Synthesis and enhanced DNA cleavage activities of bis-tacnorthoamide derivatives

Article abstract of DOI:10.1039/c2ob25743b

A new metal-free DNA cleaving reagent, bis-tacnorthoamide derivative 1 with two tacnorthoamide (tacnoa) units linked by a spacer containing anthraquinone, has been synthesized from triazatricyclo[5.2.1.0^4,10]decane and characterized by NMR and mass spectrometry. For comparison, the corresponding compounds mono-tacnorthoamide derivative 2 with one tacnorthoamide unit and 6 with two tacnorthoamide units linked by an alkyl (1,6-hexamethylene) spacer without anthraquinone have also been synthesized. The DNA-binding property investigated via fluorescence and CD spectroscopy suggests that compounds 1 and 2 have an intercalating DNA binding mode, and the apparent binding constants of 1, 2 and 6 are 1.3 × 10⁷ M^-1, 0.8 × 10 ⁷ M^-1 and 8 × 10⁵ M^-1, respectively. Agarose gel electrophoresis was used to assess plasmid pUC19 DNA cleavage activity promoted by 1, 2, 6 and parent tacnoa under physiological conditions, which gives rate constants k_obs of 0.2126 ± 0.0055 h^-1, 0.0620 ± 0.0024 h^-1, 0.040 ± 0.0007 h^-1 and 0.0043 ± 0.0002 h^-1, respectively. The 50-fold and 15-fold rate acceleration over parent tacnoa is because of the anthraquinone moiety of compound 1 or 2 intercalating into DNA base pairs via a stacking interaction. Moreover, DNA cleavage reactions promoted by compound 1 give 5.3-fold rate acceleration over compound 6, which further demonstrates that the introduction of anthraquinone results in a large enhancement of DNA cleavage activity. In particular, DNA cleavage activity promoted by 1 bearing two tacnoa units is 3.3 times more effective than 2 bearing one tacnoa unit and the DNA cleavage by compound 1 was achieved effectively at a relatively low concentration (0.03 mM). This dramatic rate acceleration suggests the cooperative catalysis of the two positively charged tacnoa units in compound 1. The radical scavenger inhibition study and ESI-MS analysis of bis(2,4-dinitrophenyl) phosphate (BDNPP) and adenylyl(3′-5′) phosphoadenine (APA) cleavage in the presence of compound 1 suggest the cleavage mechanism would be via a hydrolysis pathway by cleaving the phosphodiester bond of DNA.

Full text of DOI:10.1039/c2ob25743b

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PAPER
Synthesis and enhanced DNA cleavage activities of bis-tacnorthoamide
derivatives†
Li Wei,a Ying Shao,*b Mi Zhou,b Hong-Wen Hua and Guo-Yuan Lu*a
Received 18th April 2012, Accepted 25th August 2012
DOI: 10.1039/c2ob25743b
A new metal-free DNA cleaving reagent, bis-tacnorthoamide derivative 1 with two tacnorthoamide
(tacnoa) units linked by a spacer containing anthraquinone, has been synthesized from triazatricyclo-
[5.2.1.04,10]decane and characterized by NMR and mass spectrometry. For comparison, the
corresponding compounds mono-tacnorthoamide derivative 2 with one tacnorthoamide unit and 6 with
two tacnorthoamide units linked by an alkyl (1,6-hexamethylene) spacer without anthraquinone have also
been synthesized. The DNA-binding property investigated via fluorescence and CD spectroscopy suggests
that compounds 1 and 2 have an intercalating DNA binding mode, and the apparent binding constants of
1, 2 and 6 are 1.3 × 107 M−1, 0.8 × 107 M−1 and 8 × 105 M−1, respectively. Agarose gel electrophoresis
was used to assess plasmid pUC19 DNA cleavage activity promoted by 1, 2, 6 and parent tacnoa under
physiological conditions, which gives rate constants kobs of 0.2126 0.0055 h−1, 0.0620 0.0024 h−1
,
0.040 0.0007 h−1 and 0.0043 0.0002 h−1, respectively. The 50-fold and 15-fold rate acceleration over
parent tacnoa is because of the anthraquinone moiety of compound 1 or 2 intercalating into DNA base
pairs via a stacking interaction. Moreover, DNA cleavage reactions promoted by compound 1 give
5.3-fold rate acceleration over compound 6, which further demonstrates that the introduction of
anthraquinone results in a large enhancement of DNA cleavage activity. In particular, DNA cleavage
activity promoted by 1 bearing two tacnoa units is 3.3 times more effective than 2 bearing one tacnoa
unit and the DNA cleavage by compound 1 was achieved effectively at a relatively low concentration
(0.03 mM). This dramatic rate acceleration suggests the cooperative catalysis of the two positively
charged tacnoa units in compound 1. The radical scavenger inhibition study and ESI-MS analysis of
bis(2,4-dinitrophenyl) phosphate (BDNPP) and adenylyl(3′-5′)phosphoadenine (APA) cleavage in the
presence of compound 1 suggest the cleavage mechanism would be via a hydrolysis pathway by cleaving
the phosphodiester bond of DNA.
pharmic use of some metal complexes, such as Cu complexes, is
Introduction
hampered by concerns over the lability and toxicity in the treat-
ment of cancer due to free radical generation via a redox
pathway.4 Recently, metal-free cleaving reagents have been put
forward by Göbel and co-workers.5 In fact, DNA cleavage is
considered to be safer for therapy in the absence of transition
metal ions. Some organic molecules, such as guanidinium
derivatives,6 macrocyclic polyamines,7 pseudorotaxane com-
posed of cucurbituril8 and peptides,9 have been studied as DNA
cleaving reagents in recent years. In particular, Yu and co-
workers10 described a metal-free peptide nucleic acid (PNA)–
cyclen conjugate as a DNA-cutting agent with efficient and site-
selective DNA hydrolysis activity. Yavin’s group11 successfully
designed a cyclic peptide scaffold conjugated to anthraquinone
as a metal-free DNA nuclease. DNA cleavage was promoted at
micromolar concentration under physiological conditions. We
have also synthesized a metal-free DNA nuclease containing
guanidinoethyl and hydroxyethyl side arms, and DNA cleavage
DNA cleaving reagents have attracted continuous and extensive
interest due to their potential applications in the fields of molecu-
lar biological technology and drug development.1 Among them,
DNA cleavage promoted by transition metal complexes has been
widely investigated in the last decade.2 In particular, transition
metal dinuclear complexes are found to be quite efficient due to
synergistic catalysis of the two metal centers,3 but the clinical
aDepartment of Chemistry, State Key Laboratory of Coordination
Chemistry, Nanjing University, Nanjing 210093, Jiangsu, P.R. China.
E-mail: lugyuan@nju.edu.cn; Fax: +86-25-83317761;
Tel: +86-25-83685613
bKey Laboratory of Fine Petrochemical Engineering, Changzhou
University, Changzhou 213164, Jiangsu, P. R. China
†Electronic supplementary information (ESI) available: Spectra for all
new compounds, agarose gel electrophoretogram, and kinetic data. See
DOI: 10.1039/c2ob25743b
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promoted by the compound without metal ions was achieved
effectively at a relative low concentration (0.1 mM), giving
107-fold rate acceleration over uncatalyzed double-stranded
DNA.12 However, the DNA phosphodiester bond is very stable
under physiological conditions with a half-life hydrolysis rate of
about 200 million years, which is primarily due to the repulsion
between the negatively charged backbone and potential nucleo-
philes.13 In nature, the cleavage rate of DNA via a hydrolytic
pathway is about 1015–1016-fold accelerated by a natural nucle-
ase such as staphylococcal nuclease (SNase).14 Therefore, the
cleaving efficiency of artificial nuclease is still far from that of
natural nuclease. Thus, to develop highly efficient metal-free
DNA cleaving reagents via a non-oxidative pathway is not only
necessary for practical value but also is a challenging subject.
It is well known that an organic positively charged group such
as ammonium or guanidinium is the key functional group at the
active site in a natural nuclease such as staphylococcal nuclease
(SNase) and bovine pancreatic ribonuclease (RNase A).15 Some
compounds with a positively charged group as nuclease mimics
for cleavage of phosphodiester have been reported, a few of
them were identified as efficient cleavers of RNA.16 The
tacnorthoamide is a rigid tribridge circular structure containing
both an organic positively charged ammonium group and proto-
nated or deprotonated tertiary amino groups.17 Therefore, the
tacnoa moiety is expected to be a cleaving group, which has not
been reported so far. In addition, anthraquinone derivatives are
well-known effective DNA intercalators.18 The introduction of
anthraquinone would enhance the binding affinity of a com-
pound to the substrate (DNA). Thus we report here, for the first
time, the design and synthesis of a novel metal-free DNA cleav-
ing reagent bis-tacnorthoamide derivative 1, in which two tacnoa
units and anthraquinone are linked by two alkyl (1,6-hexamethy-
lene) spacers (Scheme 1). Compound 1 has two side arms
bearing cationic tacnorthoamide like a pair of long-chain drop
earrings, a similar model to dinuclear synergetic metallo-
nuclease. It would be expected to catalyze the cleavage of DNA
cooperatively via a hydrolytic mechanism in the absence of
metal ions. For comparison, the corresponding compounds
mono-tacnorthoamide derivative 2 with one tacnorthoamide
unit and 6 with two tacnorthoamide units linked by an alkyl
(1,6-hexamethylene) spacer without anthraquinone have also
been synthesized (Scheme 1). Binding behaviors of these com-
pounds with calf thymus DNA (CT-DNA) were investigated by
fluorescence and circular dichroism (CD) spectroscopy, and
plasmid pUC19 DNA cleaving behavior was assessed via the
agarose gel electrophoresis. The DNA cleavage activity of the
parent compound tacnoa was studied as well. The mechanism of
the cleavage process was suggested on the basis of a radical sca-
venger inhibition study and ESI-MS analysis of bis(2,4-dinitro-
phenyl) phosphate (BDNPP) and adenylyl(3′–5′)phosphoadenine
(APA) cleavage in the presence of compound 1.
Results and discussion
Synthesis of compounds 1, 2 and 6
Syntheses of compounds 1, 2 and 6 were achieved via a series of
nucleophilic substitution reactions from 1,8-dihydroxyanthraqui-
none and 1,6-dibromohexane (Scheme 2). Triazatricyclo
[5.2.1.04,10]decane (tacnoa)3a and compound 319 were prepared
according to the literature method. All the target compounds are
ammonium salts, so they gradually precipitated from anhydrous
tetrahydrofuran, and then were filtered off. The purified com-
pound was obtained after washing with THF and ether.
1H NMR, 13C NMR, ESI-MS spectra (ESI,† p2–9) and elemen-
tal analysis data of compounds 1, 2 and 6 are in accord with the
1
assigned structure. H NMR spectra of compounds 1, 2 and 6
show the methine single peak at δ 5.4. In the 13C NMR spectrum
of compound 1, the peaks at δ 116.7, 118.3, 120.0, 122.3, 133.3,
134.5, 158.1 are assigned to the central carbon atom of tacnoa
and aryl carbon atoms; the peaks at δ 183.2, 183.8 are assigned
to the signals of carbonyl carbon atoms in anthraquinone. In the
13C NMR spectrum of compound 2, the peaks at δ 118.2, 118.9,
118.9, 119.0, 119.2, 119.5, 123.3, 124.0, 124.1, 134.0, 134.8,
158.1, 159.2 and 182.7, 183.9 are ascribed to signals of the
tacnoa central carbon atom, aryl carbon atoms and carbonyl
carbon atoms in anthraquinone, respectively. In the 13C NMR
spectrum of compound 6, the peak at 116.5 is assigned to the
central carbon atom of tacnoa. In the ESI-MS spectra, the signal
at m/z 342.42 is assigned as [1 − 2Br−]2+ (calcd 342.22), m/z
Scheme 2 Synthesis of compounds 1, 2 and 6. Reagents and con-
ditions: (a) 1,6-dibromohexane, K2CO3, N2, 110 °C, 72 h, 55.3%; (b)
tacnorthoamide, NaI, THF, N2, rt, 18 h, 77.4%; (c) iodomethane,
K2CO3, N2, 55 °C, 24 h, 75.6%; (d) the same as (a), 75.0%; (e) the
same as (b), 79.0%; (f) tacnorthoamide, NaI, THF, N2, rt, 24 h, 86.9%.
Scheme 1 Chemical structure of compounds 1, 2, 6 and tacnoa.
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476.58 corresponds to [2 − Br−]+ (calcd 476.25) and m/z 181.15
corresponds to [6 − 2Br−]2+ (calcd 181.25), respectively.
concentration is the value at a 50% reduction of the fluorescence
intensity of EB and KEB = 1.0 × 107 M−1 ([EB] = 3.9 μM).21
The Kapp values for 1, 2 and 6 are 1.3 × 107 M−1, 0.8 × 107 M−1
and 8 × 105 M−1, respectively. The calculated value illustrates
that both compounds 1 and 2 have stronger affinity to CT-DNA
than that of compound 6 as a result of the presence of anthraqui-
none in their chemical structure which facilitates DNA binding
propensity. The DNA-binding mode could be evaluated from the
magnitude of the binding constants: a value above 106 M−1 is an
indication of classical intercalation (ethidium and daunomycin
bind DNA with an affinity over 106 M−1), while values in the
range of 104–105 M−1 imply a groove binding mode or electro-
static interaction.22 Therefore, we concluded that the binding of
compounds 1 and 2 to CT-DNA could be accomplished via the
intercalation binding mode, while compound 6 to DNA is via
groove or electrostatic interaction. In fact, the anthraquinone ring
is very common as a DNA intercalating group and potential anti-
tumour agent.23 In addition, there was no obvious difference
between 1 and 2 in their quenching constant K and the apparent
binding constant Kapp, only that 1 was slightly higher than 2.
This result illustrates that the anthraquinone group, present in
both their structures, plays a dominant role in the process of
binding with DNA.
DNA binding assays
DNA binding is an essential step for DNA cleavage in most
cases. Therefore, the binding behaviors of 1, 2 and 6 to calf
thymus DNA (CT-DNA) have been studied by fluorescence and
CD spectroscopy.
Fluorescence spectroscopic studies
The fluorescence spectral method is one of the convenient tools
for examining the interaction between small molecules and
nucleic acids. The binding of the compounds to CT-DNA was
studied by evaluating the fluorescence emission intensity of the
ethidium bromide (EB)–DNA system with the addition of these
compounds. The emission intensity of ethidium bromide (EB) is
used as a spectral probe as EB shows a significant enhancement
of the intensity when bound to DNA. Binding of the compounds
to DNA decreases the emission intensity and the extent of the
reduction of the emission intensity gives a measure of the DNA
binding propensity of the investigated compounds. The fluor-
escence quenching effect of EB bound to DNA induced by 1, 2
and 6 is shown in Fig. S1† (ESI, p10–11), in which the fluor-
escence intensity is at 604 nm (λex = 530 nm) for EB in bound
form. Addition of compounds caused a reduction in the emission
intensity. The relative binding propensity of compound to
CT-DNA was determined from the classical Stern–Volmer
equation I0/I = 1 + Kr, where I0 and I are the fluorescence inten-
sities in the absence and presence of the quencher; K is the
linear Stern–Volmer quenching constant dependent on the ratio
of rbE (the ratio of the bound concentration of EB to the concen-
tration of DNA); r is the ratio of total concentration of quencher
to that of DNA.20 Fig. 1 is the plot of I0/I versus [quencher]/
[DNA]; the quenching constant K is given by the gradient of the
slope. The quenching constant K values obtained for 1, 2 and 6
are 1.3 0.02, 0.8 0.01 and 0.08 0.001, respectively.
Circular dichroism studies
Circular dichroism is a very sensitive, powerful technique for
diagnosing changes in DNA morphology during drug–DNA
interactions, as the positive band due to base stacking (275 nm)
and the negative band due to right-handed helicity (248 nm) are
quite sensitive to the mode of DNA interaction with small mo-
lecules.24 The change in CD signal of DNA observed on inter-
action with a compound may often be assigned to the
corresponding change in DNA structure.25 Thus, simple groove
binding and electrostatic interaction of small molecules show
little or no perturbation on the base-stacking and helicity bands,
while intercalation enhances the intensities of both the bands
stabilizing the right-handed B conformation of CT-DNA as
observed for the classical intercalator methylene blue.26 Fig. 2
displays CD spectra of CT-DNA treated with 1, 2 and 6 with the
ratio of 0.4 ([compound]/[DNA]), the positive band (∼275 nm)
The apparent binding constant (Kapp) is also calculated from
the equation KEB[EB] = Kapp[compound], where the compound
Fig. 1 Stern–Volmer quenching plots of EB bound to DNA by 1 (▲),
2 (□) and 6 (●), which give the quenching constants K. Experiments
were conducted by adding 0–3.6 μM 1, 2 or 6 to the EB-bound
CT-DNA solution in 5 mM Tris-HCl buffer (pH 7.0) at room
temperature.
Fig. 2 CD spectra of CT-DNA (0.129 mM) in alone and its interaction
with 1, 2 and 6 at the ratio of [compound]/[DNA] = 0.4. All the spectra
were recorded in 5 mM Tris-HCl buffer (pH 7.0) at room temperature.
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of CT-DNA decreases in intensity with the addition of the com-
pound, while the negative band (∼247 nm) undergoes the
obvious reduction. The results suggest that compound 6 shows a
slight perturbation of DNA while compounds 1 and 2 unwind
the DNA helix and lead to the loss of helicity possibly due to
the partial intercalation of 1 and 2 into the DNA base pairs.27
From the results of these fluorescence and CD spectroscopic
studies, it is concluded that the binding interaction ability of the
compounds to CT-DNA follows the order 1 > 2 ≫ 6, and the DNA
binding constants of 1 and 2 indicate that both of the compounds
might bind strongly with DNA via the intercalation binding mode.
Fig. 4 Plots of % cleaved DNA vs. different concentrations of 1, 2, 6
and tacnoa. (The agarose gel of pUC19 DNA (0.025 mM bp) was incu-
bated with different concentrations of 1, 2, 6 and tacnoa for 16.0 h at
37 °C in pH 7.25 buffer (50 mM Tris-HCl–5 mM NaCl) (Fig. S5,† ESI,
p13–14)).
DNA cleavage activity
pH Dependence of DNA cleavage promoted by 1 and 2. Incu-
bation of supercoiled pUC19 DNA with compound 1 or 2 for
16 h at 37 °C results in a different extent of cleavage of DNA
depending on the corresponding pH value of the buffer. Fig. 3
shows the bell-shaped pH-dependent profiles for DNA cleavage
which indicate that pH 7.25 is the optimal pH for DNA cleavage
in the presence of compound 1 or 2. Therefore, pH 7.25 was
selected for all of the following DNA cleavage reactions. The
two compounds have almost the same change of cleavage
activity at various pH values at 37 °C. According to the structure
of the compounds, the pH value of the buffer affects the protona-
tion or deprotonation of the nitrogen atom on the tacnoa moiety
which may result in nucleophilic attack of compounds to sub-
strate, and then determines cleavage efficiency of DNA. Conse-
quently, we infer that function of tacnoa moiety in compounds is
as the cleavage part. In order to verify this conclusion, we con-
ducted a control experiment: incubation of supercoiled pUC19
DNA with compound 3, which had no tacnoa in its structure, for
16 h at 37 °C. The experimental result is shown in Fig. S3†
(ESI, p12) and indicates that compound 3 has almost no cleavage
activity on DNA, which confirms the inference. The impact of
ionic strength on cleavage activity for compound 1 was also
investigated (Fig. S4,† ESI, p13). The results showed that the
cleavage activity decreases with an increase in the ionic strength,
and therefore a relatively low NaCl concentration (5 mM) was
used for controlling ionic strength in all experiments.
Concentration dependence assay of DNA cleavage promoted by
1, 2, 6 and tacnoa
Cleavage reactions that create relaxed circular DNA from super-
coiled DNA over various concentrations of 1, 2, 6 and tacnoa
(0.0006–0.067 mM) and constant DNA concentration (25 μM,
bp) were carried out for 16.0 h at 37 °C in pH 7.25 buffer
(50 mM Tris-HCl–5 mM NaCl). Fig. 4 displays the plots of %
cleaved DNA vs. different concentrations, which shows that clea-
vage activities on DNA of these compounds increase with the
increase of their concentrations. Under the same concentration
condition, DNA cleavage abilities of these compounds are in the
following order: 1 > 2 > 6 > tacnoa. However, a further increase
concentration of 1 (larger than 0.067 mM) results in DNA
becoming stuck in the well of the gel (the agarose gel electro-
phoretogram is given in Fig. S6,† ESI, p13–14). This phenom-
enon may due to the formation of relatively high molecular
weight DNA-1 complexes from the strong binding between
DNA and the anthraquinone group.
Kinetics assays
The kinetics of pUC19 DNA degradation have been studied.
Fig. 5 shows the time course of the supercoiled plasmid DNA
cleavage promoted by 1 (0.033 mM) into nicked form in Tris-
HCl buffer (pH 7.25) at 37 °C. The rate of conversion from
form I to form II increases with increased reaction time. The
graph shows that the extension of supercoiled DNA cleavage
varies exponentially with the reaction time giving pseudo first-
order kinetics with an apparent initial first-order rate constant
(kobs) of 0.183 0.0104 h−1. The apparent initial first-order rate
constants of DNA cleavage reactions promoted by a series of
various concentrations of 1, 2 and 6 under the same conditions
as described above are summarized in Table S1† (ESI, p15). The
kinetics profiles of the supercoiled DNA cleavage at various con-
centrations of 1, 2 and 6 are presented in Fig. 6. In order to
clarify the impact of introduction of anthraquinone and the load
of positive charge on DNA cleavage efficiency, the DNA-cleaving
behavior of parent tacnoa was also investigated under the same
conditions in 50 mM Tris-HCl–5 mM NaCl buffer (pH 7.25).
Fig. 3 pH-Dependent profile for pUC19 DNA (0.025 mM bp) clea-
vage promoted by 0.033 mM 1 (■) and 2 (▲) in Tris-HCl buffer of
different pH values. (The agarose gel (Fig. S2,† ESI, p12) of pUC19
DNA (0.025 mM bp) was incubated for 16 h in buffer of different pH
values (50 mM Tris-HCl) at 37 °C.)
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Studies on the mechanism of DNA cleavage
The anthraquinone moiety usually undergoes a redox pathway,
which could directly produce a cytotoxic effect.28 To confirm if
reactive oxygen species (ROS) are, at least partly, responsible for
the cleavage of DNA promoted by compound 1, reactions were
carried out in the presence of typical scavengers29 for singlet
oxygen (NaN3), for superoxide (KI), and for hydroxyl radical
(DMSO and t-BuOH) (Fig. 7; Fig. S7 and Table S3† of the ESI,
p31–32). Obviously, there is no significant inhibition effect on
DNA cleavage in the presence of any of the scavengers (NaN3,
KI, DMSO and t-BuOH), which rules out the involvement of
these reactive oxygen species, at least in a free and diffusible
form, and implies a non-oxidative pathway.
Generally, the oxidative DNA cleavage process is due to the
oxidation of the ribose or base group of DNA by the reactive
oxygen species.30 Thus, the DNA samples containing 1 were
incubated in the presence of four nucleosides (adenosine,
uridine, guanosine and cytidine, respectively) with the ratio of
[nucleoside]/[DNA bp] = 1 : 1, followed by electrophoresis and
quantitation (Fig. S8,† ESI, p32). No inhibition in the DNA clea-
vage was detected after the treatment with 1 in the presence of
each of the four nucleosides. This suggests a non-oxidative
process (Table 1). On the other hand, in the DNA cleavage
Fig. 5 Time course of pUC19 DNA (0.025 mM bp) cleavage pro-
moted by 1 (0.033 mM). Inset is the agarose gel (1%) of the time-vari-
able reaction products. lanes 1–7, reaction times of 0, 1.15, 2.30, 3.45,
4.60, 5.75 and 6.90 h, respectively. The reactions were carried out at
37 °C in 50 mM Tris-HCl–5 mM NaCl buffer (pH 7.25).
Fig. 6 Kinetics plot of kobs versus various concentrations of com-
pounds 1, 2, 6 and tacnoa. Every reaction was carried out at 37 °C in
50 mM Tris-HCl–5 mM NaCl buffer (pH 7.25).
Fig. 7 Histogram representing cleavage of pUC19 plasmid DNA
(0.025 mM bp) by 1 (0.04 mM) in the presence of standard radical sca-
vengers for singlet oxygen (NaN3, 10 mM), for superoxide (KI,
10 mM), and for hydroxyl radical (1 mM DMSO and 1 mM t-BuOH),
incubated for 16 h at 37 °C in pH 7.25 buffer (50 mM Tris-HCl/5 mM
NaCl).
The apparent initial first-order rate constants kobs of DNA clea-
vage reactions promoted by 1, 2, 6 and tacnoa are 0.2126
0.0055 h−1, 0.062 0.0024 h−1, 0.040 0.0007 h−1 and 0.0043
0.0002 h−1, respectively. The 50-fold and 15-fold rate accelera-
tion over parent tacnoa is because of the anthraquinone moiety
of compound 1 or 2 intercalating into DNA base pairs via a
stacking interaction. This indicates that the intercalating subunit
– anthraquinone – evidently increases binding ability to the sub-
strate (DNA) and then increases DNA cleavage activity effec-
tively. Moreover, DNA cleavage reactions promoted by
compound 1 give 5.3-fold rate acceleration over that of com-
pound 6 despite both of them have two tacnoa units. This further
demonstrates that the introduction of anthraquinone in compound
1 results in a large enhancement of DNA cleavage activity. In
particular, the DNA cleavage activity promoted by 1 bearing two
tacnoa units is 3.33 times more effective than that of 2 bearing
one tacnoa unit. And the binding affinity and mode of the two
compounds to DNA are almost the same from the study of fluor-
escence and circular dichroism spectroscopy. Therefore, the rate
acceleration of DNA cleavage promoted by 1 over 2 indicates
that the two positively charged tacnoa units in compound 1 cata-
lyze cleavage of DNA cooperatively.
Table 1 DNA cleavage promoted by 1 in the presence of nucleoside
monophosphate or BDNPPa (Fig. S8, ESI, p32)
DNA %
Added compounds
Form I
Form II
DNA control
1 only
1 + Adenosine
1 + Uridine
1 + Guanosine
1 + Cytidine
97.35
19.29
19.70
20.31
24.23
26.22
58.40
76.88
2.65
80.71
80.30
79.69
75.77
73.78
41.60
23.12
1 + BDNPP (0.10 mM)
1 + BDNPP (0.20 mM)
a Cleavage reactions were carried out in pH 7.25 Tris-HCl buffer for
10 h at 37 °C.
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reactions promoted by 1, when the active phosphates bis(2,4-
dinitrophenyl) phosphate (BDNPP) without the pentose and the
pyrimidine or purine base were added, the reactions of DNA
cleavage were partially inhibited (Table 1) (Fig. S8,† ESI, p32).
The inhibition can be ascribed to the preferential hydrolyzed
phosphodiester of BDNPP. It implies that the hydrolysis pathway
for the DNA cleavage process is possible.
peaks. The generation of DNPP and DNP indicates that a poss-
ible cleavage mechanism of BDNPP promoted by compound 1
is via a hydrolysis pathway of the phosphodiester bond. Thus,
similar to BDNPP, the hydrolysis of the phosphodiester bond is a
possible mechanism for the DNA cleavage promoted by 1.
In addition, mechanistic profiles of DNA cleavage by 1 were
also evaluated in the presence of excess EDTA to scavenge
adventitious transition metal ions. (Fig. S11 and Table S4,† ESI,
p37). There was almost no inhibition of DNA cleavage, which
rules out the involvement of adventitious transition metal ions.
To study the cleavage mechanism, a small dinucleotide model
system, adenylyl(3′–5′)phosphoadenine (APA) was used as the
nucleic acid mimic. ApA (0.10 mM) and 1 (0.05 mM) were dis-
solved in deionized water (1 : 1), and after reaction for 16 h at
37 °C, ESI-MS analysis was carried out. In the ESI-MS spec-
trum (Fig. S9(1),† ESI, p33), besides the peak at m/z 342.42
indicating the signal of species [1 − 2Br−]2+ (calcd m/z 342.42),
the signals at m/z 268.17 and 346.00 show the presence of ApA
cleavage products adenosine (A) ([A + H]+, calcd m/z 268.10)
and adenosine monophosphate (AMP) ([AMP − H]−, calcd m/z
346.06); no sign of ApA was found. An ESI-MS analysis of
ApA alone was also carried out as a control experiment under
the same conditions (Fig. S9(2),† ESI, p34), showing only the
signal of ApA and no signs of A and AMP. The generation of
adenosine and AMP indicates that the possible cleavage mecha-
nisms of ApA promoted by compound 1 would be via an intra-
molecular transphosphorylation to form cAMP, and then
hydrolysis, or for the phosphodiester bond of ApA hydrolyse
directly.
To further study the cleavage mechanism, bis(2,4-dinitrophenyl)
phosphate (BDNPP) was used as a DNA mimic (Scheme 3).
BDNPP and 1 were dissolved in DMF–deionized water (v : v =
1 : 1) at 37 °C. ESI-MS analysis was carried out. At the begin-
ning, the peaks of ESI-MS spectra at m/z 342.35 and 428.90
(Fig. S10(1),† ESI, p35) show the signals of [1 − 2Br−]2+ (calcd
342.22) and [BDNPP − H+]− (calcd 429.18), respectively. After
3 h, besides the peaks at m/z 342.35 ([1 − 2Br−]2+) and 428.90
([BDNPP − H+]−), the signals at m/z 182.95 and 263.05
(Fig. S10(2),† ESI, p36) show the presence of BDNPP cleavage
products ([DNP − H+]−, calcd m/z 183.11) and ([DNPP − H+]−,
calcd m/z 263.09), respectively. After 5 h, the peak at 428.90
([BDNPP − H+]−) disappears, ESI-MS spectra (Fig. S10(3),†
ESI, p36) show only peaks (m/z 182.95 and 263.05) of BDNPP
cleavage products. An ESI-MS analysis of BDNPP alone was
also carried out as a control experiment under the same con-
ditions, showing only the signal of BDNPP and no signs of new
Experimental
Materials
Plasmid pUC19 DNA was purchased from TaKaRa Biotechnol-
ogy Co. Ltd., and the purity was checked by agarose gel electro-
phoresis and the concentration was determined by UV
spectroscopy using the extinction coefficient appropriate for
double-stranded DNA (1.0 OD260 = 50 μg mL−1). Agarose was
from Oxoid Limited of Basingstoke, ethidium bromide (EB) was
from Amresco. Inc., and tris(hydroxymethyl)amino-methane
(Tris-Base) was from Robiot Co. Ltd. Dinucleotide (APA) and
bis(2,4-dinitrophenyl) phosphate (BDNPP) were purchased from
Sigma Aldrich. Bromophenol blue, glycerol and ethyldiamine-
tetraacetic acid (EDTA) were commercially available. Deionized
water was obtained by ionized column from double distilled
water. All reagents and chemicals were of analytical grade and
used without further purification. All solvents were purified by
standard procedures.
The stock solution of CT-DNA (stored at 4 °C and used for
not more than 2 d) was prepared in 5 mM Tris-HCl in water, pH
7.0. The concentration of CT-DNA was determined according to
its absorption intensity at 260 nm with a known molar extinction
coefficient value of 6600 M−1 cm−1. The ratio of the UV absor-
bance at 260 and 280 nm, A260/A280 = 1.8–1.9, indicated that
DNAwas sufficiently free of protein.31
Apparatus
1H NMR and 13C NMR data were recorded on a Brucker AM
300 spectrometer (Germany). Mass spectra were obtained on an
electrospray mass spectrometer (LCQ, Finnigan). Elemental ana-
lyses were carried out using Perkin Elmer 240C. The fluorescent
spectra and CD spectra were carried out using AMINCO
Bowman Series 2 luminescence spectrometer and Jasco J-810
automatic recording spectropolarimeter, respectively. The pH
value was confirmed by ORION868 pH meter with an Ag/AgCl
electrode as the reference electrode in saturated KCl solution at
room temperature. The agarose gel electrophoresis was con-
ducted by DYY-5 electrophoresis apparatus. Bands were visual-
ized by UV light and photographed using DigiDoc–ItTM gel
imaging and documentation system (version 1.1.23, UVP, Inc.
Unpland, CA). The intensity of the DNA bands was estimated
by TotalLab image analysis software (version 2.01).
Fluorescence measurements. The fluorescent spectral studies
were performed by the measurement of the emission intensity of
ethidium bromide (EB) on an AMINCO Bowman Series 2
luminescence spectrometer. The experiments were done by
Scheme 3 The cleavage reaction of BDNPP in the presence of com-
pound 1.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8484–8492 | 8489

View Article Online
adding 0–3.6 μM 1 or 2 into the EB-bound CT-DNA (3.9 μM)
solution in 5 mM Tris-HCl buffer (pH 7.0), and the fluorescence
was measured and normalized to 100% relative fluorescence.
Circular dichroism measurements. All CD spectroscopic
studies were carried out with a continuous flow of nitrogen
purging the polarimeter, and the measurements were performed
at room temperature with 1 cm pathway cells. The CD spectra
were run from 320–220 nm at a speed of 20 nm min−1 and the
buffer background was automatically subtracted. Data were
recorded at an interval of 0.1 nm. The CD spectrum of CT-DNA
alone (129 μM) was recorded as control experiment.
calcd 439.06, found 439.08; [M(81Br) + Na+] calcd 441.06,
found 441.08.
1,8-Bis(1-dihexyloxy-1-azonia-4,7-diazatricyclo[5.2.1.04,10]-
decane)anthracene-9,10-dione dibromide (1). Triazatricyclo
[5.2.1.04,10]decane (tacnoa) (0.74 g, 5.32 mmol) and NaI
(1.2 mg, 0.008 mmol) were added to 5 mL anhydrous THF. An
anhydrous THF (20 mL) solution of 3 (0.30 g, 0.53 mmol) was
then added dropwise with stirring at room temperature under an
atmosphere of dry N2. The mixture was refluxed for 18 h and
then cooled to 30 °C, stirred for another 48 h. A yellow solid pre-
cipitated, and was filtered off, washed with THF (2 × 1 mL) and
ether (2 × 2 mL), and then taken to dryness under reduced
pressure. A yellow solid (0.35 g, 0.41 mmol) was obtained.
General procedures for the synthesis of compounds
1
Yield 77.4%; H NMR (300 MHz, D2O) δ 1.44–1.76 (m, 8H,
Triazatricyclo[5.2.1.04,10]decane (tacnoa). Triazatricyclo
[5.2.1.04,10]decane was prepared according to the literature
method.3a 1H NMR (300 MHz, CDCl3) δ 2.67–2.74 (m, 6H,
3CH2), 2.92–3.00 (m, 6H, 3CH2), 4.93 (s, 1H, CH); 13C NMR
(75 MHz, CDCl3) δ 51.8 (CH2), 104.0 (CH).
4 × CH2), 1.91–2.04 (m, 8H, 4 × CH2), 3.21–3.33 (m, 8H, 4 ×
NCH2), 3.55–3.65 (m, 8H, 4 × NCH2), 3.77–3.84 (m, 4H, 2 ×
NCH2), 3.91–4.06 (m, 8H, 4 × NCH2), 4.12–4.16 (m, 4H, 2 ×
OCH2), 5.47 (s, 2H, 2 × methine H), 7.27–7.30 (d, J = 8.5 Hz,
2H, 2 × ArH), 7.58–7.64 (m, 2H, 2 × ArH), 7.79–7.81 (d, J =
7.0 Hz, 2H, 2 × ArH); 13C NMR (75 MHz, D2O) δ 24.0 (CH2),
24.8 (CH2), 25.6 (CH2), 28.0 (CH2), 51.0 (NCH2), 51.7 (NCH2),
53.3 (NCH2), 57.9 (NCH2), 60.0 (NCH2), 69.2 (OCH2), 116.7,
118.3, 120.0, 122.3, 133.3, 134.5, 158.1 (tacnoa central C and
Ar C), 183.2 (CvO), 183.8 (CvO); ESI-MS m/z [M − 2Br−]2+
calcd 342.22, found 342.42; Anal. Calcd for C40H56Br2N6O4:
C, 56.87; H, 6.68; N, 9.95; Found: C, 56.79; H, 6.55; N, 9.73%.
1-Hydroxy-8-methoxy-9,10-anthraquinone (4). The com-
pound 4 was synthesized according to the literature procedure
with some modification.32 1,8-Dihydroxyanthraquinone (1.5 g,
6.2 mmol), anhydrous potassium carbonate (2.76 g, 20 mmol)
and iodomethane (0.3 mL, 4.65 mmol) were added to 60 mL
acetone and the mixture stirred at 55 °C under an atmosphere of
dry N2. The reaction was monitored by TLC. After 24 h, the
reaction mixture was cooled and filtered, and the residue was
washed with acetone (3 × 20 mL). All of the organic layers were
merged and the solvent was removed under vacuum. Column
chromatography (silica gel, petrol ether–ethyl acetate, 4 : 1, v/v)
afforded 1-hydroxy-8-methoxy-9,10-anthraquinone (1.2 g,
1-Methoxy-8-(1-hexyloxy-1-azonia-4,7-diazatricyclo[5.2.1.04,10]-
decane)anthracene-9,10-dione bromide (2). Compound 2 was
synthesized in a similar procedure to compound 1 using 1,4,7-
triazatricyclo[5.2.1.04,10]decane (tacnoa) (0.57 g, 4 mmol), NaI
(1.2 mg, 0.008 mmol) and 1-methoxy-8-(6-bromohexyloxy)-
anthraquinone (5) (0.22 g, 0.53 mmol). A yellow solid (0.23 g,
1
4.72 mmol) as an orange solid. Yield 75.6%; mp 95–96 °C; H
1
NMR (300 MHz, CDCl3) δ 4.08 (s, 3H, OCH3), 7.27–7.31 (dd,
J = 8.2 Hz, 0.9 Hz, 1H, ArH), 7.35–7.39 (dd, J = 8.2 Hz,
0.9 Hz, 1H, ArH), 7.62 (t, J = 8.1 Hz, 1H, ArH), 7.73–7.79
(m, 2H, 2 × ArH), 7.95–7.99 (m, 1H, ArH).
0.42 mmol) was obtained. Yield 79.0%; H NMR (300 MHz,
CDCl3) δ 1.27–2.00 (m, 8H, 4 × CH2), 3.03–3.04 (m, 2H,
NCH2), 3.23–3.26 (m, 2H, NCH2), 3.41–3.45 (m, 2H, NCH2),
3.47–3.50 (m, 2H, NCH2), 3.67–3.74 (m, 2H, NCH2), 3.75–3.78
(m, 2H, NCH2), 4.01 (s, 3H, OCH3), 4.15–4.17 (m, 2H, NCH2),
4.25–4.30 (m, 2H, OCH2), 5.47 (s, 1H, methine H), 7.30–7.33
(m, 2H, 2 × ArH), 7.60–7.67 (m, 2H, 2 × ArH), 7.78–7.85 (m,
2H, 2 × ArH); 13C NMR (75 MHz, CDCl3) δ 24.5 (CH2), 25.3
(CH2), 25.7 (CH2), 28.1 (CH2), 52.2 (NCH2), 52.6 (NCH2), 56.0
(NCH2), 56.8 (NCH2), 57.7 (NCH2), 58.9 (OCH2), 69.1
(OCH3), 118.2, 118.9, 118.9, 119.0, 119.2, 119.5, 123.3, 124.0,
124.1, 134.0, 134.8, 158.1, 159.2 (tacnoa central C and Ar C),
182.7 (CvO), 183.9 (CvO); ESI-MS m/z [M − Br−]+ calcd
476.25, found 476.58; Anal. Calcd for C28H34BrN3O4: C, 60.43;
H, 6.16; N, 7.55; Found: C, 60.38; H, 6.04; N, 7.34%.
1-Methoxy-8-(6-bromohexyloxy)anthraquinone (5). 1-Hydroxy-
8-methoxy-9,10-anthraquinone (4) (1.2 g, 4.72 mmol) and anhy-
drous potassium carbonate (3.0 g, 22 mmol) were added to
1,6-dibromohexane (20 mL, 130 mmol) and the mixture stirred
at 110 °C under an atmosphere of dry N2. A color change from
orange to brown was observed. After 72 h, the reaction mixture
was cooled and filtered, and the residue was washed with CHCl3
(3 × 10 mL). All of the organic layers were merged and the
solvent was removed under vacuum. Column chromatography
(silica gel, petrol ether–dichloromethane, 1 : 1, v/v) followed by
recrystallization from EtOAc afforded 5 (1.35 g, 2.4 mmol) as
1
orange solid. Yield 75.0%; mp 96–97 °C; H NMR (300 MHz,
1,1′-(Hexamethylene)bis(1-azonia-4,7-diazatricyclo[5.2.1.04,10]-
decane) dibromide (6). A solution of (0.61 g, 2.5 mmol)
1,6-dibromohexane in anhydrous tetrahydrofuran (5 mL) was
added to a stirred solution of 1,4,7-triazatricyclo[5.2.1.04,10]-
decane (0.696 g, 5 mmol) and NaI (1.2 mg, 0.008 mmol) in
anhydrous tetrahydrofuran (25 mL) under an atmosphere of dry
N2, resulting in a pale yellow precipitate. After stirring for 24 h,
the precipitate was filtered off; washed with tetrahydrofuran
(25 mL), and then ether (25 mL), dried in a vacuum desiccator.
A off-white solid (1.13 g, 2.17 mmol) was obtained. Yield
CDCl3) δ 1.55–1.67 (m, 4H, 2 × CH2), 1.92–2.00 (m, 4H, 2 ×
CH2), 3.39 (t, J = 7.0 Hz, 2H, CH2Br), 3.90 (s, 3H, OCH3), 4.07
(t, J = 6.5 Hz, 2H, CH2O), 7.18–7.24 (m, 2H, 2 × ArH),
7.49–7.56 (m, 2H, 2 × ArH), 7.71–7.75 (m, 2H, 2 × ArH);
13C NMR (75 MHz, CDCl3) δ 25.1 (CH2), 27.8 (CH2), 28.8
(CH2), 32.6 (CH2), 33.9 (CH2Br), 56.6 (OCH3), 69.4 (OCH2),
118.0 (Ar C), 118.8 (Ar C), 119.4 (Ar C), 124.1 (Ar C), 124.2
(Ar C), 133.7 (Ar C), 134.7 (Ar C), 158.8 (Ar C), 159.3 (Ar C),
182.3 (CvO), 184.0 (CvO); ESI-MS m/z [M (79Br) + Na+]
8490 | Org. Biomol. Chem., 2012, 10, 8484–8492
This journal is © The Royal Society of Chemistry 2012

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86.9%; 1H NMR (300 MHz, D2O) δ 1.37–1.38 (m, 4H, 2 ×
CH2), 1.79–1.81 (m, 4H, 2 × CH2), 3.08–3.15 (m, 8H, 4 ×
NCH2), 3.36–3.55 (m, 16H, 8 × NCH2), 3.70–3.74 (m, 4H, 2 ×
NCH2), 5.44 (s, 2H, 2 × methine H); 13C NMR (75 MHz, D2O)
δ 23.9 (CH2), 25.4 (CH2), 51.1 (NCH2), 53.2 (NCH2), 58.0
(NCH2), 60.0 (NCH2), 116.5 (tacnoa central C); ESI-MS m/z
[M − 2Br−]2+ calcd 181.25, found 181.15; Anal. Calcd for
C20H38Br2N6: C, 45.99; H, 7.33; N, 16.09; Found: C, 45.79;
H, 7.42; N, 15.97%.
Acknowledgements
We acknowledge support of this work by the National Science
Foundation of China (no. 20872061), the National Basic
Research Program of China (no. 2007CB925103) and the Pri-
ority Academic Program Development of Jiangsu Higher Edu-
cation Institutions (PAPD) for financial support.
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Agarose gel electrophoresis assays
The plasmid DNA cleavage experiments were performed using
pUC19 DNA in Tris-HCl buffer. Reactions were carried out by
incubating DNA (0.025 mM bp) at 37 °C in 50 mM Tris-HCl–
5 mM NaCl buffer with a total volume of 15 μL in the dark for
the indicated time. All reactions were quenched by loading
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In conclusion, 1,8-bis(1-dihexyloxy-1-azonia-4,7-diazatricyclo-
[5.2.1.04,10]decane)anthracene-9,10-dione dibromide 1, 1-methoxy-
8-(1-hexyloxy-1-azonia-4,7-diazatricyclo[5.2.1.04,10]decane)-
anthracene-9,10-dione bromide 2 and 1,1′-(hexamethylene)bis-
(1-azonia-4,7-diazatricyclo[5.2.1.04,10]decane) dibromide 6 were
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0.0055 h−1
,
0.0620
0.0024 h−1, 0.040 0.0007 h−1 and 0.0043
0.0002 h−1, respectively. The 50-fold and 15-fold rate accelera-
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