Phosphodiester Cleavage Promoted by an Asymmetric Dinuclear Zinc Complex: Synthesis, Structure, and Catalytic Activity

Article abstract of DOI:10.1002/zaac.201500196

The dinuclear Zn^II complex [Zn₂L(DNBA)₂]BPh₄·EtOH (1) (DNBA = 3,5-dinitrobenzonic acid) with an asymmetric dinuclear ligand, N-4-methyl-homopiperazine-N′-[N-(2-pyridylmethyl)-N-2-(2-pyridylethyl)amine]-1,3-diami

Full text of DOI:10.1002/zaac.201500196

Journal of Inorganic and General Chemistry
www.zaac.wiley-vch.de
ARTICLE
Zeitschrift für anorganische und allgemeine Chemie
DOI: 10.1002/zaac.201500196
Phosphodiester Cleavage Promoted by an Asymmetric Dinuclear Zinc
Complex: Synthesis, Structure, and Catalytic Activity
[a]
[a]
[a]
Ai-Zhi Wu,* Long Chen, and Tao Wang
Keywords: Asymmetric dinuclear zinc complex; Crystal structure; Phosphodiester cleavage; Catalytic activity; Zinc; Hydrolysis
II
Abstract. The dinuclear Zn complex [Zn
2
L(DNBA)
2
]BPh
4
·EtOH (1) nitrophenyl) phosphate (BNPP) as substrate. The pH dependence of
the BNPP cleavage in aqueous buffer media reveals a bell-shaped
gand, N-4-methyl-homopiperazine-NЈ-[N-(2-pyridylmethyl)-N-2-(2- pH-kobs profile with an optimum at about pH 7.9, which is parallel to
pyridylethyl)amine]-1,3-diamino-propan-2-ol (HL), was synthesized the formation of the dinuclear species Zn L-OH obtained from the
and characterized. Single crystal X-ray crystallographic analysis shows potentiometric titration. The catalytic rate constant (kcat is
at pH 7.9 and 25 °C, which is approx. 10 -fold higher
than that of the uncatalyzed reaction. The homopiperazine bound de-
close to the Zn···Zn distance in related natural dinuclear metallo- protonated Zn-OH group is responsible for the hydrolysis reaction.
enzymes. Phosphodiesterase activity of Zn L in situ formed from The possible mechanism for the BNPP cleavage promoted by Zn L is
a 2:1 mixture of Zn2+ ion and HL was investigated using bis(4- proposed on the basis of kinetic and spectral analysis.
(DNBA = 3,5-dinitrobenzonic acid) with an asymmetric dinuclear li-
2
)
II
–4 –1
8
that the coordination around the two Zn ions in 1 is significantly 6.30ϫ10
asymmetric, and the distance between both atoms is 3.426 Å, which is
s
2
2
1
Introduction
have been studied extensively in the context of the hydrolysis
of nucleic acid backbones or phosphate ester models,
[
6–17]
Hydrolases are a class of ubiquitous enzymes that catalyze
however, in which less attention has been paid to asymmetric
ligands and their metal complexes. Synthesis and comparison
of asymmetric dinuclear model complexes will investigate the
different roles of the two ions in the catalytic hydrolysis and
provide deeper insight into the structure and mechanism of
phosphoesterases. Recently, we have developed a convenient
and versatile procedure for the synthesis of a new asymmetric
alkoxide-based dinucleating ligand, N-4-methyl-homopiper-
the hydrolytic cleavage of target bond in a substrate-specific
manner. Most of these enzymes contain metal ions, typically
Zn , Mg , and Fe in their active centers.
II
II
III
[1–3]
For example,
in many natural phosphoesterase, such as nuclease P1, purple
acid phosphatase and alkaline phosphatases, the two or three
zinc ions are bridged by a carboxyl group from an aspartate
residue and a water molecule. The catalytic role of the central
zinc atom in these enzymes is ascribed to the orientation and
activation of substrates; the bridging groups keep the ions at a
suitable distance and give a fundamental contribution to sub-
strate activation.[
Functional mimics of phosphoesterases are important for un-
derstanding the role of metal ions in the hydrolytic mecha-
nisms and for developing artificial nucleases for biochemical
and medicinal applications. Dinuclear transition metal com-
plexes are of particular interest, and the divalent zinc ion is
the preferred metal ion for both natural enzymes and hydrolase
azine-NЈ-[N-(2-pyridylmethyl)-N-2-(2-pyridylethyl)amine]-
18]
1
,3-diaminopr-opan-2-ol (HL).[ This ligand can force two
nickel ions into proximity with different chemical environ-
ments, as in related natural dinuclear metalloenzymes. At
4]
2
5 °C, the Ni L species formed in situ from a 2:1 mixture of
2
2+
Ni and HL in aqueous solution at pH 8.4 shows efficient
phosphodiesterase activity. As a continuing study, in this paper,
synthesis and structure of a dinuclear zinc complex are re-
ported. The cleavage activity of the in situ prepared complex
[5]
Zn L on the phosphate diester bond was examined using bis(4-
2
+
2
models. This is due to a variety of factors: Zn is a good
Lewis acid, exchanges ligands rapidly, redox inactivity, and
admittedly physiological relevancy. Moreover, it has no ligand
field stabilization energy and as a consequence, it can easily
adapt its coordination arrangement to best fulfil the structural
nitrophenyl) phosphate (BNPP) as the substrate, and a possible
cleavage mechanism is proposed on the basis of kinetic, pH
titration, and spectral analysis.
requirement of a reaction. Over the past years, many dinuclear 2 Experimental Section
II
Zn complexes derived from phenol-based, alkoxide-based,
carboxylate-based, phthalazine-based, pyrazolyl-based ligands
2.1 Methods and Materials
The FT-IR spectrum was recorded with a PE Spectrum-One infrared
spectrophotometer using KBr pellets (4000–400 cm ). P NMR spec-
tra were measured with a Bruker 400 spectrometer. Elemental analysis
was performed with a Vario EL CHNS-O elemental analyzer. Mass
spectra were measured with a Bruker HCT-plus mass spectrometer.
*
Dr. A.-Z. Wu
–1 31
E-Mail: wuaizhi@gzucm.edu.cn
[
a] School of Chinese Materia Medica
Guangzhou University of Chinese Medicine
Guangzhou, 510006, P. R. China
Z. Anorg. Allg. Chem. 2015, 641, (11), 1941–1947
1941
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The potentiometric titration and pH measurements were carried out
with a Orion 420A pH meter with an Aldrich combination pH elec-
trode, the electrode was calibrated with standard buffers (pH = 4.01,
Table 1. Crystal data and structure refinement for 1.
Zn L(DNBA)
[
2
2
]BPh
4
·EtOH (1)
7
.00, and 10.01). Kinetic measurements were recorded using quarts Empirical formula
62 9 2
C H64B N O14Zn
1300.77
0.40ϫ0.25ϫ0.20
100(2)
0.71073
Formula weight
Crystal size /mm
T /K
Wavelength /Å
Crystal system
Space group
a /Å
cuvettes (1 cm) with Teflon stopper on a U-3100 UV/Vis spectropho-
tometer. The buffers were prepared with MES (2-[N-morpholino]-
ethanesulfonic acid, pK
yl]piperazine-NЈ-[2-ethanesulfonic], pK
(2-cyclohexylamino)ethanesulfonic, pK
a
= 6.1 at 25 °C), HEPES (N-[2-hydroxyeth-
a
= 7.5 at 25 °C), and CHES
a
= 9.5 at 25 °C] and adjusted
triclinic
[
¯
P1
to desired pH by adding freshly prepared NaOH solution. Ligand HL
14.1342(9)
14.3285(9)
16.2746(10)
106.4460(10)
98.5390(10)
104.1030(10)
2980.9(3)
2
1.449
1352
0.880
11419/0/793
1.53 to 26.00
17382
11419 (Rint = 0.0270)
0.976
[
18]
was synthesized as described in previous literature.
HBNPP pur-
b /Å
chased from Aldrich was recrystallized from ethanol/water before use. c /Å
Other regents were reagent grade and were used without further purifi-
cation, unless otherwise noted. Zn stock solution was titrated against
EDTA using standard procedures.
α /°
β /°
γ /°
V /Å
Z
Density /g·cm
F(000)
II
3
–
1
2
.2 Synthesis of [Zn L(DNBA) ]BPh ·EtOH (1)
2 2 4
–
1
Absorption coefficient /mm
4 2 2
A solution of Zn(ClO ) ·6H O (58.2 mg, 0.156 mmol) in methanol
1 mL) was added to a stirred solution of HL (30 mg, 0.078 mmol) in
Data / restraints / parameters
θ range for data collection /°
Reflections collected
(
methanol (1 mL). Afterwards, a solution of Et N (11 μL, 0.078 mmol)
3
in methanol (1 mL) was added and the mixture was stirred at room
temperature for 1 h. A solution of 3,5-dinitrobenzonic acid (DNBA)
Independent reflections
2
GOF on F
(
(
33 mg, 0.156 mmol) and Et
3
N (22 μL, 0.146 mmol) in methanol
Final R indices[I Ͼ 2σ(I)]
R indices (all data)
R
1
2
= 0.0437, wR = 0.0971
1 mL) was added to the reaction solution, and this solution was stirred
R = 0.0654, wR = 0.1043
1
2
–
3
for 1 h. A solution of NaBPh
4
(107 mg, 0.312 mmol) in methanol Largest diff. peak and hole /e·Å
0.950 and –0.393
(1 mL) was added and then slight yellow precipitate was formed. The
crude product was collected by filtration and then washed with meth-
anol and diethyl ether. Colorless block shaped crystals suitable for Table 2. Selected bond lengths /Å and angles /° for 1.
single-crystal X-ray study were obtained by recrystallization from 3:1
MeCN-EtOH solution (4 mL) with a yield of 72%. C62H64BN O14Zn :
9 2
found (calcd.): C, 57.89 (57.23); H, 4.61 (4.92); N, 10.05% (9.69)%.
IR (KBr): ν˜ = 3447, 3104, 3054, 2867, 1619, 1580, 1541, 1479, 1459,
Zn(1)–O(1)
Zn(1)–N(10)
Zn(1)–O(41)
Zn(1)–N(20)
Zn(1)–N(1)
Zn(2)–O(1)
Zn(2)–O(51)
Zn(2)–O(42)
Zn(2)–N(34)
Zn(2)–N(30)
Zn(1)···Zn(2)
1.923(2)
2.045(2)
2.051(2)
2.069(2)
2.213(2)
1.988(2)
1.995(2)
2.026(2)
2.128(2)
2.153(2)
3.426
O(1)–Zn(1)–N(10)
O(1)–Zn(1)–O(41)
N(1)–Zn(1)–N(10)
O(1)–Zn(1)–N(1)
N(1)–Zn(1)–N(20)
O(1)–Zn(2)–O(51)
O(1)–Zn(2)–O(42)
O(42)–Zn(2)–O(51) 96.34(8)
O(1)–Zn(2)–N(30) 79.22(8)
N(30)–Zn(2)–N(34) 74.88(9)
126.56(9)
92.53(7)
97.75(9)
80.69(8)
79.26(9)
99.62(8)
92.99(7)
1
7
446, 1426, 1400, 1345, 1267, 1121, 1067, 1030, 919, 742, 790, 730,
06, 649, 613, 556, 475 cm .
–1
2.3 X-ray Crystallography
The colorless single crystal of complex 1 was mounted on glass fibers
and used for data collection. Single X-ray diffraction data were ob-
tained at 100 K with a Bruker Smart Apex CCD area detector using
Zn(1)–O(1)–Zn(2)
122.33(10)
graphite monochromated Mo-K
α
radiation (λ = 0.71073 Å). The struc-
with the ionic strength maintained at 0.1 m by NaClO
4
. After stirring
2
ture was solved by direct methods and refined on F by full-matrix
least-squares techniques with SHELXTL-97. All non-hydrogen atoms
were refined anisotropically, and all hydrogen atoms of the ligands
were located and included at their calculated positions. The crystal
for 30 min, the titration was initiated by adding 20 μL of fresh NaOH
solution (0.040 m) with a syringe to the titrated solution. Three titration
experiments were performed in the pH rage 3.5–10.0. The species
formed in the system were characterized by the following equilibrium
data and structure refinement results are summarized in Table 1, and process of Equation (1), Equation (2), and Equation (3). The corre-
selected bond lengths and angles are listed in Table 2.
sponding dissociation constants were calculated using BEST pro-
gram.
[
19]
Crystallographic data (excluding structure factors) for the structure in
this paper have been deposited with the Cambridge Crystallographic
Data Centre, CCDC, 12 Union Road, Cambridge CB21EZ, UK.
Copies of the data can be obtained free of charge on quoting the de-
+
+
Zn
2
2
2
HL = Zn
L = Zn
2
L + H ; Ka1 = [Zn
2
L][H ]/[Zn
2
L]
(1)
(2)
+
+
Zn
2
L-OH + H ; Ka2 = [Zn
2
L-OH][H ]/[Zn
2
L]
+
+
pository number CCDC-1027685 (Fax.: +44-1223-336-033; E-Mail: Zn
L-OH = Zn
2
L-(OH)
2
+H ; Ka3 = Zn
2
L-(OH)
2
][H ]/[Zn
2
L-OH] (3)
deposit@ccdc.cam.ac.uk, http://www.ccdc.cam.ac.uk)
2.5 Kinetic Measurements
The cleavage of BNPP was monitored in a buffered water solution (I
0.1 m NaClO , T = 25 °C) by detecting the increase in the maximum
The protonation and coordination equilibria of the dinuclear zinc com- absorbance at 400 nm of the 4-nitrophenolate anion (NPat), using an
2.4 Potentiometric Titration
=
4
–
1 [20]
plex (Zn
potentiometric titration at 25 °C. The cell contained HL (1.0 mM), the solution of Zn
4 2 4 2
HClO (0.5 mM), and Zn(ClO ) (2.0 mM) in a water solution (10 mL) stock solution of HL (50 mM in water) and Zn(ClO ) (100 mM in
2
L) synthesized in situ in water solution were investigated by
extinction coefficient of 18,700 L mol .
In a typical experiment,
2
L was prepared by thoroughly mixing 40 μL of
4
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water) in containing 1.92 mL of 0.05 m buffer solution (HEPES and
CHEM) for 30 min at 25 °C. 50 μL of BNPP (40 mM in water) was
added to the above complex solution in a cuvette and mixed well.
2
The final concentration of Zn L and BNPP were all 1 mM. In the
determination of the pH-dependent cleavage reaction in an aqueous
solution, pH values vary in the region of 6.5–9.5. After addition, the
mixture was allowed to equilibrate for 5 min and the reaction was
followed up to 5% of BNPP cleavage. The absorbance values were
converted into the concentration of the NPat ion, and the total analytic
In 1, the two zinc atoms are bridged by the ionized alkoxide
oxygen of L and one carboxyl from DNBA in μ bridging
mode. Both zinc atoms are five-coordinate. In the bipyridyl
pendant, the five-coordinate environment around Zn(1) is com-
1,2
prised of the N O donor box from L and one oxygen donor
3
[
22]
form the bridging carboxyl. The calculated structural index,
τ, is 0.51, indicating that the arrangement around the central
Zn(1) atom is somewhat intermediate between trigonal-bipy-
ramidal and square-pyramidal. In the homopiperazine pendant,
4
-nitrophenol product was calculated using the buffer pH and the pK
a
[
21]
of 4-nitrophenol (7.15).
The pseudo-first-order rate constant the N O donor box from L, one oxygen donor from the bridg-
2
–1
k
obs (s ) for cleavage of BNPP were generally determined from the ing carboxyl, and one oxygen donor from the monodentate
slopes of semilogarithmic plots of reaction progress against time by
the method of initial rates which was linear with R Ͼ 0.995. In all
cases, the standard deviation for the values of kobs was Ͻ6%. Second-
carboxyl of another DNBA molecule finish a distorted square-
pyramidal arrangement around Zn(2) (τ = 0.26) with the atom
O(51) occupying the apical position in the coordination sphere.
This difference in the environment around each zinc atom can
be ascribed to the asymmetry of the ligand. The Zn–O dis-
tances [Zn(1)–O(1), 1.923 (2) Å; Zn(2)–O(1), 1.988 (2) Å;
Zn(1)–O(41), 2.051(2) Å; and Zn(2)–O(42), 2.026(2) Å] for
the bridging alkoxide and carboxyl groups indicate the bridges
in 1 are asymmetric. The bond length of Zn(1)–O(1) is obvi-
ously shorter than that of Zn(2)–O(1), partly because the coor-
dination potency of the bipyridyl pendant is stronger than that
of the homopiperazine pendant. The distance between the two
zinc atoms is 3.426 Å, and the ionized alkoxide bridging angle
order rate constants kZn (M s–1) for the catalyzed reactions were de-
–
1
2
termined as the slope of the linear plots of kobs vs. Zn L concentration.
All experiments were run in triplicate, and the average values were
adopted. To correct for the spontaneous cleavage of BNPP, each reac-
tion was measured against a reference cell, which was identical to the
sample cell in composition except for the absence of Zn
2
L.
3
Results and Discussion
3.1 Crystal Structure of Complex 1
Crystal structure analysis reveals that
1
crystallizes
[Zn(1)–O(1)–Zn(2)] is 122.33(10)°. In order to chelate a metal
¯
in a triclinic P1 space group with a mono-cation of
ion, the seven-membered homopiperazine ring has to adopt a
less stable boat conformation. The bond angle N(30)–Zn(2)–
N(34) is 74.88(9)°. This value deviates significantly from the
normal bond angle observed in complexes with square-pyrami-
dal arrangement, suggesting that the homopiperazine pendant
+
2 2 4
–
[
Zn L(DNBA) ] , and one BPh acting as the counter anion.
There is one ethanol molecule in the lattice for each complex.
This is also confirmed by the result of element analysis. An
ellipsoid drawing for the crystal structure of the cationic core
in 1 with numbering scheme is depicted in Figure 1.
[18]
is in a strained fashion.
3.2 Potentiometric Studies of Zn2L
The potentiometric titration was carried out in a pH range
of 3.5–10.0. The solution started to turn turbid when the pH
value was above pH 10.0 because of the precipitation of zinc
hydroxide which impeded the data processing. The pH titration
of HL (1 mm), HClO (0.5 mm), and Zn(ClO ) (2 mm) in
4
4 2
water at 25 °C and I = 0.1 m (NaClO ) gave three well-defined
4
inflections and revealed the formation of stable zinc complexes
at pH Ͼ 4 with simultaneous deprotonation of the alcohol OH
(
i.e., Zn HL to Zn L). A conclusion derived from the observa-
2 2
–
tion of the neutralization break at eq(OH ) = 3.5 (see Figure 2)
can be drawn that the zinc complex Zn L-(OH) will form and
2
2
remains stable up to pH 10. Moreover, this specie was also
proved by the ESI-MS spectra, in which one peak at m/z
5
65.2 can be ascribed to the positively charged ion of
+
[Zn L(OH) (H O)] .
2 2 2
From the analysis of the pH titration data, the deprotonation
constants pKa1 of 5.45, pKa2 of 7.02, and pKa3 of 8.73 were
obtained [see Equations (1)–(3)]. The extremely small pK Ͻ6
a1
observed for alcohol linker in Zn L is attributed to double co-
2
ordination of the alkoxide to two zinc(II) ions. Facile deproton-
II
ation of an alcohol upon binding of two Zn was also observed
Figure 1. ORTEP drawing of the cationic core of 1. Thermal ellipsoids
for the non-hydrogen atoms are drawn at the 30% probability level.
All hydrogen atoms are omitted for clarity.
[23]
[24]
by Valtancoil
and Morrow
for similar alkoxide-bridged
II
dinuclear Zn complexes. The pK of Zn L corresponds to the
a2
2
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3.3 Phosphodiesterase Activity of Zn2L
BNPP is often used as a DNA model compound in the in-
vestigation of phosphodiesterase activity. The test was carried
out in buffers in order to mimic biological conditions. The
reaction was monitored by following the increase of NP from
BNPP. The dependence of the reaction rate on pH for the
BNPP cleavage promoted by Zn L (1 mM) in aqueous solution
2
at 25 °C shows a bell-shaped profile with an optimum at about
pH 7.9 (Figure 3). Notable cleavage activity (kobs) was ob-
served at pH Ͼ 6, parallel with the formation of the dinuclear
species Zn L-OH. These results strongly suggest that the
2
monohydroxo form of the complex is the activity species re-
sponsible for cleaving BNPP. Based on the pH/rate constant
profile, the successively formed Zn L-(OH) complex is also
2
2
able to promote the cleavage, and its activity is almost half of
the former Zn L-OH species partly due to the leaving tendency
2
Figure 2. pH titration curve of HL (1 mM) and Zn(ClO
water (10 mL).
4 2
) (2 mM) in
of the OH group of Zn-OH in bipyridyl pendant becomes
lower, which reduce the binding ability of BNPP to the central
metal atoms.
deprotonation of zinc coordinated water in the homopiperazine
pendant. Although a value of 7.02 is somewhat like the usual
Figure 4 shows the effect of Zn L concentrations on the k
2
obs
for the cleavage of BNPP at pH 7.9 and 25 °C. The rate of
BNPP cleavage initially increases linearly with the increase of
Zn L, indicating the reaction is first-order for Zn L concentra-
[25]
pK (Ͻ8) of a bridge water in dinuclear zinc(II) complexes,
a
it is still comparable with the pK value of 6.7 determined for
a
2
2
the unbridged zinc-coordinated water in an alcohol-linked 1,3-
bis(bis-pyridin-2-ylmethylamino)propan-2-ol dizinc com-
tion. The calculated pseudo-second-order rate constant for
–2 –1 –1
Zn L determined from the plot is 7.75ϫ10 m ·s .
2
plex.[ The theoretical calculations on the relationships of
26]
II
pK , coordination numbers, and nucleophilicity among Zn
a
complexes indicate that lower coordination numbers at the cen-
[27]
tral metal atom lower pK values of bound water. Therefore,
a
the relatively lower pKa2 value may be partly the results of the
lower coordination number of zinc ion in the homopiperazine
pendant. The higher pKa3 of Zn L corresponds to the further
2
deprotonation of another zinc-coordinated water in bipyridyl
3
+
–
pendant to form Zn L -(OH ) , and it is comparable with that
2
2
observed for some dizinc(II) complexes containing dinclear
compartmental ligands.[ A typical diagram for the species
distribution as a function of pH (5.5–10) at [total zinc(II)] =
6]
2
mM and [HL] = 1 mM is displayed in Figure 3.
Figure 4. Dependence of the pseudo-first-order rate constants on the
2 4
concentration of Zn L at pH 7.9 and 25 °C. I = 0.10 m (NaClO ),
[BNPP] = 1 mM, and [HEPES buffer] = 50 mM.
The initial rate of BNPP cleavage was also determined as a
function of the substrate concentration at pH 7.9. The data
depicted in Figure 5 reveals saturation kinetics with Michaelis-
Menten-like behavior in the system, typical for native metallo-
enzymes. The experiment data were fitted to the Michaelis-
Menten model by evaluating from the Lineweave-Burk
double-reciprocal plot, which resulted in a Michaelis constant
–
3
(
[
K ) of 7.22ϫ10 m and a catalytic rata constant (k = Vmax/
M cat
–4
–1
Zn L]) of 6.30ϫ10 s . The second order rate constant
KBNPP (k /K ) for BNPP is 8.73ϫ10 m ·s . The recipro-
2
–2
–1 –1
cat
M
Figure 3. Species distribution curves (solid line) and the effect of pH
on the hydrolysis of BNPP (filled square) for zinc(II)-HL system (T =
cal of the Michaelis-Menten constant could be treated as the
2+
–1
25 °C, [HL] = 1 mM, [Zn ] = 2 mM, [BNPP] = 1 mM).
substrate binding constant, that is, K = 1/K = 138.5 m . The
b
M
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low K is fit for the observations that BNPP is a weak ligand
b
[
28,29]
–
–7
for Zn L.
At pH 7.9 ([OH ] = 6.31ϫ10 m]) and 25 °C,
2
–
the first-order rate constant (k = kOH[OH ]) of automatic hy-
drolysis of BNPP is calculated to be 3.66ϫ10
5.8ϫ10 m ·s for BNPP (Vauto = kOH[BNPP][OH ]).
–
12 –1
s
with k
OH
[30]
–
6
–1 –1
–
=
Therefore, it can be concluded that in pH 7.9 buffer solution
of 50 mM HEPES, 0.1 m NaClO in water and at 25 °C, com-
4
plex Zn L can promote the hydrolysis of BNPP by a factor of
2
8
1
.7ϫ10 . Under the same conditions, the catalytic activity of
complex 1 was also measured, and it can promote the hydroly-
5
sis of BNPP by a factor of 5.6ϫ10 , indicating that the bridg-
ing carboxyl group is an inhibitor of the catalytic species and
reduce the catalytic hydrolysis rate. These results further inter-
pret that BNPP coordinates the two central zinc atoms by a
bidentate bridging mode in the catalytic reaction. In general,
since different reaction conditions such as catalysts loading,
pH value of reaction system, reaction medium, and models of
phosphodiester were used, it is hard to critically evaluate and
II
compare the reactivity of the reported di-Zn based systems.
Figure 6. 31P NMR spectra recorded during the cleavage reaction of
2+
Nevertheless, complex Zn L reported upon herein are competi- BNPP by the Zn
2
L formed by mixing Zn ion and ligand HL in a
2
2/1 molar ratio at 25 °C as a function of time: (a) t = 0; (b) t = 15 h;
tive with most synthetic dinuclear metallohydrolase mod-
els.[31–33]
(c) t = 2 d.
3.4 Mechanism of Phosphodiester Hydrolysis Promoted by
Zn2L
On the basis of the kinetics data, pH potentiometric titration
studies and crystal structure of 1, a double Lewis acid acti-
vation mechanism is proposed for the catalytic hydrolysis of
[
34]
BNPP by Zn L, which is similar to the reported cases.
As
2
shown in Scheme 1, the catalytic hydrolysis cycle may contain
the following steps: firstly, the active species B is formed
through the dissociation of a zinc-bound water in homopiper-
azine pendant at pH 7.9, and the phosphodiester BNPP coordi-
nates to the two central zinc atoms as a bidentate ligand by
replacing two coordination water molecules, forming the
BNPP adduct C. This species was observed in the ESI-MS
spectrum of mixture solution of in situ prepared complex Zn2L
and HBNPP, in which one positively charged peak at m/z 869.4
+
corresponds to [Zn L(OH)(BNPP)] (C); secondly, the zinc-
2
bound deprotonated water molecule group acts as a nucleo-
phile and attacks BNPP, forming a transition complex D;
and buffer solution (50 mM of HEPPS). Inset: Lineweaver-Burk thirdly, the P–O bond in D breaks to release p-nitrophenolate
Figure 5. Saturation kinetic experiments for the cleavage of BNPP by
Zn L at pH 7.9 and 25 °C. I = 0.10 m (NaClO ), [Zn L] = 0.02 mM,
2 4 2
double-reciprocal plot.
followed by reestablishment of the starting complex A, ac-
complished by the replacement of NPP with water. In the first
The 31P NMR spectroscopic method was used to further in- step, an equilibrium may exist between B and C since BNPP
vestigate the cleavage reaction at 25 °C with a solution of is a weak ligand for Zn L.
2
BNPP and Zn L complex formed in situ from ligand L and
The intrinsic activity of dinuclear catalyst is highly depend-
2
2+
Zn in a molar ratio of 1/2 in D O (Figure 6). The increase ent upon the metal–metal distance. The appropriate intermet-
2
3
1
of the P resonance for the cleavage product (NP) and the allic separation (3.426 Å in 1) can facilitate the cooperative
decrease of the 31P resonance for BNPP were monitored with action for the phosphate bridging, and thus BNPP is activated
[
1–3]
time. After 15 h, a new signal attributed to the cleavage prod- by the zinc atoms as in other dinuclear metallohydrolases.
uct NP was abserved at –6.2 ppm, and this signal became more In addition, acting as Lewis acid, one zinc ion of complex
intense after 2 d. No further hydrolysis of NP producing free Zn L can promote the deprotonation of a water molecule to
2
phosphate could be detected even after 2 weeks. The observa- form nucleophile to attack phosphate group. Furthermore, the
tion suggested that the phosphate monoester NP produced by two zinc ions connect the two reactants (substrate BNPP and
–
the hydrolysis of BNPP binding to the dinuclear zinc complex, nucleophile OH ), neutralizing the electrostatic repulsion be-
and that no further hydrolysis occurs.
tween the two negatively charged species.
Z. Anorg. Allg. Chem. 2015, 1941–1947
1945
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of Inorganic and General Chemistry
www.zaac.wiley-vch.de
ARTICLE
Zeitschrift für anorganische und allgemeine Chemie
2
Scheme 1. Proposed mechanism for hydrolysis of BNPP by Zn L.
[
[
[
6] J. W. Chen, X. Y. Wang, Y. G. Zhu, J. Lin, X. L. Yang, Y. Z. Li,
4
Conclusions
Y. Lu, Z. J. Guo, Inorg. Chem. 2005, 44, 3422.
7] A. O’Donoghue, S. Y. Pyun, M. Y. Yang, J. R. Morrow, J. P. Rich-
ard, J. Am. Chem. Soc. 2006, 128, 1615.
The asymmetric dinuclear complex Zn L holds essential
2
physical features in common with the nuclease P1, phospholi-
pase C, and purple acid phosphatases. The complex can ef-
8] C. T. Liu, A. A. Neverov, R. S. Brown, J. Am. Chem. Soc. 2008,
130, 16711.
ficiently catalyze the hydrolysis of BNPP with a rate approxi- [9] M. F. Mohamed, A. A. Neverov, R. S. Brown, Inorg. Chem. 2009,
8
mately 10 -fold higher than that of the uncatalyzed reaction.
The hydrolysis of BNPP promoted by Zn L may be triggered
by the phosphodiester coordination to two central zinc atoms
in a bidentate bridging mode, followed by an attack of the
48, 11425.
[
[
10] A. Z. Wu, C. C. Zhu, T. Wang, Chin. J. Struct. Chem. 2010, 29,
2
1
127.
11] V. Lombardo, R. Bonomi, C. Sissi, F. Mancin, Tetrahedron 2011,
7, 2189.
6
–
deprotonated Zn-bound water (Zn-OH ) to the phosphorus [12] H. Gao, Z. F. Ke, N. J. DeYonker, J. P. Wang, H. Y. Xu, Z. W.
atom, and finally resulting in cleavage of the P–O bond. Since
the correlation between the phosphatase activity and the coor-
dination environment of a complex is an important factor to
be considered in designing efficient artificial phosphatases, this
research provides some insights into this issue and affords us
the knowledge to obtain more efficient phosphodieserase mim-
ics in the future.
Mao, D. L. Phillips, C. Y. Zhao, J. Am. Chem. Soc. 2011, 133,
904.
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[
[
[
[
[
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1
1
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Acknowledgements
5
We thank for the support of this work by the Special Funds from
Central Finance of China in Support of the Development of Local
Colleges and University [Educational finance Grant No. 276(2014)].
[19] A. E. Martell, R. J. Motekaitis, Determination and Use of Sta-
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[20] In respect to a pH-dependent equilibrium between p-nitrophenol
(
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tion of the reaction product can be calculated (pH = pK + log
NPat]/[NP]).
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