Ester hydrolysis by a cyclodextrin dimer catalyst with a tridentate N,N′,N″-zinc linking group

Article abstract of DOI:10.1002/asia.200900108

A new β-cyclodextrin dimer, 2,6-dimethylpyridine-bridged-bis(6- monoammonio-β-cyclodextrin) (pyridyl BisCD, L), is synthesized. Its zinc complex (ZnL) is prepared, characterized, and applied as a catalyst for diester hydrolysis. The formation constant (log K_ML = 7.31 ±0.04) of the complex and deprotonation constant (pK_a1 = 8.14 ±0.03, pK_a2 = 9.24 ±0.01) of the coordinated water molecule were determined by a potentiometric pH titration at (25±0.1)°C, indicating a triden-tate N,N′',N′′-zinc coordination. Hydrolysis kinetics of carboxylic acid esters were determined with bis(4-nitrophe-nyl) carbonate (BNPC) and 4-nitro-phenyl acetate (NA) as the substrates. The resulting hydrolysis rate constants show that ZnL has a very high rate of catalysis for BNPC hydrolysis, yielding an 8.98 × 10³-fold rate enhancement over uncatalyzed hydrolysis at pH 7.00, compared to only a 71.76-fold rate enhancement for NA hydrolysis. Hydrolysis kinetics of phosphate esters catalyzed by ZnL are also investigated using bis(4-nitrophenyl)phosphate (BNPP) and disodium 4-nitrophenyl phosphate (NPP) as the substrates. The initial first-order rate constant of cata- lytic hydrolysis for BNPP was 1.29 × 10^-7 s^-1 at pH 8.5, 35°C and 0.1 mm catalyst concentration, about 1600-fold acceleration over uncatalyzed hydrolysis. The pH dependence of the BNPP cleavage in aqueous buffer was shown as a sigmoidal curve with an inflection point around pH 8.25, which is nearly identical to the pK_a value of the catalyst from the potentiometric titration. The k_BNPP of BNPP hydrolysis promoted by ZnL is found to be 1.68 × 10^-3 m^-1 S^-1, higher than that of NPP, and comparatively higher than those promoted by its other tridentate N,N′,N′′-zinc analogues.

Full text of DOI:10.1002/asia.200900108

FULL PAPERS
DOI: 10.1002/asia.200900108
Ester Hydrolysis by a Cyclodextrin Dimer Catalyst with a Tridentate N,N’,N’’-
Zinc Linking Group
Si-Ping Tang,[a, b] Ying-Hua Zhou,[c] Huo-Yan Chen,[a] Cun-Yuan Zhao,[a]
Zong-Wan Mao,*[a] and Liang-Nian Ji[a]
Abstract: A new b-cyclodextrin dimer,
2,6-dimethylpyridine-bridged-bis(6-
The resulting hydrolysis rate constants
show that ZnL has a very high rate of
catalysis for BNPC hydrolysis, yielding
an 8.98ꢀ103-fold rate enhancement
over uncatalyzed hydrolysis at pH 7.00,
compared to only a 71.76-fold rate en-
hancement for NA hydrolysis. Hydroly-
sis kinetics of phosphate esters cata-
lyzed by ZnL are also investigated
lytic hydrolysis for BNPP was 1.29ꢀ
10ꢁ7 sꢁ1 at pH 8.5, 358C and 0.1 mm
catalyst concentration, about 1600-fold
acceleration over uncatalyzed hydroly-
sis. The pH dependence of the BNPP
cleavage in aqueous buffer was shown
as a sigmoidal curve with an inflection
point around pH 8.25, which is nearly
identical to the pKa value of the cata-
lyst from the potentiometric titration.
The kBNPP of BNPP hydrolysis promot-
ed by ZnL is found to be 1.68ꢀ
10ꢁ3 mꢁ1 sꢁ1, higher than that of NPP,
and comparatively higher than those
promoted by its other tridentate
N,N’,N’’-zinc analogues.
monoammonio-b-cyclodextrin) (pyridyl
BisCD, L), is synthesized. Its zinc com-
plex (ZnL) is prepared, characterized,
and applied as a catalyst for diester hy-
drolysis. The formation constant
(log KML =7.31ꢀ0.04) of the complex
and deprotonation constant (pKa1 =
8.14ꢀ0.03, pKa2 =9.24ꢀ0.01) of the
coordinated water molecule were de-
termined by a potentiometric pH titra-
tion at (25ꢀ0.1)8C, indicating a triden-
tate N,N’,N’’-zinc coordination. Hydrol-
ysis kinetics of carboxylic acid esters
were determined with bis(4-nitrophe-
nyl)carbonate (BNPC) and 4-nitro-
phenyl acetate (NA) as the substrates.
using
bis(4-nitrophenyl)phosphate
(BNPP) and disodium 4-nitrophenyl
phosphate (NPP) as the substrates. The
initial first-order rate constant of cata-
Keywords: cyclodextrins · hydrol-
ysis · kinetics · tridentate ligands ·
zinc complex
Introduction
During the past two decades, many b-cyclodextrin dimers
(bisCDs) have been constructed with various linkers.[1] Com-
pared with native cyclodextrin and monomodified CDs,
bridged bisCDs show significantly higher binding abilities
and molecular selectivities through the cooperative binding
of two adjacent CD units.[2–4] This fascinating property ena-
bles bisCDs to be successfully utilized in molecular recogni-
[a] S.-P. Tang, H.-Y. Chen, Prof. C.-Y. Zhao, Prof. Z.-W. Mao,
Prof. L.-N. Ji
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry and Chemical Engineering
Sun Yat-Sen University
Guangzhou, 510275 (China)
Fax : (+86)20-8411-2245
tion,[5–7] and as drug carriers,[8] such as bis
complex displaying a higher antitumor activity and satisfac-
tory water solubility than the parent paclitaxel. The bis(b-
ACHTUNGTREN(NUNG b-CD)s/paclitaxel
E-mail: cesmzw@mail.sysu.edu.cn
ACHTUNGTRENNUNG
[b] S.-P. Tang
CD)s with a carbon–carbon double bond in the linker have
Department of Chemistry and Material Science
Hengyang Normal University
Hengyang, Hunan 421008 (China)
a potential use in photodynamic therapy.[9]
Furthermore, the pioneering work of Breslow et al. pro-
vided valuable insights into the design of metallo-bisCDs
and their applications in enzyme mimics.[10,11] He and his co-
workers synthesized metallo-bisCDs bridged by a bipyridine
[c] Y.-H. Zhou
College of Chemistry and Materials Science
Anhui Normal University
Wuhu, Anhui 241000 (China)
unit with
a N,N’-bidentate ligand as metallohydrolase
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900108.
models, which held the functional group of the substrate di-
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Chem. Asian J. 2009, 4, 1354 – 1360

rectly above the metal ion bound to the linker through the
cooperative binding of the substrate with two b-CD cavities,
and remarkably accelerated catalytic hydrolysis of carboxyl-
ic acid diesters and phosphate diesters. Another b-cyclodex-
trin dimer linked by telluroxides exhibits good catalytic hy-
drolysis activity of carboxylic acid diesters, and bridged by
ditellurium shows enzymatic specificity and mimics the glu-
tathione peroxidase, exhibiting a high efficiency of catalyz-
ing the reduction of cumene peroxide and hydroperoxide in
the presence of thiol substrates.[12] Other kinds of enzyme
mimics involving metallo-bisCDs linked by porphyrins show
selective oxidation of substrates with good catalytic turnover
numbers.[13]
We have recently reported several cyclodextrin inclusion
complexes constructed by metal complexes binding into b-
CDs or their derivatives as supramolecular metalloenzyme
models.[14–17] Their catalytic activities are greatly improved
since the b-CD or its derivatives are able to mimic the envi-
ronment of the second coordination sphere in the active site
of an enzyme. On the basis of the above work, we have just
reported that the zinc complex of a b-cyclodextrin dimer,
linked by phenanthroline with a tetradentate N,N’,N’’,N’’’
ligand, was demonstrated as a promising catalyst for diester
hydrolysis.[18] In a systematic study, we designed and synthe-
sized a cyclodextrin dimer bridged by 2,6-bis(ammoniometh-
yl) pyridine (pyridyl BisCD, L) and its zinc complex (ZnL)
with a tridentate N,N’,N’’-zinc coordination, which was rea-
soned to have a more appropriate catalytic site for hydra-
tion or hydrolysis.[19] The investigation of esterase activity of
ZnL was performed to promote hydrolysis of the carboxylic
acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-ni-
trophenyl acetate (NA), and phosphate esters, bis(4-nitro-
phenyl)phosphate (BNPP) and disodium 4-nitrophenyl
phosphate (NPP).
Scheme 1. Synthetic Scheme of the catalyst ZnL.
(L) was characterized by NMR spectroscopy and Mass
Spectrometry (MS) (Figures S1–S2 in the Supporting Infor-
mation).
The further reaction of L and zinc(II) perchlorate gave
the zinc(II) complex in a moderate yield (60%). ESI-MS
spectrometry of ZnL is a very important method to investi-
gate the complex formation in aqueous/MeOH solution.
Figure 1 shows both the experimental and calculated isotop-
ic distribution for the peak at m/z: 1217.8, which was as-
signed to [ZnL]2+. This clearly indicates the existing binding
between the zinc cation and L. To probe the complexation
process, H NMR was used to investigate and distinguish the
difference before and after L complexation. Some changes
in chemical shifts of the pyridine and amine protons in ZnL
were observed relative to those in L: the Ha, Hb, and Hc pro-
tons shifted by approximately 0.32, 0.20, and 1.0 ppm, re-
spectively (Figure S2 in the Supporting Information), which
also indicated that a complex had formed between the L
and zinc(II) cation. Moreover, protons of two coordinated
water molecules Hd were observed.
Results and Discussion
Synthesis and Characterization of ZnL
Formation and Deprotonation Constants of ZnL
As illustrated in Scheme 1, the dimer (L) was synthesized in
35% yield by the reaction of 6-monodeoxy-6-monoamino-b-
cyclodextrin with 2,6-bis(bromomethyl)pyridine. The dimer
The protonation constants (Kn) of the ligand L, its complex
formation constants (KML), and the deprotonation constant
(pKa) of the coordinated water molecules as well as species
distribution in solution were determined by the pH poten-
tiometric titration at I=0.10m NaClO4 and (25ꢀ0.1)8C.
The pH profiles of the titration curves, which include the
distribution curves of the ZnII species as a function of pH
(Figure 2), were analyzed by the Hyperquad program. The
calculated results are summarized in Table 1, including data
Abstract in Chinese:
from the [Zn
dine) complex,[20] a simple analogue of ZnL.
ACTHNGUTERNNUG
Three ZnII species, [ZnL(H2O)2]2+, [ZnL(H2O)(OH)]+,
ACHTUNGTRENUN(NG bamp)] (bamp=2,6-bis(aminomethyl)pyri-
G
and [ZnL(OH)2], corresponding to Equations (1–3), respec-
tively, are involved in the complex formation at pH 3–11
(Figure 2). Since the addition of NaClO4 did not cause spec-
tral changes, it was also confirmed that the remaining coor-
dination sites of ZnII were occupied by two water mole-
cules.[16]
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Z.-W. Mao et al.
FULL PAPERS
Figure 1. ESI-MS spectra of ZnL. a) Full range spectra. b) Bivalence detected ion isotopes spectra. c) Bivalence simulated ion isotopes spectra .
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
tence of cyclodextrins which strongly enhances the stability
of the zinc complex. Additionally, the pKa values of ZnL
(pKa1 =8.14ꢀ0.03, pKa2 =9.24ꢀ0.01) are similar to those of
[Zn
N
Hydrolysis of Carboxylic Acid Esters
To determine the effect of hydrophobic interactions on the
catalytic activities of ZnL, BNPC and NA were selected as
testing substrates because of their structural features. Stud-
ies on the hydrolysis kinetics of BNPC and NA were per-
formed in a 10% MeCN solution of Tris-HCl (50 mm,
pH 7.00) at (25ꢀ0.1)8C (Figure 3). Monitoring the forma-
tion of 4-nitrophenolate by UV at 400 nm (eobs
=
Figure 2. Distribution plots of species with ZnL (1.0 mm) as a function of
pH at 0.1m NaClO4 and (25ꢀ0.1)8C.
8700mꢁ1 cmꢁ1),[21] the initial hydrolysis rates of BNPC
(50 mm) and NA (400 mm) in the presence of the catalysts
were calculated (Table 2).
2þ
Zn2þþL ¼ ½ZnLðH2OÞ2ꢂ
KZnL
Ka1
ð1Þ
ð2Þ
ð3Þ
The measured initial rate of spontaneous cleavage of
BNPC (50 mm) was very slow (ucontrol =2.45ꢀ10ꢁ10 msꢁ1),
which was consistent with the reported value.[12a] Almost no
rate enhancement in the hydrolysis was observed when only
the zinc cation was added to the solution of BNPC or NA.
However, under identical conditions, a remarkable enhance-
ment was observed when ZnL was added, which was 530-
fold higher than that of BNPC self-hydrolysis. In the case of
NA, however, the hydrolysis
½ZnLðH2OÞ2ꢂ ¼ ½ZnLðH2OÞðOHÞꢂþþHþ
2þ
½ZnLðH2OÞðOHÞꢂþ ¼ ½ZnLðOHÞ2ꢂþHþ
Ka2
It might be interesting to find out that the formation con-
stant (logKML) of ZnL is much higher than that of the [Zn-
rate for the reaction catalyzed
by ZnL is just 2.09-fold higher
Table 1. Equilibrium constants of the ligand and its zinc complex.
Chemical equilibrium
Equilibrium constant
than that of the self-hydrolysis.
To fully assess the hydrolysis
ability of ZnL for BNPC, a de-
tailed kinetic study was under-
taken. Saturation kinetics were
observed (Figure 4) and thus,
kinetic parameters deduced
bamp[a]
pyridylBisCD
H3L3+ =H2L2++H+
H2L2+ =HL++H+
pK1
pK2
logKML
pKa1
pKa2
7.50ꢀ0.04
8.32ꢀ0.02
5.56ꢀ0.02
7.94ꢀ0.1
9.63ꢀ0.09
7.52ꢀ0.06
8.54ꢀ0.05
7.31ꢀ0.03
8.14ꢀ0.03
9.24ꢀ0.01
Zn2++L=[ZnL
G
A
ACHTUNGTRENUNG ACHTUNGTRENNUNG
(H2O)2]2+ =[ZnL
A
ACHTUNGTRENUNG
[a] Ref. [20], bamp=2,6-bis(aminomethyl)pyridine.
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Chem. Asian J. 2009, 4, 1354 – 1360

Ester Hydrolysis by a Cyclodextrin Dimer Catalyst
Figure 3. Plots of absorbance versus time during BNPC hydrolysis (a)
and NA hydrolysis (b) catalyzed by ZnACHTNUTRGEN(UNG ClO4)2 and ZnL in 10% MeCN
Figure 4. Saturation kinetics of BNPC hydrolysis (a) and NA hydrolysis
(b) catalyzed by ZnL. Each reaction mixture contained ZnL (50 mm),
Tris-HCl buffer (50 mm, pH 7.00) with 0.10m NaClO4 at (25ꢀ0.1)8C.
solution of pH 7.00 Tris-HCl buffer at 0.1m NaClO4 and (25ꢀ0.1)8C.
([catalyst]=50 mm, [BNPC]=50 mm and [NA]=400 mm).
Table 2. Initial rate (v) for ester hydrolysis promoted by different cata-
lyst.
Table 3. Kinetic parameters for the ester hydrolysis in the presence of
ZnL (50 mm) in 10% MeCN solution of Tris-HCl (50 mm pH 7.00) buffer
at (25ꢀ0.1)8C.
Catalyst[a]
BNPC
NA
v [10ꢁ9 msꢁ1
]
v/vcontr
v [10ꢁ9 msꢁ1
]
v/vcontr
Substrate
kuncat [sꢁ1
kcat [sꢁ1
BNPC
NA
Buffer
ZnII
ZnL
(2.45ꢀ0.03)ꢀ10ꢁ1[b]
(2.62ꢀ0.03)ꢀ10ꢁ1
(1.30ꢀ0.30)ꢀ102
1.00
1.35ꢀ0.10
1.45ꢀ0.13
2.82ꢀ0.30
1.00
1.07
2.09
]
(4.83ꢀ0.16)ꢀ10ꢁ6
(4.34ꢀ0.20)ꢀ10ꢁ2
(9.60ꢀ0.30)ꢀ10ꢁ2
4.52ꢀ102
(3.40ꢀ0.20)ꢀ10ꢁ6
(2.44ꢀ0.30)ꢀ10ꢁ4
1.72ꢀ0.02
1.07
]
5.30ꢀ102
Km [mm]
kcat/Km [mꢁ1 sꢁ1
kcat/kuncat
]
1.42ꢀ10ꢁ1
[a] Reaction condition: 50 mm BNPC or 400 mm NA, 50 mm catalyst, 0.1m
NaClO4, 50 mm pH 7.00 Tris-HCl buffer, (25ꢀ0.1)8C. [b] Ref. [12a], v=
8.98ꢀ103
71.76
0.0147 mmminꢁ1
.
appearance of 4-nitrophenolate at 400 nm.[22] Since the sub-
strate concentration was essentially constant during the
measurement, the initial first-order rate constant (kin, in=
initial) of the total catalyst was calculated as in Equa-
tion (4):[16]
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 it showed
values up to 8.98ꢀ103 for BNPC hydrolysis. For NA hydrol-
ysis, nevertheless, the value of kcat/kuncat was found to be
71.76, which is two orders of magnitude lower than that of
v ¼ kin½BNPPꢂ ¼ ðkBNPP½ZnLꢂtotalþkOHꢁ½OHꢁꢂÞ½BNPPꢂ
ð4Þ
BNPC. Furthermore, a much better catalytic efficiency kcat
/
Km was obtained for BNPC hydrolysis (4.52ꢀ102 mꢁ1 sꢁ1) in
contrast to NA hydrolysis (1.42ꢀ10ꢁ1 mꢁ1 sꢁ1), which was
about a 3.18ꢀ103-fold higher activity than that of NA.
where v is the 4-nitrophenolate releasing rate. At a given
pH value, the kin values were measured at different concen-
trations of the catalyst. Koike et al. even reported the initial
first-order rate constant of BNPP spontaneous hydrolysis
was 8.0ꢀ10ꢁ11 sꢁ1 in water at pH 8.5 and 358C.[23] In the
presence of 0.1 mm ZnL, however, the initial first-order rate
constant of the phosphate diester hydrolysis is enhanced up
to 1.29ꢀ10ꢁ7 sꢁ1 under the same conditions, which is about a
1600-fold acceleration over the uncatalyzed hydrolysis.
Figure 5a shows the effect of ZnL concentrations on the
kin for the cleavage of BNPP at pH 8.85 and (35ꢀ0.1)8C.
Hydrolysis of Phosphate Esters
BNPP is often used as a DNA model compound in the in-
vestigation of phosphodiesterase activity. The test was car-
ried out in buffers to mimic biological conditions. The initial
phosphorylation rate in aqueous solution at (35ꢀ0.1)8C and
pH 6.50–9.50 (50 mm Goodꢂs buffer) was monitored by the
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Z.-W. Mao et al.
FULL PAPERS
sulted in an inflection point at 8.25 for BNPP and 8.15 for
NPP. This is almost the same as the pKa1 value of the coordi-
nated water deprotonation in [ZnLACHTUNGTRNEUNG
(H2O)2]2+ obtained from
the potentiometric pH titration (Table 1). The catalytic ac-
tivity of ZnL to phosphate diester BNPP is higher than that
to the monoester NPP (kBNPP =1.68ꢀ10ꢁ3 mꢁ1 sꢁ1, kNPP
=
5.75ꢀ10ꢁ4 mꢁ1 sꢁ1); the main reason is that the binding ability
of the ditopic hydrophobic substrate BNPP with the two
cavities of cyclodextrin is more stronger than that of the
monotopic hydrophobic substrate NPP.
Moreover, for BNPP hydrolysis, the kBNPP value is compa-
ratively higher than triamine and macrocyclic polyamine
ZnN3 analogues[24–27] with a similar pKa in the catalytic hy-
drolysis of BNPP (see Table 4), and is slightly higher than
that of our previous reported Zn-phenCD2 complex with N4
ligand.[18]
The rate of hydrolysis of the phosphate esters increased
with pH increase. According to the species distribution de-
termined by the pH potentiometric titration and analysis of
the kBNPP/pH and kNPP/pH profiles, the active species should
ꢁ
be [ZnL(OH)ACTHNUTRGNEUNG
(H2O)]+ and [ZnL(OH)2], and the Zn OH in
ZnL acts as the nucleophile. The second-order rate con-
stants, kBNPP and kNPP, exhibiting a sigmoidal curve versus
pH, however, is different from that of our previous work, in
which the second-order rate constant kNA increased expo-
nentially with an increase in pH, and NA hydrolysis was cat-
alyzed by a supramolecular inclusion complex [Zn(L1)-
Figure 5. a) Dependence of the initial first-order rate constants on the
concentration of ZnL at pH 8.85 and (35ꢀ0.1)8C (I=0.10m NaClO4,
[BNPP]=0.20 mm, [buffer]=50 mm). b) The pH dependence of the
second-order rate constants of BNPP hydrolysis ([ZnL]=0.10 mm,
[BNPP]=1.00 mm, [buffers]=50 mm, I=0.10m NaClO4, T=(35ꢀ0.1)8C.
A
(ClO4)2·9.5H2O (L1 =4-(4’-tert-butyl)benzyl-
2ACHUTGTNERNNUG(b-CD)]ACHTUGNTRENNUGN
The rate of BNPP cleavage ini-
Table 4. The second-order rate constants kBNPP of BNPP hydrolysis promoted by ZnII complexes at (35ꢀ
0.1)8C.[a]
tially increases linearly with the
increase of ZnL concentration
but gradually deviates from the
Species
type
kBNPP [mꢁ1 sꢁ1
]
pKa
Ref.
[Zn
[Zn
[Zn
[Zn
[Zn
[Zn
N
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’-Zn
N,N’,N’’,N’’’-Zn
2.3ꢀ10ꢁ5
6.4ꢀ10ꢁ5
2.3ꢀ10ꢁ5
9.7ꢀ10ꢁ5
1.2ꢀ10ꢁ4
1.1ꢀ10ꢁ4
8.5ꢀ10ꢁ5
8.5ꢀ10ꢁ5
1.0ꢀ10ꢁ5
1.68ꢀ10ꢁ3
9.9ꢀ10ꢁ4
8.93
8.90
8.58
8.13
8.56
8.51
7.3
[24]
[24]
[24]
[24]
[24]
[24]
[25]
[26]
linearity.
The
calculated
second-order rate constant of
BNPP hydrolysis catalyzed by
ZnL, kBNPP, was determined
from the slope of the linear
plot. Thus, the slope of kin
versus [ZnL]total from Equa-
tion (4) resulted in the second-
order rate constant (kBNPP). For
comparison, phosphate mono-
ester NPP hydrolytic cleavage
was performed under the same
conditions. The dependence of
the second-order rate (kBNPP
and kNPP) on pH for BNPP and
[Zn(OH)(N,N’,N’’-[12]ane)]+
[Zn
[Zn(L)(OH)]+
[Zn
(phenCD2)(OH)]+
7.2
6.94
8.14
8.18
[27]
this work
[18]
[a] dien=diethylenetriamine, epd=N-(2-aminoethyl)-1,3-propanediamine, dpt=dipropylenetriamine, tach=
1,3,5-triaminocyclohexane, taci=1,3,5-triamino-1,3,5-trideoxy-cis-inositol), tmca=2,4,6-trimethoxycyclohex-
ane-1,3,5-triamine, N,N’,N’’-[12]ane=1,5,9-triazacyclododecan, bmxd=3,6,9,17,20,23-hexaazatricyclotriaconta-
1,11,13,15,25,27-hexaene.
ꢁ
NPP cleavage promoted by ZnL are illustrated in Figure 5b
and Figure S3 in the Supporting Information, respectively.
kBNPP increases sharply as the pH increases from 7.50 to 8.85
and then slows down at higher pH, displaying a sigmoidal
curve for the cleavage reaction, whereas kNPP shows a slow
increase as pH increases from 7.50 to 8.85. The results indi-
cate a kinetic process controlled by an acid–base equilibri-
um. The data were fitted by a Boltzman model, which re-
should be only one of the Zn OH groups and the other hy-
droxyl group in [ZnL(OH)2] is not functional during the cat-
alysis. This may be ascribed to the orientation of the second
hydroxyl deviated from the phosphorus atom of the esters,
resulting in the inappropriate distance between the second
hydroxyl group and the phosphorus atom of the esters.
The above results show that under identical conditions,
the hydrolysis rate of BNPC is much higher than that of
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Chem. Asian J. 2009, 4, 1354 – 1360

Ester Hydrolysis by a Cyclodextrin Dimer Catalyst
physical measurement experiments
was Milli-Q grade. The materials of 6-
monodeoxy-6-monoamino-b-cyclodex-
trin was prepared from 6-monodeoxy-
6-monoazido-b-cyclodextrin according
to a previous procedure reported by
Jicsinszky et al.,[30] with a minor modi-
fication, whereas 6-mono(p-toluenesul-
fonyl)-b-cyclodextrin was prepared in
dry pyridine solution rather than in
aqueous solution.[31] All compounds
were confirmed by their elemental
analyses and ESI-MS and 1H NMR
spectra.
General Methods
1H NMR spectra were recorded on a
Varian INOVA-300NB or Mercury
plus 300 spectrometers. Elemental
contents were analyzed by a Perkin-
Elemer 240 elemental analyzer. ESI-
MS spectra were performed on
a
Thremo LCQ-DECA-XP spectrome-
ter. UV/Vis spectra were monitored
with a Varian Cary 300 UV/Vis spec-
trophotometer equipped with a tem-
perature controller (ꢀ0.18C).
Scheme 2. Suggested intermediates for ester hydrolysis catalyzed by ZnL.
NA, and the BNPP hydrolysis rate is higher than that of
NPP. It is well-known that the 4-nitrophenolate group can
bind into the hydrophobic cavity of b-cyclodextrin,[28,29] and
thus BNPP and BNPC with two ditopic groups of 4-nitro-
phenol can strongly bind into both cavities of b-cyclodex-
trins.[11c,12a,18] However, such binding hardly exists for the
one hydrophobic group substrates NA and NPP. Therefore,
the enhanced second-order rate constant should be contrib-
uted to the cooperative binding action of both hydrophobic
cavities and zinc hydroxyl active species of the complex, and
a possible intermediate of the esters hydrolysis catalyzed by
ZnL is proposed in Scheme 2.
Syntheses
2,6-dimethylpyridyl-bridged-bis(6-monoammonio-b-cyclodextrin) (L): A
solution of 2,6-bis(bromomethyl)pyridine (0.261 g, 0.985 mmol) in dry
DMF (5 mL) was added to a solution of 6-monodeoxy-6-monoamino-b-
cyclodextrin (2.461 g, 2.168 mmol) and anhydrous potassium carbonate
(1.193 g, 8.63 mmol) in dry DMF with stirring. The mixture was then
heated to 808C for 8 h under an argon atmosphere. The reaction was
cooled to room temperature, and then mixture was evaporated to dryness
under reduced pressure. The resulting residue was dissolved in a small
amount of hot water, and the aqueous solution was poured into acetone
(200 mL) to give a light-yellow precipitate. The crude product was dried
and purified by column chromatography over Sephadex G-25 with dis-
tilled deionized water as the eluent to give the pure compound in 35.2%
yield. 1H NMR (300 MHz, [D6]DMSO): d=7.62–7.68 (m, 1H; pyridine-
H-4), 7.23 (d, J=7.8 Hz, 2H; pyridine-H-3,5), 5.84–5.72 (m, 28H; OH-
2,3), 4.82 (m, 14H; H-1), 4.56–4.46 (m, 12H; OH-6), 3.63–3.31 (m, 88H;
H-3,5,6, pyridine-CH2), 2.05 ppm (brs, 2H; NH); MS (ESI, H2O/
CH3OH): m/z: calcd:1186.5 [L+2H]2+, 1204.5 [L+2H+2H2O]2+; found:
1186.2, 1204.9; elemental analysis calcd (%) for C91H147N3O68·12H2O:
C 42.24, H 6.66, N 1.62; found: C 42.54, H 7.071, N 1.819.
Conclusions
A new cyclodextrin dimer with a tridentate ligand (N,N’,N’’)
and its tridentate N,N’,N’’-zinc complex have been synthe-
sized and characterized. The zinc complex exhibits a good
catalytic ability for bis(4-nitrophenyl) substrates in the hy-
drolysis of carboxylic acid ester and phosphate esters, espe-
cially for the carboxylic acid diester. The hydrophobic inter-
actions between catalyst and substrate play important roles
in the hydrolysis.
Zinc complex (ZnL): A solution of pyridyl BisCD (0.101 g, 0.040 mmol)
in water (2 mL) was added dropwise to a dilute aqueous solution of a
slight excess of ZnACHTUNGTRNEG(UN ClO4)2·6H2O (0.020 g, 0.053 mmol) with stirring at
room temperature. The resulting solution was stirred for 3 h, and then
the solution was evaporated under reduced pressure. Ethanol was added
to form a precipitate which was collected by filtration and washed succes-
sively with a small amount of ethanol, and then dried in vacuo to give
the pure complex as a white solid in 60% yield. 1H NMR (300 MHz,
[D6]DMSO): d=8.00–7.95 (m, 1H; pyridine-H-4), 7.46–7.42 (m, 2H; pyr-
idine-H-3,5), 5.82–5.77 (m, 28H; OH-2,3), 4.84 (m, 14H; H-1), 4.52–4.34
(m, 12H; OH-6), 3.65–3.32 (m, 88H; H-3,5,6, pyridine-CH2), 1.05 ppm
(brs, 6H; NH; H2O); MS (ESI, H2O/CH3OH): m/z: calcd: 1217.4
[ZnL]2+; 1253.4 [ZnL+4H2O]2+ found: 1217.8; 1253.8; elemental analysis
calcd (%) for C91H151N3O78ZnCl2·22H2O: C 35.63, H 6.41, N 1.37; found:
C 35.55, H 6.072, N 1.499.
Experimental Section
Materials
NA, BNPC, BNPP, and 2,6-bis(bromomethyl)pyridine were purchased
from Aldrich. NPP (disodium 4-nitrophenyl phosphate) was purchased
from TCI. b-CD (reagent grade) was recrystallized twice from H2O and
dried in vacuo for 12 h at 1008C, 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. Water used in all
Potentiometric pH Titration
An automatic titrator (Metrohm 702GPD Titrino) coupled to a Metrohm
electrode was used and calibrated according to the Gran method.[32] The
electrode system was calibrated with buffers and checked by titration of
Chem. Asian J. 2009, 4, 1354 – 1360
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www.chemasianj.org
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Z.-W. Mao et al.
FULL PAPERS
HClO4 with NaOH solution (0.10m). The thermostated cell contained
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and initiated by adding fixed volumes of 0.10m standard NaOH in small
increments to the titrated solution. Duplicate measurements were per-
formed, 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 ligand protonation constants Kn, the complex formation
constant KML, and the deprotonation constants of the coordinated water
pKa.
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The hydrolysis rates of BNPC and NA in the presence of ZnL complex
were measured by an initial slope method following the increase in the
400 nm absorption of the released 4-nitrophenolate.[18] The reaction solu-
tion was maintained at (25ꢀ0.1)8C. Tris-HCl (pH 7.00, 8.85) buffers
were used (50 mm), and the ionic strength was adjusted to 0.10m with
NaClO4. In a typical experiment, after substrate (NA or BNPC) and ZnL
complex in 10% (v/v) CH3CN solution at an appropriate pH were mixed,
the UV absorption decay was recorded immediately and was followed
generally until 2% decay of 4-nitrophenyl acetate. Errors on kobs values
were about 5%.
Kinetics of BNPP and NPP Hydrolysis
The rate of hydrolysis of BNPP and NPP to give mono(4-nitrophenyl)-
phosphate and 4-nitrophenolate was measured by an initial slope method
following the increase in the 400 nm absorption of the released 4-nitro-
phenolate in aqueous solution at (35ꢀ0.1)8C.[22] At this wavelength, the
absorbance of the ester substrate was negligible. MES (pH 6.00–6.60),
MOPS (pH 6.60–7.40), HEPES (pH 7.40–8.20), TAPS (pH 8.20–8.90),
and CHES (pH 8.90–9.50) buffers were used (50 mm), and the ionic
strength was adjusted to 0.10m 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, NPP, buffers, and ZnL in aqueous solu-
tion were freshly prepared. The reactions were initiated by injecting a
small amount of BNPP and NPP into the buffer solutions of ZnL and fol-
lowed by fully mixing at (35ꢀ0.1)8C. The visible absorption increase was
recorded immediately and was followed generally until 2% formation of
4-nitrophenolate, in which
e values for 4-nitrophenolate were 7106
(pH 6.80), 9137 (pH 7.00), 11715 (pH 7.26), 13720 (pH 7.50), 15270
(pH 7.75), 16306 (pH 8.00), 16953 (pH 8.25), 17340 (pH 8.50), 17626
(pH 8.85), 17694 (pH 9.00), and 17810 (pH 9.50) at 400 nm. The initial
first-order rate constants, kin (sꢁ1), for the cleavage of BNPP and NPP,
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, kNPP) for the catalyzed re-
actions were determined as the slope of the linear plots of kin versus ZnL
concentration. To correct the spontaneous cleavage of BNPP and NPP,
each reaction was measured against a reference cell that was identical to
the sample cell in composition except for the absence of ZnL. Errors on
kBNPP and kNPP values were about 5%.
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Acknowledgements
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This work was supported by the National Natural Science Foundation of
China, the National Basic Research Program of China (No.2007C
B815306) and the Natural Science Foundation of Guangdong Province
(No.071176 37).
[32] G. Gran, Acta Chem. Scand. 1950, 4559.
Received: March 20, 2009
[1] Y. Liu, Y. Chen, Acc. Chem. Res. 2006, 39, 681.
Published online: July 3, 2009
1360
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Chem. Asian J. 2009, 4, 1354 – 1360