DNA cleavage by the photocontrolled cooperation of ZnII centers in an azobenzene-linked dizinc complex

Article abstract of DOI:10.1021/ic201244y

We synthesized a new photoactive dinuclear zinc(II) complex by linking two zinc centers with a ligand containing an azobenzene chromophore and investigated the DNA cleavage activities of its trans and cis forms. The trans structure of the dinuclear zinc c

Full text of DOI:10.1021/ic201244y

Article
pubs.acs.org/IC
DNA Cleavage by the Photocontrolled Cooperation of ZnII Centers in
an Azobenzene-Linked Dizinc Complex
Anangamohan Panja, Takashi Matsuo, Satoshi Nagao, and Shun Hirota*
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan
S
* Supporting Information
ABSTRACT: We synthesized a new photoactive dinuclear
zinc(II) complex by linking two zinc centers with a ligand
containing an azobenzene chromophore and investigated the
DNA cleavage activities of its trans and cis forms. The trans
structure of the dinuclear zinc complex was determined by X-
ray crystallography, where each zinc center is situated in an
octahedral coordination environment comprised of three
nitrogen atoms from the ligand and three oxygen atoms
from two nitrate ions. The dinuclear zinc complex containing
the azobenzene chromophore was photoisomerizable between the trans and cis forms. The binding affinities of the trans and cis
complexes with calf thymus (CT)-DNA were similar. Although the DNA cleavage activity of the trans complex was negligible,
the cis complex was able to cleave DNA. We attribute the efficient activity of the cis complex to the cooperation of the two
closely located zinc centers and the inactivity of the trans complex to the two metal centers positioned far away from each other.
The DNA cleavage activity of the cis complex exhibited a pH-dependent bell-shaped profile, which has been observed in the
hydrolytic cleavage of DNA by zinc complexes. The DNA cleavage activity was not inhibited by a major groove binder, methyl
green, but decreased significantly by a minor groove binder, 4′,6-diamidino-2-phenylindole, indicating that the dinuclear zinc
complex binds to the minor groove of DNA. The present work shows the importance of the cooperation of two zinc ions for
hydrolytic DNA cleavage, which can be photoregulated by linking the two metal centers with a photoisomerizable spacer, such as
an azobenzene chromophore.
their corresponding monometallic complexes for hydrolysis of a
phosphodiester linkage because the metal centers in the di- and
multinuclear complexes exhibit cooperation.37,38 In fact, the
extraordinary catalytic efficiencies of many natural nucleases
rely on the cooperative actions of two or more metal ions.39−45
Among various physiologically relevant metal ions, ZnII is
suitable for artificial metallonucleases because of the virtues of
its strong Lewis acidity, high charge density, and rapid ligand-
exchange ability. ZnII exhibits a d10 electronic configuration, and
the redox inertness rules out the possibility of oxidative cleavage.
In addition, it generally has no ligand-field stabilization energy and,
as a consequence, it can easily attain variable coordination
numbers for water coordination during hydrolytic cleavage.
The intermetallic distance and rigidity of a dinuclear complex
are important for the cooperation of the metal centers in DNA
cleavage. Because azobenzene has been used to regulate the
structure and distance in various systems,46−57 we have previously
linked two CuII-bound dipeptides with an azobenzene group,
which could be photoisomerized between the trans and cis
forms.51 Two CuII centers were positioned relatively far away from
each other in the trans complex, where no appreciable DNA
cleavage activity was detected. In the cis complex, the CuII centers
INTRODUCTION
■
Over the past few decades, the development of artificial DNA
cleaving reagents has attracted considerable attention because
of their practical applications in the fields of molecular biology,
biotechnology, and chemotherapy.1−7 The DNA cleavage
reactions proceed by targeting various constituents of DNA,
such as nucleic bases, the deoxyribose sugar moiety, and
phosphodiester linkage. The oxidative DNA cleavage reaction
generally involves nucleobase oxidation and/or degradation of
the sugar moiety by the toxic radical species, which is generated
by abstraction of the sugar hydrogen atom(s).8−10 Oxidative
cleavage also requires an additional reducing reagent, such as
hydrogen peroxide or ascorbic acid. These properties limit the
use of oxidative cleavage reagents to in vitro applications,11−17
in which the modified DNA fragments produced by oxidative
cleavage are not suitable for further enzymatic manipulation. In
hydrolytic cleavage, the phosphodiester backbone is hydro-
lyzed.18−21 The nucleic bases and deoxyribose sugar moiety are
not modified, and no additional reagent is necessary when they
are hydrolytically cleaved, which allows the cleaved fragments
to be regulated enzymatically.22−24 Therefore, small metal
complexes that undergo hydrolytic DNA cleavage are useful in
genetic engineering and molecular biotechnology.25−27
Many mono-, di-, and multinuclear metal complexes have
been reported for DNA hydrolytic cleavage reactions.28−36 Di-
and multinuclear complexes are typically more reactive than
Received: June 10, 2011
Published: October 26, 2011
© 2011 American Chemical Society
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After the addition of triethylamine (0.80 g, 8.0 mmol) and NaI (1.19 g,
8.0 mmol), the mixture was stirred at room temperature for 2 days. The
solution was poured into ice−water, yielding a crude product.
Recrystallization from a hexane−dichloromethane mixture afforded
were closely located, and the complex exhibited excellent DNA
cleavage activity.
In this study, we linked two ZnII centers with an azobenzene
derivative to produce a photoactive hydrolytic DNA cleavage
reagent 1·Zn-trans/1·Zn-cis (Scheme 1). A neutral ligand was
1
ligand 1 as a brownish-orange powder. Yield: 1.14 g (45%). H NMR
(400 MHz, CDCl3): δ 2.96 (4H, t, J = 7.6 Hz, −NCH2CH2C6H4N),
3.04 (4H, t, J = 7.6 Hz, −NCH2CH2C6H4N), 3.76 (4H, s, −NCH2Py),
3.84 (4H, s, −NCH2C6H4NN−), 7.07−7.14 (4H, dd, J = 11.5 and 6.3
Hz, Py5,5′), 7.26−7.36 (4H, m, Py3,3′), 7.42 (4H, d, J = 8.4 Hz, phenyl
protons near the azo group), 7.55−7.59 (4H, m, Py4,4′), 7.81 (4H, d, J =
8.4 Hz, phenyl protons far from the azo group), 8.49 (4H, dd, J = 11.5
and 6.3 Hz, Py6,6′). 13C NMR (100 MHz, CDCl3): δ 36.77
(−NCH2CH2Py), 54.85 (−NCH2CH2Py), 58.92 (−NCH2Py), 60.81
(−NCH2C6H4NN−), 121.80 (Py5 or 5′), 122.58 (Py5 or 5′), 123.29
(Py3 or 3′), 123.35 (Py3 or 3′), 123.38 (phenyl carbons near the azo
group), 123.43 (phenyl carbons near the azo group), 129.97 (phenyl
carbons far from the azo group), 130.38 (phenyl carbons far from the azo
group), 136.81 (Py4 or 4′), 137.06 (Py4 or 4′), 143.42 (ipso-carbon
bonding with the azo group), 149.50 (Py6 or 6′), 149.85 (Py6 or 6′),
152.52 (ipso -carbon far from the azo group), 160.65
a
Scheme 1. Photoisomerization of Complex 1·Zn
1
(Py 2 or 2′), 161.15 (Py2 or 2′). H NMR (400 MHz, CD3CN): δ
2.87 (4H, t, J = 7.2 Hz, −NCH2CH2C6H4N), 3.00 (4H, t, J = 7.2 Hz,
−NCH2CH2C6H4N), 3.76 (4H, s, −NCH2Py), 3.79 (4H, s,
−NCH2C6H4NN−), 7.07−7.14 (4H, m, Py5,5′), 7.33−7.39 (4H, m,
Py3,3′), 7.46 (4H, d, J = 11.7 Hz, phenyl protons near the azo group),
7.61−7.68 (4H, m, Py4,4′), 7.79 (4H, d, J = 11.7 Hz, phenyl protons far
from the azo group), 8.41 (2H, d, J = 6.4 Hz, Py6 or 6′), 8.46 (2H, d, J =
6.4 Hz, Py6 or 6′). ESI-MS (m/z, positive mode). Calcd for C40H41N8
([M + H]+): 633.35. Found: 633.35.
Synthesis of the Zinc(II) Complex 1·Zn. Ligand 1 (0.632 g, 1.0
mmol) in 20 mL of methanol was added to Zn(NO3)2·6H2O (0.595 g,
2.0 mmol) in 30 mL of acetonitrile. The resulting orange-yellow
solution was refluxed for 1 h and filtered to remove the insoluble
materials. By incubation of the solution at ambient temperature, a
dark-yellow powder was generated. Yellow microcrystalline material
was obtained by the slow evaporation of an acetonitrile−methanol
a
Two ZnII centers are interlinked with an azobenzene chromophore.
The 1·Zn-trans and 1·Zn-cis isomers are converted to 1·Zn-cis and
1·Zn-trans by irradiation with UV and visible light, respectively.
synthesized to enhance the binding ability of the complex with
DNA by increasing the electrostatic interaction. The cis form of
the complex showed efficient DNA cleavage activity, while no
significant DNA cleavage activity was observed for the trans
complex. The higher activity of the cis complex is due to the
cooperation of two closely associated ZnII centers (Scheme 1).
To the best of our knowledge, this is the first example for
photoregulation of the cooperation between two ZnII centers in
DNA cleavage.
1
solution after 1 week. H NMR (400 MHz, CD3CN): δ 3.23−3.33
(8H, m, −NCH2CH2Py), 3.91 (2H, d, J = 15 Hz, −NCHαPy), 3.98 (2H,
d, J = 15 Hz, −NCHβPy), 4.14 (2H, d, J = 14 Hz, −NCHαC6H4N
N−), 4.31 (2H, d, J = 14 Hz, −NCHβC6H4NN−), 7.42 (4H, d, J = 8.3
Hz, phenyl protons near the azo group), 7.51 (2H, d, Py3 or 3′), 7.59
(4H, m, Py5 or 5′), 7.65 (2H, m, Py3 or 3′), 7.94 (4H, d, J = 8.3 Hz,
phenyl protons near the azo group), 8.02 (2H, m, Py4 or 4′), 8.13 (2H,
m, Py4 or 4′), 8.75 (4H, m, Py6 and 6′). ESI-HR-MS (m/z, positive
mode). Calcd for C40H40N11O9Zn2 ([1·Zn − NO3]+): 946.15934. Found:
946.15948.
X-ray Analysis. Single crystals suitable for X-ray diffraction were
obtained by the slow diffusion of ether into an acetonitrile solution of
the dinuclear complex 1·Zn. X-ray diffraction intensity data were collected
with a Rigaku RAXIS RAPID (3 kW) imaging plate area detector with
graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å) in the ω−2θ
scanning mode. A single crystal was mounted at the top of a glass fiber
and collected at 123 K in a stream of gaseous nitrogen. The reflection data
were corrected for Lorentz and polarization effects. The structure was
solved by direct methods and refined on F2 by full-matrix least-squares
techniques with the SHELXL-97 program.59 All of the non-hydrogen
atoms were refined anisotropically, and all of the hydrogen atoms
belonging to the complex were fixed in their calculated positions and
refined using a riding model. Calculations were performed with the
Rigaku Crystal Structure 4.0 software package. A summary of the
crystallographic data for complex 1·Zn is listed in Table 1.
Photoisomerization Experiments. The UV light (320 nm < λ <
400 nm) was obtained by a 150-W xenon lamp (Hamamatsu Photonics)
using a combination of UV pass (280 nm < λ < 400 nm) and sharp-cut
filters (λ < 320 nm). The visible light (λ > 420 nm) was obtained by the
same light source using a sharp-cut filter (λ < 420 nm). Irradiation of
ligand 1 in CDCl3 and complex 1·Zn in D2O with UV and visible
light was performed for the trans-to-cis and cis-to-trans photo-
isomerizations, respectively. Determination of the trans/cis ratio of
ligand 1 was done by calculating the relative intensities of the shifted
EXPERIMENTAL SECTION
■
Materials and Spectroscopic Measurements. Zinc nitrate
hexahydrate, 4-nitrobenzyl alcohol, 2-pyridinecarboxaldehyde, 2-
(aminoethyl)pyridine, and tris(hydroxymethyl)aminomethane (Tris)
were purchased from Wako Pure Chemicals Industries, Ltd., Japan.
Calf thymus (CT)-DNA, ethidium bromide (EB), methyl green, and
4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma.
Other chemicals and solvents were of analytical grade and were used
without further purification.
Measurements of 1H and 13C NMR spectra were conducted using a
JNM-ECP400 spectrometer (JEOL) in CDCl3 or CD3CN. If
necessary, HMQC and HMBC measurements were carried out for
the peak assignments. Electrospray ionization mass spectrometry (ESI-
MS) was performed using a JMS-T100LC mass spectrometer (JEOL).
Absorption spectra were measured using a UV-2450 spectropho-
tometer (Shimadzu) with a 1-cm-path-length quartz cell. Fluorescence
experiments were performed with an F-4500 spectrofluorometer
(Hitachi, Japan).
Synthesis of Ligand 1. 4,4′-Bis(chloromethyl)azobenzene and
(2-pyridylmethyl)(2-pyridylethyl)amine were synthesized according
to the reported methods.52,58 4,4′-Bis(chloromethyl)azobenzene (1.11 g,
4.0 mmol) and (2-pyridylmethyl)(2-pyridylethyl)amine (1.71 g, 8.0
mmol) were dissolved in 100 mL of N,N-dimethylformamide (DMF).
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solution was used to quench the DNA cleavage reaction, where 4 μL of
the mixed solution was added to 18 μL of the reaction solution. The
quenched samples were loaded to 1% agarose gel, and electrophoresis
was carried out for 40−60 min at 100 V using a Tris-acetate-EDTA
buffer (pH 8.0). After electrophoresis, the gel was transferred to an EB
solution (1 μg/1 μL) and stained. The bands of the supercoiled and
nicked-circular DNA forms were visualized using a gel document
instrument (ChemiDoc XRS, Biorad). The intensities of the
supercoiled plasmid DNA bands were multiplied by a factor of 1.3,
to compensate for its weaker staining capability with EB compared to
the nicked-circular form.61,62 To determine the DNA interaction
center of complex 1·Zn, the supercoiled plasmid DNA was pretreated
with a major groove binder, methyl green (50 μM),63 or a minor
groove binder, DAPI (50 μM),64 at room temperature for 30 min
before the addition of complex 1·Zn.
Table 1. Crystallographic Data and Structure Refinement for
Complex 1·Zn in the Trans Form
empirical formula
fw
C40 H40 N12 O12 Zn2
1011.59
temperature (K)
wavelength (Å)
cryst syst
space group
unit-cell dimens
a (Å)
123(2)
0.710 73
monoclinic
P21/n
8.387(6)
b (Å)
14.336(8)
c (Å)
18.687(10)
β (deg)
volume (Å3)
96.537(3)
2234.6(4)
Z
2
RESULTS AND DISCUSSION
■
Dcalc (g cm−3
)
1.503
Synthesis and Characterization. The synthetic route for
the preparation of ligand 1 is depicted in Scheme 2. The
μ (mm−1
)
1.149
F(000)
1040
θ range (deg)
3.04−25.38
31 196
Scheme 2. Syntheses of Ligand 1 and Its Dinuclear Zinc
Complex 1·Zn
reflns collected
indep reflns
4075
refinement method
data/restraints/param
GOF on F2
full-matrix least squares on F2
4075/0/298
1.054
final R indices [I > 2σ(I)]
largest diff peak and hole (e Å−3
R1 = 0.102, wR2 = 0.229
0.57 and −0.43
)
signals at about 6.75 (corresponding to the cis form, four protons) and
7.42 ppm (trans form, four protons) against the less-sensitive ones at
about 8.49 ppm (cis and trans forms, four protons) by photo-
isomerization. The stability of the cis form (cis-to-trans thermal
isomerization rate) of complex 1·Zn in the absence and presence of
DNA was elucidated by observation of the UV−vis spectral changes in
an aqueous solution or a 20 mM phosphate buffer (pH 7.5) at 37 °C.
Binding of Zinc(II) Complex to CT-DNA. The binding proper-
ties of dinuclear complex 1·Zn to DNA were examined by UV−vis and
fluorescence spectroscopies. The ratio between the UV absorbances of
CT-DNA in a 20 mM phosphate buffer (pH 7.5) at 260 and 280 nm
was about 1.9, indicating that DNA was sufficiently free from protein.
The concentration of CT-DNA in base pairs was calculated using its molar
absorption coefficient value of 6600 M−1 cm−1 at 260 nm.60 Complex
1·Zn (2.5 × 10−5 M) was titrated with varying concentrations of CT-
DNA (up to about 1.2 × 10−4 M), and the changes in the absorption
spectra were recorded after incubation for 5 min.
The relative binding affinity of the complex to CT-DNA was
studied using the fluorescence spectral technique with EB-bound CT-
DNA (2.5 × 10−5 M) in a 20 mM phosphate buffer (pH 7.5). The
changes in the fluorescence intensity at 600 nm (526 nm excitation) of
EB-bound CT-DNA were recorded at each incremental addition of the
complex [(0−5.0) × 10−5 M].
DNA Cleavage Experiments. Plasmid DNA (pUC19) was
extracted by overexpression in Escherischia coli DH5α cells, and the
obtained DNA was purified with a QIAGEN kit (QIAprep spin
miniprep kit-250). A DNA stock solution was prepared in a 10 mM
Tris buffer (pH 8.0). The purity of DNA was checked by gel
electrophoresis. Then, DNA was stored at 4 °C until use. The DNA
cleavage activity of complex 1·Zn was determined by monitoring the
conversion of supercoiled plasmid DNA (form I) to nicked-circular
DNA (form II) using agarose gel electrophoresis. 1·Zn-cis-pss was
obtained by irradiation of 4.0 mM 1·Zn-trans with UV light in an
aqueous solution for 1 h and used immediately after irradiation. The
experiments were carried out by incubation of the reaction mixtures
containing about 1 μg of DNA and compound 1·Zn, in a 20 mM
phosphate buffer (∼350 ± 50 μL) at 37 °C. A mixture of equal
volumes of a 10 × DNA loading buffer (Takara-bromophenol blue)
and a 10 mM ethylenediaminetetraacetic acid (EDTA) aqueous
nucleophilic substitution of the chloride atoms of 4,4′-bis-
(chloromethyl)azobenzene with (2-pyridylmethyl)(2-pyridylethyl)-
amine in the presence of sodium iodide and triethylamine resulted
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in the production of ligand 1 in good yield. The ligand was
the M−O distance (Δd) to classify the nitrate denticity: Δd <
0.3 Å for bidentate; 0.3 Å < Δd < 0.6 Å for anisobidentate;
Δd > 0.6 Å for monodentate.65 Applying this methodology, we
concluded that one nitrate is bound in an anisobidentate
fashion in 1·Zn-trans (Δd = 0.37 Å), while the other nitrate is
undoubtedly a monodentate ligand (0.66 Å). Therefore, each
zinc center in 1·Zn-trans is six-coordinate, and the coordination
structure at the metal is best described as a distorted octahedral
geometry. The major structural distortion arises from the small
biting angle [O1−Zn1−O2 55.1(3)°] of the bidentate nitrate
ion. The bond lengths around the ZnII centers were in the
common range of similar complexes.66,67 However, the Zn−N
bond distances ranged between 2.058(8) and 2.228(7) Å, while
the Zn−O lengths varied in a wide range from 2.091(7) to
2.468(7) Å.
The azobenzene linker of 1·Zn-trans was eventually planar
with respect to the PhNNPh unit, as expected from the π
conjugation, and the maximum deviation from the mean plane
observed for the azo group was 0.086 Å. The N−N distance of the
azobenzene linker was 1.240(10) Å, which was similar to that
reported for an azobenzene complex.68 The distance between the
benzylic carbon atoms of the ligand was 12.08 Å, and that between
the two ZnII centers in the molecule was 17.87 Å. Each dinuclear
unit was interlinked with the neighboring complexes through π−π
stacking between the pyridine rings, constructing a two-dimen-
sional supramolecular sheet along the ac plane (Figure S2 in the
Supporting Information).
1
characterized by ESI-MS, H NMR, and UV−vis methods. The
treatment of ligand 1 with zinc nitrate produced complex 1·Zn.
1
Zinc complexation was confirmed by the ESI-MS and H NMR
measurements (see the Experimental Section and Figure S1 in the
Supporting Information): The peaks for the aromatic protons in
the NMR spectrum showed a downfield shift, indicating that the π-
electron density was distributed to the added Zn2+ cations. Several
pyridine protons became magnetically anisotropic. Furthermore,
geminal coupling was observed for the pyridylmethyleneamine and
azobenzenemethylene moieties. These properties suggest that the
conformational restriction of the ligand increased by incorporation
of the Zn2+ cation. The obtained complex was highly soluble in
dimethyl sulfoxide and DMF and partly soluble in water, methanol,
and acetonitrile. The complex exhibited a characteristic absorption
band at 337 nm, indicating a trans configuration around the NN
bond (vide infra).
Crystal Structure of 1·Zn in the Trans Form. The
structure of complex 1·Zn in the trans form (1·Zn-trans) was
determined by the single-crystal X-ray diffraction technique.
Crystallographic data and details of data collection and
refinement for 1·Zn-trans are listed in Table 1. The important
bond lengths and angles are summarized in Table 2. Complex
Table 2. Selected Bond Lengths (Å) and Angles (deg) of Com-
plex 1·Zn in the Trans Form
ZnII ions in mononuclear complexes with similar ligands have
been reported to be five-coordinate, with varying geometry at
the metal center from trigonal-bipyramidal to square-pyramidal
geometry, depending on the position of the solvent and/or the
counterion.66,67 In 1·Zn-trans, each zinc ion was situated in an
octahedral environment, which was regulated by the bidentate
coordination mode of one of the nitrate ions. Therefore, we can
expect at least two water molecules to coordinate to each zinc
center of 1·Zn-trans in an aqueous solution.
Zn1−N1
Zn1−N3
Zn1−O2
2.228(7)
2.058(8)
2.468(7)
Zn1−N2
Zn1−O1
Zn1−O4
2.068(8)
2.091(8)
2.128(8)
O1−Zn1−O4
O1−Zn1−N2
O4−Zn1−N1
O4−Zn1−N3
N1−Zn1−N3
O1−Zn1−O2
O2−Zn1−N1
O2−Zn1−O4
113.1(3)
90.7(3)
94.6(3)
100.7(3)
90.7(3)
55.1(3)
96.4(3)
167.4(3)
O1−Zn1−N1
O1−Zn1−N3
O4−Zn1−N2
N1−Zn1−N2
N2−Zn1−N3
O1−Zn1−O4
O4−Zn1−N1
150.8(3)
93.1(3)
94.1(3)
77.6(3)
161.8(3)
113.1(2)
94.5(2)
Photoisomerization of Ligand 1 and Complex 1·Zn. The
trans form of ligand 1 (1-trans) exhibited π−π* (337 nm) and
n−π* (429 nm) absorption bands because of the azobenzene
chromophore (Figure S3 in the Supporting Information).
Irradiation of 1-trans with UV light (320 nm < λ < 400 nm) led
to the formation of its cis congener (1-cis), with a decrease in
the intensity of the π−π* band and a concomitant increase in
that of the n−π* band with isosbestic points at 278 and
420 nm.46,49,50,53−55,57 Simultaneously, the absorption maximum
of the π−π* band shifted slightly to a shorter wavelength in the cis
form. The trans-to-cis photoisomerization of ligand 1 reached a
photostationary state (PSS) after about 30 min of irradiation with
UV light. Several 1H NMR signals of the PSS state obtained after
UV irradiation had shifted upfield compared to those of 1-trans, as
reported previously.51 The ratio of the trans and cis forms of the
ligand was calculated from the relative intensities of the shifted
signals against the unaffected ones by photoisomerization. The
trans/cis ratio of ligand 1 at the PSS obtained by UV irradiation of
1-trans was estimated to be 20:80 (Figure S4 in the Supporting
Information), where the cis form was the major isomer
(abbreviated to 1-cis-pss). Subsequent irradiation of 1-cis-pss
with visible light (λ > 420 nm) resulted in a PSS with the trans
form as the predominant isomer (∼80%; 1-trans-pss), according
to a similar NMR spectral analysis (Figure S4 in the Supporting
Information). The trans/cis ratio at 1-trans-pss was also estimated
from the absorption spectra and the trans/cis ratio of 1-cis-pss
Zn1···Zn1a
17.872 (1)
C14···C14a
12.08(1)
1·Zn crystallized in the monoclinic P21/n space group, where
the asymmetric unit corresponded to half of the molecule. Each
ZnII ion of the two centrosymmetrical metal centers was
coordinated with two pyridine nitrogen atoms and one tertiary
amine nitrogen atom from one arm of ligand 1 and three
oxygen atoms from two nitrate ions (Figure 1).
A nitrate ion can bind to the metal center in monodentate
and bidentate fashions. We were able to use the difference in
Figure 1. Molecular structure of complex 1·Zn in the trans form. The
structure is shown in an atom-labeling scheme. Hydrogen atoms are
omitted for clarity (ORTEP, 30% ellipsoids).
1
obtained from the H NMR spectra. The absorption spectrum of
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100% 1-cis was calculated from the absorption spectra of 1-trans
and 1-cis-pss and the trans/cis ratio at 1-cis-pss obtained from the
1H NMR spectra (Figure S3 in the Supporting Information). Next,
the absorption spectrum of 1-trans-pss was reproduced with the
absorption spectrum of 1-trans and the estimated spectrum of
1-cis, resulting in a trans/cis ratio of 20:80, which matched very well
that obtained from the 1H NMR spectral analysis described above.
UV−vis spectroscopy was used to evaluate photoisomeriza-
π−π* absorption bands of the azobenzene chromophore at 337
and 323 nm, respectively (Figure 3). By the addition of CT-DNA
1
tion of complex 1·Zn (Figure 2) because its H NMR signals
Figure 2. Absorption spectra of complex 1·Zn (4.0 × 10−5 M). 1·Zn-
trans (black) is converted to a PSS by irradiation with UV light (1·Zn-
cis-pss, red), and the obtained 1·Zn-cis-pss is converted to another PSS
by subsequent irradiation with visible light (1·Zn-trans-pss, blue). The
estimated spectrum of 100% 1·Zn-cis (broken pink) is calculated from
the 1·Zn-trans and 1·Zn-cis-pss spectra and the cis/trans ratio of 1·Zn-
cis-pss obtained from the NMR data. The calculated spectrum of a
mixture of 80% 1·Zn-trans and 20% 1·Zn-cis (broken purple) is also
depicted.
Figure 3. Absorption spectral changes upon the incremental addition
of CT-DNA (1.5 × 10−3 M, 3.3−13.3 μL per scan) to complex 1·Zn
(2.5 × 10−5 M, 1.0 mL) in a 20 mM phosphate buffer (pH 7.5) at
room temperature: (A) 1·Zn-trans and (B) 1·Zn-cis-pss. Inset: Plot of
[DNA]/Δε versus [DNA] obtained by the absorption titration of CT-
DNA with (A) 1·Zn-trans and (B) 1·Zn-cis-pss, respectively. Each
absorption spectrum was normalized with the concentration of
complex 1·Zn at 2.5 × 10−5 M.
were highly overlapped (Figure S1 in the Supporting
Information). The trans/cis ratios of complex 1·Zn at the PSSs
obtained by UV (1·Zn-cis-pss) and visible light irradiation (1·Zn-
trans-pss) were estimated to be trans/cis = 20:80 and 80:20,
respectively. The trans-to-cis photoconversion of 1·Zn-trans and
cis-to-trans conversion of 1·Zn-cis proceeded to the same extent as
their corresponding forms of ligand 1 by UV and visible light
irradiation, respectively. Photoisomerization of ligand 1 and
complex 1·Zn could be performed repeatedly, as reported for
other compounds with azobenzene groups.46,49,50,53−55,57 These
results show that the azobenzene chromophore of the ligand
undergoes effective photoisomerization, and therefore the spatial
orientation of the coordinated ZnII centers can be externally
controlled by light. However, 1·Zn-cis converts to the
thermodynamically stable 1·Zn-trans over time. A rate constant
of 6.7 × 10−6 s−1 was obtained for the cis-to-trans conversion of
complex 1·Zn in an aqueous solution at 37 °C, where about 30%
of the cis form converted to the trans form after incubation for
24 h (Figure S5 in the Supporting Information). No significant
difference in the rate of cis-to-trans thermal conversion was
observed between the absence and presence of DNA (Figure S6 in
the Supporting Information), suggesting that electrostatic
interactions between 1·Zn-cis and DNA are not strong enough
to affect thermal isomerization of the azo group (vide infra).
Complex 1·Zn Binding to DNA. Because binding of a
metallocomplex to DNA is considered to be an important step
in DNA cleavage, the binding abilities of 1·Zn-trans and 1·Zn-
cis-pss to CT-DNA were measured by intensity changes in their
to either form of the complex in a 20 mM phosphate buffer (pH
7.5), only a moderate hypochromism was observed. The intrinsic
binding constant (Kb) was determined with the following
equation:69
where [DNA] is the concentration of the base pairs, εa is the
observed extinction coefficient for the π−π* absorption band of
the azobenzene moiety at each DNA concentration, εf is the
extinction coefficient of the free complex, and εb is the extinction
coefficient of the complex when fully bound to DNA. The Kb
values for 1·Zn-trans and 1·Zn-cis-pss were obtained as (4.6 ±
0.1) × 104 and (4.9 ± 0.1) × 104 M−1, respectively. The Kb values
were similar between the trans and cis forms but were significantly
lower than those reported for typical intercalators, indicating that
complex 1·Zn does not bind to DNA through an intercalation
mode for either the trans or cis form.70 However, in Figure 3, the
increase in the absorbance with the addition of DNA was lower
than expected, especially at the initial stages of DNA addition. For
example, the difference between the absorbances at 260 nm of the
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initial and final spectra in Figure 3B was 0.57, whereas it was
estimated as 0.73 from the DNA concentration difference of 111
μM and ε = 6600 M−1 cm−1. Because complexes 1·Zn-trans and
1·Zn-cis-pss show absorption at 260 nm (Figure 2), the decrease
in the absorbance, mainly at the initial stage of DNA addition, may
be due to the decrease in the absorption of the complex at 260 nm
by the interaction with DNA.
To investigate the DNA binding affinity of 1·Zn-cis-pss in
more detail, its binding affinity with DNA was compared with
that of EB by fluorescence spectroscopy. The emission intensity
of EB decreases at the outside of the DNA strand because of
the quenching effect by the solvent molecules, whereas it
increases significantly by intercalating to DNA. Interaction of a
complex with EB-bound DNA may decrease the emission
intensity of EB by releasing EB from DNA, and therefore the
reduction of the emission intensity can be used as a measure of
the DNA binding propensity.71 However, the fluorescence
intensity did not decrease effectively by the addition of 2 equiv
of 1·Zn-cis-pss to EB-bound DNA, indicating that the complex
does not bind to DNA through an intercalation mode (Figure
S7 in the Supporting Information). These results support the
hypothesis that compound 1·Zn does not bind through an
intercalation mode because the obtained binding constants of
complex 1·Zn to DNA were much smaller than that of EB.
Electrostatic interaction is another major feature for small metal
complexes to interact with DNA. The positive charges in
complex 1·Zn could lead the complex to interact with the
anionic phosphodiester backbone of DNA. In fact, the binding
constant of 1·Zn-cis-pss to CT-DNA decreased from (4.9 ±
0.1) × 104 to (2.4 ± 0.2) × 104 M−1 by the addition of 200 mM
NaCl (Figure S8 in the Supporting Information).
Figure 4. Time courses of cleavage of pUC19 DNA by complex 1·Zn
(100 μM) in a 20 mM phosphate buffer (pH 7.5) at 37 °C. Agarose
gel electrophoresis patterns of pUC19 DNA (1 μg) are shown at
various reaction times for (A) 1·Zn-trans and (B) 1·Zn-cis-pss,
respectively: Lanes 1−11: reaction times of 0, 1, 2, 3, 4, 6, 8, 10, 12, 16,
and 24 h, respectively. (C) Plots of percentages of supercoiled (red)
and nicked-circular (blue) DNA versus reaction time. I and II
represent DNA forms 1 and 2, respectively.
The binding constants of 1·Zn to DNA were smaller than
that reported for a nonintercalative dinuclear ruthenium
complex with a charge of 4+ in total.72 In aqueous solutions,
water molecules and/or OH− ions may replace the nitrate
ligands in 1·Zn, and two water/OH− molecules would be
coordinated to each Zn ion in 1·Zn. The pKa of the coordinated
water/OH− is about 7.9−8.35 in zinc complexes with one
coordinated water molecule,73 whereas the pKa value of a zinc
ion with two coordinated water molecules may show relatively
dispersed values (for example, pKa = 7.8 and 9.3).74 An OH−
ion instead of a water molecule may coordinate to the zinc ion
in 1·Zn under the experimental conditions (pH 7.5). Therefore,
1·Zn may be less charged than 4+ because of deprotonation of
the coordinating water, and the binding constant to DNA may
be smaller than that of the nonintercalative 4+ complex.
DNA Cleavage by Complex 1·Zn. The DNA cleavage
properties of 1·Zn-trans and 1·Zn-cis-pss were studied using
agarose gel electrophoresis with supercoiled pUC19 plasmid
DNA. Although only a slight amount of nicked-circular DNA
(form II) was produced even after incubation of supercoiled
DNA (form I) with 100 μM 1·Zn-trans at 37 °C for 24 h,
1·Zn-cis-pss showed efficient DNA cleavage activity under the
same conditions (Figure 4). These results demonstrate that the
cis complex has a higher DNA cleavage activity than its trans
congener. In the cis form, two metal centers are sufficiently
close to each other, and therefore they can play a cooperative
role in DNA cleavage. We attribute the higher DNA cleavage
activity of the cis complex to the cooperation of two closely
associated ZnII centers, while the metal centers are positioned
relatively far way from each other in the trans form (17.87 Å).
Although 1·Zn-cis converts to 1·Zn-trans thermodynamically
(rate constant, 6.7 × 10−6 s−1), about 50% of the supercoiled
DNA converted to the nicked-circular DNA after 8 h of
incubation with 100 μM 1·Zn-cis-pss (Figure 4C). However, no
linear DNA was produced even after incubation of 1·Zn-cis-pss
with DNA for 24 h.
The DNA cleavage activity was remarkably different between
the cis and trans forms of complex 1·Zn, although both forms
had similar binding affinities toward DNA. Our previous molecular
modeling study showed that the intramolecular separation
between the benzylic carbon atoms of the azobenzene
chromophore in the cis form is ∼8 Å, whereas they are ∼12 Å
away from each other in the trans form.51 The CuII-bound
dipeptides in an azobenzene-linked complex were far away from
each other in the trans form and were not favorable for
cooperation and DNA cleavage, while they were oriented toward
each other in the cis form and exhibited effective cooperation to
cleave DNA. In fact, the X-ray crystal structure of 1·Zn-trans
showed that the intramolecular distance between the benzylic
carbon atoms of its azobenzene group was 12.08 Å. One can
expect a cooperation effect in the cis configuration of complex
1·Zn similar to that in the CuII-bound dipeptides linked with an
azobenzene group. The higher reactivity of the cis form of
complex 1·Zn shows the importance of the cooperation of the two
ZnII centers for DNA cleavage.
DNA Interaction of Complex 1·Zn in the Cis Form. The
concentration dependence of 1-cis-pss from 10 to 150 μM
on the DNA cleavage activity exhibited a saturation curve
(Figure S9 in the Supporting Information). These results
suggested that the DNA cleavage reaction proceeds through a
DNA−complex adduct, where interaction of the metal center
with DNA is important for DNA cleavage. In order to obtain
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information on the interaction between 1·Zn-cis and DNA,
DNA cleavage experiments were carried out in the presence of
a major groove binding agent, methyl green, and a minor
groove binder, DAPI. DNA cleavage was not affected by methyl
green but was affected by DAPI, suggesting that 1·Zn-cis binds
at the minor grooves of DNA (Figure 5).
The present results indicate that the nonenzymatic hydrolysis
of a phosphodiester bond of DNA can be performed effectively
by orienting two zinc ions appropriately. In fact, the DNA
cleavage activity of the cis form of complex 1·Zn was higher
than that of its trans congener, and its cleavage activity was
affected by the pH and the addition of NaCl. To explain the
cooperative role of the two zinc centers in the cis form on DNA
cleavage, a possible mechanism is proposed (Scheme 3): The
Scheme 3. Proposed Scheme for DNA Hydrolytic Cleavage
by the Cis Form of Complex 1·Zn
Figure 5. Agarose gel electrophoresis patterns of pUC19 DNA (1 μg)
after 24 h of incubation with 1·Zn-cis-pss (100 μM) in a 20 mM
phosphate buffer (pH 7.5) at 37 °C. Lane 1: in the absence of methyl
green and DAPI. Lane 2: in the presence of methyl green (50 μM).
Lane 3: in the presence of DAPI (50 μM). I and II represent DNA
forms 1 and 2, respectively.
The binding constant of 1·Zn-cis-pss to CT-DNA decreased
with the addition of NaCl (Figure S8 in the Supporting
Information), indicating the importance of electrostatic
interaction between the positively charged ZnII centers and
the anionic phosphate backbone of DNA. The DNA cleavage
efficiency was also decreased by the addition of NaCl (Figure
S10A in the Supporting Information). A similar phenomenon
has been observed in other positively charged metallocom-
pounds,26,38 and electrostatic interaction may play a significant
role in the binding of 1·Zn-cis at the minor groove region of
DNA, which is important for the DNA cleavage activity.75
Hydrolytic DNA Cleavage by Complex 1·Zn in the Cis
Form. The use of the ZnII ion allows us to discard the possibility
of metal-ion-driven oxidative cleavage, and therefore the complex
may cleave DNA hydrolytically. The pH dependence of the DNA
cleavage activity by 1·Zn-cis-pss exhibited a bell-shaped curve,
possessing a maximum at pH 8.5. The pH value of the maximum
activity was close to the pKa values of reported zinc complexes
having similar coordination environments (pKa, about 7.9−8.35
for a single-coordinated water molecule;73 Figure S11 in the
Supporting Information).66,72 This pH-dependence profile is
typical for artificial nucleases, which promote hydrolytic DNA
cleavage.33,37,76 These results suggest that metal hydroxide
coordination of the complex is involved in the course of hydrolytic
cleavage. Such species may be formed by deprotonation of at least
one water molecule, which is coordinated to the metal center.77
However, the hydroxide bound to the metal, consequently,
reduces the charge on the metal complex, which is presumably
unfavorable for the binding of the complex to the negatively
charged nucleic acid.78 Therefore, the decrease in the hydrolytic
activity at higher pH values is possibly due to the decrease in the
charge of the complex. It is noteworthy that the cleavage activity
decreased significantly in the presence of nitrate ions (Figure S10B
in the Supporting Information). The X-ray crystal structure
revealed that the nitrate ions bind firmly to the zinc ions in
complex 1·Zn (Figure 1). Therefore, nitrate ions may coordinate
to the zinc centers in an aqueous solution at high NaNO3
concentrations and block the coordination sites for water
molecules, resulting in a significance decrease in the DNA
cleavage activity. These results also show the importance of water/
hydroxide ion coordination to the zinc centers for DNA cleavage.
two ZnII centers are positioned close to each other in the cis
complex, and the positively charged zinc centers guide the
complex to interact with the negatively charged phosphate
groups of the DNA backbone by electrostatic interaction. In
this conformation, the two zinc centers both interact with the
same phosphate group in the DNA backbone. While the water
molecule(s) at one zinc center interact with the oxygen atom of
the phosphate group, a hydroxide ion bound at the other zinc
center operates a nucleophilic attack to the phosphorus atom of
the phosphate group, initiating DNA cleavage.79,80
CONCLUSIONS
■
We synthesized a novel photoactive dinuclear zinc(II) complex
with an azobenzene linker. The X-ray crystal structure of its trans
form showed that each zinc center exhibits a distorted octahedral
geometry, in which two metal centers were situated far away
from each other (17.87 Å). The spatial disposition of the two
metal centers was externally controlled by photoisomerization
of the azobenzene chromophore. The binding affinities of both
the cis and trans forms of the complex to DNA were similar,
where the positively charged complex presumably interacted
electrostatically with the negatively charged phosphate groups
of DNA. However, the complex in the cis form exhibited
hydrolytic DNA cleavage activity, whereas the activity in the trans
form was negligible. The efficient DNA cleavage activity of the
cis form is attributed to the cooperation of two zinc centers
closely located between each other. The DNA cleavage activity
of the cis complex was decreased by the addition of a minor
groove binder, DAPI, showing that the complex binds to the
minor groove of DNA. These results show the importance of
the cooperation of zinc ions, which can be photoregulated by a
photoisomerizable linker such as an azobenzene chromophore.
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(21) Bonomi, R.; Selvestrel, F.; Lombardo, V.; Sissi, C.; Polizzi, S.;
Mancin, F.; Tonellato, U.; Scrimin, P. J. Am. Chem. Soc. 2008, 130,
15744−15745.
ASSOCIATED CONTENT
■
S
* Supporting Information
X-ray crystallographic data in CIF format and additional results of
photoisomerization, DNA binding, and cleavage experiments. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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AUTHOR INFORMATION
(25) Boseggia, E.; Gatos, M.; Lucatello, L.; Mancin, F.; Moro, S.;
Palumbo, M.; Sissi, C.; Tecilla, P.; Tonellato, U.; Zagotto, G. J. Am.
Chem. Soc. 2004, 126, 4543−4549.
■
Corresponding Author
*E-mail: hirota@ms.naist.jp. Fax: +81-743-72-6110.
(26) Nomura, A.; Sugiura, Y. J. Am. Chem. Soc. 2004, 126, 15374−
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ACKNOWLEDGMENTS
■
We thank Syouhei Katao and Yuriko Nishiyama, Nara Institute of
Science and Technology, for carrying out X-ray diffraction and
ESI-MS measurements. We are also grateful to Leigh McDowell,
Nara Institute of Science and Technology, for his advice on the
manuscript preparation. This work was partially supported by
Grants-in-Aid for Scientific Research from MEXT (Priority Areas,
No. 23107723), JSPS (Category B No.21350095), JST, Sankyo
Foundation of Life Science, and Toray Science Foundation (all to
S.H.). A.P. is thankful to JSPS for a fellowship.
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