Effective homogeneous hydrolysis of phosphodiester and DNA cleavage by chitosan-copper complex

Article abstract of DOI:10.1002/cjoc.201190145

We aimed to explore the role of chitosan-based metal complexes in catalyzing the hydrolysis of phosphodiesters. To this end, we performed detailed studies on the kinetics of the chitosan copper complex (CSCu)-catalyzed hydrolysis of bis(4-nitrophenol) pho

Full text of DOI:10.1002/cjoc.201190145

FULL PAPER
Effective Homogeneous Hydrolysis of Phosphodiester and
DNA Cleavage by Chitosan-copper Complex
Zhang, Qia(张琦)
Xiang, Yan*,a,b(相艳)
Yang, Ruxinb(杨汝新)
Si, Jiangjua(司江菊)
Guo, Hongb(郭红)
a School of Materials Science and Engineering, Beihang University, Beijing 100191, China
b School of Chemistry and Environment, Beihang University, Beijing 100191, China
We aimed to explore the role of chitosan-based metal complexes in catalyzing the hydrolysis of phosphodiesters.
To this end, we performed detailed studies on the kinetics of the chitosan copper complex (CSCu)-catalyzed hy-
drolysis of bis(4-nitrophenol) phosphate (BNPP) in Tris-H＋ buffer and in an organic solvent. A significant en-
hancement in the rate of reaction (up to 3×105-fold acceleration) was observed at pH 8.0 (25 ℃). The pH depend-
ence of BNPP hydrolysis at pH 5.5—9.5 and the UV spectra revealed that the copper-bounded water molecules un-
derwent deprotonation to form the active catalytic species CSCu-OH. The kinetic behavior of BNPP catalytic hy-
drolysis in the Tris-H＋ buffer was consistent with that predicted by the Michaelis-Menten kinetics model. An in-
tramolecular nucleophilic attack by the copper-bonded hydroxide group on the same activated phosphodiester sub-
strate was proposed as the catalytic mechanism for CSCu-catalyzed reaction system. The results of DNA binding
and cleavage experiments indicated electrostatic binding mode of CSCu to DNA as well as the strong capability of
CSCu to disturb the supercoiled strand of DNA and cleave it to nicked circular form.
Keywords homogeneous catalysis, chitosan, copper, kinetics, phosphodiester, hydrolysis
Introduction
metal ions to polarize water molecules; these polarized
water molecules are more acidic than the free water
molecules, facilitating an attack on the phosphorus cen-
ter. These studies have identified the features of the
model compounds that contribute to faster phosphate
ester cleavage. In the model compounds, the ligand is
expected to provide a site for the metal ions and facili-
tate the catalytic reaction. As the results of previous
studies have indicated, metals usually combine with a
variety of macrocyclic and acyclic polydentate ligands.7
The metal center is expected to possess a site for coor-
dination with the phosphate ester and a water ligand,
which undergoes deprotonation to form a coordinated
hydroxide8 that acts as a nucleophile to attack the phos-
phate ester.
Chitosan, a natural polysaccharide, has been widely
used in biotechnological, pharmaceutical, as well as
enzyme immobilization procedures.9 However, the vi-
ability of using chitosan as a ligand for supporting a
hydrolytic catalyst has been rarely studied. Recently,
heterogeneous hydrolysis of phosphodiester by cop-
per-chelated chitosan magnetic nanocarrier was studied
by Dong-Hwang Chen.10 We previously reported cata-
lytic kinetic studies of N-cetyl-O-sulfated-chitosan cop-
per complex as an artificial hydrolase of phosphodi-
ester.11 However, the chitosan-based catalysts10,11 in
The phosphodiester bonds of DNA are exceptionally
resistant to hydrolysis under physiological conditions,
and it is extremely difficult to find new catalysts that
can accelerate the rate of hydrolysis. Mimicking the
activities of nucleases is an attractive research area in
molecular biology and therapy, since nucleases play an
essential role in the activity of artificial restriction en-
zymes and the conformational probes of nucleic acids.1
In the past decade, efforts to mimic the activities of
nucleases have led to the development of numerous
functional models for the hydrolysis of phosphodiester
bonds.2 The reaction mechanism of hydrolase and the
effect of metal ions on the active center are important
aspects in inorganic chemistry studies. Many recent
studies have sought to understand the mechanism of the
rate enhancement induced by these reagents. Most of
the model systems for phosphate-ester cleavage utilize
inner- or outer transition metal complexes. Indeed,
compounds containing metals from the transition series,
particularly Zn(II),3 Cu(II),4 and Co(III),5 and the lan-
thanide series6 have been used as model compounds.
Previous studies have shown that the observed en-
hancement in the rate of phosphate-ester hydrolysis can
be primarily attributed to the ability of the ligand-bound
* E-mail: xiangy@buaa.edu.cn; Tel./Fax: 0086-010-82339539
Received September 25, 2011; revised November 26, 2011; accepted December 13, 2011.
Project supported by the National Natural Science Foundation of China (Nos. 20403002, 20773008), the Beijing Novel Program (No. 2008B12) and
the National High-Tech R&D Program (No. 2007AA05Z146).
Chin. J. Chem. 2011, 29, 711— 718
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711

Zhang et al.
FULL PAPER
previous reports are water-insoluble and the catalytic
activity might be limited by the heterogeneous system.
In order to extend the potential of using chitosan as the
backbone of an artificial enzyme, we prepared wa-
ter-soluble chitosan-based copper complex (CSCu) and
investigated the kinetics of a series of hydrolytic reac-
tions of bis(4-nitrophenyl) phosphate (BNPP) that were
catalyzed by CSCu. Chitosan showed sufficient coordi-
nation with the copper ions; moreover, its molecular
chain may construct a suitable microenvironment for the
catalytic reaction. In this paper, we have analyzed the
result of kinetic studies and discussed a proposed hy-
drolytic mechanism for the catalytic reaction. The cop-
per ions in the catalytic system may be responsible for
the activation of the coordinated water molecule, which
is followed by a nucleophilic attack of the copper-
hydroxo complex on the substrate. We also identified
the fine ability of CSCu to DNA binding and cleavage
by UV-Vis scan and gel electrophoresis.
weight measurement of chitosan was Shimadzu LC-20A
Series Gel Permeation Chromatography system
equipped with a pump (LC-20-AD) and a refractive in-
dex (RI) detector (RID-10A). The analysis was per-
formed on a PL Gel column (Agilent) using water as the
mobile phase. The flow rate was 1.0 mL/min, and the
temperature was maintained at 40 ℃. The concentra-
tion of the sample used for the analysis was 2 mg/mL.
The average molecular weight of chitosan was tested to
be 6300 g/mol.
Fourier-transform infrared was used to characterize
the differences between chitosan and CSCu FTIR spec-
tra were obtained with a Perkin-Elmer FTIR 1600 series
spectrometer, and all the samples were prepared as po-
tassium bromide pellets. IR (－KBr) for chitosan v: 3415,
1
2920, 1630, 1520, 1018 cm . IR (KBr)－for CSCu v:
1
3435, 2920, 1630, 1510, 1020, 470 cm . XPS data
were recorded using a PHI 1600 XPS Surface Analysis
System (Physical Electronics, Eden Prairie,－MN). The
－8
7
samples were placed in vacuum (10 —10 Pa), and
the analyzed area was Ø 6 µm—Ø 6 mm. We used the
following scanning conditions: 6.80 ms/step, 0.6
eV/step, and 6 sweeps. The survey spectra were ob-
tained using an average of 10 scans with a pass energy
of 26.95 eV and running from 0 to 1100 eV. XPS data
for CSCu see Table 1.
Experimental
Materials
Chitosan powder [water-soluble, made in China by
Hai De Bei Co. Ltd.; average molecular weight deter-
mined by gel-permeation chromatography (GPC), 6300
g/mol] was used without further purification. The de-
gree of deacetylation of the amino groups was 75%; the
moisture content in the chitosan powder was 3% (w/w).
Bis(4-nitrophenyl) phosphate (BNPP), tris(hydroxy-
methyl)aminomethane (Tris-base) and calf thymus
DNA (CT DNA) were purchased from Sigma Aldrich.
Plasmid pUC19 DNA (0.5 µg/µL) and Loading buffer
for gel electrophoresis were purchased from Takara Co.
Ltd. GelRed (gene green) was purchased from Biotium.
Inc. Agarose was from Gene Tech Co. Ltd. Dialysis
membrane (molecular weight cut off: 3500) was from
Beijing Reida Henghui Tech. Co. Ltd. All reagents,
unless otherwise indicated, were of analytical g＋rade and
were used without further purification. Tris-H buffer
was used to avoid the influence of the chemical compo-
nents, and in each experimental run, the pH of the buffer
was adjusted by adding analytically pure hydrochloric
acid. The buffer system was not sensitive to the polarity
of solvent system, and its pKa value did not show any
significant changes.
Table 1 Binding energy of N, O and Cu for CS and CSCu
Sample
CS
N1s
O1s
531.4
531.6
—
Cu2p1/2
—
Cu2p3/2
—
399.2
399.7
—
400.8
401.6
—
CS—Cu
952.4
954.2
932.7
934.2
CuCl2•2H2O
The copper content in the complex was detected by
inductively coupled plasma-atomic emission spectros-
copy (ICP-AES) by using Intrepid II XSP with the fol-
lowing parameters: RF power, 1150 W; nebulizer flow,
26.0 PSI; and auxiliary air pressure, 1.0 LPM. The cop-
per content w of the chitosan copper complex was
2.82%.
Kinetics of BNPP hydrolysis
The pH of the reaction solutions was adjusted with
－1
Tris-H＋ buffer (0.01 mol•L ) and appropriate amounts
of sodium hydroxide stock solution. The chitosan cop-
per complexes were dissolved in the solutions, and the
pH value of the reaction solutions was measured at
room temperature (25 ℃). The samples were prepared
by using the procedure described in a previous section
and incubated at (25.0±0.2) ℃ prior to the analysis.
Preparation of chitosan copper complex
A specific amount of chitosan was dissolved in wa-
ter. Then, copper chloride solution was added to the
reaction vessel. The mixed solution was stirred con-
tinuously for 4 h at 50 ℃; the solution was then dia-
lyzed for 48 h to remove any unreacted copper ions. The
dialyzate was freezed and vacuum dried to obtain the
multinuclear chitosan copper complex.
The initial substrate concentration was 0.05—1 mmol•
－ 1
L
. The ligand and metal-ion concentration varied
－1
from 0.0—2.0 mmol•L . The rea－ctions were initiated
1
by adding 20 µL of a 1.5 mmol•L solution of BNPP
(solid substrate dissolved in acetonitrile) into an effi-
ciently stirred sample solution. The reactions were
monitored by detecting the increase in the absorption
Characterization
The GPC system model used for the molecular
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Chin. J. Chem. 2011, 29, 711— 718

Effective Homogeneous Hydrolysis of Phosphodiester and DNA Cleavage
band f－or the－p-nitrophenolate anion at 400 nm (ε, 16980
Table 2 Kinetic data for BNPP hydrolysis by different catalysts
1
1
mol•L •cm ; pKa, 6.98), and this increase was moni-
tored immediately after injection of the substrate. The
reported data are the average of duplicate measurements,
with reproducibility ＞50%. The initial-slope method
was used to determine the pseudo-first-order rate con-
stants. The initial rate of the reaction was obtained by
calculating the slope of a linear plot of 4-nitrophenolate
concentration versus time (R＞0.995). Reaction rate was
tested with respect to both metal complex and BNPP,
and the rate constants were determined from the initial
rates.
Spontaneous
＋
Catalyst
Buffer
Cu2
CS
CSCu
hydrolysis13
－1
－10
－7
－6
－
－
－1
kobs/s
1.1×10
4.8×10 9.1×10 7 4.2×10 7 5.0×10
－4
－1
＋
a [BNPP], 1×10 mmol•L ; [Cu2 ], 0.1 mmol•L ; 10 mmol•
＋
－1
L
Tris-H ; pH, 8.0; temperature, 25 ℃.
that there was a continuous increase in the absorption at
400 nm versus the reaction time, which suggested that
the catalytic function of the CSCu complex was en-
hanced when an excess of the phosphodiester substrate
was present in the reaction system. The CSCu-catalyzed
hydrolysis of BNPP showed a significant rate enhance-
ment (4.5×104-fold) in comparison with the rate of
spontaneous hydrolysis. Moreover, in the Tris-H ＋
buffer solution, the CSCu-catalyzed reaction showed a
10-fold rate enhancement, which was relative to the rate
of the uncatalyzed reactions.
DNA binding studies
Solution of calf thymus DNA (CT DNA) gave a ra-
tio of UV absorbance, A260/A280 of 1.8—1.9∶1, indi-
cating that the DNA was sufficiently free of protein.4g,12
The stock solution of CT-DNA was prepared in
Tris-HCl/NaCl (Tris-H＋) buffer, pH 8.0 (stored at 4 ℃
and used within 7 d). The DNA concentration per nu-
cleotide was determined by absorption spectroscopy
using t－he molar absorption coefficient (6600 mol•
The order of the reaction with respect to the catalyst
was determined. CSCu-catalyzed phosphodiester hy-
drolysis showed a pseudo-first-order dependence, which
has been explored in detail (Figure 1). The rate of hy-
drolysis of BNPP showed linear dependence on catalyst
concentration at low concentrations (Figure 1, inset),
and we obtained a pseudo-first-order rate constant (kobs)
－1
1
L •cm ) at 260 nm.
All CD spectroscopic studies were carried out with a
continuous flow of nitrogen purging the polarimeter,
and the measurements were performed at room tem-
perature with 1 cm pathway cells. The CD spectra were
run from 800 to 200 nm at 50 nm/min and the buffer
background was subtracted automatically. The average
of three independent scans was taken as the final CD
spectrum of each sample. Data were recorded at 0.1 n－m
－5 －1
of 3.6×10 s , which was calculated using the rate
law (v0＝kobs[S]). At catalyst concentrations greater than
－4
－1
2×10 mol•L , the reaction showed a decreased re-
action order (less than 1). This observation can be ex-
plained by the competition between the substrate and
water to coordinate with the catalyst. Similar kinetic
behavior was observed when the substrate was in excess
in the reaction system.
1
intervals. The CD spectrum of CT-DNA (100 µmol•L )
alone was recorded as the control experiment.
pUC19 DNA cleavage
Cleavage of supercoiled pUC19 DNA by CSCu wa＋s
investigated by agarose gel electrophoresis. Tris-H
－1
(pH＝8.0, 10 mmol•L ) buffer was used. Reactions
were performed by incubating DNA at 25 ℃ in the
presence/absence of increasing CSCu for 1 h. All reac-
tions were quenched by loading buffer (4 µL). Agarose
gel electrophoresis was carried out on a 1.0% agarose
gel in 0.5×TBE (Tris-boracic acid-EDTA) buffer con-
taining 0.5 µg/mL GelRed at a constant voltage of 100
V for 120 min. The gels were visualized in an electro-
phoresis documentation and analysis system 120.
Results and discussion
Phosphodiester hydrolysis promoted by the chitosan
copper complex
Figure 1 Effect of CSCu concentration on the initial rate of
BNPP hydrolysis. The initial concentration of BNPP was 5×
－4
－1
－1
10 mol•L in 10 mmol•L Tris-H＋ (pH 8.0, 25 ℃). The
inset is a graph of the data points at low catalyst concentrations;
the data points were fit to the line by using the linear least-
squares method (correlation coefficient, 0.995).
We tabulated the representative pseudo-first-order
rate constants for the hydr＋olysis of BNPP by CS, CSCu,
and copper salts in Tris-H buffer at 25 ℃ (Table 2).
The spontaneous hydrolytic rate of BNPP is－ ex-
－10
1
tremely low, wi－th a rate constant of 1.1×10
s
at
In normal enzyme catalytic reactions, the reaction
starts with the binding of the substrate with the enzyme
pH 8.0, and OH is the nucleophile in the spontaneous
hydrolysis.13 The pseudo-first-order kinetics showed
Chin. J. Chem. 2011, 29, 711— 718
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713

Zhang et al.
FULL PAPER
to form an intermediate with a binding constant k1, a
process that has been described with Eq. (1). This is
followed by hydrolysis, and the products are released,
with kcat as the decomposition rate constant of the
rate-limiting step [Eq. (2)]. The rate law for the reaction
can be obtained by using Eq. (3), with an assumption
that the concentration of the bound substrate (S) in the
solution is much less than that of the free substrate.
the alkaline solution.
kcat[CSCu][S]
(2)
v0＝
[S]＋KM
Figure 2 Dependence of v0 on the concentration of BNPP. The
－ 4
－ 1
initial concentration of CSCu was 1×10
mol•L
in 10
k1kcat [CSCu][S]
－ 1
(3)
mmol•L
Tris-H＋ ; pH, 8.0; temperature, 25 ℃. When the
v0＝
－4
－1
k1[S]＋k−1＋kcat
BNPP concentration was greater than 5.0×10 mol•L , the
CSCu-catalyzed hydrolysis of BNPP occurred in the satura-
tion-kinetics mode. The data were consistent with those obtained
by the Michaelis-Menten equation; the inset is the Lineweaver-
burk plot, with a linear correlation coefficient of 0.995.
In order to gain a better insight into the mechanism
of the CSCu-catalyzed reaction, the initial rate was
measured as a function of the substrate concentrations.
The hydrolysis of BNPP by CSCu, which is a first-order
reaction at lower concentrations of BNPP, was slightly
saturated at higher concentrations (Figure 2). The satu-
ration-kinetics plot represents the substrate coordination
to the CSCu complex, which occurred according to Eq.
(2). Considering that the binding of the catalyst to the
substrate must have occurred rather quickly (k1>>
k >>kcat), the initial rate can be expressed by Eqs. (3)
－1
and (4). The experimental data were consistent with the
results obtained by using Eq. (4), the Lineweaver-Burk
function [Eq. (5) and Figure 2 inset], and the results
from the equation KM＝(k ＋kcat)/k1. We also deter-
－1
mined the following values from this analysis: Micha-
－1
elis constant (KM), (2.3±0.1) mmol•L ; maximal ve-
－8
locity (vmax), (3.6±0.2)×10 mol/L•s; binding con-
－1
stant (k1), 1355 mol•L ; reverse-reaction constant (k ),
－1
－1
Figure 3 UV-Vis spectra of the reaction system at different pH
values. UV-Vis (CSCu-OH) λmax: 233 nm. The amount of
CSCu—OH was enhanced with an increase pH values.
3.0 s ; and first-order rate constant for the decomposi-
tion of the intermediate－Cu(II)-hydroxide-phosphate
－5
1
complex (kcat), 5.2×10 s .
1
KM
1
We studied the catalytic cleavage of BNPP at pH
values between 5.5 and 9.5 and found that the initial
rates of cleavage increased with an increase in pH val-
ues, especially when pH＞6.5. The graph of pH versus
v0 showed a typical sigmoidal curve (Figure 4). The
kinetic behavior was consistent with the previous ob-
servation that the water molecules coordinating with
copper ions underwent deprotonation in an alkaline
environment, leading to the generation of Cu—OH as
the catalytic species or nucleophile.10-11,14 On the basis
of these results, we hypothesized the ionization kinetics
represented in Scheme 1, in which CSCu—H2O and
CSCu—OH are the active species involved in the reac-
(4)
＝
＋
v
vmax
vmax [S]
KH O[H＋]＋KOH Ka
2
(5)
kobs＝
[H＋]＋Ka
Effects of pH on the hydrolysis of BNPP
The pH value is an important factor for the active
species in catalytic hydrolysis system. In order to iden-
tify the reactive species, we obtained UV-Vis spectra of
CSCu in aqueous systems with different pH values. As
shown in Figure 3, the absorption peak at 233 nm was
enhanced with an increase in the pH values. This phe-
nomenon indicated the formation of hydroxyl species in
tion, and KH O and KOH represent the rate constants
2
for BNPP catalytic hydrolysis of the respective species.
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Chin. J. Chem. 2011, 29, 711— 718

Effective Homogeneous Hydrolysis of Phosphodiester and DNA Cleavage
－5 －1
Ka was the disassociation constant for the chelated water
molecule. The relationship bet＋ween pseudo rate con-
stant and the concentration of H in the reaction system
has been expressed in Eq. (5). The experimental data
were consistent with the results obtained using Eq. (5)
7.3×10 s , respectively. The k1' value was more
than 10 times higher than the corresponding value for
the aqueous solution (k1), showing that the association
between Cu and the substrate in the acetonitrile system
was much faster than that in the aqueous solvent. How-
ever, there was no significant difference between the
kcat' value and the corresponding value (kcat) of the
aqueous system. These results suggested that the water
molecule might compete with the substrate during the
coordination with the copper ion. However, water
molecules did not show any significant effect on both
kcat value and the cleavage of the leaving groups, which
suggested that the intramolecular attack was mainly
caused by the copper-bound hydroxyl groups.15
and the representation of data in the inset of Figure －4,
6
and the obtained KH O and KOH values were 1.5×10
2
－1
－5 －1
s
and 1.6×10 s , respectively. We also derived a
pKa value of (8.0±0.2), corresponding to the CSCu-
bound water molecule, from Eq. (5). It was apparent
that the coordinated water molecule was less active than
the deprotonated product. Therefore, in our studies, the
CSCu-bound hydroxyl group was expected to be the
most effective nucleophile in a weak alkaline solution.
When pH＜6.5, the deprotonation was inhibited; con-
sequently, the CSCu-H2O would be the primary cata-
lytic species, with a lower hydrolytic rate.
Figure 5 Effect of catalyst concentration on BNPP hydrolysis
－1
in organic solvent in the presence of 0.5 mol•L H2O. Initial
－4
－1
Figure 4 Graph of the influence of pH on the catalytic hydroly-
concentration of the substrate was 1×10 mol•L . pH 8.0, 25
－4
－1
－4
sis of BNPP. [CSCu], 1×10 mol•L ; [BNPP], 1×10 mol•
℃.
－1
－1
L
; 10 mmol•L Tris-H＋; temperature, 25 ℃. The data are
fitted to a theoretical curv－e that is shown in the inset, with pKa,
Mechanism of BNPP hydrolysis
－5
－6 －1
8.0±0.2; kOH, 1.6×10
s
1; and KH O , 1.5×10
s .
2
On the basis of the results of XPS and FT-IR, we
proposed structure of the CSCu complex was one in
which the copper ion coordinates with the two glucosa-
mine groups on the chitosan-backbone chains; two hy-
droxyl groups occupy two other empty orbits, forming a
tetra-coordinated structure, as shown in Figure 6. The
result was consistent with the DFT study conducted by
Terreux.16
Scheme 1 Proposed deprotonation of the CSCu-bound water
molecule
In order to achieve the better understanding of the
role of water molecules in the catalytic process, we per-
formed the catalytic hydrolysis in an acetonitrile system
with a fixed concentration of water. The initial rate for
BNPP hydrolysis (v0) was obtained, as shown in Figure
5. On the basis of the previously obtai－ned resul－ts, we
4
1
Figure 6 Proposed coordinated structure of CSCu.
used a substrate concentration of 1×10 mol•－L and
－5
4
a －catalyst concentration of 2.5×10 —2×10 mol•
1; the reaction behavior in the aqueous solution
In the CSCu-promoted hydrolysis reaction, the high
affinity between the PO bond of the substrate and the
metal active-center is demonstrated by the binding con-
L
showed that v0 was related to the concentration of CSCu.
Using Eq. (3), the binding constant (k1') and decomposi-
tion rate constant (kcat') for the reaction in the acetoni-
trile system were calculated to be 1.6×104 mol•L－1 and
－ 1
stant (k1 ＝ 1355 mol•L ). The association of the
CSCu-bonded hydroxyl with the substrate, after which
Chin. J. Chem. 2011, 29, 711— 718
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715

Zhang et al.
FULL PAPER
the copper ions activate the substrate and the bound wa-
ter molecule, further polarizing the P—O bonds for the
next nucleophilic attack, forms the intermediate. Equi-
librium exists between the deprotonation and protona-
tion of Cu-H2O; at higher pH values, deprotonation is
the primary pathway, leading to the generation of more
efficient nucleophile Cu—OH, which attacks the active
P—O bond, cleaving the nitrophenyl anion. The reac-
tion ended and restarted simultaneously. The proposed
intramolecular nucleophilic mechanism has been repre-
sented in Scheme 2. The interaction between the copper
ions and the phosphorus atom can be enhanced by the
hydroxyl groups on the chitosan pyranose ring and the
two chelated hydroxyl groups, which would be helpful
in the following nucleophilic attack. The experimental
data for the organic solvent also provided the evidence
for the intramolecular nucleophilic attack.
DNA molecular by the strong electrostatic interaction
and the dimensional configuration of DNA might be
altered.
Scheme 2 Proposed mechanisms for the BNPP hydrolysis by
CSCu. Due to the high affinity, the substrate rapidly coordinated
with the active copper center, which was followed by the internal
hydroxide attack on the P atom.
Figure 7 Absorption spectra of CT-DNA in the presence of
－1
increasing amounts of CSCu. ([DNA]＝20 µmol•L , [CSCu]＝
－1
－1
0—25 µmol•L , 10 mmol•L Tris-H＋ buffer, pH 8.0, 25 ℃).
The Arrows shows the absorbance enhanced with increasing
CSCu concentration.
The further studies of circular dichroism (CD) also
proved that CSCu has effect on the structure of CT
DNA. As shown in Figure 8, the CD spectra of
CT-DNA were changed after it was treated by CSCu
([CSCu]/[DNA]＝1∶1) for 5 min, the negative band
(245 nm) and the positive band (276 nm) decreased with
addition of CSCu which means CSCu is effective in
perturbing the secondary structure of DNA. This sug-
gests CSCu can unwind the DNA helix and lead to the
loss of helicit. Additionally, the result of CD also indi-
cated the fine ability of CSCu to combine with DNA
molecular which was described by Eq. (2) we men-
tioned in kinetics studies.
Interaction between CSCu and DNA molecular
UV-Vis spectra are used to investigate interaction
between the DNA molecular and CSCu. As shown in
Figure 7, CT-DNA in Tris-H＋ buffer has absorption
bands at 260 nm. Upon addition of an increasing
－1
amount of CSCu (from 5 to 25 µmol•L ) to DNA solu-
tion (20 µg/mL), an obvious increasing absorption at
260 nm was observed. This change indicates there was
stereo configuration variation of the DNA molecular
which was caused by the binding of DNA with CSCu.
As a natural cationic polymer, there are abundant active
groups, e.g., hydroxyl, amino groups in the molecular
chain, chitosan itself has fine ability to bind with nega-
tive charged phosphate groups in the backbone of DNA
molecular. And its high affinity of CSCu to DNA could
be enhanced by the deprotonation of copperbound water
molecular in alkaline solution. Thus, we demonstrated
that the association of CSCu to DNA mainly was caused
by the strong electrostatic interaction. Additionally, part
of hydrogen bonds between the base pairs of DNA
might be breached by this strong interaction. The dis-
tinct hyperchromicity of the UV spectra suggests that
supercoiled molecular chain of DNA became relaxed
after it interacted with CSCu and more and more base
pairs were exposed that induced the increasing absorp-
tion. Thus we proposed that CSCu might bind with
－
－4
Figure 8 CD spectra of CT DNA (1.0×10 mol•L 1) and the
interaction with CSCu ([CSCu]/[DNA]＝1). The spectra were
＋
－1
recorded in 10 mmol•L Tris-H buffer; solid line was recorded
by incubating the CT-DNA with CSCu for 5 min, pH 8.0 and 25
℃.
716
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Chin. J. Chem. 2011, 29, 711— 718

Effective Homogeneous Hydrolysis of Phosphodiester and DNA Cleavage
DNA cleavage
efficiency under mildness conditions. On the basis of
kinetics studies and spectrum analysis as well as gel
electrophoresis, electrostatic of DNA binding is sug-
gested for CSCu, the active species of copper-bound
hydroxide group and the intramolecular attack mecha-
nism are proposed for CSCu-catalyzed reaction system.
We concluded that chitosan-based metal complexes
have potential application as a catalyst for cleavage of
phosphodiester bonds, since they show relative high
catalytic efficiency, and are relatively cheaper and
environment-friendly.
Considering CSCu as artificial phosphodiesterase,
the cleavage experiment on natural DNA has been car-
ried by agarose gel electrophoresis, supercoiled pUC19
DNA used as the substrate. We adopted CSCu with dif-
ferent concentration to cleave pUC19 DNA molecular.
The reactions were carried out by incubat＋ing the com-
plex with pUC 19 DNA for 1 h in Tris-H (pH＝8.0).
The result in Figure 9 indicated that the supercoiled
DNA molecular can be cleaved into nicked and linear
DNA by CSCu complex, which was evidenced by the
disappearance of form I (supercoiled form) of the plas-
mid and the appearance of the form II (nicked circular
form). The observed rate constant kobs for pUC19 DNA
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© 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2011, 29, 711— 718