Full Papers
doi.org/10.1002/ejic.202001170
to verify the mechanism of DNA cleavage by the complexes,
Table 5. Experimental conditions applied in catecholase activity tests at
external agents were added to the reaction mixtures before each
complex. The ionic strength of the reaction was modified by
replacing water with NaCl or LiClO4 solutions at 15, 30, 75 and
125 mmolLÀ 1. The reactions were also performed with groove
binders by replacing 4 μL of water with groove binder stock
solutions (50 μmolLÀ 1). The compounds used to bind to the major
and minor groove of DNA were methyl green (MG) and netropsin
(Net), respectively.[37] In this case, binders were incubated with DNA
at pH 6.0 for 30 min in the absence of light. In addition, the effect
of reactive oxygen species (ROS) was evaluated by replacing 4 μL of
water with an ROS sequestrant solution: ethanol (0.4 mmolLÀ 1),
potassium iodide (0.5 mmolLÀ 1) or sodium azide (0.5 mmolLÀ 1).
Modulation of plasmid DNA cleavage was performed by adding
10 μL of sodium ascorbate stock solution (100 μmolLÀ 1) instead of
water. Reactions occurred in the presence of 10 μmolLÀ 1 of the
°
25 C.
Effect
pH[a]
[3,5-DTBC] Saturation gas [Complex]
[b]
[mmolLÀ 1
]
[μmolLÀ 1
]
1
2
3
pH
Substrate
Inert atmosphere 9.0
Presence of H2O2 9.0
6.0–9.0 5.0
9.0 0.5–14.5
O2
O2
Ar
O2
59 16 27
60 60 60
50 50 50
50 50 50
0.2
60.0
[a] For all experiments, [buffer]=30.3 mmolLÀ 1
conducted with only one complex: 1, 2, or 3.
.
[b] Each test was
DTBC were performed by monitoring reactions under the same
conditions, but in the absence of a complex.
°
°
complexes for 1 h at 37 C or 4 h at 50 C.
The presence of H2O2 in the oxidation reactions was investigated
through a modified iodometry method.[5g] The reaction mixture was
prepared similarly to that mentioned for the kinetic procedure, but
with different experimental parameters (Table 5). After one hour, an
equal volume of water was added and the 3,5-DTBQ was extracted
with dichloromethane. The aqueous layer was acidified with sulfuric
acid (pH�2). A 2 mL aliquot was collected, and 1 mL of aqueous
potassium iodide solution (0.3 molLÀ 1) was added to this sample. If
hydrogen peroxide is present in the aqueous phase, the following
reaction occurs: H2O2 +2IÀ +2H+!2H2O+I2. Iodide excess in the
solution generates the triiodide ion (I2(aq) +IÀ !I3À ), which can be
monitored by UV/Vis spectroscopy due to the appearance of an
intense band at 353 nm (ɛ=26000 LmolÀ 1 cmÀ 1).[32]
Anaerobic tests were conducted and, to maintain an oxygen-free
environment, all experimental procedures were performed in a
glove bag. For these assays, two controls were performed: one with
CH3CN as the negative cleavage control and one containing 2 μL
Fe-EDTA (1 mmolLÀ 1 / 2 mmolLÀ 1) and 2 μL DTT (10 mmolLÀ 1) as a
positive control to replace the complex solution.[38]
The DNA cleavage kinetics was investigated with
a mixture
containing 14 μL pBSK-II DNA (25 μmolLÀ 1), 14 μL MES buffer
(10 mmolLÀ 1), 77 μL water and 35 μL complex solution in different
concentrations ([1]=[2]=1.0, 2.5, 5.0, 10.0 and 15.0 μmolLÀ 1; [3]=
0.05, 1.0, 2.5, 5.0 and 10.0 μmolLÀ 1). This reaction system was kept
°
at 50 C and aliquots were taken at regular times. After each aliquot
had been removed, 5 μL of running buffer was added to it to
complete the cleavage reaction. The samples were then submitted
to agarose gel electrophoresis. In all experiments, reactions under
the same conditions without the presence of complexes were
performed as a control to monitor the spontaneous degradation of
plasmid DNA.
DNA cleavage activity. The DNA cleavage experiments were
performed as described by Ausubel et al..[33] Plasmid DNA, used as
the substrate, was extracted from plasmid pBSK-II (2961 bp,
Stratagene). The plasmid was amplified by transformation into
competent cells of the bacterium DH5-α Escherichia coli according
to a procedure previously described[34] and purified using the
manufacturer‘s protocol (Qiagen Plasmid Maxi Kit protocol). The
DNA substrate was quantified via UV-Vis spectroscopy at 260 nm
Circular dichroism spectra were obtained with the JASCO J-815 CD
spectropolarimeter (Jasco, USA). The experiments were performed
with a 2 mm-optical path quartz cuvette containing 40 μL MES
(ɛ=13200 LmolÀ 1 cmÀ 1
) and its integrity was verified via gel
electrophoresis.[35]
buffer (10 mmolLÀ 1), 80 μL CT-DNA stock solution (200 μmolLÀ 1
)
and 280 μL water. CT-DNA was titrated with 200 μM of solutions
with [complex]/[DNA] ratios from 0.05 to 1. The CD spectra were
obtained in triplicate, with each scan resulting from the accumu-
lation of three spectra. Spectra were also obtained in the absence
of CT-DNA and complex (blank). Spectra of the complexes in the
absence of DNA were also obtained and showed no significant
signals.
In a typical DNA cleavage reaction, 2 μL of pBSK-II DNA (330 ng;
25 μmol LÀ 1), 2 μL of biological buffer, 5 μL of complex solution
and 11 μL of autoclaved Milli-Q water were added. After 4 h at
°
50 C, the reaction was terminated by the addition of 5 μL of
running buffer (0.25 molLÀ 1 EDTA at pH 8.0, 50% glycerol and
0.01% bromophenol blue). The samples were applied to an agarose
gel (1%) containing ethidium bromide (0.3 μgmLÀ 1) and electro-
phoresed for 100 min at 90 V in 0.5×TBE buffer (44.5 mmolLÀ 1 TRIS,
44.5 mmolLÀ 1 boric acid, 1 mmolLÀ 1 EDTA at pH 8.0). After the runs,
the gels were visualized and recorded using the DigiDoc-It photo-
documentation system (UVP, USA). The fractions of each plasmid
DNA shape were quantified by densitometry using the KODAK
Molecular Imaging Software 5.0 (Carestream Health, USA). Since the
ability of ethidium bromide to intercalate into supercoiled DNA
(form I) is less efficient than into other forms, that is, open circular
(form II) and linear (form III), the values found for form I were
multiplied by a correction factor of 1.47 and the values obtained for
the other forms were corrected by proportionality.[36] The amount
of cleaved DNA (%) was considered to be the sum of DNA fractions
in form II and form III.
Theoretical predictions. Optimization of the geometry of com-
plexes 1, 2 and 3 was carried out in vacuum with the Orca 4.2.1
software package[39] at the DFT level using the BP86 functional.[40]
The basis set chosen was Def2-TZVP for the copper atom and Def2-
SVP for the other atoms.[41] Our calculations also included Grimme’s
dispersion correction (D3) with Becke-Johnson damping (BJ).[42] The
vibrational frequencies for complexes 1, 2 and 3 showed only one
small imaginary frequency for complex 3 due to rotation of the
aromatic rings. The 3,5-DTBC conjugates were also optimized and
the vibrational frequencies showed one small negative frequency
due to rotation of the aromatic rings. In order to simulate the
absorption spectra, time-dependent density functional theory
under the Tamm-Dancoff approximation (TD-DFT/TDA)[43] was
employed to obtain the first 30 excitations, using the same
calculation protocol, differing only at the functional, which in this
case was PBE0[44] and the basis set for coordinated atoms chosen
was the same used for the copper atom (Def2-TZVP). To include the
solvent effects in the excited state energies, the conductor-like
The effect of pH on DNA cleavage reactions was evaluated for each
complex (10 μmolLÀ 1) using different buffers: MES (pH 6.0), 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid – HEPES (pH 7.0 and
8.0), 2-(cyclohexylamino)ethanesulfonic acid – CHES (pH 9.0) and 3-
(cyclohexylamino)-1-propanesulfonic acid – CAPS (pH 10.0). Thus,
Eur. J. Inorg. Chem. 2021, 1710–1721
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