Efficient promotion of phosphate diester cleavage by a face-to-face cyclodextrin dimer without metal

Article abstract of DOI:10.1039/c2cc31490h

An organic face-to-face cyclodextrin dimer promotes the cleavage of bis(4-nitrophenyl) phosphate efficiently in neutral pH without the addition of metal. Both of the phosphate diester bonds can be cleaved.

Full text of DOI:10.1039/c2cc31490h

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COMMUNICATION
Efficient promotion of phosphate diester cleavage by a face-to-face
cyclodextrin dimer without metalw
Ping Hu, Gao-Feng Liu, Liang-Nian Ji and Zong-Wan Mao*
Received 28th February 2012, Accepted 11th April 2012
DOI: 10.1039/c2cc31490h
An organic face-to-face cyclodextrin dimer promotes the
cleavage of bis(4-nitrophenyl) phosphate efficiently in neutral
pH without the addition of metal. Both of the phosphate diester
bonds can be cleaved.
The synthesis and characterization of L and CuL (as shown
in Scheme 1) have been described in the ESI.w The species
distribution plot of L is shown in Fig. 1 which is based on the
data from potentiometric titrations. The NH groups on the
bridge have lower pK (Table 1) values than normal primary
amines. So this bridge is deprotonated at high pH. The first
deprotonation constant of free L (pK = 11.34) is slightly lower
than that of the OH group on native CD (pK = 12).11 The
species distribution, the formation constant (Kf) and pKa of
CuL are shown in the ESI (Fig. S1 and Table S1w). The 1 : 1
complex of CuL is found in electrospray ionization mass
spectrometry (ESI-MS) (Fig. S2, ESIw) and the Kf (log Kf =
12.40) is similar to the CD dimer Cu(II) complex with the same
bridge, so the Cu2+ could coordinate with the bridge group on
L as reported.8e
To investigate the cleavage product, 31P-NMR spectroscopy
was used and the existence of the phosphorylated L monoester
(PL) was found in the 31P-NMR spectra12 (Fig. S3, ESIw) and
confirmed by ESI-MS (Fig. S4, ESIw). After we determined
that the final ratio of [4-nitrophenoxide (NPÀ)]/[PL] was 1.98
in the mixture, it was confirmed that both of the diester bonds
on BNPP were cleaved.
Phosphate diesters in nucleic acids are stable under physio-
logical conditions1 and a series of phosphodiesterases2 have
been developed by nature to maintain the metabolism of nucleic
acids. To investigate the mechanism and structure–activity
relationship (SAR) of native enzymes, chemists have developed
abundant bio-inspired mimics.3 Many of them are based on the
mimicking of first4 and second5 coordination spheres. Recently,
various cyclodextrin (CD) metal complexes with notable
phosphatediester cleavage activity have been reported because
CD can support a hydrophobic environment and a substrate-
binding site to mimic the second coordination sphere.
In these CD derivates, the CD dimers possess higher
substrate affinities than the CD monomer.6 Most of them
are linked on the primary face (‘‘back to back’’ like, see
Scheme S1, ESIw) and they exhibit considerable promotion
to bis(4-nitrophenyl)phosphate (BNPP)7 cleavage in the
presence of one8 or more9 transition metal ions. Nevertheless,
in some cases the face-to-face10 dimers linked on the secondary
face demonstrated higher activities than back-to-back CD
dimers as mentioned by Breslow et al.8a,c
To find the active species, we investigated the pH–kobs
relationship and the results are shown in Fig. 1. Then we fitted
kobs and pH to Eqn 1 (ESIw) and got the kBNPP of all the
species (Table 1). They are comparable with high activity first
transition metal complexes5a,b whose kBNPP lie in the range
Based on this inspiration, we synthesize a face-to-face CD
dimer ligand (L) (L = 2,6-bis(3-mono-amino-b-cyclodextrin-
methyl)-pyridine). Unexpectedly, without the addition of metal
ions, this ligand itself can exhibit the efficient promotion of
BNPP cleavage. Its cleavage activity is not only more than
that of its Cu(II) complex but also comparable with those of
transition metal complexes. Furthermore, it can also cleave
both of the phosphate diester bonds of BNPP, which is rarely
found in organic mimics. All of these are different from the
back-to-back CD dimers with the same bridge8d,e which
promote the cleavage of BNPP only in the presence of metal.
So we present this unexpected result in the details below.
10À2–10À1 MÀ1 À1. In particular, the LÀ exhibits a remark-
s
ably high activity which could be due to the deprotonated
OH groups. As far as we know it is rare that an organic
compound, without the addition of a metal, can promote the
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
Scheme 1 L and CuL with the phosphate ester substrate (BNPP)
À
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc31490h
used in this work. X would be water, ClO4 or OH groups on CD.
c
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Chem. Commun., 2012, 48, 5515–5517 5515

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Table 2 The kinetic parameters from the Michaelis–Menten equation
pH = 7.0
pH = 9.0
kcat (sÀ1
KM (M)
)
(2.7 Æ 0.04) Â 10À5
(5.8 Æ 0.4) Â 10À5
(1.7 Æ 0.1) Â 104
(4.7 Æ 0.3) Â 10À1
6.8 Â 105
(2.9 Æ 0.1) Â 10À5
(2.4 Æ 0.3) Â 10À4
(3.7 Æ 0.5) Â 103
(1.1 Æ 0.1) Â 10À1
7.2 Â 105
Kb (MÀ1 a
)
kcat/KM (MÀ1 sÀ1
kcat/kuncat
)
b
Fig. 1 Species distribution of L (left) and second order rate constants
(kobs) of BNPP cleavage at pH 6–11 in buffer 50 mM, I = 0.1 M
(NaClO4) and T = 308 Æ 0.1 K (right).
a
b
Kb = 1/KM
.
kuncat = 4.0 Â 10À11
s
À1, ref. 9b.
is trapped by L (Fig. S5, ESIw), then activated; (2) the OH
groups on L and their conjugated base (deprotonated OH
groups) act as nucleophiles to attack the phosphate diester to
form a new diester and one NPÀ is released. This is the rate-
determining step. (3) This new phosphate diester transforms
into the PL monoester and releases another NPÀ. Further
cleavage of PL does not happen.
Table 1 The pK and kBNPP of the L species
Species
pK
6.28
kBNPP (MÀ1 sÀ1
)
LH2
(1.7 Æ 0.2) Â 10À2
(2.4 Æ 0.3) Â 10À2
(1.8 Æ 0.1) Â 10À2
(1.2 Æ 0.04) Â 10À1
2+
LH+
free L
LÀa
7.16
11.34
—
b
a
b
The OH-deprotonated L. The determination of this pK needs a
very high pH which is hard to reach in aqueous potentiometric
titrations.
To exclude the possibility of metal-induced activity, we
added Cu2+, Zn2+, Co2+, Ni2+ and Pb2+, which are usually
used to construct hydrolase mimics, to the solution of L
separately with molar ratio = 1 : 1 in neutral pH. We found
that all the additions of metal ions reduce the rate constant
(Fig. 4) and indicate that the bridge is not metallized by these
metals in our experimental conditions.
cleavage both of the diester bonds of BNPP as efficiently as
metal complexes.
However, it is unexpected that LH2+, LH+ and free L have
similar activities. To gain a further understanding, the
Michaelis–Menten behavior of L is also investigated at different
pH at which the dominant species is not same (Fig. 2). As is
shown in Table 2, the kcat/KM of L is higher than that of
organic compounds13 and CD dimer metal complexes.9 How-
ever, notably the kcat (sÀ1) at pH 7.0 (LH+ is dominant) and
9.0 (free L is dominant) are almost identical, but the substrate
binding constant (Kb, Kb = 1/KM) at pH = 7.0 is higher than
that at pH = 9.0. It is clear that whether the bridge group is
protonated or not, it could not affect the cleavage rate of the
ester bonds significantly, though a positively charged bridge
by protonation could enhance the substrate attraction.
A probable explanation is proposed: BNPP is activated
mainly by the numerous serried OH groups on CD groups,14,15
because the number of OH groups is more than that of the NH
groups and they are stronger H-bond donors than NH. They
occupy the H-bond acceptor sites of the phosphate groups on
BNPP so the activation of NH is limited.
The SAR of L is also investigated after the mechanism is
primarily demonstrated and we focus on: the roles of the
bridge group and the CD groups. To investigate the role of
the bridge, Cu2+ is used as a probe because it could coordinate
with the N atoms on the bridge. We find that the kobs of CuL
are lower than those of L (Fig. S6, ESIw) and the addition of
Cu2+ to L reduces the initial rate constant gradually (Fig. 5).
To try to explain this, we propose that the Cu2+ coordinates
with the bridge which could induce the unsuitable location of
substrate on L just like the enzymes inhibited by metals. We
also find that the CD monomer, 3-mono-amino-b-cyclodextrin
(3-ACD), has no significant activity at neutral pH and more
basic conditions until the CD groups are linked suitably
(Fig. S7, ESIw). According to these results, we propose that
the bridge could adjust the position of two CD groups and
gain an optimized conformation in the process of the cleavage
reaction.
The role of the CD cavities of L is also studied and we used
meso-tetra(4-sulfonatophenyl)porphine (TPPS) as the inhibitor
Based on a comprehensive consideration of the time depen-
dence of 31P-NMR, ESI-MS and UV-vis spectra, a probable
reaction process is proposed as shown in Fig. 3: (1) the BNPP
Fig. 2 The effect of BNPP concentration on V0 in the presence of
0.1 mM L in buffer 50 mM, I = 0.1 M (NaClO4) and T = 308 Æ 0.1 K
(left: pH = 7.0, right: pH = 9.0). The solid lines show the fit of data to
the Michaelis–Menten equation. Inset: the Lineweaver–Burk double-
reciprocal plot.
Fig. 3 The suggested mechanism (left) and the time-dependant
31P-NMR spectra (right) of the cleavage of BNPP promoted by L in
buffer (50 mM CHES, with 10% v/v D2O), I = 0.1 M (NaClO4),
pH = 9.0 and T = 308 Æ 0.1 K. CHES, N-cyclohexyl-2-aminoethane-
sulfonic acid.
c
5516 Chem. Commun., 2012, 48, 5515–5517
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since it can form a complex with CD through the secondary
face.16 We found that the addition of TPPS to L reduced the
initial rate constant gradually (Fig. 5) when the L can also trap
TPPS (Fig. S8, ESIw). So we propose that the cavities of L
could catch the substrate in the cleavage process just like
native enzymes.
In conclusion, we present the first organic CD dimer which can
cleave both of the diester bonds of BNPP at neutral pH without
the addition of metal. Its rate constants are comparable with
those of high activity metal complexes. Further investigations to
understand the SAR of L are still in progress and it could be
useful to improve the design of phosphodiesterase mimics.
We are grateful for the financial support from the National
Natural Science Foundation of China (No.s, 20725103,
20831006, 20821001 and 21172274), the Guangdong Provincial
Natural Science Foundation (No. 9351027501000003),
National Basic Research Program of China (973 Program
No. 2007CB815306) and the Fundamental Research Funds
for the Central Universities.
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c
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Chem. Commun., 2012, 48, 5515–5517 5517