaH2PO4/Na2HPO4
buffer (100 mM, pH 7.0) at 37 ꢁC. (A) Agarose gel electrophoresis
diagram: lane 1, DNA control, 48 h; lane 2, DNA control, 0 h; lane 3,
2 h; lane 4, 4 h; lane 5, 8 h; lane 6, 12 h; lane 7, 24 h; lane 8, 48 h. (B)
Quantitation of % plasmid relaxation relative to plasmid DNA per
lane.

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X.-Y. Wang et al. / Bioorg. Med. Chem. 14 (2006) 6745–6751
More in-depth studies about the mechanism are under-
way in our laboratory.
3. Experimental
3.1. General
1
2
3
4
5
6
7
Figure 4. Effect of radical scavengers on the cleavage reaction of pUC
19 DNA (7 lg/mL) with complex 7b (0.143 mM) in NaH2PO4/
Na2HPO4 buffer (100 mM, pH 7.0) at 37 ꢁC for 24 h. Agarose gel
electrophoresis diagram: lane 1, DNA control, 24 h; lane 2, DNA
control, 0 h; lane 3, 7b; lane 4, 7b + DMSO (1.43 mM); lane 5,
7b + methol (1.43 mM); lane 6, 7b + glycerol (1.43 mM); lane 7,
7b+NaN3 (1.43 mM).
ESI-MS and HRMS spectral data were recorded on a
Finnigan LCQDECA and a Bruker Daltonics Bio
TOF mass spectrometer, respectively. The 1H NMR
spectra were measured on a Varian INOVA-400 spec-
trometer and the d scale in ppm was referenced to resid-
ual solvent peaks or internal tetramethylsilane (TMS).
Polarimetric measurements were taken on a Perkin-El-
mer-341 automatic polarimeter. UV–vis spectra were
measured on a TU-1901 spectrophotometer. Melting
points were determined using a micro-melting point
apparatus and are uncorrected. DNA Gel Extraction
Kit was purchased from V-gene Biotechnology limited.
5-Hydroxymethyluracil37 and 1,4,7-tris(tert-butyloxy-
system (Fig. 4A). Decreasing the concentration of 7b in
the order of 0.072 mM, 28.6 lM, and 14.3 lM results in
79%, 34%, and 25% nicked DNA, respectively. While
increasing the concentration of 7b to 0.572 mM, almost
100% nicked DNA was observed after 24 h (Fig. 3B).
carbonyl)-1,4,7,10-tetraazacyclododecane
(3Boc-cy-
clen)38 were prepared according to the literature. All
other chemicals and reagents were obtained commercial-
ly and were used without further purification.
The mechanism of the DNA cleavage catalyzed by 7b
was studied. Hydroxyl radical scavengers (1.43 mM
DMSO, methol, glycerol) and oxygen scavenger
(1.43 mM NaN3) were introduced to the system. The
free radical scavengers do not present any observable
inhibition on the DNA cleavage reaction (Fig. 4). There-
fore, DNA cleavage promoted by 7b might occur
through a hydrolytic pathway instead of an oxidative
pathway.
3.2. Preparation of S-thyminyl-L-cysteine hydrochloride 1
A
solution of L-cysteine hydrochloride (0.852 g,
6.00 mmol) and 5-hydroxymethyluracil (0.942 g,
6.00 mmol) in 2 N HCl (30 mL) was stirred at 50 ꢁC
for 48 h. After cooling, filtering, and evaporating the
solvent under reduced pressure, the S-thyminyl-L-cys-
The enzymatic religation of the nicked plasmid DNA
with T4 DNA ligase was also studied, but we were un-
able to confirm any religation products. We tried to
use TBARS-method35 to detect hydroxyl radical, and
no 532 nm absorbance was observed. It indicated that
no hydroxyl radical emerged in this process.
teine hydrochloride (1.59 g) was obtained as a white
20
D
powder. Yield: 94%, mp 238–240 ꢁC; ½aꢂ þ 9:8
1
(c 1.0, H2O); H NMR (400 MHz, D2O, TMS) d: 7.64
(s, 1H, uracil-6-CH), 4.36–4.33 (m, 1H, NH2CH),
3.59 (s, 2H, S–CH2–uracil), 3.28–3.23 (m, 1H,
NCHCH2S), 3.15–3.09 (m, 1H, NCHCH2S); ESI-MS:
m/z = 244.0 [Mꢁ1ꢁHCl]; HRMS (ESI) calcd for
In the Cu(II) complex 7b, the uracil moiety in the PNA
benefits the binding of the ligand to DNA, while the
introduction of phenyl group in the side chain may open
a way to stabilize the pretransition state. The role of
copper ions in the cleavage of DNA may be multi-facet-
ed, including the orientation of both the substrate and
the water molecule, which acts as a nucleophile, and
the activation of the water molecule via coordination.
According to the experiment results, 7b is an efficient
catalyst, which could accelerate the DNA cleavage pro-
cess under physiological conditions to produce selective-
ly nicked DNA (Form II, no Form III produced) with
high yields.
C8H11N3O4SNa
Found: 268.0355.
[M+NaꢁHCl]+:
m/z = 268.0362.
3.3. Preparation of N-tert-butoxycarbonyl-S-thyminyl-L-
cysteine (2)
To an aqueous solution of sodium hydrogen carbonate
(1.68 g, 20.0 mmol) was added the solution of S-thymi-
nyl-L-cysteine hydrochloride (1.12 g, 4.00 mmol) in 1,4-
dioxane (60 mL). The solution was then cooled to 0 ꢁC
with an ice bath. Di-tert-butyl carbonate (2.62 g,
12.0 mmol) which was dissolved in 1,4-dioxane
(20 mL) was added dropwise. After being kept at this
temperature for another 20 h, the solution was allowed
to warm to room temperature. The reaction mixture
was then concentrated under reduced pressure and the
pH was adjusted to 3–4 with 1 N KHSO4. After cooling
and filtering, compound 2 (1.324 g) was obtained as a
It has been reported that for mononuclear metal com-
plexes, two water molecules coordinating to central met-
al are needed to achieve satisfying catalytic result of
DNA cleavage.36 However, copper(II) could provide
only five-coordination structure, which means that in
complex 7b, the central metal can coordinate to only
one water molecule. Therefore, it is interesting that
mononuclear Cu(II) complex 7b shows considerably
high catalytic activity in the DNA cleavage process.
white powder. Yield: 96%, mp 118–119 ꢁC;
20
½aꢂ þ 11:1 (c 1.0, CH3OH); 1H NMR (400 MHz,
D
DMSO) d: 12.82 (s, 1H, COOH), 11.14 (s,1H, uracil-1-
NH), 10.85 (s, 1H, uracil-3-NH), 7.41 (s, 1H, uracil-6-
CH), 6.92 (s, 1H, OCONH), 4.04–3.99 (m, 1H, Boc-

X.-Y. Wang et al. / Bioorg. Med. Chem. 14 (2006) 6745–6751
6749
NHCH), 2.89–2.79 (m, 2H, S–CH2–uracil), 2.73–2.65
(m, 2H, NCHCH2S), 1.38 (s, 9H, C(CH3)3); ESI-MS:
m/z = 344.4 [Mꢁ1]ꢁ; HRMS (ESI) calcd for
C13H19N3O6SNa [M+Na]+: m/z = 368.0887. Found:
368.0889.
1.10 mmol) and i-C4H9OCOCl (0.134 mL, 1.00 mmol)
were added at about ꢁ15 ꢁC. After 10 min, a solution
of 3Boc-cyclen (0.472 g, 1.00 mmol) in anhydrous
THF (30 mL) was poured into the reaction mixture.
The mixture was stirred for another 0.5 h at ꢁ15 ꢁC
and left overnight at room temperature. The mixture
was then evaporated under reduced pressure to remove
the solvent. The solid residue was dissolved in ethyl ace-
tate and washed with saturated NaHCO3, saturated
brine, and 2 N citric acid, the organic layer was dried
with anhydrous Na2SO4. After the solvent was evapo-
rated, the residue was purified by column chromatogra-
phy on silica gel (eluent: ethyl acetate/petroleum ether/
acetone = 1:1:1, v/v/v) to afford compounds 5 (0.615 g)
3.4. Preparation of compound 3
To a solution of N-tert-butyloxycarbonyl-S-thyminyl-L-
cysteine 2 (1.03 g, 3.00 mmol) in anhydrous tetrahydro-
furan (THF, 40 mL) was added N-methyl morphine
(NMM) (0.390 mL, 3.30 mmol) and i-C4H9OCOCl
(0.402 mL, 3.00 mmol) sequentially at about ꢁ15 ꢁC.
After stirring for 10 min, a solution of L-phenylalanine
methyl ester (3.30 mmol) and NMM (0.390 mL,
3.30 mmol) in anhydrous THF (60 mL) was poured into
the reaction mixture. The mixture was stirred for anoth-
er 0.5 h at ꢁ15 ꢁC and left overnight at room tempera-
ture. After the solvent was evaporated under reduced
pressure, the solid residue was dissolved in ethyl acetate
and washed with saturated NaHCO3, saturated brine
and 2 N citric acid, the organic layer was dried with
anhydrous Na2SO4. After the solvent was removed,
the residue was purified by recrystallization from
acetone and ethyl ether. Compound 3 (1.366 g) was
obtained as a white powder. Yield: 90%, mp 210–
1
as a white solid. Yield: 65%, mp 141–143 ꢁC; H NMR
(400 MHz, CDCl3) d: 10.28 (s, 1H, uracil-1-NH), 9.88
(s, 1H, uracil-3-NH), 7.75 (s, 1H, CONH), 7.57 (s, 1H,
uracil-6-CH), 7.37–7.24 (m, 5H, Ph-H), 5.59 (s, 1H,
OCONH), 5.10–5.05 (m, 1H, CHCON), 4.54–4.50 (m,
1H, Boc-NHCH), 3.57–3.02 (m, 18H, Ph-CH2, cyclen–
CH2), 2.81–2.79 (m, 2H, S–CH2–uracil), 2.73–2.68 (m,
2H, NCHCH2S), 1.50–1.41 (m, 36H, Boc-H); ESI-MS:
m/z = 970.1 [M+Na]+.
3.7. Preparation of compound 6
1
212 ꢁC; H NMR (400 MHz, DMSO) d: 11.17 (s, 1H,
To a solution of compound 5 (0.500 mmol) in ethanol
(10 mL), 47.5% aqueous HBr (5 mL) was added drop-
wise. After being stirred at room temperature overnight,
the reaction mixture was concentrated under reduced
pressure to give the crude product, which was crystal-
lized from ethanol/24% aqueous HBr to afford
compound 6 (0.333 g) as a white powder. Yield: 77%,
uracil-1-NH), 10.85 (d, 1H, J = 4.0 Hz, uracil-3-NH),
8.29 (d, 1H, J = 7.6 Hz, CONH), 7.40 (d, 1H,
J = 5.6 Hz, uracil-6-CH), 7.28–7.18 (m, 5H, Ph-H),
6.92 (d, 1H, J = 8.4 Hz, OCONH), 4.49–4.44 (m, 1H,
CHCOOCH3), 4.15–4.09 (m, 1H, Boc-NHCH), 3.57 (s,
3H, OCH3), 3.30–3.27 (m, 2H, Ph-CH2), 3.05–2.91 (m,
2H, S–CH2–uracil), 2.66–2.49 (m, 2H, NCHCH2S),
1.37 (s, 9H, C(CH3)3); ESI-MS: m/z = 505.1 [Mꢁ1]ꢁ.
1
mp 239–241 ꢁC; H NMR (400 MHz, D2O) d: 7.63 (s,
1H, uracil-6-CH), 7.35–7.22 (m, 5H, Ph-H), 4.47–4.44
(m, 1H, CHCON), 4.30–4.24 (m, 1H, NH2CH), 3.65–
3.06 (m, 18H, Ph-CH2, cyclen–CH2), 3.01–2.95 (m,
2H, S–CH2–uracil), 2.92–2.87 (m, 2H, NCHCH2S);
ESI-MS: m/z = 547.6 [Mꢁ4HBr+1]+.
3.5. Preparation of compound 4
2N Aqueous sodium hydroxide (5 mL, 10 mmol) was
added dropwise to a suspension of compound 3
(2.00 mmol) in methanol (20 mL) at 0 ꢁC. The mixture
was stirred for 2 h at room temperature and the pH
was adjusted to 7 with 1 N HCl. After removing most
of the methanol, the pH was adjusted to about 2. The
mixture was then extracted with ethyl acetate (3·
30 mL) and the organic layer was dried with anhydrous
Na2SO4. After the solvent was evaporated under
reduced pressure, compound 4 (0.964 g) was obtained
3.8. Preparation of PNA–cyclen metal complexes (7)
To an aqueous solution (5 mL) of compound 6 (0.304 g,
0.350 mmol), an aqueous solution (5 mL) of
Zn(ClO4)2Æ6H2O (0.134 g, 0.360 mmol) was added
slowly. The pH of the solution was adjusted to 8–9
with 1 N NaOH. After being stirred overnight, the
reaction mixture was concentrated under reduced pres-
sure. The obtained residue was crystallized from water
to afford zinc(II) complex 7a (0.215 g) as a white sol-
1
as a white solid. Yield: 98%, mp 211–212 ꢁC; H NMR
(400 MHz, DMSO) d: 12.80 (s, 1H, COOH), 11.17 (s,
1H, uracil-1-NH), 10.84 (d, 1H, J = 5.6 Hz, uracil-3-
NH), 8.05 (d, 1H, J = 7.6 Hz, CONH), 7.46 (d, 1H,
J = 8.4 Hz, uracil-6-CH), 7.39–7.19 (m, 5H, Ph-H),
6.94 (d, 1H, J = 8.4 Hz, OCONH), 4.47–4.40 (m, 1H,
CHCOOH), 4.16–4.12 (m, 1H, Boc-NHCH), 3.31–3.27
(m, 2H, Ph-CH2), 2.56–2.54 (m, 2H, S–CH2–uracil),
2.52–2.45 (m, 2H, NCHCH2S), 1.37 (s, 9H, C(CH3)3);
ESI-MS: m/z = 515.5 [M+Na]+.
1
id. Yield: 76%, mp 391–393 ꢁC; H NMR (400 MHz,
D2O) d: 7.50–7.46 (m, 1H, uracil-6-CH), 7.13–7.05
(m, 5H, Ph-H), 4.18–4.15 (m, 1H, CHCON), 4.10–
4.06 (m, 1H, NH2CH), 3.49–2.88 (m, 18H, Ph-CH2,
cyclen–CH2), 2.82–2.75 (m, 2H, S–CH2–uracil), 2.70–
2.66 (m, 2H, NCHCH2S); ESI-MS: m/z = 609.6
[Mꢁ2ClO4]+.
To an aqueous solution (5 mL) of compound 6 (0.304 g,
0.350 mmol), 1 N NaOH was added to adjust pH 8–9.
Cu(NO3)2 (0.068 g, 0.360 mmol) was then added. The
reaction mixture was stirred overnight and blue precipi-
tate was formed. The reaction mixture was concentrated
3.6. Preparation of compound 5
To a solution of compound 4 (1.00 mmol) in anhydrous
THF (20 mL), N-methyl morphine (NMM) (0.130 mL,

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X.-Y. Wang et al. / Bioorg. Med. Chem. 14 (2006) 6745–6751
under reduced pressure. The precipitate was centrifuged
and washed by EtOH to afford 7b (0.174 g) as blue solid.
Yield: 68%, ESI-MS: m/z = 608.2 [M+CuꢁH]+, HRMS
(ESI) calcd for C25H37N8O4SCu [M+CuꢁH]+:
m/z = 608.1949. Found: 608.1966.
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3.10. Procedure for DNA ligation experiments
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DNA Gel Extraction Kit. The plasmid was then incu-
bated for overnight at 16 ꢁC with 10 U of T4 ligase in
the buffer provided by the supplier.
3.11. TBARS-method
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contained the following reagents at the final concen-
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Na2HPO4/NaH2PO4 buffer (pH 7.0). The reaction
mixture was incubated at 37 ꢁC for 48 h. TBA (2 mL
of 1% (w/v)) was then added, plus 2 mL of 2.8% (w/
v) trichloroacetic acid. The whole was heated at
100 ꢁC, cooled, and the absorbance of 532 nm was
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much better in the DNA cleavage than the complexes
without aromatic side chain (serine and glycine were
studied). When using the complexes derived from serine
and glycine, very low conversions of substrate were
observed.
Acknowledgments
This work was financially supported by the National
Science Foundation of China (Nos. 20132020,
20372051, and 20471038), Program for New Century
Excellent Talents in University, Specialized Research
Fund for the Doctoral Program of Higher Education,
and Scientific Fund of Sichuan Province for outstanding
Young Scientist.
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