Synthesis and DNA cleavage activity of triazacrown-anthraquinone conjugates

Article abstract of DOI:10.1039/c0nj00347f

1,4,7-Triazacyclononane (TACN), with DNA cleaving ability, was appended to anthraquinone via different spacers to construct the new compounds 1,8-[2,2′-(1,4,7-triazacyclonon)diethoxy] anthracene-9,10-dione hydrochloride (1) and 1,8-[2,2′-(1,4,7-triazacyclonon)dihexyloxy] anthracene-9,10-dione hydrochloride (2) as new agents for metal-free DNA cleavage. Fluorescence and CD spectroscopic studies suggest an intercalating DNA binding mode, and the apparent DNA binding constants of 1 and 2 are 3.93 × 10⁷ and 6.07 × 10⁷ M^-1, respectively. Compound 2, bearing the longer spacer, exhibits the higher DNA binding ability. The apparent initial first-order rate constant (k _obs) of DNA cleavage promoted by 1 and 2 (0.05 mM) in physiological media are 0.077 ± 0.0028 and 0.123 ± 0.0027 h^-1, respectively. The 51-fold and 82-fold rate accelerations over parent TACN (the k_obs is 0.0015 ± 0.00003 h^-1 (0.05 mM) under the same conditions) are due to the anthraquinone moiety of compounds 1 and 2 intercalating into the DNA base pairs via stacking interactions. ESI-MS analysis of the dinucleotide cleavage promoted by 1 and 2, and radical scavenger inhibition studies suggest that the cleavage process is a hydrolytic mechanism.

Full text of DOI:10.1039/c0nj00347f

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Synthesis and DNA cleavage activity of triazacrown-anthraquinone
conjugatesw
Weihong Xu, Xiaoli Yang, Lian Yang, Zhao-Li Jia, Li Wei, Fang Liu and
Guo-Yuan Lu*
Received (in Montpellier, France) 6th May 2010, Accepted 16th June 2010
DOI: 10.1039/c0nj00347f
1,4,7-Triazacyclononane (TACN), with DNA cleaving ability, was appended to anthraquinone
via different spacers to construct the new compounds 1,8-[2,20-(1,4,7-triazacyclonon)diethoxy]
anthracene-9,10-dione hydrochloride (1) and 1,8-[2,20-(1,4,7-triazacyclonon)dihexyloxy]
anthracene-9,10-dione hydrochloride (2) as new agents for metal-free DNA cleavage. Fluorescence
and CD spectroscopic studies suggest an intercalating DNA binding mode, and the apparent
DNA binding constants of 1 and 2 are 3.93 ꢀ 107 and 6.07 ꢀ 107 Mꢁ1, respectively. Compound
2, bearing the longer spacer, exhibits the higher DNA binding ability. The apparent initial
first-order rate constant (kobs) of DNA cleavage promoted by 1 and 2 (0.05 mM) in physiological
media are 0.077 ꢂ 0.0028 and 0.123 ꢂ 0.0027 hꢁ1, respectively. The 51-fold and 82-fold rate
accelerations over parent TACN (the kobs is 0.0015 ꢂ 0.00003 hꢁ1 (0.05 mM) under the same
conditions) are due to the anthraquinone moiety of compounds 1 and 2 intercalating into the
DNA base pairs via stacking interactions. ESI-MS analysis of the dinucleotide cleavage promoted
by 1 and 2, and radical scavenger inhibition studies suggest that the cleavage process is a
hydrolytic mechanism.
metal ions.8 Recently, we also developed a new artificial DNA
Introduction
cleaving agent via combining the triazacrown ether 1,4,7-
DNA recognition, binding, cleavage and modification, or
triazacyclononane (TACN) with guanidinoethyl and
cross-linking by small molecules, have attracted extensive
hydroxyethyl side arms. The promoted kinetic data kmax of
interest due to their potential applications in the fields of
molecular biological technology and drug development.1 The
DNA cleavage via transphosphorylation disclosed via
Michaelis–Menten fitting is B 0.160 hꢁ1, giving a 107-fold
rate acceleration over uncatalyzed DNA.9 This is a successful
transition metal complexes of macrocyclic polyamines as
efficient cleaving agents of nucleic acids have been widely
investigated.2 However, their medicinal application is still
example of an azacrown ether derivative as an artificial
nuclease for cleaving the phosphodiester bond of DNA via
limited due to the distinct toxicity of the free radicals
non-redox ‘‘metal-free catalysis’’. However, compared with
generated in the oxidative cleaving processes of these metal
natural nucleases such as staphylococal nuclease (SNase, the
complexes,3 such as copper complexes. Hence, it is of great
rate acceleration for the hydrolysis of the phosphodiester of
DNA is up to 1016-fold),10 the efficiency of DNA cleavage
significance to develop small organic molecules to promote the
cleavage of nucleic acids via a non-oxidative pathway.
promoted by the azacrown ether derivatives is still very low.
Recently, metal-free DNA cleaving reagents have been put
The anthraquinone group, as a fine intercalator of DNA,
forward by Gobel and co-workers.4 These compounds are
¨
has been frequently adopted in certain anticancer drugs, such as
doxorubicin, anthracyclines, mitoxantrone and anthrapyrazoles.11
thought of as safer agents for cleaving the P–O bond of
phosphodiesters in nucleic acids, showing clinical potential.
Many small organic molecules, such as guanidinium derivatives,4,5
Teilla, Boseggia and co-workers reported that the compound
formed by conjugating the cis,cis-triaminocyclohexane-Zn2+
cyclodextrin derivatives,6 dipeptides7 and especially macro-
complex (cleaving moiety) with anthraquinone (intercalating
cyclic polyamines,8 have also been used as cleaving agents of
moiety) via an alkyl spacer led to a 15-fold increase in DNA
nucleic acids. The macrocyclic polyamines reported by Liang
cleavage efficiency when compared with the cis,cis-triamino-
cyclohexane-Zn2+ complex without the anthraquinone moiety.12
and co-workers, such as 1,7-dimethyl-1,4,7,10-tetraazacyclo-
dodecane (DMC), can cleave DNA via the hydrolysis pathway
Yu and co-workers also reported that macrocyclic polyamine
in physiological media (37 1C, pH 7.2) without the presence of
bis-anthracene conjugates showed higher DNA binding and
photocleaving abilities than their corresponding mono-anthracene
conjugates.13a All these reports suggest that the conjugation of
Department of Chemistry, State Key Laboratory of Coordination
the intercalating group to a normal DNA cleaving agent is an
efficient strategy to develop DNA cleaving agents of enhanced
DNA cleavage rate.
Chemistry, Nanjing University, Nanjing 210093, P. R. China.
E-mail: lugyuan@nju.edu.cn; Fax: +86 25-83317761;
Tel: +86 25-83685613
w Electronic supplementary information (ESI) available: Spectra for
all new compounds, agarose gel electrophoretogram and kinetic data.
See DOI: 10.1039/c0nj00347f
In this work, we report the design and synthesis of two
triazacrown-anthraquinone conjugates linked by bis-alkyl spacers,
c
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spectra of compounds 1 and 2, the signals at d 183.1, 186.2 and
182.2, 183.5 are assigned to the signals of the carbonyl carbon
atoms. In the ESI mass spectra, the signals at m/z 422.17
and 444.25 can be assigned as (M ꢁ Clꢁ)+ (calc. 422.20) and
(M ꢁ HCl + Na)+ (calc. 444.18) of 1, respectively. The
signals at m/z 534.33 and 556.33 correspond to (M ꢁ Clꢁ)+
(calc. 534.33) and (M ꢁ HCl + Na)+ (calc. 556.32) of 2,
respectively.
DNA binding assays
The DNA binding of compounds 1 and 2 is essential for DNA
cleavage. Therefore, the DNA binding behaviors of 1 and 2 to
calf thymus DNA (CT-DNA) have been studied by using
fluorescence and CD spectroscopy.
Scheme 1 Chemical structures of compounds 1, 2 and 3.
1,8-[2,20-(1,4,7-triazacyclonon)diethoxy] anthracene-9,10-dione (1)
and 1,8-[2,20-(1,4,7-triazacyclonon)dihexyloxy] anthracene-
9,10-dione (2) (Scheme 1). In these two compounds, the
TACN moiety and the anthraquinone moiety are employed
as DNA cleaving group and intercalating group, respectively.
Moreover, the length of the alkyl spacers involved in the two
compounds is adjusted to altering their DNA binding and
cleaving ability. Besides the calf thymus DNA binding
behavior of the two compounds investigated by fluorescence
and circular dichroism (CD) spectroscopy, the plasmid pUC
19 DNA cleaving behavior of the two compounds is assessed
via agarose gel electrophoresis. For comparison, the DNA
cleavage activity of the parent TACN (Scheme 1) is studied
as well.
Fluorescence spectroscopic studies. The CT-DNA binding
ability of the two compounds was studied by evaluating the
fluorescence emission intensity of the ethidium bromide (EB)-DNA
system upon the addition of the two compounds, respectively.
Normally the emission intensity of the EB-DNA system
undergoes a distinct reduction when the intercalated EB is
replaced by the investigated compounds. The fluorescence
quenching effect of EB bound to DNA induced by the addition
of 2 and 1 is shown in Fig. 1 and Fig. S13 (ESIw), respectively,
according to the fluorescence intensity at 604 nm (lex = 530 nm)
of EB in the bound form. Under the same conditions, the
fluorescence of compounds 1 and 2 is very weak when
measured in the DNA system without EB (Fig. S24w).
The relative binding propensity to CT-DNA of the two
compounds was determined by fitting the emission intensity to
the classical Stern–Volmer equation
Results and discussion
Synthesis of compounds 1 and 2
I0/I = 1 + Kr
(1)
Synthesis of target compounds 1 and 2 were achieved via a
two-step reaction from 1,8-dihydroxyanthracene-9,10-dione
(5) (Scheme 2). Therefore, reacting 5 with 1,2-dibromoethane
or 1,6-dibromohexane in the presence of base afforded
compounds 4a or 4b. The crude compounds were purified by
silica gel column chromatography using petroleum ether–
dichloromethane (v/v = 1 : 1) as eluent and recrystallized from
EtOAc. Purified compounds 4 were transformed into 1 or 2 via
nucleophilic substitution by TACN in anhydrous chloroform
in the presence of base. 1H NMR, 13C NMR and ESI-MS
analytical characterization was carried out for compounds 1, 2
and all intermediates (Fig. S1–S12, ESIw). In the 13C NMR
where I0 and I are the fluorescence intensities in the absence
and the presence of the quencher, respectively, K is the linear
Stern–Volmer quenching constant, dependent on the ratio of
rbE (the ratio of [EB]bound to [DNA]), and r is the ratio of
[quencher]total to [DNA].14 Fig. 2 shows the plot of I0/I vs.
[quencher]/[DNA], and the quenching constant K is given by
Scheme 2 Synthesis of compounds 1 and 2. Reagents and conditions:
(i) 1,2-dibromoethane or 1,6-dibromohexane, K2CO3, N2, 110 1C, 72 h.
Yield: 55% for 4a and 80% for 4b; (ii) (1) 1,4,7-triazacyclonon,
K2CO3, anhydrous chloroform, reflux, 4 h; (2) r.t., ethanol, conc.
HCl. Yield: 30% for 1 and 24% for 2.
Fig. 1 Emission spectra of EB bound to DNA in the absence (TT) and
presence (---) of 2 (r = 0, 0.05, 0.07, 0.10, 0.13, 0.15, 0.21, 0.23, from
top to bottom), r = [compound]/[CT-DNA], [CT-DNA] = 0.039 mM,
[EB] = 3.9 mM, lex = 530 nm.
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in their chemical structures. Normally, the binding constants
can be adopted to evaluate the DNA-binding modes: a value
above 106 Mꢁ1 is an indication of intercalation (ethidium and
daunomycin bind DNA with an affinity over 106 Mꢁ1), while
values in the range 104–106 Mꢁ1 imply the groove binding
mode.16 Therefore the DNA binding of compounds 1 and 2
could be accomplished via the intercalation binding mode. In
fact, the anthraquinone rings are normal in reported DNA
intercalating agents.12,15b–g Besides, according to the fluorescence
changes, both conjugates 1 and 2 exhibit almost equal affinity
for the ethidium bromide (EB)-DNA system. This is because
both have the same intercalating group, anthraquinone rings,
which play a fundamental role in the intercalation.
The apparent binding constant (Kapp) of 1 and 2 to calf
thymus DNA in the presence of various concentrations of
sodium chloride was investigated in the EB-DNA system in
5 mM Tris-HCl buffer (pH 7.0) at room temperature. The Kapp
changing with the ionic strength of the solvent medium
expressed as a total positive ion concentration, [M+], is shown
in Table S1 (ESIw). The effect of ionic strength on the apparent
binding constant (Kapp) is small at low concentrations of
sodium chloride, which implies that the compounds bind to
DNA mainly by intercalation rather than by electrostatic
interactions. The apparent binding constant decreased gradually
as the ionic strength increased when the concentration of
sodium chloride was more than 10 mM. The addition of a
relatively high concentration of Na+ resulted in a decrease in
the negative charge density of DNA. The DNA double helix
in solution becomes tighter and it becomes difficult for
compounds intercalating to DNA.13b
Fig. 2 Stern–Volmer quenching plots of EB bound to DNA by 2 (m)
and 1 (&), The experiments were conducted by adding 0–1.43 mM 2 or 1
to the EB-bound CT-DNA solution in 5 mM Tris-HCl buffer (pH 7.0).
the gradient of the slope. The quenching constant K values
obtained for 1 and 2 are 4.01 ꢂ 0.11 and 6.32 ꢂ 0.18,
respectively, much greater than that observed for TACN
(0.053 ꢂ 0.010).2a These results demonstrate that anthraquinone-
based compounds 1 and 2 possess much better DNA binding
abilities than the free macrocyclic polyamine, TACN.
Moreover, compound 2 exhibits a slightly higher affinity to
DNA than compound 1.
The apparent binding constant (Kapp) is also calculated
from the equation
K
EB[EB] = Kapp[compound]
(2)
where the compound concentration is the value at a 50%
Circular dichroism (CD) studies. CD spectroscopy is a useful
technique to monitor the conformation alteration of nucleic
acids upon complex formation, since 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 interaction
between DNA and small molecules.17 The simple groove
binding mode and electrostatic interactions between small
molecules and DNA normally induce little or no perturbation
of the base stacking and helicity bands, while the intercalation
mode enhances the intensities of both bands via stabilizing the
right-handed B conformation of CT-DNA, just as observed
for the classical intercalator methylene blue.18
reduction of the fluorescence intensity of EB and KEB =
1.0 ꢀ 107 Mꢁ1 ([EB] = 3.9 mM).15a The Kapp values for
1 and 2 are 3.93 ꢀ 107 and 6.07 ꢀ 107
M
ꢁ1, respectively.
The significantly high Kapp value for the two compounds
might be correlated to the presence of anthraquinone rings
In the CD spectrum of CT-DNA treated with 1 or 2
([compound]/[DNA] = 0.4 : 1) (Fig. 3), the positive band
(B 275 nm) of CT-DNA decreases in intensity upon the
addition of 1 or 2, while the negative band (B 247 nm)
undergoes an obvious reduction (Fig. 3). Moreover, a
bathochromic shift to 250 nm of the negative band is observed
upon the addition of 2. The results suggest that 1 and 2 could
unwind the DNA helix and reduce the helicity possibly due to
the partial intercalation of 1 or 2 into the DNA base-pairs.19
The larger decrement in the CD bands induced by 2 than 1
implies that 2 is more effective in perturbing the secondary
structure of DNA at the same concentration because
compound 2 has a longer spacer than 1, resulting in a stronger
interaction between the triazacrown subunit and the
phosphate backbone.12
Fig. 3 CD spectra of CT-DNA (0.129 mM) in black and the
interaction with 1 and 2 at ratio of [compound]/[DNA] = 0.4. All
the spectra were recorded in 5 mM Tris-HCl buffer at pH 7.0.
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From the results of the fluorescence and CD spectroscopic
studies, it is concluded that compound 2 binds more strongly
to DNA than 1, and the DNA binding constants of 1 and 2
indicate that these compounds bind with DNA through the
intercalation binding mode.
DNA cleavage activity
The cleavage reaction of supercoiled pUC 19 DNA
(0.025 mM bp) promoted by 1 and 2 was investigated in the
dark at 37 1C in 50 mM Tris-HCl buffer with a total volume of
15 mL. The results indicated that supercoiled DNA (Form I)
could be degraded to relaxed circular DNA (Form II) in the
presence of 1 and 2.
pH-dependence of the promoted DNA cleavage. Incubation
of supercoiled pUC 19 DNA with compounds 1 or 2 for 16 h
at 37 1C resulted in a different extent of DNA cleavage,
depending on the pH of the buffer. Fig. S14 (the agarose gel
electrophoretograms, ESIw) shows the decrease in supercoiled
pUC 19 DNA and the appearance of relaxed DNA. Fig. 4
presents the pH-dependence profile, which indicates that the
DNA cleavage ability of 2 is stronger than that of 1, and the
optimal pH for DNA cleavage in the presence of 1 or 2 in 50 mM
Tris-HCl at 37 1C is pH 7.0–7.5. This accords with the result
reported by Liang and co-workers.8 They found that macrocyclic
polyamines such as 1,7-dimethyl-1,4,7,10-tetraazacyclododecane
(DMC) can cleave DNA via the hydrolysis pathway and that
the optimal pH for DNA cleavage is 7.25. Therefore, pH 7.25
was chosen for the following agarose gel electrophoresis
assays.
Fig. 5 Plot of % cleaved DNA vs. different concentrations of 1 (’)
and 2 (.). The reactions were carried out at 37 1C in 50 mM Tris-HCl
buffer (pH 7.25).
concentrations, and no other forms were observed. Fig. 5
shows the plots of % cleaved DNA vs. different concentrations
of 1 and 2, which indicates that the DNA cleavage activities of
1 and 2 are increased with an increase in their concentrations.
However, further increases of the concentrations of 1 and 2
(larger than 0.05 mM) could lead to the formation of relatively
high molecular weight DNA–1 (or –2) complexes.
Consequently DNA is stuck in the well (the agarose gel
electrophoretogram is shown in Fig. S16, ESIw).
For comparison, the DNA-cleaving behaviors of
compounds 4a and 4b (0.027 mM), lacking TACN, were also
investigated. It was found that the promotion of DNA cleavage
induced by these two compounds is very minor (Table S2, ESIw),
similar to the result reported by Teilla, Boseggia and
co-workers.12
Concentration dependence assays of DNA cleavage promoted
by 1 and 2. Reactions that lead to the formation of relaxed
circular DNA from supercoiled DNA over various concentrations
of 1 or 2 (6.7–40 mM) and constant DNA concentration
(25 mM, bp) were carried out for 16.0 h at 37 1C (the agarose
gel electrophoretograms are shown in Fig. S15, ESIw). Under
the experimental conditions, form II was observed, even at low
Kinetic assays. The kinetics of pUC 19 DNA degradation
have been studied. Fig. 6a shows the agarose gel electro-
phoretogram of the supercoiled plasmid DNA cleavage promoted
by 2 (0.04 mM) in Tris-HCl buffer (pH 7.25) at 37 1C into
nicked forms. The rate of conversion of form I to form II
increases with the increase reaction time. The time course
plot (Fig. 6b) indicates that the extent of supercoiled DNA
cleavage varies exponentially with the reaction time, giving
pseudo first-order kinetics (the first-order rate constant is
given by the gradient of the slope) with an apparent initial
first-order rate constant (kobs) of 0.113 ꢂ 0.0071 hꢁ1. Then, the
apparent initial first-order rate constants of DNA cleavage
reactions promoted by a series of various concentrations of 2
and 1 under the similar conditions, as described above, are
summarized in Table S2 (ESIw). Fig. 7 shows the kinetics
profiles of the supercoiled DNA cleavage at various
concentrations of 2 and 1. The kobs promoted by 1 and 2
(0.05 mM) in physiological media are 0.077 ꢂ 0.0028 and
0.123 ꢂ 0.0027 hꢁ1, respectively (Table S3, ESIw).
As a comparison, the DNA-cleaving behavior of parent
TACN, without the anthracenedione moiety, was also
investigated under the same conditions, and showed a low
cleavage activity ( kobs was 0.0015 ꢂ 0.00003 hꢁ1 (0.05 mM) in
Tris-HCl/NaCl buffer at 37 1C) (Fig. S18 and Table S4, ESIw).
Fig. 4 pH-dependent profile for pUC DNA (0.025 mM bp) cleavage
promoted by 0.033 mM 1 (’) and 2 (m) in different pH buffers. The
incubation time was 16 h at 37 1C.
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two compounds is adjusted to alter their DNA binding and
cleaving abilities. After intercalation of the anthraquinone
moiety, the spacer must be free to fold in such a way as to
position the TACN moiety close to a phosphate group, and
too-short a spacer may prevent correct folding. This accords
with the result reported by Teilla, Boseggia and co-workers.12
They investigated a series of conjugates, a cis,cis-triamino-
cyclohexane (TACH) Zn2+ complex and an anthraquinone
intercalator linked by alkyl spacers of different lengths, and
demonstrated that the length and flexibility of the spacer
played a fundamental role in the interaction between the
catalytic subunit and the phosphate backbone.
Studies on mechanism of DNA cleavage
The anthracenedione moiety is known to undergo redox
processes, which could directly produce cytotoxic effects.20
To verify if reactive oxygen species (ROS) are, at least in part,
responsible for the cleavage of DNA promoted by compounds
1 and 2, reactions were carried out in the presence of typical
scavengers for singlet oxygen (NaN3, 10 mM), for superoxide
(KI, 10 mM) and for hydroxyl radicals (1 mM DMSO and
1 mM t-BuOH) (Fig. S19 and Table S5, ESIw).21a Fig. 8 and
Fig. S20w show that there is no evident inhibition effect on
the DNA cleavage in the presence of all the scavengers
(NaN3, DMSO, t-BuOH and KI), which rules out the involvement
of these reactive oxygen species, at least in a free and diffusible
form. Therefore, DNA cleavage promoted by compounds 1
and 2 might occur by a non-oxidative pathway.
Fig. 6 Time course of pUC19 DNA (0.025 mM bp) cleavage
promoted by 2 (0.040 mM). (a) Agarose gel (1%) of the time-variable
reaction products: lanes 1–8, reaction times of 0, 2.00, 4.00, 5.50, 7.00,
8.00, 9.00 and 10.00 h, respectively. (b) Plot of ln (% supercoiled
DNA) vs. reaction time. The inset of Fig. 6b is the plot of % DNA vs.
time. The reactions were carried out at 37 1C in 50 mM Tris-HCl/10 mM
NaCl buffer (pH 7.25).
Mechanistic profiles of DNA cleavage by 1 and 2 were also
evaluated in the presence of excess EDTA to scavenge adventitious
transition metal ions (Fig. S19(c) and Table S6, ESIw). The
inhibition of DNA cleavage was very small, which rules out
the involvement of adventitious transition metal ions.
To further study the pathway of DNA cleavage promoted
by 1 and 2, dinucleotide adenylyl(30-50)phosphoadenine (ApA)
was used as the nucleic acid mimic. ApA (0.10 mM) and 1
Fig. 7 Kinetics plot of kobs vs. various concentrations of 2 (’) and
1 (m).
The cleavage efficiency of compounds 1 and 2 gave 51- and
82-fold rate accelerations over 3, respectively, which indicates
that the anthraquinone moiety effectively increases the DNA
cleavage activity.
Furthermore, kinetic data for DNA cleavage promoted by 1
and 2 show that 2, with C6 spacers, has a rate acceleration
over 1, with C2 spacers. In the two compounds, the TACN
moiety and the anthraquinone moiety are employed as DNA
cleaving group and intercalating group, respectively.
Moreover, the length of the alkyl spacers involved in the
Fig. 8 Histogram representing of pUC19 plasmid DNA (0.025 mM bp)
cleaved by 2 (0.04 mM) in the presence of standard radical scavengers for
singlet oxygen (NaN3, 10 mM), for superoxide (KI, 10 mM) and for
hydroxyl radicals (1 mM DMSO and 1 mM t-BuOH), incubated for 18 h
at 37 1C in pH 7.25 buffer (50 mM Tris-HCl/10 mM NaCl) (the agarose
gel electrophoretogram is shown in Fig. S19(a), ESIw).
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(0.05 mM) or 2 (0.05 mM) were dissolved in de-ionized water
(1 : 1), and after 16 h equilibration time at 37 1C, ESI-MS
analysis was carried out. In the ESI-MS spectrum of ApA
treated with 2 (Fig. S21(a), ESIw), the signals at m/z 268.08
and 345.99 show the ApA cleavage products A (adenosine)
([A + H]+, calc. 268.10) and AMP (adenosine monophosphate)
([AMP ꢁ H]ꢁ, calc. 346.06); no sign of ApA was found. Under
the same conditions, the ESI-MS spectrum of ApA treated
with 1 (Fig. S21(b), ESIw) showed not only the signal of A and
AMP, but also signs of ApA ([ApAꢁ H]ꢁ; m/z 595.17, calc.
m/z 595.14). As a control experiment, an ESI-MS analysis of
ApA alone was also carried out under the same conditions
(Fig. S21(c), ESIw), showing only the signal of ApA and no
sign of A or AMP. The generation of adenosine and AMP
indicates that the phosphodiester bonds of ApA were cleaved
by 1 or 2 via the hydrolysis pathway.21b Therefore, similar to
ApA, the hydrolysis pathway is the likely mechanism for DNA
cleavage promoted by 1 and 2.
The stock solution of CT-DNA (stored at 4 1C and used for
not more than 2 d) was prepared in 5 mM Tris-HCl/10 mM
NaCl in water, pH 7.0. The concentration of the CT-DNA was
determined according to its absorption intensity at 260 nm
with
a known molar extinction coefficient value of
6600 Mꢁ1 cmꢁ1. The ratio of the UV absorbance at 260 and
280 nm, A260/A280 = 1.8–1.9, indicated that the DNA was
sufficiently free of protein.22
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
analyses (C, H, N) were performed on a VARIO EL instrument.
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. Agarose gel electrophoresis was
conducted with a DYY-5 electrophoresis apparatus. Bands were
visualized by UV light and photographed using a DigiDoc-It
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).
Conclusions
In conclusion, 1,8-[2,20-(1,4,7-triazacyclonon)diethoxy]anthracene-
9,10-dione hydrochloride (1) and 1,8-[2,20-(1,4,7-triazacyclonon)-
dihexyloxy]anthracene-9,10-dione hydrochloride (2) as metal-free
DNA cleaving reagents were prepared. Fluorescence and CD
spectroscopic studies suggest an intercalating DNA binding
Synthesis of the compounds
1,4,7-triazacyclononane
(3).
1,4,7-Triazacyclononane
(TACN) was prepared according to the reported procedure.23
1H NMR (300 MHz, CDCl3) d: 2.00 (s, 3H, 3 ꢀ NH), 2.79 (s,
12H, 6 ꢀ NCH2); 13C NMR (300 MHz, D2O) d: 41.2 (CH2).
mode. Kinetic data of DNA cleavage promoted by
1
(0.05 mM) and 2 (0.05 mM) under physiological conditions
give kobs of 0.077 ꢂ 0.0028 and 0.123 ꢂ 0.0027 hꢁ1
,
1,8-Bis(2-bromoethoxy)anthraquinone (4a). 1,8-Dihydroxy-
anthraquinone (2.00 g, 8.3 mmol), anhydrous potassium
carbonate (14.00 g, 101.3 mmol) were added to 1,2-dibro-
moethane (40 mL, 464 mmol) and the mixture stirred at 110 1C
under an atmosphere of dry N2. A color change from orange
to deep purple 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 chromato-
graphy (silica gel, petrol ether-dichloromethane, 1 : 1, v/v)
followed by recrystallization from EtOAc afforded 4a
(2.09 g, 55%) as long orange needles. mp 154–156 1C; 1H NMR
(300 MHz, CDCl3) d: 3.78 (t, J = 6.7 Hz, 4H, 2 ꢀ CH2Br),
4.44 (t, J = 6.7 Hz, 4H, 2 ꢀ OCH2), 7.33 (d, J = 8.2 Hz, 2H,
2 ꢀ ArH), 7.61–7.67 (m, 2H, 2 ꢀ ArH), 7.91 (d, J = 7.7 Hz,
2H, 2 ꢀ ArH); 13C NMR (300 MHz, CDCl3) d: 28.8 (CH2Br),
70.4 (OCH2), 120.5 (Ar C), 121.6 (Ar C), 125.2 (Ar C), 133.9
(Ar C), 134.9 (Ar C), 157.9 (Ar C), 182.0 (CQO), 183.5
(CQO); Anal. calc. for Cl8H14Br2O4: C, 47.61; H, 3.11.
Found: C, 47.51; H, 3.13%.
respectively, which shows that 1 and 2 give 51- and 82-fold
rate accelerations over parent TACN, respectively, due to the
anthraquinone moiety of these compounds intercalating into
the DNA base-pairs via stacking interactions. Furthermore, 2,
with C6 spacers, has a rate acceleration over 1, with C2
spacers. ESI-MS analyses and radical scavenger inhibition
studies suggest that the DNA cleavage promoted by 1 and 2
in physiological conditions is by a hydrolytic mechanism.
Experimental
Materials
All reagents and chemicals, purchased from commercial
sources, were of analytical grade and used without further
purification. pUC 19 plasmid DNA was purchased from
TaKaRa Biotechnology (Dalian) Co. Ltd., and the purity
was checked by agarose gel electrophoresis and their
concentration was determined by UV spectroscopy using the
extinction coefficient appropriate for double-stranded DNA
(1.0 OD260 = 50 mg mLꢁ1). Agarose was from Oxoid Limited
of Basingstoke (UK). Ethidium bromide (EB) was from
Amresco. Inc., and tris(hydroxymethyl)aminomethane
(Tris-Base) was from Robiot Co. Ltd. Dinucleotide (ApA)
was purchased from Sigma-Aldrich. Bromophenol blue,
glycerol and ethyl diamine tetraacetic acid (EDTA) were
commercially available. De-ionized water was obtained by
an ionized column from double distilled water. All solvents
were purified by standard procedures. All aqueous solutions
were prepared from de-ionized water.
1,8-Bis(6-bromohexyloxy)anthraquinone (4b). Compound 4b
was synthesized in a similar procedure to compound 4a using
1,8-dihydroxyanthraquinone (2.00 g, 8.3 mmol), anhydrous
potassium carbonate (10.35 g, 74.9 mmol), 1,6-dibromohexane
(40 mL, 260 mmol). A orange solid (3.77 g, 80%) was
obtained. mp 62–63 1C; 1H NMR (300 MHz, CDCl3) d:
1.53–1.69 (m, 8H, 4 ꢀ CH2), 1.89–1.99 (m, 8H, 4 ꢀ CH2),
3.45 (t, J = 6.8 Hz, 4H, 2 ꢀ CH2Br), 4.14 (t, J = 6.4 Hz, 4H,
2 ꢀ OCH2), 7.28 (d, 2H, 2 ꢀ ArH), 7.57–7.63 (m, 2H, 2 ꢀ ArH),
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2010 New J. Chem., 2010, 34, 2654–2661 2659

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7.82 (d, J = 7.7 Hz, 2H, 2 ꢀ ArH); 13C NMR (300 MHz,
CDCl3) d: 25.1 (CH2), 27.8 (CH2), 28.9 (CH2), 32.7 (CH2),
34.0 (CH2Br), 69.4 (OCH2), 118.8 (Ar C), 119.4 (Ar C), 124.5
(Ar C), 133.6 (Ar C), 134.8 (Ar C), 158.7 (Ar C), 182.0 (CQO),
184.1 (CQO); Anal. calc. for C26H30Br2O4: C, 55.14; H, 5.34.
Found: C, 55.11; H, 5.36%.
256 and 393 nm for 1, and 195, 222, 257 and 403 nm for 2,
respectively. The weak absorption bands at about 400 nm are
caused by the carbonyl groups. The molar extinction coefficients
of compounds 1 and 2 at about 400 nm are 5.59 ꢀ 103 and
5.14 ꢀ 103 Mꢁ1 cmꢁ1, respectively. Fig. S23 (ESIw) shows the
emission spectra of compounds 1 and 2 excited at 430 nm,
where the maximum emission peaks are at about 592 nm.
1,8-[2,20-(1,4,7-triazacyclonon)diethoxy] anthracene-9,10-dione
hydrochloride (1). Compound 3 (0.42 g, 3.3 mmol) was
dissolved in 5 mL dry chloroform, and to this solution was
added 6.70 g of K2CO3 (6.70 g, 48.5 mmol). A dry chloroform
(15 mL) solution of 1,8-bis(2-bromoethoxy)anthraquinone
(0.30 g, 0.66 mmol) was then added dropwise with stirring to
this suspension. The mixture was refluxed for 4 h, cooled to
room temperature and then filtered and washed with chloroform.
The filtrate was evaporated to dryness in vacuo. The residue
was dissolved in ethanol (5 mL) and standing for 4 h after
concentrated HCl (10 drops) was added dropwise. The mixture
was then filtered and the crude product was dissolved in water
(30 mL), and the pH adjusted to 11 using a 1 M sodium
hydroxide solution. The desired product was extracted into
chloroform (30 mL ꢀ 3). The organic layer was then dried
over sodium sulphate and evaporated to dryness in vacuo.
Ethanol (5 mL) and concentrated HCl (2 mL) was added.
Followed by filtration, the desired product 1 was obtained
(90 mg, 30%). 1H NMR (300 MHz, D2O) d: 3.38–3.58
(m, 16H, 8 ꢀ NCH2), 4.36–4.38 (m, 4H, 2 ꢀ OCH2),
7.31–7.34 (m, 2H, 2 ꢀ ArH), 7.52–7.59 (m, 4H, 4 ꢀ ArH);
13C NMR (300 MHz, D2O) d: 42.7 (CH2NH), 48.1 (NCH2),
52.3 (NCH2), 58.6 (NCH2), 66.9 (OCH2), 121.4 (Ar C), 123.9
(Ar C), 125.5 (Ar C), 133.0 (Ar C), 136.3 (Ar C), 157.8 (Ar C),
183.1 (CQO), 186.2 (CQO); ESI-MS m/z 422.17 (M ꢁ Clꢁ.
C24H28N3O4 requires 422.20), 444.18. Anal. calc. for
C24H28N3O4Cl: C, 62.95; H, 6.16; N, 9.18. Found: C, 63.05;
H, 6.18; N 9.15%.
Fluorescence measurements
The fluorescent spectral studies were performed by measurements
of the emission intensity of ethidium bromide (EB) on an
AMINCO Bowman Series 2 luminescence spectrometer. The
experiments were carried out by adding 0–1.43 mM 1 or 2 into
the EB-bound CT-DNA (3.9 mM) solution in 5 mM Tris-HCl
buffer (pH 7.0). The measured fluorescence was 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 320B220 nm at a speed of
20 nm minꢁ1 and the buffer background was subtracted
automatically. Data were recorded at an interval of 0.1 nm.
The CD spectrum of CT-DNA alone (129 mM) was recorded
as the control experiment.
Agarose gel electrophoresis assays
The plasmid DNA cleavage experiments were performed using
pUC19 DNA in Tris-HCl buffer. Reactions were performed
by incubating DNA (0.025 mM bp) at 37 1C in a total volume
of 15 mL in the dark for the indicated time. All reactions were
quenched by loading buffer (3.5 mL) (30 mM EDTA, 0.05%
(w/v) glycerol, 36% (v/v) bromophenol blue). Agarose gel
electrophoresis was carried out on a 1% agarose gel in
0.5 ꢀ TAE (Tris-acetate–EDTA) buffer containing 1 mg mLꢁ1
EB at 80 V for 1.5 h. The resolved bands were visualized with a
UV transilluminator and quantified using TotalLab 2.01 soft-
ware. The supercoiled plasmid DNA values were corrected by
a factor of 1.3 on the basis of average literature estimates of
lowered binding of EB to this structure.24
1,8-[2,20-(1,4,7-triazacyclonon)dihexyloxy]anthracene-9,10-dione
hydrochloride (2). Compound 2 was synthesized in a similar
procedure to compound 1 using 1,4,7-triazacyclononane (3)
(0.42 g, 3.3 mmol), potassium carbonate (6.70 g, 48.5 mmol)
and 1,8-bis(6-bromohexyloxy)anthraquinone (0.35 g, 0.62 mmol).
A tan solid (85.0 mg, 24%) was obtained. 1H NMR (300 MHz,
D2O) d: 1.11–1.63 (m, 16H, 8 ꢀ CH2), 2.78–3.34 (m, 16H,
8
ꢀ
NCH2), 3.83 (m, 4H,
2
ꢀ
OCH2), 7.03–7.29
Acknowledgements
(m, 6H, 6 ꢀ ArH); 13C NMR (300 MHz, D2O) d: 25.5
(CH2), 26.4 (CH2), 28.3 (CH2), 35.3 (CH2), 42.0 (CH2NH),
44.5 (NCH2), 48.7 (NCH2), 57.2 (NCH2), 62.5 (NCH2), 69.3
(OCH2), 118.1 (Ar C), 119.7 (Ar C), 122.7 (Ar C), 133.4
(Ar C), 134.1 (Ar C), 158.2 (Ar C), 182.2 (CQO), 183.5
(CQO); ESI-MS m/z 534.33 (M-Clꢁ. C32H44N3O4 requires
534.33), 556.33. Anal. calc. for C32H44N3O4Cl: C, 67.41; H,
7.78; N, 7.37. Found: C, 67.22; H, 7.81; N 7.41%.
This work was supported by the National Science Foundation
(Grant no. 20872061) and National Basic Research (Grant no.
2007CB925103) of China.
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