Phosphate ester hydrolysis catalyzed by a dinuclear cobalt(II) complex equipped with intramolecular β-cyclodextrins

Article abstract of DOI:10.1016/j.molcata.2014.10.020

Dinuclear metallohydrolases are ubiquitously involved in biological transformations. For understanding and application purpose, significant efforts have been dedicated to the structural or/and functional mimics. Herein, a dinuclear Co(II) complex, Co

Full text of DOI:10.1016/j.molcata.2014.10.020

Journal of Molecular Catalysis A: Chemical 396 (2015) 346–352
Contents lists available at ScienceDirect
Journal of Molecular Catalysis A: Chemical
journal homepage: www.elsevier.com/locate/molcata
Phosphate ester hydrolysis catalyzed by a dinuclear cobalt(II)
complex equipped with intramolecular ␤-cyclodextrins
Meng Zhaoa,b, Shan-Shan Xue , Xue-Qin Jiang , Limin Zhengb,∗, Liang-Nian Jia,
Zong-Wan Mao
a
a
a
a,∗∗
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275,
China
b
Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University,
Guangzhou 510275, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 May 2014
Received in revised form 14 October 2014
Accepted 19 October 2014
Available online 31 October 2014
Dinuclear metallohydrolases are ubiquitously involved in biological transformations. For understand-
ing and application purpose, significant efforts have been dedicated to the structural or/and functional
mimics. Herein, a dinuclear Co(II) complex, Co2L, with intramolecular ␤-CDs is presented. The ␤-CDs
confine a hydrophobic microenvironment that contributes to the valence stability of the ligated Co(II)
ions. Species of Co2L in aqueous solution is clarified according to the results of potentiometric titration
and UV–vis titration experiments. A highly positive-charged species [Co2HL] is present at quasi-neutral
condition, which contributed significantly to the binding and activity toward bis(4-nitrophenyl) phos-
phate (BNPP) assisted by the hydrophobic interactions exerted by intramolecular ␤-CDs. The kcat/KM value
4
+
Keywords:
Dinuclear cobalt(II) complex
Cyclodextrin
Phosphate ester
Hydrolysis
Kinetic
−
2
−1 −1
of Co2L is determined to be 9.8 × 10
M s at pH 6.5 and 308 K, which exceeds that of the reported
zinc analog (Zn2L) by three orders of magnitude. Unexpectedly, Co2L also catalyzes hydrolysis of a phos-
phate monoester, 4-nitrophenyl phosphate (NPP). The observed phosphate esterase activities are rarely
reported among synthetic Co(II) complexes.
©
2014 Elsevier B.V. All rights reserved.
1
. Introduction
residues of Glu235 and Asp108 and a hydroxide (or water). In
another case, glycerophosphodiesterase (GpdQ) probably employs
two Co(II) ions to catalyze the hydrolysis of phosphate esters.
Recent studies on GpdQ indicated the nucleophilic agent was the
terminal metal-bound hydroxide (M-OH) that was activated by the
non-covalent interactions within the second coordination sphere
[10–13].
To gain insights into the enzymatic catalysis, a large number of
simple dinuclear cobalt complexes were synthesized and explored
as the structural or/and functional models. Amongst those, the
functional ones were almost exclusively Co(III)-involved com-
plexes, which took advantages of the high positive charge of Co(III)
to activate substrates and/or nucleophiles [14–20]. In contrast, the
Co(II)-involved ones that performed activity on hydrolysis of phos-
phate ester bonds were rarely reported [21,22], although there
were some Co(II) complexes explored to probe the electronic and
magnetic properties [7,8,11,23–26]. A common problem for Co(II)
model complexes is their oxidation in air. To address this issue,
some studies incorporated intramolecular hydrophobic groups in
Co(II) complexes to keep Co(II) from oxidation [25].
Dinuclear metal centers are ubiquitous at the active sites of
metallohydrolases [1–3]. They uniformly comprise two first row
transition metal ions that are positioned in close proximity. The
advantage of the dinuclear organization rests in the cooperativ-
ity between the metal ions [4], leading to the highly accelerated
hydrolysis of inert biological macromolecules such as nucleic acids
and protein under mild conditions. The most commonly encoun-
tered metal ions in native metallohydrolases are zinc ions [5,6].
In many cases, replacing zinc ions with Co(II) ions leads to the
fully restored activity [7,8]. There are indeed native metallohydro-
lases that employ Co(II) ions as the catalytic cofactors. For example,
methionine aminopeptidase (MetAP) is a kind of metallohydro-
lase that removes the N-terminal methionine of nascent proteins
[
9]. Its dinuclear Co(II) core (Fig. 1) is bridged by two carboxylic
∗
Corresponding author. Tel.: +86 20 84115531; fax: +86 20 84112169.
Corresponding author. Tel.: +86 20 84113788; fax: +86 20 84112245.
E-mail addresses: ce04zhm@mail2.sysu.edu.cn (M. Zhao),
∗
∗
Over the past years, strategies were proposed to construct a sec-
ond coordination sphere that adjusted properties and functions of
zhenglm@mail.sysu.edu.cn (L. Zheng), cesmzw@mail.sysu.edu.cn (Z.-W. Mao).
http://dx.doi.org/10.1016/j.molcata.2014.10.020
1
381-1169/© 2014 Elsevier B.V. All rights reserved.

M. Zhao et al. / Journal of Molecular Catalysis A: Chemical 396 (2015) 346–352
347
100
+
Co(II)
[Co L(OH) ]
2
2
8
6
4
2
0
0
0
0
0
2+
[
Co L(OH)]
2
Fig. 1. The protein structure (left) and the coordination structure at the catalytic
center (right) of MetAP (PDB: 2MAT). Color codes: blue, N; red, O; cyan, C. (For
interpretation of the references to color in this figure caption, the reader is referred
to the web version of this article.)
3
4
5
6
7
8
9
10 11
pH
Fig. 3. Species distribution plots of Co2L as a function of pH in aqueous solution
containing 0.1 M NaClO4 at 298 ± 0.1 K. (For interpretation of the references to color
in the text, the reader is referred to the web version of this article.)
metal complexes [27]. An applicable way was to build hydrophobic
domains to shield metal centers [28,29]. Alternatively, employ-
ing the supramolecular hosts such as cyclodextrins (CDs) [30–32],
calixarenes [33–35], and cucurbiturils [36–38] was feasible and
applicable. Our past work [27] indicated, by covalently or non-
covalently coupling metal complexes to ␤-CDs, the properties and
functions of metalloenzyme mimics were improved, reflected in
enhancement of catalytic activity [39–42], increase of substrate
specificity [43] and activation of reactive species [44–46]. Specifi-
cally, the first dinuclear zinc complex conjugated with bis-␤-CDs,
dinuclear Co(II) site [25]. By adjusting the solution pH in a wide
range in air, the spectrum could be fully recovered and no charac-
teristic Co(III) LMCT assigned at 350–600 nm was observed [25],
excluding the oxidation of Co(II). The static magnetic moment
of polycrystalline samples of Co L was measured using a SQUID
magnetometer in the temperature range 2–300 K at 5000 Oe
Fig. S3, supporting information). At room temperature, ꢀT
value of 4.21 cm K mol
of 3.75 cm K mol
2
(
3
−1
was higher than the spin-only value
−1
expected for two uncoupled (S = 3/2, g = 2),
Zn L (Fig. 2), performed effective phosphodiesterase activity and
2
3
specificity [43], and the copper complex, Cu L (Fig. 2), possessed a
2
which revealed that there existed a considerable contribution
from the orbital angular momentum. Upon cooling, ꢀT decreased,
first slowly and then faster, reaching a minimum value of ca.
surprisingly low pKa of the metal-bound hydroxide (M-OH), con-
tributed by the non-covalent interactions exerted by ␤-CDs [46].
Herein, we present a valence-stable dinuclear Co(II) complex, Co2L
3
−1
0
.99 cm K mol
at 2.0 K. The observed decrease of ꢀT was due
(Fig. 2), which performs not only phosphodiesterase activity but
to the antiferromagnetic interactions between Co(II) ions and the
zero field splitting effect of the Co(II) ion. Referring to the reported
case [25], we proposed that in our case it was the hydrophobic
microsphere confined by the intramolecular ␤-CDs that stabilized
the dinuclear Co(II) center.
also phosphatase activity that is even lesser observed for divalent
metal complexes [1].
2
. Results and discussions
2.1. Synthesis and characterization
2
.2. Species of Co L in aqueous solution
2
Co L was synthesized by mixing the dinucleating ligand HL
2
[
43,46] with two equivalents of Co(ClO4)2 in aqueous solution in
2.2.1. Potentiometric titration
dinitrogen atmosphere. The IR spectra of Co L (Fig. S1, supporting
Owing to the valence stability of Co L, it was legal to inves-
2
2
information) showed the absorption band of the p-methyl phenol
moiety shifted relative to that of HL, which was indicative of the
phenol deprotonation in parallel with the coordination of Co(II)
ions. Elemental analysis result (Section 4.4) was in consistent
with the deprotonation of phenol group and the coordination
of two Co(II) ions. ESI MS characterization (Fig. S2, supporting
information) gave the solid evidence of dinuclear Co(II) species in
tigate the species in aqueous solution in a broad pH range by
means of potentiometric titration method. The calculation model
turned out to be very well fitted to the experimental data (Fig.
S4, supporting information). The derived species distribution plots
(Fig. 3) showed the complexation of HL with two equivalents of
Co(II) ions occurred at pH > 4. Three tandem deprotonation pro-
cesses were observed: the first corresponded to the proton release
from the phenolic group of ligand HL; the latter two cases corre-
sponded to the deprotonations of the metal-bound water ligands.
solution. The UV–vis spectrums (Section 2.2.2) of Co L showed the
2
absorption at 303 nm, the diagnostic LMCT of phenoxide-bridged
Fig. 2. Synthesis of the dinuclear divalent metal complexes explored as phosphate esterase mimics.

3
48
M. Zhao et al. / Journal of Molecular Catalysis A: Chemical 396 (2015) 346–352
Fig. 4. (A) pH-dependent UV–vis spectrums of Co2L; (B) Varied absorbance at 252 and 305 nm of Co2L at adjusted pH from 3.1 to 10.9. Conditions: [Co2L] = 1.1 mM and
T = 298 ± 0.1 K. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
Table 1
2
.2.2. pH-dependent UV–vis spectrums of Co L in aqueous
2
Equilibrium constants of Co2L and Zn2L in aqueous solution at 298 K.
solution
To gain more evidences of Co2L species in aqueous solution, the
Equilibriums
Constants
Metal ions
Co(II)
pH-dependent UV–vis spectrums were taken into account. Absorp-
tion changes at 252 and 305 nm that were assigned to pyridine and
phenol moieties were plotted in Fig. 4. In ranges of pH 3.0–6.5 and
8.0–10.5, the absorption changes at 252 and 305 nm were in paral-
lel. The “S” curves (shown in black curve in Fig. 4B) corresponded
Zn(II)a
MII + HL ↔ [M2HL]
4+
8.9
6.9
8.9
8.4
5.5
7.5
9.9
2
logKM2
L
M2HL] ↔ [M2L] + H+
4
+
3+
pKa1
pKa2
pKa3
[
[
[
M2L]3 + H2O ↔ [M2L(OH)] + H+
+
2+
M2L(OH)] + H2O ↔ [M2L(OH)2] + H+
2
+
+
10.5
4
+
to the complex formation equilibrium 2Co(II) + HL ↔ [Co HL]
,
a
2
The data were selected from Ref. [43].
log K
= 8.9, representing the coordination of pyridine and phe-
Co
L
2
nolic donors to Co(II) ions. The linear increase (shown in blue line
in Fig. 4) at pH > 8.0 corresponded to the two tandem deprotona-
3
+
2+
+
The whole distribution picture of Co L was akin to that of Zn L
tion processes: [Co L] + H O ↔ [Co L(OH)] + H , pK = 8.9 and
2
2
2
2
2
a2
2+ + +
[
43], making it reasonable to evaluate the metal effect on the
[Co L(OH)] + H O ↔ [Co L(OH) ] + H , pK = 10.5. The constant
2
2
2
2
a3
thermodynamic.
As shown in Table 1, the metal complex stability constant
log KM2L of Co L is slightly higher than that of Zn L, indicating
the similar binding affinity toward the dinucleating ligand HL. The
deprotonation constants pKas of Co L are higher than those of
linear increase suggested the two deprotonation processes hap-
pened at two symmetrical Co(II) sites in order. A notable difference
is observed between pH 6.5 and pH 8.0 (shown in red line in Fig. 4B)
2
2
4+
3+
+
which corresponded to [Co HL] ↔ [M L] + H , pK = 6.9. In this
2
2
a3
2
range, the absorption at 305 nm kept constant while the absorption
at 252 nm decreased, indicating that the coordination of phenoxide
was not perturbed by the deprotonation while the coordination of
pyridine was affected. This was consistent with the above specula-
tion that an intramolecular proton shift was present in prior to the
Zn L. It could be rationalized by the weaker Lewis acidity of Co(II)
2
ions than Zn(II) ions. In principle, the phenol group of HL should
deprotonate into L for adopting the two Co(II) ions. However, the
substantial speciation of [Co HL] at neutral pH (red line in Fig. 3)
−
4+
2
4+
signified there should be intramolecular proton shift from the phe-
nol moiety to the other sites of the ligand in prior to the metal
coordination. With respect to basicity and proximity, the tertiary
amine (marked in blue in Fig. 5) would accept the phenolic proton.
In response to this speculation, Hernandez-Gil et al. [22] recently
reported the protonation of the tertiary amine of a binucleating
ligand, which gave the additional supports to the intramolecular
proton shift. The resulted tertiary ammonium was deprotonated at
deprotonation of [Co HL] .
2
Gathering up the threads, the acid–base equilibriums of Co2L
in aqueous solution was proposed in Fig. 5. In free ligand HL, an
intramolecular hydrogen bond (dashed line) that complemented
a six-member ring might be present, leaving the phenolic proton
readily shifting to the proximate nitrogen (marked in blue) in paral-
4
+
lel with the coordination of Co(II) ions. At pH < 7.0 where [Co HL]
2
was predominant, the protonated ammonium dissociated from the
Co(II) ion, leaving pyridine-Co(II) coordination bond strengthened
(shown in bold). As pH increased, the deprotonation of the ammo-
nium (pKa1) proceeded. The resultant tertiary amine recovered
its coordination to Co(II) ion, weakening the pyridine-Co(II) bond,
3
+
pH > 7.0, yielding [Co L] . As pH further increased, the water lig-
ands were activated, forming the metal-bound hydroxide species,
2
2
+
+
[
Co L(OH)] and [Co L(OH) ] . These nucleophile species implied
2 2 2
that Co L should be an active hydrolytic catalyst.
2
Fig. 5. Intramolecular proton shift, complex formation and acid–base equilibriums of Co2L in aqueous solution. Water/hydroxide ligands involved in the acid–base equili-
briums are only shown. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)

M. Zhao et al. / Journal of Molecular Catalysis A: Chemical 396 (2015) 346–352
349
reflected in the changed absorption at 252 nm (Fig. 4B). In brief,
the tertiary amine nitrogen switched its role from a proton relay to
a ligand donor. Further deprotonation (pKa2 and pKa3) at higher
pH leads to the activation of metal-bound water ligands, yield-
ing the potentially nucleophilic metal-bound hydroxide species,
1.00
0.75
0.50
0.25
0.00
2
+
+
[
Co L(OH)] and [Co L(OH)] , which were conventionally defined
2 2
as the active species of metallohydrolases [47].
2.3. Kinetic studies on phosphate ester hydrolysis
Phosphodiesterase activities of Zn L and Cu L were previously
2
2
studied on bis(4-nitrophenyl) phosphate (BNPP) [43,46]. It was
found the cooperativity between the double Lewis-acid activations
of metal ions and the non-covalent interactions of ␤-CDs played
important roles in catalytic hydrolysis of phosphate esters. To
further investigate the relationship between metal catalysis and
assistant intramolecular non-covalent interactions, kinetic studies
0
1
2
3
[Co(II)]/ [HL]
Fig. 6. The metal-to-ligand ration effect on BNPP hydrolysis catalyzed by Co2L. v
represents the rates determined at [Co(II)]/[HL] = 2.
0
of phosphodiesterase activity of Co L were carried out by examin-
2
ing the same substrate.
Fig. 7. Saturation kinetics of BNPP hydrolysis catalyzed by Co2L at I = 0.1 M (NaClO4) and 308 ± 0.1 K. Solid line: fitting according to Michaelis–Menten equation. Inset:
Lineweavor–Burk double-reciprocal plots.

3
50
M. Zhao et al. / Journal of Molecular Catalysis A: Chemical 396 (2015) 346–352
ESI MS was applied to characterize the species of Co L upon
2
incubation with BNPP. The hydrolytic product NP (4-nitrophenol)
and the adductive Co L-NPP (4-nitrophenyl phosphate) were
2
observed (Fig. S5, supporting information), confirming the
hydrolytic cleavage of the phosphate diester bonds of BNPP. Con-
trol experiment indicated activity of ligand HL could be neglected as
compared to Co L (Fig. S6, supporting information). By changing the
2
metal-to-ligand ratio (Fig. 6), it was found the reaction rate reached
the highest until the ratio was increased to two. Further addition
of Co(II) ions did not increase the activity. This result confirmed
the cooperativity between the two intramolecular Co(II) ions in
hydrolyzing the phosphate diester bonds.
By changing the substrate concentrations, further insights into
the catalytic mechanisms could be gained. In Fig. 7, rates increased
non-linearly as the substrate concentration increased. Characteris-
tic Michaelis–Menten kinetics were observed, indicating Michaelis
Fig. 8. The logarithmic values of second-order-rate constants of BNPP hydrolysis
complex Co L-BNPP was pre-formed in prior to the catalytic cleav-
catalyzed by Cu2L, Zn2L and Co2L.
2
age step. This result was in good response to the characterized
adductive complex Co L-NPP (Fig. S5, supporting information) that
should be the downstream product of the labile Michaelis com-
2
2+
[
Cu L(OH)] involved in the catalysis, which was prevalent in a
2
wide pH range [46].
plex Co L-BNPP. Michaelis–Menten kinetics were also observed
2
in cases of Zn L and Cu L [43,46]. This common character under-
2
2
2
.5. Phosphatase-like activity
lined the advantage of substrate-binding gained by incorporating
-CDs. The kinetic parameters of Co L-catalyzed BNPP hydrol-
␤
2
There were a great number of synthetic metal complexes capa-
ysis under different pHs were gathered in Table 2. In column
−
1
ble of hydrolyzing phosphate diester bonds [52–55]. In contrast,
quite less metal complexes were capable of cleaving phosphate
monoester bonds which was indispensable for biological phos-
phate metabolism [1]. Koike et al. [56] reported on the dinuclear
zinc cryptate that cleaved the phosphate monoester bond. Recently,
Cao and Lippard et al. used tripodal trinuclear metal complexes
to anchor phosphate anion. Thereby, the phsophate monoester
could be cleaved [57,58]. We also reported on a mononuclear zinc-
bis-␤-cyclodextrins complex that performed the phosphatase-like
of kcat (s ), the values did not change much as pH increased,
4+
3+
2+
implying the present species [Co HL] , [Co L] and [Co L(OH)]
2
2
2
in the tested pH range are comparably active [48]. Difference
was noted in column of Michaelis constants KM (M), where the
value examined at pH 6.5 was an order of magnitude lower
than those examined at higher pHs. This result indicated that
Co L bound to BNPP at pH 6.5 much stronger than it did at
2
higher pHs. A reasonable interpretation was the highly coordinate-
4+
unsaturated Co(II) ion of the predominant species [Co HL]
2
activity [41]. In this work, Co L was found to catalyze the hydrol-
ysis of the phosphate monoester bond of 4-nitrophenyl phosphate
(NPP).
2
(
marked in green in Fig. 5) at pH 6.5 interacted with the phosphoryl
oxygens of BNPP strongly. In addition, the high apparent posi-
4
+
tive charge of [Co HL] enhanced the electrostatic interaction or
2
In kinetic studies of Co L on NPP hydrolysis, increasing the
2
hydrogen bonds with the negatively charged phosphate substrate
concentrations of NPP did not lead to notable saturated curves,
which was in contrast to the results in Fig. 7. This difference indi-
cated that the binding affinity between NPP and Co L was lower
[
22,49–51]. As a result of the combined effect, the pH dependency
−1
−1
s ) turned out to be sharply decreased as pH
of kcat/KM (M
2
increased.
than that between BNPP and Co L, suggesting that the hydropho-
2
bic interactions exerted by the intramolecular ␤-CDs played an
important role in the substrate-binding step. By comparing the
second-order rate constants (Fig. 9), it was found the catalyzed
BNPP hydrolysis was generally 5 times faster than the catalyzed
2
.4. Metal ion effect
Attributed to the same ligand and the same number and valence
of the metal ions, the metal ion effect on the phosphodiesterase
activity of Co L, Zn L and Cu L could be drawn. The logarithmic
values of kcat/KM (M
NPP hydrolysis by Co L, which was probably due to the stronger
2
2
2
2
−1
−1
hydrophobic interactions between ␤-CDs and BNPP. Moreover, the
s
) of Co L, Zn L and Cu L are plotted
2
2
2
in Fig. 8. Interestingly, Co L and Zn L, the two complexes that
2
2
shared analogous species distribution in aqueous solution, exhib-
ited opposite trends of pH dependency. At pH < 7.0, the catalytic
constants of Co L exceeded those of Zn L by more than three orders
-
-
-
1.0
1.5
2.0
NPP
BNPP
2
2
of magnitude. The divergence gradually weakened as pH increased.
According to the trend, their catalytic constants were expected to
−
2
−3
−1 −1
converge to a level between 10 and 10
M s at higher pHs.
The behavior could be rationalized by several aspects. At low pHs
where the strong nucleophiles were absent, the substrate binding
4+
was crucial. The species [Co HL] that had a highly coordinate-
2
unsaturated Co(II) ion (marked in green in Fig. 5) and a highly
positive charge interacted with the negatively-charged substrate
strongly, leading to the low values of KM (M). At basic pHs where
highly positive-charged species was absent, nucleophilic attack by
the metal-bound hydroxides were prevalent. Co(II) and Zn(II) both
enabled the fully activation of nucleophiles at pH > 10.0 (refer to
Table 1). Then, the metal types would make no difference. As to the
6
.5
7.0
7.5
8.0
8.5
9.0
pH
Fig. 9. The logarithmic values of second-order rate constants of BNPP and NPP
flatness of Cu L dependency, it was because of a sole active speices
hydrolysis catalyzed by Co2L at different pH.
2

M. Zhao et al. / Journal of Molecular Catalysis A: Chemical 396 (2015) 346–352
351
Table 2
Kinetic parameters of BNPP hydrolysis catalyzed by Co2L at 308 K.
kcat (10 5
−
s
−1
)
KM (10 M)
−4
1/KM (103
M
−1
)
kcat/KM (10
−2
M
−1 −1
s
)
−log (kcat/KM M−1
−1
s )
pH
6
7
7
8
9
.50
0.83
1.29
0.61
0.77
0.29
0.85
7.30
7.10
9.30
6.50
11.75
1.37
1.40
1.07
1.55
9.79
1.77
0.85
0.83
0.44
1.01
1.72
1.75
2.03
2.35
.20
.60
.10
.00
Fig. 10. Proposed mechanisms of BNPP hydrolysis catalyzed by [Co2HL]4+, [Co2L]3+ and [Co2L(OH)]2+. Water ligands is ommitted unless the nucleophile. Dashed lines denote
weak ligation; bold lines denote strong ligation.
pH dependency trends of the two substrates were in parallel, imply-
activity of Co L exceeded that of Zn L by three orders of magnitude
2 2
ing Co L-catalyzed hydrolysis of BNPP and NPP should be governed
at quasi-neutral pH. In addition, the unexpected activity of Co L on
2
2
by the same acid–base equilibriums.
phosphate monoester was observed, which was rarely witnessed
with synthetic divalent metal complexes.
In general, Co L possessed a collection of intramolecular non-
2
2
.6. Catalytic mechanisms
covalent interactions that cooperated with dinuclear Co(II) center
to catalyze hydrolysis of phosphate esters. This work represents
an example of enriching functions of a metal complex by equip-
ping a second coordination sphere, implicating the applications in
a broader scope of metal-based catalysts.
The mechanisms of the phosphate diester bond hydrolysis cat-
alyzed by different species of Co L in aqueous solution were
proposed in Fig. 10. (i) The protonated tertiary amine weakened
the interaction with Co(II), resulting in the strengthened bonds
2
of Co(II)-OPO (C H NO ) (marked in bold). By the cooperativity
3
6
4
2 2
4+
between metal anchoring and hydrophobic interactions, [Co HL]
4. Experimental
2
bound to substrate with a high affinity, as reflected in the low value
of KM (M) at pH 6.5 (Table 2). The tertiary amine nitrogen atom (in
blue) was assigned to activate the local water molecule (in pink),
coordinated or not [22]. This assumption was also supported by
an extreme case where a nitrogen ligand donor was nucleophilic
enough even to attack phosphate ester [56]. (ii) The deprotona-
tion made the tertiary amine restore the coordination to Co(II), and
subsequently the interaction strength between Co(II) and phospho-
ryl group was considerably decreased, resulting in the high values
of KM (M) at pH > 7.2. (iii) At higher pH, the more reactive metal-
bound hydroxides formed. In respects of nucleophilicity and steric
hindrance, the hydroxides were more likely to be the nucleophilic
agent. Upon the nucleophilic attack, the leaving group NP dissoci-
ated.
4.1. Materials
Bis(4-nitrophenyl) phosphate (BNPP) were purchased from
Aldrich Chemical Company, and used without further purification.
␤-CD of reagent grade was recrystallized twice from H2O and dried
in vacuum for 12 h at 373 K. DMF were dried over CaH2 for 2 days
and then distilled under reduced pressure prior to use. Water used
in all physical measurement experiments was Milli-Q grade.
4.2. Spectrum measurements
1H NMR and 31P NMR spectra were recorded on a Varian INOVA-
00NB or Mercury plus 300 spectrometers. Elemental contents
3
were analyzed by a Perkin-Elemer 240 elemental analyzer. ESI-MS
spectra were performed on a Thremo LCQ-DECA-XP spectrometer.
UV–vis spectra were monitored with a Varian Cary 100 UV/Vis spec-
trophotometer equipped with a temperature controller (± 0.1 K).
3
. Conclusions
A dinuclear metallohydrolase mimic, Co L, with intramolecular
-CDs was synthesized and characterized. The divalence of Co(II)
2
␤
in Co L was stable in air, owing to the effect of hydrophobic
4.3. Potentiometric titration
2
microenvironment confined by ␤-CDs, which implicated a feasible
method to prepare stable divalent Co(II) complexes. By means of
potentiometric titration and UV–vis titration experiments, species
An automatic titrator (Metrohm 702GPD Titrino) coupled to a
Metrohm electrode was used and calibrated according to the Gran
method [60]. The electrode system was calibrated with buffers and
checked by titration of HClO4 with NaOH solution (0.10 M). The
thermostated cell contained 25 mL of 1.0 mM species in aqueous
solutions with the ionic strength maintained at 0.10 M by sodium
perchlorate. All titrations were carried out on the aqueous solutions
under nitrogen at 298 ± 0.1 K, and initiated by adding fixed vol-
umes of 0.10 M standard NaOH in small increments to the titrated
distribution of Co L in aqueous solution was clarified. The tertiary
2
amine nitrogen atom of the dinucleating ligand switched its role
from a proton acceptor [22,50,59] to a coordination donor as pH
4+
increased. The highly positive-charged species [Co HL] resulted
2
in the high binding affinity toward BNPP, probably arisen from the
combined effect of metal anchoring, hydrogen bonds, electrostatic
interactions and hydrophobic interactions. The phosphodiesterase

3
52
M. Zhao et al. / Journal of Molecular Catalysis A: Chemical 396 (2015) 346–352
solution. Triplicate measurements were performed, for which the
experimental error was below 1%. The titration data were fitted
from the raw data with the Hyperquad 2000 program to calculate
the complex formation constant KML, and the pKa values.
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4.4. Synthesis of Co2L
The supramolecular dinucleating ligand HL was prepared
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[
[
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according to our previously reported procedures [43,46]. The syn-
thesis of the dinuclear Co(II) complex was carried out in the
thermostated cell of the potentiometric titration equipment. To
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[
[
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an aqueous solution (25 mL, I = 0.1 M NaClO ) of the dinucleating
4
ligand HL (21.6 mg, 7.5 ␮mol), two equivalents of Co(ClO ) ·6H O
4
2
2
(
5.5 mg, 15.0 ␮mol) was added with magnetic stirring at room
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temperature under N2 atmosphere. The solution was acidified
with perchloric acid to pH ≈ 3 prior to the titration experiment.
Then, the solution was titrated with 0.10 M NaOH solution to
pH ≈ 11, during which no precipitate was observed. After the
titration data were collected, the solution was neutralized with
perchloric acid, and evaporated under vacuum. Acetone (20 mL)
was poured, and then the precipitate appeared. The precipitate
was collected with centrifuge, followed by three times wash with
acetone. After being dried overnight under vacuum, light pink
powder (22.2 mg, 87%) was obtained (Found: C 36.98, H 5.81, N
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.59. C105H159N O ·Co (ClO ) ·23H O requires C 36.96, H 6.06,
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69
2
4
3
2
N 1.64%); IR (KBr): ꢁ = 3410, 2926, 1795, 1630, 1431,1381, 1263,
−
1
1
7
155, 1082, 1032, 876, 756, 706, 607, 575, 472 cm ; ꢂmax(pH
.2, H O)/nm 253 (ε/M cm
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1341) and 302 (537); m/z (ESI-
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[
[
Acknowledgements
(
1
We thank Dr. Fu-Sheng Guo for the magnetic experiment analy-
sis. This work is supported by National Natural Science Foundation
of China (21172274, 21201183 and 21231007), Key Project of
Chinese Ministry of Education (313058), Program for Changjiang
Scholars and Innovative Research Team in University of China
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IRT1298), State High-Tech Development Program (863 Program:
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4
Appendix A. Supplementary data
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Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcata.2014.10.020.
4
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