Binuclear copper(II) complexes 1: Synthesis, characterization and evaluation of a new complex in phosphatase-like activity

Article abstract of DOI:10.1016/j.ica.2012.04.019

A new binuclear Cu(II) complex derived from a pamoic type ligand has been synthesized and characterized by X-ray crystallography. This complex was checked as catalysts in the hydrolysis of bis(p-nitrophenyl)phosphate. Its catalytic properties were studied

Full text of DOI:10.1016/j.ica.2012.04.019

Inorganica Chimica Acta 391 (2012) 189–194
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Inorganica Chimica Acta
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Binuclear copper(II) complexes 1: Synthesis, characterization and evaluation
of a new complex in phosphatase-like activity
Olivier Schicke a, Bruno Faure a, Michel Giorgi b, A. Jalila Simaan a, Marius Réglier a,
⇑
a Institut des Sciences Moléculaires de Marseille/BiosCiences UMR CNRS 7313, Aix-Marseille Université, Campus Scientifique de St-Jérome, avenue Escadrille Normandie-Niemen,
13397 Marseille Cedex 20, France
b Spectropole FR CNRS 1739, Aix-Marseille Université, Campus Scientifique de St-Jérome, avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new binuclear Cu(II) complex derived from a pamoic type ligand has been synthesized and character-
ized by X-ray crystallography. This complex was checked as catalysts in the hydrolysis of bis(p-nitro-
phenyl)phosphate. Its catalytic properties were studied at various pH and compared to the
mononuclear Cu(II) complex. At pH 5.38, the binuclear Cu(II) complex exhibited a phosphatase-like activ-
ity with 11 TON and 24% conversion and was found to be 41-fold more active than the mononuclear
counterpart.
Received 20 January 2012
Received in revised form 11 April 2012
Accepted 11 April 2012
Available online 3 May 2012
Keywords:
Copper
Ó 2012 Elsevier B.V. All rights reserved.
Binuclear copper(II) complex
Phosphatase-like activity
1. Introduction
this metal exhibits interesting features rendering it attractive for
artificial nucleases: (i) Cu(II) is substitutionally labile and at the
The development of dinuclear metal ion complexes that pro-
mote the hydrolysis of phosphate ester linkages has been a subject
of great interest during the last decade [1,2]. On one hand, the de-
sign of functional models for hydrolytic metalloenzymes that con-
tain two metal ion in their active site [3–5] is of great importance
in biotechnology for the prospect of synthetic nucleases which
could be used as artificial restriction enzymes [6]. On the other
hand, since a number of phosphate esters and related P(V) com-
pounds are used in agriculture as pesticides or, unfortunately, as
potent nerve agents in chemical weapons, the development of cat-
alytic systems able to hydrolyze and destroy such compounds is of
considerable environmental and medicinal importance. Due to its
high natural abundance and good Lewis acid properties, Mg(II) is
the most important metal encountered in nucleases [7], but multi-
nuclear transition metal centers with Zn(II), Mn(II) and Fe(II/III) are
often present too [3]. In these binuclear systems, hydrolysis occurs
generally by a double Lewis acid activation: one of the metal ions
renders the coordinated water more acidic while the second metal
activates the phosphate ester group thus favoring an intramolecu-
lar hydrolysis of the phosphoester group (Scheme 1).
same time a strong Lewis acid necessary for activation of the phos-
phodiester bond via nucleophilic attack and (ii) Cu(II) lowers the
pKa of coordinated water, thereby providing metal-bound hydrox-
ide at near-neutral pH. That is the reason why binuclear Cu(II)
complexes have also been described as efficient catalysts in phos-
phoester hydrolysis [17–23]. We report on the synthesis and the
characterization of a new binuclear copper(II) complex based on
a pamoic moiety which exhibit a remarkable catalytic phospha-
tase-like activity compared to mononuclear copper(II) complexes.
2. Experimental
2.1. General
All chemicals were obtained from commercial sources and used
as received. THF and dichloromethane were respectively distilled
from sodium-benzophenone and calcium hydride. Silica gel TLC
and column chromatography were performed using Meck Kieselgel
60F254 and silica gel 60 (0.063–0.200). Each compound which mass
spectrometry data is given had its structure confirmed by (ESI)MS/
MS. All NMR spectra were recorded with Brüker Avance DPX200
and DPX300 using TMS as internal reference. X-band ESR spectra
were obtained using a BRUKER EMX9/2.7 spectrometer equipped
with a B-VT2000 digital temperature controller (100–400 K). The
simulations with automatic parameter fitting were performed for
axial symmetry [24]. The contribution of naturally abundant 63Cu
Nuclease mimics incorporating a dinuclear metal center with Co
[8,9], Ln [10–12], Zn [13–15], and Fe [16] were recently published
as bioinspired efficient catalysts in phosphoester hydrolysis. While
Cu(II) has never been identified as cofactor in natural nucleases,
⇑
Corresponding author. Tel.: +33 4 91 28 8823; fax: +33 4 91 28 8440.
E-mail address: marius.reglier@univ-amu.fr (M. Réglier).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.04.019

190
O. Schicke et al. / Inorganica Chimica Acta 391 (2012) 189–194
Scheme 1. Double Lewis acid activation at bimetallic center.
and 65Cu was considered, but the values given in the text refer to
63Cu. All principal axes were supposed parallel.
2.2. Methyl pamoate, 1
Methyl pamoate 1 was synthesized starting from the commer-
cially available pamoic acid according to a literature procedure
[25]. Recrystallization in methanol affords compound 1 as white
crystals. Crystal data for compound 1, together with details of
the single crystal X-ray diffraction experiment are reported in
Table 1 and an ORTEP representation in Fig. 1A.
2.3. Methyl 2,20-[1,2-ethanediylbis(oxy)]-1,10-dinaphthylmethane-
3,30-dicarboxylate, 2
To a stirred solution of methyl pamoate 1 (1.1 g, 2.64 mmol) in
dry acetone (500 cm3) was successively added anhydrous potas-
sium carbonate (2.92 g, 21.1 mmol) and 1,2-dibromoethane
(0.22 cm3, 2.64 mmol). The suspension was stirred and refluxed
for a week, then vacuum evaporated. The solid obtained was dis-
solved in 50 cm3 dichloromethane and washed several times with
distilled water. The organic phase was dried over MgSO4, and vac-
uum evaporated. Purification was done by fractional crystallization
removal of product 1 using ethyl acetate (or chloroform) as solvent
Fig. 1. ORTEP drawings of (A) and 4 (B) (displacement ellipsoids are drawn at 50%
probability level).
123.50, 124.01, 125.19, 128.63, 129.64, 129.98, 131.74, 134.59,
154.83, 166.48 ppm; MS m/z (ESI): 443 [M+H+].
to afford a white solid (0.16 g, 14%); mp 211 °C;
m
max/cmꢀ1 2941
2.4. 2,20-[1,2-Ethanediylbis(oxy)]-1,10-dinaphthylmethane-3,30-
(CH), 1718 (CO), 1622 and 1589 (C–Carom), 1190 and 1148 (CO);
dH (300 MHz, CDCl3, Me4Si) 3.92 (6H, s, OCH3), 4.50 (4H, m,
OCH2–CH2O), 5.09 (2H, s, naph-CH2-naph), 7.45 (4H, m, naph),
7.86 (2H, d, J = 7.80, naph), 8,08 (2H, d, J = 7.97, naph), 8.29 (2H,
s, naph) ppm; dC (75.47 MHz, CDCl3, Me4Si) 20.50, 52.65, 75.08,
methyl alcohol, 3
A solution of 2 (0.090 g, 0.2 mmol) in THF (5 cm3) was added
cautiously to a stirred suspension of lithium aluminum hydride
(0.190 g, 5 mmol) in THF (10 cm3). After 3 h of reaction the mixture
Table 1
Crystal data for the compounds 1, 4, 6 and 7.
1
4
6
7
Formula
MW
C
25H20O6
C25H20Br2O2
512.23
C56H58Cu2F12N6O19S4
1600.45
C23H24CuF6N4O7S2
710.12
416.41
Crystal system
Space group
a (Å)
b (Å)
c (Å)
orthorhombic
Pbcn
10.3290(7)
14.7250(9)
26.376(1)
90
4011.6(4)
8
0.099
monoclinic
P21/c
monoclinic
P21/c
monoclinic
P21
4.8000(2)
23.571(2)
18.571(2)
101.927(5)
2055.8(3)
4
18.3967(1)
13.8680(1)
26.5326(2)
98.1940(4)
6700.04(8)
4
11.0690(6)
8.641(7)
16.481(1)
108.812(2)
1492(1)
b (°)
V (Å3)
Z
2
l(Mo K
a
) (mmꢀ1
)
3.962
0.867
0.957
Unique reflections, Rint
Reflections with I > 2(I)
Final R indices for I > 2(I)
4792, 0.076
3211
R = 0.066
wR = 0.116
R = 0.109
wR = 0.134
3780, 0.108
2677
R = 0.061
wR = 0.096
R = 0.097
wR = 0.1102
11369, 0.053
9472
R = 0.0648
wR = 0.1712
R = 0.0782
wR = 0.1815
2880, 0.040
2659
R = 0.039
wR = 0.088
R = 0.045
wR = 0.093
0.074(18)
R indices (all data)
Flack parameter

O. Schicke et al. / Inorganica Chimica Acta 391 (2012) 189–194
191
was hydrolyzed by successive addition of distilled water
(0.190 cm3), NaOH 1 M (0.380 cm3), then distilled water
(0.190 cm3) and left under stirring for 1 h. The reaction mixture
was centrifuged, the supernatant dried through a microcolumn of
anhydrous Na2SO4. Vacuum evaporation of THF affords compound
3 as a white solid (0.075 g, 98%); mp 293 °C; m
max/cmꢀ1 3342 (OH),
2954 (CH), 1624 and 1598 (C–Carom), 1192 (CO); dH (200 MHz,
DMSO, Me4Si) 4.36 (4H, m, OCH2–CH2O), 4.65 (4H, s, CH2OH),
4.98 (2H, s, naph-CH2-naph), 5.22 (2H, s, OH), 7.38 (4H, m, naph),
7.81 (2H, s, naph), 7.87 (2H, m, naph), 8.13 (2H, m, naph) ppm;
dC (50.32 MHz, DMSO, Me4Si) 22.40, 58.55, 73.19, 123.31, 124.31,
125.11, 125.74, 127.38, 128.35, 130.48, 131.47, 135.03,
154.20 ppm.
2.5. 2,20-[1,2-Ethanediylbis(oxy)]-1,10-dinaphthylmethane-3,30-
bromomethane, 4
To a suspension of 3 (0.080 g, 0.21 mmol) in dichloromethane
(10 cm3) was added phosphorus tribromide (0.04 cm3, 0.43 mmol)
via micro-syringe. The mixture was stirred and refluxed for 24 h.
After cooling the solution was washed by a saturated aqueous
NaHCO3 solution (2 ꢁ 50 cm3), distilled water (50 cm3) then dried
over anhydrous Na2SO4. Dichloromethane was vacuum evaporated
to give a solid that was purified by column chromatography using
diethyl ether as solvent. Compound 4 was recrystallized from chlo-
roform to give white crystals (0.054 g, 51%); mp 235 °C (decompo-
Fig. 2. ORTEP drawings of 6, the H-atoms and the three un-coordinated triflate
counter ions have been removed for clarity (displacement ellipsoids are drawn at
the 50% probability level).
93 l
mol) in acetone (2 cm3). After 3 h of reaction, precipitation
with diethylether affords compound 6 as blue solid (0.034 g). The
slow evaporation at room temperature of a saturated acetone solu-
tion of compound 6 affords deep blue crystals suitable for a single
crystal X-ray analysis. Crystal data for complex 6, together with de-
tails of the X-ray diffraction experiment are reported in Table 1 and
an ORTEP representation in Fig. 2. The X-structure reveals the pres-
ence of a non-coordinated acetone molecule and four water mole-
sition);
m
max/cmꢀ1 2910 (CH), 1621 and 1595 (C–Carom), 1195
(CO), 700 (CBr); dH (200 MHz, CDCl3, Me4Si) 4.67 (8H, m, CH2Br
and OCH2–CH2O), 5.03 (2H, s, naph-CH2-naph), 7.38 (4H, m, naph),
7.78 (2H, m, naph), 7.81 (2H, s, naph), 8.03 (2H, m, H8) ppm; dC
(50.32 MHz, CDCl3, Me4Si) 23.73, 29.15, 74.41, 123.54, 124.98,
127.20, 128.56, 128.88, 129.91, 130.65, 130.98, 133.25,
154.68 ppm; MS m/z (ESI): 513[M+H+], 511 (51%), 515 (48%). Crys-
tal data for compound 4, together with details of the single crystal
X-ray diffraction experiment are reported in Table 1 and an ORTEP
representation in Fig. 1B.
cules; mp = 257 °C (decomposition); m
max/cmꢀ1 3429 (OH), 1613
and 1575 (C–Carom), 1222 (CF), 1025 (SO). Elemental Anal. Calc.
for C56H58Cu2F12N6O19S4 (MW 1602.42): C, 41.97; H, 3.65; N,
5.24. Found: C, 40.73; H, 3.46; N, 5.39%.
2.6. N,N,N-Bis(2-picolyl)-(2,20-[1,2-ethanediylbis(oxy)]-1,10-
2.8. Mononuclear copper(II) complex, 7
dinaphthylmethane-3,30-methyl) amine, 5
To
a stirred solution of N-benzyl-bis-2-picolylamine [26]
(0.300 g, 1.03 mmol) in chloroform (10 cm3) was added a solution
of copper(II) triflate (0.375 g, 1.03 mmol) in chloroform (10 cm3).
After three hours of stirring at room temperature the mixture
was precipitated with diethylether and filtered to give a blue solid
(0.360 g, 50%). The slow evaporation at room temperature of a sat-
To a solution of 4 (0.056 g, 0.10 mmol) in ethyl acetate (2 cm3)
was successively added
a
solution of bis(2-picolyl)amine
(0.057 cm3) in ethyl acetate (2 cm3), then a solution of N,N-diiso-
propylethylamine (0.025 cm3, 0.21 mmol) in ethyl acetate
(2 cm3). The mixture was refluxed under stirring for 15 h. Ethyl
acetate was removed by vacuum evaporation. The resulting crude
solid was solubilized in dichloromethane (50 cm3), washed succes-
sively by distilled water (50 cm3), saturated aqueous NaHCO3 solu-
tion (50 cm3), distilled water (50 cm3) then dried over anhydrous
Na2SO4. Dichloromethane was vacuum evaporated to give brown
product purified by column chromatography using acetone then
an acetone/MeOH (95/5) mixture as eluent. Vacuum evaporation
affords compound 5 as a white solid (0.035 g, 47%); dH (200 MHz,
acetone-d6, Me4Si) 3.81 (8H, s, N–CH2–Pyr ꢁ4), 3.89 (4H, s, naph-
CH2–N), 4.21 and 4.33 (4H, m, O–CH2–CH2–O), 5.00 (2H, s, naph-
CH2-naph), 7.13 (4H, m, pyr), 7.30 (4H, m, naph), 7.59 (8H, m,
pyr), 7.82 (2H, m, naph), 8.07 (4H, m, naph), 8.44 (4H, m, pyr)
ppm; dC (50.32 MHz, acetone-d6, Me4Si) 24.92, 55.30, 62.01,
75.57, 123.75, 124.69, 125.41, 126.19, 127.74, 129.94, 130.17,
130.53, 133.02, 133.87, 134.24, 138.04, 150.73, 157.88,
161.16 ppm.
2.7. Binuclear copper(II) complex, 6
Fig. 3. ORTEP drawings of 7, the H-atoms and the un-coordinated triflate counter
ion has been removed for clarity (displacement ellipsoids are drawn at the 50%
probability level).
To a stirred solution of compound 5 (0.035 g, 46
lmol) in ace-
tone (5 cm3) was added a solution of copper(II) triflate (0.033 g,

192
O. Schicke et al. / Inorganica Chimica Acta 391 (2012) 189–194
urated acetonitrile solution of compound 7 affords deep blue crys-
tals suitable for a single crystal X-ray analysis. Crystal data for
complex 7, together with details of the X-ray diffraction experi-
ment are reported in Table 1 and an ORTEP representation in
Fig. 3. The X-structure reveals the presence of a coordinated aceto-
nitrile molecule and one water molecule; m
max/cmꢀ1 3436 (OH),
1612 and 1576 (C–Carom), 1223 (CF), 1027 (SO). Elemental Anal.
Calc. for C23H24CuF6N4O7S2 (MW 711.14): C, 38.85; H, 3.54; N,
7.88. Found: C, 38.78; H, 3.49; N, 7.91%.
2.9. Hydrolysis of BNPP catalyzed by copper complexes
In a typical kinetic experiment, 50
stock solution (20 mM) in buffer (50 mM MES for pH 5.38 and 6.24
and HEPES for pH 7.26) were added to 950 l of same buffer con-
taining copper(II) complex (6 50 M; 7 100 M) at 40 °C. Hydroly-
sis of BNPP was monitored by following the visible absorption
change at 400 nm (
= 18500 Mꢀ1 cmꢀ1) due to the release of p-
ll of a freshly prepared BNPP
l
l
l
e
nitrophenolate anion (PNPate). Conversion from absorbance to
concentration was performed by using the Lambert–Beer law (Eq.
(1)). To take into account the concentration change of PNPate in
function of pH, the [PNP]tot was calculated following Eqs. (1)–(4),
where pKa of p-nitrophenol (PNP) is 7.15 [27]. The activity of the
complex was determined in triplicate by the initial rate method.
OD
18500
½PNPateꢂmeas
¼
ð1Þ
ð2Þ
½PNPateꢂtot ¼ ½PNPateꢂmeas þ ½PNPꢂ
½PNPateꢂ
½PNPꢂmeas
pH ¼ 7:15 þ log
ð3Þ
ꢀ
ꢀ
ꢁ
½PNPꢂ
½PNPꢂtot
¼
¼
1 þ
1 þ
½PNPateꢂmeas
½PNPateꢂmeas
ꢁ
1
OD
18500
ð4Þ
10pHꢀ7:15
2.10. Crystallographic data
Scheme 2. Synthesis of the ligand 5 and the Cu(II) complex 6 (reagents and
conditions: (i) (CH2)2Br2, K2CO3, acetone reflux, 14%; (ii) AlLiH4, THF, 98%; (iii) PBr3,
CH2Cl2, 51%; (iv) BPA, diisopropylethylamine, ethyl acetate/CH2Cl2 (1/1), 47% and
(v) Cu(CF3SO3)2, acetone, 90%).
Single crystal X-ray diffraction data were collected on four cir-
cles Nonius Kappa-CCD diffractometer at 293 K, using monochro-
mated Mo
Ka radiation, k = 0.71073 Å. The structures were
refined using SHELXL-97 [28]. H-atoms of water molecules com-
plexed on copper ions of 6 were determined experimentally. One
triflate anion in compound 6 was found to be strongly disordered
and refined on several sites. The co-crystallized acetone molecule
in this compound was also disordered and the oxygen was refined
on 2 sites of occupancy equal to 0.5.
since methyl pamoate 1 is cheap and the by-products of the reac-
tion can be recovered. The cyclic ether 2 was reduced with LiAlH4
into alcohol 3 and then transformed into the dibromo compound 4
by a bromination step using phosphorus tribromide. Compound 4
afforded suitable crystal for X-ray structure determination after
recrystallized from chloroform. As expected, the 1,2-dioxoethano
bridge linking the two naphthyl moieties maintains the bromome-
thane groups in a close vicinity and confers an envelop shape to the
molecule (Fig. 1B). Finally, ligand 5 was prepared by N-alkylation
of N,N-bis(2-picolyl)amine (BPA) of the dibromo compound 4 in
the presence of N,N-diisopropylethylamine as base in a 1:1 mixture
of ethyl acetate:CH2Cl2 as solvent.
The diCu(II) complex 6 was obtained in good yield by treatment
of 1 equiv. of ligand 5 in acetone with 2 equiv. of copper(II) triflate.
A deep blue powder was collected by filtration and single crystals
suitable for X-ray structure determination were obtained by slow
evaporation of a saturated solution of 6 in acetone at room temper-
ature. Crystal data for complex 6, together with details of the X-ray
diffraction experiment are reported in Table 1 and selected bond
lengths and angles in Table 2. In compound 6 one copper complex
moiety is pointing inside the hydrophobic pocket, whereas the
3. Results and discussion
3.1. Synthesis and characterization
The ligand 5 was synthesized in four steps from methyl pamo-
ate 1 as described in Scheme 2. Methyl pamoate 1 was obtained by
esterification in methyl alcohol of the commercially available pa-
moic acid [25]. As revealed by the X-ray structure (Fig. 1A), methyl
pamoate 1 exhibits an important flexibility around the methano
bridge linking the two naphthyl moieties that takes away the
two ester groups. In order to rigidify the ligand skeleton and to
constrain the ester groups in close vicinity, the compound 1 was
transformed into the cyclic ether 2 by reaction with 1,2-dibromo-
ethane in the presence of potassium carbonate in refluxing ace-
tone. The poor yield (14%) in this step is nevertheless acceptable

O. Schicke et al. / Inorganica Chimica Acta 391 (2012) 189–194
193
Table 2
Cu(1)–N(1), N(1)–Cu(1)–N(2), N(6)–Cu(2)–N(4), and N(4)–Cu(2)–
N(5).
Selected bond lengths (Å) and angles (°) for 6.a
The mononuclear Cu(II) complex 7 was prepared in a similar
fashion by treatment of 1 equiv. of N-benzyl-bis-2-picolylamine
[26] in acetone with 1 equiv. of copper(II) triflate in good yield. A
deep blue powder was collected by filtration and single crystals
suitable for X-ray structure determination were obtained by slow
evaporation of a saturated solution of 7 in acetone at room temper-
ature (Fig. 3).
Cu(1) environment
Cu(2) environment
Bond lengths
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–N(1)
Cu(1)–O(3)w
Cu(1)–O(13)t
Cu(1)–O(1)
2.008(3)
1.990(3)
2.019(3)
1.971(3)
2.451(4)
2.593(3)
Cu(2)–N(5)
1.989(4)
1.987(4)
2.030(3)
1.959(3)
2.356(3)
2.561(3)
Cu(2)–N(6)
Cu(2)–N(4)
Cu(2)–O(4)w
Cu(2)–O(5)w
Cu(2)–O(2)
The X-band ESR spectra of 6 and 7 were recorded at 120 K in a
frozen acetone solution (Fig. 4). The simulations with automatic
parameter fitting were performed for axially symmetry environ-
ment and allowed us to get parameters corresponding to copper(II)
environment (g|| = 2.264, g1 = 2.050). The simulations with auto-
matic parameter fitting were performed for axial symmetry envi-
ronment and the obtained ESR constants are (hyperfine coupling
constants expressed in 10ꢀ4 T): complex 6: g|| = 2.264, A|| = 163.2,
Angles
N(2)–Cu(1)–O(13)t
O(13)t–Cu(1)–N(3)
N(3)–Cu(1)–O(1)
O(1)–Cu(1)–N(2)
N(2)–Cu(1)–O(3)w
O(3)w–Cu(1)–N(3)
N(3)–Cu(1)–N(1)
N(1)–Cu(1)–N(2)
N(1)–Cu(1)–O(13)t
O(13)t–Cu(1)–O(3)w
O(3)w–Cu(1)–O(1)
O(1)–Cu(1)–N(1)
91.91(16)
86.65(16)
88.73(14)
93.82(14)
98.94(14)
95.62(14)
83.14(14)
82.35(14)
95.76(15)
85.25(14)
90.25(14)
88.63(14)
N(5)–Cu(2)–O(5)w
O(5)w–Cu(2)–N(6)
N(6)–Cu(2)–O(2)
O(2)–Cu(2)–N(5)
N(5)–Cu(2)–O(4)w
O(4)w–Cu(2)–N(6)
N(6)–Cu(2)–N(4)
N(4)–Cu(2)–N(5)
N(4)–Cu(2)–O(5)w
O(5)w–Cu(2)–O(4)w
O(4)w–Cu(2)–O(2)
O(2)–Cu(2)–N(4)
87.29(16)
89.74(15)
89.91(14)
93.59(14)
98.84(18)
95.28(18)
83.42(15)
82.75(15)
92.84(14)
92.54(15)
85.22(14)
89.39(14)
g
g
= 2.057, A = 27.5 and complex 7: g|| = 2.260, A|| = 161.5,
1
1
1
= 2.054, A = 16.7. These constants are consistent with cop-
1
per(II) ions in pseudo-octahedral geometries elongated along z-
axis in accordance with the solid-state structures [29].
a
w and t stand respectively for water and triflate.
3.2. Phosphoesterase activity
other is pointing outside the cavity (Fig. 2). The naphthyl-rings
form a 79° angle and the Cu(1)–Cu(2) distance is 7.28 Å. Each cop-
per ion shows a pseudo octahedral geometry with equatorial posi-
tions occupied by the three nitrogen atoms of BPA and a water
molecule. Both copper(II) ions have an oxygen from the ether
bridge in axial position, the sixth coordination being occupied by
a triflate and a molecule of water for Cu(1) and Cu(2) respectively.
The axial Cu–O distances (ranging from 2.36 to 2.59 Å) observed
for both complexes are significantly longer than the equatorial
ones and are relevant of a strong Jahn–Teller distortion. The equa-
torial angles sum is equal to 360.05° for Cu(1) and 360.29° for
Cu(2) indicating no deviation from planarity. Due to the structure
of BPA (one methyl group between amine and pyridines) con-
straint angles are present (between 82.59° and 83.64°) for N(3)–
The bis(p-nitrophenyl)phosphate (BNPP) hydrolysis was used to
check the phosphoesterase activity of the copper complex 6. Since,
hydrolysis of BNPP by 6 proceeds cleanly to give p-nitrophenol
(PNP) and p-nitrophenyl phosphate (MNPP) as revealed by thin-
layer chromatography, the progress of the reaction was monitored
by the visible absorbance change at 400 nm (e )
= 18500 Mꢀ1 cmꢀ1
due to the release of p-nitrophenolate anion (PNPate). The activity
of the complex was determined by the initial rate method. The effi-
ciency of the copper complex 6 was studied in the 7.26–5.38 pH
range with respect to BNPP hydrolysis. Values of the observed ini-
tial rate for the appearance of PNPate are reported in Table 3 and in
Fig. 5 as a function of the pH.
We observed first that no noticeable hydrolysis occurs without
a copper complex in the 7.26–5.38 pH range (MES 50 mM for pH
5.38 and 6.24; HEPES 50 mM for pH 7.26; 10 min; 40 °C). While,
complex exhibited a moderate catalytic activity at pH 6.24 and
7.26, it hydrolyzed rapidly BNPP at pH 5.38 (Fig. 5A). Unfortu-
nately, the reaction stops after few minutes at 40 °C, probably
due to an inhibition of the catalyst by the reaction product (MNPP).
This phenomenon was already observed with other binuclear
1 106
5 105
Cu(II) complexes for which an inactive l-phosphato diCu(II) com-
plex could be isolated [21]. Activity of complex 6 was compared to
the one of complex 7. The result is remarkable since the mononu-
clear Cu(II) complex 7 exhibited a poor activity in the same range
of pH (Fig. 5B). The binuclear complex 6 is 41-fold more active
(Vobs6/Vobs7) than the mononuclear one 7 (Table 3).
The kinetic of BNPP hydrolysis by complex 6 deserves some
commentaries. First of all, the kinetic is not classical (Fig. 5A).
The dependence of initial rate (Vobs) on the BNPP concentrations
0
-5 105
-1 106
Table 3
pH dependence of 6 vs. 7 in BNPP hydrolysis.a
Complex 6
Complex 7
-1.5 106
pH
Vobs
(l
M sꢀ1
)
TONb
Conv. (%)
Vobs (l )
M sꢀ1
Vobs6/Vobs
7
5.38
6.24
7.26
85
13.8 0.9
5.1 0.1
3
11
1.6
1.2
24
3
2
2.09 0.05
2.05 0.09
1.99 0.06
41
7
3
Fig. 4. ESR spectra of complexes 6 (blue) and 7 (red) in acetone. ESR conditions:
120 K, 9.445 GHz, microwave power 20 mW, modulation frequency 100 kHz,
modulation amplitude 2G. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
a
Reaction conditions: [BNPP] = 2.25 mM; [6] = 50 M or [7] = 100 M; buf-
fer = 50 mM (HEPES pH 7.26, MES pH 6.24 and 5.38); 40 °C).
b
TON = [BNPP]trans/[6].

194
O. Schicke et al. / Inorganica Chimica Acta 391 (2012) 189–194
Another important feature of the copper(II) complex 6 is its
(A)
maximum activity at pH 5.38. With another binuclear copper(II)
complex, we observed a maximum of activity at pH 5.5–6 and
we showed that a bis(aqua)copper complex is responsible for BNPP
hydrolysis according to a double Lewis acid activation mechanism
(Scheme 1) [11].
4. Conclusion
We described the synthesis and characterization of a new dinu-
clear Cu(II) complex 6 which exhibits a remarkable activity in the
hydrolysis of activated phosphodiester BNPP with good conversion
yields (24%) and TON (11). These results are very promising for the
development of future more efficient metal catalysts for hydrolysis
of phosphodiesters.
Appendix A. Supplementary material
CCDC 655474, 655475, 655476 and 655477 contain the supple-
mentary crystallographic data for compounds 1, 4, 6 and 7, respec-
tively. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. Supplementary data associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.ica.2012.04.019.
(B)
NO2
O
O
O
OH
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P
O
O2N
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is not a hyperbolic curve as classically observed with other copper
complexes [11] and complex 7, but a sigmoid curve with a strong
inhibition by the substrate at high BNPP concentrations. This
behavior is generally encountered with allosteric enzymes that
have multiple binding sites. In these cases, the affinity for ligand
to one binding site is increased, upon the binding of ligand to an-
other binding site [30]. Cooperativity is also observed in oligomeric
proteins, when they undergo unfolding or unwinding during catal-
ysis [31].
The second commentary regards the remarkable enhancement
of the hydrolysis rate by 41 which clearly demonstrates a certain
cooperativity between the two Cu(II) centers during the hydrolysis
of BNPP. The X-ray structure of Cu(II) complex 6 revealed that Cu–
Cu distance is about 7.287 Å, which is a too long distance to envis-
age a cooperativity between both copper(II) centers as depicted in
Scheme 1 [3]. However, we can envisage that the binding of BNPP
to the copper centers favors a conformational change which allows
the shortening of the Cu–Cu distance to be compatible for a coop-
erativity in phosphate hydrolysis.