Carboxy and diphosphate ester hydrolysis promoted by di- or tri-nuclear zinc(II) complexes based on β-cyclodextrin

Article abstract of DOI:10.1016/j.molcata.2010.11.037

A new ligand (L), 6-mono-(2-(2-hydroxy-3-(hydroxymethyl)-5-methyl benzylamino)-ethylamino)-β-cyclodextrin, based on β-cyclodextrin derivatives with dinucleating units was synthesized and used to prepare a trimetallic bis-ligands zinc complex (Zn₃(L^2-) ₂). The esterase activity of the complex was investigated by the hydrolysis of two carboxylic acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-nitrophenyl acetate (NA), and a DNA model bis(4-nitrophenyl)phosphate (BNPP) as a phosphate ester. The catalytic rate for BNPC was very high, which was found to be a 5.63 × 10³-fold rate enhancement over uncatalyzed hydrolysis and 1.62 × 10²-fold rate enhancement over uncatalyzed hydrolysis for NA hydrolysis at pH = 7.0. For the catalytic hydrolysis of BNPP, the initial first-order rate constant of 0.1 mM catalyst was 5.85 × 10^-8 s^-1 at pH = 8.50 and 35 °C, which is a 731-fold acceleration over uncatalyzed hydrolysis. The second rate constant (k_BNPP) was found to be 1.22 × 10^-3 M^-1 s^-1 at pH = 10.0. According to the potentiometric titration study, the zinc complex exists in a dinuclear single ligand coordinated mode and a trinuclear bis-ligands system at pH ≥ 7.0. The ester hydrolysis activity was attributed to the cooperative interaction of the two metal centers and the hydrophobic cavity of β-cyclodextrin with substrates.

Full text of DOI:10.1016/j.molcata.2010.11.037

Journal of Molecular Catalysis A: Chemical 335 (2011) 222–227
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Journal of Molecular Catalysis A: Chemical
journal homepage: www.elsevier.com/locate/molcata
Carboxy and diphosphate ester hydrolysis promoted by di- or tri-nuclear zinc(II)
complexes based on ␤-cyclodextrin
Si-Ping Tanga,b, Ping Hua, Huo-Yan Chena, Sha Chena, Zong-Wan Maoa,c,∗, Liang-Nian Jia
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 Functional Organometallic Materials, Department of Chemistry and Material Science, Hengyang Normal University, Hengyang 421008, China
c Department of Applied Chemistry, College of Sciences, South China Agricultural University, Guangzhou 510642, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new ligand (L), 6-mono-(2-(2-hydroxy-3-(hydroxymethyl)-5-methyl benzylamino)-ethylamino)-␤-
cyclodextrin, based on ␤-cyclodextrin derivatives with dinucleating units was synthesized and used
to prepare a trimetallic bis-ligands zinc complex (Zn3(L2−)2). The esterase activity of the complex was
investigated by the hydrolysis of two carboxylic acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-
nitrophenyl acetate (NA), and a DNA model bis(4-nitrophenyl)phosphate (BNPP) as a phosphate ester.
The catalytic rate for BNPC was very high, which was found to be a 5.63 × 103-fold rate enhancement
over uncatalyzed hydrolysis and 1.62 × 102-fold rate enhancement over uncatalyzed hydrolysis for NA
hydrolysis at pH = 7.0. For the catalytic hydrolysis of BNPP, the initial first-order rate constant of 0.1 mM
catalyst was 5.85 × 10−8 s−1 at pH = 8.50 and 35 ◦C, which is a 731-fold acceleration over uncatalyzed
hydrolysis. The second rate constant (kBNPP) was found to be 1.22 × 10−3 M−1 s−1 at pH = 10.0. According
to the potentiometric titration study, the zinc complex exists in a dinuclear single ligand coordinated
mode and a trinuclear bis-ligands system at pH ≥ 7.0. The ester hydrolysis activity was attributed to
the cooperative interaction of the two metal centers and the hydrophobic cavity of ␤-cyclodextrin with
substrates.
Received 19 July 2010
Received in revised form
23 November 2010
Accepted 28 November 2010
Available online 8 December 2010
Keywords:
Trinuclear zinc complex
Bis-cyclodextrins
Ester hydrolysis
Kinetics
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
cation of the RNA model substrate 2-hydroxypropyl-p-nitrophenyl
phosphate. However, the complexes based on calix[4]arenas are
Many metalloenzymes such as phospholipase C, nuclease P1
and exonuclease site of DNA polymerase use two or three metal
ions in the active site to catalyze the hydrolytic cleavage of phos-
phate diester bonds in nucleotides (e.g., RNA and DNA). The metal
ions act cooperatively as Lewis acid sites in the activation of a
nucleophile and the substrate, and in the stabilization of the pen-
tacoordinate phosphorus transition state and the leaving group. In
the last two decades, many model systems for small molecule dinu-
clear or trinuclear hydrolytic metalloenzymes have been designed
and studied for the hydrolysis of carboxylate esters and phosphate
esters. Examples include macrocyclic polyamine dinuclear [1–7]
and trinuclear metal complexes [8,9], rigid pyridine [10] or pyra-
zol [11–14] bridged polyamine ligands dinuclear metal complexes.
Reinhoudt and co-workers reported synthetic dinuclear and trinu-
clear metallophosphodiesterases based on calix[4]arenes [15–18],
which exhibited a very high catalytic activity in the transesterifi-
not active in the hydrolysis of the phosphate triester diethyl
p-nitrophenyl phosphate (DEPNP), the diester EPNP and the
monoester p-nitrophenyl phosphate (PNP). Metalloenzyme mod-
els of mononuclear complexes based on cyclodextrin (CD) dimers
exhibit high hydrolytic activities due to the cooperative binding
of two ␤-CD cavities with the substrates. For example, Breslow
and Zhang synthesized metallo-bisCDs bridged by a bipyridine
unit with a N,Nꢀ-bidentate ligand as metallohydrolase models,
which remarkably accelerated catalytic hydrolysis of carboxylic
acid diesters and phosphate diesters [19,20]. ␤-Cyclodextrin dimer
linked by telluroxides prepared by Liu et al. [21] exhibited good
catalytic hydrolysis activity of carboxylic acid diesters.
We previously reported the zinc complexes of ␤-CD dimers,
either linked by phenanthroline with tetradentate N4 or pyri-
dine with tridentate N3 ligands, which demonstrated satisfactory
activities for diester hydrolysis [22,23]. However, changing the
bridge ligands from tetradentate to tridentate had no obvious
rate enhancement in esters hydrolysis. Research on the hydrolytic
activity influenced by poly nuclear metal complexes based on
cyclodextrins will aid the design of highly active artificial hydro-
lase. Therefore, we designed and synthesized a dinucleating ligand
based on a cyclodextrin, and prepared its trinuclear bis-ligand
∗
Corresponding author at: MOE Key Laboratory of Bioinorganic and Synthetic
Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University,
Guangzhou 510275, China. Tel.: +86 20 84113788; fax: +86 20 84112245.
E-mail address: cesmzw@mail.sysu.edu.cn (Z.-W. Mao).
1381-1169/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcata.2010.11.037

S.-P. Tang et al. / Journal of Molecular Catalysis A: Chemical 335 (2011) 222–227
223
zinc complex. We also investigated the hydrolytic activities of
this zinc complex affected by the cooperative action between
metal centers and cooperative binding of two ␤-CD cavities with
substrates. The investigation of esterase activity of the zinc com-
plex was performed to promote hydrolysis of the carboxylic
acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-nitrophenyl
acetate (NA), and phosphate ester, bis(4-nitrophenyl)phosphate
(BNPP).
into acetone (200 mL) to give a light-yellow precipitate. The crude
product was dried and purified by column chromatography over
Sephadex G-25 and eluted with distilled deionized water to give
0.450 g (26.3%) of the pure compound as white solid. 1H NMR
(300 MHz, DMSO-d6): ı = 6.78–6.74 (m, 2H; phenyl-H), 6.58–6.61
(brs, 1H; phenyl-OH), 5.69–5.68 (m, 14H; OH-2,3), 4.82 (m, 9H;
H-1, hydroxymethylene-CH2), 4.56–4.46 (m, 6H; OH-6), 3.63 (m,
30H; H-3,5,6, pyridine–CH2, methylene–CH2), 3.36 (m, 18H; H-
2,4, methylene–CH2), 2.16(s, 3H; methyl–CH3), 1.07 (brs, 2H; NH,
CH2OH). MS (ESI, H2O/CH3OH): m/z: calcd: 1327.3 [L+H]+, 1349.3
[L+Na]+; found: 1327.6, 1349.3; elemental analysis calcd (%) for
C53H86N2O36·10H2O: C, 41.98; H, 7.11; N, 1.85. Found: C, 41.57;
H, 6.60; N, 2.08.
2. Experimental
2.1. Materials
2-Hydroxy-5-methylisophthalaldehyde, NA, BNPC, and BNPP
were purchased from Sigma Aldrich. Ethylene diamine was pur-
chased from Acros. ␤-CD (reagent grade) was recrystallized twice
from H2O and dried in vacuo for 12 h at 100 ◦C. DMF was dried
over CaH2 for 2 days and then distilled under reduced pressure
prior to use. Common organic reagents were reagent grade and
redistilled before use. 6-Mono(p-toluenesulfonyl)-␤-cyclodextrin
was synthesized by a literature procedure [24]. Water used in all
physical measurement experiments was Milli-Q grade. All com-
pounds were confirmed by elemental analyses, ESI-MS and 1H NMR
spectroscopy.
2.3.3. Preparation of trinuclear zinc complex (Zn3(L2−)2)
A solution of L (0.047 g, 0.035 mmol) in water (2 mL) was
added dropwise to a dilute aqueous solution of Zn(ClO4)2·6H2O
(0.020 g, 0.054 mmol) with stirring at room temperature. The mix-
ture was adjusted to pH = 9–10 and stirred for 2 h, and then
concentrated. Acetone was added to precipitate the product, which
was collected by centrifugation and washed with 100 mL ace-
tone, followed by drying in vacuo to give the pure complex as
a white solid (0.030 g, 47.6%). 1H NMR (300 MHz, DMSO-d6):
ı = 6.86 (m, 2H; phenyl–H), 6.65 (m, 2H; phenyl–H), 5.82–5.64
(m, 28H; OH-2,3), 4.82 (m, 18H; H-1, hydroxymethylene–CH2),
4.56–4.46 (m, 6H; OH-6), 3.62 (m, 60H; H-3,5,6, pyridine–CH2,
methylene–CH2), 3.32 (m, 36H; H-2,4, methylene–CH2), 2.12 (s, 3H;
methyl–CH3), 2.07 (s, 3H; methyl–CH3); elemental analysis calcd
(%) for C106H232Cl2N4O112Zn3: C, 35.15; H, 6.46; N, 1.55. Found: C,
34.74; H, 6.33; N 1.63. ICP, Zn, calcd: 4.98%; found: 5.33%.
2.2. Instrumentation
1H NMR spectra were recorded on a Varian INOVA-300NB or
Mercury plus 300 spectrometers. Elemental analyses were per-
formed on a Perkin-Elemer 240 elemental analyzer. ESI-MS spectra
were performed on a Thremo LCQ-DECA-XP spectrometer. UV–vis
spectra were obtained on a Varian Cary 300 UV/Vis spectropho-
tometer equipped with a temperature controller ( 0.1 ◦C).
CAUTION: Zinc perchlorate is potentially explosive. It should be
prepared in small quantities and handled with care.
2.4. Potentiometric titration
2.3. Synthesis
An automatic titrator (Metrohm 702GPD Titrino) coupled to a
Metrohm electrode was used and calibrated according to Gran’s
method [25], then checked by titrating HClO4 with NaOH solu-
tion (0.10 M). The thermostated cell contained 25 mL of 1.00 mM
species in aqueous solutions with the ionic strength maintained at
0.10 M by sodium perchlorate. All titrations were carried out in the
aqueous solutions under argon atmosphere at 25 0.1 ◦C, and ini-
tiated by adding fixed volumes of 0.10 M standard NaOH in small
increments to the titrated solution. Duplicate measurements were
performed and the experimental error was below 1%. The titration
data were fitted from the raw data using the Hyperquad 2000 pro-
gram to calculate the ligand protonation constants Kn, the complex
formation constant KML, and the deprotonation constants of the
coordinated water pKa.
2.3.1. Preparation of 6-mono-(N-aminoethyl) amino-ˇ-
cyclodextrin
A
solution of 6-mono(p-toluenesulfonyl)-␤-cyclodextrin
(4.000 g, 3.10 mmol) in dry DMF (12 mL) was added to 10 mL
ethylene diamine with stirring. The mixture was heated to 65 ◦C
for 3 h under argon atmosphere. After cooling to room tempera-
ture, excess ethylene diamine was removed by evaporation under
reduced pressure, and then 400 mL acetone was poured into the
resulting residue to give a white precipitate. The crude product was
recrystallized twice from H2O to give the pure compound (3.898 g)
in 98.4% yield. 1H NMR (300 MHz, DMSO-d6): ı = 5.70–5.69 (m,
14H; OH-2,3), 4.82–4.81 (m, 7H; H-1), 4.45–4.44 (m, 6H, OH-6),
3.31–3.62 (m, 46H; H-2,3,4,5,6; CH2), MS (ESI, H2O/CH3OH): m/z:
calcd: 1177.3 [M+H]+; found: 1177.6; elemental analysis calcd (%)
for C44H76N2O34·7H2O: C, 40.55; H, 6.96; N, 2.15. Found: C, 40.69;
H, 6.93; N, 2.22.
2.5. Kinetics of BNPC and NA hydrolysis
The hydrolysis rates of BNPC and NA in the presence of zinc
complex were measured by an initial slope method that follows the
increase in absorption at 400 nm of the released 4-nitrophenolate
[22]. 50 mM Tris–HCl (pH = 7.00) buffers were used; the ionic
strength was adjusted to 0.10 M with NaClO4 and the reaction
solution was maintained at 25 0.1 ◦C. In a typical experiment,
after substrate (NA or BNPC), zinc(II) cation and ligand in 10%
(v/v) CH3CN solution at pH = 7.0 were mixed, the UV absorption
decay was recorded immediately and monitored until 2% decay of
4-nitrophenyl acetate. Errors on kobs values were about 5%.
2.3.2. Preparation of 6-mono-(2-(2-hydroxy-3-(hydroxymethyl)-
5-methylbenzylamino)-ethylamino)-ˇ-cyclodextrin (L)
A solution of 2-hydroxy-5-methylisophthalaldehyde (0.210 g,
1.30 mmol) in 5 mL dry DMF was added to the solution of 6-
mono-(N-aminoethyl) amino-␤-cyclodextrin (1.521 g, 1.29 mmol)
in 20 mL dry DMF and methanol with stirring. The mixture was
heated to 50 ◦C for 48 h under argon. After cooling to room tem-
perature, the mixture was chilled in an ice-water bath and added
with NaBH4 (0.284 g, 7.51 mmol), then allowed to slowly warm
to room temperature and stirred overnight. A small amount of
hot water was then added to the mixture and filtered. The filtrate
was evaporated to dryness under reduced pressure. The resulting
residue was dissolved in a small amount of hot water and poured
2.6. Kinetics of BNPP hydrolysis
The rate of hydrolysis of BNPP to give mono(4-
nitrophenyl)phosphate and 4-nitrophenolate was measured

224
S.-P. Tang et al. / Journal of Molecular Catalysis A: Chemical 335 (2011) 222–227
Scheme 1. Synthetic scheme of ligand and its trinuclear zinc complex.
by an initial slope method that follows the increase in the
400 nm absorption of the released 4-nitrophenolate in aqueous
solution at 35 0.1 ◦C [26]. At this wavelength, the absorbance
of the ester substrate was negligible. Buffer solutions (50 mM)
of HEPES (pH = 7.40–8.20), TAPS (pH = 8.20–8.90), and CHES
(pH = 8.90–10.50) were used, and the ionic strength was adjusted
to 0.10 M with NaClO4. The pH of the solution was measured after
each run, and all kinetic runs with pH variation larger than 0.1
were excluded. BNPP, buffers, zinc(II) cation and ligand in aqueous
solution were freshly prepared. The reactions were initiated by
injecting a small amount of BNPP into the zinc(II) cation and
ligand buffer solutions, and then mixed at 35 0.1 ◦C. The increase
in visible absorption was recorded immediately and monitored
until 2% formation of 4-nitrophenolate, in which ε values for
4-nitrophenolate were 13,720 (pH = 7.50), 15,270 (pH = 7.75),
16,306 (pH = 8.00), 17,340 (pH = 8.50), 17,626 (pH = 8.85), 17,694
(pH = 9.00), 17,810 (pH = 9.50), 17,846 (pH = 10.0) and 17,858
(pH = 10.50) at 400 nm. The initial first-order rate constants, kin
(s−1), for the cleavage of BNPP were obtained directly from a plot of
the 4-nitrophenolate concentration versus time by the method of
initial rates, which was linear with R > 0.996. The second-order rate
constants (kBNPP) for the catalyzed reactions were determined as
the slope of the linear plots of kin versus (Zn3(L2−)2) concentration.
To correct for the spontaneous cleavage of BNPP, each reaction was
measured against a reference cell that was identical to the sample
cell in composition except for the absence of catalyst. Errors on
kBNPP values were about 5%.
dehyde and 6-mono-(N-aminoethyl)amino-␤-cyclodextrin, and
was further characterized by NMR spectroscopy and mass spec-
trometry (MS) (Fig. S1–S2 in the Supporting Information (SI)). By
reacting L with zinc(II) perchlorate at pH = 9.0–10.0, a trinuclear
zinc(II) complex was obtained in a moderate yield (47.6%), which
was characterized by ICP and NMR spectroscopy. Compared with
the ligand, the 1H NMR spectrum of the complex showed two kinds
of protons on the phenyl and two kinds of methyl protons with
the same peak areas; however, the phenolic proton peak had dis-
appeared. Moreover, the obvious peak splitting of the protons of
OH–2,3 and OH–6 were observed (Fig. S3 in the SI), which indicated
that the trimetalic bis-ligands complex was formed.
3.2. Zn(II) coordination in aqueous solution
Artificial metallohydrolase activity is preferentially studied in
buffered aqueous solutions to most closely mimic biological con-
ditions. Hence, knowledge of the species distribution in solution is
crucial for understanding the trends in hydrolytic reactivity. Poten-
tiometric titrations were performed to determine the pKa values of
the ligand as well as the stability constants of its zinc complexes and
the pKa values of zinc-bound water molecules in these complexes
(Table 1).
The deprotonation steps of the material could be derived
from their titration curves by titrating the corresponding acidic
solutions with a sodium hydroxide solution. In the case of the
ligand, three deprotonation steps were found in the pH range
of 2.0–11.5, including the deprotonation of phenolic proton. For-
mation of both dinuclear single ligand and trinuclear bis-ligands
complexes were detected in aqueous solution (Fig. 1). In the pH
range of 6.0–8.0, deprotonation of the ligand’s phenolic hydroxyl
3. Results and discussion
3.1. Characterization of Zn3(L2−
)
2
leads to [Zn2L−]3+, and then deprotonation of the hydroxymethyl
2+
As illustrated in Scheme 1, the ligand (L) was synthesized in
26.3% yield by the reaction of 2-hydroxy-5-methylisophthalal-
induced by the metallic zinc ion would lead to [Zn2L2−
]
species.
At higher pH (8.0–11.5), three more ZnII species are fromed:

S.-P. Tang et al. / Journal of Molecular Catalysis A: Chemical 335 (2011) 222–227
225
Table 1
Equilibrium constants of the ligand and its zinc complexes.
Table 2
Initial rate (v) for ester hydrolysis promoted by different catalyst.
Chemical equilibrium
Equilibrium constant
Catalysta
BNPC
NA
H2L2+ = HL+ + H+
HL+ = L + H+
pK1
pK2
pK3
logK Zn2L
pKa1
logKZn3L2
pKa1
pKa2
1.30 0.03
5.52 0.02
8.55 0.02
6.96 0.04
6.54 0.02
16.32 0.06
9.09 0.01
10.79 0.01
ꢀ (×10−9 M s−1
)
ꢀ/ꢀcontr
ꢀ (×10−9 M s−1
)
ꢀ/ꢀcontr
Buffer
Zn(II)
Zn2L
(2.45 0.03) × 10−1[21] 1.00
1.35 0.10
1.45 0.13
1.00
1.07
4.82
L = L− + H+
(2.65 0.03) × 10−1
1.10
2Zn2+ + L− = [Zn2L−
]
3+
(2.12 0.30) × 102
8.65 × 102 6.52 0.30
[Zn2L− 3+ = [Zn2L2− 2+ + H+
] ]
3Zn2+ + 2L2− + 2H2O = [Zn3(L2−)2(H2O)2]2+
a
Reaction condition: 50 ␮M BNPC or 400 ␮M NA, 150 ␮M catalyst, 0.1 M NaClO4,
[Zn3(L2−)2(H2O)2]2+ = [Zn3(L2−)2(H2O)(OH)]+ + H+
[Zn3(L2−)2(H2O)(OH)]+ = [Zn3(L2−)2(OH)2] + H+
50 mM pH = 7.00 Tris–HCl buffer, 25 0.1 ◦C.
[Zn3(L2−)2(H2O)2]2+, [Zn3(L2−)2(H2O)(OH)]+ and [Zn3(L2−)2(OH)2].
The higher stability constant of trizinc complex compared to dinu-
clear single ligand complex indicates that trinuclear system is more
stable at higher pH, which can be ascribed to distribution in two
terminals of the two cyclodextrins from two ligands.
3.3. Hydrolysis of carboxylic acid esters
BNPC and NA hydrolysis promoted by the zinc complex were
monitored by the appearance of the p-nitrophenate anion at
400 nm (εobs = 8700 M−1 cm−1) [27] in a 10% MeCN solution in
Tris–HCl (50 mM, pH = 7.00) at 25 0.1 ◦C (Fig. 2). The initial
hydrolysis rates of BNPC (50 ␮M) and NA (400 ␮M) in the pres-
ence of the catalysts were calculated (Table 2). The measured
initial rate of spontaneous cleavage of BNPC (50 ␮M) was very
slow (ꢁcontrol = 2.45 × 10−10 M s−1), which was consistent with the
reported value [21]. Almost no hydrolysis rate enhancement was
observed when only zinc cation was added to either BNPC or
NA solution. However, under identical conditions, a remarkable
865-fold increase in hydrolysis rate over BNPC self-hydrolysis was
observed when the zinc complex catalyst was added to BNPC. For
NA, the catalyzed hydrolysis rate was 4.82-fold higher than that
Fig. 1. Distribution plots of species with zinc complex as a function of pH at 0.1 M
NaClO4 and 25 0.1 ◦C.
0.030
b
0.25
0.20
0.15
0.10
0.05
0.00
a
0.025
0.020
0.015
0.010
0.005
0.000
Zn L
2
Zn L
2
Zn2+
Zn2+
100
0
100
200
300
400
500
0
25
50
75
Time/s
Time/s
Fig. 2. Plots of absorbance vs time during BNPC hydrolysis (a), NA hydrolysis (b) catalyzed by Zn(ClO4)2, and Zn2L in 10% MeCN solution of pH = 7.00 Tris–HCl buffer at 0.1 M
NaClO4 and 25 0.1 ◦C. ([L] = 150 ␮M, [Zn2+] = 300 ␮M, [BNPC] = 50 ␮M and [NA] = 400 ␮M).
a
5
b 2.0
4
1.5
3
1.0
2
0.5
1
2
4
6
8
10
M
12
14
2
4
6
8
10
12
14
[BNPC]/10-5
[ p-NA]/10-4
M
Fig. 3. Saturation kinetics of BNPC hydrolysis (a) and p-NA hydrolysis (b) catalyzed by Zn2L. Each reaction mixture contained Zn2L (150 ␮M), Tris–HCl buffer (50 mM pH = 7.00)
with 0.10 M NaClO4 at 25 0.1 ◦C.

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S.-P. Tang et al. / Journal of Molecular Catalysis A: Chemical 335 (2011) 222–227
Table 3
1.4
Kinetic parameters for the ester hydrolysis in the presence of Zn2L (150 ␮M) in 10%
MeCN solution of Tris–HCl (50 mM pH = 7.00) buffer at 25 0.1 ◦C.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Substrate
BNPC
NA
kuncat (s−1
)
(4.83 0.16) × 10−6
(2.72 0.10) × 10−2
1.01 0.03
(3.40 0.20) × 10−6
(5.52 0.15) × 10−4
42.58 0.02
kcat (s−1
)
Km (mM)
kcat/Km (M−1 s−1
kcat/kuncat
)
26.7
1.29 × 10−2
5.63 × 103
1.62 × 102
of self-hydrolysis. To fully assess the hydrolysis ability of the cat-
alyst for BNPC, a detailed kinetic study was carried out. Saturation
kinetics was observed (Fig. 3) and thus kinetic parameters deduced
from the Michaelis–Menten equation for the hydrolysis are listed
in Table 3. The value of kcat/kuncat was used to describe the catalytic
ability of hydrolase mimics, and the values for BNPC hydrolysis and
NA hydrolysis were 5.63 × 103 and 1.62 × 102, respectively.
7.5
8.0
8.5
9.0
PH
9.5
10.0
10.5
Fig. 4. The pH dependence of the second-order rate constants of BNPP hydroly-
sis ([L] = 0.20 mM, [Zn2+] = 0.30 mM, [BNPP] = 1.00 mM, [buffers] = 50 mM, I = 0.10 M
NaClO4, T = 35 0.1 ◦C).
3.4. Hydrolysis of phosphate esters
The DNA model compound BNPP was used as substrate to inves-
tigate phosphodiesterase activity. The initial phosphorylation rate
in 50 mM Good’s buffer at 35 ◦C and pH = 7.50–10.50 was moni-
tored by the appearance of 4-nitrophenolate at 400 nm. Since the
substrate concentration was essentially constant during the mea-
surement, the initial first-order rate constant (kin) of the total
catalyst was calculated using Eq. (1) [28]:
cation, the initial first-order rate constant of the phosphate diester
hydrolysis was 5.85 × 10−8 s−1 under the same conditions, which
is about 731-fold acceleration over the uncatalyzed hydrolysis.
Fig. 4 displays the dependence of the second-order rate (kBNPP
)
on pH for BNPP hydrolysis promoted by Zn3(L2−
)
2
as a sigmoid
curve for the cleavage reaction. The results indicate a kinetic pro-
cess controlled by an acid/base equilibrium. The data were fitted
by a Boltzman model, which resulted in an inflection point at
9.00 for BNPP. This is almost the same as the pKa1 value of the
coordinated water deprotonation in [Zn3(L2−)2(H2O)2]2+ obtained
from the potentiometric pH titration (Table 1). The second-order
ꢀ = kin [BNPP] = (kBNPP[Zn3(L2−)2]total + kOH− [OH−])[BNPP]
(1)
where v is the 4-nitrophenolate releasing rate. At a given pH value,
the kin values were measured at different catalyst concentrations.
Koike et al. reported the initial first-order rate constant of BNPP
spontaneous hydrolysis was 8.0 × 10−11 s−1 in water at pH = 8.5 and
35 ◦C [29]. In the presence of 0.20 mM ligand and 0.30 mM zinc(II)
rate constants of Zn3(L2−
) for phosphate diester BNPP (kBNPP) is
2
1.22 × 10−3 M−1 s−1 at pH = 10.0.
Scheme 2. Suggested intermediates of ester hydrolysis catalyzed by zinc complexes.

S.-P. Tang et al. / Journal of Molecular Catalysis A: Chemical 335 (2011) 222–227
227
The hydrolysis rate of the phosphate diester increased with
Appendix A. Supplementary data
increasing pH. According to the species distribution as determined
by the pH potentiometric titration and analysis of the kBNPP/pH
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcata.2010.11.037.
profiles, the active species should be [Zn3(L2−
)
2
(H2O)(OH)]+
and [Zn3(L2−)2(OH)2], while the Zn–OH in Zn3(L2−
)
2
acted as
the nucleophile. The second-order rate constants (kBNPP) were
plotted as a sigmoid curve over pH; however, this is differ-
ent from of our previous work in which the second-order
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A trinuclear bis-cyclodextrins zinc complex based on a new
␤-cyclodextrin derivatized ligand was synthesized and character-
ized. The zinc complex displayed good hydrolytic activities for both
monoester and diesters, which could be attributed to the special
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Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (20821001, 20831006, 20725103 and 30770494),
the National Basic Research Program of China (No. 2007C
B815306), the Natural Science Foundation of Guangdong Province
(9351027501000003), the Scientific Research Fund of Hunan
Provincial Education Department (09K099), and the Foundation of
Hengyang Normal University (No. 10B66).
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