Functional mimics of catechol oxidase by mononuclear copper complexes of sterically demanding [NNO] ligands

Article abstract of DOI:10.1016/j.ica.2011.01.081

Several mononuclear copper complexes 1(a-b) and 2(a-b) supported over sterically demanding [NNO] ligands namely, N-(aryl)-2-[(pyridin-2-ylmethyl) amino]acetamide [aryl = 2,6-diethylphenyl (1) and mesityl (2)], exhibit catecholase-like activity in performing the aerial oxidation of 3,5-di-t-butylcatehol (3,5-DTBC) to 3,5-di-t-butyl-catequinone (3,5-DTBQ) under ambient conditions. The 1(a-b) and 2(a-b) complexes were directly synthesized from the reaction of the respective ligands 1-2 with CuX₂· nH₂O (X = Cl, NO₃, n = 2, 3) in 55-85% yield. Mechanistic insights on the catalytic cycle as obtained by density functional theory studies for a representative complex 1a suggest that an intramolecular hydrogen transfer, from a catechol-OH moiety to a copper bound superoxo moiety, form the rate-determining step of the oxidation process, displaying an activation barrier of 18.3 kcal/mol (ΔG^?) [6.9 kcal/mol in Δ(PE + ZPE)^? scale].

Full text of DOI:10.1016/j.ica.2011.01.081

Inorganica Chimica Acta 372 (2011) 145–151
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Functional mimics of catechol oxidase by mononuclear copper complexes
of sterically demanding [NNO] ligands
Manas K. Panda a, Mobin M. Shaikh b, Ray J. Butcher c, Prasenjit Ghosh a,
⇑
a Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India
b National Single Crystal X-ray Diffraction Facility, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India
c Department of Chemistry, Howard University, 525 College Street, NW, Washington DC 20059, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 4 February 2011
Several mononuclear copper complexes 1(a–b) and 2(a–b) supported over sterically demanding [NNO]
ligands namely, N-(aryl)-2-[(pyridin-2-ylmethyl)amino]acetamide [aryl = 2,6-diethylphenyl (1) and
mesityl (2)], exhibit catecholase-like activity in performing the aerial oxidation of 3,5-di-t-butylcatehol
(3,5-DTBC) to 3,5-di-t-butyl-catequinone (3,5-DTBQ) under ambient conditions. The 1(a–b) and 2(a–b)
complexes were directly synthesized from the reaction of the respective ligands 1–2 with CuX2ꢀnH2O
(X = Cl, NO3, n = 2, 3) in 55–85% yield. Mechanistic insights on the catalytic cycle as obtained by density
functional theory studies for a representative complex 1a suggest that an intramolecular hydrogen trans-
fer, from a catechol-OH moiety to a copper bound superoxo moiety, form the rate-determining step of the
Dedicated to Prof. S.S. Krishnamurthy.
Keywords:
Copper
Catechol oxidase
Oxidation
Density functional theory
Catalysis
oxidation process, displaying an activation barrier of 18.3 kcal/mol (
scale].
D D
Gà) [6.9 kcal/mol in (PE + ZPE)à
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
complexes needed to be less than 5 Å for an effective electron
transfer during the catalytic process to display catecholase activity
[16,17] while a non-planer geometry around the metal center is
necessary for mononuclear complexes to be active for catechol oxi-
dation [18]. The catalytic activity of the mononuclear complexes
can be tailored by varying the geometry and the sterics around
the metal center, thus providing with valuable opportunities to
probe the structure–activity relationship in these model com-
plexes. Several mechanism and theoretical investigations have
been reported for different binuclear catechol oxidase models
[19–21] while the same for the mononuclear counterparts remain
surprisingly rare [22] and because of which we became interested
in studying the mononuclear model complexes.
Towards our overall objective of obtaining a deeper under-
standing of various chemical and biological oxidation processes,
we took recourse to synthetic small molecule modeling studies
for obtaining valuable insights about these reactions. In this regard,
on the chemical oxidation front, we have recently reported the use
of a series of titanium isopropoxide complexes for the sulfoxida-
tion of aryl sulfides using hydrogen peroxide as a terminal oxidant
[23] and on the biological oxidation reactions front, we have re-
ported a series of high-spin iron complexes as functional mimics
of catechol dioxygenase [24] and a copper complex of a sterically
demanding ligand as a both structural and functional model for
galactose oxidase [25]. Thus, extending further along this theme
of research, we setout to design functional mimics of yet another
important copper enzyme namely the catechol oxidase.
Being synonymous with life, the role of oxygen in support and
sustain of myriad of life forms has long evoked human curiosity
and its interactions with the biological world have thus been stud-
ied with great interests [1,2]. In this regard notable are the iron and
copper metalloenzymes that engage in numerous critically impor-
tant activities from the dioxygen transport and storage to various
oxidation reactions. Catechol oxidase, an enzyme ubiquitous in
plants, insects and crustaceans [3], is a type III copper protein con-
taining a binuclear active site specializing in the two-electron oxi-
dation of a broad range of o-diphenols (catechols) to the highly
reactive o-quinones [4–6] that auto polymerizes producing the pig-
ment, melanin, which in turn, guards the damaged tissues against
pathogens and insects among many of its protective functions [7].
The catechol oxidase enzyme thus plays an important role in dis-
ease resistance in higher plants.
Significant understanding of the structural and the functional
aspects of catechol oxidase has been obtained through synthetic
modeling studies [8]. Though several dinuclear copper complexes
successfully emulate the enzyme [9–12], both structurally and
functionally, a great number of mononuclear copper complexes
too are known to exhibit significant catecholase activity [13–15].
It has been observed that the Cu. . .Cu distance in dinuclear copper
⇑
Corresponding author. Fax: +91 22 2572 3480.
E-mail address: pghosh@chem.iitb.ac.in (P. Ghosh).
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.01.081

146
M.K. Panda et al. / Inorganica Chimica Acta 372 (2011) 145–151
[NNO] ligand, 1 or 2, was seen chelated to the metal center through
the pyridyl and amine–Ns and amide–O atoms. The following Npyr
–
Cu [1.981(2)–1.999(4) Å], Namine–Cu [1.965(2)–1.993(2) Å] and Oa-
mide–Cu [1.976(2)–2.030(3) Å] bond distances were observed in
these 1(a–b) and 2(a–b) complexes. Notably, the two Cu–Cl dis-
tances were found to be inequivalent in the 1a [2.5598(15) Å,
2.2353(13) Å] and 2a [2.239(2) Å, 2.560(3) Å] complexes. Along
the same lines the nitrate moieties too were seen unsymmetrically
bound to the metal center displaying Onitrate–Cu distance of
2.207(2) Å (1b) and 1.9857(18) Å (2b) in these complexes.
The 1(a–b) and 2(a–b) complexes exhibited moderate to little
catecholase activity towards a convenient model substrate, 3,5-
di-t-butylcatehol (3,5-DTBC), using aerial oxygen as an oxidant
(Eq. (1)) in methanol, so performed due to the satisfactory solubil-
ity of both the substrate (3,5-DTBC) and its product, 3,5-di-t-butyl-
catequinone (3,5-DTBQ), in the methanol solvent. The oxidation of
3,5-DTBC to the corresponding quinone, 3,5-DTBQ, was monitored
by the growth of the strong absorption band at 390 nm. In order to
determine the kinetic parameters for the oxidation reaction, the
Michaelis–Menten approach for enzyme kinetics was applied and
the initial rates of the reactions were determined by monitoring
the absorption at 390 nm for the first 2 min.
A linear relationship for the initial rates and the concentration
was observed for the 1b and 2(a–b) complexes and the subsequent
Lineweaver–Burke plots (Supplementary material, Figs. S9–S11)
yielding the kinetic parameters (Table 2) with the exception of
1a whose rates were observed to be too slow for initial rate analy-
sis study. Quite interestingly, the nitrato derivatives (1–2)b were
consistently more active than the chloro analogs (1–2)a presum-
ably owing to subtle variation in the coordination geometry
around the metal center [16,32–35].
Important is the comparison of 1b and 2(a–b) with the other
reported ones like, a mononuclear complex [1,3-bis(N-methyl-2’-
pyridylimino)isoindoline]CuI2 [(kcat) 64 hꢁ1][22] and the binuclear
complexes, {[4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-{(2-
pyridylmethyl)aminomethyl}phenoxo]Cu2 (OMe)}[ClO4]2ꢀ2MeOH,
[(kcat) 33 hꢁ1] [36], [4-bromo-2-(4-methylpiperazin-1-ylmethyl)-
6-{2-(2-pyridyl)-ethyl}aminomethyl}phenoxo]Cu2(OMe)(MeOH)
(ClO4)2 [(kcat) 48 hꢁ1] [36] and [4-bromo-2-(4-methylpiperazin-1-
ylmethyl)-6-{{2-(1-methyl-2-imidazolyl)ethyl}aminomethyl}-
phenoxo]Cu2(OMe)(MeOH)(ClO4)2 [(kcat) 43 hꢁ1] [36] that exhib-
ited slightly higher catecholase activity while the other binuclear
ð1Þ
Here in this contribution, we report a series of mononuclear
copper complexes 1(a–b) and 2(a–b) supported over sterically
demanding [NNO] ligands that exhibit catecholase-like activity
by efficiently oxidizing 3,5-di-t-butylcatehol (3,5-DTBC) to 3,5-di-
t-butyl-catequinone (3,5-DTBQ) under ambient conditions using
aerial oxygen as an oxidant (Fig. 1 and Eq. (1)). Further understand-
ing of the mode of action of these model complexes was obtained
by performing density functional theory studies on a representa-
tive complex 1a.
2. Results and discussion
The copper chloro (1–2)a and the nitrato (1–2)b complexes of
the N-(aryl)-2-[(pyridin-2-ylmethyl)amino]acetamide [aryl = 2,6-
diethylphenyl (1) and mesityl (2)] ligand was synthesized by the
direct reaction of the ligand 1 or 2 with CuX2ꢀnH2O (X = Cl, NO3)
in 55–85% yield (Scheme 1). All of the 1a–b and 2a–b complexes
were characterized by various spectroscopic techniques including
elemental analysis.
The electronic spectra of the 1(a–b) and 2(a–b) complexes in
CH3OH showed a weak d–d transition band at ca. 704–735 nm hav-
ing an extinction coefficient of ca. 19–40 Mꢁ1 cmꢁ1, similar to that
observed in other reported Cu(II) complexes [26,27]. In addition, to
the d–d transition band, two more ligand based high energy tran-
sitions were observed at 213–218 nm (10528–15026 Mꢁ1 cmꢁ1
)
and as a shoulder at 256–258 nm (1165–2982 Mꢁ1 cmꢁ1). The
absorption at 256–258 nm is attributed to the
p⁄ transition of
p
–
the ligand [28,29] (Supplementary material, Figs. S4–S7 and
Table S2).
Both the magnetic susceptibilities as well as the EPR studies
indicated the presence of a Cu(II) center in the 1(a–b) and 2(a–b)
complexes. The room temperature magnetic susceptibilities (lB
,
1.61–2.11 BM) of 1(a–b) and 2(a–b), as determined in the solid
state, were in the range normally reported for other mononuclear
Cu(II) complexes (Table 1) [30,31]. Similarly, the X-band EPR spec-
trum of the 1(a–b) and 2(a–b) complexes (Table 1 and see Supple-
mentary material, Fig. S8), recorded in the frozen state in (CH3)2SO
at 77 K, showed well resolved axial signals consisting of three g||
lines and a broad g\ line that are characteristics of mononuclear
copper(II) complexes [27]. Specifically, all of the 1(a–b) and 2(a–
b) complexes exhibited analogous g|| (2.37–2.39 G), g\ (2.07–
2.08 G), and hyperfine splitting A|| (130–160 G) values (Table 1).
Lastly, the observation of g|| > g\ > 2.0032 indicates the presence
of singly occupied dx2 ꢁ dy2 orbital in 1(a–b) and 2(a–b).
complexes
namely,
{[N-(2-oxybenzyl)aminomethanesulfonic
acid]2Cu2(H2O)2}ꢀH2O [(kcat) 1140 hꢁ1] [37] and {[N-(2-oxyben-
zyl)aminoethanesulfonic acid]2Cu2}ꢀ2H2O [(kcat) 4612 hꢁ1] [37]
displayed much superior activity than 1b and 2(a–b). The moder-
ate to low catecholase activity observed for 1b and 2(a–b) may be
due to mononuclear nature of these complexes in contrast to the
presence of a binuclear active site in the catechol oxidase enzyme.
Further insight into the mechanism of catechol oxidation could
be obtained by performing the density functional theory (DFT)
studies on a suitably modified mechanism proposed based on prior
studies [38]. Specifically, the oxidation of 3,5-di-t-butylcatechol
was modeled for a representative complex 1a at UB3LYP level of
theory using LANL2DZ, 6-31G(d) basis sets. The intermediates
and the transition states involved in the catalytic cycle were com-
puted by geometry optimization followed by single point calcula-
tions of the appropriately modified input geometry of the parent
complex 1a, whose atomic coordinates were obtained from the
X-ray analysis data.
The molecular structures of all of the 1(a–b) and 2(a–b) com-
plexes revealed a distorted trigonal bipyramidal geometry around
copper with the [NNO] ligand, 1 or 2, occupying the equatorial
plane while the chlorides in 1a and 2a and the nitrates in 2b and
a nitrate along with an water molecule in 1b residing in the axial
positions (Fig. 2 and Supplementary material, Figs. S1–S3). The
H
H
N
N
R1
1a
R1 = Et, R2 = H, X = Cl (
)
The catalytic cycle is proposed to initiate with a catecholate
bound Cu(I) species 1B, formed from the starting precatalyst 1a
(Scheme 2), reacting with aerial oxygen to generate a superoxide
species 3C by undergoing an oxidation of the Cu(I) center in 1B to
a Cu(II) center in 3C (Scheme 3). Indeed, the Mulliken spin-density
analysis showed increased spin density on copper (+0.384) in 3C
R1
N
R1 = Et, R2 = H, X = NO3 (1b)
R1 = R2 = Me, X = Cl (2a)
2b
O
R2
Cu
R1 = R2 = Me, X = NO3 (
)
X
X
Fig. 1. Copper complexes of sterically demanding [NNO] ligands.

M.K. Panda et al. / Inorganica Chimica Acta 372 (2011) 145–151
147
Scheme 1.
Table 2
Table 1
EPR parameters and magnetic moments (
l
B) of 1(a,b) and 2(a,b).
Kinetic parameters of the catalytically active 1b and 2(a,b) complexes.
Complex
A||
A\
g||
g\
Magnetic moment (lB, BM)
Complex
kcat (hꢁ1
)
KM (mol dmꢁ3
)
Vmax (mol dmꢁ3 mꢁ1
)
1a
1b
2a
2b
130
150
150
160
NRa
NRa
NRa
NRa
2.368
2.385
2.376
2.394
2.079
2.066
2.066
2.078
1.61
2.11
1.88
1.95
1b
2a
2b
3
0.2
6
1.2 ꢂ 10ꢁ3
2.6 ꢂ 10ꢁ3
2.7 ꢂ 10ꢁ2
2.83 ꢂ 10ꢁ5
2.87 ꢂ 10ꢁ6
3.59 ꢂ 10ꢁ5
a
NR = Not resolved.
O2 = 2.181 Å, and Cu1–O3 = 1.970 Å) were found to be unsymmet-
rical (Supplementary material, Figs. S12–S13).
from that in 1B (0.000) (Supplementary material, Table S7). It is
worth noting that prior investigation suggests the formation of a
superoxide species in the catalytic cycle of catecholase oxidase
[22]. Quite interestingly, in the 3C intermediate, the two C–O
bond distances of the catechol moiety (C19–O2 = 1.415 Å and
C21–O3 = 1.322 Å) as well as two Cu–O bond distances (Cu1–
The next step of the catalytic cycle involves a critical intramo-
lecular proton transfer process the occur from the O2 oxygen atom
of the catechol moiety to the superoxide O5 oxygen atom in the 3C
intermediate proceeding via a transition state 3DTS to give a peroxo
intermediate 3E (Scheme 3). The intramolecular proton transfer in
the superxo 3C species yielding a peroxo 3E intermediate is also
N2
N3
N1
Cu1
O1
Cl2
Cl1
Fig. 2. ORTEP of 1a with thermal ellipsoids drawn at 50% probability level. Selected bond lengths (Å) and angles (°) are given: Cu1–N1 1.999(4), Cu1–N2 1.989(4), Cu1–O1
2.030(3), Cu1–Cl1 2.5598(15), Cu1–Cl2 2.2353(13), N(1)–Cu1–O1 162.74(15), N2–Cu1–Cl2 155.82(13), N1–Cu1–Cl2 97.81(12), N1–Cu1–Cl1 94.61(12), N2–Cu1–Cl1
95.24(13), Cl2–Cu1–Cl1 108.88(5).
H
N
2
2 X
H
N
Et
t-Bu
t-Bu
H
N
H
N
Et
Et
N
HO
HO
HO
HO
O
N
H
N
CuII
H
N
Et
Et
N
t-Bu
O
CuI
t-Bu
2
Et
t-Bu
2
N
O
O
CuII
O
HO
t-Bu
O
X
X
2 HX
t-Bu
CuII
NH
t-Bu
O
X = Cl, NO3
1a-b, 2a-b
Et
O
O
t-Bu
Et
N
H
(
)
t-Bu
3A
1B
(
)
(
)
Scheme 2.

148
M.K. Panda et al. / Inorganica Chimica Acta 372 (2011) 145–151
Scheme 3.
coincidentally the overall rate-determining step of the catalytic cy-
of 13 kcal/mol has been determined by measuring the rate of cat-
echol conversion in the catecholase enzyme in sweet potato
3
cle with the DTS transition state (Fig. 3) exhibiting an activation
3
barrier of 18.3 kcal/mol (
D
Gà) [6.9 kcal/mol in
D
(PE + ZPE)à scale]
[38,39]. The DTS transition state displayed an elongated O4–O5
(Fig. 4). Interestingly enough, a slightly lower activation barrier
bond of 1.402 Å compared to the 1.281 Å in 3C. The decrease in spin
Fig. 3. Geometry optimized structure of 3DTS. Selected bond lengths (Å) and angles (°): Cu1–O3 1.920, Cu1–O4 1.923, Cu1–O5 2.021, Cu1–N1 2.283, Cu1–N2 2.122, N1–Cu1–
N2 53.7, N1–Cu1–O3 34.0.

M.K. Panda et al. / Inorganica Chimica Acta 372 (2011) 145–151
149
Fig. 4. Computed free energy (
DG) profile (in red) and total energy (E) with zero-point energy (ZPE) correction (E + ZPE) profile (in blue) along reaction coordinate of the of
the catalytic of oxidation of catechol by a proposed active species(1B) of 1a (DTBC = 3,5-di-t-butyl catechol and DTBQ = 3,5-di-t-butyl catequinone).
density of the two oxygen atoms of superoxide moiety [+0.308
ylmethyl)amino]acetamide [aryl = 2,6-diethylphenyl (1) and mesi-
3
(O5), +0.416 (O4)] in transition state DTS relative to that of the
tyl (2)] ligands exhibited catecholase activity as evidenced from
the oxidation of 3,5-DTBC with molecular oxygen to give 3,5-DTBQ.
Kinetic studies reveal better activity of the nitrato (1–2)b deriva-
tives compared to the chloro (1–2)a ones. The catechol oxidation
mechanism as modeled using density functional theory suggests
that the catalytic cycle proceeds via an important intramolecular
hydrogen transfer step that represents the rate-determining step
of the oxidation reaction.
starting 3C intermediate [+0.779 (O5), +0.715 (O4)] further attests
toward the intramolecular proton transfer (Supplementary mate-
rial, Table S7). As expected, a slight decrease in spin density was
3
observed in copper atom of transition state DTS. In this regard it
is noteworthy that the similar proton transfer process has been
suggested to be the rate-determining step in the catechol oxidation
catalytic cycle [22].
The peroxo intermediate 3E exhibited a much elongated O4–O5
(1.433 Å) bond as characteristics of a peroxo moiety and which was
found bound to the metal center exhibiting a Cu1–O4 bond dis-
tance of 1.915 Å (Supplementary material, Fig. S14). Quite expect-
edly, the spin densities on the oxygen atoms of peroxo moiety was
found to diminish [+0.067 (O5), +0.293 (O4)] in 3E, in comparison
3. Experimental section
3.1. General procedures
3
to the transition state DTS [+0.308 (O5), +0.416 (O4)] and the
All manipulations were carried out using standard Schlenk
techniques. Solvents were purified and degassed by standard pro-
cedures. The 2,6-diethylaniline, 2,4,6-trimethylaniline and 2-picol-
ylamine were purchased from Sigma Aldrich, Germany, and the
chloroacetyl chloride was purchased from Spectrochem, India,
and were used without any further purification. The N-(2,6-dieth-
ylphenyl)-2-[(pyridin-2-ylmethyl)amino]acetamide (1) and N-
mesityl-2-(pyridine-2-ylmethylamino)acetamide (2) were pre-
pared by the procedure reported in literature [24]. 1H and
13C{1H} NMR spectra were recorded on a Varian 400 MHz NMR
spectrometer. 1H NMR peaks are labeled as singlet (s), doublet
(d), triplet (t) and septet (sept). Infrared spectra were recorded
on a Perkin Elmer Spectrum One FT-IR spectrometer. Mass spec-
trometry measurements were done on a Micromass Q-Tof spec-
trometer. X-ray diffraction data for the compounds 1(a–b) and
2(a–b) were collected on a Bruker P4 diffractometer equipped with
a SMART CCD detector and crystal data collection and refinement
parameters are summarized in Table S1 (Supplementary material).
The structures were solved using direct methods and standard dif-
superoxo 3C intermediate [+0.779 (O5), +0.715 (O4)] as a result
of the intramolecular proton transfer. The quinone character of
the catechol moiety was further corroborated by the observation
of much shorter bond distances C19–O2 (1.2857 Å) and C21–O3
(1.2852 Å) in 3E as compared to the previous superoxo intermedi-
ate 3C [C19–O2 = 1.415 Å and C21–O3 = 1.322 Å].
Finally, the last step involves the binding of another molecule of
catechol substrate, 3,5-DTBC, to the copper center in 3E regenerat-
ing the starting 1B species with concomitant release of the desired
3,5-DTBQ product and H2O2, which in turn would decompose to
oxygen and water. More significantly, the disproportionation of
H2O2 would render the catechol oxidation cycle thermodynami-
cally favorable by making the overall process exergonic by
ꢁ35.4 kcal/mol (
catalytic cycle by itself, up until the generation of H2O2, remains
endergonic by 16.7 kcal/mol ( G) (Fig. 4).
DG) as without which the catechol oxidation
D
In summary, a series of copper chloro (1–2)a and nitrato (1–2)b
derivatives supported over [NNO] type N-(aryl)-2-[(pyridin-2-

150
M.K. Panda et al. / Inorganica Chimica Acta 372 (2011) 145–151
ference map techniques, and were refined by full-matrix least-
squares procedures on F2 with SHELXTL (version 6.10) [40,41]. CCDC
609847, CCDC 612608, CCDC 796629 and CCDC 619910 contain the
supplementary crystallographic data for the copper 1(a–b) and
2(a–b) complexes, respectively. The EPR measurements were made
with a Varian model 109C E-line X-band spectrometer fitted with a
quartz Dewar for measurements at 77 K. The spectra were cali-
brated by using tetracyanoethylene (tcne) (g = 2.0037). The elec-
tronic spectra were recorded in CH3CN by using a JASCO V-570
UV–Vis spectrophotometer. Elemental analysis was carried out
on Thermo Quest FLASH 1112 SERIES (CHNS) Elemental Analyzer.
63%). leff (298 K): 1.95 BM. UV–Vis (CH3OH, 1 ꢂ 10ꢁ4 M) kmax
,
nm
(e,
Mꢁ1 cmꢁ1): 212.5 (10 842), 256 (1613), 727.5 (9,
1 ꢂ 10ꢁ2 M). IR (cmꢁ1, KBr pallet): 3457 (m), 3147 (m), 3072 (w),
2915 (w), 1616 (s), 1597 (s), 1563 (m), 1482 (m), 1466 (w), 1438
(m), 1384 (w), 1285 (w), 1212 (w), 1159 (w), 1108 (w), 1032
(m), 1018 (m), 951 (w), 856 (w), 761 (m), 714 (w), 656 (w), 492
(w), 468 (w). Anal. Calc. for C17H21Cl2CuN3OꢀH2O: C, 46.85; H,
5.32; N, 9.64. Found: C, 46.29; H, 4.79; N, 9.66%.
3.5. Synthesis of [N-mesityl-2-(pyridine-2-ylmethylamino)-
acetamide]Cu(NO3)2ꢀ2H2O (2b)
3.2. Synthesis of [N-(2,6-diethylphenyl)-2-[(pyridin-2-ylmethyl)-
amino]acetamide]CuCl2ꢀH2O (1a)
To a solution of N-mesityl-2-(pyridine-2-ylmethylamino)acet-
amide (2) (0.285 g, 1.01 mmol) in methanol (ca. 10 mL), a solution
of Cu(NO3)2ꢀ3H2O (0.241 g, 1.00 mmol) in methanol (ca. 10 mL)
was added slowly and then the resulting mixture was stirred at
room temperature for 12 h. Addition of excess diethyl ether (ca.
30 mL) resulted in the formation of a precipitate. The precipitate
was isolated by filtration, washed with diethyl ether (3 ꢂ 20 mL)
and vacuum dried to obtain the product 2b as a deep blue solid
(0.263 g, 55%). LRMS (ES): m/z 345 [Mꢁ2NO3ꢁH]+. leff (298 K):
To a solution of N-(2,6-diethylphenyl)-2-[(pyridin-2-ylmethyl)-
amino]acetamide (1) (0.298 g, 1.00 mmol) in methanol (ca. 20 mL),
a solution of CuCl2ꢀ2H2O (0.172 g, 1.01 mmol) in methanol (ca.
10 mL) was added slowly and then the resulting mixture was stir-
red at room temperature for 12 h. It was filtered and all the vola-
tiles were removed under reduced pressure. The residue was
dissolved in a small amount of CH2Cl2 (ca. 5 mL). Addition of excess
diethyl ether (ca. 40 mL) to the CH2Cl2 solution resulted in the for-
mation of a precipitate. The precipitate was isolated by filtration,
washed with diethyl ether and vacuum dried to obtain the product
1a as a green solid (0.372 g, 85%). LRMS (ES): m/z 395 [MꢁCl]+. leff
e
1.88 BM. UV–Vis (CH3OH, 1 ꢂ 10ꢁ4 M) kmax, nm ( , Mꢁ1 cmꢁ1):
217.5 (15 026), 257.5 (1165), 704 (19, 1 ꢂ 10ꢁ2 M). IR (cmꢁ1, KBr
pallet): 3446 (s), 2923 (w), 2068 (w), 1625 (s), 1486 (m), 1384
(s), 1291 (m), 1108 (w), 1018 (m), 769 (m). Anal. Calc. for C17H21Cu-
N5O7ꢀ2H2O: C, 40.28; H, 4.97; N, 13.81. Found: C, 40.29; H, 3.89; N,
14.58%.
(298 K): 1.61 BM. UV–Vis (CH3OH, 1 ꢂ 10ꢁ4 M) kmax, nm (
e,
Mꢁ1 cmꢁ1): 212.5 (10 528), 257.5 (2982), 734.5 (40, 1 ꢂ 10ꢁ2 M).
IR (cmꢁ1, KBr pallet): 3481 (m), 3208 (m), 2966 (m), 2931 (m),
2874 (w), 1655 (s), 1617 (s), 1585 (m), 1449 (m), 1377 (w), 1283
(w), 1262 (w), 1106 (w), 1054 (w), 1029 (m), 974 (w), 808 (w),
767 (m), 725 (w). 653 (m). Anal. Calc. for C18H23Cl2CuN3OꢀH2O: C,
48.06; H, 5.60; N, 9.34. Found: C, 48.19; H, 5.24; N, 8.67%.
3.6. Computational methods
Density functional theory (DFT) calculations were performed
using GAUSSIAN 03 [42] suite of quantum chemical programs on
the various intermediates and transition states namely, 1B, 3C,
3DTS and 3E pertaining to a proposed mechanistic pathway and de-
rived from a representative complex 1a whose atomic coordinates
were obtained from X-ray analysis data. The Becke three parameter
exchange functional in conjunction with Lee–Yang–Parr correla-
tion functional (UB3LYP) has been employed in this study
[43,44]. The LANL2DZ basis set was used for the Cu atom while
all other atoms are treated with 6-31G(d) basis set [45,38]. The
geometry of the transition states structures were optimized in
the first order saddle point using GAUSSIAN 03 program suite. Station-
ary points were characterized as minima by evaluating Hessian
indices on the respective potential energy surfaces using tight
SCF convergence (10ꢁ8 a.u). Fully optimized transition state struc-
tures have been characterized by a single imaginary frequency,
while rest of the optimized structures possesses only real fre-
quency. Natural bond orbital (NBO) analysis [46] was performed
using the NBO 3.1 programs implemented in the GAUSSIAN 03 pack-
age (Supplementary material, Tables S3–S6).
3.3. Synthesis of [[N-(2,6-diethylphenyl)-2-[(pyridin-2-
ylmethyl)amino]acetamide]Cu(NO3)(OH2)]NO3ꢀH2O (1b)
To
a
solution
of
N-(2,6-diethylphenyl)-2-[(pyridin-2-
ylmethyl)amino]acetamide (1) (0.298 g, 1.00 mmol) in methanol
(ca. 20 mL), a solution of Cu(NO3)2ꢀ3H2O (0.241 g, 1.00 mmol) in
methanol (ca. 20 mL) was added slowly and then the resulting
mixture was stirred at room temperature for 12 h. Addition of ex-
cess hexane (ca. 30 mL) to the product mixture resulted in the for-
mation of a blue precipitate which was isolated by filtration,
washed with hexane (3 ꢂ 20 mL) and vacuum dried to obtain the
product 1b as a blue solid (0.358 g, 74%). LRMS (ES): m/z 422
[MꢁNO3]+. leff (298 K): 2.11 BM. UV–Vis (CH3OH, 1 ꢂ 10ꢁ4 M)
kmax, nm (e
, Mꢁ1 cmꢁ1): 216.5 (14 754), 256 (1871), 708.5 (22,
1 ꢂ 10ꢁ2 M). IR (cmꢁ1, KBr pallet): 3444 (m), 3221 (w), 3072 (w),
2969 (w), 2934 (w), 2874 (w), 1624 (s), 1586 (m), 1463 (m),
1445 (w), 1384 (s), 1318 (w), 1202 (w), 1160 (w), 1108 (w), 1056
(w), 1033 (w), 1016 (m), 980 (w), 823 (w), 781 (m), 735 (m), 723
(w). Anal. Calc. for C18H23CuN5O7ꢀ2H2O: C, 41.50; H, 5.22; N,
13.44. Found: C, 41.65; H, 5.20; N, 14.15%.
3.7. General procedure for the aerobic oxidation of catechol to quinone
In order to determine the kinetics parameters of catecholase
activity of copper complexes, the aerial oxidation of 3,5-di-t-butyl
catechol (3,5-DTBC) were measured at 25 °C by the method of ini-
tial reaction rates by monitoring the increase of the absorption
band at 390 nm of the product 3,5-di-t-butyl catequinone (3,5-
DTBQ) (Eq. (1)) following a modified procedure reported in the lit-
erature [37]. In a typical run, 0.3 mL of 1.0 ꢂ 10–3 M solution of the
1(a–b) and 2(a–b) complex in methanol was added to a 0.3 mL
methanol solution of 3,5-di-t-butyl catechol (3,5-DTBC) of different
concentrations 1.0 ꢂ 10ꢁ3–1.5 ꢂ 10ꢁ2 M and the volume was made
to 3 mL so that the final cuvette concentration of the complex was
maintained at 10ꢁ4 M. The growth of the absorption band at
390 nm was monitored for first 10 min of the reaction time and a
3.4. Synthesis of [N-mesityl-2-(pyridine-2-
ylmethylamino)acetamide]CuCl2ꢀH2O (2a)
To a solution of N-mesityl-2-(pyridine-2-ylmethylamino)acet-
amide (2) (0.283 g, 1.00 mmol) in methanol (ca. 10 mL), a solution
of CuCl2ꢀ2H2O (0.171 g, 1.00 mmol) in methanol (ca. 10 mL) was
added slowly and then the resulting mixture was stirred at room
temperature for 12 h. Addition of excess diethyl ether (ca. 30 mL)
resulted in the formation of a precipitate. The precipitate was iso-
lated by filtration, washed with diethyl ether (3 ꢂ 20 mL) and vac-
uum dried to obtain the product 2a as a deep blue solid (0.263 g,

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Acknowledgments
We thank BRNS, Mumbai, for financial support of this research.
We are grateful to the National Single Crystal X-ray Diffraction
Facility and Sophisticated Analytical Instrument Facility (SAIF) at
IIT Bombay, India, for the crystallographic and other characteriza-
tion data. Computational facilities from the IIT Bombay Computer
Center are gratefully acknowledged. M.P. thanks CSIR, New Delhi,
for research fellowship.
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Supplementary material (CCDC 609847, CCDC 612608, CCDC
796629 and CCDC 619910 contain the supplementary crystallo-
graphic data the title compounds 1(a,b) and 2(a,b). These data
can be obtained free of charge from The Cambridge Crystallo-
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TEP plots of 1b and 2(a,b), geometry optimized structures of 1B, 3C,
and 3E along with the B3LYP coordinates of the optimized geome-
tries for 1B, 3C, and 3E, X-ray crystallographic parameters table, EPR
and Mulliken spin density data) associated with this article can be
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