F.A. Beckford et al. / Inorganic Chemistry Communications 15 (2012) 225–229
227
Table 2
all relative to Ag/AgCl. This couple is assumed to be the metal-based
oxidation Cu(II)/Cu(III). While these values might appear low, other
works have reported similar values and have suggested that the
redox process associated with the oxidation of Cu(II) to Cu(III) may
probably originate due to the presence of the thione sulfur center
[13]. We have noted for other copper complexes in our labs (unpub-
lished work) similar electrochemical behavior and it appears that the
Cu(II)/Cu(III) redox couple can merge with the redox couple of the
oxidized ligand around +0.75 V. We are also carrying out extensive
square wave voltammetry in order to ascertain the separate redox
couple for the ligand and the Cu(II)/Cu(III) redox process.The second
peak is ill-defined and occur at +1.33 V for all the complexes except
for 1 where it occurs at +1.44 V. We are not exactly sure of the nature
of this couple but based on the constancy of the potential we propose
that it might be ligand based [13]. The single oxidation peak observed
for complex 1 would appear to support the EPR data that it is monomeric
in the solution phase. It should be noted that the lower scan rate
(100 mV/s) of the first peak is hardly noticeable but becomes more
pronounced as the scan rate is increased (see Supplemental Information).
Cyclic voltammetric studies of thiosemicarbazone ligands have shown
the existence of an irreversible cathodic peak at −1.06 V, assignable
to the reduction of the thione group of the thiosemicarbazone moiety
[13]. In our complexes this peak appears at −0.659 V, −0.609 V,
−0.598 V and −0.587 V for 1, 2, 3 and 4 respectively.
Ethidium bromide competition experiment. We have investigated the
reaction of the complexes with calf thymus DNA via a fluorescence com-
petition experiment. Ethidium bromide (EB) is a well-known dye that is
commonly used as a marker for nucleic acids by intercalating between
the base pairs and generating a fluorescent adduct. The fluorescence
from the adduct may be quenched by addition of a compound that
can displace the EB from the binding sites on the DNA. This quenching
may be taken as evidence that the compound can bind to DNA. Using 3
as a typical example, we can see from Fig. 2 that the complex can indeed
reduce the fluorescence of the EB–DNA solution by as much as 63%. A
quantitative estimate of this quenching behavior can be obtained by
treating the data according to the Stern–Volmer Eq. (1):
Binding constants for the interaction of the complexes with the EB-DNA adduct at
303 K.
1
2
3
4
104 KSV (M−1
104 Kapp (M−1
1012 Kq (M−1 s−1
)
)
–
5.35
1.79
2.43
4.26
1.91
1.94
0.773
0.406
0.351
2.23
–
)
For complex 3 Kapp =1.53×104 M−1 at 298 K.
KSV is the Stern–Volmer quenching constant which is the measure of
the effectiveness of the complex as a quencher. The inset of Fig. 2
shows the Stern–Volmer plots and the linearity of the plots for 2
and 3 confirm the quenching behavior. The values of the quenching
constant seen in Table 2 are on the order of 104 M−1 indicating that
these are moderately strong quenchers. The Stern–Volmer plot for
complex 1 shows a positive deviation from linearity at high concen-
trations of the complex. The quenching of the fluorescence can
occur by two common mechanisms — dynamic and static quenching.
The deviation from linearity is usually explained by suggesting that
there are multiple binding sites on the fluorophore or that both
quenching mechanisms are occurring simultaneously. The static
contribution may be calculated from the following Eq. (2):
F0
F
¼ ð1 þ KD½QꢀÞð1 þ KS½QꢀÞ
ð2Þ
where KD and KS are the dynamic and static quenching constants.
(KS would actually represent the formation constant for a dark complex
between the quencher and the chromophores). However a completely
unambiguous assignment of KD and KS requires fluorescence lifetime
measurements [14] (though varying temperature can be used to
characterize the quenching as predominantly dynamic or static). We
propose that for 1 the static contribution is significant. This is inferred
from the bimolecular quenching constant (Kq in Eq. 1) calculated by
using τ0=22 ns [15] for the EB–DNA complex. Keq for the reactions
are on the order of 1012 M−1 s−1 which is two orders of magnitude
larger than the limiting value of 1010 M−1 s−1 [16] considered the
largest possible value in aqueous solution.
F0
F
¼ 1 þ KSV½Ruꢀ ¼ 1 þ Kqτ0½Ruꢀ:
ð1Þ
To assess the strength of the binding Eq. 3 was employed to calculate
the apparent binding constant.
In this equation F0 and F are the fluorescence intensities of the
reaction solution in the absence and presence of the metal compound.
KEB½EBꢀ
Kapp
¼
ð3Þ
½Cuꢀ50%
In this equation KEB is the binding constant for ethidium bromide,
taken as 1.2×106 M−1 [17], and [Cu]50% is the concentration of the
complex that causes a 50% reduction of the initial fluorescence.
These values are shown in Table 2 and range from 1.79×104 to
2.23×104 M−1 which suggest that the complexes are moderate to
strong binders.
Reaction of the complexes with HSA. Human serum albumin (HSA)
is the most abundant of the blood serum proteins occurring to the
extent of 0.63 mM [7]. The protein serves as a transport unit for a
wide variety of endogenous substances including drugs. In this
study we investigated the binding of the complexes to HSA using
fluorescence spectroscopy. HSA has a well-known structure that
contains a single tryptophan residue that is responsible for the majority
of the intrinsic fluorescence of the protein. On excitation at 295 nm HSA
has strong fluorescence emission at 350 nm. This emission can be
attenuated by a small molecule binding at or near the tryptophan as
this amino acid unit is quite susceptible to changes in its environment.
As a representative example Fig. 3a shows that addition of 2 to HSA
can very efficiently reduce the fluorescence of the solution. Moreover
the wavelength of maximum fluorescence increased to 369 nm from
344 nm after addition of 22.5 μM of the complexes. It has been noted
[18] that the polarity of the molecular environment around the
Fig. 2. Fluorescence emission spectra of the EB-DNA adduct in the absence and presence of
increasing amounts of 2, λex=520 nm, [EB]=0.33 μM, [DNA]=10 μM, [2] (μM): 0–32.5
in 2.5 μM increments. Temperature=303 K. Inset: Stern–Volmer plots for the complexes.