Oxidative Release of Copper from Pharmacologic Copper Bis(thiosemicarbazonato) Compounds

Article abstract of DOI:10.1021/acs.inorgchem.8b00853

Intracellular delivery of therapeutic or analytic copper from copper bis-thiosemicabazonato complexes is generally described in terms of mechanisms involving one-electron reduction to the Cu(I) analogue by endogenous reductants, thereby rendering the metal ion labile and less strongly coordinating to the bis-thiosemicarbazone (btsc) ligand. However, electrochemical and spectroscopic studies described herein indicate that one-electron oxidation of Cu^II(btsc) and Zn^IIATSM (btsc = diacetyl-bis(4-methylthiosemicarbazonato)) complexes occurs within the range of physiological oxidants, leading to the likelihood that unrecognized oxidative pathways for copper release also exist. Oxidations of Cu^II(btsc) by H₂O₂ catalyzed by either myeloperoxidase or horseradish peroxidase, by HOCl and taurine chloramine (which are chlorinating agents generated primarily in activated neutrophils from MPO-catalyzed reactions), and by peroxynitrite species (ONOOH, ONOOCO₂^-) that can form under certain conditions of oxidative stress are demonstrated. Unlike reduction, the oxidative reactions proceed by irreversible ligand oxidation, culminating in release of Cu(II). 2-Pyridylazoresorcinol complexation was used to demonstrate that Cu(II) release by reaction with peroxynitrite species involved rate-limiting homolysis of the peroxy O-O bond to generate secondary oxidizing radicals (NO₂^?, ^?OH, and CO₃^?-). Because the potentials for Cu^II(btsc) oxidation and reduction are ligand-dependent, varying by as much as 200 mV, it is clearly advantageous in designing therapeutic methodologies for specific treatments to identify the operative Cu-release pathway.

Full text of DOI:10.1021/acs.inorgchem.8b00853

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Oxidative Release of Copper from Pharmacologic Copper
Bis(thiosemicarbazonato) Compounds
John J. Sirois,†,⊥ Lillian Padgitt-Cobb,‡ Marissa A. Gallegos,† Joseph S. Beckman,‡,§
Christopher M. Beaudry,† and James K. Hurst*,‡
†Department of Chemistry, ‡Department of Biochemistry & Biophysics, §Linus Pauling Institute, Oregon State University, Corvallis,
Oregon 97331-7305, United States
S
* Supporting Information
ABSTRACT: Intracellular delivery of therapeutic or analytic
copper from copper bis-thiosemicabazonato complexes is
generally described in terms of mechanisms involving one-
electron reduction to the Cu(I) analogue by endogenous
reductants, thereby rendering the metal ion labile and less
strongly coordinating to the bis-thiosemicarbazone (btsc) ligand.
However, electrochemical and spectroscopic studies described
herein indicate that one-electron oxidation of CuII(btsc) and
ZnIIATSM (btsc = diacetyl-bis(4-methylthiosemicarbazonato))
complexes occurs within the range of physiological oxidants,
leading to the likelihood that unrecognized oxidative pathways
for copper release also exist. Oxidations of CuII(btsc) by H2O2
catalyzed by either myeloperoxidase or horseradish peroxidase, by HOCl and taurine chloramine (which are chlorinating agents
generated primarily in activated neutrophils from MPO-catalyzed reactions), and by peroxynitrite species (ONOOH,
−
ONOOCO2 ) that can form under certain conditions of oxidative stress are demonstrated. Unlike reduction, the oxidative
reactions proceed by irreversible ligand oxidation, culminating in release of Cu(II). 2-Pyridylazoresorcinol complexation was
used to demonstrate that Cu(II) release by reaction with peroxynitrite species involved rate-limiting homolysis of the peroxy
O−O bond to generate secondary oxidizing radicals (NO2 , •OH, and CO3•−). Because the potentials for CuII(btsc) oxidation
•
and reduction are ligand-dependent, varying by as much as 200 mV, it is clearly advantageous in designing therapeutic
methodologies for specific treatments to identify the operative Cu-release pathway.
E° = −0.23 to −0.47 V (in DMSO vs NHE) being estimated
by cyclic voltammetry, so that efficient Cu(II) reduction in
these complexes should be thermodynamically limited to
highly reducing biological systems. Correspondingly, the low-
potential redox sites in several NADH or NADPH-dependent
electron transport chains have been shown to be capable of
reducing CuII(btsc) complexes,5−7 whereas weaker biological
reductants such as ascorbate and biological thiols are much less
effective, and reactivity is observed only in instances where the
medium contains strongly complexing Cu(I) ligands that can
drive the reactions forward.8 An almost overlooked feature of
these CuII(btsc) complexes is that they can also be oxidized at
potentials that are well within the range of biological oxidants
(∼0.81−0.94 V in DMSO vs NHE); see below). We
demonstrate herein that oxidations by peroxidase-catalyzed
H2O2, biological chlorinating agents, or peroxynitrite-derived
radicals lead to rapid oxidative degradation of the btsc ligand
with concomitant release of Cu(II) (e.g., oxidative pathway,
Scheme 1).
INTRODUCTION
■
Copper(II) bis-thiosemicarbazonato complexes (CuII(btsc))
(Chart 1) show promise as radioimaging agents and tissue-
Chart 1. Generic Chemical Structure of CuII(btsc)
Compounds
specific therapeutic agents for treatment of ailments as diverse
as solid cancerous tumors and neurological disorders.1−4 Based
primarily upon the observation that copper accumulates in
hypoxic cells to a greater extent than normoxic cells,1 a model
has been advanced that ascribes delivery to the cell interior by
passive transmembrane transport of charge-neutral CuII(btsc),
followed by intracellular reduction to Cu(I) and its release and
sequestration at biological copper-binding sites (e.g., reductive
pathway, Scheme 1).1,5 The one-electron reduction potentials
for CuII(btsc) complexes are low (see below), however, with
Received: March 29, 2018
© XXXX American Chemical Society
A
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Cu(I) reduction. From the relative amplitudes of the oxidation
and reduction waves exhibited by CuATSM, which are nearly
equal, it follows that the oxidation process is also a one-
electron event. Analogous electrochemical behavior was
observed for an additional 18 CuII(btsc) and ZnII(btsc)
complexes which differed in the identities of alkyl and aromatic
substituents in the R1 and R2 positions, as well as the extent of
alkylation on R′ and R′′ (Chart 1). The oxidation and
reduction half-wave potentials measured for the CuII(btsc)
analogues are given in Figure 2 as an electrochemical series,
Scheme 1. Proposed Reductive and Oxidative Pathways for
Intracellular Delivery of Cu from CuATSM
a
a
ROS/RNS = cellularly generated reactive oxidative species and
reactive nitrosative species, respectively.
RESULTS AND DISCUSSION
■
Electrochemistry. The electrochemical redox properties of
the btsc free ligands and metal ion complexes are illustrated in
the cyclic voltammograms for ATSM (diacetyl-bis(N4-
methylthiosemicarbazone) (R1, R2, R′ = CH3, R′′ = H)
shown in Figure 1. Massive irreversible degradation of
Figure 2. Electrochemical series for representative CuII(btsc)
complexes in DMSO (this work). Reduction potentials estimated
from cyclic voltammetry from E1/2 values using Fc+/o as an internal
standard and referenced to NHE by assuming E°(Fc+/o) = 680 mV vs
NHE.11 Specific ligands are designated according to the key, where
substituent positions are identified in Chart 1 (Me = CH3; Et =
CH3CH2; ϕ = C6H5).
Figure 1. Cyclic voltammograms of 5 mM H2ATSM, ZnATSM, and
CuATSM in DMSO vs Ag/AgCl.
uncoordinated H2ATSM began at ∼0.9 V (vs Ag/AgCl);
binding of either redox-active Cu(II) or redox-inactive Zn(II)
ions introduced a new oxidation peak at ∼0.6 V. For ZnATSM,
this was closely followed by additional irreversible oxidation
processes, and a corresponding reduction half-wave was not
observed. For CuATSM, oxidation was quasi-reversible;
repetitive cycling through the oxidation wave led to the
appearance of new reduction peaks at ∼0 to −0.2 V, suggestive
of accumulation of degradation products (not shown). This
behavior is consistent with earlier accounts of the inability to
isolate stable oxidized species from constant potential
electrolysis.9 A quasi-reversible reduction wave was also seen
at −0.67 V in the CuATSM voltammogram; because the
ZnATSM voltammogram was devoid of reduction waves, it can
be unambiguously assigned as the metal-centered Cu(II) →
where the experimental potentials measured against an Ag/
AgCl reference electrode were adjusted to NHE as indicated in
the figure caption. The oxidation and reduction E1/2 values
obtained varied from 0.81 to 0.94 V and −(0.23−0.46) V (vs
NHE), respectively, over the series. Oxidation of ZnATSM
must be ligand-centered, giving rise to a ligand radical cation,
i.e., [ZnII-ATSM•]+, as the initial one-electron oxidized species.
Given that CuATSM is oxidized at essentially the same
potential (Figure 1), the ligand may also be the reaction site in
this complex. However, the CuII(btsc) complexes possess
structural features (anionic strong donor and highly polarizable
B
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lead-in atoms in a tetradentate square planar geometry) that
would stabilize the d8 Cu(III) center and could thereby lower
its reduction potential into the observed range. Numerous
similarly structured cupric complexes have in fact been
described for which E°(CuIII/II) ∼ 0.7−0.9 V (NHE).10 The
experiments described herein do not allow discrimination
between metal-centered and ligand-centered oxidation of
CuII(btsc), although one might speculate that the greater
stability of its oxidized state evident in the CuATSM and
ZnATSM voltammograms (Figure 1) relates to its capacity to
undergo metal-centered oxidation.
Chemical Oxidations: General Considerations. The
optical properties of btsc compounds are well-suited for
spectrochemical analysis, exhibiting intense, clearly resolved
absorption bands for the free ligand and its Cu(II) and Zn(II)
complexes; typical spectra (for ATSM) are shown in Figure 3a.
Figure 4. Representative kinetic traces for MPO/H2O2/M-ATSM
reactions. Conditions: 0.020 mM CuATSM, 0.10 mM H2O2, 55 nM
MPO (red); 0.020 mM ZnATSM, 0.10 mM H2O2, 37 nM MPO
(black); 0.030 mM CuATSM, 0.10 mM H2O2, 110 nM MPO (blue).
In 5 mM MOPS, pH7.2, at 23 °C. Absorbances of the lower two
traces have been offset to allow simultaneous display; also, for
CuATSM, absorbance shown at 465 nm is 3× actual value.
addition of H2O2 and MPO or HRP was immaterial to the
catalytic reaction rates. The reactions were unaffected by
inclusion of 1 mM DTPA to scavenge adventitious metal ions,
but were partially inhibited when 0.1−3.0 mM of the heme
peroxidase inhibitor, NaN3, was added to the medium.
Irreversible enzyme inactivation also accompanied turnover;
typically, standard guaiacol assays indicated that ∼50% MPO
activity was lost following catalysis of CuATSM degradation
under these conditions. Copper release was probed using 4-(2-
pyridylazo)resorcinol (PAR), which under the experimental
conditions rapidly coordinates Cu(II) (Figure S2) to form a
tightly bound terdentate 1:1 complex16 (Figure S3, TOC
Figure 3. Optical spectra. Panel a: H2ATSM and its complexes in 20
mM MOPS, pH 7.2, plus 50 mM SDS; panel b: PAR and CuPAR in
50 mM phosphate, pH 7.2, plus 20 mM SDS.
To study oxidative reactions, we generally used 3-(N-
morpholino)propanesulfonic acid (MOPS) or phosphate-
buffered media containing sodium dodecyl sulfate (SDS) at
concentrations above its critical micelle concentration;12 the
presence of micelles promoted solubilization of the btsc
compounds while allowing use of aqueous oxidant systems.
The micellar environment also provides a hydrophobic
microphase which may mimic the actual internal locus of
CuII(btsc) compounds adsorbed by cells; specifically, recent
confocal fluorescence microscopic studies using BODIPY-
tagged CuIIATSM have suggested that the compound resides
predominantly within intracellular lipid droplets and the
membrane ultrastructure of cells.13
Peroxidase-Catalyzed Oxidation by Hydrogen Per-
oxide. CuATSM is unreactive toward H2O2 (Figures 4 and
S1). However, when a peroxidase was introduced to solutions
containing CuATSM or ZnATSM and H2O2, rapid decoloriza-
tion occurred over the entire optical spectrum, indicating that
the complex had been oxidatively degraded. These reactions
were demonstrated for both myeloperoxidase (MPO)14
(E7°′(compoundI/compoundII) = 1.35 V; E7°′(compoundII/
FeIII-MPO) = 0.97 V) (Figure 3), and the less strongly
oxidizing horseradish peroxidase (HRP)15 (E7°′(compoundI/
compoundII) = 0.92 V; E7°′(compoundII/FeIII−HRP) = 0.94
V) (Figure S1). Based upon their one-electron reduction
potentials, compounds I and II of both peroxidases are
themodynamically competent to drive oxidation of CuII(btsc)
and ZnII(btsc) ligands. All components of the enzymatic
systems were required for reaction to occur, and the order of
graphic) possessing a strong visible absorption band (λmax
=
511 nm, ε511 = 28 mM−1 cm−1) (Figure 3b). Transmetalation
from CuATSM to PAR does not occur under these conditions
(Figure S4). Because the ε-value for CuPAR at its absorption
maximum is ∼6-fold greater than that for CuATSM (Figure 3),
it is easy to detect free Cu(II) in the presence of CuATSM
from spectral changes accompanying addition of PAR. Cu(II)
release from the CuATSM/MPO/H2O2 reaction was demon-
strated in this fashion by adding PAR to the product solution,
upon which formation of CuPAR corresponding to ∼40% of
the CuATSM originally present was calculated from the
spectral changes (Figure S5). These data set a conservative
lower limit on MPO turnover of 100-fold under the
experimental conditions of Figure 4. Unlike its metal
complexes, the H2ATSM free ligand was unreactive in the
presence of HRP/H2O2 (Figure S1), consistent with
thermodynamic expectations.
The capacity of these compounds to modulate the reactivity
of HRP under standard assay conditions using 2,2′-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a one-
electron donor17 was also briefly examined. As shown in Figure
5, addition of CuATSM increased the initial velocity of the
HRP-catalyzed H2O2 oxidation product (ABTS•+) in a dose-
dependent manner, accelerating the reaction by ∼3.5-fold at
the highest concentrations used. This unexpected result
suggests that the complex can mediate electron transfer
between ABTS and HRP compounds I and II via its own
one-electron oxidized state. In contrast, addition of ZnATSM
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Figure 6. Oxidation of CuATSM by HOCl. Spectra are for 30 μM
CuATSM in 20 mM phosphate, pH 7.2, plus 25 mM SDS (black) and
product solutions obtained following addition of 30 μM (blue), 60
μM (green), 120 μM (purple), 195 μM (orange), and 240 μM (red)
HOCl.
Figure 5. CuATSM acceleration of HRP-catalyzed oxidation of ABTS
by H2O2 under steady-state conditions. CuATSM added at the
indicated concentrations to 50 μM H2O2, 50 μM ABTS, and 4 nM
HRP in 10 mM phosphate, pH 7.2, at 23 °C. The order of addition of
reactants and catalysts is indicated by the vertical arrows. Apparent
discontinuities in the kinetic traces at the points of addition are
artifactual and arise because it was necessary to briefly stop recording
the data to manually add reagents. Thus, spectral changes occurring
during these intervals are not recorded, but appear as instantaneous
“jumps” once the kinetic traces are restarted.
were seen when taurine chloramine was used in place of HOCl
(Figure S8), although the reactions in this case were
considerably slower, requiring nearly an hour following
chloramine addition to reach completion. This dynamic
behavior is consistent with the much lower intrinsic reactivity
of primary chloramines relative to HOCl. Release of Cu(II) at
various points in the titration was probed by adding PAR to
the solution and determining the extent of CuPAR formation.
From these studies, it was evident that free Cu(II) appeared
very early in the course of the titration, e.g., after addition of
one molar equivalent HOCl, the CuPAR formed was 16%, and
after addition of two equivalent, it was 29% of the CuATSM
initially present (Figure S9).
or, to a lesser extent, free H2ATSM to the assay medium led to
reduction in the initial rates of ABTS•+ formation under
otherwise identical reaction conditions (Figures S6 and S7).
Accordingly, neither oxidized ZnATSM nor H2ATSM appear
capable of reacting with ABTS. These reactivity differences are
consistent with the electrochemical redox behavior displayed in
Figure 1, where CuATSM oxidation was fully reversible on a
voltammetric sweep time scale comprising several seconds, but
degradation of the other species was too rapid to permit re-
reduction of the initially oxidized forms, leading to irreversible
voltammetric peaks.
Peroxynitrite Reactions. The peroxynitrite anion
(ONOO−) is formed via radical coupling in tissues that are
−
simultaneously generating superoxide anion (O2 ) (e.g., as a
Oxidation by Chlorinating Agents. Hypochlorous acid
is widely regarded as a primary MPO-generated component of
mammalian cellular defense systems against invading patho-
gens.18 The prospect therefore exists that HOCl, as well as
secondary chlorinating agents such as chloramines formed by
its reaction with physiological amines and amino acids, could
promote release of Cu from CuII(btsc) complexes at sites of
infection where phagocytes are activated. Indeed, we found
that HOCl and taurine chloramine (N-chloro-2-aminoethane-
sulfonate) oxidatively degrade CuATSM under conditions that
mimic the physiological milieu. Shown in Figure 6 are spectral
changes accompanying incremental additions of HOCl to
CuATSM in an aqueous micellar medium. One observes
initially a progressive hypsochromic shift of the visible band
with decreasing absorbance in the UV band, followed at higher
oxidant levels a hypsochromic shift in all bands accompanied
by the appearance and subsequent decline of new bands in the
near-UV region. These changes, together with the absence of
any isosbestic points, indicate that multiple products are
formed over the course of the titration. Complete bleaching of
the CuATSM spectrum requires addition of ∼10-fold molar
excess (∼20 oxidizing equivalents) HOCl. The reactions are
complete within the time of mixing of reactant solutions.
Preliminary stopped-flow studies suggest that the initial
reaction step has a bimolecular rate constant of ∼2 × 107
M−1 s−1 under these conditions. Very similar spectral changes
byproduct of respiratory electron transport or via phagocyte
NADPH oxidases) and nitric oxide (NO) (e.g., by aerobic
oxidation of L-arginine catalyzed by constitutive or inducible
forms of nitric oxide synthetase).19,20 In the absence of
oxidizable substrates, the anion is relatively stable, but addition
of Lewis acids (H+, CO2, metal ions) to the terminal oxygen
weakens the peroxo O−O bond, giving rise to net
−
decomposition to O2 and NO2 and/or isomerization to
nitrate.21−23 Decomposition occurs via homolysis of the
peroxo O−O bond, leading to formation of inorganic radicals
21,22
•
(OH•, CO3•−, and NO2 ),
which are powerful one-
electron oxidants.24 Oxidations by peroxynitrite can therefore
be complex, involving either direct bimolecular reaction of the
reductant with ONOOH (or, less frequently, ONOO−) or
indirect reactions involving the secondary radicals formed
during peroxynitrite decomposition.25 These two pathways can
easily be distinguished from the kinetic patterns of the
oxidative reactions, which for the direct pathway is simple
mixed-second order and for the indirect pathway is first-order
in peroxynitrite and zero-order in reductant concentration.
Moreover, in the indirect pathway, the oxidation rate constant
coincides with the peroxynitrite decomposition rate constant
under the prevailing conditions, and maximal product yields
are ≤30% of stoichiometric, reflecting the minor fraction of
radicals that escape recombination within the solvent cage to
23,25
−
give the isomerized oxidant, i.e., NO3 .
As described
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below, all of the CuII(btsc) complexes examined reacted with
peroxynitrite via the indirect pathway. Additionally, for those
complexes that are relatively easily oxidized, evidence for a
competing direct reaction between ONOOH and the
complexes was found.
Peroxynitrous acid (ONOOH) and the peroxynitrite-CO2
−
adduct (ONOOCO2 ) rapidly degraded ZnATSM and
CuII(btsc) complexes (including CuATSM and analogues for
which R1, R2 = H (CuGTSM) or R1 = C6H5, R2 = CH3
(CuϕMTSM)). Stopped-flow spectrophotometric analyses
established the following properties of the reactions: (1) the
oxidation pathway involved first-order reactions whose rate
constants were pH-dependent and nearly identical to
independently measured constants for pH-dependent perox-
ynitrite decomposition (Table 1, Group A);22 (2) similarly,
Figure 7. Kinetic traces at selected wavelengths of the reaction of
CuATSM with peroxynitrite in the presence of PAR. Conditions:
0.020 mM CuATSM, 0.25 mM ONOOH, and 0.015 mM PAR in 25
mM phosphate (final pH 7.44) plus 50 mM SDS, 23 °C. CuPAR
formation at 511 nm (red); PAR consumption at 410 nm (black);
CuPAR/PAR isosbestic point at 450 nm (blue). Reactions at 511 and
410 nm are first-order with identical k = 0.20 s−1; for comparison,
independently measured first-order peroxynitrite decay under the
same conditions gives kd = 0.19 s−1. The reaction at 450 nm can be
attributed to CuATSM + ONOOH, which shows a characteristic fast
component corresponding to direct reaction with ONOOH and
slower reactions involving decay of oxidized CuATSM (see text for
discussion).
Table 1. Comparison of Experimentally Determined (kd)
and Calculated (kcalc) First-order Rate Constants for
Peroxynitrite Decomposition with Oxidation (A−C and E)
a
II
ox
and Cu(II) Release (D) from Cu (btsc) Complexes (k )
b
kox (s−1
)
kd (s−1
)
kcalc (s−1
)
A. CuATSM + ONOOH → products
pH 6.3
pH 7.4
pH 9.8
0.30−0.67
0.22−0.23
very slow
0.58
0.11
0.0006
c
0.19
d
−
B. CuATSM + ONOOCO2 → products
c
pH 7.5
pH 7.4
9.1−10.5
C. CuGTSM + ONOOH → products
0.25−0.40 0.19
29
9.1
bleaching of the complex in neutral solutions. At this point,
∼70% of the Cu(II) present was released and could be
recovered as CuPAR upon addition of PAR to the product
solution. Peroxynitrite involvement was confirmed by control
studies in which ONOOH was allowed to decompose to
nitrate and nitrite/O2 for several minutes prior to addition to
CuATSM-containing solutions;22 under these conditions, no
reaction was observed. Assuming that this reaction is carried by
generation of inorganic radicals derived from O−O bond
homolysis, this corresponds maximally to about 8 oxidizing
equivalents (i.e., 4 ONOOH molecules) per complex. Clearly,
then, the reaction is a multistep oxidation process.
Intermediate species were not spectrally detected during the
redox titration. Spectral bandshapes and peak maxima (308
and 462 nm) remained unchanged over the course of the
titration, with absorbency changes that corresponded linearly
to the amount of added oxidant. However, a transiently
generated red intermediate that slowly faded within several
seconds was visually apparent following bolus addition of
ONOOH to CuATSM (Figure 8). Stopped-flow kinetic
analysis with ONOOH in 10-fold excess indicated that
formation of the red intermediate was faster than peroxynitrite
decomposition (Figure 8) and therefore must involve direct
bimolecular reaction of ONOOH with CuATSM. Intermediate
decay occurred on time scales that approximated those for
peroxynitrite decomposition and may therefore represent
reaction between initially formed partially oxidized complexes
and peroxynitrite-derived oxidizing radicals (Figure 8).
Formation of this transient is apparently not obligatory for
net oxidation of CuII(btsc) complexes, however. No inter-
mediates were detected (either visually or by stopped-flow
analysis) when CuGTSM was used in place of CuATSM. A
survey of various CuII(btsc) complexes using visual detection
indicated that only the more easily oxidized members of the
series (with E1/2(ox) ≤ 0.92 V) (Figure 1)) generated red
0.11
D. CuATSM + ONOOH + PAR → CuPAR + other products
pH 7.4
pH 7.3
0.20
0.19
0.11
E. PAR + ONOOH → products
0.23
0.16
a
b
In 25 mM phosphate, 25 mM SDS, 23 °C, except where noted. Ref
c
d
22. SDS absent. Medium contained 12.5 mM total carbonate (as
−
CO2 + HCO3 ).
decomposition and CuATSM oxidation rate constants were
accelerated by equal amounts in the presence of CO2 (Table 1,
Group B); and (3) the rate constants for CuII(btsc) oxidation
were independent of the identity of the complex (cf. Table 1,
Groups A (CuATSM) and C (CuGTSM)); (4) when PAR
was present in the reaction solutions, the rate constant for
CuPAR formation was also first-order and equal to that for
peroxynitrite decomposition (Figure 7; Table 1, Group D).
Independent measurements established that Cu(II)-PAR
association rates are considerably more rapid than the redox
reactions under investigation (Figure S2), that free PAR reacts
only with the peroxynitrite-generated radicals (Table 1, Group
E, Figure S9), and that CuPAR is unreactive toward all of the
peroxynitrite-derived oxidants. This set of circumstances,
including the previously mentioned absence of CuII(btsm)
→ CuIIPAR transmetalation, allow one to conclude that Cu(II)
release occurs by reaction of CuATSM with the secondary
radicals and not from direct bimolecular reaction between
CuATSM and peroxynitrite species. Overall, these reactions
exhibit the characteristics of indirect reactions of peroxynitrite
for which the actual oxidants are NO2 and OH• or CO3
•
•−
formed by rate-limiting homolysis of the ONOOH or
ONOOCO2 O−O bonds.21
−
Titrimetric addition indicated that a ∼14-fold excess of
peroxynitrite over CuATSM was required to achieve complete
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intermediates could be temporally resolved in reactions of
CuϕMTSM with peroxynitrite and, although ZnATSM was
extensively degraded by peroxynitrite, no discrete intermedi-
ates could be detected in that reaction.
Biomedical Implications. Mechanisms for intracellular
release of copper are yet to be identified. However, it is likely
that both reductive and oxidative pathways (Scheme 1) can
prevail under different physiological conditions. Thus, for
example, the reductive pathway seems well-established in
positron-emission tomography (PET) applications to detect
hypoxia, where the extent of intracellular copper accumulation
parallels the reduction potentials of CuII(btsc) complexes.1 In
similar PET imaging studies to detect regions of oxidative
stress in tissues from patients suffering from neurodegenerative
disorders, enhanced copper release has been attributed to
increased reducing capacity in cells as a consequence of
NADH accumulation caused by impaired mitochondrial
electron transport,28,29 although these conditions also promote
formation of reactive oxygen species (ROS) that are
thermodynamically competent to oxidize the complexes.
Moreover, disease progression in neurological disorders for
which CuII(btsc) treatment may have therapeutic benefit is
characterized by oxidative damage by ROS and reactive
nitrosative species, the latter possibly including ONOOH. The
antineoplastic activity of certain CuII(btsc) complexes (notably
CuGTSM) has recently been attributed to their capacity for
intracellular generation of ROS via redox cycling between the
Cu(II) and Cu(I) states of the complexes; notably, in these
studies, a marked dependence of cellular toxicity upon the
CuII/I(btsc) reduction potential was observed.30 The accel-
eration of HRP-catalyzed ABTS oxidation by H2O2 observed
in this study when CuATSM was introduced into the assay
medium (Figure 5) suggests that cycling between CuII(btsc)
and oxidized Cu(btsc) states might also occur in biological
settings. Biological precedents for this type of reactivity can be
found in the reactions of galactose oxidase (GAO), a protein
containing a single active site copper atom that catalyzes
aerobic oxidation of primary alcohols to the corresponding
aldehydes with stoichiometric formation of H2O2,31 and
copper complexes that have been developed as functional
models of the catalytic reaction center.26 A pertinent example
is the study by Wieghardt and co-workers on the oxidative
chemistry of N,N′-bis(3,5-di-tert-butyl-2-hydroxyphenyl)-1,2-
phenylenediamine.32 This ligand forms monomeric tetraden-
tate square-planar complexes with Cu(II) and Zn(II) with
N2O2 coordination sets that are structurally quite similar to the
MII(btsc) complexes studied here. Five different oxidation
states are accessible in the GAO model complex; catalytic
aerobic oxidation of primary alcohols to aldehydes, i.e.,
RCH2OH + O2 → RCHO + H2O2, is proposed to occur by
net two-electron cycling between two ligand radical states of
the complexes, namely, a reduced state containing a
di(hydroxyphenyl)-diiminosemiquinone ligand and an oxi-
dized state containing a hydroxyphenyl(phenoxyl)-diiminoqui-
none ligand. Notably, both the Cu(II) and Zn(II) complexes
are effective catalysts. Collectively, the studies on GAO
catalysis and CuATSM cocatalysis of the HRP/H2O2/ABTS
reaction suggest a scenario in which intracellular release of
copper might occur via a reaction sequence in which
transmembrane diffusion of CuII(btsc) is followed by
complex-catalyzed generation of the oxidant (H2O2) that
leads to its own oxidative destruction. Upregulation of MPO in
damaged neuronal tissues has also been documented, opening
Figure 8. Stopped-flow traces of the CuATSM-ONOOH reaction.
Conditions: 0.025 mM CuATSM + 0.25 mM ONOOH in 25 mM
SDS plus 25 mM phosphate, pH 7.5, at 23 °C. Overlay of biphasic
traces for CuATSM decay at 465 nm (black) and intermediate
formation and decay at 590 nm (red, 4× actual absorbance).
Photographs above the kinetic traces give a visual record of
intermediate formation and decay in the reaction. Conditions: 0.03
mM CuATSM with 0.5 mM peroxynitrite in 50 mM phosphate plus
20 mM SDS, pH 7.2, 23 °C.
intermediates upon exposure to peroxynitrite; moreover, the
extent of formation of the CuATSM intermediate was highly
pH-dependent, decreasing with decreasing acidity from pH 6.3
to 9.8, where it was barely detectable by stopped-flow analysis.
The kinetic waveforms obtained at pH 7.2 were accurately
reproduced by fitting to a double exponential form, suggesting
that the reactions proceeded by concurrent pathways. Overall,
these data are consistent with simultaneous expression of two
pathways in which (1) thermodynamically limited direct
reaction of the more easily oxidized complexes within the
CuII(btsc) series with ONOOH, but not with the less strongly
oxidizing ONOO− (pKa = 6.6), runs in parallel with (2)
indirect reactions of peroxynitrite-derived oxidizing radicals
that can rapidly oxidize all complexes within the series. Finally,
transient diode array spectrophotometry of CuATSM
indicated that the spectrum of the intermediate has a relatively
intense near UV band at 340 nm, and very broad weak
absorption above ∼500 nm that accounts for the red
coloration. These spectral features are consistent with either
ligand radical formation (e.g., CuII(ATSM)•+) or oxidation to
Cu(III)ATSM. Specifically, strikingly similar spectra have been
observed for multidentate Cu(II) and other metal complexes
containing phenoxyl and N-heterocyclic ligand radicals,26,27
but many tetradentate square-planar Cu(III) complexes also
exhibit intense LMCT absorption bands in the near-UV
region.10 Stopped-flow analyses also indicated that several
F
DOI: 10.1021/acs.inorgchem.8b00853
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
thiosemicarbazone)40 (H2ϕMTSM), 1H NMR (700 MHz, d6-
pathways for formation of additional oxidizing species as part
of the neurodegenerative processes.33−36 In this context, the
present studies have clearly demonstrated facile cupric ion
1
1
1
DMSO): 10.704 (s, H), 8.7402 (m, H), 8.6505 (s, H), 7.592−
7.541 (m, 3H), 7.295−7.265 (m, 2H), 6.979 (m, 1H), 3.043 (d, 3H, J
= 5), 2.819 (d, 3H, J= 5), 2.373 (s, 3H); M.S.: calcd for
CuC13H17N6S2 [(M + H+)] 384.03, found 384.08. Cupric and zinc
complexes were generally prepared as stock solutions obtained by
adding a slight excess of a concentrated solution of Cu(OAc)2 or
Zn(OAc)2 to the free ligand dissolved in DMSO; these were
monitored spectrally using a Shimadzu UV-2041PC recording
spectrophotometer to confirm complete formation of 1:1 complexes.
During the course of subsequent experimentation, it was noted that
the presence of even small amounts (∼2%) of DMSO partially
inhibited release of Cu(II) (measured as formation of CuIIPAR in
experiments using peroxynitrite as oxidant). This protection almost
certainly arose from scavenging of secondary peroxynitrite-derived
oxidizing radicals by the DMSO. Additional experiments with
peroxynitrite were therefore done using solid CuATSM that was
solubilized in aqueous-based media devoid of DMSO; these studies
indicated that the presence of DMSO at concentration levels present
in the reaction medium caused only minor alteration of the reaction
course. Solid CuATSM was obtained following a general procedure41
that involved dropwise addition of aqueous CuSO4 to a suspensions
of H2ATSM in methanol and isolating the CuATSM that precipitated.
A more critical issue was the highly effective scavenging of HOCl by
DMSO, which completely masked its reaction with CuATSM at even
the millimolar concentration levels present following dilution of
concentrated DMSO stock solutions of CuATSM into the reaction
medium. This problem was circumvented by preparing CuATSM in
N-methyl-2-pyrrolidone, which control studies showed did not reduce
HOCl when diluted into the reaction medium for periods exceeding 1
h.
−
release following oxidation by either ONOOH/ONOOCO2
or MPO-catalyzed peroxide systems, including HOCl, the
physiological product of neutrophil MPO-catalyzed reactions.
Our demonstration that reactions of major ROS and RNS
oxidants effect efficient Cu(II) release from CuII(btsc)
complexes establish the plausibility of similar reactions
occurring in biological tissues, particularly in regions under-
going oxidative stress. Distinguishing between oxidative and
reductive pathways in biological environments may prove
difficult, but we note that the fate of the btsc ligand is
fundamentally different in the two general pathways outlined
in Scheme 1, where it remains intact following reduction of the
complex to continually cycle metal ions, but is modified with
loss of metal-binding capacity in the oxidative pathway. Thus,
the pathways could presumably be distinguished in specific
instances by quantitative determination of the fate of the
ligand. Finally, we note that cupric ion in the presence of H2O2
is potently bactericidal.37 Use of CuII(btsc) complexes as
copper delivery systems offers potential for developing new
antibiotics, as has been demonstrated in recent studies.38,39
Interestingly, one proposed bactericidal mechanism is
inhibition of respiration through direct binding of intact
CuII(btsc) to the bacterial electron transport chain. Presum-
ably, for this application, one would seek to identify CuII(btsc)
complexes that underwent minimal intracellular degradation.
Myeloperoxidase (MPO) was isolated from bovine spleens by
column chromatography as previously described;42 the purified
enzyme had an RZ value of 0.70 and a specific activity of 345
units/mg protein, as measured by the guaiacol assay.43 Type VI
horseradish peroxidase (HRP) was obtained from Sigma, and its
activity was determined by HRP-catalyzed oxidation of 2,2′-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with H2O2.17
Reactants. To investigate oxidative reactions, we used where
possible aqueous buffered media containing 20−50 mM SDS. Under
these conditions, most of the SDS is present as microphase-separated
micelles. Specifically, the SDS critical micelle concentration is ∼8
mM, and its aggregation number in aqueous solutions is ∼60,12 so
that the effective concentration of micelles in these mixtures was
200−700 μM. Because the MII(btsc) concentrations never exceeded
50 μM in these experiments, the average occupancy number, i.e.,
MII(btsc)/micelle, was always less than 1. We believe that this
medium effectively mimics the biological milieu inside eukaryotic
cells, which contain numerous phase-separated microdomains.13
Inclusion of SDS also serves to increase the effective solubility of
the neutral CuII(btsc) complexes in aqueous media. As noted above,
some of the reactions studied are incompatible with all system
components. Most prominently, SDS inactivated the peroxidases
(HRP, MPO) used in studying catalyzed oxidation by H2O2 and
could not be included in the reaction medium. These studies utilized
the Good’s buffer, 3-(N-morpholino)propanesulfonic acid (MOPS), a
neutral tertiary amine that does not strongly coordinate Cu(II).
However, MOPS also protected CuATSM from reaction with
peroxynitrite, so that it was necessary to substitute phosphate ion as
the buffer component in reactions involving ONOOH and
EXPERIMENTAL SECTION
■
Materials. All reagents and solvents used in preparation and
characterization of synthetic compounds were purchased through TCI
America or Sigma-Aldrich and used directly without purification.
Bisthiosemicarbazone ligands used in these studies were prepared
following the general procedure outlined below:30
Typically, 2.0 equiv of 4-methyl-thiosemicarbazide was added to an
oven-dried 250 mL round-bottom flask containing 50 mL anhydrous
EtOH, and the mixture was heated to 65 °C while stirring until the
reactant completely dissolved. One equivalent of the appropriate
dicarbonyl compound was then added dropwise to the stirring
solution, followed by 5 drops of conc H2SO4. Within 5 min a
precipitate formed; following overnight stirring, the mixture was
filtered and sequentially washed with deionized water, MeOH and
EtOH. Product analyses included (1) 1H NMR and 13C NMR
spectra, recorded in d6-DMSO on a Bruker 700 MHz Avance III
spectrometer and a Bruker 400 MHz DPX-400 spectrometer,
respectively and (2) mass spectra acquired on a Thermo LTQ-FT
Ultra hybrid linear ion trap-Fourier transform ion cyclotron resonance
mass spectrometer with a Finnigan Ion Max API source configured for
ESI in positive ion mode. NMR and mass spectra for each compound
agreed with those previously reported in the literature.30 Specifically,
for diacetyl-bis(4-methylthiosemicarbazone) (H2ATSM), 1H NMR
(700 MHz, d6-DMSO): 10.215 (s, 2H), 8.372 (q, 2H, J = 5, J = 13),
3.023 (d, 6H, J = 5), 2.209 (s, 6H); 13C NMR (176 MHz, d6-
DMSO): 178.9587, 148.4411, 31.6750, 12.1330; MS: calcd for
CuC8H15N6S2 [(M+H)+] 322.01, found 322.08; for glyoxal-bis(4-
methylthiosemicarbazone) (H2GTSM), 1H NMR (700 MHz, d6-
DMSO): 11.7317 (s, 2H), 8.4736 (q, 2H, J = 5, J = 13), 7.7182 (s,
2H), 2.9576 (d, 6H, J = 5); 13C NMR (176 MHz, d6-DMSO):
178.0312, 140.5007, 31.3725; MS: calcd for CuC6H11N6S2 [(M+H)+]
293.90, found 294.00; for 1-phenyl-1,2-propanedione bis(4-methyl-3-
−
ONOOCO2 as oxidants.
Reactant concentrations of free and metalated bis-thiosemicarba-
zones were determined spectrophotometrically in buffered aqueous/
SDS micellar suspensions using independently measured molar
absorptivities from serially diluted samples in the same medium.
These standard solutions followed Beer’s law over the entire
concentration range encompassed in subsequent experiments. The
extinction coefficients (in mM−1 cm−1) at the peak maxima are as
follows: H2ATSM (ε326 = 45); CuATSM (ε309 = 21, ε465 = 5.6);
ZnATSM (ε413 = 8.56); H2GTSM (ε335 = ε346 = 41); CuGTSM (ε314
G
DOI: 10.1021/acs.inorgchem.8b00853
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
= 16.7, ε485 = 6.4); H2ϕMTSM (ε333 = 45); CuϕMTSM (ε311 = 22,
ε479 = 6.4); PAR (ε413 = 33); CuPAR (ε511 = 34). Reactant solutions
of ONOOH were prepared by diluting into alkaline (pH > 12) water
portions of frozen concentrated stocks (∼0.1 M) that had been made
by flow-mixing sodium nitrite with hydrogen peroxide;44 concen-
trations were determined spectrophotometrically using ε302 = 1.67
mM−1 cm−1.45 Reactant solutions of hypochlorous acid were prepared
by dilution of commercial bleach into H2O and determining their
concentrations spectrophotometrically in alkaline (pH > 12) solutions
using ε292(OCl−) = 350 M−1 cm−1.46 Reactant solutions of taurine
chloramine were prepared by dropwise addition of HOCl to a final
concentration of 10 mM into a rapidly stirred aqueous solution
containing 2-fold excess taurine; spectrophotometric analysis
confirmed the complete conversion of HOCl to the monochloramine
without formation of detectable amounts of taurine dichloramine.
Instrumental Methods. Cyclic voltammograms were measured
in DMSO with an EG&G Model 273 potentiostat/galvanostat using
an electrochemical cell in a standard 3-electrode configuration with
glassy carbon, Pt wire, and Ag/AgCl/KNO3 working, counter and
reference electrodes, respectively; 0.1 M tetrabutylammonium
tetrafluoroborate was the inert electrolyte and ferrocene (Fc) was
added internally as a reference standard to determine peak potentials.
The Fc+/o potential in DMSO was taken to be 0.68 V vs NHE.11
Sweep rates were typically 50 mV/s. Peroxidase-catalyzed oxidations
of MII(btsc) complexes were monitored spectroscopically by
following the time course of bleaching of their optical bands using
a Shimadzu UV-2041PC recording spectrophotometer interfaced to
Shimadzu UVProbe software. This instrument was also used to make
routine spectrophotometric analyses. Rapid reactions, including
ACKNOWLEDGMENTS
■
This work was supported by the Office of the Assistant
Secretary of Defense for Health Affairs through the Congres-
sionally Directed Medical Research Program on ALS
(AL140108, Award Number W81XWH-15-1-0289) and
funding from the Oregon Chapter of the ALS Association
(ALSA 16-320).
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ASSOCIATED CONTENT
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S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b00853.
Additional electrochemical, spectroscopic and kinetic
information involving CuII(btsc) oxidations, Cu-PAR
association dynamics, and ATSM inhibition of the HRP-
catalyzed ABTS-H2O2 reaction (PDF)
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: hurstja@onid.orst.edu.
ORCID
Hanss, J.; Beckmann, A.; Kruger, H.-J. Stabilization of Copper(III)
̈
James K. Hurst: 0000-0003-1070-2690
Present Address
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Structures, and Electronic Properties of Copper(II) and Copper(III)
Complexes. Eur. J. Inorg. Chem. 1999, 1999, 163−172. Fritsky, I. O.;
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Gumienna-Kontecka, E.; Meyer, F. Efficient Stabilization of Copper-
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with Adjustable Cavity Size. Chem. Commun. 2006, 4125−4127.
Pratesi, A.; Zanello, P.; Giorgi, G.; Messori, L.; Laschi, F.; Casini, A.;
⊥J.J.S.: Department of Chemistry, Matanuska-Susitna College
University of Alaska, Anchorage, Palmer, Alaska 99645, United
States.
Notes
The authors declare no competing financial interest.
H
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