Tuning the DNA binding and cleavage of bpa Cu(II) complexes by ether tethers with hydroxyl and methoxy groups

Article abstract of DOI:10.1016/j.ica.2016.02.034

Five bpa (bis(2-picolyl)amine) derivatives (1b, 2e, 3f, 4c, 5d) carrying ether tethers with hydroxyl and methoxy groups, have been synthesized from pyridine aldehyde and the respective amines by reductive amination. Their Cu(II) complexes carrying additio

Full text of DOI:10.1016/j.ica.2016.02.034

Inorganica Chimica Acta xxx (2016) xxx–xxx
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Inorganica Chimica Acta
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Tuning the DNA binding and cleavage of bpa Cu(II) complexes by ether
tethers with hydroxyl and methoxy groups
⇑
Airlangga Arya Janitra Sudarga Tjakraatmadja, Carsten Lüdtke, Nora Kulak
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Fabeckstr. 34/36, 14195 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Five bpa (bis(2-picolyl)amine) derivatives (1b, 2e, 3f, 4c, 5d) carrying ether tethers with hydroxyl and
methoxy groups, have been synthesized from pyridine aldehyde and the respective amines by reductive
amination. Their Cu(II) complexes carrying additional nitrato (6a–b) and perchlorato (7a–e) ligands,
respectively, have been characterized and subjected to DNA cleavage studies. Since the hydrolytic DNA
cleavage activity of 6a is known from the literature, further experiments comprising the novel derivatives
have been carried out. These reveal that derivative 7e was able to generate 10 times more linear DNA in a
standard cleavage experiment than 6a. Considering the redox activity of the Cu(II) center, a possible
oxidative cleavage activity of this kind of bpa complexes was also investigated.
Received 31 December 2015
Received in revised form 16 February 2016
Accepted 18 February 2016
Available online xxxx
Keywords:
Copper(II) complexes
Metallonucleases
Bis(2-picolyl)amine (bpa) ligands
DNA cleavage
Ó 2016 Elsevier B.V. All rights reserved.
1
. Introduction
The development of artificial metallonucleases with the aim of
electrophoresis [16]. Among the metals investigated, Cu(II) and
Co(II) showed the highest DNA cleavage activity. It has been pro-
posed that cleavage occurs hydrolytically based on a crystal struc-
ture of complex 6a ([Cu(1b)(OH )(ONO )]NO ). The reason for the
using them as potential enzyme mimetics and chemotherapeutic
agents plays a key role in the field of inorganic medicinal chemistry
2
2
3
comparatively high hydrolytic cleavage activity was attributed to
the close proximity between the pendant hydroxyl group (proton
acceptor) and an aqua ligand. The coordinated water molecule
can thus get deprotonated and cleave the phosphodiester bond
through an addition–elimination mechanism (Scheme 1) [16].
In the present work we describe the synthesis and characteriza-
tion of bpa derivatives with different side chain lengths and func-
tionalities (Scheme 2), aiming to optimize the nuclease activity of
the respective Cu(II) complexes. In addition, our purpose herein
was also to gain a better insight into the aforementioned proposed
hydrolytic DNA cleavage mechanism, into a possible oxidative
pathway and into the overall interactions of the described
complexes with DNA.
[
1,2]. Since the well-established DNA binder cisplatin shows strong
side effects in cancer treatment, DNA cleaving agents and other
biologically active agents containing endogenous transition metals
like Cu and Zn have been widely explored [3–7].
Copper complexes with aromatic N-donor ligands can act as
artificial nucleases [8]. Phenanthroline was established as one of
the first N-donor ligands with high oxidative DNA cleavage activ-
ity. Furthermore similar systems such as bipyridine (bpy), ter-
pyridine (tpy) and bis(2-picolyl)amine (bpa) metal complexes
have been studied [9–11]. The mechanism of dephosphorylation
and DNA hydrolysis caused by bpa complexes has been described
in the late 1990s [12–14].
Cu(II)-coordinating bpa ligands with different hydroxyalkyl
substituents (Fig. 1) at the aliphatic amino group and their hydro-
lytic cleavage activity towards bis(2,4-dinitrophenyl)phosphate
2
. Experimental
(
BNPP) as a DNA model have been described by Chin and co-
workers [17]. Metzler-Nolte et al. presented metal complexes (M
II) = Co, Ni, Zn, Cu) of ligand 1b which contains an elongated
2
.1. General information
(
If not stated otherwise all chemicals and solvents were obtained
hydroxyalkyl substituent with an ether group. They showed that
these bpa complexes are able to cleave the phosphodiester bonds
of plasmid DNA with higher efficiency compared to the complexes
described by Chin et al. This fact could be confirmed by gel
from commercial sources and used without further purification.
The Dowex resin used was 50WX8 hydrogen form, 100–200 mesh
(
Sigma-Aldrich). For the characterization of the synthesized com-
1
13
pounds H NMR and C NMR measurements were recorded on a
JEOL ECS 400, JEOL ECP500 or Bruker AVANCE500 spectrometer
in solutions of CDCl or CD OD with the solvent signal as an
3 3
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.ica.2016.02.034
020-1693/Ó 2016 Elsevier B.V. All rights reserved.
0
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2
A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
O
HO
HO
HO
N
N
M2+
-
N
N
N
NO
3
N
Cu2
+
Cu2+
N
N
N
H
2
O
2
H O
O
O
N
O
M = Co, Ni, Cu, Zn
Chin et al. [17]
Metzler-Nolte et al. [16]
O
O
HO
O
N
N
N
N
Cu2+
NO -
Cu
O
2+
3
N
N
H
2
O
O
O
N
O
O
O
N
O
N
O
O
6a
6b
HO
O
HO
OH
OH
O
O
O
O
O
O
O
HO
O
OH
O
N
N
N
N
N
N
N
N
N
Cu2+
N
ClO -
Cu2+
N
Cu2+
N
Cu2+
N
ClO -
-
-
Cu2+
ClO -
4
4
ClO
4
ClO
4
4
N
MeOH
MeOH
N
MeOH
MeOH
MeOH
O
O
O
O
O
O
O
O
O
O
O
O
O
Cl
Cl
Cl
Cl
O
O
Cl
O
O
O
O
O
7a
7b
7c
7d
7e
Fig. 1. Overview of literature-known bpa complexes and bpa complexes presented within this work. 7a carries a methanol solvent molecule as an equatorial ligand as derived
from X-ray crystallography analysis [15], whereas elemental analysis pointed to an aqua ligand at this coordination site. 6a has been reported to carry an aqua ligand [16].
Elemental analysis, however, did not show any water.
O
M
O
M
O
M
O
M
HO
H
HO
HO
HO
H
(
RO)
2
PO
2
N
N
N
N
N
N
N
N
N
NO -
N
- H+
N
N
-
3
-ROH
O
O
O
O
H
H
O
O
O
O
O
RO
RO
P
O
RO
RO
P
OH
RO P
O
N
O
Scheme 1. Hydrolytic DNA cleavage mechanism as proposed by Metzler-Nolte and co-workers [16].
internal standard. Mass spectral data were measured with the
device Agilent 6210 ESI-TOF, Agilent Technologies, Santa Clara,
chloroform. The collected organic phase was dried over Na
2 4
SO , fil-
tered and evaporated under reduced pressure. 2b was obtained as
1
CA, USA. The flow rate was 4
kV. The desolvation gas was set at 1 bar. All other parameters
were optimized for a maximal abundance of the respective
l
L/min and the spray voltage was
3
a colorless oil. Yield: 91%. H NMR (400 MHz, CDCl ): d 2.40 (s, 3H,
4
H-16), 3.30 (s, 3H, H-8), 3.41–3.46 (m, 2H, H-5), 3.51–3.55 (m, 2H,
H-4), 3.62–3.67 (m, 2H, H-2), 4.08–4.18 (m, 2H, H-1), 7.30 (d,
+
1
[
M+H] . Elemental analysis was carried out on a varioEL CHNS
J = 8.4 Hz, 2H, Ar), 7.75 (d, J = 8.3 Hz, 2H, Ar) ppm. H NMR data
elemental analyzer (C, H, N).
are in agreement with data reported in the literature [18].
2.2. Synthesis of amino ethers, bpa ligands and their Cu(II) complexes
2.2.1.2. Synthesis of N-[2-(2-methoxyethoxy)ethyl]phthalimide
(2c). 2-(2-Methoxyethoxy)ethyl tosylate (2b) (55 mmol) and
2
2
.2.1. Synthesis of amino ethers
.2.1.1. Synthesis of 2-(2-methoxyethoxy)ethyl tosylate (2b). To a
potassium phthalimide (55 mmol) were dissolved in DMF
(45 mL) and the reaction mixture was refluxed at 115 °C for 14 h.
After cooling down to room temperature the mixture was diluted
with water and extracted with ethyl acetate. The organic phase
solution of 2-(2-methoxyethoxy)ethanol (2a) (127 mmol) in THF
10 mL) a 24% NaOH aqueous solution (30 mL) was slowly added
(
at 0 °C, and the mixture was stirred for 5 min. Afterwards, a
solution of tosyl chloride (134 mmol) in THF (35 mL) was added
dropwise. After stirring at 0 °C for 3 h, the reaction mixture was
poured into ice-cold water (100 mL) and stirred until the ice
melted. Subsequently, the aqueous layer was extracted with
2 4
was dried over Na SO , filtered and evaporated under reduced
pressure. The residue was diluted with chloroform and washed
with brine (3 Â 50 mL) until DMF was removed completely as ver-
1
ified through H NMR. The organic layer was dried over Na
SO
2 4
,
and the solvents were removed under reduced pressure. 2c was
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A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
3
O
O
O
O
Cl + HO
3a
O
bpa=
H
2
N
OH
HO
O
N
N
N
1
a
2a
TsCl, THF
aq. NaOH
TBAB, NaOH
Benzene
0
°C, 3 h
0 °C, 48h
O
O
O
O
O
TsO
O
O
2
b
3b
O
NaN3, NH
EtOH
4
Cl
+
-
DMF
N K
O
1
10°C, 3 h
100 °C, 18 h
KOH, MeI
DMSO
OH
O
O
O
rt, 45 min
O
O
O
N
3
O
N
3
O
N
O
O
2
c
3c
5a
Dowex
Dowex
+
+
MeOH, H
MeOH, H
8
5 °C, 16 h
85 °C, 16 h
KOH, MeI
DMSO
OH
OH
OH
O
O
O
OH
rt, 45 min
N
3
O
OH
N3
O
O
O
N3
O
OH
N H x H O
3d
4a
5b
2
4
2
EtOH
1
00 °C, 3 h
H
2, Pd/C
H
2, Pd/C
H2, Pd/C
MeOH
rt, 24 h
MeOH
rt, 24 h
MeOH
rt, 24 h
OH
O
O
O
OH
O
2
H N
O
OH
2
H N
O
2
H N
O
OH
H
2
N
O
2
d
3e
4b
5c
O
O
N
O
O
O
N
N
N
N
NaBH(OAc)
AcOH, THF/MeOH
NaBH(OAc)
NaBH(OAc)
AcOH, THF/MeOH
NaBH(OAc)
AcOH, THF/MeOH
NaBH(OAc)
3
AcOH, THF/MeOH
rt, 48 h
3
3
3
3
AcOH, THF/MeOH
rt, 48 h
rt, 48 h
rt, 48 h
rt, 48 h
OH
OH
O
O
O
OH
O
O
bpa
O
OH
bpa
O
O
bpa
O
OH
bpa
OH
bpa
O
1
b
2e
3f
4c
5d
Scheme 2. Synthesis of the bpa derivatives 1b, 2e, 3f, 4c and 5d.
obtained as a yellow oil. Yield: 49%. 1H NMR (400 MHz, CDCl
.29 (s, 3H, H-8), 3.43–3.51 (m, 2H), 3.60–3.66 (m, 2H), 3.73 (t,
stirred for 48 h. The reaction mixture was diluted with diethyl
ether and washed with water (3 Â 75 mL), saturated NaHCO solu-
tion (3 Â 75 mL) and brine (3 Â 75 mL). The collected organic
phase was dried over Na SO , filtered and evaporated under
3
): d
3
3
J = 5.9 Hz, 2H), 3.89 (t, J = 5.9 Hz, 2H), 7.69 (dd, J = 5.5, 3.0 Hz, 2H,
Ar), 7.82 (dd, J = 5.4, 3.1 Hz, 2H, Ar) ppm; HR ESI-MS (in MeOH):
2
4
+
m/z [M+H] Calc. 250.1073, found 250.1098.
reduced pressure. Afterwards, the product was distilled at 165 °C
and 1.2 mbar (b.p. 90 °C). 3b was obtained as a colorless liquid.
1
Yield: 29%. H NMR (500 MHz, CDCl
3
): d 1.34 (m, 3H, CH
3
), 1.40
2
.2.1.3. Synthesis of 2-(2-methoxyethoxy)ethylamine (2d). A mixture
(
(
1
m, 3H, CH
m, 1H, H-7), 3.08–3.18 (m, 1H), 3.40 (ddd, J = 17.6, 11.7, 6.0 Hz,
H), 3.46–3.64 (m, 2H), 3.71 (td, J = 8,3, 6.4 Hz, 1H), 3.80 (ddd,
J = 13.4, 11.7, 2.9 Hz, 1H), 4.04 (ddd, J = 8.0, 6.4, 1.5 Hz, 1H), 4.27
3
), 2.58 (ddd, J = 5.0, 3.9, 2.7 Hz, 1H, H-7), 2.73–2.82
of N-[2-(2-methoxyethoxy)ethyl]phthalimide (2c) (27 mmol) and
hydrazine monohydrate (43 mmol) in EtOH (30 mL) was refluxed
at 100 °C for 3 h. After cooling down to room temperature, the mix-
ture was filtered and the filtrate was evaporated. Acetone (50 mL)
was added to the residue and the precipitate was filtered. Subse-
quently, the filtrate was concentrated under reduced pressure. 2d
was obtained as an orange oil. Yield: 62%. 1H NMR (500 MHz,
1
(
dq, J = 11.6, 5.8, 5.3 Hz, 1H, H-1) ppm. H NMR data are in agree-
ment with data reported in the literature [19].
3
CDCl ): d 1.83 (s, 1H), 2.00 (s, 1H), 2.15 (s, 1H), 2.87 (t, J = 5.3 Hz,
2
.2.1.5. Synthesis of 1,2-isopropylidene glyceryl (1-azido-2-propanol)
1
1
H), 3.35–3.39 (m, 2H), 3.41 (t, J = 6.4 Hz, 1H), 3.50 (t, J = 5.3 Hz,
H), 3.52–3.55 (m, 2H), 3.60 (dd, J = 5.7, 3.3 Hz, 1H), 3.64 (dd,
ether (3c). A mixture of 3b (29 mmol), sodium azide (144 mmol)
and ammonium chloride (66 mmol) in 80% EtOH (150 mL) was
refluxed at 100 °C for 18 h. The reaction mixture was poured into
ice-cold water. The solution was extracted with dichloromethane
and washed with water (3 Â 50 mL) and brine (3 Â 50 mL). The
J = 5.6, 3.8 Hz, 1H), 3.73 (t, J = 6.5 Hz, 1H) ppm; HR ESI-MS (in
+
MeOH): m/z [M+H] Calc. 120.0973, found 120.1047.
2.2.1.4. Synthesis of 1,2-isopropylidene glyceryl glycidyl ether (3b). A
2 4
organic phase was dried over Na SO , filtered and evaporated
mixture of solketal 3a (98 mmol), epichlorohydrine (198 mmol),
benzene (20 mL), 50% NaOH (20 mL) and tetra-n-butylammonium
bromide (TBAB, 10 mmol) was cooled down to 0 °C and then
under reduced pressure. 3c was obtained as a light yellow oil.
1
3 3
Yield: 85%. H NMR (500 MHz, CDCl ): d 1.33 (m, 3H, CH ), 1.40
(m, 3H, CH ), 2.92 (dd, J = 7.8, 4.9 Hz, 1H), 3.26–3.40 (m, 2H),
3
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A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
3
1
.47–3.61 (m, 4H), 3.69 (ddd, J = 8.3, 6.4, 2.9 Hz, 1H), 3.88–3.97 (m,
reaction was quenched by addition of ice-cold water and further
extraction with DCM. The combined organic layers were washed
with water and dried over Na SO , filtered and evaporated. Subse-
H), 3.99–4.06 (m, 1H), 4.21–4.31 (m, 1H, H-1) ppm; 1 C NMR
): d 25.4 (CH ), 26.8 (CH ), 53.4 (2x), 66.4 (2x),
9.7 (2x), 72.6 (2x), 73.0 (2x), 74.8 (2x), 109.7 ppm. Both H NMR
3
(
126 MHz, CDCl
3
3
3
2
4
1
6
quently the residue was filtered through a silica plug (MeOH/DCM
1
3
and C NMR data are in agreement with data reported in the liter-
ature [20].
1:4) and the filtrate was concentrated under reduced pressure. 5a
1
was obtained as a light-yellow liquid. Yield: 92%. H NMR
(
3 3 3
500 MHz, CDCl ): d 1.37 (s, 3H, CH ), 1.43 (s, 3H, CH ), 3.35 (dd,
2
.2.1.6. Synthesis of propylene glycol (1-azido-2-propanol) ether
J = 12.8, 6.0 Hz, 1H), 3.38–3.44 (m, 1H), 3.48 (d, J = 1.9 Hz, 3H, H-
14), 3.49–3.65 (m, 4H), 3.74 (ddd, J = 8.3, 7.5, 6.3 Hz, 1H), 4.06
(3d). 3c (24 mmol) was stirred with Dowex (15 g) in MeOH
(
150 mL) and refluxed at 85 °C for 16 h. After the mixture was
(ddd, J = 8.2, 6.4, 0.9 Hz, 1H), 4.23–4.31 (m, 1H, H-1) ppm; 13
C
cooled down to room temperature the ionic exchange material
was filtered and the filtrate was concentrated. Then, the residue
was treated again with Dowex (10 g) in MeOH (150 mL) and the
3 3 3
NMR (126 MHz, CDCl ): d 24.68 (CH ), 26.05 (CH ), 50.72 (C-6),
57.26, 65.93 (2x), 69.93 (2x), 71.86 (2x), 73.92 (2x), 78.50 (2x),
+
108.75 ppm; HR ESI-MS (in MeOH): m/z [M+Na] Calc. 268.1292,
reaction was repeated. 3d was obtained as a dark yellow oil. Yield:
found 268.1323.
1
9
3
3
3%. H NMR (500 MHz, CDCl
.48–3.65 (m, 8H), 3.69 (dd, J = 11.7, 3.6 Hz, 1H), 3.90 (dq, J = 6.3,
3
): d 3.35 (dd, J = 5.9, 2.5 Hz, 2H),
2
.2.1.11. Synthesis of propylene glycol (1-azido-2-methoxypropanol)
ether (5b). 5a (23 mmol) was stirred with Dowex (20 g) in MeOH
150 mL) and refluxed at 85 °C for 16 h. After the mixture was
.0, 2.6 Hz, 1H), 3.96 (ddt, J = 9.4, 6.1, 3.4 Hz, 1H) ppm; HR ESI-
+
MS (in MeOH): m/z [M+H] Calc. 192.0973, found 192.0982.
(
cooled down to room temperature, the ionic exchange material
was filtered and the filtrate was concentrated. Then, the residue
was treated again with Dowex (10 g) in MeOH (150 mL) and the
2
.2.1.7. Synthesis of propylene glycol (1-amino-2-propanol) ether
(3e). 10% Pd/C (0.12 g) was added to a solution of 3d (6 mmol) in
MeOH (24 mL). The reaction mixture was stirred under hydrogen
atmosphere for 24 h. After the catalyst was removed completely
via repeated filtration through Celite and standard filters, the fil-
trate was evaporated under reduced pressure. 3e was obtained as
reaction was repeated. 5b was obtained as a dark orange oil. Yield:
1
8
1
7%. H NMR (500 MHz, CDCl
3
): d 2.39 (s, 2H), 3.36 (d, J = 5.9 Hz,
H), 3.40 (ddd, J = 12.9, 4.3, 2.1 Hz, 1H), 3.46 (d, J = 0.9 Hz, 3H,
), 3.50 (q, J = 4.8 Hz, 1H), 3.52–3.64 (m, 5H), 3.70 (ddd,
CH
J = 11.4, 3.9, 2.3 Hz, 1H), 3.85–3.90 (m, 1H) ppm;
126 MHz, CDCl ): d 51.3 (C-6), 58.0 (2x), 36.9, 70.6 (2x), 70.7,
3
a light-yellow oil. Yield: 97%. 1H NMR (500 MHz, MeOH-d
): d
.63 (ddd, J = 13.1, 7.4, 1.0 Hz, 1H), 2.74 (dd, J = 13.1, 4.3 Hz, 1H),
.42–3.51 (m, 3H), 3.51–3.60 (m, 3H), 3.69–3.79 (m, 2H) ppm;
4
13
C NMR
2
3
(
7
3
+
3.3, 79.3 (2x) ppm; HR ESI-MS (in MeOH): m/z [M+Na] Calc.
1
3
4
C NMR (126 MHz, MeOH-d ): d 44.0 (C-6), 48.5, 63.0, 70.9 (2x),
2
28.0992, found 228.0968.
+
7
2.5, 73.5 ppm; HR ESI-MS (in MeOH): m/z [M+H] Calc.
1
66.1073, found 166.1211.
2.2.1.12. Synthesis of propylene glycol (1-amino-2-methoxypropanol)
2
.2.1.8. Synthesis of 1,2-methoxy propylidene (1-azido-2-methoxy-
ether (5c). 10% Pd/C (0.4 g) was added to a solution of 5b
(20 mmol) in MeOH (50 mL). The reaction mixture was stirred
under hydrogen atmosphere for 24 h. After double-filtration of
the catalyst, the filtrate was concentrated under reduced pressure.
propanol) ether (4a). A solution of 3d (11 mmol) and KOH
126 mmol) in DMSO (30 mL) was stirred for 10 min under argon
(
atmosphere. Iodomethane (63 mmol) was added to the solution
and the reaction mixture was stirred for additional 45 min. The
reaction was quenched by addition of ice-cold water and further
extraction with DCM. The combined organic layers were washed
1
5c was obtained as a dark yellow oil. Yield: 99%. H NMR (500 MHz,
CDCl
3
): d 2.56 (s, 4H), 2.80 (ddd, J = 13.1, 5.6, 2.4 Hz, 1H), 2.89 (dd,
), 3.48–
3.66 (m, 6H), 3.81–3.87 (m, 1H) ppm; C NMR (126 MHz, CDCl ):
J = 4.5, 2.5 Hz, 1H), 3.33 (t, J = 5.1 Hz, 1H), 3.41 (s, 3H, CH
3
1
3
with water, dried over Na
2
SO
4
, filtered and evaporated. Subse-
3
quently the residue was filtered through a silica plug (MeOH/
DCM 1:4) and the filtrate was concentrated under reduced pres-
sure. 4a was obtained as a light yellow oil. Yield: 87%. 1H NMR
d 42.3 (C-6), 57.7 (2x), 63.8, 70.6 (2x), 70.8, 70.9 (2x), 80.6 (2x)
ppm; HR ESI-MS (in MeOH): m/z [M+H] Calc. 180.2272, found
180.1415.
+
(
3
8
500 MHz, CDCl
3
): d 3.26–3.35 (m, 1 H), 3.36 (s, 3H, CH
.44 (m, 2H), 3.45 (s, 3H, CH ), 3.46 (s, 3H, CH ), 3.46–3.60 (m,
H) ppm; HR ESI-MS (in MeOH): m/z [M+Na] Calc. 256.1292,
3
), 3.37–
3
3
+
2.2.2. General procedure for the synthesis of the bpa ligands
Pyridine-2-aldehyde and acetic acid were added to a solution of
found 256.1293.
the respective amino ether (1a, 2d, 3e, 4b, 5c) in dry THF/MeOH
under argon atmosphere. Subsequently sodium triacetoxy borohy-
dride was added to the mixture. The reaction mixture was stirred
at room temperature for 48 h. The solvent was removed under
reduced pressure. The residue was dissolved in DCM and washed
2
.2.1.9. Synthesis of 1,2-methoxy propylidene (1-amino-2-methoxy-
propanol) ether (4b). 10% Pd/C (0.13 g) was added to a solution of
a (5 mmol) in MeOH (24 mL). The reaction mixture was stirred
4
under hydrogen atmosphere for 24 h. After filtration of the cata-
lyst, the filtrate was concentrated under reduced pressure. 4b
was obtained as a light-yellow oil. Yield: 95%. 1H NMR (500 MHz,
with a saturated aqueous NaHCO
3
solution (2 Â 100 mL). The
organic phase was dried over Na
under reduced pressure.
2
SO , filtered and evaporated
4
CDCl
H), 3.26–3.33 (m, 1H), 3.34 (s, 3H, CH
s, 3H, CH ), 3.43 (s, 3H, CH ), 3.43–3.59 (m, 8H) ppm; C NMR
126 MHz, CDCl ): d 43.1 (C-6), 57.9 (2x), 58.1 (2x), 59.4, 71.0,
1.1, 71.6 (2x), 72.3, 79.3 (2x), 81.5 ppm; HR ESI-MS (in MeOH):
3
): d 2.72 (dd, J = 13.3, 6.5 Hz, 1H), 2.84 (dd, J = 13.3, 4.1 Hz,
1
3
), 3.34–3.41 (m, 2H), 3.42
13
(
(
3
3
2.2.2.1. Synthesis of 1b. Pyridine-2-aldehyde (42 mmol), 1a
(21 mmol), sodium triacetoxy borohydride (56 mmol), acetic acid
(42 mmol) and 100 mL THF were used for the synthetic procedure
3
7
+
1
m/z [M+H] Calc. 208.1573, found 208.1597.
of 1b, which was obtained as a dark yellow oil. Yield: 76%. H NMR
(
3
500 MHz, CDCl ): d 2.84 (t, J = 5.0 Hz, 2H, H-2), 3.53 (dd, J = 5.0,
2
.2.1.10. Synthesis of 1,2-isopropylidene glyceryl (1-azido-2-methox-
ypropanol) ether (5a). A solution of 3c (25 mmol) and KOH
298 mmol) in DMSO (75 mL) was stirred under argon atmosphere
3.8 Hz, 2H, H-5), 3.64–3.68 (m, 2H, H-6), 3.71 (dd, J = 5.0, 3.8 Hz,
2H, H-3), 3.96 (s, 4 H, H-9, H-11), 7.17 (ddd, J = 7.4, 4.9, 1.1 Hz,
2H, py), 7.56 (d, J = 7.8 Hz, 2H, py), 7.67 (td, J = 7.7, 1.8 Hz, 2H,
py), 8.53 (ddd, J = 4.9, 1.7, 0.9 Hz, 2H, py) ppm. H NMR data are
in agreement with data reported in the literature [16].
(
1
for 10 min. Iodomethane (149 mmol) was added to the solution
and the reaction mixture was stirred for additional 45 min. The
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A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
5
2
.2.2.2. Synthesis of 2e. Pyridine-2-aldehyde (17 mmol), 2d
to room temperature. The vial was placed in a screw-capped con-
tainer (100 mL), filled with diethyl ether (10 mL) and closed. After
standing overnight at room temperature the precipitated product
was washed with hexane and diethyl ether and dried under
reduced pressure.
(
(
8 mmol), sodium triacetoxy borohydride (22 mmol) acetic acid
17 mmol) and 25 mL THF were used for the synthetic procedure
1
of 2e, which was obtained as a dark brown oil. Yield: 71%.
NMR (500 MHz, CDCl
H-8), 3.50–3.54 (m, 2H), 3.55–3.58 (m, 2H), 3.65 (t, J = 6.1 Hz,
H), 3.92 (s, 4H, H-9, H-11), 7.12–7.18 (m, 2H, py), 7.58 (d,
J = 7.8 Hz, 2H, py), 7.65 (td, J = 7.6, 1.8 Hz, 2H, py), 8.53 (d,
J = 5.8 Hz, 2H, py) ppm; 13C NMR (126 MHz, CDCl
): d 53.6, 59.1,
1.0, 69.6, 70.3, 72.0, 121.9 (py), 122.9 (py), 136.4 (py), 149.0
H
3
): d 2.86 (t, J = 6.1 Hz, 2H, H-2), 3.37 (s, 3H,
7a–e: A solution of Cu(ClO
4
)
2
2
Á6H O in MeOH was added to a
2
solution of the bpa ligand (1b, 2e, 3f, 4c, 5d) in MeOH and the reac-
tion mixture was stirred for 2 h at room temperature. Upon addi-
tion of the metal salt, the solution turned deep blue. Diethyl
ether was added to the mixture and the flask was cooled in the
freezer for 5 h. The formed precipitate was dried under reduced
pressure.
3
6
+
(
py), 159.9 (py) ppm; HR ESI-MS (in MeOH): m/z [M+H] Calc.
3
02.1873, found 302.1880.
2
.2.2.3. Synthesis of 3f. Pyridine-2-aldehyde (17 mmol), 3e
2.2.3.1. Complex 6a. Cu(NO
3
)
2
Á3H
2
O (1.5 mmol), 1b (1.5 mmol) and
(
(
8 mmol), sodium triacetoxy borohydride (22 mmol), acetic acid
17 mmol), 10 mL MeOH and 100 mL THF were used for the syn-
10 mL EtOH were used for the synthesis of 6a, which was obtained
as a blue solid. Yield: 35%. HR ESI-MS (in MeOH): m/z [[Cu(1b)]À
+
thetic procedure of 3f. The residue was dissolved in water and a
H] Calc. 349.0857, found 349.0903; Elemental analysis (%): Calc.
pH of 3 was adjusted with 2 M HCl. After extraction with DCM
for C16
5 8
H21CuN O : C, 40.47; H, 4.46; N, 14.75. Found: C, 40.20; H,
(
5 Â 100 mL), the aqueous phase was neutralized with 2 M NaOH.
4.49; N 14.73.
The solution was evaporated under reduced pressure. The residue
was dissolved in 2-propanol and excess NaCl was removed by fil-
2.2.3.2. Complex 6b. Cu(NO
3
)
2
Á3H
2
O (1.8 mmol), 2e (1.8 mmol) and
tration. Solvents were removed under reduced pressure. 3f was
10 mL EtOH were used for the synthesis of 6b, which was obtained
as a blue solid. Yield: 30%. HR ESI-MS (in MeOH): m/z [[Cu(2e)]]+
Calc. 364.1086, found 364.1138; Elemental analysis (%): Calc. for
1
obtained as a dark brown oil. Yield: 4%. H NMR (500 MHz, CDCl
3
):
d 3.23–3.31 (m, 1H), 3.36 (d, J = 5.6 Hz, 2H), 3.37–3.43 (m, 1H), 3.56
dtd, J = 17.2, 5.9, 2.1 Hz, 3H), 3.64 (td, J = 11.4, 10.4, 4.1 Hz, 3H),
.67–3.78 (m, 2H), 3.84–3.91 (m, 1H), 4.46–4.75 (m, 4H), 7.27
dd, J = 7.5, 5.0 Hz, 2H, py), 7.58–7.64 (m, 2H, py), 7.74 (td, J = 7.6,
(
3
(
C
17
H
23CuN
5 8
O : C, 41.76; H, 4.74; N, 14.32. Found: C, 41.76; H,
4.85; N 14.02.
1
3
1
.6 Hz, 2H, py), 8.58 (dt, J = 5.1, 2.6 Hz, 2H, py) ppm; C NMR
): d 55.6 (2x), 57.4, 59.0, 59.1, 63.6 (2x), 70.3,
2.2.3.3. Complex 7a. Cu(ClO
4
)
2
2
Á6H O (0.4 mmol), 1b (0.4 mmol) and
(
126 MHz, CDCl
3
3 mL MeOH were used for the synthesis of 7a, which was obtained
as a blue solid. Yield: 34%. HR ESI-MS (in MeOH): m/z [[Cu(1b)]À
7
1
3
0.5, 70.7 (2x), 73.2, 73.6, 76.0, 123.7 (2x), 124.6 (2x), 137.9,
48.9 (2x) ppm; HR ESI-MS (in MeOH): m/z [M+H] Calc.
48.1872, found 348.2016.
+
+
H] Calc. 349.0857, found 349.0846; Elemental analysis (%): Calc.
for C16
2 3
H23Cl CuN O11: C, 33.84; H, 4.08; N, 7.40. Found: C, 34.07;
H, 3.76; N 7.35.
2.2.2.4. Synthesis of 4c. Pyridine-2-aldehyde (5 mmol), 4b
(
(
2.5 mmol), sodium triacetoxy borohydride (7 mmol), acetic acid
2.2.3.4. Complex 7b. Cu(ClO
4
)
2
Á6H
2
O (0.3 mmol), 2e (0.3 mmol) and
5 mmol) and 15 mL THF were used for the synthetic procedure
3 mL MeOH were used for the synthesis of 7b, which was obtained
as a dark green solid. Yield: 21%. HR ESI-MS (in MeOH): m/z [[Cu
1
of 4c, which was obtained as a dark yellow oil. Yield: 87%.
NMR (500 MHz, CDCl ):
J = 5.6 Hz, 2H), 3.36 (s, 3H, CH
H
+
3
d
3.29 (d, J = 3.7 Hz, 3H), 3.32 (d,
), 3.37 (s, 3H, CH ), 3.38–3.52 (m,
H), 3.84 (d, J = 2.6 Hz, 4H), 7.10 (ddd, J = 7.4, 4.9, 1.1 Hz, 2H, py),
.49 (d, J = 7.7 Hz, 2H, py), 7.61 (td, J = 7.7, 1.8 Hz, 2H, py), 8.47
): d
(2e)]] Calc. 364.1086, found 364.1094; Elemental analysis (%):
3
3
Calc. for C18
2 3
H29Cl CuN O12: C, 35.22; H, 4.76; N, 6.84. Found: C,
8
7
(
35.55; H, 4.47; N 6.76.
dd, J = 5.8, 1.7 Hz, 2H, py) ppm; 13C NMR (126 MHz, CDCl
2.2.3.5. Complex 7c. Cu(ClO
)
3
4
2
2
Á6H O (0.2 mmol), 3f (0.2 mmol) and
5
1
(
5.2, 57.8 (2x), 58.0 (2x), 59.3, 61.3, 71.0, 72.4, 79.2, 121.8, 122.0,
3 mL MeOH were used for the synthesis of 7c, which was obtained
as a dark blue–green solid. Yield: 40%. HR ESI-MS (in MeOH) m/z
23.0, 127.9, 136.4, 137.1, 149.0, 150.3, 159.8 ppm; HR ESI-MS
+
+
in MeOH): m/z [M+H] Calc. 390.2373, found 390.2940.
[[Cu(3f)]ÀH] Calc. 409.1068, Found 409.1111; Elemental analysis
(
2 3
%): Calc. for C19H29Cl CuN O13: C, 35.55; H, 4.55; N, 6.55. Found: C,
2.2.2.5. Synthesis of 5d. Pyridine-2-aldehyde (40 mmol), 5c
35.69; H, 4.28; N 6.69.
(
(
20 mmol), sodium triacetoxy borohydride (52 mmol) acetic acid
40 mmol) and 100 mL THF were used for the synthesis of 5d.
2.2.3.6. Complex 7d. Cu(ClO
4
)
2
Á6H
2
O (0.3 mmol), 4c (0.3 mmol) and
Additional work-up of the residue was performed as described in
the synthesis of 3f. 5d was obtained as a dark brown oil. Yield:
1
J = 5.6 Hz, 3H, CH
2
3
3 mL MeOH were used for the synthesis of 7d, which was obtained
as a dark blue–green solid. Yield: 38%. HR ESI-MS (in MeOH): m/z
6%. 1H NMR (500 MHz, CDCl
): d 3.23–3.31 (m, 1H), 3.36 (d,
), 3.37–3.43 (m, 1H), 3.56 (dtd, J = 17.2, 5.9,
.1 Hz, 4H), 3.64 (td, J = 11.4, 10.4, 4.1 Hz, 3H), 3.67–3.78 (m, 2H),
.84–3.91 (m, 1H), 4.46–4.75 (m, 4H), 7.27 (dd, J = 7.5, 5.0 Hz, 2H,
[[Cu(4c)]] Calc. 452.1611, found 452.1727; Elemental analysis
+
3
3
2 3
(%): Calc. for C22H35Cl CuN O13: C, 38.63; H, 5.16; N, 6.14. Found:
C, 38.57; H, 5.43; N 6.26.
py), 7.58–7.64 (m, 2H, py), 7.74 (td, J = 7.6, 1.6 Hz, 2H, py), 8.58
dt, J = 5.1, 2.6 Hz, 2H, py) ppm; 13C NMR (176 MHz, CDCl
): d
5.6 (2x), 57.5, 59.1 (2x), 63.6 (2x), 70.4, 70.6, 70.7 (2x), 73.3,
3.6, 76.1, 123.7 (2x), 124.7 (2x), 137.9, 148.9 (2x) ppm; HR ESI-
2.2.3.7. Complex 7e. Cu(ClO
4
)
2
Á6H
2
O (0.3 mmol), 5d (0.3 mmol) and
(
5
7
3
3 mL MeOH were used for the synthesis of 7e, which was obtained
as a dark blue–green solid. Yield: 39%. HR ESI-MS (in MeOH): m/z
+
[[Cu(5d)]ÀH] Calc. 423.1225, found 423.1287; Elemental analysis
+
MS (in MeOH): m/z [M+H] Calc. 362.2073, found 362.2257.
2 3
(%): Calc. for C20H31Cl CuN O13: C, 36.62; H, 4.76; N, 6.41. Found: C,
3
6.76; H, 4.83; N 6.36.
2
.2.3. Synthesis of copper(II) complexes
a–b: Cu(NO O was added to a solution of the bpa ligand
Á3H
1b, 2e) in EtOH and the reaction mixture was refluxed for 1 h.
6
3
)
2
2
2.3. X-ray crystallographic data collection and refinement
(
Upon addition of the metal salt, the solution turned deep blue.
The reaction mixture was filtered into a vial and allowed to cool
Single crystals suitable for X-ray analysis were mounted on a
Bruker APEX-II CCD diffractometer. The crystals were kept at
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6
A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
1
2
00.09 K during data collection. For data correction SADBS (Bruker,
014) and SAINT (Bruker, 2013) were used. Using Olex2 [21], the
suggested has been proven to unnecessary [19]. The epoxide
opening of 3b [20] and further acetal deprotection of 3c led to
the formation of azide 3d, which could be confirmed by the disap-
structures were solved with the ShelXS [22] structure solution pro-
gram using direct methods and refined with the ShelXL [23] refine-
ment package using least squares minimization.
1
pearance of the acetal methyl peaks within the H NMR spectra.
The reduction of the azide 3d was carried out using hydrogen
gas with palladium on charcoal as a catalyst, since a procedure
using lithium aluminum hydride as described in the literature
was unsuccessful [25]. The desired compound 3e [20] and its
methoxylated equivalent 4b [20] were obtained in high yields
2.4. DNA cleavage experiments
Plasmid DNA pBR322 was purchased from Carl Roth GmbH.
(
97% for 3e; 95% for 4b). Moreover, the novel amino ether 5c with
DNA cleavage experiments were performed with 0.2
0 mM Tris–HCl buffer (pH 7.4) at 37 °C for 24 h in case of hydro-
lytic cleavage reactions.
lg DNA in
two hydroxyl groups and one methoxy group was successfully
synthesized (Yield: 99%).
5
Oxidative cleavage was examined by DNA incubation for 2 h in
the presence of the reducing agent ascorbate (1 mM). For the per-
formance of ROS studies, DNA was incubated for 2 h in the pres-
ence of ascorbate (1 mM) and either 200 mM tert-butanol,
3.1.2. Synthesis of bpa ligands
The synthesis of the bpa ligands was accomplished following a
reductive amination mechanism [16]. Both bpa ligands 1b and the
novel compound 2e were obtained in moderate yields (76% for 1b,
Lit. [16] 79%; 71% for 2e). Derivatisation of 1b through introduction
of different OH and OMe functionalities was achieved easily over
few reaction steps. Nevertheless, as discussed below, these differ-
ent substitution patterns play a clear role for the DNA cleavage
activity of the corresponding complexes.
The synthesis of 3f was carried out following the same proce-
dure as for 1b but due to the insolubility of 3e in THF, MeOH
was added as a solvent. Furthermore, another kind of work-up
was carried out following the work published by Metzler-Nolte
and co-workers [26] Compound 3f was isolated with a yield of only
4%. The yield for 4c was surprisingly high (98%). The synthesis of
5d was carried out using the same work-up process as performed
for 3f and yielded 16% of the desired product 5d.
2
00 mM DMSO, 10 mM NaN
liver, 2–5 units/ L) or 5 units/
bovine erythrocytes. Addition of 2Â phosphate buffered saline
PBS) to all samples (except for the reference) was necessary
3
, 2.5 mg/mL catalase (from bovine
l
lL of superoxide dismutase from
(
because superoxide dismutase was kept in 10Â PBS and catalase
had to be pre-incubated at 37 °C in 1Â PBS for 30 min. The result-
ing PBS concentration in every incubation mixture in quenching
experiments amounts to 0.125Â.
All cleavage experiments except for the quenching reactions
were carried out three times, the error bars result from the stan-
dard deviation.
After incubation, DNA samples were run on horizontal agarose
gels (1%) containing ethidium bromide (0.2
lg/mL) in 0.5Â TBE
buffer for 2 h at 40 V. The bands of supercoiled (Form I), open cir-
cular (Form II), and linear (Form III) DNA were visualized by fluo-
rescence imaging of ethidium bromide on a Bio-Rad GelDoc EZ
Imager. Data analysis was performed with Bio-Rad’s Image Lab
Software (Version 3.0). The intensity of the bands was measured
using the supercoiled control DNA as standard. Taking into account
that the supercoiled form I of plasmid DNA has a smaller affinity to
ethidium bromide, its intensity was multiplied with a correction
factor of 1.22 [24].
3.1.3. Synthesis of complexes
The synthesis of bpa Cu(II) nitrate and perchlorate complexes
was performed with compounds 1b, 2e, 3f, 4c, and 5d in EtOH
and MeOH, respectively [15,16]. Nitrate complexes of the bpa
ligands 1b and 2e (6a–b) could be isolated. The perchlorate com-
plexes 7a–e were precipitated with diethyl ether at À40 °C instead
of slow ether diffusion at room temperature (as suggested for the
nitrate bpa complex 6a) [16] or at 4 °C (as suggested for bpa
perchlorate complex 7a) [15] because no precipitate could be
formed at the aforementioned temperatures. The perchlorate com-
plexes were hygroscopic, and were thus stored under inert
atmosphere.
2.5. CD spectroscopy
The circular dichroism (CD) spectra were measured on a Jasco
J-810 Spectropolarimeter with a continuous flow of nitrogen at
room temperature with 1 cm pathway cells. The CD spectra were
À1
run from 320 to 220 nm at 100 nm min and the buffer back-
3.2. Description of crystal structures
ground was subtracted automatically. Data were recorded at
0
.1 nm intervals. The CD spectrum of calf thymus DNA (100
alone was recorded as the control experiment. 50 M of complex
concentration was used for recording the CD spectra.
lM)
X-ray crystal structures of [Cu(1b)(OH
2
)(ONO
2 3
)]NO 6a (CCDC
l
2
[
3
18710) [16] and [Cu(1b)(MeOH)(OClO )]ClO 7a (CCDC612379)
4
15] have been reported before, but are included in the following
discussion due to their similarity with the complexes presented
herein.
3
. Results and discussion
Single crystals of copper(II) complexes [Cu(2e)(ONO
2 2
) ] 6b and
3
3
2
.1. Synthesis
[Cu(2e)(MeOH)(OClO )]ClO 7b were obtained by slow diffusion of
3
4
diethyl ether into a methanolic solution of the complexes. Table 1
summarizes crystal data and structure refinement parameters of
6b and 7b.
The structure of 6b is shown in Fig. 2 and selected bond lengths
and angles are shown in Table 2. The complex crystallized in the
.1.1. Synthesis of amino ethers
The synthetic pathways for the preparation of compounds 1b,
e, 3f, 4c, and 5d are shown in Scheme 2. For these bpa derivatives,
amino ethers 2d, 3e, 4b and 5c were synthesized, whereas for 1b a
commercially available amino ether was used. Within the synthe-
sis of the amino ether 2d [18], the first reaction (2a ? 2b) was car-
ried out as described by Inagaki and co-workers. After substitution
of the tosyl group of 2b with a phthalimide group, 2c was depro-
tected to yield amine 2d (62%).
1
monoclinic space group P2 /c (as 6a [16]) and exhibits a neutral
Cu(II) complex without any additional solvent molecule in the
asymmetric unit. The Cu(II) center is surrounded in a distorted
octahedral fashion by six donor atoms including three nitrogen
and one oxygen atom of 2e. Two additional coordinated oxygen
atoms arise from two nitrato ligands. This is in contrast to complex
6a that contains the non-methylated ether tether ligand 1b, where
Compound 3b was synthesized according to a literature proce-
dure, however, purification via column chromatography as
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A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
7
Table 1
Crystal data and structure refinement parameters of complexes 6b and 7b.
6
b
7b
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C
17
H
23CuN
5
O
8
C
18
H
27Cl
595.86
monoclinic
P2 /c
2 3 11
CuN O
488.94
monoclinic
P2 /c
8.1392(8)
28.827(3)
8.4997(9)
90
98.922(3)
90
1970.1(4)
4
1
1
8.6944(3)
26.5046(12)
11.3350(5)
90
110.0202(14)
90
2454.21(18)
4
1.613
b (Å)
c (Å)
a
(°)
b (°)
(°)
c
3
V (Å )
Z
q
l
À3
(g cm
(Mo K
)
1.648
À1
a
) (mm
)
0.71073
1012.0
100.09
2.53–26.40
0.71073
1228.0
100.0
F(000)
T (K)
h minimum–maximum (°)
Data set [h, k, l]
2.45–26.49
À10 6 h 6 8, À35 6 k 6 36, À10 6 l 6 10
À10 6 h 6 10, À33 6 k 6 33, À14 6 l 6 14
Refinement
Least squares
0.0311 (4038)
0.0263 (3651)
0.0648
Least squares
0.0412 (5056)
0.0324 (4403)
0.0727
R
R
1
(reflections) [all data]
(reflections) [IP2r(I)]
1
wR
2
2
Goodness-of-fit (GOF) on F
Largest difference peak and hole (e Å
1.061
0.37 and À0.39
1.071
0.45 and À0.46
À3
)
Fig. 2. Comparison of ORTEP-III views of complexes 6a [16] and 6b (50% ellipsoid probability, hydrogen atoms have been omitted for clarity).
Table 2
Selected bond lengths (Å) and angles (°) of complexes 6b and 7b, as well as 6a and 7a for comparison.
6
a [16]
6b
7a [15]
7b
Cu–N1
Cu–N2
Cu–N3
Cu–O1
Cu–O4
Cu–O7
N1–Cu–N2
N1–Cu–N3
N2–Cu–N3
O1–Cu–N1
O1–Cu–N2
O1–Cu–N3
O4–Cu–O7
1.996
1.990
2.044
1.981
2.338
2.562 (calc.)
165.7
83.4
82.3
98.5
95.6
171.2
163.169 (calc.)
1.9950(14)
1.9940(14)
2.0460(13)
1.9590(12)
2.3672(12)
2.633 (calc.)
165.06(6)
81.41(5)
83.69(5)
92.47(5)
102.21(5)
163.80(5)
161.269 (calc.)
Cu–N1
Cu–N2
Cu–N3
Cu–O1
Cu–O2
Cu–O4
N1–Cu–N2
N1–Cu–N3
N2–Cu–N3
O1––Cu–N1
O1–Cu–N2
O1–Cu–N3
O2–Cu–O4
1.985
1.981
2.013
1.954
2.452
2.478
165.2
83.5
82.4
98.8
95.6
172.1
176.2
1.9756(18)
1.9642(18)
2.0133(18)
1.9603(16)
2.4787(16)
2.4716(16)
166.62(8)
84.81(7)
83.45(7)
96.66(7)
95.60(7)
174.70(7)
164.66(6)
Please cite this article in press as: A.A.J. Sudarga Tjakraatmadja et al., Inorg. Chim. Acta (2016), http://dx.doi.org/10.1016/j.ica.2016.02.034

8
A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
the second oxygen is provided by an aqua ligand, and the second
nitrate ion belongs to the outer coordination sphere.
of the ether tether to the copper center (Cu–O2) in the perchlorate
complexes 7a and 7b when compared to the nitrato complexes 6a
and 6b (Cu–O7: 2.562 and 2.633 Å, respectively), whereas the
nitrato ligand is closer to the copper center (Cu–O4: 2.338 and
2.367 Å, respectively). Generally, the structures regarding the
mean plane and the copper atom related to the basal plane are less
distorted compared to 6b with a maximum deviation for N3 of
À0.117 Å in 7a and À0.103 Å in 7b concerning the basal plane.
The copper atom in both molecules lies just slightly above the
square plane with distances of 0.030 (7a) and 0.005 Å (7b), respec-
tively. Both complexes exhibit an intramolecular hydrogen bond
O1–H1Á Á ÁO3 between the coordinated methanol and an oxygen
atom of the chain in the bpa ligand (Table S1). In contrast to 6a
The basal plane in 6b includes three nitrogen atoms from the
bpa ligand and one oxygen atom from a nitrato ligand (in 6a from
an aqua ligand, respectively). Bond lengths in 6b differ just slightly
from each other and range from 1.959 to 2.046 Å. The copper atom
lies 0.138 Å above the mean plane, defined by N1, N3, N2 and O1.
The maximum deviation is shown by N1 (À0.130 Å) and N3
(
+0.137 Å). Both facts indicate a significant deformation in the
octahedral arrangement around the copper center. The axial bonds
are elongated with a Cu–O4 distance of 2.367 Å and 2.633 Å for
Cu–O7, significantly less than the sum of the van der Waals radii
of copper and oxygen (2.92 Å). The numbers for 6a deviate only
slightly from the ones reported above, indicating that despite the
methyl group at O8 and an aqua ligand in place of the nitrato
ligand, the structures are very similar. Differences are as expected
only observed for the angles in the equatorial plane that accommo-
dates the aqua and one nitrato ligand, respectively. Angles differ
remarkably from the ideal 90° of an undistorted octahedron (e.g.
and 6b, no intermolecular p–p interaction between pyridine moi-
eties is observable.
In conclusion, the crystal system seems to depend on the substi-
tution of the bpa ligand. All complexes are Jahn–Teller distorted as
indicated by a value of 0.80 (0.82 for 6a) for the tetragonality T
[27,28]. Furthermore, the molecular structure is more dependent
on the anion resulting from the used copper salt, but the way of
coordination of the bpa ligand to the copper center does not differ
significantly. The extent of hydrogen bonding, however, depends
on both, the type of bpa ligand and the anion used.
O1–Cu–N3 171° for 6a, 163° for 6b). Intermolecular p–p stacking
can be observed between two adjacent pyridine planes for both
complexes (Fig. S1, Table S2 for 6b). The structure of 6a is stabi-
lized by two hydrogen bonds (O1–H1aÁ Á ÁO8, O1–H1bÁ Á ÁO9
(
2
ONO )) of the aqua ligand, whereas no hydrogen bonding is possi-
ble in the methylated species 6b because of an additional nitrato
ligand occupying this coordination site instead of water.
The perchlorate complex 7b crystallizes like its nitrate analog
3.3. Gel electrophoretic studies
3.3.1. Hydrolytic cleavage
6
b in the monoclinic space group P2
1
/c, whereas 7a crystallizes
In order to assess the hydrolytic DNA cleavage ability of the
complexes, supercoiled (sc) pBR322 DNA (Form I) was incubated
with 5 mM of 6a–b and 7a–e in Tris–HCl buffer (50 mM) at pH
7.4 for 24 h (Fig. 4).
As it can be seen in lane 1 of Fig. 4, even the reference DNA was
converted into 35% of Form II due to the long incubation time. Nev-
ertheless, percentages of cleaved plasmid, were still higher for the
employed complexes (55–80% of Form II and 1–10% of linear plas-
mid DNA (Form III)).
At 1.25 mM concentration and under identical conditions, the
complexes 6a–b and 7a–d (lanes 2–7, Fig. S2) exhibited similar
DNA cleavage activity, but only up to 1% of linear plasmid was
obtained. Metzler-Nolte and co-workers reported [16] complex
6a showing cleavage activity within the same order of magnitude,
however, the experimental conditions were slightly different
(pUC19 plasmid, 5 mM NaCl, 5 mM Tris HCl buffer).
Although only small differences in cleavage activity are
observed for the complexes tested, the study produced high repro-
ducibility with small error bars and thus allows to some extent to
in the orthorhombic space group Pbca [15]. The structures of 7a
and 7b are shown in Fig. 3 and selected bond lengths and angles
of 7a and 7b are shown in Table 2. Both molecules contain a catio-
+
+
nic species, [Cu(1b)(MeOH)(OClO
3
)] (7a) [Cu(2e)(MeOH)(OClO
3
)]
(7b) and one perchlorate counter anion. The center atom is also
coordinated by six surrounding atoms in a distorted octahedral
fashion but compared to 6b one anion is exchanged by a solvent
molecule (MeOH). A reason for that could be the steric influence
of the perchlorate anion compared to the smaller nitrato ligand
which leads to the smaller methanol occupying the sixth position
in the octahedral environment of Cu(II). Regardless of the length
and substitution of the ether tether, both complexes, 7a and 7b
show almost equal binding lengths for the Cu(II) coordinating
atoms (Table 2). Bond lengths in the basal plane for 7a and 7b
are in the same range compared to 6b and axial ligands exhibit a
distance of 2.472 Å for the Cu–O4 bond and 2.479 Å for Cu–O2
(
(
7b), respectively, and 2.478 Å for Cu–O4 and 2.452 Å for Cu–O2
7a). There is a considerably shorter binding length for the oxygen
Fig. 3. Comparison of ORTEP-III views of complexes 7a [15] and 7b including intramolecular H bonding (50% ellipsoid probability).
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A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
9
1
-
2
-
3
-
4
-
5
-
6
-
7
-
8
-
7e with a 3-hydroxypropyl-propoxy tether outreaches the litera-
ture-known complex 6a carrying a 2-hydroxyethyl-ethoxy tether
by a factor of 10 with respect to the yield of linear DNA. Also 7b
and 7c still yield twice as much linear DNA in comparison to 6a.
The longer alkyl chain analog 7d of the methylated complex 7b,
however, is less active, which might be attributed to the additional
methoxy group in position 2 of the propoxy linker.
Interestingly, Chin and co-workers [17] have observed two dif-
ferent mechanisms for the two tethers mentioned above. Whereas
for the hydroxypropyl tether a fast transesterification process is
postulated, the hydroxyethyl tethered complex showed slower
hydrolysis of BNPP due to an exchange of the tether by a hydroxide
anion. Thus, depending on which mechanism applies to the com-
plexes presented here, also differences in DNA cleavage activity
might be observed. On the other hand, as also stated by Metzler-
Nolte and co-workers [16]. BNPP might not be a realistic model
for DNA, and comparisons between BNPP-based and plasmid-
DNA-based studies might be problematic.
A
B
ascorbate
nicked
linear
supercoiled
1
00
90
80
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
Lane
Nicked Plasmid DNA
Linear Plasmid DNA
3.3.2. Oxidative cleavage
In order to assess the oxidative DNA cleavage ability of the com-
plexes, supercoiled (sc) pBR322 DNA (Form I) was incubated with
varying concentrations of 6a–b and 7a–e in Tris–HCl buffer
Fig. 4. (A) Cleavage activity of the complexes monitored by 1% agarose gel
electrophoresis after incubation at 37 °C for 24 h. Every lane contained 0.2
plasmid DNA in 50 mM Tris–HCl buffer (pH 7.4) (representative gel). Lane 1:
lg
reference DNA; lane 2: 6a (5 mM); lane 3: 6b (5 mM); lane 4: 7a (5 mM); lane 5: 7b
(
50 mM) at pH 7.4 for 2 h in the presence of ascorbate. At concen-
trations of 50 M and 12.5 M, respectively, all complexes showed
similar activity leading to complete cleavage of DNA (70–75% of
Form II and 25–30% of Form III at 50 M, Fig. 5, and about 90% of
Form II and 5–10% of Form III at 12.5 M, Fig. S3). This is to be
(
5 mM); lane 6: 7c (5 mM); lane 7: 7d (5 mM); lane 8: 7e (5 mM). (B) Percentage of
l
l
degraded DNA.
l
draw conclusions connecting DNA cleavage activity and the struc-
tures of the complexes applied. According to the mechanism pos-
tulated by Metzler-Nolte and co-workers [16], the hydroxyl
group (cf. Scheme 1) abstracts a proton of a coordinated water
molecule. Although in the crystal structure in some cases MeOH
occupies this coordination site, it should be uncontroversial that
in aqueous solution an aqua ligand can replace MeOH.
Complex 6a possibly is a more efficient DNA cleaving agent
than 6b due to the hydrogen bonds that can be formed by the aqua
ligand as deduced from the crystal structure (Fig. 2) and promote
hydrolytic DNA cleavage. This situation corroborates the mecha-
nism as reported by Metzler-Nolte and co-workers [16]. It can be
assumed, however, that the nitrato ligands of 6b are also
exchanged with aqua ligands in aqueous solution, enabling hydro-
gen bonding also in this case. Additionally, the lower proton accep-
tor ability of the methyl ethyl ether in 6b compared to the free
hydroxyl group of 6a might be a reason for the lower activity of
l
expected since oxidative cleavage activity should not depend on
the linker type used, but only on the redox activity of the copper
center.
A very recent study by Kim et al. has shown that the substitu-
tion pattern of bpa Cu(II) complexes did influence the oxidative
cleavage activity. The cleavage efficiencies of complexes of the
3 2 3 2
type [Cu(R-benzyl-bpa)(NO ) ] (R = OMe, CH , H, F and NO )
were therein compared with the unsubstituted bpa complex.
Electron-donating groups like CH
3
in the para-position of the
1
+
2
+
3
+
4
+
5
+
6
+
7
+
8
+
ascorbate
nicked
A
B
linear
supercoiled
6
b. Indeed, ethanol (as a simplification of the 2(3)-hydroxyl-
ethoxy(propoxy) tether in 6a, but also in 7a, 7c and 7e) is a better
proton acceptor, i.e. Brønsted base (pK
100
A
of the conjugate acid À2.3)
29], in comparison to methyl ethyl ether (as a simplification of 2
3)-methoxy-ethoxy(propoxy) tethers as in 6b, but also 7b and
d, pK
of the conjugate acid À3.8 [30]).
When 6a is compared to its perchlorate equivalent 7a, as
90
80
70
[
(
7
A
60
50
expected, the counter ion does not have any influence and results
in similar DNA cleavage activity of the two complexes. However,
when 6b and 7b are compared, the perchlorate complex is a more
efficient DNA cleaving agent. The structures of complexes 6b and
40
30
20
10
0
7
b are too similar as indicated by characteristic bond lengths and
angles (Table 2) to serve as a basis of discussion for the differences
in cleavage activity. In particular, it is to be expected that in aque-
ous solution they relate to each other structurally.
Chin and co-workers [17] have shown before that hydrox-
ypropyl substituents at the bpa moiety lead to more efficient cleav-
ing agents of phosphate ester bonds than ligands with
hydroxyethyl tethers. This was justified with the closer proximity
of the reactive hydroxyl group to the aqua ligand to be deproto-
nated and the substrate to be cleaved, respectively, due to the
bulkiness of the longer tether. Indeed, at a 5 mM concentration
1
2
3
4
5
6
7
8
Lane
Nicked Plasmid DNA
Linear Plasmid DNA
Fig. 5. (A) Cleavage activity of the complexes monitored by 1% agarose gel
electrophoresis after incubation at 37 °C for 2 h. Every lane contained 0.2
plasmid DNA in 50 mM Tris–HCl buffer (pH 7.4) in the presence of ascorbate
l
g
(
(
1 mM) (representative gel). Lane 1: reference DNA; lane 2: 6a (50
lM); lane 3: 6b
50
lM); lane 4: 7a (50
l
M); lane 5: 7b (50
lM); lane 6: 7c (50 lM); lane 7: 7d
(50
lM); lane 8: 7e (50
lM). (B) Percentage of degraded DNA.
Please cite this article in press as: A.A.J. Sudarga Tjakraatmadja et al., Inorg. Chim. Acta (2016), http://dx.doi.org/10.1016/j.ica.2016.02.034

1
0
A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
benzyl substituent led to an enhancement, whereas electron-with-
drawing groups lead to a reduction in DNA cleavage activity [31].
Such substitutions might indeed have a direct effect on the redox
potential of the copper complexes, whereas the alkyl linkers used
in this study should not have such an influence.
redoxinactive or redoxactive [16]. There are, however, several liter-
ature examples for complexes with redoxactive metals [36,37] and
even redoxinert metals [38], where oxidative cleavage can occur in
the absence of reducing agents. The term ‘‘self-activating” nucle-
ases was coined for such complexes that carry out DNA cleavage
by using their redoxactive ligand systems as redox partners.
In order to find out, if ROS could also be generated in the
absence of ascorbate, DNA cleavage studies were carried out with
the same complex concentration used in the hydrolytic cleavage
studies and in absence of any reducing agent. For the DNA cleavage
of 7a, 7c and 7d the following scavengers were used (shown in
Fig. 7 for 7a representatively):
3.3.3. ROS studies for oxidative cleavage
In order to characterize the essential reactive oxygen species
responsible for the oxidative DNA cleavage of the complexes,
exemplarily 7a, 7c and 7d were investigated and incubated in
the presence of DNA and ascorbate with different scavengers for
the following species [32–35] (Fig. 6):
(
(
(
(
a) tert-Butanol for hydroxyl radicals.
b) DMSO for hydroxyl radicals.
c) Sodium azide (NaN ) for singlet oxygen.
d) Catalase for hydrogen peroxide.
e) Superoxide dismutase for superoxide.
(a) tert-Butanol for hydroxyl radicals.
(b) DMSO for hydroxyl radicals.
(c) Sodium azide (NaN ) for singlet oxygen.
3
3
(
The enzymes catalase and superoxide dismutase were not
applied as above (Fig. 6) due to the long incubation time, which
might have resulted in an inactivation/decomposition of the
enzymes.
Compared to the reference (lane 2), i.e. 7a without any scav-
engers, the DNA cleavage was surprisingly increased in the pres-
ence of catalase, for which we do not have
a
reasonable
No significant change of DNA cleavage activity occurred for all
the complexes 7a, 7c and 7d indicating the absence of hydroxyl
radicals and singlet oxygen (Figs. 7 and S6, S7). Although this
experiment did not catch hydrogen peroxide and superoxide as
potential ROS, it can be assumed that hydrolytic DNA cleavage is
the predominant mechanism in the absence of reducing agents.
explanation at the moment. Only a small scavenging effect was
t
observed in the presence of BuOH and DMSO. A distinct quench-
ing, however, as indicated by the reappearance of supercoiled
DNA happened only in the case of NaN
3
and superoxide dismutase
(
lanes 5 and 7). Most probably, singlet oxygen and superoxide are
involved in the DNA cleaving process. These species have recently
also been identified as the active species in the oxidative DNA
3.4. CD spectroscopy
cleavage by the complexes [Cu(R-benzyl-bpa)(NO
above [31]. Complexes 7c and 7d showed a similar behavior
Figs. S4 and S5).
3 2
) ], mentioned
Circular dichroism (CD) spectroscopy was used in order to
investigate the interaction between the complexes and CT-DNA.
The CD spectrum of CT-DNA consists of a positive band at
277 nm due to base stacking and a negative band at 245 nm due
to right-handed helicity of DNA. In case of intercalation, the inten-
sities of the bands might be enhanced, which would be attributed
(
3.3.4. Investigation of possible ROS species in the absence of ascorbate
Hydrolytic cleavage activity has been proposed for bpa com-
plexes before, regardless of the metal present in the complex, i.e.
1
+
2
+
3
+
4
+
5
+
6
+
7
+
8
+
ascorbate
nicked
linear
supercoiled
1
-
2
-
3
-
4
-
5
-
6
-
A
B
ascorbate
nicked
A
linear
supercoiled
B 100
100
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
2
3
4
5
6
7
8
Lane
Nicked Plasmid DNA
2
3
4
5
6
Linear Plasmid DNA
Lane
Nicked Plasmid DNA Linear Plasmid DNA
Fig. 6. (A) Quenching effects on DNA cleavage by 7a monitored by 1% agarose gel
electrophoresis after incubation at 37 °C for 2 h. Every lane contained 0.2
plasmid DNA. Lane 1–7 contained 1 mM ascorbate and 0.125Â PBS in 50 mM Tris–
HCl buffer (pH 7.4). Lane 2–7 contained 50 M of 7a. Lane 1: DNA ladder; lane 2:
reference; lane 3: 200 mM BuOH; lane 4: 200 mM DMSO, lane 5: 10 mM NaN ; lane
: 2.5 mg/mL catalase; lane 7: 313 U/mL superoxide dismutase; lane 8: reference
DNA. (B) Percentage of degraded DNA.
l
g
Fig. 7. (A) Quenching effects on DNA cleavage by 7a monitored by 1% agarose gel
electrophoresis after incubation at 37 °C for 24 h. Every lane contained 0.2
lg
l
plasmid DNA. Lane 1–6 contained 0.125Â PBS in 50 mM Tris–HCl buffer (pH 7.4).
t
3
Lane 2–5 contained 5 mM of 7a. Lane 1: DNA ladder; lane 2: reference; lane 3:
t
6
200 mM BuOH; lane 4: 200 mM DMSO, lane 5: 10 mM NaN
DNA. (B) Percentage of degraded DNA.
3
; lane 6: reference
Please cite this article in press as: A.A.J. Sudarga Tjakraatmadja et al., Inorg. Chim. Acta (2016), http://dx.doi.org/10.1016/j.ica.2016.02.034

A.A.J. Sudarga Tjakraatmadja et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
11
Acknowledgements
4
2
0
0
We gratefully acknowledge financial support by Deutsche
Forschungsgemeinschaft (grant GR 3585/3-1). We thank the Kulak
Group for proofreading of the manuscript.
0
20
40
60
80
-
-
-
-
Appendix A. Supplementary material
CCDC 1440789 and 1440791 contains the supplementary crys-
tallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data_request/cif. Supplementary data associated
with this article can be found in the online version, at http://
dx.doi.org/10.1016/j.ica.2016.02.034.
CT DNA
CT DNA + 7a
CT DNA + 7c
CT DNA + 7d
-
100
220
240
260
280
300
320
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Please cite this article in press as: A.A.J. Sudarga Tjakraatmadja et al., Inorg. Chim. Acta (2016), http://dx.doi.org/10.1016/j.ica.2016.02.034