Ester catalytic hydrolysis by a tridentate N,N′,N″-copper bridged cyclodextrin dimer

Article abstract of DOI:10.1016/j.inoche.2010.10.018

A new pyridine bridged cyclodextrin dimer mono-copper complex (CuL) was synthesized and characterized. The hydrolysis of carboxylic acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-nitrophenyl acetate (NA), and phosphate ester, a DNA model bis(4-nitrophenyl)phosphate (BNPP), promoted by CuL has been investigated. The resulting hydrolysis rate constants showed that CuL had a very high rate of catalysis for BNPC hydrolysis, yielding a 2.73 × 10 ³-fold rate enhancement over uncatalyzed hydrolysis at pH 7.00, compared to only a 78.2-fold rate enhancement for NA hydrolysis. The initial first-order rate constant of catalytic hydrolysis for BNPP was 1.01 × 10^-7 s^-1 at pH 8.5, 35 °C and 0.1 mM catalyst concentration, about 1260-fold acceleration over uncatalyzed hydrolysis. The second rate constant k_BNPP of BNPP hydrolysis promoted by CuL was found to be 5.94 × 10^-4 M^-1 s^-1.

Full text of DOI:10.1016/j.inoche.2010.10.018

Inorganic Chemistry Communications 14 (2011) 184–188
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Inorganic Chemistry Communications
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Ester catalytic hydrolysis by a tridentate N,N′,N″-copper bridged cyclodextrin dimer
a,b
a
a
a
a,
a
Si-Ping Tang , Sha Chen , Guang-Fei Wu , Huo-Yan Chen , Zong-Wan Mao ⁎, Liang-Nian Ji
a
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry School of Chemistry and Chemical Engineering, Sun Yat-Sen University Guangzhou, 510275, China
Department of Chemistry and Material Science Hengyang Normal University Hengyang, Hunan 421008, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2010
Accepted 21 October 2010
Available online 28 October 2010
A new pyridine bridged cyclodextrin dimer mono-copper complex (CuL) was synthesized and characterized.
The hydrolysis of carboxylic acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-nitrophenyl acetate (NA),
and phosphate ester, a DNA model bis(4-nitrophenyl)phosphate (BNPP), promoted by CuL has been
investigated. The resulting hydrolysis rate constants showed that CuL had a very high rate of catalysis for
3
BNPC hydrolysis, yielding a 2.73×10 -fold rate enhancement over uncatalyzed hydrolysis at pH 7.00,
Keywords:
compared to only a 78.2-fold rate enhancement for NA hydrolysis. The initial first-order rate constant of
Cyclodextrin dimer
Hydrolysis
Kinetics
catalytic hydrolysis for BNPP was 1.01×10−
7
s
−1
at pH 8.5, 35 °C and 0.1 mM catalyst concentration, about
1260-fold acceleration over uncatalyzed hydrolysis. The second rate constant kBNPP of BNPP hydrolysis
promoted by CuL was found to be 5.94×10−
4
M
−1 −1
s
.
Ester
Copper complex
© 2010 Elsevier B.V. All rights reserved.
Phosphodiester bond is highly stable kinetically at physiological
pH. The halftimes for hydrolysis of RNA and DNA at pH 7.0 and 25 °C
are reported to be 110 and up to 100 billion years, respectively.
Nature employs suitable enzymes like phosphodiesterases to
enhance the hydrolytic rate. However, in general, enzymes are
expensive, need purification, and are sensitive to the environment.
So, development of artificial metallonucleases would have potential
applications in biotechnology and drug development. In the past two
geneous catalyst, copper(II)-chelated chitosan magnetic nanocarrier
(CMN–Cu(II)), and its calatytic rate constant for BNPP hydrolysis is
higher than that of the other carrier catalysts and small molecular Cu
3
[9]aneN complexes. However, these carrier catalysts can't mimic
the microenvironment of nature enzymes such as hydrophobic
property and recognition to substrate very well. Compounds based
on β-cyclodextrins as the models of enzyme have been paid
extensive close attention due to the hydrophobic cavity and
hydrophilic external surface of β-cyclodextrin, in which copper
complexes have been reported. Li and Liu et al. constructed copper
(II) complexes of tris(2-aminoethyl)amino-cyclodextrin (Cu-tren-
CD) [34] and of CD-modified gold nanoparticles as supramolecular
hydrolase models [35], both mimics showed preferable activities for
BNPC hydrolysis. Breslow group designed and synthesized a
cyclodextrin dimer catalyst with a N,N′-bidentate bipyridine-copper
linking group, which remarkably accelerated catalytic hydrolysis of
carboxylic acid diesters and phosphate diesters [36,37].
2
+
3+
2+
decades, many metal complexes, including Cu , Co , Zn , and
3
+
La
complexes, have been synthesized as models of metallohy-
drolases and/or as catalysts for the hydrolysis of DNA, RNA and
peptides in which copper complexes were the most studied as
artificial nucleases. Examples include small molecule copper com-
plexes with N,N′-bidentate bipyridyl [1–5], N,N′,N′′-tridentate
terpyridyl [6–16] and 1,4,7-triazacyclononane ligands [17–26] and
their derivatives. The catalytic activities of small molecule copper
complexes for ester hydrolysis are high. However, the main
disadvantage is that the dimer bridged by hydroxyl group with no
hydrolytic reactivity is easily formed at high pH. Recently, few
investigations demonstrated copper complexes show the effective-
ness of heterogeneous catalysis for phosphodiester hydrolytic
reaction using polymer [27–31] or polymer-silica [32] particles as
catalyst carriers. Dong-Hwang Chen et al. [33] prepared a hetero-
In previous papers, we reported the zinc complexes of
β-cyclodextrin dimers, respectively linked by phenanthroline with
4 3
tetradentate N and pyridine with tridentate N ligands, which
demonstrated satisified activities for diester hydrolysis [38,39].
However, structure change of the bridge from tetradentate to
tridentate has no obvious rate enhancement for esters hydrolysis.
So, on the basis of our works, we designed and synthesized a new
copper complex based on cyclodextrin dimer bridged by diamino-
methyl pyridine(CuL) to investigate the hydrolytic activities affected
by copper cation.
The copper complex (CuL) was prepared from the ligand (L) and
copper perchlorate (Scheme 1) [40]. ESI-MS spectrometry of CuL is a
⁎
Corresponding author. Tel.: +86 20 84113788; fax: +86 20 84112245.
E-mail address: cesmzw@mail.sysu.edu.cn (Z.-W. Mao).
1387-7003/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2010.10.018

S.-P. Tang et al. / Inorganic Chemistry Communications 14 (2011) 184–188
185
Scheme 1. Synthetic scheme of copper complex CuL.
very important method to investigate the complex formation in
aqueous/MeOH solution. Fig. S1 (see Supporting Information) shows
both the experimental and calculated isotopic distribution for the
[Cu(bamp)] (bamp=2,6-bis(aminomethyl)pyridine) complex [42], a
II
2+
simple analogue of CuL. Two copper species, [CuL(H
(OH)] , are involved in the complex formation at pH 3.0–11.5. Since
2
O)] and [CuL
+
2+
peak at m/z: 1216.6 and 1269.1, which were assigned to [CuL] and
the addition of NaClO
confirmed that the remaining coordination site of copper was
occupied by one water molecule. The formation constant (logKML
and pK values of CuL are similar to that of [Cu(bamp)] complex
4
did not cause spectral changes, it was also
2
+
II
[
CuL+4H
2
O+CH
3
OH]
respectively. This clearly indicates the
existing binding between the copper cation and L. According to the
result of thermal analysis (Fig. S2, see Supporting Information), when
temperature was lower than 120 °C, the copper complex lost its
solvent water molecules included in the cavities of cyclodextrin
with 10.8% weight( theoretical value 11.2%). And as the temperature
was heated to 190 °C, the coordinated water molecular was lost and
the coordinated environment of metal copper atom became
unstable obviously, which caused structure collapseand of CuL
directly. So the copper complex was tetra-coordinated with one
coordinated water molecular. And this structure was confirmed
again by the result of potentiometric pH titration (Fig. S3, see
Supporting Information).
)
a
(logKML 12.53± 0.03; pKa1 8.13± 0.06 for CuL and logKML 12.20±
0.04; pKa18.11± 0.09 for [Cu(bamp)]).
To determine the effect of hydrophobic interactions on the
catalytic activities of CuL, BNPC and NA were selected as testing
substrates because of their structural features. Studies on the
hydrolysis kinetics of BNPC and NA were performed in a 10% MeCN
solution of Tris–HCl (50 mM, pH 7.00) at (25± 0.1)°C (Fig. 1).
Monitoring the formation of 4-nitrophenolate by UV at 400 nm
−1
−1
(εobs=8700 M cm ) [43], the initial hydrolysis rates of BNPC
(50 μM) and NA (400 μM) in the presence of the catalysts were
calculated (Table 1). The measured initial rate of spontaneous cleavage
Caution: Copper perchlorate is a potentially explosive chemical,
and it should be prepared in small quantities and handled with
care.
−10
−1
of BNPC (50 μM) was very slow (υcontrol =2.45×10
M s ), which
was consistent with the reported value [44]. Almost no rate enhance-
ment in the hydrolysis was observed when only the copper cation was
added to the solution of BNPC or NA. However, under identical
conditions, a remarkable enhancement was observed when CuL was
added, which was 412-fold higher than that of BNPC self-hydrolysis. In
the case of NA, however, hydrolysis rate catalyzed by CuL is just 2.87-
fold higher than that of the self-hydrolysis. To fully assess the hydrolysis
ability of CuL for BNPC, a detailed kinetic study was undertaken.
The potentiometric pH titrations were performed starting at acidic
pH, using a sodium hydroxide solution as the titrant. The deprotona-
tion steps could be derived from the titration curves. The pH profiles
of the titration curves, which include the distribution curves of the
II
copper species as a function of pH (Fig. S3, see Supporting
Information), were analyzed by the Hyperquad program [41]. The
calculated results are summarized in Table S1, including data from the

186
S.-P. Tang et al. / Inorganic Chemistry Communications 14 (2011) 184–188
Fig. 2. Saturation kinetics of BNPC hydrolysis (a) and p-NA hydrolysis (b) catalyzed by
CuL. Each reaction mixture contained CuL (150 μM), Tris–HCl buffer (50 mM pH 7.00)
with 0.10 M NaClO at (25± 0.1)°C.
4
Fig. 1. Plots of absorbance vs time during BNPC hydrolysis (a), NA hydrolysis (b) catalyzed
by Cu(ClO and CuL in 10% MeCN solution of pH 7.00 Tris–HCl buffer at 0.1 M NaClO and
25± 0.1)°C. ([catalyst]=150 μM, [BNPC]=50 μM and [NA]=400 μM).
)
4 2
4
(
was essentially constant during the measurement, the initial first-
order rate constant (kin, in=initial) of the total catalyst was calcu-
lated as in Eq. (1) [46]:
À
Á
−
Saturation kinetics was observed (Fig. 2) and thus kinetic parameters
deduced from the Michaelis–Menten equation for the hydrolysis are
listed in Table 2. The value of kcat/kuncat was used to describe the catalytic
v = k ½BNPPꢀ = kBNPP½CuLꢀtotal + kOH
−
½OH ꢀ ½BNPPꢀ
ð1Þ
in
where v is the 4-nitrophenolate releasing rate. At a given pH value, the
3
ability of hydrolase mimics, and it showed values up to 2.72×10 for
k
in values were measured at different concentrations of the catalyst.
BNPC hydrolysis. For NA hydrolysis, nevertheless, the value of kcat/kuncat
was found to be 78.2, two orders of magnitude lower than that of BNPC.
Koike et al. even reported the initial first-order rate constant of BNPP
−11 −1
spontaneous hydrolysis was 8.0×10
s
in water at pH 8.5 and
Furthermore, a much better catalytic efficiency kcat/K
m
was obtained for
35 °C [47]. In the presence of 0.1 mM CuL, however, the initial first-
−
1 −1
BNPC hydrolysis (18.30 M
s
) in contrast to NA hydrolysis
order rate constant of the phosphate diester hydrolysis enhances up
−2
−1 −1
2
−7 −1
(
6.30×10
M
s
), which was about 2.90×10 -fold higher activity
to 1.01×10
s
at the same conditions, which is about 1260-fold
than that of NA.
acceleration over the uncatalyzed hydrolysis.
The DNA model compound BNPP was used as substrate to
investigate phosphodiesterase activity. The test was carried out
in buffers to mimic biological conditions. The initial phosphor-
ylation rate in aqueous solution at (35± 0.1)°C and pH 6.50–9.00
Fig. 3(a) shows the effect of CuL concentrations on the kin for the
cleavage of BNPP at pH 8.85 and (35± 0.1)°C. The rate of BNPP
cleavage initially increases linearly with the increase of CuL
concentration but gradually deviates from the linearity. The
calculated second-order rate constant of BNPP hydrolysis catalyzed
by CuL, kBNPP, was determined from the slope of the linear
plot. Thus, the slope of kin versus [CuL]total from Eq. (1) resulted
(
50 mM Good's buffer) was monitored by the appearance of
-nitrophenolate at 400 nm [45]. Since the substrate concentration
4
Table 1
Table 2
Initial rate (v) for ester hydrolysis promoted by different catalyst.
Kinetic parameters for the ester hydrolysis in the presence of CuL (150 μM ) in 10%
MeCN solution of Tris–HCl (50 mM pH 7.00) buffer at (25± 0.1)°C.
Catalysta
BNPC
NA
v (10− M s−1
9
)
v/vcontr
v (10−9 M s−1
)
v/vcontr
Substrate
BNPC
NA
1
uncat (s−1)
−6
−2
−6
−4
Buffer
Cu(II)
CuL
(2.45± 0.03)×10− [44]
(2.70± 0.03)×10
(1.02± 0.30)×102
1.00
1.35± 0.10
1.37± 0.20
3.88± 0.40
1.00
1.01
2.87
k
k
K
k
k
(4.83± 0.16)×10
(1.32± 0.10)×10
0.72± 0.03
(3.40± 0.20)×10
(2.65± 0.15)×10
4.68± 0.20
6.30×10
78.2
−
1
−1
1.10
cat (s
)
4.12×102
(mM)
m
(M− s−1
1
)
18.30
−2
cat/K
cat/kuncat
m
a
Reaction condition: 50 μM BNPC or 400 μM NA, 150 μM catalyst, 0.1 M NaClO
0 mM pH 7.00 Tris–HCl buffer, (25± 0.1)°C.
4
,
2.73×103
5

S.-P. Tang et al. / Inorganic Chemistry Communications 14 (2011) 184–188
187
Acknowledgements
This work was supported by the National Natural Science
Foundation of China, the National Basic Research Program of China
(
No. 2007C B815306), the Natural Science Foundation of Guangdong
Province (No. 071176 37), the Foundation of Hunan Province
Education Office (No. 08C178) and the Found of Hengyang Normal
University (10B66).
Appendix A. Supplementary material
Supplementary material to this article can be found online at
doi:10.1016/j.inoche.2010.10.018.
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.94×10
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[
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(
4
[
[
[
[
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4
. In a typical experiment, after
CN solution at an
3
appropriate pH were mixed, the UV absorption decay was recorded immediately
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4
)
2
2
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of the linear plots of kin versus CuL concentration. To correct 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 CuL.
Errors on kBNPP values were about 5%.
and then dried in vacuo to give the pure complex as a blue solid in 70% yield. MS (ESI,
2+
H
2
O/CH
3
OH): m/z:calcd: 1216.8 [CuL]2+; 1269.3 [CuL+4H
2
O+CH
3
OH] found:
1
216.6; 1269.1. elemental analysis calcd (%) for C91
H
195
N
3
O
100CuCl : C, 35.63; H,
2
6.41; N, 1.37. Found: C, 35.23; H, 5.94; N 1.48.
[
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