molecules
Article
DNA Cleavage and Condensation Activities of
Mono- and Binuclear Hybrid Complexes and
Regulation by Graphene Oxide
Shuo Li 1,2 , Mingxing Dai 2 , Chunping Zhang 3, *, Bingying Jiang 2 , Junqiang Xu 2 , Dewen Zhou 2
and Zhongwei Gu 1, *
1
2
3
*
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China;
[email protected]
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, China;
[email protected] (M.D.); [email protected] (B.J.); [email protected] (J.X.);
[email protected] (D.Z.)
Jiangsu Key Laboratory of Target Drug and Clinical Application, Xuzhou Medical College,
Xuzhou 221004, China
Correspondence: [email protected] (C.Z.); [email protected] (Z.G.);
Tel.: +86-516-8326-2140 (C.Z.); +86-28-8541-2923 (Z.G.); Fax: +86-28-8541-0653 (Z.G.)
Academic Editor: Derek J. McPhee
Received: 7 June 2016; Accepted: 8 July 2016; Published: 15 July 2016
Abstract: Hybrid complexes with N,N1 -bis(2-benzimidazolylmethyl)amine and cyclen moieties are
novel enzyme mimics and controlled DNA release materials, which could interact with DNA through
three models under different conditions. In this paper, the interactions between plasmid DNA and
seven different complexes were investigated, and the methods to change the interaction patterns
by graphene oxide (GO) or concentrations were also investigated. The cleavage of pUC19 DNA
promoted by target complexes were via hydrolytic or oxidative mechanisms at low concentrations
ranging from 3.13 ˆ 10´7 to 6.25 ˆ 10´5 mol/L. Dinuclear complexes 2a and 2b can promote the
cleavage of plasmid pUC19 DNA to a linear form at pH values below 7.0. Furthermore, binuclear
hybrid complexes could condense DNA as nanoparticles above 3.13 ˆ 10´5 mol/L and partly release
DNA by graphene oxide with π-π stacking. Meanwhile, the results also reflected that graphene oxide
could prevent DNA from breaking down. Cell viability assays showed dinuclear complexes were
safe to normal human hepatic cells at relative high concentrations. The present work might help to
develop novel strategies for the design and synthesis of DNA controllable releasing agents, which
may be applied to gene delivery and also to exploit the new application for GO.
Keywords: cyclen; N,N1 -bis(2-benzimidazolylmethyl)amine; DNA condenstion; DNA release;
enzyme mimic; graphene oxide
1. Introduction
Artificial nucleases have attracted continuous and extensive interest due to their potential
applications in the fields of molecular biology, biotechnology and drug development [1,2]. The
design and synthesis of small-molecule catalysts as artificial nucleases for highly effective hydrolytic
cleavage of the P–O bond in DNA are becoming a crucial tool in biotechnology. Metal complexes
are the main part of artificial nucleases. Acting as Lewis acids, they stabilize the developing negative
charge of the transition state and assist the departure of the leaving group [3]. Although the
cations are the catalytic centers, the ligands are the key factors that influence the properties of
artificial nucleases, and sometimes ligands participate in the catalytic process directly [4]. In general,
nitrogen-containing compounds and their metal complexes display a wide range of biological activities,
Molecules 2016, 21, 920; doi:10.3390/molecules21070920
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Molecules 2016, 21, 920
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especially benzimidazole-substituted derivatives. For example, the benzimidazole moiety is the key
constituent of vitamin B12 and is found in antibacterial, antifungal, antiviral [5–7] and antitumor
agents as inhibitors of cyclin-dependent kinase and is useful for inhibiting cell proliferation [8,9].
N,N1 -Bis(2-benzimidazolylmethyl)amine (IDB) derivatives, a kind of benzimidazole complex reported
by our group recently, showed good antimicrobial properties and selective recognition toward
cytosine [10].
At the same time, DNA protection and condensation are also fundamental life processes, and
there are many studies in this field. Besides cationic polymers, cationic metal complexes are under
evaluation as a kind of promising non-viral nucleic acid carrier. The early studies indicated that
hexamine cobalt(III) cation (Co(NH3 )6 3+ ), a complex that was nonreactive to DNA, effectively induced
DNA condensation [11]. Many other metal complexes which were used alone [12], as part of
multicomponent systems [13] or conjugated with polymers [14] could deliver gene availability. Up to
now, the design and synthesis of new cationic molecules for the purpose of controlling condensate size
and morphology is still an important and attractive objective. Some novel complexes were developed,
including AMD3100 polymer coordinated with Cu(II), [15] bis(zinc(II)-dipicolylamine)-functionalized
material [16], acetylated 1,4,7,10-tetraazacyclotetradecane (cyclen) complex, acetylated IDB metal
complexes [17] and [Ca(IDB)2 ]2+ derivatives [18]. Above all, cyclen, 1,4,8,11-tetraazacyclotetradecane,
IDB, DTPB (1,1,4,7,7-penta(21 -benzimidazol-2-yl-methyl)-triazaheptane) and EGTB (N,N,N1 ,N1 -tetrakis
(21 -benzimidazol-2-yl-methyl)-1,4-bis(ethylamino)-bis-(ether)) are high performance ligands for small
molecular DNA condensing agents. On the other hand, the release process of DNA nanoparticles also
plays a crucial role in determining the effectiveness of the complex to deliver a therapeutic gene to the
target cell nucleus. Cu2+ -EGTB or its derivatives release genes through unknown mechanisms. Zhao
and co-workers reported that azaheterocyclic-based metal complexes could release DNA controlled by
temperature [17], and not through pH changing, “proton sponge” effect or compound degradation.
Although acylation of cyclen reduced the cleavage properties of cyclen, the cleavage activity of the
ring-Cu2+ complex would still result in a range of smaller DNA fragments at concentrations above
0.8 mM, and the condensation temperature was relatively high and the configuration of DNA changed
by release [19].
To our best knowledge, there are few papers reporting hybrid complexes with cyclen and IDB and
their bioorganic DNA interaction properties, so this attracted our interest. In the work presented here,
we designed and investigated a serial of hybrid complexes composed of cyclen-ethyl and IDB-ethyl,
and the results showed that there were synergetic effects and totally prior to their assemblies. However,
few experimental studies have addressed how to modulate DNA condensation-release by convenient
external additives besides matrix metalloproteinase [20] or GSH [21]. Graphene oxide (GO) was often
used as a DNA cleavage promoter [22] or Cu2+ /GO nuclease in some cases [23], and some kinds
of cationic-modified graphene were used as gene delivery which release DNA by “proton sponge”
effect [24]. At the same time, it is important to point out that the sp2 -hybridized carbon backbone in
GO maintains a high degree of planarity, which is much larger than that of most planar organic DNA
intercalators or the aromatic ligands of inorganic DNA intercalators [25], so GO might offer a new kind
controlled release method by π-π stacking, but there are few papers that report the direct use of GO
was as regulation agent for gene release. Notably, this work was the first time using the classic planar
aromatic structures of GO as a switch for DNA complexes.
Meanwhile, when these binuclear hybrid complexes were used as artificial nucleases in low
concentration they showed interesting properties, but they were also seldom reported. To our surprise,
the cleavage mechanisms of those complexes were different for their varied constructs, and some
target materials cleaved DNA by an oxidation model and the others cleaved DNA by a hydrolysis
model. Although several addition agents were also used to reduce the intensity of interactions between
DNA and complexes, for example cyclodextrin was applied to wrap up naphthalene to avoid DNA
breakdown [26] and GO is a newcomer to protect DNA when the concentration of target material is
suitable for cleavage. From these results, it could be concluded that this kind novel hybrid compound
Molecules 2016, 21, 920
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could condense DNA in aqueous solution and that divalent metal ions enhance the efficiency of
condensation. Importantly, GO may be used to regulate DNA condensation and release. The data is
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presented
in21,
this
2.
2. Results
Resultsand
andDiscussion
Discussion
2.1. Concentration
Concentration Effect
Effect of
of the
the Complex
Complex for
for DNA
DNA Cleavage
Cleavage and
and Condensation
Condensation
2.1.
DNA cleavage
cleavage is
is controlled
controlled by
by relaxation
relaxation of
of supercoiled
supercoiled form
form DNA
DNA (Form
(Form I).
I). Plasmid
Plasmid pUC19
pUC19
DNA
DNA, aa widely
cleavage
substrate,
is a circular
double
stranded
DNA (Form
The cleavage
DNA,
widelyused
usedDNA
DNA
cleavage
substrate,
is a circular
double
stranded
DNAI).(Form
I). The
agent canagent
convert
I to nicked
(Form form
II) or linear
(Form
In gel
electrophoresis,
cleavage
canForm
convert
Form Iform
to nicked
(Formform
II) orDNA
linear
formIII).
DNA
(Form
III). In gel
the migration rate
the threerate
DNA
forms
is DNA
usually
in the
order: Form
> FormForm
III > IForm
II. III
In
electrophoresis,
the of
migration
of the
three
forms
is usually
in theI order:
> Form
the to
DNA
cleavage
activities
of these
complexes,
cleavage of
pUC19
DNA
>order
FormtoII.assess
In order
assess
the DNA
cleavage
activities
of thesethe
complexes,
theplasmid
cleavage
of plasmid
assays were
agarose gel
All these target
compounds
(structures
pUC19
DNA investigated
assays were by
investigated
byelectrophoresis.
agarose gel electrophoresis.
All these
target compounds
shown in Scheme
1) were
tested.
Figures
1 and
2, Figures
S1Figures
and S2 S1
in the
Materials
(structures
shown in
Scheme
1) were
tested.
Figures
1 and 2,
andSupplementary
S2 in the Supplementary
showed that
the plasmid
DNA cleavage
promoted
by mononuclear
complexes
at different
Materials
showed
that pUC19
the plasmid
pUC19 was
DNA
cleavage
was promoted
by mononuclear
´ 5 mol/L,
concentrations
from 3.13
ˆ 10´ 7 to 6.25from
ˆ 103.13
and
pUC19
degraded
fromDNA
Formdegraded
I to Form
complexes
at different
concentrations
× 10−7 to
6.25
× 10−5DNA
mol/L,
and pUC19
II. Meanwhile,
out that
Figure
form I out
would
bein
convert
form
II inconsistently
from
Form I toit must
Formbe
II.pointed
Meanwhile,
it in
must
be 1B
pointed
that
Figureto1B
form
I would be
by increasing
theIIcomplex
1c. Weby
supposed
thatthe
compounds
with
rings
would
interact with
with
convert
to form
inconsistently
increasing
complex 1c.
Webenzene
supposed
that
compounds
each other
strongly,
that the
interactions
between
compounds
interfere
with their
cutting
benzene
rings
wouldso
interact
with
each other
strongly,
so that thewould
interactions
between
compounds
actions,interfere
but interferential
inconsistent
upon increasing
theinconsistent
complexes. upon
To our
surprise,
would
with their effects
cuttingwere
actions,
but interferential
effects were
increasing
increasing
the complex
concentration
did not
result
in more conversion
of plasmid
DNA
from
the
complexes.
To our surprise,
increasing
thealways
complex
concentration
did not always
result
in more
´ 5 mol/L, some part
Form
I
to
Form
II.
Furthermore,
when
the
concentration
approached
3.13
ˆ
10
conversion of plasmid DNA from Form I to Form II. Furthermore, when the concentration approached
−5 mol/L,
of the
DNA
was some
condensed
dinuclear
complex,byand
Form I complex,
and Formand
II bands
became
3.13
× 10
part ofby
thethe
DNA
was condensed
the its
dinuclear
its Form
I and
indefinite
(shown
in Figures
1 and (shown
2). Moreover,
when1the
concentration
became
10´ 5 mol/L,
Form
II bands
became
indefinite
in Figures
and
2). Moreover,
when6.25
the ˆ
concentration
these bands
disappeared
mononuclear
complexes
1b,mononuclear
1c and dinuclear
complexes
became
6.25 nearly
× 10−5 mol/L,
these with
bandsboth
nearly
disappeared
with both
complexes
1b, 2a,
1c
2b, 2c
and 2d.complexes
Taken together,
results
indicate
that the
condensation
interactions
DNA
and
dinuclear
2a, 2b,these
2c and
2d. Taken
together,
these
results indicate
that thebetween
condensation
and dinuclear
complexes
than
those ofare
mononuclear
complexes,
and complexcomplexes,
2c showed
interactions
between
DNAare
andstronger
dinuclear
complexes
stronger than
those of mononuclear
the strongest
compression
The compression
details are discussed
below.
and
complex 2c
showed theability.
strongest
ability. The
details are discussed below.
H
N
HN
M2+ N
R
N
N
N
H
nClO4 1a-1c
a: R = m-xylyl ; M = Cu; n = 2;
b: R = m-xylyl ; M = Zn; n = 2;
c: R = 2,6-dimethyl pyridyl ; M = Zn; n = 3;
HN
H
N
HN
M2+
N
H
N
R
N
N
M2+
NH
nClO4-.mOH -.XH2 O
N
2a-2d
a: R = m-xylyl ; M = Cu ; n = 4;
b: R = p-xylyl ; M = Zn ; n = 4; X = 2;
c: R = p-xylyl ; M = Cu ; n = 4; X = 2;
d: R = 2,6-dimethyl pyridyl; M = Zn ;
n = 3; m = 1; X = 1.
Scheme 1. Structures of target benzimidazole complexes.
Scheme 1. Structures of target benzimidazole complexes.
2.2. pH Effect on DNA Cleavage
pH-dependence profiles for DNA cleavage are shown in Figures 3 and 4. From these figures, it
can be seen that the pH-rate curve presents an “M-shape” (Figure 3) or 11 bell-shape11 (Figure 4) profile
with the acidity change of the solution of mononuclear and dinuclear complexes from pH = 6.5–8.3,
and the optimal pH value for DNA cleavage is around 7.0 to 7.2 for both mononuclear and dinuclear
complexes. This indicates that the cleavage efficiency of supercoiled DNA by complex is correlated
to the pH value of the reaction system. In addition, the slightly alkaline conditions match humans’
normal physiological conditions, so major DNA cleavage assays in this work were performed in slightly
Figure 1. Histograms and electropherograms (the right side figure) representing cleavage of pUC19
plasmid DNA (0.008 µg/µL) by different concentrations of 1b (pH = 7.4) and 1c (pH = 7.4) in buffer (5 mM
Tris-HCl/10 mM NaCl) at 37 °C for 6 h. (A) Complex 1b; Lanes 1–6: 6.25 × 10−5, 3.13 × 10−5, 6.25 × 10−6,
3.13 × 10−6, 6.25 × 10−7, 3.13 × 10−7 mol/L, Lane 7 = DNA control, respectively; (B) Complex 1c; Lanes
1–6: 6.25 × 10−5, 3.13 × 10−5, 6.25 × 10−6, 3.13 × 10−6, 6.25 × 10−7, 3.13 × 10−7 mol/L, Lane 7 = DNA control,
the complexes. To our surprise, increasing the complex concentration did not always result in more
conversion of plasmid DNA from Form I to Form II. Furthermore, when the concentration approached
3.13 × 10−5 mol/L, some part of the DNA was condensed by the dinuclear complex, and its Form I and
Form II bands became indefinite (shown in Figures 1 and 2). Moreover, when the concentration
became 2016,
6.25 21,
× 10
Molecules
920−5 mol/L, these bands nearly disappeared with both mononuclear complexes 1b,
4 of 1c
15
and dinuclear complexes 2a, 2b, 2c and 2d. Taken together, these results indicate that the condensation
interactions between DNA and dinuclear complexes are stronger than those of mononuclear complexes,
alkaline solution (pH = 7.4). To our surprise, when the pH was lower than 7.0, dinuclear complexes
and complex 2c showed the strongest compression ability. The details are discussed below.
showed stronger cleavage properties for Form III band formation, and mononuclear complexes had
the same cleavage properties at pH below 7.0 as their cleavage properties at physiological conditions.
HN
It was proposed that H
for the benzimidazole group whichH contains two nitrogen atoms, one with
N
N
N = 11 could capture H+
pKa of 5.6 and another with
aRpKa
ofNabout 11, so the nitrogen atom
2+
R with pKa
N
HN M N
2+ N
M2+
N
M
H
N
and enhance the interaction with DNA with negative charge. It must be pointed out that the pKa
NH
of pyridine is 6.2, and N
the nitrogen atom in the pyridine could
not be protonated at pH values from
N
H
H
N
pH = 6.5 to 8.0. Furthermore,
IDB greatly
nClO4 - the linkage between cyclen andnClO
-. influenced the pH sensitivity
4 .mOH XH2 O
of dinuclear complexes.
For example, compounds 2a, 2b and 2d were
2a-2d linked with a m-xylyl, p-xylyl
1a-1c
and pyridyl group, respectively, and the pH-rate curves ofa:compounds
R = m-xylyl ; 2a,
M = 2b
Cu or
; n 2d
= 4;displayed different
b:
R
=
p-xylyl
;
M
=
Zn
;
n
=
= 2; coordination
a:
R
=
m-xylyl
;
M
=
Cu;
n
=
2;
“M-shape” profiles (shown in Figure 4), the pyridyl group might participate 4;
inXthe
c: R = p-xylyl ; M = Cu ; n = 4; X = 2;
b: R = m-xylyl
; M = Zn; n = 2;
2+
and reduce the
activity pyridyl
to result
cleavage
regardless
of
d: R ability
c: RZn
= 2,6-dimethyl
pyridyl;of
M =the
Zn protonation
;
= 2,6-dimethyl
; M =in
Zn;reduced
n = 3;
= 1; Xform
= 1. of DNA and showed that
beneimidazole in IDB. As for compounds 2a and 2b, Form IIIn =is3;a m
linear
dinuclear complexes possess
stronger
cleavage
properties
at lowercomplexes.
pH value.
Scheme
1. Structures
of target
benzimidazole
Figure 1.
1. Histograms
Histograms and
representing cleavage
cleavage of
of pUC19
pUC19
Figure
and electropherograms
electropherograms (the
(the right
right side
side figure)
figure) representing
plasmid
DNA
(0.008
µg/µL)
by
different
concentrations
of
1b
(pH
=
7.4)
and
1c
(pH
=
7.4)
in
buffer
(5 mM
plasmid DNA (0.008 µg/µL) by different concentrations of 1b (pH = 7.4) and 1c (pH = 7.4)
in
−5, 3.13 × 10−5, 6.25 × 10
−6
Tris-HCl/10
mM
NaCl)
at
37
°C
for
6
h.
(A)
Complex
1b;
Lanes
1–6:
6.25
×
10
˝
buffer (5 mM Tris-HCl/10 mM NaCl) at 37 C for 6 h. (A) Complex 1b; Lanes 1–6: 6.25 ˆ 10´5,,
−6, 6.25 × 10−7, 3.13
´7 , 3.13
´7 mol/L, Lane
× 10−7
Laneˆ7 =10DNA
control,
(B) Complex
Lanes
3.13 ˆ
× 10
3.13
10´5
, 6.25 ˆ 10´6 , 3.13
ˆ mol/L,
10´6 , 6.25
ˆ 10respectively;
7 = DNA1c;control,
−5
−5
−6
−6
−7
−7
1–6: 6.25 × 10 (B)
, 3.13
× 10 , 6.25
× 10 , 1–6:
3.13 ×6.25
10 , ˆ
6.25
, 3.13ˆ× 10
10´5mol/L,
control,
respectively;
Complex
1c; Lanes
10×´510
, 3.13
, 6.25 Lane
ˆ 107´6=,DNA
3.13 ˆ
10´6 ,
´7
´7
respectively.
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2016,
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920
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6.25 ˆ 10 , 3.13 ˆ 10 mol/L, Lane 7 = DNA control, respectively.
Figure
Figure 2.
2. Histograms
Histogramsand
andelectropherograms
electropherograms(the
(theright
rightside
side figure)
figure) representing
representing cleavage
cleavage of
of pUC19
pUC19
plasmid
DNA
(0.008
µg/µL)
by
different
concentrations
of
2b
(pH
=
7.4),
2c
(pH
=
7.4)
and
2d
(pH
=
7.4) 2d
in
plasmid DNA (0.008 µg/µL) by different concentrations of 2b (pH = 7.4), 2c (pH = 7.4) and
−5, 3.13 × 10−5,
˝
buffer
(5
mM
Tris-HCl/10
mM
NaCl)
at
37°C
for
6
h.
(A)
Complex
2b;
Lanes
1–6:
6.25
×
10
(pH = 7.4) in buffer (5 mM Tris-HCl/10 mM NaCl) at 37 C for 6 h. (A) Complex 2b; Lanes 1–6:
−7, 3.13
´7 , 3.13
´7 mol/L, Lane
, 3.13
× 10
6.25
× 10ˆ
10−7 ˆ
mol/L,
DNA
control,
(B) Complex
6.25
6.25 ׈10
10−6´5
, 3.13
ˆ −6
10, ´5
, 6.25
10´6 ,×3.13
10´6 ,Lane
6.25 7ˆ=10
ˆ 10respectively;
7 = DNA
−5
−5
−6
−6
−7
−7 mol/L,
´5
´5
× 10 ,2c;
6.25
× 10 1–6:
, 3.13
× 10
, 6.25, ×3.13
10 ˆ, 3.13
= DNA
2c;
Lanesrespectively;
1–6: 6.25 × 10(B), 3.13
control,
Complex
Lanes
6.25
ˆ 10
10 ×, 10
6.25
ˆ 10´6Lane
, 3.137 ˆ
10´6 ,
−5
−5
−6
−6,
´7 , 3.13 ˆ 10
´7 Complex
control,
(C)
2d; Lanes
1–6:control,
6.25 × respectively;
10 , 3.13 × 10
6.25 × 10 2d;
, 3.13
× 10
6.25 ˆ 10respectively;
mol/L, Lane
7 = DNA
(C), Complex
Lanes
1–6:
´5 , 6.25
6.25
, 3.13
× 10
mol/L,
Lane
7 ´6
= DNA
6.25 ׈10
10−7´5
, 3.13
ˆ−710
ˆ 10
, 3.13control,
ˆ 10´6 ,respectively.
6.25 ˆ 10´7 , 3.13 ˆ 10´7 mol/L, Lane 7 = DNA
control, respectively.
2.2. pH Effect on DNA Cleavage
pH-dependence profiles for DNA cleavage are shown in Figures 3 and 4. From these figures, it
can be seen that the pH-rate curve presents an “M-shape” (Figure 3) or ′′bell-shape′′ (Figure 4) profile
with the acidity change of the solution of mononuclear and dinuclear complexes from pH = 6.5–8.3,
and the optimal pH value for DNA cleavage is around 7.0 to 7.2 for both mononuclear and dinuclear
complexes. This indicates that the cleavage efficiency of supercoiled DNA by complex is correlated
Molecules2016,
2016,21,
21,920
920
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15
55ofof15
Zn2+2+complex
complexat
atthe
thesame
sameZn
Zn2+2+concentration.
concentration.Furthermore,
Furthermore,from
fromFigures
Figures66and
and7,7,ititcould
couldbe
befigured
figured
Zn
out that
that Cu
Cu2+2+ complex
complex 2c
2c was
was more
more effective
effective than
than Zn
Zn2+2+ complex
complex 2b
2b at
at the
the same
same concentration
concentration and
and
out
Molecules 2016, 21, 920
5 of 15
individualoptimal
optimalcleavage
cleavagepHs.
pHs.
individual
Figure
3.
Histograms
andelectropherograms
electropherograms(the
rightside
sidefigure)
figure) representing
representing
cleavage
of
pUC19
Figure3.
3.Histograms
Histogramsand
(theright
representingcleavage
cleavageof
ofpUC19
pUC19
Figure
´7−7−7
´6
−6−6mol/L)
plasmid
buffer
ofof
1a1a
(6.25
ˆ 10
ˆ××10
mol/L)
mol/L),1b
1b(3.13
(3.13
10
mol/L)
plasmidDNA
DNA(0.008
(0.008µg/µL)
µg/µL)in
indifferent
differentpH
pH
buffer
of
1a
(6.25
10 mol/L),
mol/L),
1b
(3.13
10
plasmid
(0.008
µg/µL)
in
different
pH
buffer
(6.25
××10
˝ C for 6 h. (A)
−7mol/L)
and
1c
(6.25
10
mol/L)
mMTris-HCl/10
Tris-HCl/10mM
mMNaCl)
NaCl)atat
at37
1´5:
and1c
1c(6.25
(6.25ˆ
10−7´7
mol/L)
mM
Tris-HCl/10
mM
NaCl)
37°C
°Cfor 66h.h.(A)
(A)Complex
Complex1a;
1a;Lanes
Lanes1−5:
1−5:
and
××10
(5(5(5
mM
pH
=
6.5,
7.0,
7.4,
8.0,
8.3,
Lane
6
=
DNA
control,
respectively;
(B)
Complex
1b;
Lanes
1–7:
pH
=
6.8,
7.0,
pH==6.5,
6.5, 7.0,7.4,
7.4,8.0,
8.0,8.3,
8.3,Lane
Lane66==DNA
DNAcontrol,
control,respectively;
respectively;(B)
(B)Complex
Complex1b;
1b;Lanes
Lanes1–7:
1–7:pH
pH==6.8,
6.8,
pH
7.2,
7.4,
7.6,
7.8,
8.0;
Lane
8 = DNA
control,
respectively;
(C) (C)
Complex
1c; Lanes
1–8:1–8:
pH pH
=pH
6.5,
6.8,
7.0,
7.0,7.2,
7.2,7.4,
7.4,7.6,
7.6,7.8,
7.8,8.0;
8.0;Lane
Lane
DNA
control,
respectively;
(C)Complex
Complex
1c;Lanes
Lanes
1–8:
6.5,6.8,
6.8,
7.0,
88==DNA
control,
respectively;
1c;
==6.5,
7.2,
7.4,
7.6,
7.8,
8.0;
Lane
9 = 9DNA
control,
respectively.
7.0,7.2,
7.2,7.4,
7.4,7.6,
7.6,7.8,
7.8,8.0;
8.0;Lane
Lane
9==DNA
DNA
control,
respectively.
7.0,
control,
respectively.
Figure4.
4.Histograms
Histogramsand
andelectropherograms
electropherograms(the
(theright
rightside
sidefigure)
figure)representing
representingcleavage
cleavageof
ofpUC19
pUC19
Figure
Figure
4.
Histograms
and
electropherograms
(the
right
side
figure)
representing
cleavage
of
pUC19
−7−7mol/L)
´7
´7
mol/L),2b
2b(3.13
(3.13
10
mol/L)
plasmidDNA
DNA(0.008
(0.008µg/µL)
µg/µL)in
indifferent
differentpH
pH
buffer
of
2a
(6.25
10−7−7
mol/L),
2b
(3.13
10
plasmid
(0.008
µg/µL)
in
different
pH
buffer
(6.25
××10
plasmid
buffer
ofof
2a2a
(6.25
ˆ 10
mol/L),
ˆ××10
mol/L)
−7mol/L)
−7
´7
˝
mol/L)
(5
mM
Tris-HCl/10
mM
NaCl)
at
37
°C
for
6
h.
(A)
Complex
2a;
Lanes
1–8:
and
2d
(6.25
×
10
(5
mM
Tris-HCl/10
mM
NaCl)
at
37
°C
for
6
h.
(A)
Complex
2a;
Lanes
1–8:
and
2d
(6.25
×
10
and 2d (6.25 ˆ
mol/L) (5 mM Tris-HCl/10 mM NaCl) at 37 C for
pH
=
6.5,
6.8,
7.0,
7.2,
7.4,
7.6,
7.8,
8.0;
Lane
9
=
DNA
control,
respectively.
(B)
Complex
2b
and
Lanes
pH
7.2,
7.4,7.4,
7.6,7.6,
7.8,7.8,
8.0;8.0;
Lane
9 = DNA
control,
respectively.
(B) Complex
2b and2b
Lanes
pH = 6.5,
6.5,6.8,
6.8,7.0,
7.0,
7.2,
Lane
9 = DNA
control,
respectively.
(B) Complex
and
1–8:pH
pH
6.5,
6.8,
7.0,
7.2,
7.4,
7.6,
7.8,
8.07.8,
Lane
DNA
control,
respectively;
(C)complex
complex
2d;Lanes
Lanes
1–8:
==6.5,
7.0,
7.2,
7.4,
7.6,
7.8,
8.0
Lane
==DNA
respectively;
(C)
2d;
Lanes
1–8:
pH6.8,
= 6.5,
6.8,
7.0,
7.2,
7.4,
7.6,
8.099Lane
9 =control,
DNA control,
respectively;
(C) complex
2d;
1–8:pH
pH
6.5,
6.8,
7.0,6.8,
7.2,7.0,
7.4,7.2,
7.6,7.4,
7.8,7.6,
8.0;7.8,
Lane
DNA
control,
respectively.
1–8:
==6.5,
7.0,
7.2,
7.4,
7.6,
7.8,
8.0;
Lane
==DNA
respectively.
Lanes
1–8:
pH6.8,
= 6.5,
8.0;99Lane
9 =control,
DNA control,
respectively.
2.3. Reaction Time Effect on DNA Cleavage
Agarose gel electrophoretogram of time courses of pUC19 DNA cleavage by complexes 1b and
2b and 2c were selected for comparing ligands and cations, and results are shown in Figures 5–7.
These figures show that the longer the reaction time at 37 ˝ C, the higher the conversion efficiency of
plasmid DNA from Form I to Form II. The fluorescence intensity of Form II increases markedly with
a corresponding decrease in the intensity of Form I. This result indicates that these complexes can
promote the cleavage of plasmid pUC19 DNA from supercoiled Form I to linear Form II efficiently
and the catalytic efficiency of DNA cleavage is correlated to the reaction time. From Figures 5 and 6, it
was found that both mononuclear complex and dinuclear complex would take more than 6 hours to
cleave DNA, and the cleavage ability of dinuclear Zn2+ complex is weaker than that of
mononuclear
Figure5.5.Time
Timecourse
courseof
ofpUC19
pUC19DNA
DNA(0.008
(0.008µg/µL)
µg/µL)cleavage
cleavagepromoted
promotedby
by1b
1b(6.25
(6.25××10
10−7−7mol/L)
mol/L)in
in
Figure
Zn2+ complex at the same Zn2+ concentration. Furthermore, from Figures 6 and 7, it could be figured
pH==7.4
7.4buffers
buffers(5(5mM
mMTris-HCl/10
Tris-HCl/10mM
mMNaCl)
NaCl)atat37
37°C.
°C.Lanes
Lanes1–6,
1–6,reaction
reactiontime
time6,6,5,5,4,4,3,3,2,2,11h,h,
pH
out that
Cu2+ complex 2c was more effective than Zn2+ complex 2b at the same concentration and
respectively.
respectively.
individual optimal cleavage pHs.
Figure 4. Histograms and electropherograms (the right side figure) representing cleavage of pUC19
plasmid DNA (0.008 µg/µL) in different pH buffer of 2a (6.25 × 10−7 mol/L), 2b (3.13 × 10−7 mol/L)
and 2d (6.25 × 10−7 mol/L) (5 mM Tris-HCl/10 mM NaCl) at 37 °C for 6 h. (A) Complex 2a; Lanes 1–8:
pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively. (B) Complex 2b and Lanes
1–8:
pH
6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 Lane 9 = DNA control, respectively; (C) complex 2d; Lanes
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1–8: pH = 6.5, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0; Lane 9 = DNA control, respectively.
−7 mol/L) in
Figure
Timecourse
courseofof
pUC19DNA
DNA
(0.008
µg/µL)cleavage
cleavagepromoted
promotedby
by1b
1b(6.25
(6.25ˆ×10
10´7
Figure
5. 5.Time
pUC19
(0.008
µg/µL)
mol/L)
mM
Tris-HCl/10
mM
NaCl)
at 37
1–6, 1–6,
reaction
time time
6, 5, 6,
4, 3,
inpH
pH= =7.4
7.4buffers
buffers(5(5
mM
Tris-HCl/10
mM
NaCl)
at °C.
37 ˝Lanes
C. Lanes
reaction
5, 2,
4, 13,h,
respectively.
2, 1 h, respectively.
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2016,
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2016,
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920
6 of
6 of
1515
´7mol/L)
Figure
Time
course
of
pUC19
DNA
(0.008
µg/µL)
cleavage
promoted
(3.13
× 10
−7
Figure
Time
course
pUC19
DNA
(0.008
µg/µL)
10−7
mol/L)
Figure
6.6.6.
Time
course
ofof
pUC19
DNA
(0.008
µg/µL)
cleavage
promoted
byby
2b2b
(3.13
׈
10
mol/L)
inin
˝
pH
= 7.4
buffers
mM
Tris-HCl/10
mM
NaCl)
Lanes
1–6,
reaction
time
5,
1 3,
h,
in
=buffers
7.4
buffers
(5 Tris-HCl/10
mM
Tris-HCl/10
mM
NaCl)
at°C.
37
C. 1–6,
Lanes
1–6, reaction
time
pH
=pH
7.4
(5(5
mM
mM
NaCl)
atat
3737
°C.
Lanes
reaction
time
6, 6,
5,
4, 4,
3,6,3,
2,5,2,
14,h,
respectively.
2,
1
h,
respectively.
respectively.
´7mol/L)
Figure
Time
course
of
pUC19
DNA
(0.008
µg/µL)
cleavage
promoted
× 10
−7
Figure
Time
course
pUC19
DNA
(0.008
µg/µL)
(3.13
ˆ
10−7
mol/L)
Figure
7.7.7.
Time
course
ofof
pUC19
DNA
(0.008
µg/µL)
cleavage
promoted
byby
2c2c
(3.13
× 10
mol/L)
inin
˝
pH
=
6.0
buffers
(5
mM
Tris-HCl/10
mM
NaCl)
at
37
°C.
Lanes
1–6,
6,
5,
4,
3,
2,
1
h
reaction
time,
in =pH
6.0 buffers
(5 mM
Tris-HCl/10
mM NaCl)
at 37
C. Lanes
2, 1 h reaction
pH
6.0=buffers
(5 mM
Tris-HCl/10
mM NaCl)
at 37 °C.
Lanes
1–6, 6,1–6,
5, 4,6,3,5,2,4,1 3,
h reaction
time,
respectively.
time,
respectively.
respectively.
2.4.
DNA
Cleavage
on
the
Presence
Typical
Radical
Scavengers
2.4.
DNA
Cleavage
the
Presence
Typical
Radical
Scavengers
2.4.
DNA
Cleavage
onon
the
Presence
ofofof
Typical
Radical
Scavengers
Asisisisknown,
known,DNA
DNAcleavage
cleavagegenerally
generallyproceeds
proceedsvia
viatwo
twomajor
majorpathways:
pathways:one
oneisisisthe
thehydrolytic
hydrolytic
As
known,
DNA
cleavage
generally
proceeds
via
two
major
pathways:
one
the
hydrolytic
As
pathway
involving
the
phosphate
group
[4];
the
other
is
oxidative
cleavage
of
the
sugar
and/or
pathwayinvolving
involvingthe
thephosphate
phosphategroup
group[4];
[4];the
theother
otherisis oxidative
oxidativecleavage
cleavageof
of the
the sugar
sugar and/or
and/or
pathway
nucleobase
moiety
due
to
the
oxidation
of
the
ribose
or
base
group
of
DNA
by
reactive
oxygen
species
nucleobase
moiety
due
the
oxidation
the
ribose
base
group
DNA
by
reactive
oxygen
species
nucleobase
moiety
due
toto
the
oxidation
ofof
the
ribose
oror
base
group
ofof
DNA
by
reactive
oxygen
species
resulting
in
the
oxidative
DNA
cleavage
pathway
[27].
To
explore
the
DNA
cleavage
mechanism
by
resulting
in
the
oxidative
DNA
cleavage
pathway
[27].
To
explore
the
DNA
cleavage
mechanism
by
resulting in the oxidative DNA cleavage pathway [27]. To explore the DNA cleavage mechanism by
these
complexes,
the
cleavage
of
DNA
by
mononuclear
and
dinuclear
complexes
was
carried
out
thesecomplexes,
complexes,the
thecleavage
cleavage
DNA
mononuclear
and
dinuclear
complexes
carried
these
ofof
DNA
byby
mononuclear
and
dinuclear
complexes
waswas
carried
out out
inin
thepresence
presenceofoftypical
typicalradical
radicalscavengers
scavengersforforsinglet
singletoxygen
oxygen(NaN
(NaN
forsuperoxide
superoxide(KI),
(KI),and
andfor
for
the
3),3),
for
hydroxyl
radical
(DMSO
and
t-BuOH)
(shown
in
Figures
8–10
and
Supplementary
Information).
hydroxyl radical (DMSO and t-BuOH) (shown in Figures 8–10 and Supplementary Information). AsAs
evidently
depicted
Figures
8–10,
the
presence
any
these
scavengers
(NaN
, DMSO,
t-BuOH,
evidently
depicted
inin
Figures
8–10,
inin
the
presence
ofof
any
ofof
these
scavengers
(NaN
3, 3DMSO,
t-BuOH,
KI),there
therewere
weresignificant
significantinhibition
inhibitioneffects
effectsononthe
theDNA
DNAcleavage
cleavagebybynearly
nearlyallall
thesecomplexes,
complexes,
KI),
ofofthese
except
for
1a,
which
rules
out
the
involvement
of
reactive
oxygen
species.
Figure
8
shows
thatthe
the
except for 1a, which rules out the involvement of reactive oxygen species. Figure 8 shows that
Molecules 2016, 21, 920
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in the presence of typical radical scavengers for singlet oxygen (NaN3 ), for superoxide (KI), and for
hydroxyl radical (DMSO and t-BuOH) (shown in Figures 8–10 and Supplementary Information). As
evidently depicted in Figures 8–10, in the presence of any of these scavengers (NaN3 , DMSO, t-BuOH,
KI), there were significant inhibition effects on the DNA cleavage by nearly all of these complexes,
except for 1a, which rules out the involvement of reactive oxygen species. Figure 8 shows that the
cleavage property of complex 1a was barely affected by these scavengers, which mean free radicals
were slightly involved in the cleavage process, and that was to say DNA cleavage by 1a was via a
hydrolytic pathway, but other mononuclear compounds use oxidative cleavage to destroy DNA.
Figure 9 shows that sample 2a with added NaN3 did suppress the cleavage efficiency, which was
reduced over 60% by superoxide (KI) and hydroxyl radical (t-BuOH). Similar situations accompanied
the cleavage processes of 2b, 2c and 2d, and NaN3 and KI were the most effective individual
suppressors. These results reflected that these target materials destroyed DNA via different processes
and ligands affected these mechanisms and mononuclear Cu2+ complex 1a had more probability
to mimic hydrolytic enzymes. Furthermore, Cu2+ complexes always cleave DNA by an oxidative
pathway, but the ligand structure was the key factor for artificial nucleases and some examples
Molecules
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920
of[28]
15 and
showed
Cu2+
complexes
could hydrolyse DNA for special ligands, such as diaza-crown ether
1,3-bis(1,4,7-triaza-1-cyclononyl)propane
[29]. However,
compounds
with IDBthese
always cleave
DNA by
3+
by
for
by an
an oxidative
oxidative pathway
pathway expect
expect3+
for Fe
Fe3+ complexes,
complexes, and
and it
it was
was proposed
proposed that
that these two
two centers
centers of
of
an oxidative
pathway
expect
for
Fe
complexes,
and
it
was
proposed
that
these
two
centers
of
positive
positive
positive charge
charge synergistically
synergistically interacted
interacted with
with DNA
DNA units
units to
to destroy
destroy DNA
DNA for
for relative
relative faster
faster
charge
synergistically
interacted
with concentration.
DNA units toTherefore,
destroy DNA
for relative
faster cleavage
speed at
cleavage
speed
same
the
mechanisms
of
cleavage
speed at
at the
the
same cationic
cationic
concentration.
Therefore,
the cleavage
cleavage
mechanisms
of this
this kind
kind
of
benzimidazole
cyclen
complexes
were
influenced
by
cations
and
IDB
group,
especially
by
the same
cationic
concentration.
Therefore,
the
cleavage
mechanisms
of
this
kind
of
benzimidazole
of benzimidazole cyclen complexes were influenced by cations and IDB group, especially by the
the
benzimidazole
ligand.
cyclen
complexes were
influenced by cations and IDB group, especially by the benzimidazole ligand.
benzimidazole
ligand.
Figure
8.
representing
cleavage
of
pUC19
(0.008
µg/µL)
by
1a
Figure
8. Histograms
Histograms
representingcleavage
cleavage of
of pUC19
pUC19 plasmid
plasmid
DNA
(0.008
µg/µL)
by compound
compound
1a
Figure
8. Histograms
representing
plasmidDNA
DNA
(0.008
µg/µL)
by compound
1a
−7 mol/L, pH = 8.0). Lane 1: DNA control, Lane 2:
with
different
typical
radical
scavengers
(6.25
×
10
−7
´7
different
typical
radical
scavengers(6.25
(6.25ˆ× 10
10 mol/L,
pHpH
= 8.0).
Lane
1: DNA
control,
Lane 2:
with with
different
typical
radical
scavengers
mol/L,
= 8.0).
Lane
1: DNA
control,
Lane 2:
no
Lane
3:
3, Lane 4: KI, Lane 5: DMSO, Lane 6: t-BuOH.
no inhibitor,
inhibitor,
Lane
3: NaN
NaN
3, Lane 4: KI, Lane 5: DMSO, Lane 6: t-BuOH.
no inhibitor,
Lane
3: NaN
3 , Lane 4: KI, Lane 5: DMSO, Lane 6: t-BuOH.
Figure 9. Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 1b
Figure 9. Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 1b
Figure
9. Histograms representing cleavage
of pUC19 plasmid DNA (0.008 µg/µL) by compound 1b
−4 mol/L, pH = 7.4). Lane 1: DNA control, Lane 2: no inhibitor,
typical
−4 mol/L, pH = 7.4). Lane 1: DNA control, Lane 2: no inhibitor,
typical radical
radical scavengers
scavengers (3.13
(3.13 ×× 10
10
´4
typical
radical
scavengers
(3.13 ˆ5:10
mol/L, pH
= 7.4). Lane 1: DNA control, Lane 2: no inhibitor,
Lane
Lane 4:
4: KI,
KI, Lane
Lane 5: t-BuOH,
t-BuOH, Lane
Lane 6:
6: DMSO.
DMSO.
Lane 3:
3: NaN
NaN33,, Lane
Lane 3: NaN3 , Lane 4: KI, Lane 5: t-BuOH, Lane 6: DMSO.
Figure 9. Histograms representing cleavage of pUC19 plasmid DNA (0.008 µg/µL) by compound 1b
typical radical scavengers (3.13 × 10−4 mol/L, pH = 7.4). Lane 1: DNA control, Lane 2: no inhibitor,
Molecules
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3, Lane 4: KI, Lane 5: t-BuOH, Lane 6: DMSO.
Lane
3: NaN
Figure 10.
plasmid
DNA
(0.008
µg/µL)
by by
compound
2a
10. Histograms
Histogramsrepresenting
representingcleavage
cleavageofofpUC19
pUC19
plasmid
DNA
(0.008
µg/µL)
compound
´7−7mol/L, pH = 7.4). Lane 1:
typical
radical
scavengers
(6.25
ˆ 10
no inhibitor,
inhibitor,
2a typical
radical
scavengers
(6.25
× 10
mol/L, pH =
DNA control, Lane 2: no
Lane 3: NaN3,, Lane
Lane 4:
4: KI,
KI, Lane
Lane 5:
5: DMSO,
DMSO, Lane
Lane 6:
6: t-BuOH.
t-BuOH.
Molecules 2016, 21, 920
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On the
above
DNA-cutting
experimental
results,
a tentative
mechanism
of DNA
the basis
basisofofthethe
above
DNA-cutting
experimental
results,
a tentative
mechanism
of
cleavage
catalyzed
by the metal
complexes
1a as an example
proposed
Scheme 2.
macrocyclic
DNA cleavage
catalyzed
by the
metal complexes
1a as anisexample
is in
proposed
inLike
Scheme
2. Like
2+ complexes
polyamine
Zn2+binuclear
complexesZn
[30]
and Cu2+ -IDB
or
oxygen
participate
macrocyclicbinuclear
polyamine
[30][31],
andHCu
-IDB
[31],atoms
H2O usually
or oxygen
atoms
2 O 2+
in
coordination.
With
water in the unit
of water
complex
1a confirmed
by elemental
analysis
it was
usually
participate
in coordination.
With
in the
unit of complex
1a confirmed
by[32],
elemental
released
groupsthat
from
crystallization
be the attacking
groupbeatthe
pHattacking
8.0. This
analysis that
[32], hydroxyl
it was released
hydroxyl
groupswater
from might
crystallization
water might
scheme
that the
positive
metal ion
in the
the positive
metal complex
chargedattracted
oxygen
group atindicated
pH 8.0. This
scheme
indicated
that
metal attracted
ion in thenegatively
metal complex
in
the DNAcharged
phosphate
groupinbythe
electrostatic
interaction,
andby
then
nucleophilic
hydroxyl produced
negatively
oxygen
DNA phosphate
group
electrostatic
interaction,
and then
from
water molecules
metal
ion attacked
the phosphorus
DNA
and then
nucleophilic
hydroxylassociated
produced with
fromthe
water
molecules
associated
with the atom
metalofion
attacked
the
promoted
theatom
cleavage
of P-O
to generate
product.
phosphorus
of DNA
andbonds
then promoted
the
cleavage of P-O bonds to generate product.
Scheme
2. Possible
cleavage catalyzed
catalyzed by
by the
the complex
complex 1a.
1a.
Scheme 2.
Possible mechanism
mechanism of
of DNA
DNA cleavage
2.5. DNA
and Regulation
Regulation by
by GO
GO
2.5.
DNA Condenstion
Condenstion and
Compound 2c
2c was
was selected
selected to
to investigate
investigate the
the DNA
DNA condensation
condensation properties
properties for
for its
its representative
representative
Compound
structure with
with the
the strongest
strongest compression
compression ability
ability according
according to
to Figure
Figure 2.
2. At
At the
the same
same time,
time, the
the particle
particle
structure
sizes
of
2c/DNA
complex
with
weight
ratios
3.9
and
7.8
were
280
nm
and
645
nm
respectively.
sizes of 2c/DNA complex with weight ratios 3.9 and 7.8 were 280 nm and 645 nm respectively.
Meanwhile, the ξ potentials were 15 mV and 26 mV. Figure 11 reflects that GO alone could not
condense or cleave DNA, and complex 2c could effectively cleave DNA at 6.25 × 10−7 mol/L.
Scheme 2. Possible mechanism of DNA cleavage catalyzed by the complex 1a.
2.5. DNA Condenstion and Regulation by GO
Compound 2c was selected to investigate the DNA condensation properties for its representative
9 of 15
strongest compression ability according to Figure 2. At the same time, the particle
sizes of 2c/DNA complex with weight ratios 3.9 and 7.8 were 280 nm and 645 nm respectively.
Meanwhile, the ξ potentials were 15 mV and 26 mV. Figure 11 reflects that GO alone could not
Meanwhile, the ξ potentials were 15 mV and 26 mV. Figure 11 reflects that GO alone could not
−7 mol/L.
condense or cleave DNA, and complex 2c could effectively cleave DNA at 6.25 × 10´
condense or cleave DNA, and complex 2c could effectively cleave DNA at 6.25 ˆ 10 7 mol/L.
Molecules
2016,
21, 920
structure
with
the
Figure
Figure 11.
11. Electropherograms
Electropherograms representing
representing cleavage
cleavageof
ofpUC19
pUC19plasmid
plasmidDNA
DNA(0.008
(0.008µg/µL)
µg/µL) in
in buffer
buffer
´7 mol/L, 5 mM Tris-HCl/10 mM NaCl,
of
ratios of
ofGO
GOtotocomplex
complex2c2c(6.25
(6.25× ˆ
10mol/L,
5 mM Tris-HCl/10 mM NaCl, pH =
of different weight ratios
10−7
˝ C for 8 h. Lanes 1–7: complex 2c alone, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, respectively and Lane
pH
6.0)=at6.0)
37 at
°C37
for
8 h. Lanes 1–7: complex 2c alone, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, respectively and Lane 8 is
8DNA
is DNA
control
with
(0.67
µg/µL),
respectively.
control
with
GOGO
(0.67
µg/µL),
respectively.
However, when
when the
the weight
weight ratio
ratio of
of 2c/GO
2c/GO was
was above
above 0.5,
0.5, the
the regulation
regulation function
function of
of GO
GO was
was
However,
obvious
and
it
inhibited
the
cleavage
ability
of
2c
to
maintain
most
DNA
in
superhelical
form.
At
obvious and it inhibited the cleavage ability of 2c to maintain most DNA in superhelical form. the
At
same
time,time,
FormForm
I shown
in Figure
12 disappeared
when the
concentration
of 2c wasof
above
3.13above
× 10−5
the
same
I shown
in Figure
12 disappeared
when
the concentration
2c was
´5 when GO
mol/L,
but
was added after the DNA complex was formed, Form I was partly released.
3.13
ˆ 10
Molecules
2016, mol/L,
21, 920 but when GO was added after the DNA complex was formed, Form I9 was
of 15
Furthermore,
the
amount
of 2c was
important,
andwas
when
the concentration
was
× 10−5 mol/L,was
the
partly released. Furthermore,
the amount
of 2c
important,
and when
the6.25
concentration
5 mol/L,
release
of ´DNA
was weak,
so theofbest
control
releaseso
concentrations
of 2c
and GO
were 3.13 × 10
mol/L
6.25 ˆ 10
the release
DNA
was weak,
the best control
release
concentrations
of−52c
and
´
5
and
1.34 µg/µL,
GO were
3.13 ˆ respectively.
10 mol/L and 1.34 µg/µL, respectively.
Figure 12. Electropherograms
Electropherograms representing
representing condensation
condensation and
and release
release of
of pUC19
pUC19 plasmid
plasmid DNA
(0.008 µg/µL)
µg/µL) in
Tris-HCl/10 mM
in buffer of different weight ratios of GO to complex 2c (5 mM Tris-HCl/10
mM NaCl,
NaCl,
−5 mol/L), 2c (3.13 × 10−5 ´5
´5
mol/L),
2c
pH = 7.4) at 37 ˝°C.
Lane
1
is
DNA
control,
Lanes
2–8:
2c
(6.25
×
10
C.
1 is DNA control, Lanes 2–8: 2c (6.25 ˆ 10 mol/L), 2c (3.13 ˆ 10 mol/L),
−5
−5
−5
´5
´5
(6.25
× 10 ˆmol/L)/GO
(0.67 µg/µL),
2c (6.25
× 10 2cmol/L)/GO
2c (3.13(1.34
× 10 µg/µL),
mol/L)/GO
2c (6.25
10
mol/L)/GO
(0.67
µg/µL),
(6.25 ˆ (1.34
10 µg/µL),
mol/L)/GO
2c
mol/L)/GO
µg/µL),
respectively.
(0.67
µg/µL),
2c (3.13 × 10−5(0.67
(3.13 ˆ
10´5 mol/L)/GO
µg/µL),(1.34
2c (3.13
ˆ 10´5
mol/L)/GO (1.34 µg/µL), respectively.
As
between
DNA
andand
complex,
the the
π-ππ-π
stacking
waswas
the key
As for
for GO
GOregulation
regulationofofthe
theinteraction
interaction
between
DNA
complex,
stacking
the
2+-IDB which led to DNA release. Although the mononuclear
factor,
and
complex
adhered
to
GO
by
Cu
2+
key factor, and complex adhered to GO by Cu -IDB which led to DNA release. Although the
compounds
have thealso
benzimidazole
group shown
in shown
Figure in
1,Figure
they cannot
DNA.
mononuclearalso
compounds
have the benzimidazole
group
1, they condense
cannot condense
Furthermore,
cyclen
complex
conjugated
with
one
or
two
big
aromatic
ring
structures,
such
DNA. Furthermore, cyclen complex conjugated with one or two big aromatic ring structures, such as
as
anthracene
[33]
and
acridine
groups
[34],
always
show
DNA
cleavage
abilities.
IDB
had
advantages
anthracene [33] and acridine groups [34], always show DNA cleavage abilities. IDB had advantages
over
tridentate ligands
ligands like
like tris(benzimidazol-2-ylmethyl)amine
tris(benzimidazol-2-ylmethyl)amine (NTB)
possessing four
over tridentate
(NTB) or
or ligands
ligands possessing
four
benzimidazole
groups
like
N,N,N′,N′-tetrakis(benzimidazol-2-ylmethyl)-1,2-ethanediamine
benzimidazole groups like N,N,N1 ,N1 -tetrakis(benzimidazol-2-ylmethyl)-1,2-ethanediamine (EDTB),
(EDTB),
2+-IDB was nearly 180°, as verified
because
the internal
internal angle
angle between
between two
two benzimidazoles
benzimidazoles of
of Cu
Cu2+
because of
of the
-IDB was nearly 180˝ , as verified
by
the
single
crystal
structure
[31]
and
the
internal
angles
of
benzimidazoles
by the single crystal structure [31] and the internal angles of benzimidazoles in
in NTB
NTB and
and EDTB
EDTB were
were
2+-IDB and
about
120°
[18],
so
it
was
even
greater
in
IDB
than
in
NTB
and
EDTB.
As
we
know,
Cu
˝
2+
about 120 [18], so it was even greater in IDB than in NTB and EDTB. As we know, Cu -IDB and
2+
Cu
Cu2+-cyclen
-cyclencoordinated
coordinatedwith
withone
oneH
H22OOeach
eachas
asconfirmed
confirmedby
byelemental
elemental analysis
analysis to
to maintain
maintain individual
individual
architecture
stabilization,
and
the
p-xylyl
group
as
linker
forced
two
nuclei
away
architecture stabilization, and the p-xylyl group as linker forced two nuclei away from
from each
each other,
other, so
so
there
might
be
no
interference
between
them.
The
benzyl
group
as
linker
also
did
not
participate
there might be no interference between them. The benzyl group as linker also did not participate in
in
coordination,
coordination, and
and each
each cationic
cationic nucleus
nucleus of
of the
the hybrid
hybrid complex
complex 2c
2c would
would keep
keep its
its original
original configuration.
configuration.
Moreover,
the benzyl
benzyllinker
linkermight
mightprovide
provideextra
extraπ-π
π-π
stacking.
It also
is also
important
to point
Moreover, the
stacking.
It is
important
to point
outout
thatthat
the
the
π-π
stacking
between
two
independent
benzimidazoles
has
been
proven
[12]
and
the
effect
π-π stacking between two independent benzimidazoles has been proven [12] and the effect should
should be strong enough for GO to conjugate with dinuclear complexes. Figures 11 and 12 also show
that when complex 2c and DNA were incubated in an EP tube together, no matter the DNA complex
formed or approaching cleavage by electrostatic interaction, the GO could hinder the two interacting
models. There was nearly no Form II or Form III DNA present and structures at the concentrations
for cleavage were almost superhelical. Furthermore, it also can be found that the electrostatic
about 120° [18], so it was even greater in IDB than in NTB and EDTB. As we know, Cu2+-IDB and
Cu2+-cyclen coordinated with one H2O each as confirmed by elemental analysis to maintain individual
architecture stabilization, and the p-xylyl group as linker forced two nuclei away from each other, so
there might be no interference between them. The benzyl group as linker also did not participate in
coordination, and each cationic nucleus of the hybrid complex 2c would keep its original configuration.
Molecules 2016, 21, 920
10 of 15
Moreover, the benzyl linker might provide extra π-π stacking. It is also important to point out that
the π-π stacking between two independent benzimidazoles has been proven [12] and the effect
be
strong
for GO for
to conjugate
with dinuclear
complexes.
Figures
11 and1112
also
that
should
beenough
strong enough
GO to conjugate
with dinuclear
complexes.
Figures
and
12show
also show
when
complex
2c
and
DNA
were
incubated
in
an
EP
tube
together,
no
matter
the
DNA
complex
that when complex 2c and DNA were incubated in an EP tube together, no matter the DNA complex
formed
formed or
or approaching
approaching cleavage
cleavage by
by electrostatic
electrostaticinteraction,
interaction,the
theGO
GOcould
could hinder
hinder the
the two
two interacting
interacting
models.
Form
IIIIII
DNA
present
andand
structures
at the
concentrations
for
models. There
Therewas
wasnearly
nearlyno
noForm
FormII IIoror
Form
DNA
present
structures
at the
concentrations
cleavage
were
almost
superhelical.
Furthermore,
it
also
can
be
found
that
the
electrostatic
interaction
for cleavage were almost superhelical. Furthermore, it also can be found that the electrostatic
which
is the which
key effect
forkey
commonly
used
gene delivery
to condense
DNA
was relative
weaker
interaction
is the
effect for
commonly
used agents
gene delivery
agents
to condense
DNA
was
than
the
π-π
stacking
action.
The
possible
mechanism
of
DNA
release
and
cleavage
hindrance
by
the
relative weaker than the π-π stacking action. The possible mechanism of DNA release and cleavage
complex
2cby
is shown
in Scheme
3.
hindrance
the complex
2c is shown
in Scheme 3.
Scheme 3.
3. Possible
Possible mechanism
mechanism of
of DNA
DNA release
release and
and cleavage
cleavage blockage
blockageby
bycomplex
complex2c.
2c.
Scheme
To our surprise, the condensation concentrations of cyclen-ethyl and IDB-ethyl complexes at
2.0 ˆ 10´ 3 mol/L were only about 11.1% and 12.3%, respectively (CDNA = 3.1 ˆ 10´3 µg/µL) [19].
However, most hybrid complexes at 3.13 ˆ 10´5 mol/L (CDNA = 8.0 ˆ 10´3 µg/µL) could block nearly
all superhelical DNA in gel as shown in Figure 12, and the condensation concentration was nearly
1/60 with regard to cyclen and IDB. From the results above, it can be concluded that there would be
synergetic effects and the reinforcement between the two different cationic nuclei. This kind of unique
synergetic structures of binuclear compounds might produce more stable condensation with DNA by
stronger electrostatic interactions with two cationic nuclei. The DNA complex formed by acetylated
cyclen at high temperature would disintegrate with decreasing temperature. [17] From the literature,
it is also important to point out that the acetylated cyclen merged into the double-stranded of DNA
and drew the outer sphere of DNA to form nanoparticles [17], so this kind of DNA complex formed
with acetylated cyclen was negatively charged and zeta potentials were around ´34 mV to ´10 mV,
and it was hard to adhere to the cells. In contrast, target materials could form complexes with positive
charges which were blocked in the sample holes in the electrophoresis experiments could fulfill the
needs of gene delivery.
2.6. Fluorescence Spectra of the Interaction between Complex and GO
Guo1 s group recently demonstrated by using fluorescence spectroscopy the existence of single
atomic layered graphene oxide (GO) sheets, due to their unique structural properties [35], so to further
confirm the interactions between complex and GO, fluorescence spectra were used. From Figure 13, it
could be seen that the peaks of emission spectra a–e were quenched by adding GO solutions. This
confirmed that there was valid interaction between the GO and complex.
Guo′s group recently demonstrated by using fluorescence spectroscopy the existence of single
atomic layered graphene oxide (GO) sheets, due to their unique structural properties [35], so to further
confirm the interactions between complex and GO, fluorescence spectra were used. From Figure 13, it
could be seen that the peaks of emission spectra a–e were quenched by adding GO solutions. This
Molecules 2016, 21, 920
11 of 15
confirmed that there was valid interaction between the GO and complex.
60000
Fluorescence intensity (a.u)
50000
Compound 2c
a
40000
e
30000
20000
10000
0
260
280
300
320
340
360
Wavelength (nm)
−5 mol/L in 5 mM Tris,
´5
Figure 13.
13. Emission spectra a–e
(a–e)the
thetarget
target
material2c2c(1(1ˆ×1010
Figure
material
mol/L in 5 mM
50 mM NaCl,
´5mg/mL).
pH == 7.4
7.4 buffer
buffer with
with increasing
increasing GO
GO concentration
concentration (from
(from00to
to6.68
6.68ˆ
× 10−5
pH
mg/mL).
2.7. Cytotoxicity
Cytotoxicity
2.7.
The cytotoxic
cytotoxic activities
activities of
of these
these complexes
complexes were
were tested
tested by
by the
the CCK8
CCK8method,
method, and
and evaluated
evaluated in
in
The
HL-7702
cells.
As
shown
in
Figure
14,
none
of
them
displayed
serious
cytotoxicity
in
this
cell-line.
The
HL-7702 cells. As shown in Figure 14, none of them displayed serious cytotoxicity in this cell-line. The
relative cell
cellviability
viabilityofof2b
2boror2d2dwas
was
more
than
70%
when
concentration
mg/mL.
relative
more
than
70%
when
thethe
concentration
waswas
overover
100 100
mg/mL.
To
2+ complexes showed relative high cytotoxicity in the cell-line. Data obtained
2+
To
our
surprise,
two
Cu
our surprise, two Cu complexes showed relative high cytotoxicity in the cell-line. Data obtained
1 s capacity
from in
in vitro
vitro and
and cell
cell culture
culturestudies
studieswere
werelargely
largelysupportive
supportiveof
ofcopper
copper′s
capacity to
to initiate
initiate oxidative
oxidative
from
damage
and
interfere
with
important
cellular
events
[36],
but
the
results
also
reflected
that
all
of these
these
damage and interfere with important cellular events [36], but the results also reflected that all of
complexes
2a–d
showed
relative
low
toxicity
to
normal
human
hepatic
cells
at
high
concentration,
complexes 2a–d showed relative low toxicity to normal human hepatic cells at high concentration, and
and dinuclear
safefurther
for further
applications.
dinuclear
compounds
werewere
safe for
applications.
Molecules
2016,
21, compounds
920
11 of 15
Figure
Figure 14.
14. Relative
Relative cell
cell viabilities
viabilities of
of compounds
compounds 2a–d
2a–d in
in HL-7702
HL-7702 cells.
cells.
3. Experimental Section
3. Experimental Section
3.1.
3.1. Materials
Materials
Plasmid
DNA (TaKaRa
(TaKaRa Biotechnology,
Biotechnology, Dalian,
Dalian, China),
China), 50
50 ˆ
× TAE,
TAE, 6ˆ
6× loading
loading buffer,
buffer, gold
gold
Plasmid pUC19
pUC19 DNA
view
dye,
and
agarose
were
purchased
from
Beijing
Changsheng
Biotechnology
Co.,
Ltd.
(Beijing,
China).
view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. (Beijing, China).
Cell
CountingKit-8
Kit-8
(CCK8)
from
Yeasen
Company
(Shanghai,
trishydroxymethylaminoCell Counting
(CCK8)
from
Yeasen
Company
(Shanghai,
China),China),
trishydroxymethylamino-methane
methane
(Tris),
HCl,
NaCl,
and
NaOH
were
analytical
grade
products
and
used
asAll
supplied.
All other
(Tris), HCl, NaCl, and NaOH were analytical grade products and used as supplied.
other chemicals
chemicals
purchased
fromChemical
Chongqing
Co.China),
(Chongqing,
China), unless
otherwise
purchased from
Chongqing
Co. Chemical
(Chongqing,
unless otherwise
indicated,
were of indicated,
analytical
were of analytical grade. The water used for experiments was doubly distilled water. All chemicals
were reagent grade and were used without further purification, unless otherwise noted. Graphene
oxide solution was purchased from the Chinese Academy of Sciences Institute of Coal Chemistry
(Taiyuan, China). 1-(3-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)benzyl)-1H-benzo[d]imidazole Cu2+
complex (1a), 1-(3-((1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)-1H-benzo[d]imidazole Zn2+
complex (1b), 1-((6-((1,4,7,10-tetraazacyclododecan-1-yl)methyl)pyridin-2-yl)methyl)-1H-benzo[d]
Molecules 2016, 21, 920
12 of 15
grade. The water used for experiments was doubly distilled water. All chemicals were reagent grade
and were used without further purification, unless otherwise noted. Graphene oxide solution was
purchased from the Chinese Academy of Sciences Institute of Coal Chemistry (Taiyuan, China).
1-(3-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)benzyl)-1H-benzo[d]imidazole Cu2+ complex (1a),
1-(3-((1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)-1H-benzo[d]imidazole Zn2+ complex (1b),
1-((6-((1,4,7,10-tetraazacyclododecan-1-yl)methyl)pyridin-2-yl)methyl)-1H-benzo[d]imidazole Zn2+
complex (1c), N-(3-((1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)-N-((1H-benzo[d]imidazol
-2-yl)methyl)-1-(1H-benzo[d]imidazol-2-yl)methanamine Cu(II) complex (2a), N-(4-((1,4,7,10tetraazacyclododecan-1-yl)methyl)benzyl)-N-((1H-benzo[d]imidazol-2-yl)methyl)-1-(1H-benzo[d]
imidazol-2-yl)methanamine Zn(II) complex (2b), N-(4-((1,4,7,10-tetraazacyclododecan-1-yl)methyl)
benzyl)-N-((1H-benzo[d]imidazol-2-yl)methyl)-1-(1H-benzo[d]imidazol-2-yl)methanamine Cu(II)
complex (2c) and 1-(6-((1,4,7,10-tetraaza-cyclododecan-1-yl)methyl)pyridin-2-yl)-N,N-bis((1H-benzo[d]
imidazol-2-yl)methyl)methanamine Zn(II) complex (2d) were synthesized according to published
procedures [10,32]. Because the solubilities of 1b, 1c, 2b and 2d were poor in doubly distilled water, so
they were dissolved in DMSO to form 1 ˆ 10´3 mol/L solutions, and then dissolved with doubly
distilled water to get the test samples.
3.2. Instrumentation
The pH of the solution was determined by using a Sartorius PB-10 pH meter (Sartorius Scientific
Instrument Co., Ltd., Beijing, China). The fluorescence spectra was recorded on a Lumina instrument
(ThermoFisher, Waltham, MA, USA). The DNA cleavage was analyzed by gel electrophoresis on
a DYY-12 electrophoresis meter (Beijing Liuyi Biotechnology Co., Ltd., Beijing, China) and a gel
image analyzing system (Vilber Lourmat BIO-1D, Eberhardzell, Germany). Nano-ZS 3600 (Malvern
Instruments, Westborough, MA, USA). Elix Advantage ultrapure water system (Merck Millipore,
Darmstadt, Germany).
3.3. DNA Cleavage Experiments
The cleavage of pUC19 DNA was studied according to a reported procedure [28]. The cleavage in
the presence of standard quenchers or promoters has also been investigated. In these experiments,
DMSO (hydroxyl radical scavenger, final percentage 10%), 10 mM NaN3 (singlet oxygen scavenger),
10 mM KI (hydrogen peroxide scavenger) or was added to the solution containing SC DNA and
complexes, respectively.
3.4. DNA Condensation, Cleavage and Regulation by GO Experiments
The condensation and regulation of pUC19 DNA was studied according to methods described
before. Compound 2c was selected as an example and the concentrations of 2c were changed from
3.13 ˆ 10´ 5 mol/L to 6.25 ˆ 10´ 7 mol/L. As for DNA condensation and regulation experiments, DNA
and compound 2c were mixed and incubated for 20 min in room temperature before GO was added.
3.5. Fluorescent Spectrum Analysis
The interaction experiments were carried out according to the literature [22] with a Lumina
instrument (Thermo Fisher) equipped with 1.0 cm quartz cells, the widths of both the excitation
and emission slit were set as 5.0 nm, and the emission wavelength was 300 nm, connected to a
temperature controller.
3.6. Particle Size and ξ Potential Measurements
Particle size and ξ potential measurements of polyplexes were carried out using a Nano-ZS
3600 (Malvern Instruments) with a He-Ne Laser beam (633 nm, fixed scattering angle of 901) at 25 ˝ C.
Complex 2c/DNA at weight ratio were 3.9 and 7.8 prepared by the same method with abovementioned
Molecules 2016, 21, 920
13 of 15
agarose gel electrophoresis. After 30 min incubation in 100 mL ultrapure water, polyplex solutions
were diluted to a final volume of 1 mL before measurements.
3.7. Cell Viability Assay
Toxicity of 11a,e,f toward HL-7702 cells was determined using a Cell Counting Kit-8 based on
a (4-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl)benzene-1,3-disulfonate)
WST-8 reduction assay following literature procedures [37]. The HL-7702 cells (6000 cells per well)
were seeded into 96-well plates. The cells were then incubated in a culture medium containing 2a–d
with a particular concentration for 24 h. After that, 10 mL of CCK8 was added to each well. After 4 h,
the unreacted dye was removed by aspiration. The OD value were measured spectrophotometrically
in an ELISA plate reader (model 550, Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm. The cell
survival was expressed as follows: cell viability = (OD treated/OD control) ˆ 100%. The results are
shown in Figure 14.
4. Conclusions
The studied novel benzimidazole mononuclear and dinuclear complexes exhibited nuclease
activities towards DNA at low concentrations ranging from 3.13 ˆ 10´6 to 6.25 ˆ 10´7 mol/L.
Compound 1a showed almost similar DNA cleavage efficiency in the presence and absence of typical
radical scavengers indicating that DNA cleavage by a hydrolytic cleavage mechanism. Most complexes
showed good cleavage properties under lower pH as well as under physiological condition, and
dinuclear complexes 2a and 2b can promote the cleavage of plasmid pUC19 DNA from supercoiled to
the nicked and linear forms at pH = 7.0. It could be concluded that this kind of hybrid compounds
could be used as novel artificial nucleases. More importantly, the most important functions of dinuclear
compounds were to condense DNA, and this is the first time this novel method for DNA controlled
release by GO is reported. Dinuclear compounds were relative safe for potential use in vivo.
Supplementary Materials: Figures about reaction time effects of DNA cleavage of some target materials, figures
of some complexes about DNA cleavage on the presence of typical radical scavengers, and figures to depict
concentration effect of the complex for DNA cleavage and condensation. This material is available free of charge
via the Internet. Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/7/920/s1.
Acknowledgments: This research is funded by Chongqing Research Program of Basic Research and Frontier
Technology (No. cstc2013jcyjA50012, cstc2016jcyjA0508), China Postdoctoral Science Foundation funded project
(2014M562326, 2016T90851), Program for Innovative Research Team in Chongqing university of technology
(2015TD22), National Natural Science Foundation of China (No. 31300222, 21206202), Natural Science Foundation
of Jiangsu Province (No.BK20130214), and the Scientific and Technical Research Program for Chongqing Education
Commission (No. KJ1400915).
Author Contributions: S.L., C.Z. and Z.G. conceived and designed the experiments; S.L. and M.D. performed the
experiments; S.L., B.J., C.Z., J.X. and D.Z. analyzed the data; Z.G. contributed reagents/materials/analysis tools;
S.L. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1a–1c are available from the authors.
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