Effect of hydrophobic interaction cooperating with double Lewis acid activation in a zinc(ii) phosphodiesterase mimic

Article abstract of DOI:10.1039/c0cc00838a

The novel dinuclear Zn(ii) complex (1) containing a β-CD dimer could accelerate BNPP (a DNA substitute) hydrolysis more efficiently than catalyze HPNP (a RNA substitute) transesterification with different mechanisms involved; the β-CDs played remarkably different roles.

Full text of DOI:10.1039/c0cc00838a

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Effect of hydrophobic interaction cooperating with double Lewis acid
activation in a zinc(II) phosphodiesterase mimicw
Meng Zhao, Li Zhang, Huo-Yan Chen, Han-Lu Wang, Liang-Nian Ji and Zong-Wan Mao*
Received 9th April 2010, Accepted 15th July 2010
DOI: 10.1039/c0cc00838a
The novel dinuclear Zn(II) complex (1) containing a b-CD dimer
could accelerate BNPP (a DNA substitute) hydrolysis more
efficiently than catalyze HPNP (a RNA substitute) transesterifi-
cation with different mechanisms involved; the b-CDs played
remarkably different roles.
Zn(II)-water and the nearby C6-hydroxyl group of b-CD, which
could assist the Zn(II) center to facilitate the deprotonation.
8
The pKa3 of 9.91 Æ 0.05 assigned to a terminal Zn(II) bond was
somewhat higher due to the reduced positive charge at the
9
metal core. This analysis revealed that 1 generated two sets of
Zn(II) bound hydroxyl groups in solution which might be the
active species responsible for the hydrolysis of phosphoric
10
Under normal circumstances, the phosphodiester bonds in DNA
1
and RNA are very stable, but in the presence of phospho-
ester bonds.
diesterase, they could be hydrolyzed very easily. Significant
efforts have been dedicated to build bio-inspired and smart
Then, BNPP was used as a substrate to test the catalytic
activity of 1 mimicking metallohydrolases. The Machaelis
complex formed by 1 with BNPP (1 : 1) was detected using
ESI-MS (Fig. S1 and Table S2, ESIw), suggesting enhanced
2
phosphodiesterase mimics. Inorganic chemists have synthesized
simple mono- or di-nuclear complexes imitating active sites of
3
1
phosphodiesterase to understand the cooperative role of metal
3
ions and weak interaction effect between a metal site and
affinity between the catalyst and the substrate. P NMR
spectroscopy monitoring of the hydrolysis reaction showed
that the final product of the catalyzed reactions was 4-nitrophenyl
phosphate (NPP), and no further hydrolysis of NPP was
observed (Fig. S2, ESIw). The dependence of the second-order
rate constant (kobs), as determined by varying catalyst con-
centration, on pH was investigated (Fig. 2a). Compared to the
distribution plots in Fig. 1, it was assumed that the kinetic
process was controlled by two acid–base equilibria. The data
4
organic groups. Meanwhile, for the first time, the supra-
molecular method was applied to the studies of mimetic hydro-
5
lase by organic chemists. One of the most typical examples is a
type of artificial enzyme constructed by modified b-cyclodextrins
6
,7
(
b-CDs). In these studies, the b-CD molecule could effi-
ciently catch the substrate by its cavity to stabilize the transi-
tion state. Based on the above-mentioned inorganic and
organic approaches, herein we present a novel hydrolase
mimic, which is a dinuclear Zn(II) complex (1), derived from
a modified b-CD dimer, 2,6-bis{[6-mono(2-pyridylmethyl-
amino)-b-cyclodextrin]-methyl}-4-methylphenol (HL) (ESIw).
Incubation of HL with 2 equivs of Zn(II) in water at room
temperature yielded the dinuclear complex, 1 (Scheme 1),
which was structurally characterized (ESIw). Compared with
other reported hydrolase mimics, 1 is the first complex that
possesses both double Lewis activation sites and double
hydrophobic cavities, which implies that the multiple inter-
actions could function cooperatively to result in remarkable
reactivity.
was fit to eqn 1 (ESIw) in which the pK
a
values were fixed as
determined from the titration experiment, giving (1.1 Æ 0.1) Â
À3
À1 À1
À3
À1 À1
1
10
2
k
M
s
and (4.9 Æ 0.6) Â 10
M
s
for k BNPP and
2
BNPP
,
respectively. The
+
k BNPP corresponding to
1
was higher than k BNPP corresponding to
[Zn
[Zn
2
L(OH)
2
]
2+
L(OH)] , which could be explained by the fact that a
2
higher pK
for metal coordinated water usually accompanies
a
11
better nucleophilic properties. Note that the more nucleophilic
+
species, [Zn
L(OH) ] , showed even higher reactivity than the
2 2
1
2
most active Zn(II)-bound hydroxyl species ever reported.
Compared with either reported simple dinuclear Zn(II) analogues
or mononuclear Zn(II)-b-CD complex, the reactivity enhance-
ment was remarkable in our study. Thus, we concluded that
First, we investigated the thermodynamic properties of 1 in
aqueous solution using potentiometric titration to determine
the active species. In total, three deprotonation events were
observed (Fig. 1 and Table S1, ESIw). The obtained pKa1 of
5
.45 Æ 0.02 was assigned to the proton release from the acidic
phenolic hydroxyl group. The pKa2 of 7.49 Æ 0.04 assigned to
a terminal Zn(II) bound water was lower than most reported
a
ones which commonly give pK values above 8.0. The increased
acidity might be due to the hydrogen bond between
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,
School of Chemistry and Chemical Engineering,
Sun Yat-Sen University, Guangzhou 510275, China.
E-mail: cesmzw@mail.sysu.edu.cn; Fax: +86-20-84112245;
Tel: +86-20-84113788
w Electronic supplementary information (ESI) available: Synthesis,
characterization and kinetic experiment details. See DOI: 10.1039/
c0cc00838a
Scheme 1 Synthesis of catalyst 1 and the phosphate diester substrates
used in this study.
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Fig. 3 Saturation kinetics catalyzed by 0.1 mM 1 in buffer (50 mM
TAPS and HEPES), I = 0.1 M (NaClO
4
) at 308 Æ 0.1 K: (a) BNPP
hydrolysis at pH 8.82; (b) HPNP transesterification at pH 7.95.
Fig. 1 Distribution plots of species with 1.0 mM 1 as a function of
pH at 0.1 M NaClO
and 298 Æ 0.1 K.
pH (Fig. 3). Saturation kinetics were observed for both at
4
higher concentrations. The data were fitted to the Michaelis–
À5 À1
combined Lewis activation and hydrophobic interaction could
result in synergistic effects on BNPP hydrolysis.
Menten equation with K
for BNPP, and K
m
= 10 mM and kcat = 2.2 Â 10
s
À3 À1
m
= 7.4 mM and kcat = 1.9 Â 10
s
for
Another substrate, HPNP, serving as an RNA substitute,
HPNP, which were determined from Lineweaver–Burk double-
6
3
1
was also used to test the reactivity of 1. P NMR spectroscopy
showed that the catalytic reaction followed an accepted intra-
molecular pathway producing cyclic propylene phosphate
reciprocal plots. The kcat/kuncat for BNPP (2.0 Â 10 ) was one
5
order of magnitude higher than that for HPNP (1.6 Â 10 ).
Thus, 1 not only showed high catalytic activity toward phospho-
(CPP) as the final product (Fig. S3, ESIw). A complex formed
by 1 with the product CPP (1 : 1) was also detected using
diester hydrolysis, but also had a preference for BNPP. The
À1
binding constant (K = 100 M ) of BNPP was much higher
b
ESI-MS (Fig. S4 and Table S2, ESIw). The data in the pH-rate
13
than those of the simple dinuclear Zn(II) analogues, which
profile (Fig. 2b) were fitted to eqn 2 (ESIw) in which the pK
a
might be due to the additional hydrophobic interactions
value was fixed as determined from the titration experiment,
À1 À1
caused by b-CDs. However, since BNPP is a much weaker
giving a kHPNP of 0.36 Æ 0.01 M
s
. A cursory comparison
4a,13
ligand than HPNP,
the binding constant of BNPP was a
À1
with reported simple dinuclear analogues indicated that
little lower than that of HPNP (K
b
= 135 M ).
the reactivity of 1 toward HPNP was moderate but lower
To further understand the roles of b-CDs, a ditopic com-
pound di(p-tert-butylbenzyl) amine (DBBA) was employed.
6e,f
4
a
than the most efficient one. This fact implied that b-CDs in 1
might not produce efficient positive effects toward HPNP
transesterification.
7b
DBBA could form an inclusion complex with b-CD
as
characterized by ESI-MS and ROESY spectra (Fig. S5 and
S6, ESIw). Thus, the cavities were occupied. Therefore, DBBA
could be applied as a b-CD inhibitor. In each run, DBBA was
added to the reaction system at increased concentrations in the
presence of the substrates. As a result, BNPP hydrolysis was
strongly inhibited by DBBA (Fig. 4), and the profile fitted to
The dependence of initial rate of BNPP and HPNP on
substrate concentration was also measured at the quasi-optimal
eqn 3 (ESIw) gave a K
inhibition for BNPP was also observed at a lower pH with a
value of 0.014 mM (Fig. S7), which excluded the pK effects
i
value of 0.017 mM. The strong
K
i
a
of the amine inhibitor. This indicated that the hydrophobic
interaction offered by b-CDs played a crucial role during
catalyzed BNPP hydrolysis. Very unexpectedly, as for HPNP
transesterification, DBBA did not show any evident inhibition
effect (Fig. 4), indicating b-CDs were not involved in this
catalytic cycle. Thus, the metal sites provided the primary
driving force to attract and activate HPNP. In contrast, the
bulky b-CDs might introduce the steric hindrance which
decreased the binding affinity between HPNP and zinc(II)
ions. This might prevent the C2-hydroxyl chain from forming
the assumed pseudocyclic conformation, which is required for
1
4
the intramolecular nucleophilic addition to the phosphorus.
Based on the above results, we proposed possible catalytic
intermediates which are illustrated in Fig. 5. In the case of
BNPP hydrolysis, hydrophobic and electrostatic interactions
functioned cooperatively to anchor and activate substrates. In
tandem, the two hydroxyl groups coordinated to Zn(II) ions
could both intervene in the nucleophile attack. The hydro-
phobic interaction was proposed to assist the positive metal
core to stabilize the catalytic transition states. In the case of
Fig. 2 The pH dependence of the second-order rate constants for
(a) BNPP hydrolysis and (b) HPNP transesterification catalyzed by 1
at 308 Æ 0.1 K, I = 0.10 M NaClO , and [buffer] = 50 mM.
4
6
498 | Chem. Commun., 2010, 46, 6497–6499
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No. 2007CB815306) and the Fundamental Research Funds
for the Central Universities. We also thank Prof. Zhong-Lin
Lu for a gift of HPNP.
Notes and references
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Fig. 4 DBBA concentration dependence of normalized pseudo-first-
order rate constants of BNPP ( ) and HPNP ( ) cleavage catalyzed
by 1 (0.1 mM) at pH 8.82 (50 mM TAPS buffer) and pH 7.95 (50 mM
0
HEPES buffer), respectively, where k is the pseudo-first-order rate
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Fig. 5 The proposed intermediates of BNPP hydrolysis (left) and
HPNP transesterification (right).
(
(
HPNP transesterification, the binding and activation of
substrates mainly depended on the concerted double Zn(II)
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two cavities which suited the ditopic hydrophobic substrate
BNPP much better than HPNP.
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In summary, the mimic in our study is the first one
characterized by a dinuclear metal core containing hydro-
phobic b-CD domains for substrates. The kinetic investigation
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dependent on both the dinuclear core and the hydrophobic
domains, which is the major reason for the higher catalytic
acceleration. In the study of HPNP transesterification, the
catalytic rates were independent of the hydrophobic domains.
Our results provided novel insights into the development of
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This journal is ꢀc The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 6497–6499 | 6499