Remarkable reactivity of alkoxide/acetato-bridged binuclear copper(II) complex as artificial carboxylesterase

Article abstract of DOI:10.1007/s00775-017-1456-1

Abstract: Bromo-containing binuclear Schiff base copper(II) complex, Cu₂L(OAc), with an alkoxo/acetato-bridged moiety was employed as a model of carboxylesterases to promote the hydrolytic cleavage of p-nitrophenyl picolinate (PNPP). Furthermore, the reactivity of a mononuclear complex (CuHL) was evaluated for comparing it with that of binuclear one. The results reveal that the as-prepared binuclear Cu₂L(OAc) efficiently accelerated the hydrolysis of PNPP, giving rise to excess four orders of magnitude rate enhancement in contrast to the un-catalyzed reaction. Cu₂L(OAc) represented an enzyme-like bell-shaped pH-responsive kinetic behavior. Moreover, the binuclear one is more reactive than its mononuclear analogue (CuHL) by two orders of magnitude. The total efficiency of Cu₂L(OAc) is about 61-fold than that of its mononuclear analogue, CuHL. In addition, a contrast experiment reveals that binuclear Cu₂L(OAc) displayed good activity in the hydrolysis of PNPP as well another active ester, i.e., S-2-benzothiazolyl 2-amino-alpha-(methoxyimino)-4-thiazolethiolacetate (AE-active ester). Noteworthyly, it was found that mononuclear one inspired more obvious rate enhancement in the hydrolysis of AE-active ester relative to PNPP hydrolysis. The estimated pK_a1 of bound water on the binuclear Cu₂L(OAc) using second derivative method (SDM) is relatively smaller than that for CuHL by a gap of about 0.8 pK unit, which facilitates the hydrolysis of PNPP. Graphical Abstract: Four orders of magnitude rate enhancement was observed for the catalytic hydrolysis of p-nitrophenyl picolinate (PNPP) by one μ-alkoxide/acetato-bridged binuclear copper(II) complex under physiological conditions. Substrate specificity of the resulting binuclear complexes was observed for the hydrolysis of PNPP and AE-active ester.

Full text of DOI:10.1007/s00775-017-1456-1

J Biol Inorg Chem
DOI 10.1007/s00775-017-1456-1
ORIGINAL PAPER
Remarkable reactivity of alkoxide/acetato‑bridged binuclear
copper(II) complex as artificial carboxylesterase
Bin Xu1,2 · Weidong Jiang1,2 · Xiaoqiang Liu1,2 · Fuan Liu1,2 · Zheng Xiang1,2
Received: 25 January 2017 / Accepted: 28 March 2017
© SBIC 2017
Abstract Bromo-containing binuclear Schiff base
copper(II) complex, Cu2L(OAc), with an alkoxo/acetato-
bridged moiety was employed as a model of carboxylester-
ases to promote the hydrolytic cleavage of p-nitrophenyl
picolinate (PNPP). Furthermore, the reactivity of a mono-
nuclear complex (CuHL) was evaluated for comparing it
with that of binuclear one. The results reveal that the as-
prepared binuclear Cu2L(OAc) efficiently accelerated the
hydrolysis of PNPP, giving rise to excess four orders of
magnitude rate enhancement in contrast to the un-catalyzed
reaction. Cu2L(OAc) represented an enzyme-like bell-
shaped pH-responsive kinetic behavior. Moreover, the binu-
clear one is more reactive than its mononuclear analogue
(CuHL) by two orders of magnitude. The total efficiency
of Cu2L(OAc) is about 61-fold than that of its mononu-
clear analogue, CuHL. In addition, a contrast experiment
reveals that binuclear Cu2L(OAc) displayed good activity
in the hydrolysis of PNPP as well another active ester, i.e.,
S-2-benzothiazolyl 2-amino-alpha-(methoxyimino)-4-thi-
azolethiolacetate (AE-active ester). Noteworthyly, it was
found that mononuclear one inspired more obvious rate
enhancement in the hydrolysis of AE-active ester relative
to PNPP hydrolysis. The estimated pKa1 of bound water on
the binuclear Cu2L(OAc) using second derivative method
(SDM) is relatively smaller than that for CuHL by a gap of
about 0.8 pK unit, which facilitates the hydrolysis of PNPP.
Graphical Abstract Four orders of magnitude rate
enhancement was observed for the catalytic hydrolysis
of p-nitrophenyl picolinate (PNPP) by one μ-alkoxide/
acetato-bridged binuclear copper(II) complex under physi-
ological conditions. Substrate specificity of the resulting
binuclear complexes was observed for the hydrolysis of
PNPP and AE-active ester.
PNPP
1000
450
400
350
300
250
200
150
100
50
Cu2L(OAc)/PNPP
0.200mM
0.267mM
0.333mM
0.400mM
0.467mM
500
0
pKa1
Zero Baseline
pKa2
-500
-1000
-1500
0.467mM
0.400mM
0.333mM
0.267mM
0.200mM
0
6.5
7.0
7.5
8.0
8.5
pH
6.0
6.5
7.0
7.5
pH
8.0
8.5
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-017-1456-1) contains supplementary
material, which is available to authorized users.
Keywords Bimetallic cooperation · Artificial enzyme ·
Hydrolysis · Comparable kinetics
* Weidong Jiang
jwdxb@163.com
1
Introduction
School of Chemistry and Environmental Engineering,
Sichuan University of Science and Engineering,
Zigong 643000, China
Natural hydrolases containing multi-metal centers play
vital roles in some physiological activity [1], which
mainly involve in the selective and efficient cleavage of
2
Laboratory of Biomimetic Catalysis, Key Laboratory
of Green Catalysis of Sichuan Institute of High Education,
Zigong 643000, China
1 3

J Biol Inorg Chem
the esters, amides and DNA [2–4]. As noted in a number
of previous researches, remarkable catalytic efficiency
of hydrolases shall be ascribed to bimetallic synergetic
effect, hydrogen bond activation and hydrophobic effect
[5], and so on. Binuclear and poly-nuclear metal com-
plexes are dominative candidates for metallohydrolases
with enzymatic efficiency so far [6–13]. The main driving
motivation is that related researchers devoted to elucidate
the similar catalytic performances of those di- or poly-
nuclear complexes used. Moreover, major interests were
dedicated to simulating the structural features of catalytic
sites and binding sites [14]. Functional mimics of hydro-
lases are highly important for understanding the role of
metal ions in the hydrolytic mechanisms and developing
artificial hydrolases. In varied designing strategies for the
construction of multi-center moieties, bridging units (e.g.,
phenolate [15, 16], alkoxide [17], carboxylate [18], etc.)
were widely serviced as key components for the bridging
of metal ions.
as a frame of reference. To elucidate the structure–activity
relationship of the two complexes, molecular simulations
were performed by Gaussian program for comparing the
different coordination sphere of the as-prepared copper(II)
complexes. Calculated molecule structures confirm that
the binding site of binuclear Cu2L(OAc) is relatively open
relative to that of CuHL, which leads to an easier forma-
tion of reactive substrate–catalyst binary complex. Proba-
bly taking account to possibly distinguishable selectivities
and activities of the resulting catalysts towards different
substrates, herein we conducted one control experiment
concerning the hydrolysis of a selected active ester, i.e.,
S-2-benzothiazolyl 2-amino-alpha-(methoxyimino)-4-thia-
zolethiolacetate (AE-active ester) (part B of Fig. 1) which
widely acts as raw material of ceftriaxone and claforan,
mediated by the two copper(II) catalysts used in the pre-
sent work.
In our previous study [19], it was also found that an
alkoxo-bridged binuclear Cu(II) complex is more active
than its analogue with solo-metal center for excess two
orders of magnitude in the hydrolysis of scissile p-nitro-
phenyl picolinate (PNPP). Additionally, a phenoxo-
bridged binuclear Cu(II) complex with amino acid Schiff
base ligand exhibited two or three orders of magnitude rate
enhancement in comparison with spontaneous hydrolysis
of PNPP [20]. Inspired by structural properties of binu-
clear metalloenzymes, here we further investigated the
reactivity of one bromo-containing μ-alkoxo/acetato-
bridged binuclear copper(II) complex (Cu2L(OAc), Fig. 1)
towards the PNPP hydrolysis. This case is comparable for
the previous work concerning chloro-containing or non-
halogen catalysts [21]. Moreover, a mononuclear CuHL
(part A of Fig. 1) was screened during its catalytic activity
Experimental
Materials and instruments
All reagents were of analytical grade and used as sup-
plied, unless otherwise noted. 1,3-diamino-2-propanol,
Tris-H+ [tris(hydroxymethyl) aminomethane], and
potassium chloride were products from Sigma-Aldrich
Co. Triethylamine was purchased from Chengdu
Kelong Chemical Co. 5-Bromosalicylaldehyde was
prior product in our lab. The ligand, namely 1,3-bis(5-
bromosalicylideneamino)-propan-2-ol (H3L) was syn-
thesized according to the published procedure [22].
Binuclear Cu2L(OAc) and mononuclear CuHL were pre-
pared using the following modified methods described.
Highly purified-water with resistance of excess 18 MΩ,
Fig. 1 Schematic structures
of the resulting copper (II)
Part A
complexes (part A) and two
substrates (part B)
OH
Cu
N
O
N
O
N
N
Br
Br
Cu
O
Br
Br
O
Cu
O
O
N
O
(CuHL)
Cu2L(OAc)
Part B
H2N
S
S
O
O
O
N
N
S
N
O
NO2
AE-active ester
PNPP
1 3

J Biol Inorg Chem
which come from a Water Purification System (Nex
Power 1000, Human Corporation, South Korea), was
used throughout for kinetic runs. PNPP as substrate was
prepared by a prior art [23]. S-2-benzothiazolyl 2-amino-
alpha-(methoxyimino)-4-thiazolethiolacetate was pur-
chased from J and K Scientific Ltd. Ionic strength (I) was
calc. for C19H16Cu2Br2N2O5 639.80; found 640.81 (M + H),
662. 80 (M + Na). Anal. calcd. For C19H16Br2Cu2N2O5: C
35.70, H 2.52, N 4.38; found C 35.66, H 2.58, N 4.29%.
Synthesis of mononuclear CuHL
1
maintained at a constant value (0.1 M) with KCl. The H
CuHL was prepared by the reaction of 1,3-bis(5-
bromosalicylideneamino)-propan-2-ol in a 25 ml mixed
solvent of ethanol/DMSO (4:1 v/v) with 1 equiv. of
Cu(OAc)2·H2O solved in 40 ml of anhydrous ethanol. The
solution of the copper(II) acetate was added dropwise into
the solution of 1,3-bis(5-bromosalicylideneamino)-propan-
2-ol. The reaction mixture was kept stirring for 1 h and then
cooled at room temperature, yielding yellow-green powders
as crude product. Yellow-green powders as end product was
collected after further recrystallization of the crude prod-
ucts using anhydrous ethanol. CuHL: yellow-green solid,
NMR spectrum was determined at 400 MHz on a Bruker
Avance-400 NMR spectrometer (Bruke Corporation, Ger-
many). Mass spectra of the two complexes were recorded
on AGILENT 1100 LC–MS (Agilent, USA). Elemental
analysis was carried out on CHNOS Elemental Analyzer
Vario EL Cube (Germany Elementar Co.). Melting point
determination was conducted on X-5-type Digital Preci-
sion Melting Point Apparatus (Beijing Fukai Instrument
Co., Ltd., China). PNPP stock solution (0.02 M) was
freshly prepared with distilled acetonitrile. Hydrolysis
reaction of PNPP was detected by monitoring the appear-
ance of the product p-nitrophenolate anion at 400 nm on
a GBC Cintra 1010 UV–Vis spectrometer (Australia).
Optimal structures of both copper(II) complexes were
calculated by Gaussian 09 W software (Revision D.01).
yield of 94.06%. m. p. > 260 °C (dec.). IR(KBr, film)νmax
:
3396(O–H), 1621(C=N). m/z: calc. for C17H14CuBr2N2O3
516.86; found 517.85 (M + H), 539. 87 (M + Na). Anal.
calcd. For C17H14CuBr2N2O3: C 39.44, H 2.73, N 5.41;
found C 39.88, H 2.74, N 5.41%.
Synthesis
Hydrolysis kinetics
of 1,3‑bis(5‑bromo‑2‑salicylideneamino)‑propan‑2‑ol
(H3L)
Kinetic measurements of PNPP hydrolysis mediated by the
resulting copper complexes were performed in a pH range
6.50–8.50. Pseudo first-order rate constants (kob) were
determined by monitoring the absorbance changes of the
released p-nitrophenolate anions (PNP) at 400 nm for the
PNPP hydrolysis. Each kinetic run started with an injection
of substrate stock solution into a preheated buffered solu-
tion containing catalyst. For comparison, at a temperature
of 17 °C contrast kinetics of AE-active ester were carried
out. Moreover, during the current control runs, the final
concentration of AE-active ester was kept at a lower level
(0.033 mM) because AE-active ester with a higher concen-
tration is hardly soluble in the catalytic system. Hydrolysis
rates of AE-active ester were determined by the detection
of one hydrolytic product (2-mercaptobenzothiazole, abbr.
2-MBT) at 309 nm in buffered catalytic system containing
the binuclear Cu2L(OAc). The data reported are averages of
three runs with uncertainty of less than 5%.
Schiff base ligand (H3L) was synthesized by reaction of 1,
3-diamino-2-hydroxypropane and 5-bromosalicylaldehyde
in 1:2 molar ratio in anhydrous ethanol as solvent at 75 °C.
The mixture was stirred for 4 h, and cooled to room tem-
perature. The Schiff base compound (H3L) was obtained as
yellow crystals after a recrystallization of the crude products
with anhydrous methanol. H3L: yellow crystals with a yield
1
of 93.6%. m.p 161.2–161.8 °C. H NMR(DMSO) δ: 13.66
(s, 2H, ArOH), 8.51 (s, 2H, N=CH), 6.83–7.68(m, 6H, ArH),
5.26–5.27 (m, 1H, CH–CH2), 3.32–3.80(m, 4H, NCH2),
4.00(s, 1H, CH–OH); IR(KBr, film) νmax: 3432, 3060, 2895,
1637, 1476, 1274, 807. Anal. calcd. For C17H16N2Br2O3: C
44.76, H 3.54, N 6.14; found C 45.35, H 3.57, N 6.15%.
Synthesis of binuclear Cu2L(OAc)
For the preparation of the binuclear copper(II) complex, a
solution of 0.2 mmol Cu(OAc)2·H2O was prepared with
90 ml of methanol/water mixed solvent (5:1 v/v). After that
0.1 mmol Schiff base ligand solved in a 25 ml of methanol/
DMSO mixed solvent (4:1 v/v) was added into the resulting
solution containing copper salt. The mixed solution was stirred
for 2 h, and then allowed to evaporate at room temperature to
give blue-green crystals, which were collected and washed
with ethanol. Cu2L(OAc): blue-green solid, yield of 91.51%.
m. p. > 201.3 °C (dec.). IR(KBr, film)νmax: 1636(C=N). m/z:
Results and discussion
Hydrolysis rates of PNPP catalyzed by binuclear
and mononuclear copper(II) complexes
Owing to the unique characters and good performance of
bimetallic complexes in contrast with their mononuclear
analogues, many reports involved the design of binuclear
1 3

J Biol Inorg Chem
metal complexes with various ligands [24–26]. Herein, we
investigated the hydrolytic reactivity of a binuclear cop-
per (II) complex (Cu2L(OAc)) with an alkoxo-containing
compound (H3L). Shown in Fig. 2 are the pH-rate constant
profiles of PNPP hydrolysis by Cu2L(OAc) and the mono-
nuclear CuHL. The observations denote that the binuclear
Cu2L(OAc) efficiently accelerated the hydrolytic cleavage
of PNPP by a four-orders-of-magnitude rate enhancement
under almost neutral conditions (25 °C, pH 7.0) compared
To confirm if the prepared copper complexes still pos-
sess good activity in the catalytic hydrolysis of the other
esters, herein we comparatively evaluated the hydro-
lytic reactivity of binuclear copper complex towards
the hydrolysis of PNPP and AE-active ester as a con-
trast model substrate (Fig. 3) in the operated pH range
(6.50–8.50). It was found that binuclear copper complex
showed its better catalytic function in the hydrolysis of
the two substrates used here. This result may be ascribed
to good-activity of Cu2L(OAc) which results from the
bimetallic synergistic effect. Also, in term of both sub-
strates Cu2L(OAc) represents enzyme-like bell-shape
pH response. Thereupon, Cu2L(OAc) is considered as a
potential mimic model of natural carboxylases.
Additionally, under the same concentration
(0.033 mM) hydrolysis performance of AE-active ester
and PNPP by Cu2L(OAc) and CuHL was conducted at
selected pH (7.30 and 7.60), for evaluating their distin-
guishable activities of the two complexes. Experimental
observations provides us with two important informa-
tion concerning substrate selectivity and bimetallic coop-
eration (Fig. 4). That is, binuclear Cu2L(OAc) displayed
to the background rate (k0 = 7.8 × 10−6
s
−1) of PNPP
spontaneous hydrolysis [23].
Figure 2 shows that binuclear Cu2L(OAc) displays nota-
ble activity, giving rise to ca. 11031-fold rate enhance-
ment against an un-catalyzed reaction [23]. Unfortunately,
under comparable conditions the activity of the mononu-
clear one was less active than binuclear one, Cu2L(OAc).
Namely, CuHL acted as a poor catalyst only showing about
90-fold rate enhancement in comparison with the spon-
taneous hydrolysis of PNPP. The above-mentioned facts
provide evidence for the bimetallic cooperation of the two
copper ions, which are highly in agreement with previous
reports [19, 27]. Furthermore, the rate constants for the
Cu2L(OAc)-induced PNPP hydrolysis exhibit bell-shaped
curves as a function of pH, which has enzyme-like behavior
[28]. The best catalytic efficiency reached around pH 7.6,
followed by a sharp decrease in rate constant with further
increases in the pH value. This feature has an inseparable
relationship with the pH-dependent deprotonation of the
metal-bound water. As compared to that for binuclear com-
plex, a sigmoid-shaped changing tendency of hydrolysis
rates is represented for the CuHL-promoted hydrolysis of
PNPP. This observation implies that the maximum rate for
the PNPP hydrolysis induced by CuHL probably appears
only at above pH 8.50. Above changing tendency is almost
the same as in Fig. 1S that showed the pH-respondant vari-
ation of initial hydrolysis rate. Observations mentioned
above consists the pH-respond features of natural enzyme
possessing its own optimum range for pH where it will be
most active.
26
24
22
20
18
16
14
12
10
8
AE-active ester
PNPP
6
4
2
6.5
7.0
7.5
pH
8.0
8.5
Fig. 3 pH-dependent rate constants of the hydrolysis of PNPP and
AE-active ester induced by Cu2L(OAc) in the range of 6.50–8.50.
Conditions: 17 0.1 °C, [PNPP]final = 3.3 × 10−5 mol L−1, [com-
plex] = 1×10−5 mol L−1
450
400
350
300
250
200
150
100
50
Fig. 2 pH-dependent first-order
rate constants of the PNPP
hydrolysis by Cu2L(OAc)
(left) and CuHL (right) in
the PNPP concentration
30
0.200mM
0.267mM
0.333mM
0.400mM
0.467mM
0.200mM
0.267mM
25
0.333mM
0.400mM
20
0.467mM
range of 0.200–0.467 mM.
Conditions: 25 0.1 °C,
15
10
5
[PNPP]stock = 2×10−2 mol L−1
,
[complex] = 1×10−5 mol L−1
0
0
6.5
7.0
7.5
pH
8.0
8.5
6.5
7.0
7.5
pH
8.0
8.5
1 3

J Biol Inorg Chem
80
70
60
50
40
30
20
10
0
the tetrahedron transition state. As for our present study, the
divalent copper cores of the as-prepared copper(II) com-
plexes also play a similar role in the hydrolysis of PNPP as
a typical model of carboxylic acid esters.
PNPP/AE (for binuclear one)
AE/PNPP (for mononuclear one)
Bi-/Mono- (for AE)
Bi-/Mono- (for PNPP)
Part A of Scheme 1 shows a possible mechanism of the
PNPP hydrolysis promoted by Cu2L(OAc) during which
there may exist two kinds of hydrolysis modes. That is, the
PNPP hydrolysis occurs via a nucleophilic attack by H2O
bound to Cu(II) ion (not shown here) and its deprotonated
Cu(II)-hydroxyl form. As the operated pH is below the
systematic pKa1 of the bound H2O for Cu2L(OAc), lower
reaction rates were observed due to the worse nucleophi-
licity of bound water relative to nucleophilic reactivity
of the Cu(II)-linked hydroxyl forms. In contrast, sharp
increase in rate constant (see Fig. 2) appears when the
operated pH exceeds its inherent pKa1. In the hydrolytic
system, dihydrate complex undergoes a two-step deproto-
nation in which an active one-hydrated Cu(II)–OH− spe-
cies is involved. Afterwards, PNPP as substrate then links
to another Cu(II) core through a displacement of bound
H2O with PNPP. Efficient association of substrate to the
binding site of an enzyme is crucially important for gain-
ing remarkable efficiency due to a declined energy barrier
and shortened distance of PNPP to the catalytic nucleo-
philes [1]. Subsequently, an intramolecular nucleophilic
attack of the active Cu(II)–OH− on the carboxyl of PNPP
-10
7.3
7.6
Two selected pH values (pH 7.30 and pH 7.60)
Fig. 4 Relative hydrolytic rate of PNPP and AE-active ester induced
by Cu2L(OAc) and CuHL at selected pH values, i.e., 7.30, 7.60. Con-
ditions: 17 0.1 °C, [PNPP]final = 3.3 × 10−5 mol L−1, [AE-active
ester]final = 3.3 × 10−5 mol L−1, [complex] = 1×10−5 mol L−1
about 1.23-fold or 1.32-fold kinetic advantage for the
PNPP hydrolysis in contrast with AE-active ester. How-
ever, CuHL-promoted hydrolysis of AE-active ester is
approximately 4.43-fold or 5.78-fold faster than that of
PNPP, suggesting CuHL possesses much better selectiv-
ity in the hydrolysis of AE-active ester. Another aspect
represents that binuclear one is indeed a better catalyst to
accelerate the hydrolysis process of both substrates used
in this work. Moreover, Cu2L(OAc) showed more obvi-
ous advantage (about 50 or 68 time) compared to CuHL
for accelerating PNPP hydrolysis, whereas in the case of
hydrolysis of AE-active ester, declined activity differ-
ence between both catalysts was detected revealing about
ninefold difference in catalytic activity. In short, CuHL
is more efficient for catalyzing hydrolysis of AE-active
ester, and Cu2L(OAc) is able to play a more positive
role in accelerating hydrolysis of PNPP. This property
provides evidence for the substrate specificity of natu-
ral hydrolases [29–31], which confirms that the artificial
enzymes are required for favorable intrinsic structures so
that it match various substrates.
releases
a detectable chromophore (p-nitrophenolate
anion, pNP) which originates from the deprotonation of
p-nitrophenol in the operated pH region. Negative tetra-
hedron transition state (TS) can be stabilized by the posi-
tive copper(II) ion, which is beneficial to the rate accelera-
tion. Lastly, dihydrated species as the catalyst precursor is
regenerated by the displacement of picolinic acid with free
water, arousing new hydrolytic cycle. By the way, an obvi-
ous declining of rates shall be contributed to the forma-
tion of inactive dihydroxide form when the operated pH
beyond the pKa2 value.
In spite of a similar catalytic pathway for the PNPP
hydrolysis catalyzed by Cu2L(OAc) and CuHL (part B of
Scheme 1), there are distinguishable properties between
both the hydrolytic systems containing different cop-
per complexes due to their different structures. Bimetallic
sites of Cu2L(OAc) lead to a favorable catalytic mode of
the PNPP hydrolysis. First of all, the corresponding transi-
tion state is relatively stable owing to smaller steric repul-
sion for the Cu2L(OAc)-bearing system. Moreover, the two
Cu(II) ions of Cu2L(OAc) can efficiently act as the binding
site and the catalytic site, displaying a positive cooperative
bimetallic effect. As a result, Cu2L(OAc) is a highly active
catalyst for the hydrolysis of PNPP. In the case of CuHL,
however, both substrate (PNPP) and water molecule bound
to the same one copper ion, resulting in higher activation
energy (Ea) due to the bigger steric repulsion, as well as
Distinguishable structure–mechanism relationship
of PNPP hydrolysis by di‑ or mononuclear complexes
In the field of artificial metallohydrolases, it is widely
accepted that the metal-induced hydrolysis processes of
esters involve in the nucleophilic attack of the active metal-
OH species on the substrate bound to the central metal [32].
In the metal-mediated hydrolytic process, there are at least
three aspects of contributions of the central metals [33, 34],
which concerns (1) the binding activation of substrate; (2)
the activation of metal-bound H2O; (3) the stabilization of
1 3

J Biol Inorg Chem
Br
Br
N
N
O
(A)
O
O
Cu
Cu
H2O
O
O
Br
Br
Br
H+
Br
Br
H+
Br
N
N
N
N
N
N
O
O
O
Ka2
Ka1
O
O
O
O
O
O
Cu
Cu
OH2
(Dihydrate Form)
OH
Cu
H2O
(Active Form)
Br
Br
Cu
OH
Cu
Cu
OH
(Inactive Form)
H2O
HO
PNPP
Ks
N
O
Br
Br
Br
Br
fast
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
H2O
Cu
N
Cu
N
Cu
Cu
Cu
N
Cu
OH2
OH
OH
rate-determing step
O
O
OH
kcat
O
H2O
O
O
O
NO2
NO2
NO2
Transition State (TS)
(B)
OH
OH
OH
Br
Br
Br
Br
Br
Br
N
O
N
N
N
N
N
O
Cu
O
O
O
O
Cu
Cu
PNPP
Ks
N
OH
OH
H2O
OH
N
O
(Active Form)
O
O
O
NO2
NO2
Transition State (TS)
Scheme 1 Proposed mechanisms of the Cu2L(OAc)-promoted hydrolysis of PNPP. Part B only gives the formation of reactive PNPP-CuHL
binary complex and the tetrahedral transition state for clarity
a difficult cis-orientation controlling nucleophilic hydrox-
ide anion to substrate (Scheme 1). Thus, the CuHL-induced
PNPP hydrolysis is less reactive in comparison with
Cu2L(OAc).
solution in contrast with those in absolute ethanol. The
above-mentioned changed absorptions to some extent con-
firm the formation of dihydrated complexes, Cu2L(H2O)2
or CuHL(H2O)2. In short, UV–Vis spectral scans shown
in Fig. 5 demonstrate that the proposed mechanism of
PNPP by the prepared copper complexes is reasonable and
reliable.
Two characteristic absorption bands of the formed
2-MBT, which originated from the hydrolysis of AE-active
ester, respectively, locate at wavelength range of 230–240
and 308–320 nm [35]. As for the AE-active ester hydrol-
ysis mediated by Cu2L(OAc), strengthened absorption
(see Fig. 6) located at 230–240 nm indicates the forma-
tion of binary catalyst–substrate complex, which is highly
in accordance with the generation of PNPP–Cu2L(OAc)
intermediate (Fig. 5a). Moreover, UV scans show the sus-
tained increase in the strength (orange arrow) of both
characteristic peaks of 2-MBT, companying with gradual
decrease (green arrow) in the concentration of AE-active
ester. Changed absorption mentioned above suggests that
Cu2L(OAc) also efficiently catalyzes the hydrolysis cleav-
age of AE-active ester besides PNPP.
The spectral studies (Fig. 5) were performed for
grasping information about the hydrolytic routes of
PNPP mediated by Cu2L(OAc) and CuHL, respectively.
New strengthened spectra marked with PNPP-complex
(Cu2L(OAc) or CuHL) denote the formation of the reactive
Cu2L(OAc)-PNPP binary complexes. As the reaction time
was enlarged, one can find that the absorbance at 263 nm
gradually decreased suggesting the consumption of PNPP.
Meanwhile, the increased absorption at 400 nm indicates
the formation of p-nitrophenolate anions (pNP) as one of
the products. Also, two inserted pictures in Figs. 5a and c
showed distinguishable yellow with respect to the concen-
tration of the producing p-nitrophenolate anion at the same
time interval, providing direct evidence for the much bet-
ter activity of Cu2L(OAc) in comparison with CuHL. Fur-
thermore, the distinguishable spectra of the two copper(II)
complexes were obtained in absolute ethanol and aque-
ous solution, respectively. Namely, the absorption spectra
showed in Figs. 5a and d represents approximately 2.4 and
4.8 nm red shifts for Cu2L(OAc) and CuHL in aqueous
The kinetic parameters including catalytic constant
(kcat) and binding constant (Ks) were determined by the
1 3

J Biol Inorg Chem
Fig. 5 UV–Vis spectra (a and
c) for the hydrolysis of PNPP
by Cu2L(OAc) or CuHL. In
addition, the absorption spectra
(b and d) of the two copper
complexes was recorded in the
water and absolute ethanol,
respectively
(A)3.0
(B) 2.5
Cu2L(OAc)
PNPP+Cu2L(OAc)
Cu2L(OAc)
Water
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
2.0
1.5
1.0
0.5
0.0
Ethanol
pNP
PNPP
190
200
210
220
230
240
200
250
300
350
400
450
Wavelength
Wavelength (nm)
(D) 2.5
(C) 3.0
PNPP+CuHL
CuHL
CuHL
Water
Ethanol
2.0
2.5
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
pNP
PNPP
190
200
210
220
230
240
200
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
corresponding slopes and intercepts of the double-recipro-
cal plots in Fig. 7. The catalytic constant kcat (1111 s−1) for
the Cu2L(OAc)-induced PNPP hydrolysis is about 1.8 times
than that (625 s−1) for the CuHL-promoted hydrolysis of
PNPP. At the same time, the linkage of PNPP to binuclear
Cu2L(OAc) is much easier than that of PNPP to CuHL,
of esters. Meanwhile, the generation of this active spe-
cies is relevant to the systematic acid–base circumstance.
Appropriate pH is thereby required for natural hydrolases
to maintain the optimum enzyme efficiency and keep the
majority of enzymes from deactivation [37]. Conversely,
extreme pH condition may cause extensive deactivation
of enzymes due to a negative pH-dependant allosteric
structure.
In the hydrolysis of phosphate esters and carboxylates, it
is expected that the formation of the active metal-hydroxyl
species with a high concentration level at enzyme’s opti-
mum pH. According to the proposed mechanism (see
Scheme 1), there is a two-step deprotonation process of
the dihydrate copper(II) complex. The first deprotonation
of the one water bound to the divalent Cu(II) core pro-
duces the Cu(II)–OH− species with a dissociation constant,
Ka1. Furthermore, the ionization of another bound H2O to
the second central copper happens as the systematic pH
excesses its intrinsic dissociation constant (Ka2). In the cur-
rent work, Cu2L(OAc) displays good activity under almost
neutral condition, whereas CuHL is proved to be a worse
catalyst. This fact shall be traced to two main reasons.
Apart from the easier linkage of PNPP with the binding
site of Cu2L(OAc), the formation of the nucleophilic metal-
hydroxide species in the Cu2L(OAc)-containing system is
easier than that in another system in the presence of CuHL
under comparable conditions.
which can be supported by a 34-fold value (0.69 M−1
)
of binding constant compared to the latter (0.02 M−1).
According to enzymology theory, the formation of sub-
strate–catalyst binary complex intermediate highly facili-
tates the acceleration of enzyme reactions [36]. Calculated
total efficiency (kcatKs) of Cu2L(OAc) towards the PNPP
hydrolysis at pH 7.6 is approximately 769.2 s−1·M−1
,
exhibiting 61-fold kinetic advantage against CuHL. Hence,
in our case the high rates for the Cu2L(OAc)-catalyzed
hydrolysis of PNPP is partially ascribed to the relatively
convenient generation of reactive Cu2L(OAc)–PNPP inter-
mediates. The easier linkage of PNPP to Cu2L(OAc) shall
be contributed to the intrinsic structure of Cu2L(OAc),
which is confirmed by the optimal geometry shown in the
later section.
Feasible methodology for the determination of pKa
value of Cu(II)‑bound H2O
Metal-bound OH− species are the real active form of nucle-
ophilic attack on the substrate in the hydrolysis process
1 3

J Biol Inorg Chem
Fig. 6 Selected UV–
Vis absorption of the
Cu2L(OAc)-catalyzed hydroly-
sis of AE-active ester. Regions
a and b are respective enlarged
parts corresponding to those
selected regions marked with
dash square
curves for the two copper complexes-catalyzed hydrolysis
of PNPP. For each substrate concentration level, the desired
pKa1 can be obtained by the corresponding pH value which
corresponds to the second derivative with a zero value.
That is to say, each value of pKa1 can be determined by the
x-axis value of the intersection point of the depicted zero
baseline and each second derivative curve. Table 1 summa-
rizes all of pKa1 values determined with SDM.
Cu2L(OAc)
400
CuHL
300
Y =0.0016+0.0794 X
R2=0.9918
200
100
0
(pH 7.60)
The data shown in Table 1 give us at least two aspects
of information. The one is that the central copper(II) ion
efficiently activates the bound water with a lower pKa1
value, which benefits the nucleophilic attack of the Cu(II)-
bound hydroxyl on the PNPP under weak-basic condition.
Another one is that binuclear Cu2L(OAc) possesses more
powerful ability to lower the pKa1 of the bound water in
contrast with CuHL. The pKa1 value of the H2O bound to
Cu2L(OAc) around 7.20, and the pKa1 of CuHL-bound
H2O is about 8.0. Notably, the pKa1 values of bound water
in Table 1 were found to be almost coincident, regardless
of CuL(OAc) or CuHL demonstrating that the deproto-
nation process of the bound H2O prior to the linkage of
PNPP with metal complex. This experimental evidence
-100
-200
Y =0.0009+0.0013 X
R2=0.9959
2000 2500 3000 3500 4000 4500 5000
1/[PNPP] (mol-1 L)
Fig. 7 Linear curve fitting of k−ob1-[PNPP]−1 for the hydrolysis of
PNPP by Cu2L(OAc) or CuHL, respectively. Conditions are the same
as in Fig. 2
Herein, we attempt to adopt a second derivative method
(SDM) [38] acted as an applicable and convenient one for
the determination of pKa1 of bound H2O. Figure 8 gives the
respective second derivatives profiles responding to kob-pH
1 3

J Biol Inorg Chem
Table 1 Determined pKa1 values of the bound water using second
derivative method
for acting as the catalytic site and binding site of metallo-
hydrolases. This is in accordance with the feasible metal–
metal distance (3.0–4.0 Å) of natural metalloenzymes [41].
Hence, positive bimetallic synergic effect [42] plays a vital
role in the hydrolysis of PNPP and AE-active ester medi-
ated by Cu2L(OAc), leading to dominated reactivity of
Cu2L(OAc) against CuHL.
[PNPP] (mM)
pKa1 values of the Cu(II)-bound
H2O
Cu2L(OAc)
CuHL
0.200
0.267
0.333
0.400
0.467
7.20
7.16
7.15
7.15
7.15
7.98
7.99
7.99
7.97
7.98
Based on the following equation, pKa = −log(Kw/
[H2O]), in which Kw is about 1.0 × 10−13.79 [43, 44] and
[H2O] equals to 55.5 M, the determined pKa of free water
at 0.1 M ionic strength (KCl) is about 15.5 at 25 °C. As a
result, the high pKa (15.5) reveals the highly weak depro-
tonation of water. For hydrolysis reactions of some com-
pounds including esters mediated by various metallohy-
drases, the metal-bound hydroxide forms shall be the best
nucleophilic reagent rather than metal-bound water and
free water. Therefore, it is a challenge that the designs
of artificial metalloenzymes with high activity and abil-
ity of activating the bound water. In the present study,
Cu2L(OAc) gives an obvious pKa declining by about eight
pK units relative to that of free H2O, which is in favor of
the nucleophilic attack of PNPP by the Cu(II)–OH− spe-
cies even under physiological conditions (pH 7.0, 25 °C).
At some extent Cu2L(OAc) is able to mimic the binuclear
metal centers of natural metalloenzymes. As the result of
those factors mentioned above, it is comprehensible that
binuclear Cu2L(OAc) displays better activity than CuHL in
the hydrolysis of PNPP.
By the way, we can find that the catalytic activity of
the bromo-containing Cu2L(OAc) in this case is relatively
lower in comparison with that analogue [21] with chloride
substituents. Two aspects of factors should be considered.
The one may be that the much bigger steric hindrance of
bromine group blocks the association of PNPP with the
central copper, which is negative for the PNPP hydrolysis.
Another one is that the deprotonation of the bound water
on the copper site of bromo-containing Cu2L(OAc) is
more difficult than that of chloro-bearing analogue, which
can be confirmed by the bigger pKa1 (~7.2) of the bound
water on the former compared to the latter (pKa1 = 7.0).
This phenomenon probably results from the stronger
Conditions are the same as in Fig. 2
strongly supports the proposed mechanism shown in
Scheme 1. Indeed, one can draw a conclusion that the
difference in pKa1 of 0.8 pK unit probably acts as a cru-
cial factor for highly prompting the catalytic activity of
Cu2L(OAc).
Structure–activity relationship of the As‑prepared
copper(II) complexes
Those researches concerning the simulation of natural met-
alloenzymes provide invaluable resources for designing and
synthesizing more novel and efficient artificial enzymes
[39]. Especially, poly-nuclear metal complexes containing
oxygen-bridged copper(II) are of particular interest because
they might represent valuable model compounds for the
active site of a number of metalloenzymes [40].
In our work, binuclear Cu2L(OAc) is found to be more
reactive than its mononuclear analogue, CuHL. Under the
same conditions (pH 7.0, 25 °C), the relative reactivity of
Cu2L(OAc) to CuHL is up to 122.9. Additionally, optimum
efficiency of Cu2L(OAc) is achieved at pH 7.6, while that
of CuHL probably reaches at pH > 8.20. This fact shall be
attributed to their different structures of Cu2L(OAc) and
CuHL. The two oxygen-bridged copper moieties of the
former are close to each other, resulting in an appropri-
ate Cu(II)…Cu(II) distance (~3.6Å, a calculated distance
from molecule structure optimization shown in Fig. 2S)
80
1000
500
Fig. 8 Profiles of the sec-
ond derivatives of kob values
as a function of pH for the
Cu2L(OAc)- and CuHL-con-
taining systems, respectively
Cu2L(OAc)/PNPP
CuHL/PNPP
60
40
pKa1
20
0
pKa1
0
Zero Baseline
Zero Baseline
0.467mM
0.400mM
0.333mM
0.267mM
0.200mM
pKa2
-500
-1000
-1500
-20
-40
-60
-80
0.467mM
0.400mM
0.333mM
0.267mM
0.200mM
6.0
6.5
7.0
7.5
8.0
8.5
6.0
6.5
7.0
7.5
pH
8.0
8.5
9.0
pH
1 3

J Biol Inorg Chem
140
120
100
80
ester, a kind of raw material for a few medicines (ceftri-
axone and claforan). On the contrary, mononuclear CuHL
proves to be not a good catalyst for the PNPP hydrolysis
due to its instinctive structure. Interestingly, CuHL exhib-
ited much better activity towards AE-active ester hydrolysis
in contrast to PNPP. These facts highly testify that higher
substrate-catalyst matching level is beneficial to the reac-
tion acceleration. Our research will impel the construc-
tion of novel mimic enzymes with various bridged units.
Structural optimum confirms the more open binding site
of Cu2L(OAc) that results in easier linkage of PNPP with
Cu2L(OAc). In addition, we prospect that the introduction
of more eletron-withdrawing substituent groups into the
ligand molecules should upgrade the activity of designed
artificial metallohydrolases.
pH 7.0
0.200mM
0.267mM
0.333mM
0.400mM
0.467mM
60
40
20
0
6.5
7.0
7.5
pH
8.0
8.5
Fig. 9 pH-respondant relative rates of the hydrolysis of PNPP by
Cu2L(OAc) and CuHL in the PNPP concentration range of 0.200–
0.467 mM. Conditions: 25 0.1 °C, [PNPP]stock = 2×10−2 mol L−1
[complex] = 1×10−5 mol L−1
,
Acknowledgements This work gratefully thanks financial sup-
ports from the Key Project of Key Laboratory of Green Catalysis of
Sichuan Institutes of High Education (LZJ1303) and the Key Project
of Zigong Science & Technology Bureau (2014HX12, 2015HX21).
electron-withdrawing property of the chlorine atom in con-
trast with bromine atom, resulting in the increasing Lewis
acidity of the central copper inside the chloro-bearing binu-
clear complex.
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Figure 9 shows the relative values of the hydrolysis rate
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For instant, it was observed that about 123-fold difference
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1 3