Cyclodextrin trimers as receptors for arranging ester and catalyst at optimized location to achieve enhancement of hydrolytic activity

Article abstract of DOI:10.1016/j.bmcl.2004.01.035

Two novel trimer receptor molecules (AC and AD trimers) consisting of two α-cyclodextrins (α-CDs) and one β-cyclodextrin (β-CD) have been designed and synthesized in order to make an optimum arrangement between a substrate and a catalyst. In the reaction

Full text of DOI:10.1016/j.bmcl.2004.01.035

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1783–1786
Cyclodextrin trimers as receptors for arranging ester and catalyst
at optimized location to achieve enhancement of hydrolytic activity
Hiroki Nakajima,a Yuzuru Sakabe,b Hiroshi Ikedaa,* and Akihiko Uenoa
aDepartment of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
bDepartment of Chemistry, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara 228-8555, Japan
Received 25 November 2003;accepted 10 January 2004
This work is dedicated to Professor Akihiko Ueno who passed away on March 23rd, 2003
Abstract—Two novel trimer receptor molecules (AC and AD trimers) consisting of two a-cyclodextrins (a-CDs) and one b-cyclo-
dextrin (b-CD) have been designed and synthesized in order to make an optimum arrangement between a substrate and a catalyst.
In the reaction systems that use trimer receptors as host molecules, the substrate and the catalyst are thought to be accommodated
by two a-CDs and one b-CD, respectively. The rate for the hydrolytic reaction of a long-shaped ester was largely increased, when
the trimer receptors were used as receptor molecules or molecular flasks, which provided optimum location between the substrate
and the catalyst.
# 2004 Elsevier Ltd. All rights reserved.
Cyclodextrins (CDs) are cyclic oligosaccharides con-
taining 6 (a-CD), 7 (b-CD), 8 (g-CD) or more glucose
units. They have a hydrophobic cavity in their struc-
tures and can accommodate various organic molecules
into their cavities in aqueous solution. Because of this
fascinating property, several enzyme models using CDs
have widely been studied for hydrolytic reaction of ester
molecules. In those cases, imidazole moieties as cata-
lysts were fixed by covalent bonding on the edge of a
CD1ꢀ9 or on the linker of two CDs,10 and substrates
were included into the CD cavity to be arranged close to
the catalyst. These modified CDs as artificial enzymes,
however, accelerate the hydrolytic reaction but are not
useful for a wide range of reactions because of the
covalently fixed imidazole moiety as a catalyst for ester
hydrolysis. If CD receptors that can arrange the catalyst
and the substrate in the optimum location by non-
covalent bonding could be designed, they might be use-
ful for plural catalytic reactions and can act as reactors
for various reactions. In the design of such CD-based
reactor, it is very important to introduce one cavity for
the catalyst and another one for the substrate to be
accommodated. Based on this idea, two novel trimer
receptor molecules (AC and AD trimers) have been
designed and synthesized in this study (Chart 1). These
two receptor molecules are geometrical isomers with
two a-CDs attached at the AC and AD glucose units in
b-CD. The important feature of this system is that two
a-CDs can cooperatively include a long-shaped sub-
strate and b-CD can selectively include a defined cata-
lyst molecule (Scheme 1). It is well known that the
binding constants of a-CD for various guest compounds
are not so strong.11 Therefore, two a-CDs are introduced
into these trimer systems to increase the binding ability
of a-CD for a particular substrate.12 If the substrate and
* Corresponding author. E-mail: hikeda@bio.titech.ac.jp
Chart 1. Structures of compounds used in this study.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.035

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H. Nakajima et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1783–1786
Scheme 1. Illustration of the geometrical arrangement between the catalyst and the substrate in the trimer receptor system.
the catalyst are located at the optimized position with-
out covalent bonding as shown in Scheme 1, an increase in
the rate of a catalytic reaction would be observed. The
hydrolytic reaction of ester compounds as a catalytic
reaction was selected because the method of assay using
p-nitrophenolate for hydrolysis of ester compounds has
been extensively studied. To achieve this system, N-(4-
imidazolylethyl)-4-tert-butylbenzamide (Im-tBuB) was
synthesized as a catalytic compound (Chart 1), which
can be accommodated only by b-CD (Scheme 1) since
a-CD has a small sized cavity it is unfavorable for
inclusion of the bulky tert-butyl group in Im-tBuB. It
has been reported that the tert-butyl group in Im-tBuB
is held at the secondary side of the b-CD cavity and the
other end turns toward the primary side when tert-butyl
benzene derivatives are accommodated by b-CD.1,13,14
Thus, we can control the direction of Im-tBuB as the
imidazole residue turns toward the part of the ester
bond of the substrate. We found that the catalyst with
the trimers caused an increase in the activity of the
hydrolytic reaction for a relatively long-shaped sub-
strate. Therefore, we intend to report on the selectivity
of the trimers for the length of substrate and their cata-
lytic activity for the ester hydrolysis.
trimer. Because the binding constant of b-CD for a
benzene derivative having no bulky group like the sub-
strate 1 is about 1000 Mꢀ1, the affinity of b-CD for Im-
tBuB is 20-folds larger than that for the substrate 1.
Therefore, only a few of Im-tBuB were excluded by the
substrate 1, and the main structure of the reactive com-
plex of the trimer is expected to be as shown in Scheme 1,
although other miner structure will exist.
The pH 8.5, which is found to be the optimum pH
value, has been described in the following discussion.
The combination of concentrations among the sub-
strates, the catalyst and the receptors was made fixed at
the concentration where the difference of catalytic
activities for substrates in the absence and presence of
receptors was maximum.
The effects of the trimers and dimer on the rate of
hydrolysis of 1 at pH 8.5 are shown in Figure 1. It was
observed that the presence of the AD trimer caused
much higher hydrolytic activity for 1 than that of the
dimer or the AC trimer in aqueous solution. From this
result, we assume that the length and shape of 1 is suit-
able to be accommodated cooperatively by two a-CDs
in the AD trimer system. The cooperative binding by
two a-CDs is important to localize the substrate 1 at the
optimized position for hydrolysis (Scheme 1). To con-
firm the validity of the concept that a substrate is
accommodated cooperatively by two a-CDs in the tri-
mer system, p-nitrophenyl acetate (2) and p-nitrophenyl
propionate (3), which have the length shorter than 1,
Each of the trimer receptors were synthesized by a
coupling reaction of 6,60-bis(carboxymethylthio)-6,60-
dideoxy-b-CD (AC or AD isomer) and 6-amino-6-
deoxy-a-CD (NH2-a-CD).15 The dimer receptor as a
reference host compound was synthesized by a coupling
reaction of 6-carboxymethylthio-6-deoxy-b-CD and
NH2-a-CD16 (Chart 1). Histamine was coupled with p-
tert-butyl-benzoic acid to give Im-tBuB17 as a catalyst,
and hippuric acid was reacted with p-nitrophenol to give
p-nitrophenyl hippurate (1) as a substrate18 (Chart 1).
All these products were identified by a matrix assisted
laser desorption ionization time of flight mass spectro-
metry (MALDI-TOFMS) and a 1H NMR spectroscopy.
The hydrolyses of all ester compounds used as sub-
strates in this study (1 mM) with Im-tBuB (0.2 mM) in
the presence or absence of the trimers or the dimer (0.05
mM) at 25 ꢁC were recorded on a Shimadzu UV-3100
spectrophotometer at 400 nm (released p-nitrophenolate
as a product) at various pH’s (from pH 7.0 to pH 9.0) in
40 mM Tris–HCl buffer.
Before the effect of the trimer on rate enhancement is
discussed, the structure of the inclusion complex of the
trimer is examined. The binding constant of b-CD for
Im-tBuB was obtained to be 22,000 Mꢀ1 by an iso-
thermal titration calorimetry (ITC), whereas that of a-
CD was too small to be estimated. This result indicates
that Im-tBuB is accommodated only by b-CD of the
Figure 1. The changes in absorbance at 400 nm (released p-nitrophe-
nolate as a product) for hydrolysis of substrate 1 in 40 mM Tris–HCl
buffer at 25 ꢁC (pH 8.5). (a) 1 alone;(b) 1 and Im-tBuB;(c) 1, Im-tBuB
and dimer;(d) 1, Im-tBuB and AC trimer;(e) 1, Im-tBuB and AD
trimer. [1]=1.0 mM, [Im-tBuB]=0.2 mM, [dimer]=0.05 mM, [AC
trimer]=[AD trimer]=0.05 mM.

H. Nakajima et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1783–1786
1785
were examined, and the rates of hydrolytic reactions for
them with Im-tBuB were measured in the presence or
absence of the trimers or the dimer. In these experi-
ments, though the hydrolysis of 3 in the presence of the
AD trimer showed rate enhancement compared with
the AC trimer or the dimer, we found no rate enhancement
compared with the dimer for the hydrolysis of 2 in the
presence of the trimers. Figure 2 shows proportions of
increase in the initial rate of hydrolytic reactions for the
substrates used in this study in the presence of the dimer
or the trimers. The proportion of increase in the initial
rate of hydrolysis of 1 in the presence of the AD trimer
showed remarkable rate enhancement, approximately 3-
fold, compared with the dimer system, whereas the rate
of hydrolysis of 2 was not accelerated by the AD trimer
system (Fig. 2). This result implies that 1 is located
comparatively at the effective position for hydrolysis in
the AD trimer, as its length is suitable to be accom-
modated cooperatively by two a-CDs in the AD trimer
system (Scheme 1). On the other hand, the length of 2 is
thought not to be long enough for it to be accom-
modated by two a-CDs cooperatively in the trimers.
That is, instead of two, one a-CD might have accom-
modated the substrate when Im-tBuB is accommodated
by one b-CD in the trimers. Therefore, the ester bond
region of 2 might be not located at the optimized posi-
tion for the imidazole moiety of Im-tBuB to show high
hydrolytic activity. Therefore, the phenomena of inclu-
sion of 2 in the trimer systems might be similar to the
dimer. The importance of the lengths of substrates in
the trimer systems is also supported by the fact that the
rate of hydrolysis of 3, having longer length than 2, in
the presence of the AD trimer was approximately 2.4-
fold higher than that in the presence of the dimer (Fig.
2). In addition, the rate of hydrolysis of 1 and 3 in the
presence of the AD trimer was approximately 2.5-fold,
and 1.9-fold higher respectively, than that in the pre-
sence of the AC trimer (Fig. 2). This result can be
explained in terms of the geometrical arrangement
between CDs and the substrate. The CPK model shows
that the two a-CDs in the AD trimer face each other,
whereas the two a-CDs in the AC trimer face away from
each other as shown in Figure 3, and the shape of 1 or 3
is almost straight. Therefore, the geometrical positions
Figure 3. Orientations of a-CDs on the b-CD at AC and AD position.
of two a-CDs in the AD trimer are more favorable than
that of two a-CDs in the AC trimer for the inclusion of
1 or 3. This indicates that the shape as well as the length
of the substrate plays a crucial role for the hydrolysis
reaction in the presence of the CD-based receptor sys-
tems. However, the rate of hydrolysis of 1 or 3 in the
presence of the AC trimer was slightly higher than those
in the presence of dimer (Fig. 2). This result indicates
that when the longer length esters are employed as sub-
strates, two a-CDs play a role of increasing the binding
constant for the substrate and help to enhance the rate
of hydrolysis even though the geometrical positions of
two a-CDs in the trimer are not favorable for the
inclusion of the substrates.
In our dimer or trimer systems, it is difficult to estimate
various kinetic values since the process of the hydrolysis
reaction undergoes three constituent parts consisting of
the substrate, the catalyst, and the dimer or trimers. In
order to compare the catalytic activity accurately at
each condition, we will find out the way to estimate
various kinetic values in the dimer or trimer systems.
These trimer systems, however, suggest that they could
be applicable as the reactors for hydrolysis of ester
compounds with the catalyst. The combinations of
other kind of catalysts have the potential that might be
applicable not only for hydrolysis of ester compounds
but also for various reactions by controlling the geome-
trical arrangement between CDs, the substrate and the
catalyst.
In this study, we have shown that the addition of the
trimers into the solution containing ester compounds as
a substrate and Im-tBuB as a catalyst caused enhance-
ment in the activity of the hydrolytic reaction. In the
case of using the substrate 2, the addition of trimers
caused no increase in the activity of the hydrolytic
reaction. On the other hand, in the case of using the
substrate 1, the addition of the trimers showed remark-
ably higher hydrolytic activity. This observation sug-
gests that the size and shape of the substrate is
important for this trimer system because the substrate is
required to be accommodated cooperatively by two a-
CDs for achieving high hydrolytic activity. This is sup-
ported by the fact that the rates of hydrolysis of 3, hav-
ing longer length than 2, in the presence of trimers were
slightly higher than those of 2. Again, the rate of
hydrolysis of 1 or 3 in the presence of the AD trimer
showed much higher hydrolytic activity than that of the
AC trimer, indicating that the geometrical arrangement
of the CDs in the trimer receptors is also important.
Figure 2. Proportions of increase in the initial rates of hydrolytic
reactions of substrates in the presence of dimer or trimers compared
with the initial rates in the absence of dimer or trimers in Tris–HCl
buffer (40 mM, pH 8.5).

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H. Nakajima et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1783–1786
These findings lead us to improve the system by mod-
ifying the structures of trimer receptors as well as the
substrates. The work on this line is now underway
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Acknowledgements
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We are grateful to Nihon Shokuhin Kako Co., Ltd. for
a generous supply of cyclodextrins. This work was sup-
ported by a Grant-in-Aid for Science Research from the
Ministry of Education, Culture, Sports, Science and
Technology.
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15. AC trimer; 1H NMR (D2O): d=2.70 (s, 4H, –S–CH2–CO–),
2.87 (d, 2H, a-CD–H6–N), 3.05 (m, 2H, a-CD–H6–N),
3.34–3.95 (m, 116H, CDs–H2-6, b-CD–H6–S), 4.96–5.04
(m, 19H, CDs–H1), MALDI-TOF MS: m/z: 3218.3 (calcd
for M+Na+: 3213.2) AD trimer; 1H NMR (D2O):
d=2.66 (s, 4H, –S–CH2–CO–), 2.85 (d, 2H, a-CD–H6–N),
References and notes
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1
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1
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