Electron transfer mechanism of catalytic superoxide dismutation: Via Cu(II/i) complexes: Evidence of cupric-superoxo/-hydroperoxo species

Article abstract of DOI:10.1039/c6dt02220k

To understand the electron transfer mechanisms (outer versus inner sphere) of catalytic superoxide dismutation via a Cu(ii/i) redox couple such as occur in the enzyme copper-zinc superoxide dismutase, the Cu(ii/i) complexes [(L1)₂Cu](ClO_4<

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ARTICLE TYPE
Electron Transfer Mechanism of Catalytic Superoxide Dismutation via
Cu(II/I) Complexes: Evidence of Cupric-superoxo/-hydroperoxo Species
a)
b)
a)
a)
a)
Ram Chandra Maji , Partha Pratim Das , Saikat Mishra , Anirban Bhandari , Milan Maji , Apurba
K. Patra*
a)
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
To understand the electron transfer mechanisms (outer versus inner sphere) of catalytic superoxide
dismutation via a Cu(II/I) redox couple such as occurs in the enzyme copper-zinc superoxide dismutase,
the Cu(II/I) complexes [(L1)
2 4 2 3 3 2 4
Cu](ClO ) •CH CN, (1•CH CN) and [(L1) Cu](ClO ), (2) supported by a
1
1
2
0
5
0
bis-N thioether ligand, 2-pyridyl-N-(2’-methylthiophenyl)methyleneimine (L1) have been synthesized and
structurally characterised. Both 1 and 2 displays same cyclic voltammogram (CV) featuring a
quasireversible response at E1/2 = +0.33 V vs SCE that falls in the SOD potential window of -0.04 V to
2
S
+0.99 V. These complexes catalytically dismute superoxide radicals at 298 K in aqueous medium (IC50
for 1 is 2.15 µM). Electronic absorption spectra (233 K and 298 K), FTIR, ESI mass, CV (233 K and 298
.
-
+
2-
K) and DFT calculation collectively support formation of [(L1)
2
Cu(O
2 2 2
)] , [(L1) Cu(O )] and
-
+
[
(L1)
Once O
does not follow but the second step (Cu + O
2
Cu(OOH )] species, help to elucidate the electron transfer mechanism for SOD function of 1 and 2.
.-
2
II
II
.-
2
I
binds to Cu (evident at 233 K), the first step of the catalytic cycle (Cu + O
Cu + O
2
)
I
.-
2
+
II
+ 2H H O + Cu ) follows. Therefore, the catalytic
2 2
disproportionation of superoxide radicals via 1 and 2 at 298 K supports that first and second step of the
catalytic cycle proceeds through an outer and inner sphere electron transfer mechanisms respectively.
Feasibility of the first step to occur in pure aprotic solvent (then 18-Crown-6-ether is used to solubilise
2
KO ) was also tested that support too the same notion of electron transfer mechanisms as stated above.
Introduction
II/I
Employing Cu redox couple, copper proteins play vital roles in
2
5
biology such as transfer electron (Type-I Cu), activate molecular
oxygen (O
2
) for organic substrate oxidation, transport O
2
-
.
(
(
O
hemocyanin) and dismute toxic superoxide radicals (O
2
)
copper-zinc superoxide dismutase, Cu-ZnSOD) into H
O
2 2
and
1
2
.
The copper monoxygenases e.g. peptidylglycine α-
I
II
.-
2
3
0
hydroxylating monoxygenase (PHM) follows Cu -O
2
{Cu -O
II
2-
2
II
II
.-

Cu -O
}Cu -OOHCu -O pathway to activate O
2
for
substrate C-H hydroxylation, a key step towards biosynthesis of
2
physiologically active neurotransmitters and hormones, while to
.
-
combat the oxidative cellular damage of O
2
the enzyme Cu-
3,4
3
5
ZnSOD, a member of SOD family (contain Fe, Mn, Ni or Cu-
Zn), rapidly and catalytically disproportionates O
45
5
.-
2
Scheme 1. Chemdraw structures of the copper active site in (a) Cu-
ZnSOD: Cu and Zn site and (b) PHM: Cu and Cu site.
2
into O and
M
H
II/I
H O
2
2
via Cu redox couple as follows.
The active site of PHM and Cu-ZnSOD has been shown in
II
II
.-
I
II
Cu -Zn SOD + O
2
Cu -Zn SOD + O
2
(1)
II
5
0
Scheme 1. The X-ray structure of Cu-ZnSOD revealed a Cu in
4
0
II
I
II
.-
+
II
II
N
4
O
water and a Zn in N
3
OAspartate donor environment with a
Cu -Zn SOD + O
2
+ 2H  Cu -Zn SOD + H
2 2
O (2)
bridging imidazolate-Histidine between Cu and Zn (Scheme 1,
3,4
.-
II
I
.
2
-
+
2
a). In presence of O the Cu reduces to a three-coordinate Cu
2
O
+ 2H O
2
+ H
2 2
O
and the imidazolate-N bridge breaking and protonation occurs in
the first step. This reduced enzyme oxidizes back, reforming the
5
5
II
imidazolate-N bridge to Cu in the second step when another
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Page 2 of 12
.
-
II
I
.-
+
II
equivalent of O
2
and protons are supplied. Zn does not
of
bridging imidazole-N, attached to Cu , assists pH independent
however, the second step (Cu + O
2
2 2
+ 2H H O + Cu )
participate in the redox reaction but diminishing the pK
a
follows. These findings support OET and IET for the first and
second step of the catalytic cycle respectively. SOD activities of
pH = 5.5-10.5) high SOD activity of the enzyme. A positively 50 1 in pure aprotic solvent (here instead of CH OH, 18-Crown-6-
3
II
4
(
5
0
5
0
5
0
5
charged Arg-143 residue near the active site Cu ensures
ether is used to solubilise KO ) also support this notion of ET
mechanisms. We also report here the experimental and theoretical
2
.-
electrostatic guidance of O
high catalytic efficiency (rate constant, k=2x10 M s ). The
2
to Cu thus attributes to enzyme’s
9
-1 -1
6
.-
+
2-
evidences of {[(L1 Cu(O )]
2
2
⇌
[(L1) Cu(O )]}
2
2

-
+
structure-function aspect of Cu-ZnSOD has thoroughly been
[(L1)
2
Cu(OOH )] conversion that is in sharp contrast to the
reactivity of L2 bound Cu species with H O that leads to easy
thioether-S oxidation. The contrasting reactivity of L1 and L2
bound Cu species, in case of former O
later case H attacks to thioether-S, supports the choice of
neutral His-N instead of peptido-N coordination to Cu
.
-
II/I
-
II
studied, however, a little evidence of O
2
binding to Cu ion and
5
5
2 2
5
1
1
2
2
3
3
its fate towards dismutation is documented. In fact, the literature
survey reveals controversial reports on electron transfer (ET)
mechanisms, inner (IET) versus outer (OET) sphere. A recent
binding to both Cu and Cu , supports an
IET for both the steps, however, early pioneering works
16
-
II
.-
II
2
binds to Cu whereas in
2 2
O
7
.-
2
II
I
report proposed O
-
M
site of
6a,8
6
0
copper monoxygenases that leads only to substrate C-H
hydroxylation without Met-S oxidation.
support an IET and OET for the first and the second step of the
catalytic cycle respectively (eq. 1&2).
II
.-
9
The cupric-superoxo (Cu -O
(g) from a solution of such complexes
strongly support the feasibility of IET for the first step. However,
2
) complexes and generation of
9c,9i
Experimental
O
2
in aprotic solvent
II
.-
Materials and reagents.
at the same time it is also known that the Cu -O
highly nucleophilic, abstracts H to form further reduced (O
O ) and more stable cupric-hydroperoxo (Cu -OOH) species
2
when a proton source is available. Report on generation of
2
models are
.-
2
Pyridine-2-carboxaldehyde, 2-(methylthio)aniline, potassium
superoxide, nitro blue tetrazolium chloride (NBT), N-ethyl
2-
II
6
5
10
morpholine, anhydrous sodium sulphate, [Cu(H
Bu N](ClO ) and 18-crown-6-ether were purchased from Aldrich
Chemical Co. and used without further purification. CH CN,
CH OH, CHCl , (C O used either for spectroscopic studies or
2 6 4 2
O) ](ClO ) , [n-
.-
2 2 2
O (g) i.e. O O oxidation and concomitant reduction of
4
4
II
I
Cu Cu either from a cupric superoxo species in protic solvent
or from a cupric-hydroperoxo species is unknown. Therefore, if
the enzyme Cu-ZnSOD follows the first step (eq. 1) via IET in
H O, as proposed, but without forming Cu -OOH species then
2
that is unusual. The Imidazolate bridged, Cu-Zn and Cu-Cu
structural models and Cu-Zn, Cu-Cu, or Cu containing
functional models of Cu-ZnSOD are known, however, a set of
Cu complex that follow both the steps of the catalytic cycle (eq.
3
3
3
2 5 2
H )
7
0
for syntheses were purified and dried following standard
procedures prior to use. The ligand L1 was synthesized following
6-8
II
11
12
17
a reported procedure.
1
3
14
15
Syntheses
II/I
Syntheses of Ligand
15a
1
& 2) is unknown, except an example, though, that follow the
7
8
8
9
9
5
0
5
0
5
2-pyridyl-N-(2’-methylthiophenyl)-methyleneimine (L1): To a
solution of pyridine-2-carboxaldehyde (1.0 g, 9.33 mmol) in 15
mL of freshly distilled methanol was added a methanol solution
first but not the second step, lacks completing the catalytic cycle.
(
a)
(
15 mL) of 2-(methylthioaniline) (1.299 g, 9.33 mmol) and
O
N
N
refluxed under N for 6 h. Solvent removal afforded a thick
2
N
S
HN
S
yellow oil that was dissolved in 70 mL chloroform and washed
successively with distilled water, brine solution and finally with
distilled water. The organic layer separated was then dried over
H3C
H3C
L1
HL2
2 4
anhydrous Na SO for 1 h, filtered and solvent was removed
0
completely. The yellow oil kept at 4 C overnight to get a yellow
solid. The solid was then recrystallized from diethyl ether that
afforded yellow block/needle shaped crystals of the ligand, L1,
after a day (1.80 g, 7.8 mmol, yield = 84%). Selected IR
-1
1
frequencies (KBr disk, cm ): 1622(νC=N, vs), 695 (νC-S). H NMR
300 MHz, CDCl3):  8.70 (1H, d, pyridine proton adjacent to
(
N), 8.57 (1H, s, HC=N), 8.33 (1H, d, pyridine proton), 7.81 (1H,
t, pyridine proton), 7.37 (1H, t, pyridine proton), 7.28-7.16 (3H,
m, phenyl ring proton), 7.08 (1H, d, phenyl ring proton), 2.47
Scheme 2. Chemdraw depiction of (a) ligands and the (b) the SOD
catalytic cycle with 1 &2
(
3H, t, methyl group of S-Me).
4
0
Here in, we first report the spectroscopic evidences
demonstrating both the steps of the catalytic cycle (Scheme 2, b)
Syntheses of complexes
II/I
followed cleanly via
(L1) Cu](ClO •CH CN (1•CH
First time we report that once O
2
a
set
of Cu
complex,
) (2).
binds to Cu , the first step of
.
.
[
(L1)
2
Cu](ClO
4
)
2
CH
3
CN, 1 CH
3
CN. The ligand L1 (0.3 g, 1.32
OH that generted a light
yellow solution. To the resulting yellow solution a CH OH
00 solution (20 mL) of [Cu(H O) ](ClO (0.245 g, 0.657 mmol)
[
2
4
)
2
3
3
CN) and [(L1) Cu](ClO
2 4
mmol) was dissolved in 40 mL dry CH
3
.-
II
4
5
3
II
.-
I
the catalytic cycle (Cu + O
2
2
Cu + O ) does not follow,
1
2
6
4 2
)
2
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was added drop wise. The solution color was changed from
this cooled solution of 2 was then added a cooled CH
3
OH
yellow to dark green. Resulting reaction mixture was stirred for 60 solution (0.5 ml) of KO
2
(0.0057 g, 0.081 mmol), while a reddish
1
6 h at room temperature. The green solid precipitated out was
filtered, washed with ether and dried. The green solid was re-
dissolved in CH CN and kept for slow ether diffusion at 4°C.
violate color was generated immediately. It was stirred for 10
minutes and then 30 ml dry degassed pre-cooled ether was added.
The greenish red solid precipitated out was filtered and washed
with ether and dried. The FTIR spectrum of this product displays
5
0
5
0
5
0
5
0
5
0
5
3
After a day, dark green needle shaped crystals obtained were
filtered off and washed with ether and vacuum-dried (0.42 g,
0
-1
-1
-1
65
νO-H, νO-O and νCu-O at 3372 cm , 802 cm and 460 cm , similar
-
.552 mmol, yield = 84%). Elemental analysis calculated for
to the isolated [(L1)
2 4
Cu(OOH )](ClO ) obtained from Method A
.
C
28
H
27
N
5 8
O S
2
Cl
2 3
Cu (1 CH CN): C 44.24, H 3.58, N 9.21; Found:
albeit with less purity.
-
1
1
2
2
3
3
4
4
5
5
C 44.18, H 3.49, N 9.11; Selected IR frequencies (KBr disc, cm
1
)
: 3078 (w), 3036(w), 2928(w), 2853(w), 1598(CN,s), 1560(w),
Synthesis safety note:
1
1
7
CH
486(m), 1448(m), 1432(m), 1386(m), 1308(w), 1272(m),
239(m), 1199(w), 1167(w), 1094(vs), 982(w), 905(w), 833(w),
79(s), 766(s), 747(s), 695( CS, w),645(CS, w) 622(s); UV-Vis in
7
0
Transition metal perchlorates should be handled cautiously as
these are hazardous and explosive upon heating. No such
happening is encountered in the present study.
-
1
-1
3
CN, λmax, nm( in M cm ): 280(15520), 300(14495),
OH:
00(11410), 334(12850), 648 (120), 920 (sh, 35); in H O:
80(9620), 300(10530), 334(12250), 675(125); ESI mass: m/z =
3
3
2
6
34(17290), 430(2250), 635(215), 890 (105); in CH
3
Physical Measurements.
2
The FTIR spectra of the complexes were recorded on a Thermo
+
75 Nicolet iS10 spectrometer using KBr pellet in the range 4000 –
61.176 corresponds to [(L1)
](ClO ), 2. In a schlenk flask the ligand L1 (0.3 g, 1.32
mmol) was dissolved in dry and degassed CH OH (25 mL). To
the resulting yellow solution was then added solid
2 3 4
Cu(CH CN)(ClO )] .
-1
4
8
00 cm . The electronic spectra were recorded on an Agilent
453 diode array spectrophotometer. Elemental analyses were
[Cu(L1)
2
4
3
carried out on a Perkin-Elmer 2400 series-II CHN Analyzer.
Electron paramagnetic resonance (EPR) spectra were obtained
using Bruker- EMX-1444 EPR spectrometer, ESI Mass spectra
were recorded on Waters Q-Tof premier- HAB213 spectrometer
or using the Advion made compact mass spectrometer (serial
I
3 4 4
[Cu (CH CN) ]ClO (0.215 g, 0.657 mmol) one at a time under
8
8
9
9
0
5
0
5
argon gas flow. The solution color was immediately changed
from yellow to reddish brown which was then refluxed under
argon for 30 min. The resulting clean solution was then kept at
1
0
No.3013-0140). H NMR spectra were recorded on JEOL JNM
4
C. After a day, dark needle shaped crystals obtained were
LA 500 or on Bruker DPX-300. Redox potentials were measured
using CHI 1120A spectro-electrochemical analyzer. The cyclic
voltammograms (CV) were recorded in CH CN using same cell
3
filtered off and washed with ether and vacuum-dried (0.370 g,
.597 mmol, yield = 91%). Elemental analysis calculated for
ClCu (2): C 50.40, H 3.90, N 9.04; Found: C 50.34,
0
26 24 4 4 2
C H N O S
-1
set up. A three electrode cell set up such as platinum, saturated
calomel electrode (SCE) and a platinum wire as a working,
reference and auxiliary electrode respectively were.
IC50 determination. All solutions were prepared in ultrapure
water (Sartorius) that had an initial resistance of 18.2 MΩ. The
absorption spectra were obtained using 50 mM N-ethyl
morpholine (NEM) buffer of pH 7.4. The NBT/formazane assay
was done as follows. A solution of 0.030 mM NBT in 50 mM
NEM (pH 7.4) was prepared. To this a 0.010 mM water solution
H 3.84, N 8.97; Selected IR frequencies (KBr disc, cm ):
3
1
1
447(w), 3056 (w), 2978(w), 2917(w), 1588(C=N, s), 1559(w),
473(s), 1463(s), 1438(s), 1359(w), 1301(w), 1282(w), 1271(m),
231(w), 1199(m), 1154(w), 1090(vs), 1009(w), 971(w), 951(w),
909(w), 833(w), 771(s), 745(s), 695(C-S, m), 623(s); UV-Vis in
CH
5
-
1
-1
3
CN: λmax, nm( in M cm ): 300(sh, 19090), 365(6570),
+
10(640). ESI mass: m/z = 519.076 [(L1)
2
Cu] .
), Method A. The complex 1 (0.05 g,
.065 mmol) was dissolved in 10 mL of dry CH CN, while a
-
[
2 4
(L1) Cu(OOH )](ClO
0
3
of 1 was added. Then a DMSO solution of KO
2
was added to the
green solution was generated. Then the resulting solution was
cooled to -40 C. The pre-cooled (at -40 C) CH
mL) of KO (0.0046 g, 0.065mmol) was added to the solution of
compound 1, while a faint reddish violate color was generated.
The resulting reaction mixture was then stirred for 10 minutes
and to this was added a pre-cooled dry diethyl ether (30 mL) to
-1
o
o
above mentioned solution of 1 and formazan (560 ≈ 35,000 M
3
OH solution (0.5
-
1
cm ) formation was monitored at  = 560 nm using UV-Vis
2
spectroscopy. Blank experiments were run without adding
1
00 complex 1. We observed that in case of the blank experiment 50
equivalent of KO (per NBT) had to be added to the solution to
2
afford the complete conversion of NBT to formazane. To get the
same absorbance at  = 560 nm in presence of complex 1, 2000
precipitate out
a greenish red solid (the mother liquor
concentrated and then slow evaporation affords 1) which was
filtered off and washed with ether and vacuum-dried (15 mg).
Selected IR frequencies (KBr disc, cm ): 3383(νO-H, m), 3078
(w), 3036(w), 2962(w), 2924(w), 2853(w),1630(w), 1597(s),
1
1
9
6
equiv of KO
105 the solid KO
complex 1, kinetically the reduction of NBT to formazane in
presence of different concentration of were measured
2
needed (for more than 100 equiv of KO
2
addition,
-1
2
was used). For the calculation of % inhibition of
1
570(w), 1482(m), 1466(m), 1448(m), 1369(m), 1351(m),
308(w), 1298(m), 1287(m), 1262(m),1238(w), 1094(vs),
82(w), 905(w), 855(w), 802(νO-O, m), 779(s), 766(s), 747(s),
considering absorbance changes at  = 560 nm. From the %
inhibition versus –log[1] plot the IC50 value for 1 was
10 determined.
1
3
95(νCS, m), 623(s), 460(νCu-O, m); UV-Vis in CH CN: 345
Quantification of liberated H
determined by iodometry as follows. To a CH
was added an equivalent amount of KO (CH
then two equivalent of HClO was added. After completion of
15 the reaction progress (spectrum of complex 1 developed), another
2
O
2
: Amount of H
CN solution of 2
OH solution) and
2 2
O was
(21950), 425 (9480), 625 (sh, 125), 965 (41).
Method B. The complex 2 (0.05 g, 0.081 mmol) was dissolved
in 10 mL of dry CH CN under argon while a red solution was
3
2
3
3
o
4
generated. Then the resulting solution was cooled to -40 C. To
1
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Page 4 of 12
two equivalent of HClO
stable in acid medium. Then 1 mL of this final reaction mixture
4
was added as the liberated H
2
O
2
is 55 EPR signal with principle g values at 2.20, 2.08 and 2.05 (ESI †,
Fig. S2) due to the anisotropy arises out of the donor strength
variation along three axes (vide infra, see Table 1 the bond
distances of 1 along N1-Cu-N4 axis the dCu-N = 2.085 (3)Å, along
solution was diluted to 10 mL and was treated with excess NaI.
−
3
The amount of I
liberated was then quantified using Uv-Vis
−1
−1 18
5
0
5
0
5
spectroscopy (λmax = 361 nm, ε = 2.5 × 104 M cm ).
N2-Cu-N3 axis the dCu-N = 1.957(3) Å and the axial dCu-S =
I
Computational: DFT calculations were performed using the 60 2.4461(10) Å). Reaction of [Cu (CH
3
CN)
4 4
]ClO with L1 in 1:2
19
Gaussian 09 program. Geometry optimizations were performed
starting from the X-ray structural coordinates without symmetry
molar ratio under argon produces a dark red solid, 2 that exhibits
-1
-1

C=N of imine at 1588 cm and C-S at 695 cm , former is red
20
restrictions. B3LYP functional was employed together with
shifted but later is same to that of free L1, indicates coordination
2
1
22
23
1
1
2
2
LANL2DZ for Cu, 6-311+G(d) for N and S and 6-31G(d,p)
for C and H, as basis sets. Wave function stability calculations 65 in CH
of imine-N but not the thioether-S to Cu. The electronic spectrum
-1
-1
3
CN displays bands at λmax/nm (ε in M cm ) = 510 (sh,
were performed on all optimized calculations to confirm that they
corresponded to true ground states.
640), 365 (6570) and 300 (sh, 19090). The lowest energy band at
510 nm is due to the metal to ligand charge transfer transition
I
(
MLCT). The Cu complexes with -accepting N donor ligand
15a,28
Experimental setup for O
Potentials were measured using same cell setup as kept for
measurement of O (g) generation from the reaction of 1 with KO
in 6 mL CH CN. Pure O (g) (passes through P column) has
been taken using a gas tight syringe. After taking baseline under
Ar atmosphere, a measured amount of O (g) has been introduced
slowly into the CH CN solution of [n-Bu N](ClO ) inside CV
2
(g) standardization:
exhibits similar lower energy transition.
70
2
2
Table 1 Data collection and structure refinement parameters for
the complexes 1and 2.
3
2
2 5
O
Complex
formulae
mol wt
1.CH
3
CN
2
C
2
28
C H
27
N
8 2
O S Cl
2
Cu
26 24 4 4 2
H N O S ClCu
5
3
4
4
7
8
8
5
0
5
760.12
619.60
cell and the CV scans were started to collect until it reaches to a
maximum ipc at -1.05 V. For each measurement with different
amount of O (g) such as 0.1 mL, 0.2 mL, 0.4 mL and 0.6 mL the
2
final CV scan having maximum ipc was considered to get the
calibration curve (ESI †, Fig. S1). It can be assumed that the
cryst. System
Color
Monoclinic
green
Orthorhombic
Red
Space group
a (Å)
b (Å)
C2/c
43.210(2)
7.5967(3)
21.9571(11)
90.00
119.6330(10)
90.00
6264.8(5)
4
1.612
2.17-28.44
1.059
Pna21
15.749(2)
13.5061(18)
25.243(4)
90.00
90.00
90.00
5369.4(13)
4
1.533
1.99-28.37
1.109
2
O (g) injected to the CH
3
CN solvent is get dissolved and owing
c (Å)
to this reason a linear calibration curve (ESI †, Fig. S1) has been
o



( )
24a
obtained.
o
( )
o
( )
3
3
0
X-ray Diffraction Studies.
V (Å )
Z
Single crystal intensity measurements for 1 and 2 were collected
at 100(2) K with a Bruker Smart APEX II CCD area detector
using MoKα radiation (λ = 0.71073 Å) with a graphite
monochromator. The cell refinement, indexing and scaling of the
-3
d


calc(g cm )
range, deg
(mm )
-1
25
90 F(000)
3112
2544
3
4
4
5
0
5
data set were carried out using SAINT and Apex2 program. All
structures were solved by direct methods with SHELXS, and
refl./params.
unique refl.
7818/418
5217
11094/689
7923
2
26
refined by full-matrix least square based on F with SHELXL.
R
1
[I>2(I)]
2
0.0538
0.1208
1.067
0.0602
0.1402
0.902
1.382/-0.787
The positions of the C-bound H atoms were calculated assuming
ideal geometry using riding model. Figures showing displacement
wR [I>2]b
95 GOF
27
parameters were created using the ORTEP programme.
-
3
res. dens. eÅ
0.791/-0.787
Crystallographic data are summarized in Table 3. CCDC number
for the supplementary crystallographic data of 1 and 2 are
a
R = ∑||F
wR
o
|- |F
c o
||/∑|F |
b
2 2 2
2
2
1/2
2
= { ∑ [w(F
o
–F
c
) ] / ∑w [(F
o
) ]}
1
424380 and 1424381 respectively. These data can be obtained
free of charge from the Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
1
1
1
00 Structure of [(L1)
2
Cu](ClO
4
)
2
3 3
•CH CN, 1•CH CN. The X-ray
structure of 1 (Figure 1) revealed a bis-ligand mononuclear
copper in N S donor environment with a coordination geometry
intermediate between square-pyramidal and trigonal bipyramidal
trigonality index,  = 0.54 for 1;  = 0 and 1 for ideal square-
4
Results and Discussion
(
Synthesis and characterization
29
05 pyramidal and trigonal bipyramidal coordination geometry) like
N O donors provide to Cu-ZnSOD ( = 0.57). The average Cu-
3b
The ligand L1 has been synthesized following reported
4
1
7
N distance (avg. Cu-Npy and Cu-Nimine distances are 1.986(3) Å
and 2.055(3) Å respectively) of 2.020(3) Å in 1 is close to the
average Cu-NHis distance of the oxidized enzyme which is in the
10 range 2.04-2.10 Å. The Cu-S distances of 2.4462(10) Å and
procedure. Complex [(L1)
2
Cu](ClO
O) ](ClO )
6
4
)
2
•CH
with L1 in 1:2 molar
CN features
bands at λmax, nm (ε in M cm ) = 890 (105), 635 (215), 430 (sh,
250), 365 (11400), 334 (17290) and 300 (sh, 14495), typical to
3 3
CN, 1•CH CN was
5
0
synthesized reacting [Cu(H
2
4 2
ratio. Electronic absorption spectrum of 1 in CH
3
-1
-1
3.667(2) Å indicates that earlier S is coordinated to Cu but later is
2
II
not. The lability of coordinated thioether-S in 1 may serves the
Cu complex. The solid sample of 1 at 77 K displays a rhombic
4
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Dalton Transactions
purpose to binds other substrate, sacrificing its own ligation to
Complexes 1 and 2 display identical CV as shown in Figure
2. A quasireversible response at E1/2 = +0.33 V (ΔE = 100 mV)
and an irreversible response at Epc = -0.52 V are correspond to
II
Cu . In case of oxidized Cu-ZNSOD the labile H
substrate binding place by de-ligating itself. In enzymes the Cu-
distance ranges 2.26 -3.0 Å, indicate a weak bond and its
lability. To better compare the bond distances and angles crystal 35 than expected for a thermodynamically reversible system (≤60
2
O offers
p
II/I
I/0
O
w
p
Cu and Cu couple respectively. The ΔE of 100 mV, more
5
0
5
0
II
I
data of 1 and 2 have been collected at same temperature 100(2)
K.
mV), indicates structural changes upon Cu Cu conversion (or
vice versa) which is evident from their X-ray structures. Despite
of this structural change, the 1↔2 inter conversion is reversible
as no changes of peak potentials occur when repetitive CV scans
2 4
Structure of [(L1) Cu]ClO , 2. The X-ray structure revealed a
1
1
2
distorted tetrahedral coordination geometry around Cu with two 40 with various scan rates in the potential window (+)0.12 V -
pyridine- and two imine-N ligation of two L1 as shown in Figure
, bottom. No thioether-S ligation of L1 is observed. Two
molecules are present in the asymmetric unit. The average Cu-Npy
and Cu-Nimine distances of 2.061(5) and 2.065(5)
respectively are 0.076 Å and 0.009 Å longer than the 45 of its oxidized congener, 1. DFT studies revealed the same
(+)0.52 V (Figure 2, in set) were measured. To get energy
II/I
1
difference of 1 and 2, with and without thioether-S to Cu bond
respectively, the DFT calculations (Gaussian 09) were performed
-1
Å
Å
that revealed complex 2 is ~202.45 Kcal mol more stable than
II
corresponding distances of Cu complex, 1. For reduced Cu-
coordination zone of 1 and 2 and comparable bond distances and
angles (ESI †, Fig. S3-S4 and Table S1-S2) to those obtained
from their X-ray structure determination (Table 2).
ZnSOD an average Cu-NHis distance of ~2.21 Å is ~0.1 Å longer
than that of its oxidized form. The long Cu-S distances of
3
3
.173(2) Å and 3.221(2) Å indicate no coordination of thioether-
0
S to Cu. Bond distances and angles of the copper coordination 50 Table 2. Selected Bond Distances (Å) and angles ( ) of 1 and 2
zone of 1 and 2 are presented in Table 2.
2 4 2 3 3
[(L1) Cu](ClO ) •CH CN, (1•CH CN)
Cu(01)-N(1)
2.017(3)
1.956(3)
Cu(01)-N(2)
Cu(01)-N(4)
Cu(01)-S(2)
1.958(3)
2.153(3)
3.667(2)
80.29(8)
96.30(8)
100.72(11)
174.82(12)
80.39(11)
Cu(01)-N(3)
Cu(01)-S(1)
2.4461(10)
142.37(8)
95.14(8)
S(1)-Cu(01)-N(1)
S(1)-Cu(01)-N(3)
S(1)-Cu(01)-N(2)
S(1)-Cu(01)-N(4)
N(1)-Cu(01)-N(3)
N(2)-Cu(01)-N(3)
N(3)-Cu(01)-N(4)
N(1)-Cu(01)-N(2)
N(1)-Cu(01)-N(4)
N(2)-Cu(01)-N(4)
81.73(11)
119.79(11)
102.43(11)
[(L1)2Cu](ClO4), (2)
Molecule 1
Cu(1)-N(1)
2.093(6)
2.029(5)
80.7(2)
Cu(1)-N(2)
2.019(5)
2.112(4)
106.3(2)
138.7(2)
80.86(18)
Cu(1)-N(3)
Cu(1)-N(4)
N(1)-Cu(1)-N(2)
N(1)-Cu(1)-N(4)
N(2)-Cu(1)-N(4)
N(1)-Cu(1)-N(3)
N(2)-Cu(1)-N(3)
N(3)-Cu(1)-N(4)
112.8(2)
135.02(19)
Molecule 2
Cu(2)-N(5)
2.115(6)
2.056(5)
79.3(2)
Cu(2)-N(6)
2.045(6)
2.072(5)
105.0(2)
143.4(2)
79.6(2)
Cu(2)-N(7)
Cu(2)-N(8)
N(5)-Cu(2)-N(6)
N(5)-Cu(2)-N(8)
N(6)-Cu(2)-N(8)
N(5)-Cu(2)-N(7)
N(6)-Cu(2)-N(7)
N(7)-Cu(2)-N(8)
106.0(2)
135.1(2)
2
5
Fig. 1: ORTEP plots of cationic part of [(L1)
•CH CN (top) and one of the two molecules present in the asymmetric
unit of [(L1) Cu](ClO ), 2 (bottom) with atom labelling scheme, thermal
2 4 2 3
Cu](ClO ) •CH CN,
SOD activity of 1
The easy inter-convertibility of 1↔2 at E1/2 = +0.33 V which
is within the reported potential window (E1/2 = -0.04 V to +0.99
1
3
2
4
24
ellipsoids are at 50% probability level, H atoms and anions are omitted 55 V versus SCE) of superoxide dismutation, tempted us to
for clarity.
investigate their SOD activity according to the following two eq.
and 4, similar to eq. 1 and 2 of the enzyme catalytic cycle.
3
3
0
Redox properties
.
-
1
+ O
2
 2 + O
2
(g)
(3)
(4)
+
.-
6
0
2 + 2H + O
2
 1 + H O
2 2
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Dalton Transactions
Page 6 of 12
4
4
5
enzyme and shown as steps b & c in scheme 3 for the present
case. This notion of formation of 45% 1 via intermediate 2 is
confirmed by one time addition of KO2 to 1 that shows
stoichiometric formation of 2, monitored by UV-Vis
The reactions as stated above followed accordingly and cleanly in
CH OH, CH CN and H O, monitored by UV-Vis spectroscopy.
The catalytic SOD mimic activity of 1 was examined in N-ethyl
3
3
2
5
morpholine (NEM) buffer of pH = 7.4 at 298 K using NBT dye
spectroscopy. Similarly to quantify O
all at once was added instead of
adding slowly and the CV was measured that displayed a new
2
(eq. 3 or step ‘a’ in
.-
and a saturated DMSO solution of KO
S5-S7). NBT reacts with KO to form violet formazane (λmax
60 nm), thus 1 will inhibit formazane formation in presence of
2
as O
2
source (ESI †, Fig. 50 Scheme 3), an equivalent KO
2
2
=
5
.-
2
O , if it shows SOD activity. The IC50 value, the concentration
1
0
of the tested sample that inhibits formazane formation to 50%,
determined for 1 is 2.15 µM. This value indicates that 1 is less
effective than the native enzyme (IC50 = 0.04 µM for bovine
erythrocyte Cu-ZnSOD) but fairly comparable to other reported
2
0
5
6
6
7
5
0
5
0
1.00
3
34
0.75
0.50
0.25
15
10
5
14-15
efficient SOD models.
510
635
-250
-200
-150
-100
300
j
0.00
-
0.14V
4
00
600
800
1000
4
30
Wavelength(nm)
a
3
65
a
0
3
0
j
-50
+0.38V
0.2
0.3
0.4
0.5
E(V) vs SCE
0.6
0
2
80
34
50
20
10
0
3
0.4
+
0.28V
100
-
0.52V
5
55 635
0.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
890
E(V)vs SCE
9
75
1
2
5
0
0.0
30 400
600
800
1000
Fig. 2: Cyclic voltammograms of
(nBu) N]ClO as a supporting electrolyte at 298K at a Pt working
1
in CH
3
CN containing 0.1M
75
4
3
52
wavelength(nm)
[
4
4
-
1
electrode at 50 mV s scan rate using SCE as reference electrode. Inset:
CVs of 2 at various scan rates (aj = 50, 100, 200, 300, 400, 500, 600,
4
00
600 800
Wavelength(nm)
1000
-
1
700, 800, 900 mV s ).
Fig. 3, Top: UV-Vis absorption traces showing conversion of a CH
3
CN
 2 (gray traces) at 298 K,
is dissolved in 200 µl CH OH
then 20 µl aliquot is added each time and spectrum taken. Bottom: UV-
-
4
8
0
solution of 1 (1 x 10 M, black trace) + KO
2
To check SOD activity of 1 whether follows eq. 3 and 4, a
-3
inset: [1] = 1 x 10 M. One equivalent KO
2
3
3
CH CN solution of it was titrated against a CH
3
OH solution (200
µl) of KO
2
and the formation of 2 was monitored using UV-Vis
Vis absorption traces showing conversion of a CH
3
CN solution of 1 (1 x
2
3
3
4
5
0
5
0
spectroscopy (Figure 3, top). The absorption bands of the final
spectrum (gray trace) and isosbestic points at λ = 504 nm and 275
nm indicate a clean transformation of 12, however, less
extinction coefficient at  = 365 nm compared to authentic 2
indicates ~55% formation of 2. To know, whether a different
species rather than sole 2 is formed, the CV of 1 was measured at
-
4
.-
+
1
0
M, black trace) + KO
2
(CH
3
OH solution)  [(L1)
2
Cu(O
is dissolved in
3
OH then 20 µl aliquot is added each time and spectrum taken.
2
)] (gray
-
3
8
5
trace) at 233 K, inset: [1] = 1 x 10 M. One equiv. KO
00 µl CH
2
1
2
98 K following addition of KO
equivalent). After each addition of KO
shows complete retention of ΔE and the current heights (ipc and
pa) of E1/2 = +0.33 V response (ESI †, Fig. S8). This experiment
firmly support that the reaction mixture under investigation of
KO treatment contains 1 and 2 only, as both of these display the
same CV, thereby, account same ΔE and current heights
2
solution in portions (5 times 0.2
2
the CV traces taken
p
i
2
p
irrespective to their proportion in the reaction mixture. This result
strongly support that remaining 45% species is then 1 that
reformed during this slow addition of KO
reasonable that progressive addition of KO
increases formation of 2 that in turn competes with 1 to react with
2
solution. It is quite
to a solution of 1
.-
2
9
0
Scheme 3: Reaction scheme for O
(LT) and at 298 K (RT), S = µl amount CH
8-C-6-E = 18 crown-6-ether. All reactions have been performed in
CH CN.
interaction with 1 and 2 at 233 K
2
3
OH used to solubilize KO
2
,
2-
1
KO
2 2 2
to form back 1 via a cupric-peroxo, ([(L1) Cu(O )])
3
intermediate, following a similar mechanism as reported for
6
| Journal Name, [year], [vol], 00–00
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Dalton Transactions
.
- +
response at Epc = -0.93 V (Figure 4, top) corresponds to the
reduction potential of O (g). Based on [1], 97% O (g) formation
is observed (ESI †). The CV taken after removing the in-situ
formed O (g) by purging N matches exactly to that of 1 (Figure
4, top, dashed and solid lines). This experiment strongly support 60 [(L1)
an OET for step ‘a’ that proceeds much faster than IET steps b&c
scheme 3) and thereby, generated stoichiometric O (g) at 298 K.
2 2
680 mV unlike the in-situ generated [(L1) Cu(O )] species that
2
2
at 233 K shows a quasireversible response at E1/2 = + 0.27 V (Epa
= +0.315 V, Epc = +0.225 V, ΔE = 90 mV and i /i ≈1, see
Figure 5, top). Similarly the electronic spectrum of isolated
Cu(OOH )] in CH CN at 233 K features bands at max/nm
2 3
( in M cm ) = 345 (21950), 425 (9480), 625 (sh, 125) and 965
(41) (vide infra Fig. S7) dissimilar to that of the in-situ generated
24a
p
pa pc
2
2
-
+
5
0
5
0
-1
-1
(
2
.- +
[(L1)
2
Cu(O
2
)] that displays these bands at 280 (22100), 362
As the room temperature UV-Vis or electrochemical
measurement does not designate the formation of transient
cupric-superoxo species, the possibility of IET for the first step
(
6415), 555 (200) and 975 (45) (Fig. 3, Bottom).
6
5
1
1
2
(
eq. 3) could not be ruled out with certainty. To confirm this
belief of OET, generation of cupric-superoxo species
-
-
80
60
.- +
[(L1)
2 2 2
Cu(O )] and then to check O evolution electrochemically
-0.16
from this species is unequivocally necessary. If from
[(L1) Cu(O )] species O (g) generation occurs then it will be
2 2 2
conclude that first step can proceeds through IET, otherwise via
OET mechanism.
.-
+
70
-40
-0.93
-
20
0
.-
+
-
+
+0.38
2 2 2
Evidences of [(L1) Cu(O )] and [(L1) Cu(OOH )] species
Vs ET mechanisms
+0.28
-0.52
2
0
0
Keeping in mind that (i) thioether-S decoordination may offer
.-
30
75
a space for O
2
binding as reported and (ii) the thermal
4
instability of cupric-superoxo/-hydroperoxo intermediates, the
interaction of O with 1 was examined by low temperature UV-
2
Vis spectroscopic and electrochemical studies in aprotic and/or
protic solvent as described below.
-
-
-
200
150
100
.-
-0.13
2
5
8
0
In aprotic and protic mixed solvent
-
50
0
Stoichiometric KO
aliquots were added to the CH
each addition, the electronic spectra recorded were shown in
Figure 3, bottom. The d-d band of 1 at  = 890 nm and 635 nm
shifted to 975 nm and 555 nm, band at 430 nm disappeared and
the 334 nm band is red shifted to 362 nm. The final spectrum
2
was dissolved in 100 µl CH
3
OH then 20 µl
+
0.36
+
0.315
3
CN solution of 1 at 233 K. After
3
3
4
4
5
5
0
5
0
5
0
5
+
0.225
+
0.27
-
0.69
50
-0.52
-0.5
85
-
1.0
0.0
0.5
(
gray trace) appeared does not match to either 1 or 2, strongly
.
-
+
E(V) vs SCE
Fig. 4, Top: Electrochemical experiment showing generation of O
Cyclic voltammograms in CH CN containing 0.1 M [(n-Bu) N]ClO as a
electronic configuration). The isosbestic points at λ = 581 nm, 90 supporting electrolyte at 298 K at a platinum working electrode at a scan
2 2
support formation of a new species, possibly [(L1) Cu(O )] or
-
+
[(L1)
2
Cu(OOH )] . Appearance of d-d transitions at  = 975 nm
2
.
II
6
3
g
and 564 nm firmly established it a new species of Cu (t2g
e
3
4
4
-
1
rate of 100 mV s using SCE as reference electrode of 1 in CH
(Black trace), then after adding KO (100 µl CH OH solution) repeated
scans until reach to a maximum ipc (gray trace) at -0.93 V (reduction
potential of O ) then after purging of N (g) to complete remove of O (g)
mass spectrometry, possessed peaks at m/z (%) = 277.31 (70), 95 produced (dash trace). Calculated amount of O (g) evolution is 97%.
at 233 K (gray trace) and
3
CN
4
89 nm and 274 nm state that this conversion of 1 to the transient
species is clean. This observation is in sharp contrast to that of
98 K experiments where 1 is converted straight to 2 (Figure 3,
top). Attempt to identify the species generated at 233 K, using
2
3
2
2
2
2
2
2
91.0037(100), 308.0098(20) and 322.02(35) corresponds to
Bottom: of 1 at 233 K (black trace), of 1 + KO
2
+
2+
+
2+
[(L1)
2 2
Cu(O )+1H ] , [(L1)Cu] , [(L1)Cu(O
2
)Cu(L1)]
and
then the solution temperature raised to 298 K (dash trace), after N
purging (same dash trace).
2
+
[
{(L1)Cu(O )-1H}] respectively (ESI †, Fig. S9) as the isotope
2
pattern matches to that of the calculated one with these
formulations. Attempt to force precipitate this in situ generated 100
We believe that at 233 K the species generated in solution is
.-
+
.- +
species [(L1)
2
Cu(O
2
)] at 233 K, expending diethyl ether,
[(L1)
[(L1)
2
Cu(O
2
)] , but precipitation results the more stable
-
+
allowed us to isolate a reddish-green solid that displayed distinct
2
Cu(OOH )] species in presence of CH
3
OH (100 L used to
-
1
solubilize KO
stability of [(L1)
2
). DFT calculation supports this notion of more
ν
8
O-H, νO-O and νCu-O stretching in its FTIR spectrum at 3383 cm ,
-1
-1
-
+
.- +
02 cm and 460 cm respectively (ESI †, Fig. S10), very close
to those of the structurally characterized and other reported 105 ~408 Kcal Mol more ground state energy than the former.
2
Cu(OOH )] than [(L1)
2 2
Cu(O )] , the later has
31a
-1
II
15b,31
Cu -OOH species.
The CV of this isolated solid displays an
Interestingly, the electrochemical experiments revealed that none
.-
+
-
+
irreversible response with Epc=-0.46 V, Epa = +0.22 V and E
p
=
of these transient species, [(L1)
2 2 2
Cu(O )] and [(L1) Cu(OOH )]
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7

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Dalton Transactions
Page 8 of 12
.
-
+
- +
can generate O
temperature to 298 K (Fig. 4, bottom; no response at -0.93 V
corresponds to O reduction potential indicates no generation of
(g)), thereby discard the possibility of IET for the first step. 60 performed the following CV experiments and UV- Vis study in
Thus the stoichiometric O (g) evolution at 298 K (Figure 4, top) aprotic solvent (CH CN) to check whether O (g) evolution and
even in presence of same amount of CH OH reveals a more likely formation of 2 or transient species occur. Complex 1 plus
equivalent KO and 18C6E in CH CN displays same CV to that
2
(g) either at 233 K or after increasing the solution
To stop, [(L1)
occurs in presence of CH
crown-6-ether (18C6E) instead of CH
2
Cu(O
2
)] [(L1)
OH as evident, we have used 18-
OH to solubilize KO and
2
Cu(OOH )] conversion that
3
2
3
2
O
2
5
0
5
0
5
0
5
0
5
0
5
2
3
2
3
OET mechanism for the first step (eq. 3). Also the catalytic SOD
function of 1 (IC50 = 2.15 µM) in aqueous medium in NEM
buffer strongly support the OET mechanism for the first step
2
3
6
5
1
1
2
2
3
3
4
4
5
5
reaction, as plenty of N-ethyl morpholine and H
2
O molecules
.-
present in the reaction medium will compete with O
2
and will
.-
2
II
rendered binding of weak O
to Cu of 1.
7
0
-10
+
0.315
II
.-
+
[
(L1) Cu -O
]
2
2
-
5
0
5
0
75
8
0
1
II
2-
[
(L1) Cu -O
]
2
2
8
5
-30
-20
-10
0
II
.-
+
[
(L1) Cu -O
]
2
2
90
1
0
0
9
5
II
2-
[
(L1) Cu -O
]
Fig. 6: DFT optimized structure of end-on singlet cupric-superoxo
2
2
.-
+
species, [(L1)
2
Cu(O
2
)] (top) and end-on cupric-hydroperoxo species,
2
+
0.225
-
+
[
(L1) Cu(OOH )] (bottom).
2
0.0
0.1
0.2
0.3
0.4
0.5
0.6
1
1
1
1
00 of a CH
3
CN solution of authentic 2 plus equivalent 18C6E plus
(g), introduced externally using a gas tight syringe (ESI †,
Fig. S11). This observation supports the conversion, 1 + KO +
E(V) vs SCE
pure O
2
.-
+
2-
2
Fig. 5: Cyclic Voltammograms showing [(L1)
2
Cu(O
2
)] ⇌ [(L1)
2
Cu(O
2
1
8C6E2 + O
here is at -1.07 V (ESI †, Fig. S11). The -1.07 V response is for
(g) reduction, confirmed from the CV of pure O (g) plus
18C6E in CH CN (ESI †, Fig. S11) that displays the same
2 2
(g) + 18C6E. The O reduction potential observed
)
] reversibile conversion. Top: Cyclic voltammograms of 1 in CH
containing 0.1M [(nBu) N]ClO as a supporting electrolyte at 298K (dash
line), at 233 K (gray line) and of 1 + KO at 233 K (solid line) at Pt
3
CN
4
4
05
O
2
2
2
-
1
working electrode at 50 mV s scan rate using SCE as reference
electrode. Bottom: of 2 at 298 K (dash line), at 233 K (gray line) and of 2
3
potential. After complete removal of O
18C6E2 + O (g) + 18C6E reaction) by purging N
taken (ESI †, Fig. S11) is same to that of the authentic 2. The
10 straight formation of 2 from the above reaction is further
supported from the UV-Vis traces taken at 233 K when a CH CN
solution of 1 is titrated with a CH CN solution of equivalent
8C6E and KO (ESI †, Fig. S12). These observations are in
sharp contrast to those where instead of 18C6E, the protic solvent
OH was used. In case when CH OH was used formation of
2
(g) formed (from 1 + KO
2
+
2
2
the CV
+
2
KO at 233 K (solid line) with same cell set up. The black, gray and
dash traces of both the top and bottom Figures has same peak potential of
which black traces has been shown by vertical lines.
3
3
1
2
In pure aprotic solvent
15 CH
3
3
8
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

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DOI: 10.1039/C6DT02220K
Page 9 of 12
Dalton Transactions
.
- +
[
4
1
(L1)
2
Cu(O
2
)] (Fig. 3, bottom) and no generation of O
2
(g) (Fig.
Mononuclear transient species and their importance
Comparable electronic absorption coefficient of the d-d band
, bottom) were observed. Therefore, in pure aprotic solvent, the
2 + O
OET mechanism where the O
Noteworthy here that the binding of O (g) to Cu forming Cu -
2
species at -85 C in aprotic solvent and decomposition back
of the Cu -O
that can be viewed as, at high temperature no bond between O
and Cu exists, the O
its own oxidation to O
60
.-
2
.- +
+ O
2
(g) conversion occurs more likely following an
of [(L1)
stretching frequencies of [(L1)
CV current heights of [(L1)
(Figure 5, top and bottom) to that of 1 support the mononuclear
2 2
Cu(O )] (Figure 3 bottom), Intensity of FTIR
.-
- +
2
acts as just a reductant.
2
Cu(OOH )] (ESI †, Fig. S10) and
I
II
.-
+
2-
5
0
5
0
5
0
5
0
5
0
5
2
2 2 2 2
Cu(O )] and [(L1) Cu(O )]
.-
0
O
II
.-
2
I
9c
to Cu + O
2
(g) at room temperature is known
65 composition of these transient species. X-ray structure of end-on
superoxo or hydroperoxo bound copper complexes are rare,
.-
2
II
.-
II
I
2
directly reduces the Cu Cu sacrificing
(g) therefore supports OET mechanism.
though, the structure of at least one of each of these type
+
1
1
2
2
3
3
4
4
5
5
2
complexes is reported such as [(TMG
3
tren)Cu(O
TMG tren
2
)] and
is
II
-
[
Cu (bppa)(OOH )](ClO
4
)
where
3
.-
Electrochemical and theoretical evidences of [(L1)
2
Cu(O
2
70 tris(tetramethylguanidino)tren and bppa = bis(6-pivalamide-2-
pyridylmethyl)-2-pyridylmethyl amine, both are tripodal N
+
II
2- 0
)
] ⇌[(L1)
2
Cu -O
2
] conversion
4
donor ligand. However, no set of cupric-superoxo and –
.
- +
The transient [(L1)
2
Cu(O
2
)] species is expected to
hydroperoxo complexes with same ligand donors is reported. So,
II
decompose upon reduction, however, displaying a quasireversible
considering similar coordination environment surrounding Cu ,
.-
+
- +
2 2 2
CV at E1/2 = +0.27 V (Epa = +0.315 V, Epc = +0.225 V) with a 75 DFT calculation on [(L1) Cu(O )] and [(L1) Cu(OOH )] model
ΔE
p
of 90 mV (less than that of 1) indicates less structural
encourages
structures were performed. The energy optimized structures have
been shown in Figure 6. In both cases the Cu is in N O donor
changes (than 1) and no decomposition. This low ΔE
p
4
us to investigate the origin of this E1/2=+0.27 V response whether
metal or ligand centred. DFT calculation revealed that HOMO
and LUMO of [(L1)
metal orbital contribution (%) as 88.2, 8.2, 3.6 and 56.7, 17.3,
6.0 respectively (ESI †, Fig. S13) strongly supports that E1/2
+0.27 V response is of O centred in origin, corresponds to
environment like the structurally characterised complexes of their
kind. The Cu-O1, Cu-O2, O1-O2 distances and Cu-O1-O2 angle
.-
2
+
0
2
Cu(O
)] model has oxygen, ligand and 80 of 2.070 Å, 2.909 Å, 1.261 Å and 119.70 respectively of
.- +
[(L1)
2 2
Cu(O )] (ESI †, Table S3) are comparable to the
2
=
corresponding values of 1.927(2) Å, 2.842(7) Å, 1.280(3) Å and
0
+ 9c
2
123.53(2) respectively, reported for [(TMG
3 2
tren)Cu(O )] .
.-
2
+
II
2- 0
[
(L1)
shown in Figure 5, top. This O
activity is further supported from the following electrochemical
2
Cu(O
)] ⇌ [(L1)
2
Cu -O
2
]
reversible conversion as
Similarly, the Cu-O1, O1-O2 distances of 1.966 Å and 1.425 Å
0
0
2
centered reversible redox 85 and the Cu-O1-O2, O1-O2-H angles of 110.57 and 103.03 of
-
+
[(L1)
2
Cu(OOH )] (ESI †, table S4) are fairly comparable to the
0
experiment of 2 + KO
2
at 233 K (Figure 5, bottom). When KO
will get reduced to O
oxidation of Cu of 2 to Cu and more likely form [(L1)
]. If this species form then the CV scan towards anodic will
results the one electron oxidized species, [(L1) Cu(O )] .
2 2
Therefore, the same CV traces are expected from the one electron
2
is
corresponding values of 1.888(4) Å, 1.460(6) Å, 114.5 and
respectively evident from X-ray structure of
4
[Cu (bppa)(OOH )](ClO ).
.-
2-
0
added to 2, the O
2
2
with concomitant
101.8
I
II
2-
2
II
-
32
2
Cu(O
)
90
.- +
3
0
0.5
0.4
.
-
+
.
-
+
2-
____[(L1) Cu(O ) ] exp
reduction of [(L1)
2
Cu(O
2
)] [(L1)
)][(L1)
2
Cu(O
2
)] and one electron
)] , that exactly
2
2
.-
+
2-
2
.-
+
345
355
........[(L1) Cu(O ) ] Theo
2
2
oxidation of [(L1)
2
Cu(O
2
Cu(O
2
25
20
-
+
_
___[(L1) Cu(OOH )] exp
2
observed as shown in Figure 5 (top and bottom). Lower
solubility of the neutral species, [(L1) Cu(O )], in polar CH CN
2 2 3
results partial precipitation resulting a lower ipa and ipc (Figure 5,
bottom, black trace) than the CV of authentic 2 (Figure 5, bottom,
gray trace), unlike as shown in Figure 5, top for the case of
)] . Therefore, essentially this redox couple at E1/2
0.27 V (Epa = +0.315 V, Epc = +0.225 V, ΔE
)] ⇌ [(L1)
transformation (Scheme 3). The O
0
.3
.2
.1
0.0
555
-
+
2-
........[(L1) Cu(OOH )] Theo
2
0
15
975
65
465
23
0
625
4
9
.-
2
+
10
5
[
+
(L1)
2
Cu(O
=
4
25 500 600 700 800 900 1000 1100
Wavelength(nm)
p
= 90 mV)
Cu(O )] reversible
2 2
362
555
2-
2
.- +
835
corresponds to [(L1)
2
Cu(O
620
2
orbital contribution of ~88%
900
0
.-
+
.-
in HOMO of [(L1)
2
Cu(O
2
)] reveals that O
concomitant Cu Cu reduction is highly possible, provided the
2 2
O oxidation and
4
00
600
800
1000
II
I
Wavelength(nm)
.
- +
species, [(L1)
2
Cu(O
2
)]
is excited with an external energy
9i
9c
source such as laser irradiation or increasing temperature as
reported but that would be before transforming to more stable
Fig. 7: Experimental UV-Vis absorption spectrum of in-situ generated
II
+
.-
+
cupric-superoxo species, [(L1)
2
Cu(O
2
)] (black solid line), isolated
2
cupric-hydroperoxo complex, [(L1) Cu -OOH] . Once the cupric
.-
2
+
-
+
9
5
[(L1)
singlet form of [(L1)
calculated spectrum of [(L1)
2
Cu(OOH )] (gray solid line), theoretically calculated end-on
superoxo species, [(L1)
hydroperoxo, [(L1) Cu(OOH )] , the O
possible because [(L1)
2
Cu(O
)] transforms to cupric-
(g) generation is not
Cu(OOH )] require H atom or proton
-
+
.-
+
2
2
Cu(O )] (black dash line) and theoretically
2
2
-
+
-
+
2
Cu(OOH )] species (gray dash line).
2
+
.-
2-
2 2
(H ) abstraction and then re-oxidation of the reduced O or O
moiety respectively which is thermodynamically unfavorable
until or unless any external oxidizing agent is used.
.
- +
Noteworthy, the model structure of both [(L1)
2
Cu(O
2
)] and
-
+
1
2
00 [(L1) Cu(OOH )] revealed de-coordination of thioether-S donor
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DOI: 10.1039/C6DT02220K
Dalton Transactions
Page 10 of 12
30
atom like other reported complexes. The electronic spectrum of 40 1 (ESI †, Fig. S14) that strongly supports 21 conversion. The
.
-
+
- +
in-situ generated [(L1)
matches well to that of their TDDFT calculated spectrum too as
shown in Figure 7. In relation to the PHM’s Cu site modelling,
x 2 x
(L) Cu -O mononuclear (L) Cu -OOH conversion is
2
Cu(O
2
)] and isolated [(L1)
2
Cu(OOH )]
liberated H
stoichiometric formation of H
the SOD activity of 2 follows eq. 4 at 298 K. To know whether
this step can proceeds via IET, we did the same experiment but at
233 K. To a solution of 2 in CH
was added once at a time, then the CH OH solution of HClO
was added portion wise and electronic spectra were recorded as
shown in Fig. 8, bottom. Clean conversion of 2 to 1 (Figure 8,
2 2
O was quantified spectrophotometrically that shows
18
2 2
O (ESI †, Fig. S15), therefore,
M
II
.-
II
5
4
5
5
5
0
5
3
CN at 233 K, stoichiometric KO
2
unknown, rather formation of end-on peroxo or bis-µ-oxo bridged
9
3
4
dicopper species occurs.
.- +
bottom) even at 233 K (evident of [(L1)
achieved after acid addition (isosbestic points developed at 302,
05 and 586 nm after started acid addition), strongly supports that
IET mechanism is more likely for this step. Blue shifting of the
65 nm band of 2 to 334 nm is indicative of thioether-S
2 2
Cu(O )] formation),
3
00
1.8
4
10.0
1
1
2
0
3
34
3
1.2
coordination as is evident in 1. The CV trace at 233 K (Figure 5,
bottom) and DFT calculation (HOMO and LUMO of
7.5
5.0
2.5
0.0
.-
2
+
2-
2
0
.6
[(L1)
].The isolated solid from a reaction of 2 plus KO
displays similar IR peaks to that of [(L1)
isolated from plus KO
60 intermediary of [(L1)
finally converted to [(L1)
(Scheme 3). This support {[(L1)
2
Cu(O
)] , vide supra) support formation of [(L1)
2
Cu(O
365
)
2
-
at 233 K
Cu(OOH )] species
5
10
890
+
2
6
35
0.0
1
2
reaction, support further the
430 400
600
800
1000
.-
2
+
2-
Wavelength(nm)
2
Cu(O
)] and [(L1)
Cu(OOH )] in presence of CH
2
Cu(O
2
)] species that
OH
-
+
2
3
.-
+
2-
2
Cu(O
2
)] ⇌ [(L1)
2
Cu(O
2
-
+
5
)]}[(L1)
experiment also support [(L1)
2
Cu(OOH )] conversion. The ESI mass spectral
I
+
.- +
1.0
0.8
0.6
0.4
0.2
0.0
Cu -O
] {[(L1)
Cu(O
2
)] ⇌
2
0
5
0
5
0
2
2
2
2-
- +
6
5
[(L1) Cu(O )]}  [(L1) Cu(OOH )] conversion (ESI †, Fig.
2 2 2
S16). Same bunches of peaks generated, evident from the ESI
2
80
5
10
I
II
.-
2
334
mass spectra of 2 + O
(g) (corresponds to Cu -O
or Cu -O
), 1
1
2
2
.
-
II
.-
+
O
2
(solution made at low temperature corresponds to Cu -O
2
)
.
-
II
2-
3
65
and 2 + O
2 2
(corresponds to Cu -O ) strongly support this inter-
.
- +
1
635
70 conversions. The favorable conversion of [(L1)
2
Cu(O
2
)] 
-
+
8
90
9
75
[(L1)
and the observation of no generation of O
2
Cu(OOH )] in presence of a proton source (here CH OH)
3
.
- +
2
from [(L1)
2 2
Cu(O )]
5
86
- +
or [(L1)
2
Cu(OOH )] either at 233 K or at 298 K are clearly in
4
30
0
sharp contrast to the proposed IET mechanism for the first step of
the catalytic cycle follows by native enzyme Cu-ZnSOD that in
6
00
800
1000
7
5
Wavelength(nm)
.-
2
H
2
O at 303 K dismutes O
with very high rate (rate constant k =
4
00
600
800
1000
9
~
10 orders ) but generating no cupric-hydroperoxo species; that
Wavelength(nm)
is quite interesting and unusual.
Fig. 8, Top: UV-Vis absorption traces showing conversion of a CH
3
CN
solution of 2 (1 x 10 M, black trace)  1 (gray trace) at 298 K. 200 µl
CH OH solution of equivalent KO was added at a time then each scan
taken after addition of 20 µl CH OH solution of 2 equiv. HClO dissolved
in 120 µl CH OH, inset: [2] = 1 x 10 M. Bottom: UV-Vis absorption
Conclusions
-
4
8
8
9
0
5
0
In summary, first time we have shown by means of any
spectroscopy, here UV-Vis spectroscopy, that both the steps of
the catalytic cycle are followed cleanly via a set of Cu complex,
1 and 2. First time it is shown that once O
step of the catalytic cycle (Cu + O
2
5
3
2
3
4
II/I
-
3
3
.
-
II
-
4
+
2
binds to Cu , the first
traces showing conversion of 2 (10 M) + KO
2
+ 2H  1 +H
2
O
2
, black:
II
.-
2
I
+
2
Cu + O ) does not
2
at 233 K, gray: 2 + KO
2
+2H at 233 K, each scans taken after addition
I
.-
2
+
II
follow, however, the second step (Cu + O
2
+ 2H H O
2
+ Cu )
3
0
3 4
of a 20 μl aliquots of a total 120 μl CH OH solution of HClO , isosbestic
-3
follows supporting OET and IET for the first and second step
reactions of the catalytic cycle respectively. SOD activity of 1 in
aprotic solvent also supports that first step proceeds via OET.
Collective information from FTIR, ESI Mass, UV-Vis,
electrochemical measurements and DFT calculations support the
point at 586 nm started developed after acid addition, inset: [2] = 1 x 10
M.
SOD activity of 2
.-
2
+
2-
To check whether SOD activity of 2 follows eq. 4, a mixture
of 2 plus KO in CH CN was titrated with a CH OH solution of
two equivalents HClO at 298 K and the spectrum recorded after
transformation,
{[(L1)
2
Cu(O
)]
⇌
2 2
[(L1) Cu(O
-
+
3
5
2
3
3
)]}[(L1) Cu(OOH )] .
2
4
each addition is shown in Figure 8, top. After complete addition
of acid, the final spectrum obtained (Figure 8, top, gray trace)
features same absorption bands (and their ) to those of authentic
Acknowledgement:
1
0
| Journal Name, [year], [vol], 00–00
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DOI: 10.1039/C6DT02220K
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Dalton Transactions
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Notes and references
a
Department of Chemistry,National Institute of Technology Durgapur,
Mahatma Gandhi Avenue, Durgapur 713209, West Bengal, India.
Tel:+91(0343)275-4127; E-mail: apurba.patra.nitdgp@gmail.com
80
85
1
1
2
2
3
3
4
4
5
5
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6
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b
Department of Chemistry, Indian Institute of Technology Kanpur,
Kanpur 208016, India.
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†
Electronic Supplementary Information (ESI) available: CV of O
CH CN (Fig. S1), % O (g) calculation (of Fig. 4), X-Band EPR spectra
of 1 (Fig. S2), DFT optimized structures of [(L1)
2
(g) in
3
2
2
+
+
2
2
Cu] and [(L1) Cu]
2+ +
(Fig. S3-S4), Bond distances and angles of [(L1)
2 2
Cu] and [(L1) Cu] X-
ray and optimized structure (Table S1-S2), UV-Vis spectra of NBT and
NBT+ KO
Fig. S6), % inhibition and IC50 calculation (Fig. S7), CVs of 1 + KO
(Fig. S8), ESI mass of 1 + KO
2
(Fig. S5), UV-Vis spectral changes of NBT + 1 with KO
2
2
(
90
2
(Fig. S9), FTIR spectra of 1 and
(Fig. S10), CVs of 1 + 18C6E + KO at 233 K
Fig. S11), UV-Vis spectral changes of 1 with KO
Fig. S12), HOMO and LUMO plot of [(L1)
(11)
(12)
-
[
(
(
[
(L1)
2
Cu(OOH )]ClO
4
2
2
+ 18C6E at 233 K
.-
+
2
Cu(O
2
)] and
-
+
. -
(+L1)
2
Cu(OOH )] (Fig. S13), Bond distances and angles of [(L1)
2
Cu(O
2
95
-
+
-
)]
generated from 2+KO
spectrum of (a) 1 + O (g), (b) 2 + KO
and [(L1)
2
Cu(OOH )] (Table S3-S4), UV-Vis spectrum of I
+HClO +NaI (Fig.S14), ESI positive mass
and (c) 1 + KO (Fig. S 15). This
3
2
4
2
2
2
material is available free of charge via the internet See
DOI: xx.xxxx/b000000x/
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