Double-strand DNA cleavage by copper complexes of 2,2′-dipyridyl with guanidinium/ammonium pendants

Article abstract of DOI:10.1039/b801549j

Two ligands with guanidinium/ammonium groups were synthesized and their copper complexes, [Cu(L¹)Cl₂](ClO₄) ₂?H₂O (1) and [Cu(L²)Cl ₂](ClO₄)₂ (2) (L¹ = 5,5′-di[1-(guanidyl)methyl]-2,2′-bipyridyl cation and L² = 5,5′-di[1-(amino)methyl]-2,2′-bipyridyl cation), were prepared to serve as nuclease mimics. X-Ray analysis revealed that Cu(ii) ion in 1 has a planar square CuN₂Cl₂-configuration. The shortest distance between the nitrogen of guanidinium and copper atoms is 6.5408(5) A, which is coincident with that of adjacent phosphodiesters in DNA (ca. 6 A). In the absence of reducing agent, supercoiled plasmid DNA cleavage by the complexes were performed and their hydrolytic mechanisms were demonstrated with radical scavengers and T4 ligase. The pseudo-Michaelis-Menten kinetic parameters (k_cat, K_M) were calculated to be 4.42 h ^-1, 7.46 × 10^-5 M for 1, and 4.21 h^-1, 1.07 × 10^-4 M for 2, respectively. The result shows that their cleavage efficiency is about 10-fold higher than the simple analogue [Cu(bipy)Cl₂] (3) (0.50 h^-1, 3.5 × 10^-4 M). The pH dependence of DNA cleavage by 1 and its hydroxide species in solution indicates that mononuclear [Cu(L¹)(OH)(H₂O)]³⁺ ion is the active species. Highly effective DNA cleavage ability of 1 is attributed to the effective cooperation of the metal moiety and two guanidinium pendants with the phosphodiester backbone of nucleic acid. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b801549j

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Double-strand DNA cleavage by copper complexes of 2,2ꢀ-dipyridyl with
guanidinium/ammonium pendants†
Juan He,a Ping Hu,a Yu-Jia Wang,a Ming-Liang Tong,a Hongzhe Sun,b Zong-Wan Mao*a and Liang-Nian Jia
Received 28th January 2008, Accepted 4th April 2008
First published as an Advance Article on the web 7th May 2008
DOI: 10.1039/b801549j
Two ligands with guanidinium/ammonium groups were synthesized and their copper complexes,
[Cu(L1)Cl2](ClO4)2·H2O (1) and [Cu(L2)Cl2](ClO4)2 (2) (L1 = 5,5ꢀ-di[1-(guanidyl)methyl]-2,2ꢀ-bipyridyl
cation and L2 = 5,5ꢀ-di[1-(amino)methyl]-2,2ꢀ-bipyridyl cation), were prepared to serve as nuclease
mimics. X-Ray analysis revealed that Cu(II) ion in 1 has a planar square CuN2Cl2-configuration. The
˚
shortest distance between the nitrogen of guanidinium and copper atoms is 6.5408(5) A, which is
˚
coincident with that of adjacent phosphodiesters in DNA (ca. 6 A). In the absence of reducing agent,
supercoiled plasmid DNA cleavage by the complexes were performed and their hydrolytic mechanisms
were demonstrated with radical scavengers and T4 ligase. The pseudo-Michaelis–Menten kinetic
parameters (kcat, KM) were calculated to be 4.42 h−1, 7.46 × 10−5 M for 1, and 4.21 h−1, 1.07 × 10−4
M
for 2, respectively. The result shows that their cleavage efficiency is about 10-fold higher than the simple
analogue [Cu(bipy)Cl2] (3) (0.50 h−1, 3.5 × 10−4 M). The pH dependence of DNA cleavage by 1 and its
hydroxide species in solution indicates that mononuclear [Cu(L1)(OH)(H2O)]3+ ion is the active species.
Highly effective DNA cleavage ability of 1 is attributed to the effective cooperation of the metal moiety
and two guanidinium pendants with the phosphodiester backbone of nucleic acid.
such as molecular recognition and catalysis.18–24 In peptides,
Introduction
guanidine, a residue of arginine, exists in the protonated form
as a guanidinium ion, which functions as an efficient recognition
moiety of anionic functionalities, such as carboxylate, phosphate
and nitrate, through hydrogen-bonding.25 The replacement of the
amino groups of ethidium with guanidinium groups resulted
in a marked gain of both affinity and sequence selectivity in
recognition of AT binding sites in DNA.26 In alkaline phosphatase
and purple acid phosphatase,27,28 the highly efficient coopera-
tivity of a metal ion and Arg-guanidinium residues promoted
phosphate monoester hydrolysis. In addition to their biological
roles, guanidine derivatives are widely utilized in synthetic organic
chemistry as strong bases. Indeed, chiral guanidine catalysts are
attractive targets in organocatalysis.29 However, few metal com-
plexes with guanidinium groups used for DNA cleavage have been
reported.30
Transition metal complexes that cleave DNA under physiological
condition are of current interest in the development of artificial
nucleases.1–11 One of approaches is to construct multi-functional
models promoting phosphodiester hydrolysis through cooperation
of metal ions and functional groups.12–17 Of functional groups,
the guanidinium group is one of the most representative. It
contains two amines and an iminium in a plane forming a
Y shape to establish its great stability as an ion in aqueous
environment. Guanidinium, or protonated guanidine, has six
potential hydrogen bond donors available, making it highly soluble
in aqueous systems. These structural features make it an extremely
advantageous functional group for the binding of carboxylates
or phosphates in enzymes and antibodies via forming strong
ion-pairs, which play a key role in many biological activities,
We previously reported a few dipyridyl derivatives with
tetraalkylammonium pendants and their Cu(II) complexes, such as
[Cu(L)2Br](ClO4)5, where L is 5, 5ꢀ-di[1-(triethylammonio)methyl)-
2,2ꢀ-dipyridyl, as nuclease mimics.31 We found that the Cu(II)
complex exhibits strong affinity towards DNA binding by elec-
trostatic interaction and high nuclease activity. Relative to a
tetraalkylammonium ion, a guanidinium or ammonium ion has
not only electrostatic interaction but also hydrogen-bonding to the
phosphodiester backbone of nucleic acid. In view of these unique
characteristics of the guanidinium group, we recently focused our
efforts on the construction of complexes containing appended
ammonium or guanidinium functionalities on the dipyridyl ligand
to explore more fully the combination and cleavage of DNA
(Scheme 1). We report herein their synthesis, structure, species
distribution in solution, DNA affinity and nuclease activity.
aMOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of
Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou,
510275, China. E-mail: cesmzw@mail.sysu.edu.cn; Fax: +86 20 8411 2245;
Tel: +86 20 8411 3788
bDepartment of Chemistry, the University of Hong Kong, Pokfulam Road,
Hong Kong, China
1
† Electronic supplementary information (ESI) available: H NMR spec-
trum of L1. ESI-MS spectrum of L1. X-Ray crystal structure of 1 in which
each perchlorate anion binds with the copper ion and the guanidinium
group by weak coordination and hydrogen-bonding to form a network
structure. Tm curves of complexes 1, 2, 3. Agarose gel electrophoresis and
corresponding time course plots showing cleavage of pBR322 DNA by
complexes 1 and 2. Agarose gel showing the influence of catalase for the
cleavage ability of complexes 1 and 2. Kinetics for the cleavage of plasmid
pBR322 DNA by 3. Agarose gel showing cleavage of 38 lM pBR322
DNA incubated with 150 lM of 1 in 20 mM buffer of different pH. CCDC
reference number 632380. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/b801549j
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Table 1 Species distribution and equilibrium constants of L1 in the
presence of Cu(II) ions
Reaction equilibrium
Constant
HL3+ = L2+ + H+
pKa
2.19 0.05
4.23 0.04
[Cu(H2O)6]2+ + L2+
=
log KML
[CuL(H2O)2]4+ + 4 H2O
2 [CuL(OH)(H2O)]3+
[Cu2L2(OH)2(H2O)2]6+
[CuL(H2O)2]4+
[CuL(OH)(H2O)]3+ + H+
[Cu2L2(OH)2(H2O)2]6+
=
log KM
5.81 0.34
8.17 0.17
9.81 0.02
10.45 0.02
2 L2
=
pKa(ML)
pKa1(M
=
2 L2
)
)
[Cu2L2(OH)3(H2O)]5+ + H+
[Cu2L2(OH)3(H2O)]5+
[Cu2L2(OH)4]4+ + H+
=
pKa2(M
2 L2
of the coordinated water molecule as well as species distribution
in solution were determined by potentiometric pH titration at
25 0.1 ◦C. The pH profiles of the titration curves were analyzed
and the calculated results were summarized (Table 1), including the
distribution curves of the Cu(II) species as a function of pH (Fig. 2).
Since the addition of NaCl did not cause any spectral changes (Fig.
S9, ESI†), one can assume that the remaining coordination sites
of Cu(II) are occupied by 2–4 water molecules.
Scheme 1 Schematic illustration of complexes 1–3.
Results and discussion
Molecular structure of 1
A prospective view of the cationic structure of 1 is shown in Fig. 1.
along with selected bond distances and angles. The geometry
around the copper(II) ion is square-planar with the basal plane
formed by the two nitrogen atoms of the ligand and two chloride
atoms [Cu(1)–N(1) and Cu(1)–Cl(1) distances are 2.024(3) and
˚
2.2469(11) A, respectively]. Since the guanidinium plane defined
by N(2), N(3), N(4) and C(7) atoms adopts an extended conforma-
tion in the crystal stacking, the distance between Cu(1) and N(2)
˚
atoms is 6.5408(5) A, which is coincident with that of adjacent
˚
phosphodiesters in B-form DNA (ca. 6 A). Each perchlorate anion
binds with the copper ion and the guanidinium group by weak
coordination and hydrogen-bonding to form a network structure.
Fig. 2 Distribution plots of species with L1 as a function of pH at 0.1 M
NaClO4 and 25 0.1 ◦C.
In the case of the L1, it is shown in Fig. 2 that five Cu(II)
species are involved in complex formation at pH 5–11.5. Two
3+
simple mononuclear species, CuL4+ and CuLH−1 (charges of
the ligand and coordinated water molecules are omitted for
clarity), correspond to [CuL(H2O)2]4+ and [CuL(OH)(H2O)]3+, re-
spectively. Their equilibrium constants are shown in Table 1. Since
guanidinium remains protonated over a wide pH range (pKa =
Fig. 1 Molecular structure of complex 1 with H atoms omitted for clarity;
the other half of the complex is generated by the two-fold diad present in
◦
˚
the space group C2/c. Key bond lengths (A) and angles ( ): Cu(1)–N(1)
2.024(3), Cu(1)–Cl(1) 2.2469(11), C(7)–N(2) 1.324(5), C(7)–N(3) 1.304(6),
C(7)–N(4) 1.319(6); N(1)–Cu(1)–N(1A) 80.56(17), N(1)–Cu(1)–Cl(1A)
93.85(9), N(1)–Cu(1)–Cl(1) 171.05(9), Cl(1A)1–Cu(1)–Cl(1) 92.51(6),
N(4)–C(7)–N(3) 119.4(4), N(2)–C(7)–N(3) 120.7(4), N(4)–C(7)–N(2)
119.9(41).
12.5),32 it is reasonable to assume that Cu2L2H−26+, Cu2L2H−3
5+
and Cu2L2H−44+, are involved in the formation of l-dihydroxo-
structure dimer species, corresponding to [Cu2L2(OH)2(H2O)2]6+,
[Cu2L2(OH)3(H2O)]5+ and [Cu2L2(OH)4]4+, respectively. A possible
6+
l-dihydroxo Cu2L2H−2 dimer has been isolated at pH ∼12 and
Species distribution of 1
confirmed by elemental analysis (calc. (%) for: (L1H−2)2Cu2(l-
OH)2Cl2(C2H5OH) ·2H2O (910.81): C 39.46, H 5.74, N 24.56,
Found: C 39.56, H 5.31, N 24.61). The result also shows that
The protonation constants (pKn) of the ligands and their complex
formation constants (KML) and the deprotonation constant (pKa)
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Table 2 Interaction of complexes with CT-DNAa
Complex
rb
DTmc/◦C
1
2
3
0.1
0.2
0.1
0.2
0.1
0.2
4.24
9.37
1.93
3.26
0.72
1.25
a Reaction conditions: 20 mM HEPES at pH 8.0, I = 0.1 M, [DNA] =
100 lM. b r = Molar ratio of complex/nucleic acid phosphate. c DTm/◦C =
Melting temperature of DNA with complex minus the melting temperature
of DNA alone.
guanidinium group always remains protonated in range of the
measured pH.
In the presence of L2, the titration of Cu(II) species were limited
by precipitation of Cu(II) hydroxide at pH ≥ 7. This interfered
with the saturation portion of the binding isotherm, making the
curve fitting process impossible, so the data was not uploaded.
Fig. 3 Agarose gel electrophoresis of 38 lM bp pBR322 plasmid DNA
at 37 ◦C in 20 mM HEPES at pH 7.2 in the presence of 150 lM 1 (a), 2 (b)
and 3 (c). Lane 1: DNA control, lanes 2–8: DNA + complex for 0, 1, 2, 4,
8, 16, 24 h.
DNA affinity
(EDTA) and hydroxyl radical scavengers (DMSO and t-BuOH).33
As shown in Fig. 4, EDTA can efficiently inhibit the complex
activity, however, both DMSO and tBuOH have no effect. This
result can rule out the possibility of DNA cleavage by hydroxyl
radicals. The cleavage was investigated further in the presence of
catalase, which can lower solution concentrations of H2O2 (Fig. S6,
ESI†). The negative result indicates that catalase does not inhibit
DNA cleavage and so the mechanism of cleavage is not oxidative
The interaction between the two Cu(II) complexes 1 and 2 and
the analogous non-pendant [Cu(bipy)Cl2] (3) with calf thymus
(CT) DNA is characterized by measuring their varying effects on
the melting temperature of DNA (Table 2). The Tm curves were
shown as Fig. S3 (ESI†). Considerable increase in the melting
temperature in each case is observed, indicative of stabilization
of the double-stranded nucleic acids by the metal complexes. A
markedly larger stabilization effect of complex 1 over 2 is observed
(Table 2). In general, there are at least three interaction modes
between metal complexes and DNA: electrostatic interaction,
hydrophobic binding and intercalating. Both complexes with
dicationic pendants show higher affinity towards CT DNA than
their analogue without any pendants, which suggests that the
guanidinium/ammonium groups in 1 and 2 can be bound to the
oxygen atoms of phosphate backbone.
•
arising from O2 − and H2O2. In the absence of any reducing agents,
therefore, DNA cleavage by 1 and 2 is likely to proceed via a
hydrolytic degradative pathway.
Furthermore, the DTm values of 1 are similar to those of
the Cu(II) complex with tetraalkylammonium pendants reported
by us,30a but their charge numbers are different ([Cu(L1)Cl2]2+
and [Cu(L)2 Br]5+, L = 5, 5ꢀ-di[1-(triethylammonio)methyl)-2,2ꢀ-
dipyridyl). The X-ray structure analysis shows that, similarly to 1,
only two dicationic pendants of the [Cu(L)2Br]5+ ion can interact
with the DNA backbone together with Cu(II) ion although it
has four pendants in all. In the process of metal complex–DNA
interaction, therefore, appended pendants are more influential
than the ion charge.
Fig. 4 Agarose gel showing cleavage of 38 lM bp pBR322 DNA
incubated with complex (150 lM) in 20 mM HEPES, pH 7.2 at 37 ◦C
for 1 h. Lane 1: DNA control, lane 2: DNA + complex, lanes 3–6: 0.1 M
EDTA, DNA + complex + 1 M DMSO, 1 M tBuOH.
Direct evidence of DNA hydrolysis was obtained further from
ligation experiments of pBR322 DNA linearized by 1. It is well
known that in DNA hydrolytic cleavage 3ꢀ-OH and 5ꢀ-OPO3
(5ꢀ-OH and 3ꢀ-OPO3) fragments remain intact and that these
fragments can be enzymatically ligated and end-labeled.34 We
tried to recover the linear DNA from an agarose low melting
point gel by cutting off the gel fragment and subjecting it to the
DNA recovery system. The recovered linear DNA was subjected
to overnight ligation reaction with T4 DNA ligase. The result after
electrophoresis (Fig. 5) shows that the linear DNA fragments
cleaved by 1 can be religated by T4 ligase just like linear DNA
mediated by EcoRI. Hence, this result indicates that the process
DNA cleavage
Supercoiled plasmid DNA cleavage by the Cu(II) complexes and
their analogues was studied in the absence of H2O2 or any reducing
agents (Fig. 3) and a time-dependent cleavage was observed. We
found that the supercoiled DNA (form I) was completely cleaved
by 1 or 2 only after 2 h, but can not be completely cleaved by 3
even after 24 h. The cleavage activity of 1 is equivalent to 2, and
much higher than 3.
In order to clarify the DNA cleavage mechanism, complexes
1 and 2 were investigated in the presence of chelating agent
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Fig. 6 Kinetics for the cleavage of plasmid pBR322 DNA by 1 (ꢀ) and 2
(ꢁ) (60–300 lM) in 20 mM HEPES, pH 7.2 at 37 ◦C. The samples were
run on a 0.9% agarose gel and stained with ethidium bromide.
Fig. 5 Agarose gel electrophoresis for ligation of pBR322 DNA linearized
by 1: lane 1: DNA control, lane 2: DNA + 150 lM 1; lanes 3 and 4: pBR322
DNA linearized by 1 with and without T4 DNA ligase; lanes 5 and 6:
pBR322 DNA linearized by EcoRI with and without T4 DNA ligase; lane
7: kHindIII DNA markers.
DNA cleavage hydrolyzed by some Cu(II) complexes have been
reported.12,35–37 The rates are generally in the range of 10−2–
1.0 h−1.12 To our knowledge, few mononuclear complexes exhibited
very high nuclease activities: examples include Cu–dpq (dpp =
dipyrido[3,2-d:2ꢀ,3ꢀ-f ]quinoxaline) with a rate constant of 5.58 h−1
at pH = 7.2,35a Cu–tach (tach = cis,cis-1,3,5-triaminocyclohexane)
with a rate constant of 4.34 h−1 at pH = 8.136 and a Cu–neamin
complex with the rate constant of 3.57 h−1 at pH = 7.3.12
of DNA cleavage by 1 is a hydrolysis that takes place by a reaction
similar to that of the natural enzyme EcoRI.
Pseudo-Michaelis–Menten kinetics of DNA cleavage
The DNA cleavage by 1 under different pH values was
investigated. The observed pH profile can be described by a bell-
shaped curve with the maximum centered at pH ≈ 8 (Fig. 7(B)).
It has been widely accepted that metal-bound hydroxyl species
(LM–OH) are the active species in the hydration of the phosphate
backbone.1–11 As in previous research,6,38 the formation of the
active species from the precursor generally involves the steps of
rapid exchange between coordinated halide and water molecules,
followed by hydrolysis of the resulting aqua compounds to
form a coordinated hydroxyl group that acts as a nucleophile
for DNA cleavage. In solution, hydroxide complexes co-exist
in comparable concentrations: mononuclear Cu(L1)(OH) and
binuclear and Cu2(L1)2(OH)2, Cu2(L1)2(OH)3 and Cu2(L1)2(OH)4
species (Fig. 2).38,39 As one can see from the results shown in Fig. 7,
the pH-profiles of the observed fraction of DNA II and III cleaved
by 1 superimposed with the distribution curve for the mononuclear
hydroxide complexes is not consistent with the curve of the sum
of mononuclear and binuclear hydroxide complexes. As shown in
Fig. 7(B), the DNA cleavage rate Vobs and the pH dependencies
of the concentration of Cu(L1)(OH) species were in agreement,
indicating that the Cu(L1)(OH) species may be considered as a
reactive form of the catalyst. However, the maximum attainable
degree of formation of this species is only 2.8%, as is seen from
the results in Fig. 7(B). It is worth noting that even with the
active complex present at such a low amount one observes an
unusually high kcat value of 4.42 h−1 at physiological pH. This
result can explain why DNA cleavage is very low when the total
concentration of 1 is lower than 50 lM (due to too low active
species concentration).
All the complexes were tested for DNA cleavage under hydrolytic
conditions, and a concentration-dependent cleavage was observed.
Reaction that leads to formation of open circular DNA (form
II) from the supercoiled from I over various concentrations of
complexes 1/2 (60–300 lM) and 3 (150–1000 lM) and constant
DNA concentration (38 lM, bp) was followed for different times at
37 ◦C (Fig. S4, ESI†). It is not an easy task to control the reaction
conditions, and extract good quality rate data from gels. However,
the disappearance of form I with time followed pseudo-first-order
kinetic profiles and can be well fitted with a single-exponential
decay curve. The kobs for different concentrations and the degree
for data fitting (R2) are listed in Table S1, ESI.† Based on the plots
of kobs vs. concentrations of complex, saturation kinetics of DNA
cleavage were observed at high concentration of complexes. The
pseudo Michaelis–Menten kinetic parameters (kcat and KM) were
calculated to be 4.42 0.18 h−1 (R2 = 0.985) and 7.46 × 10−5
M
for 1, 4.21 0.11 h−1 (R2 = 0.968) and 1.07 × 10−4 M for 2, and
0.49 0.13 h−1 (R2 = 0.953) and 3.5 × 10−4 M for 3, respectively,
Fig. 6 and Fig. S7.
The obtained hydrolysis rate constants show that 1 and 2
have very high nuclease activities, giving ca 1.2 × 108-fold rate
enhancement over the noncatalyzed hydrolysis of double-strand
DNA. These values are also almost the same as for copper com-
plexes with tetraalkylammonium pendants although dominant
modes of their interactions with DNA are electrostatic interaction
and hydrogen-bonding, respectively.30 Likewise, the DNA cleavage
activities of 1 and 2 are also about 10-fold higher than its simple
analogue, 3. Interestingly, under the same experimental condition
◦
of 20 mM HEPES, pH 7.2 at 37 C for 1 h, DNA cleavage was
In view of the X-ray structure of 1 and the distance between
adjacent phosphorus atoms of the phosphodiester in a DNA back-
bone, we suggest a mechanism which is similar to that proposed
for [Cu[9]aneCl2] hydrolyzing phosphate diesters38a (Scheme 2). X-
Ray analysis has confirmed that the distances between coordinated
not promoted distinctly by either free Cu2+(aq) or free L1/2 alone
(Fig. S5, ESI†), which confirms that two electropositive pendants
in 1 and 2 facilitate binding of the Cu(II)–bipy moiety to DNA
and subsequently accelerates DNA cleavage. Rate constants of
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Fig. 7 Speciation diagram of Cu(II)–L1 (left y axes) and pH depen-
dence of DNA cleavage rate by 1 (●, right y axes). For (A), the
dashed line represents the sum of [CuL(OH)(H2O)]3+ and [Cu2(l2-OH)2]2+
species (Cu2(L1)2(OH)2, Cu2(L1)2(OH)3 and Cu2(L1)2(OH)4 (coordinated
water molecules and charges omitted)), the solid line corresponds
to [CuL(OH)(H2O)]3+
. (B) is the enlarged speciation diagram of
[CuL(OH)(H2O)]3+ (left y axes) and pH dependence of DNA cleavage
rate by 1 (●, right y axes). Titration conditions: [L1] = 1 mM, [Cu(II)] =
1 mM, 0.1 M NaClO4, 25 0.1 ◦C. Reaction conditions for DNA cleavage:
[DNA] = 38.0 lM bp, [1] = 150 lM, pH 6.0 (20 mM MES buffer),
pH 6.5–7.0 (20 mM MOPSO buffer), pH 7.5–8.5 (20 mM Tris buffer),
pH 9.0–9.5 (20 mM CHES buffer), pH 9.5–10.5 (20 mM CAPS buffer),
0.1 M NaClO4, 37 0.1 ◦C.
˚
hydroxyl anion and quaternary guanidyl ion are around 6.5 A in
Scheme 2 Proposed mechanism for the DNA cleavage by 1.
1, similar to the distance between adjacent phosphorus atoms
of the phosphodiester in a DNA backbone (ca. 6 A). This
30
˚
suggests that the two guanidinium groups in 1 can synchronously
interact with alternate phosphodiester groups in a DNA strand,
Scheme 2(A). The direct interaction between the neighboring
phosphoryl oxygen atoms and guanidinium groups facilitates the
formation of an intermediate, which allows the DNA to be cleaved
readily. Therefore, the higher activity of 1 can be attributed to its
structure matching with the phosphodiester backbone of nucleic
acid and cooperative interaction from the highly active bipy–Cu(II)
moiety and two positive guanidinium groups.
Experimental
Materials
5,5ꢀ-Dimethyl-2,2ꢀ-dipyridyl was purchased from Aldrich Chem-
ical Co. The pBR322 DNA was purchased from MBI. Catalase
was purchased from BBI. Ethidium bromide and HEPES were
purchased from AMRESCO. T4 DNA ligase and EcoRI enzyme
was purchased from Toyobo Co., Ltd. Other reagents of analytical
grade were obtained from commercial suppliers and used directly
without further purification. Milli-Q water was used in all physical
measurement experiments.
Conclusion
Preparation of ligands
Two new Cu(II) complexes are formed and isolated when cop-
per(II) is reacted with any molar ratio of 5,5ꢀ-dimethyl-2,2ꢀ-
bipyridyl derivatives with guanidinium/ammonium pendants. The
complexes with guanidinium/ammonium pendants exhibit very
high DNA affinity and nuclease activity. For the complex with
guanidinium pendants, the observed reactivity is entirely due to the
mononuclear hydroxide species and give 10-fold rate acceleration
for hydrolyzing the phosphate diesters than their unmodified
analogue even though its concentration in aqueous solution is less
than 3% due to strong dimerization. The enhanced acceleration
could be attributed to electrostatic interaction between the positive
pendants with guanidinium/ammonium groups of the complexes
and the phosphodiester backbone of nucleic acid.
The ligands were synthesized according to Scheme 3, while the
derivatives of 5,5ꢀ-dimethyl-2,2ꢀ-dipyridyl (b, c) were synthesized
as in the previous method.31a
5,5ꢀ-Di[1-(guanidyl)methyl]-2,2ꢀ-bipyridyl chloride (L1·2Cl).
1H-Pyrazole-1-carboxamidine monohydrochloride (1.47 g,
10 mmol) and N,N-diisopropylethylamine (3.5 mL, 20.0 mmol)
were dissolved in dry DMF (10 mL), stirred under N2 for
10 min, then L2 (1.07 g, 5 mmol) was added into the mixture.
The mixture solution was stirred for 30 h under N2 at room
temperature. 50 mL acetone was added dropwise into the mixture
and the resulting precipitate was filtered off and washed with
ethanol to give a straw yellow powder (1.3 g, 70%). ESI-MS:
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Table 3 Crystallographic data for 1
Complex
1
Empirical formula
C14H20Cl4CuN8O8
633.72
293(2)
Mr
T/K
˚
k/A
0.71073
Crystal system
Space group
Monoclinic
C2/c
17.202(3)
8.9681(13)
15.145(2)
92.609(3)
2334.0(6)
4
˚
a/A
˚
b/A
˚
c/A
b/◦
3
˚
V/A
Z
Dc/g cm−3
1.803
1.454
l/mm−1
F(000)
1284
Crystal size/mm
h range for data collection/◦
Limiting indices, hkl
Reflections collected
0.325 × 0.324 × 0.206
2.56–27.00
−12 to 21, −7 to 11, −19 to 19
4360
Independent reflections (Rint
Goodness-of-fit on F2
R/wR2 [I > 2r(I)]
)
2419 (0.0217)
1.019
0.0508/0.1301
0.0624/0.1380
0.685/−0.315
R/wR2 (all data)a
Scheme 3 Schematic view of the synthetic route for the ligands.
−3
˚
Dqmax,min/e A
ꢀ
ꢀ
ꢀ
ꢀ
299 [M + H]+, 150 [M + 2H]2+/2. Elemental analysis: calc. (%)
a R1 = ꢁFo| − |Fcꢁ/ |Fo|, wR2 = [ w(Fo − Fc2)2/ w(Fo2)2]1/2
.
2
for (C14H20N8Cl2·0.3CH3OH (380.89): C 45.07, H 5.63, N 29.34;
1
found: C 45.55, H 5.56, N 28.92. H NMR (DMSO, 300 MHz):
d 8.623 (s, 2 H, PyH), 8.386 (d, 2 H, PyH), 7.871 (d, 2 H, PyH),
4.503 (d, 4 H, NHCH2 Py).
was employed for the investigation of charged ligands in a mixture
of water and methanol.
5,5ꢀ-Di[1-(amino)methyl]-2,2ꢀ-bipyridyl
chloride
(L2·2Cl).
X-Ray crystallography
L2·2Cl was synthesized according to literature.40
Single-crystal X-ray data of 1 was collected on a Bruker Smart
Apex CCD diffractometer at 293 K with graphite-monochromated
Preparation of copper complexes
˚
Mo-Ka radiation (k = 0.71073 A). The reflections were corrected
for Lorentz and polarization effects and empirical absorption
corrections were applied using the SADABS program. The space
groups were determined from systematic absences and confirmed
by the results of refinement. The structures were solved by direct
methods using the SHELXTL software and all non-H atoms
were refined with anisotropic displacement parameters.41 The
crystallographic data are given in Table 3.
CAUTION: Although no problems were encountered in this work,
transition-metal perchlorates are potentially explosive and should
thus be prepared in small quantities and handled with care.
[Cu(L1)Cl2](ClO4)2·H2O (1).
A
methanol solution of
Cu(ClO4)2·6H2O (60 mg, 0.16 mmol) was added dropwise into a
solution of L2·2Cl (50 mg, 0.135 mmol) in 10 mL of water and a
green solution was obtained after stirring for 4 h. The solution was
filtered and allowed to stand for evaporation. Several days later,
green crystals suitable for X-ray analysis were obtained. Elemental
analysis data: calc. (%) for Cu(C14H20N8)Cl2(ClO4)2·H2O (651.73):
C 25.80, H 3.40, N 17.19; found: C 26.22, H 3.14, N 17.07.
CCDC reference number 632380.
For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b801549j
Potentiometric titration
[Cu(L2)Cl2](ClO4)2 (2). Complex 2 was prepared in the
same way as above. Elemental analysis data: calc. (%) for
Cu(C12H16N4)Cl2(ClO4)2 (549.64): C 26.22, H 2.93, N 10.19; found:
C 26.70, H 3.19, N 9.85.
An automatic titrator (Metrohm 702 Titrino) coupled to a
Metrohm electrode was used and calibrated according to the Gran
method.42,43 The electrode system was calibrated with buffers and
checked by titration of HClO4 with 0.10 M NaOH. All titrations
were carried out under a N2 flow to eliminate the presence of
atmospheric CO2, at 25.0
0.1 ◦C and 0.10 M NaClO4. The
General methods
measurements were carried out in a thermostated cell containing a
complex solution (25 mmol/25 mL) with ionic strength of 0.10 M
NaClO4. The sample was titrated by addition of fixed volumes
of a standard CO2-free NaOH solution (0.10 M). Duplicate
measurements were performed, for which the experimental error
was below 1%. The titration data were fitted with the HYPER-
QUAD program44 to calculate the ligand protonation constants
Microanalyses (C, H and N) were carried out with an Elementar
Vario EL elemental analyser. UV-vis spectroscopy was recorded
on a Varian Cary 100 spectrophotometer with a thermostatic cell
holder and NMR spectroscopy was performed on a Varian Inova
500/Mercury plus 300 NMR spectrometer with D2O or DMSO-
d6 as solvent. An LCQ DECA XP electrospray mass spectrometer
3212 | Dalton Trans., 2008, 3207–3214
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Kn, the complex-formation constant, KML, and the deprotonation
constants of the coordinated water molecules.
remaining solution containing DNA cleavage fragment was kept
at −20 ◦C.
Thermal melting curves and DTm calculation
Acknowledgements
The concentration of the calf thymus (CT) DNA was determined
spectrophotometrically on the basis of known molar extinction
coefficient (e260) 6600 dm3 mol−1 cm−1.30,45 Thermal melting curves
were obtained on a Cary 100 UV-vis spectrophotometer connected
to a temperature controller. The melting curves were recorded at
different molar ratios of compound to DNA (r) by measurement
of the changes in absorption at 260 nm as function of temperature
in the range of 55–95 ◦C. Tm values were determined from the
maximum of the first derivative or tangentially from the graph at
the midpoint of the transition curves. DTm values were calculated
by subtracting Tm of the free nucleic acid from the Tm of the nucleic
acid interacted with the complex.
This work was supported by the National Natural Science Foun-
dation of China (20725103, 30770494, 20671098, 20529101), the
Guangdong Provincial Natural Science Foundation (07117637)
and National Basic Research Program of China (973 Program).
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3214 | Dalton Trans., 2008, 3207–3214
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©