Biological catalysis regulated by cucurbit[7]uril molecular containers

Article abstract of DOI:10.1021/ja910915k

We report the synthesis of two-faced inhibitors 1-5 that contain both enzyme inhibitor and cucurbit[n]uril binding domains. The enzyme binding domains of 1 -5 bind to the active sites of bovine carbonic anhydrase (BCA) or acetylcholinesterase (AChE) and inhibit their catalytic activities. Addition of CB[7] to BCA·1 and BCA·2 results In the transient formation of the BCA·1·CB[7] and BCA·2·CB[7] ternary complexes that undergo rapid dissociation to form free catalytically active BCA along with CB[7]·1 and CB[7]·2. The on-off cycle can be performed repetitively by the sequential addition of competitive guest 8 and CB[7], The detailed origins of this on-off switching of the catalytic activity of BCA is delineated by the combined inference of UV/vis catalytic assays, fluorescence displacement assays, ¹H NMR, along with measurement of the fundamental values of K_a, K_on, and K_off for the various complexes involved. In contrast, addition of CB[7] to AChE·4₄ and AChE·5₄ results In the formation of thermodynamically stable ternary complexes AChE·4₄· CB[7]₄ and AChE·5₄·;CB[7]₄ that are catalytically inactive. We highlight some of the advantages and disadvantages of the strategy, based on the direct competition between two receptors (e.g., enzyme and CB[7]) for a common inhibitor, used In this paper to control enzyme catalytic activity compared to the strategy employed by Nature involving the binding of an allosteric small molecule remote from the enzyme active site.

Full text of DOI:10.1021/ja910915k

Published on Web 03/08/2010
Biological Catalysis Regulated by Cucurbit[7]uril Molecular
Containers
Soumyadip Ghosh and Lyle Isaacs*
Department of Chemistry and Biochemistry, UniVersity of Maryland, College Park, Maryland 20742
Received January 4, 2010; E-mail: LIsaacs@umd.edu
Abstract: We report the synthesis of two-faced inhibitors 1-5 that contain both enzyme inhibitor and
cucurbit[n]uril binding domains. The enzyme binding domains of 1-5 bind to the active sites of bovine carbonic
anhydrase (BCA) or acetylcholinesterase (AChE) and inhibit their catalytic activities. Addition of CB[7] to BCA•1
and BCA•2 results in the transient formation of the BCA•1•CB[7] and BCA•2•CB[7] ternary complexes that
undergo rapid dissociation to form free catalytically active BCA along with CB[7]•1 and CB[7]•2. The on-off
cycle can be performed repetitively by the sequential addition of competitive guest 8 and CB[7]. The detailed
origins of this on-off switching of the catalytic activity of BCA is delineated by the combined inference of UV/vis
1
catalytic assays, fluorescence displacement assays, H NMR, along with measurement of the fundamental
values of Ka, kon, and koff for the various complexes involved. In contrast, addition of CB[7] to AChE•44 and
AChE•54 results in the formation of thermodynamically stable ternary complexes AChE•44•CB[7]4 and
AChE•54•CB[7]4 that are catalytically inactive. We highlight some of the advantages and disadvantages of the
strategy, based on the direct competition between two receptors (e.g., enzyme and CB[7]) for a common inhibitor,
used in this paper to control enzyme catalytic activity compared to the strategy employed by Nature involving
the binding of an allosteric small molecule remote from the enzyme active site.
graphic and remediation applications.2,3 Concurrent with these
advances in technological applications, members of the CB[n]
Introduction
Over the past decade the cucurbit[n]uril (CB[n]) family of
macrocycles has emerged as a premiere platform for basic and
applied studies of molecular recognition and self-assembly
processes in aqueous solution.1,2 For example, the CB[n] family
of macrocycles has been used in the construction of a variety
of stimuli responsive (pH, photochemical, electrochemical,
chemical) molecular machines, in chemical sensing applications,
and as the critical stationary phase component for chromato-
family have begun to be studied as molecular containers for a
variety of biologically active pharmaceuticals4 and first steps
are now being taken to apply CB[n] compounds in drug delivery
applications.5 For many years, our group has been involved in
the elucidation of the mechanism of CB[n] formation6,7 that
lead us to the isolation of new CB[n] (i-CB[n] and CB[10]),8
nor-seco-CB[n] (bis-ns-CB[10], (()-bis-ns-CB[6], ns-CB[6]),9
and acyclic glycoluril oligomers7 that exhibit a variety of
interesting biomimetic phenomena including controlling folding
of non-natural oligomers, mimics of metalloenzymes, homo-
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9
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J. AM. CHEM. SOC. 2010, 132, 4445–4454 4445

A R T I C L E S
Ghosh and Isaacs
Scheme 1. Thermodynamic Shuttling of 1 between BCA and CB[7]
Chart 1. Compounds Used in this Study
tropic allostery, chiral recognition, and metal-ion triggered
folding and assembly reminiscent of natural RNA. The ability
of CB[n] molecular containers to participate in high-fidelity
biomimetic applications can be traced to the extremely high
affinities (Ka up to 1015 M-1) and selectivities (Krel up to 107)
that CB[n] display toward cationic species in water.10-12 Such
values of affinity and selectivity meet and exceed those typically
observed in the interaction of proteins and antibodies toward
their cognate ligands.13 Accordingly, we wondered whether it
would be possible to introduce CB[n] compounds into biological
systems and thereby influence the behavior of biological
receptors toward their guests and modulate their biologi-
cal activity.
We have previously shown that the components of a series
of well-defined aggregates from the literature are capable of
reforming those aggregates even when present within a more
complex multicomponent mixture through a self-sorting pro-
cess.14 Because of their high values of Ka and high selectivities
CB[n] molecular containers are prime components for the
construction of self-sorting systems under thermodynamic
control.11 As a strategy to develop systems that were also
capable of self-sorting under kinetic control, which are im-
mediately more relevant to biological systems that operate far
from equilibrium, we have explored the behavior of compounds
that contain two different binding epitopes that we refer to as
two-faced guests.15 Such hybrid guests are well precedented in
medicinal chemistry where two drugs are often covalently linked
in hopes of combining the desirable properties of both.16 For
example, a hybrid drug combining chloroquine with imipramine
overcomes resistance problems by inhibiting the drug efflux
transmembrane channel.17 In this paper we continue this line
of inquiry by investigating the behavior of two-faced guests
that combine an enzyme inhibitor binding epitope with a CB[n]
binding epitope.18 We were confident that such two-faced guests
would turn off the catalytic activity of their cognate enzyme
by binding of the enzyme inhibitor binding epitope to the
enzyme active site. We anticipated that CB[7] would be capable
of turning on the enzyme activity by sequestering the two-faced
inhibitor by binding to the CB[n] binding epitope. This strategy
is illustrated in Scheme 1 based on two-faced guest 1 that
contains adamantaneammonium (Ka ≈ 1012 M-1 toward CB[7])
and benzenesulfonamide binding epitopes. This strategy differs
from the one typically used by Nature to regulate enzyme
activity, allosteric regulation, in that direct competition between
two receptors (enzyme and CB[7]) is involved. In this paper
we describe the successful implementation of this strategy for
the regulation of the catalytic activity of bovine carbonic
anhydrase (BCA),19 discuss some of the limitations of this
approach illustrated by attempted regulation of acetylcholine
esterase (AChE), and finally use these results to speculate on
why Nature has evolved allosteric systems to regulate enzyme
catalysis.
(11) Liu, S.; Ruspic, C.; Mukhopadhyay, P.; Chakrabarti, S.; Zavalij, P. Y.;
Isaacs, L. J. Am. Chem. Soc. 2005, 127, 15959–15967.
(12) Rekharsky, M. V.; Mori, T.; Yang, C.; Ko, Y. H.; Selvapalam, N.;
Kim, H.; Sobransingh, D.; Kaifer, A. E.; Liu, S.; Isaacs, L.; Chen,
W.; Moghaddam, S.; Gilson, M. K.; Kim, K.; Inoue, Y. Proc. Natl.
Acad. Sci. U.S.A. 2007, 104, 20737–20742. Moghaddam, S.; Inoue,
Y.; Gilson, M. K. J. Am. Chem. Soc. 2009, 131, 4012–4021.
(13) Houk, K. N.; Leach, A. G.; Kim, S. P.; Zhang, X. Angew. Chem., Int.
Ed. 2003, 42, 4872–4897.
Results and Discussion
This results and discussion section is organized as follows.
First, we introduce the enzyme assays used to monitor the
catalytic activity of BCA and AChE. Next, we demonstrate how
(14) Wu, A.; Isaacs, L. J. Am. Chem. Soc. 2003, 125, 4831–4835.
Mukhopadhyay, P.; Wu, A.; Isaacs, L. J. Org. Chem. 2004, 69, 6157–
6164.
(15) Mukhopadhyay, P.; Zavalij, P. Y.; Isaacs, L. J. Am. Chem. Soc. 2006,
128, 14093–14102. Meunier, B. Acc. Chem. Res. 2008, 41, 69–77.
(16) Walsh, J. J.; Bell, A. Curr. Pharm. Des. 2009, 15, 2970–2985.
(17) Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe, M. K.;
Peyton, D. H. J. Med. Chem. 2006, 49, 5623–5625. Walsh, J. J.;
Coughlan, D.; Heneghan, N.; Gaynor, C.; Bell, A. Bioorg. Med. Chem.
Lett. 2007, 17, 3599–3602.
(18) Gianneschi and Ghadiri previously reported a molecular logic device
based on a DNA-inhibitor conjugate to control enzymatic activity.
See Gianneschi, N. C.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2007,
46, 3955–3958.
(19) For photochemical regulation of carbonic anhydrase see: Vomasta,
D.; Hogner, C.; Branda, N. R.; Konig, B. Angew. Chem., Int. Ed. 2008,
47, 7644–7647.
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Biological Catalysis Regulated by CB[7]
A R T I C L E S
Scheme 2. Synthesis of 1-5a
a Conditions: (a) K2CO3 or Et3N, THF, reflux, (b) RNH2, K2CO3, KI, anh. THF, reflux, (c) CH3COCH3, reflux.
Scheme 3. Chemical Reactions Used to Monitor Enzymatic
Activities
CB[7] can regenerate enzymatic activity of BCA by sequestering
compound 1 and 2 from its active site. Subsequently, we discuss
in detail the mechanism of the shuttling of 1 and 2 between
CB[7] and BCA based on results from fluorophore displacement
assays. In the final section of this paper we discuss the pitfalls
encountered when we attempted to use two-faced guest mol-
ecules to control the enzymatic behavior of AChE.
Design Aspects of the Chemical Components Used in this
Study. Chart 1 shows the chemical structures of molecular
containers CB[7] and ꢀ-CD, two-faced guests 1-5, colorimetric
substrates for AChE (compounds 6 and 12) and BCA (com-
pound 7), competitive binding guests for CB[7] and ꢀ-CD
(8-10), and dye (11) used in fluorescence displacement assays
with BCA that were used in this study. Compounds 1-3 and 4
and 5 each contain a single CB[7] binding epitope (adamantane
ammonium (Ka ≈ 1012 M-1), trimethylsilylmethyl ammonium
(Ka ≈ 109 M-1), or hexylammonium (Ka ≈ 106 M-1)) connected
by a short linker to enzyme binding epitopes (benzenesulfona-
mide for BCA, (Ka ≈ 106 M-1), tacrine for AChE (Ka ≈ 109
M-1)). We expected that by varying the strength of the CB[7]
binding interaction relative to that of the enzyme•inhibitor
binding strength that we would observe different behavior.
Synthesis of Guests 1-5. Scheme 2 shows the synthesis of
two-faced guests 1-5. For the synthesis of 1-3 we start by
the reaction of aniline 14 with acid chloride 13 to deliver 15 in
83% yield according to the literature procedure.20 Compound
15 was subsequently reacted with the appropriate amine under
Finkelstein reaction conditions to deliver 1-3 in 68-75% yield.
Compounds 4 and 5 were synthesized in two steps from 16 in
a similar manner. Compound 17 was prepared by the acylation
of 16 with 13.21 Subsequently, 17 was alkylated with adaman-
taneamine or H2NCH2SiMe3 to give 4 and 5 in 60 and 65%
yield, respectively.
measuring the absorbance at 400 nm by UV/vis spectroscopy
(Scheme 3a).22,23 Similarly, the enzymatic activity of AChE can
be measured by the Ellman assay. The Ellman assay relies on
the AChE catalyzed hydrolysis of acetylthiocholine (6) to 19
and acetate. Thiolate 19 reacts rapidly with disulfide 12 to
generate thiophenolate 21, which has a yellow color (λmax
)
412 nm). The rate of enzyme catalyzed hydrolysis can, therefore,
be monitored by measuring absorbance at 412 nm using UV/
vis spectroscopy (Scheme 3b).24
Regulation of Biological Catalysis of Bovine Carbonic
Anhydrase. As a first test of our design strategy, we monitored
the enzymatic hydrolysis of 7 by BCA alone (Figure 1, step 1)
and after the addition of 1 (Figure 1, step 2). A shut down of
the hydrolysis of 7 was observed, indicating that 1 inhibits the
enzymatic activity of BCA as expected due to formation of
BCA•1. Next (Figure 1, step 3) we added CB[7] to the solution
of BCA•1 and monitored the rate of hydrolysis by UV/vis
spectroscopy. In this instance we observed only 45% regenera-
tion of the initial enzymatic activity (Figure 1b). Given that the
binding affinity of CB[7] toward 1 (Ka ) 4.1 × 1012 M-1, Vide
infra) was much higher than that of BCA toward 1 ((1.08 (
0.10) × 108 M-1), we found this result rather surprising.
Chemical Reactions Used to Monitor Enzymatic Activities.
We used two straightforward colorimetric assays that allowed
us to quantify the catalytic activity of BCA and AChE (Scheme
3). BCA is an esterase that catalyzes the hydrolysis of
p-nitrophenylacetate (7) to form p-nitrophenolate (18), which
is yellow colored and has an UV/vis absorption maximum at
400 nm. When the active site of BCA is bound to an inhibitor
the catalytic hydrolysis is turned off and the formation of 18 is
not observed. Consequently, the catalytic activity of BCA is
reflected in the rate of hydrolysis of 7 and can be monitored by
(22) Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668–678. Rozema, D.;
Gellman, S. H. J. Am. Chem. Soc. 1995, 117, 2373–4.
(20) Turkmen, H.; Durgun, M.; Yilmaztekin, S.; Emul, M.; Innocenti, A.;
Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2005, 15, 367–372.
(23) Rozema, D.; Gellman, S. H. J. Biol. Chem. 1996, 271, 3478–3487.
(24) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Featherstone, R. M.
Biochem. Pharmacol. 1961, 7, 88–95.
(21) Ettel, V.; Neumann, J. Chem. Listy Vedu Prum. 1957, 51, 1906–1908.
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Ghosh and Isaacs
that addition of 2 to a solution of BCA (Figure 1, steps 1 and
2) nearly completely inhibits the enzymatic activity of BCA as
monitored by UV/vis spectroscopy. When we added CB[7] to
a solution of BCA•2 (Figure 1, step 3) we observed 83%
regeneration of the initial enzymatic activity. In step 4, the
addition of 8 triggers the dissociation of CB[7]•1 that results in
the release of free 1 that then inhibits the enzymatic activity of
BCA by formation of BCA•1.25 We were able to turn the
enzymatic activity of BCA off and on over several additional
cycles (Figure 1, steps 5-8) by adding CB[7] and 8 in an
alternating sequence (Figure 1, panels a and b).26
Strength of the CB[7]•Two-Faced Inhibitor Complex Is
Critically Important. If the swapping of two-faced inhibitor 2
from the active site of BCA to CB[7] was driven by the fact
that 2 has a higher affinity toward CB[7] than BCA, then the
enzymatic activity should not be regenerated if a related two-
faced inhibitor (e.g., 3) was used whose CB[7]•two-faced
inhibitor complex was weaker than the BCA•sulfonamide
interaction. Based on the literature we expected that the
hexylammonium ion tail of 3 would bind relatively weakly to
CB[7] (Ka ≈ 106 M-1).10,11 Experimentally, we did not observe
any regeneration of enzymatic activity after addition of CB[7]
to a solution containing BCA•3. As a second control experiment
we attempted to use two-faced inhibitor 1 along with ꢀ-CD to
control enzymatic activity. ꢀ-CD is known to have a modest
affinity toward adamantane derivatives (Ka ≈ 104 M-1).27 As
expected, the affinity of ꢀ-CD toward the adamantaneammonium
epitope of 1 was insufficient to cause the dissociation of BCA•1
and enzymatic activity was not regenerated. These control
experiments indicate that one important factor governing the
observed on-off cycling of enzymatic activity is the thermo-
dynamically controlled shuttling of the two-faced inhibitor
between BCA and CB[7] based on differences in the value of
Ka of the binding epitopes toward their cognate receptors.
Fluorescence Spectroscopy Provides Indirect Evidence for
Exchange of the Two-Faced Inhibitor Between BCA and
CB[7] under Thermodynamic Control. We used a competitive
fluorescence assay to further confirm the binding location of
the two-faced inhibitor inferred from the UV/vis studies. For
this process we use dye 11 that has a low quantum yield in
water that increases markedly (at 460 nm) when bound inside
the active site of BCA.28,29 Accordingly, addition of 1 or 2 to
a solution containing BCA•11 resulted in a decrease in
fluorescence intensity at 460 nm that indicates the displacement
of 11 from the active site of BCA by 1 or 2 (Scheme 4). In this
manner, fluoroscent probe 11 signals whether or not the two-
faced guest is bound to the enzyme. Interestingly, when CB[7]
Figure 1. (a) Plot of change in absorbance versus time for the hydrolysis
of 7 by BCA (1 µM) in response to the addition of 2 (4 µM) followed by
sequential additions of CB[7] (8 µM) and 8 (8 µM). (b) Plot of the relative
activity of BCA at each step determined from the initial slope data shown
in part a. Step 1 (b), step 2 (×), step 3 ([), step 4 (4), step 5 (9), step 6
(]), step 7 (2), and step 8 (3). The kinetic assays were performed as
described in the literature.22
Although we were disappointed with the 45% regeneration of
enzymatic activity, we decided to take the experiment one step
further and try to turn off the enzymatic activity of BCA by
the addition of 8. Compound 8 binds more tightly to CB[7]
than 1 does, and therefore, we expected the formation of
CB[7]•8 along with the regeneration of 1 that would fully inhibit
the enzyme by the formation of BCA•1. In the experiment we
found that addition of 8 (Figure 1, step 4) did not result in a
significant change in enzymatic activity. This result indicates
that addition of 8 failed to release 1 from CB[7]•1 over the
time scale of the experiment (45 min incubation). It is known
from the literature that dissociation of the CB[7]•
adamantaneammonium ion complex is slow (koff ) 2 × 10-5
s-1; t1/2 ) 9 h). This slow dissociation allows us to rationalize
the inability of 8 to function in Figure 1b, step 4. Although 1
appears at first glance to be ideally suited to control the
enzymatic activity of BCA in concert with CB[7] because of
its very high thermodynamic binding constant (Ka ) 4.1 × 1012
M-1) in reality the slow kinetic dissociation of CB[7]•1 limits
its utility to a single on-off cycle.
(25) Mohanty previously used competitive binding of sodium to CB[7] to
induce transfer of neutral red to bovine serum albumin. See Shaikh,
M.; Mohanty, J.; Bhasikuttan, A. C.; Uzunova, V. D.; Nau, W. M.;
Pal, H. Chem. Commun. 2008, 3681–3683.
(26) We do not fully understand the steady decrease in activity over
several cycles. We note that the entire assay shown in Figure 1
requires more than 12 h to complete and that substantial amounts
of CB[7]•8 accumulate over the various steps. From control
experiments we know that some enzymatic activity (∼15%) is lost
over this time frame and suspect that the remaining 25% may be
due to the presence of CB[7]•8.
The partial regeneration of enzymatic activity observed using
1 was intuitively promising and inspired us to modify our design
to improve the performance over multiple on-off cycles. We
synthesized two-faced guest 2 bearing a (trimethylsilyl)methyl
ammonium group. Trimethylsilylmethylammonium ion
(Me3SiCH2NH3+) is known from the literature11 to bind tightly
(Ka ≈ 8.9 × 108 M-1) to CB[7] with exchange kinetics that are
fast on the laboratory time scale. Figure 1, panels a and b, show
(27) Rekharsky, M. V.; Inoue, Y. Chem. ReV. 1998, 98, 1875–1917.
(28) Banerjee, A. L.; Tobwala, S.; Ganguly, B.; Mallik, S.; Srivastava, D. K.
Biochemistry 2005, 44, 3673–3682. Krishnamurthy, V. M.; Semetey,
V.; Bracher, P. J.; Shen, N.; Whitesides, G. M. J. Am. Chem. Soc.
2007, 129, 1312–1320.
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Biological Catalysis Regulated by CB[7]
A R T I C L E S
Scheme 4. Schematic Representation of the Fluorophore
Displacement Assay Under Thermodynamic Control
Figure 3. Plot of fluorescence intensity (at 460 nm) versus time after
addition of CB[7] (50 µM) to a mixture of 11 (2 µM), 1 (20 µM), and
BCA (80 nM). All measurements were done in 20 mM NaH2PO4 buffer,
pH 7.3, 298 K.
pletely and only the resonance for BCA•2•CB[7] was observed
1
(Figure 2d). These H NMR experiments further support the
was added to the solution of BCA•2 and 11, we observed an
increase in fluorescence intensity at 460 nm. This observation
is consistent with the formation of a mixture comprising BCA•11
and CB[7]•2. Finally, addition of 8 to this mixture of BCA•11
and CB[7]•2 results in a decrease in fluorescence intensity at
460 nm signaling the formation of a mixture consisting of
CB[7]•8, BCA•2, and free 11. The alternate increase and
decrease of fluorescence intensity at 460 nm supports our
interpretation that the regulation of the biological catalysis of
BCA by 2 and CB[7] is due to binding and dissociation of 2
from the active site of BCA triggered by the presence of CB[7].
1H NMR Provides Direct Evidence for the Guest
Exchange Process. The UV/vis and fluorescence assays de-
scribed above serve as indirect evidence of the sequential
exchange of 2 between BCA and CB[7]. Direct evidence of
guest shuttling can be obtained from 1H NMR spectroscopy by
monitoring the chemical shift of the resonance for the
swapping of 2 between BCA and CB[7] as depicted in Scheme
1 in accord with the UV/vis results.30
Kinetic Profile Observed During the CB[7] Assisted Shutting
of the Two-Faced Inhibitor from the BCA•Two-Faced Inhibitor
Complex to the CB[7]•Two-Faced Inhibitor Complex is
Unusual. While we were carrying out the fluorophore displace-
ment assay described above, we observed unexpected dynamic
(kinetic) behavior during the removal of the two-faced inhibitor
from the active site of BCA upon addition of CB[7]. For
example, when CB[7] was added to a solution of BCA•1 and
11 (also to a solution of BCA•2 and 11, Supporting Information,
Figure S20), we observed an initial rapid increase, reflecting
the formation of BCA•11, followed by a slower exponential
decrease in fluorescence intensity (Figure 3).31 To begin to
understand this complex behavior, we added CB[7]•1 to a
solution of BCA•11 and observed only the slower exponential
decrease in fluorescence intensity. This result strongly suggested
to us that CB[7]•1 (and CB[7]•2) are also capable of acting as
inhibitors of BCA (Vide infra). Given the unusual kinetic profile
displayed in Figure 3, we decided to determine the fundamental
kinetic and thermodynamic parameters controlling this process.
Determination of the Values of Ka, kon, and koff. As described
above, the successful alternate turn on and off sequence of the
biological catalysis of BCA using 2, the results obtained from
1H NMR, and control experiments suggested the shuttling of
guests operates under thermodynamic control. Conversely, the
unusual kinetic profile of the approach to equilibrium (Figure
3) suggested that a knowledge of the kinetics was important.
To fully understand the complex behavior of this system we
decided to determine the values of Ka, kon, and koff for the various
receptor•guest pairs.
1
(CH3)3SiCH2NH2 group of 2. Figure 2a shows the H NMR
spectrum of BCA in the region from +0.5 to -1 ppm. The
appearance of a new peak at -0.27 ppm upon addition of 2 to
BCA is indicative of the formation of the BCA•2 complex
(Figure 2b). Figure 2e shows the resonances for free 2 and the
CB[7]•2 complex. Figure 2c shows the 1H NMR spectrum of a
mixture containing 2, BCA•2, and BCA•2•CB[7]. After addition
of excess CB[7] the resonance for BCA•2 disappeared com-
Measurement of the Ka Values of the Two-Faced
Inhibitors to CB[7]. The association constants for 1 and 2 toward
CB[7] were measured by competition with appropriate guests
1
of known values of Ka by using H NMR competition experi-
(30) Initially, we were a little surprised that the (CH3)3Si resonance appears
at the same chemical shift for both CB[7]•2 and BCA•2•CB[7]. On
further reflection we realized that in both the CB[7]•2 and
BCA•2•CB[7] complex the (CH3)3Si group is bound within CB[7] so
its chemical shift is not influenced by the protein BCA.
(31) Initially we found this kinetic profile to be counter-intuitive since the
concentration of BCA•11 (fluorescence intensity) overshoots the
equilibrium point. This intuition, of course, was based on standard
two component systems where the approach to equilibrium occurs
asymptotically and overshooting does not occur.
Figure 2. 1H NMR spectra recorded for (400 MHz, 30 mM Na2D2PO4
buffer, pD 7.3, 298 K): (a) BCA (0.75 mM, 1 equiv); (b) BCA (0.75 mM,
1 equiv) and 2 (1.32 mM, 1.76 equiv); (c) BCA (0.75 mM, 1 equiv), 2
(1.32 mM, 1.76 equiv), and CB[7] (0.38 mM, 0.5 equiv); (d) BCA (0.75
mM, 1 equiv), 2 (1.32 mM, 1.76 equiv), and CB[7] (1.50 mM, 2.0 equiv);
and (e) 2 (2.0 mM, 2 equiv) and CB[7] (1.0 mM, 1 equiv).
9
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A R T I C L E S
Ghosh and Isaacs
Table 1. Thermodynamic and Kinetic Parameters for BCA•Inhibitor Interactions
bovine carbonic anhydrase
kon (M-1s-1
Ka (M-1
)
∆G (kcal mol-1
)
)
koff (s-1
)
t1/2 (s)
1
2
(1.08 ( 0.10) × 108
(2.73 ( 0.24) × 107
(3.87 ( 0.34) × 106
(9.14 ( 0.77) × 105
(2.56 ( 0.21) × 105
-10.92
- 10.11
- 8.96
-8.10
5.1 × 105
2.8 × 105
4.7 × 105
3.1 × 104
6.5 × 104
(4.30 ( 0.01) × 10-3
(1.40 ( 0.01) × 10-2
(1.20 ( 0.10) × 10-1
(4.81 ( 0.58) × 10-2
(2.70 ( 0.13) × 10-1
161
49.5
5.8
14.3
2.6
11
CB[7]•1
CB[7]•2
- 7.36
ments (Supporting Information, Figure S1) as described previ-
ously.11 All binding experiments were performed in 50 mM
NaO2CCD3-buffered D2O (pD 4.75) at 25 °C. The association
constants of 1 (Ka ) 4.1 × 1012 M-1) and 2 (Ka ) 2.5 × 108
M-1) to CB[7] were measured by competition with 10 and 9,
respectively, for a limiting quantity of CB[7].32 On the basis of
our previous studies of binding constants for CB[6]-CB[8] and
those of Mock and co-workers,10,11 we estimate the binding
11 was released into free solution. The decrease in fluorescence
intensity could be fitted to a single exponential decay in the
standard manner to yield an observed value of koff (Table 1).
The overall process can be described according to the equilibria
given in eq 2 controlled by the fundamental dissociation (k-1
and k-2) and association (k1 and k2) rate constants. To ensure
that the observed value of koff is equal to the fundamental
dissociation rate constant k-1, we measured koff as a function
of [1]. As the concentration of 1 is increased, we initially
observed an increase in the observed value of koff that plateaus
at a limiting value at high concentrations of 1 (Supporting
Information, Figure S15). At low concentrations of 1, addition
of 1 to BCA is the rate-determining step; hence, the observed
rate constant is a function of both concentration of 1 and 11.
At high values of [1], the dissociation of the BCA•11 complex
is the rate determining step ensuring that the observed value of
koff is equal to k-1 as given in Table 1. In a similar manner, we
calculated the value of dissociation rate constant of 1 and 2
from BCA by adding excess 11 to BCA•1 or BCA•2 to ensure
that dissociation of 1 and 2 is the rate-determining step (Table
1 and Figures S13-S16).
constant for CB[7]•3 as ∼106 M-1
.
Measurement of the Ka Values of the Two-Faced
Inhibitors to BCA. We directly measured the value of Ka for
BCA•11 by monitoring the increase in fluorescence intensity
at 460 nm of BCA with the concomitant increase of the
concentration of 11 and fitting the data by nonlinear least-squares
analysis to a 1:1 binding model (Supporting Information, Figures
S5 and S6). The binding constant values for the interaction of
BCA with two-faced inhibitors 1 and 2 were subsequently
measured by a competitive fluorophore displacement assay
where we monitored the decrease in fluorescence intensity at
460 nm of BCA•11 as the concentration of competitors 1 and
2 were increased (Figure 4). The values of Ka for BCA•1 and
BCA•2 were calculated by fitting the decrease in fluorescence
intensity to eq 1 (Table 1 and Figure S7).33
k-1
k2
BCA·11 + 1 h BCA + 11 + 1 h BCA·1 + 11
(2)
k1
k-2
F - FMin
1
Addition of CB[7] Enhances the Rate of Dissociation of 1
and 2 from the Active Site of BCA. Figure 5a shows the change
in fluorescence intensity at 460 nm upon addition of 11 to a
solution containing BCA•1. This change in fluorescence intensity
is directly related to the concentration of the fluorescent BCA•11
complex. Therefore, the observed slow change in fluorescence
reflects the rate determining dissociation of BCA•1 followed
by the fast formation of BCA•11. Somewhat surprisingly, when
we added CB[7] to a solution containing BCA•1 and 11, we
observed a significantly faster (Table 1, 11-fold) kinetic profile
(Figure 5b and Supporting Information Figure S17). In the same
)
(1)
FMax-FMin
KdDNSA
[lnh]
1 +
1 +
d
(
)(
)
[DNSA]
Klnh
Measurement of the Ka Values of CB[7]•1 and CB[7]•2 to
BCA. Based on the kinetic profiles observed in the fluorescence
assays described above we surmised that the CB[7] complexes
CB[7]•1 and CB[7]•2 might be capable of acting as inhibitors
of BCA. As described above, we observed the change in
fluorescence of a solution of BCA•11 at thermodynamic
equilibrium as a function of the concentration of added CB[7]•1
or CB[7]•2 as competitor (Table 1, Figures S8 and S9). As
expected the CB[7]•1 and CB[7]•2 bind substantially weaker
(118- and 107-fold, respectively) to BCA than do the free two-
faced inhibitors 1 and 2, presumably due to steric interactions
between the CB[7] moiety and the walls of the active site of
BCA.
Measurement of the Values of koff for the BCA•11 and
BCA•Two-Faced Inhibitor Complexes. To measure the value
of koff for the BCA•11 complex, we treated a solution of BCA•11
with 1 and observed the decrease in fluorescence intensity as
(32) In this paper we use different conditions for the catalytic processes
(30 mM Na2DPO4 buffer, (pD 7.3) at 25 °C). Because the values of
pKa of the ammonium groups of 1 and 2 should be at least two pKa
units higher than the working pH, we believe the binding constants
obtained by 1H NMR competition experiments at pD 4.75 will be
comparable at pD 7.3.
Figure 4. Plot of fluorescence intensity versus [1] in the titration of BCA•11
with 1. Concentration of BCA (67.5 nM), 11 (2 µM), and 1 (50 nM to 10
µM). All measurements were performed in 20 mM NaH2PO4 buffer, pH
7.3, 298 K.
(33) Nair, S. K.; Krebs, J. F.; Christianson, D. W.; Fierke, C. A.
Biochemistry 1995, 34, 3981–3989.
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Biological Catalysis Regulated by CB[7]
A R T I C L E S
Scheme 6. Representation of the Control of Enzymatic Activity of
BCA usnig 2 and CB[7]
Figure 5. Plot of fluorescence intensity versus time: (a) Addition of 11 to
a solution of BCA and 1. Final concentration of BCA, 11, and 1 are 76
nM, 98 µM, and 2 µM. (b) Addition of CB[7] to a solution containing
BCA, 11, and 1. Final concentrations of BCA, 11, 1, and CB[7] are 75
nM, 25 µM, 18 µM, and 50 µM, respectively. All experiments were done
in 20 mM NaH2PO4 buffer, pH 7.3, 298 K.
Scheme 5. Proposed Mechanism of CB[7] Assisted Dissociation of
1 and 2 from BCA
value of Ka for the formation of BCA•1•CB[7] from BCA and
CB[7]•1 (Ka ) 9.14 ( 0.77 × 105 M-1) and the working
concentrations of BCA (80 nM) and 1 (20 µM). Why does 2
perform better in the off-on regulation of enzymatic activity of
BCA? In addition to the slow dissociation kinetics of CB[7]•1
that effectively limits the on-off behavior to a single cycle,
the thermodynamics of the system are also important. The
catalytic activity of BCA is better regenerated using two-faced
inhibitor 2 than 1 because CB[7]•2 is a weaker inhibitor of BCA
than CB[7]•1 (Ka ) (2.56 ( 0.21) × 105 M-1 for BCA•2•CB[7]
versus Ka ) (9.14 ( 0.77) × 105 M-1 for BCA•1•CB[7]; Table
1). Scheme 6 summarizes the overall reaction scheme that uses
two-faced inhibitors 1 and 2 in concert with CB[7] to regulate
the activity of BCA. The most notable feature is the ability of
CB[7] to accelerate the dissociation of BCA•1 and BCA•2
complexes by transient formation of the corresponding ternary
complexes.
Can Two-Faced Inhibitors Be Used to Regulate the
Activity of Enzymes in General? To address the question of
whether other enzymes could be regulated in a manner similar
to that described above for BCA, we decided to investigate
acetylcholine esterase. Based on an identical design strategy,
we synthesized compounds 4 and 5 featuring a cationic amine
group linked to the well-known AChE inhibitor tacrine.34 On
the basis of our experience with BCA, we anticipated a similar
behavior under thermodynamic control where the addition of
CB[7] would regenerate activity of AChE by sequestering 4
(or 5) from AChE•44 (or AChE•54).
manner, addition of CB[7] to a mixture of BCA•2 and 11
displayed a 19-fold enhancement of the rate of dissociation of
2 from BCA (Figure S18). Scheme 5 shows our interpretation
of the mechanism of this process. Initially, CB[7] binds to the
adamantaneammonium ion binding epitope present in BCA•1
to form the ternary complex BCA•1•CB[7]. The ternary complex
BCA•1•CB[7] then undergoes dissociation to free catalytically
active BCA and CB[7]•1 as a consequence of the fact that
CB[7]•1 binds 118-fold weaker to BCA than 1 (Table 1). In
this manner the binding free energy of the CB[7]•1 interaction
(Ka ) 4.1 × 1012 M-1) pays the thermodynamic price to
transform 1 from a small and tight binding BCA inhibitor into
a sterically hindered weaker binding BCA inhibitor (CB[7]•1).
In this manner CB[7] enhances the dissociation of 1 (and 2)
from the active site of BCA.
Overall Understanding of the Regulation of BCA Catalysis
Using CB[7] and Two-Faced Inhibitors 1 and 2. Some issues
remain that require explanation. What is the cause of the biphasic
kinetics observed in Figure 3? Binding of CB[7] to BCA•1
results in ternary complex BCA•1•CB[7] that dissociates under
the experimental conditions to form free BCA and CB[7]•1.
An initial fast increase in fluorescence occurs when free 11
interacts with BCA to form BCA•11. The system overshoots
the equilibrium point because the association rate constant for
the formation of BCA•11 (4.7 × 105 M-1 s-1, Table 1) is
substantially larger than that for the formation of BCA•1•CB[7]
from BCA and CB[7]•1 (3.1 × 104 M-1 s-1, Table 1). The
slower phase reflects the transformation from the kinetically
preferred state comprising mainly BCA•11 and CB[7]•1 to the
thermodynamic state comprising containing a substantial fraction
of BCA•1•CB[7] (Figure 1, step 3, loss of 55% of initial catalytic
activity). This reduction in activity is as expected based on the
Regulation of the Biological Catalysis of Acetylcholinesterase.
The rate of hydrolysis of 6 catalyzed by AChE was measured
using Ellman’s assay before and after the addition of two-faced
inhibitors 4 or 5 (Figure 6).24 As expected, we observed a
decrease in rate of hydrolysis of 6 as the concentration of two-
faced inhibitor 4 or 5 was increased. These results demonstrate
(34) Marco, J. L.; Carreiras, M. C. Mini-ReV. Med. Chem. 2003, 3, 518–
524. Musial, A.; Bajda, M.; Malawska, B. Curr. Med. Chem. 2007,
14, 2654–2679. Villarroya, M.; Garcia, A. G.; Marco, J. L. Curr.
Pharm. Des. 2004, 10, 3177–3184.
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A R T I C L E S
Ghosh and Isaacs
Table 2. Thermodynamic Parameters for the Interaction of 4 and 5
with BCA and CB[7]
Ka (M-1
)
AChE
CB[7]
4.0 × 106
3.7 × 1012
a
a
b
4
5
1.5 × 106
5.6 × 108
c
a Measured by Lineweaver-Burk analysis. b Measured by
competition with 10 for a limiting quantity of CB[7]. c Measured by
competition with 9 for a limiting quantity of CB[7].
Scheme 7. Formation of Ternary Complex Upon Addition of CB[7]
to AChE•4
are capable of binding to both AChE and the enzyme•substrate
(e.g., AChE•6) complex. Both AChE•inhibitor complexes show
reduced rates of catalytic hydrolysis of 6. The association
1
constant of 4 and 5 with CB[7] were measured by H NMR
competition experiments as described above (Table 2 and
Supporting Information). Table 2 shows that both 4 and 5 have
a strong thermodynamic preference to bind to CB[7]. So, the
inability of CB[7] to regenerate enzymatic activity must be due
to another factor.
On the basis of the values of Ka and results of the colorimetric
assays, we considered two possibilities: either (1) CB[7] does
not interact with AChE•44 or AChE•54 under the experimental
conditions or (2) CB[7] binds to AChE•4 or AChE•5 to form a
ternary complex AChE•44•CB[7]4 or AChE•54•CB[7]4.36 In
either situation, inhibitors 4 or 5 are bound to AChE in both
the absence or presence of CB[7] and therefore the regeneration
of the biological catalysis would not be observed. Additionally,
when we added the separately prepared 1:1 complexes CB[7]•4
or CB[7]•5 to AChE, we also observed a decrease in the rate
of hydrolysis of 6. This result strongly suggests that both
CB[7]•4 and CB[7]•5 are capable of binding to AChE and
therefore inhibit the catalytic activity by formation of the ternary
complexes as indicated in Scheme 7.
Figure 6. (a) Plot of absorbance versus time due to the AChE (0.084 units)
catalyzed hydrolysis of 6 as the concentration of 4 is increased. (b)
Lineweaver-Burk plot of the activity of AChE (0.84 units) in the presence
of increasing concentrations of 4. Conditions: 20 mM potassium phosphate
buffer, pH 8.0, [6] ) 6.5-325 µM, [12] ) 353 µM. By definition, one unit
of AChE hydrolyzes 1.0 µmol of acetylcholine to choline and acetate per
minute at pH 8.0 and 37 °C.
that the tacrine subunits in 4 and 5 retain their inhibition
capabilities even after derivatization with the CB[7] binding
epitopes. Unfortunately, addition of CB[7] to a solution of
AChE•4 or AChE•5 did not result in an increase in the rate of
hydrolysis of 6. We conclude that CB[7] is incapable of driving
the dissociation of 4 or 5 from AChE. We found this result
somewhat puzzling since tacrine and most of its derivatives are
known to bind to AChE with moderate affinities (106-109
M-1)35 that are significantly weaker than the interaction of CB[7]
with adamantaneammonium derivatives.11
Measurement of Association Constants. To assess the influ-
ence of the thermodynamic parameters on the inability of CB[7]
to regenerate the activity of AChE we determined the Ka values
for AChE•inhibitor and CB[7]•guest pairs (Table 2). We
measured the association constant of the two-faced inhibitors 4
and 5 to AChE using Lineweaver-Burk analysis, which also
demonstrates the mixed inhibition behavior of 4 and 5 (Figure
6). It is well-known that AChE has two binding sites: 1) a deeply
buried active site, and 2) a peripheral site that binds certain
inhibitors including tacrine. The fact that the Lineweaver-Burk
plot indicates mixed inhibition means that inhibitors 4 and 5
Secondary Effects of CB[7] on the Enzymatic Activity of
AChE. As one of our control experiments we changed the
concentration of CB[7] used in the attempted regeneration of
AChE. In these experiments, we observed a decrease in the
enzymatic activity of AChE as assessed by Ellman’s assay as
the concentration of CB[7] was increased (Figure 7). We
considered two possibilities: (1) CB[7] acts as an inhibitor of
AChE or (2) CB[7] reduces the observed enzymatic activity of
AChE by binding to and thereby sequestering cationic substrate
6. Accordingly, we prepared a solution of CB[7] and 6 and
1
observed an upfield shift of the (CH3)3N resonance in the H
NMR spectrum indicating that the quaternary ammonium group
of 6 binds to CB[7] to form the CB[7]•6 complex. CB[7] thereby
reduces the concentration of free 6 that in turn reduces the
observed rate of hydrolysis of 6 by AChE. Similar observations
(36) The Nau group has previously observed the formation of ternary
complexes between bovine serum albumin, brilliant green, and CB[7].
See: Bhasikuttan, A. C. ; Mohanty, J. ; Nau, W. M. ; Pal, H. Angew.
Chem., Int. Ed. 2007, 46, 4120-4122.
(35) Porcelli, F.; Delfini, M.; Del Giudice, M. R. Bioorg. Chem. 1999, 27,
197–205.
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Biological Catalysis Regulated by CB[7]
A R T I C L E S
to effectively regulate the catalysis of BCA in concert with two-
faced inhibitor 2 because the CB[7]•2 complex binds 106-fold
weaker to BCA. This reduction in affinity can be attributed to
the sterically bulky CB[7]•2 complex being relatively poorly
accommodated by the rigid active site of BCA. In contrast, the
more open binding sites of AChE readily accommodate the more
bulky CB[7]•4 and CB[7]•5 resulting in the AChE•44•CB[7]4
and AChE•54•CB[7]4 complexes that are catalytically inactive.
Summary and Conclusion
In summary, we have described the use of two-faced
inhibitors (1 - 5), comprising enzyme inhibitor and cucurbit-
[n]uril binding domains, in combination with CB[7] to control
enzymatic activity. We find that the two-faced inhibitors retain
their ability to inhibit enzymatic activity by binding to the
enzyme active site (BCA) or peripheral sites (AChE). In the
case of BCA the addition of CB[7] to BCA•1 or BCA•2 results
in the regeneration of enzymatic activity due to sequestration
of the two faced inhibitor as the CB[7]•1 or CB[7]•2 complex.
One of the key controlling factors in this process is the relative
binding affinity of the two-faced inhibitors toward BCA versus
CB[7]. The high binding free energy of CB[7] toward its best
guests in water, which exceeds that of enzyme•inhibitor
complexes, pays the thermodynamic price associated with the
on-off cycling of catalytic activity. Perhaps the most intriguing
aspect of the study is the ability of CB[7] to enhance the rate
of dissociation of BCA•1 or BCA•2 by the transient formation
of the BCA•1•CB[7] and BCA•2•CB[7] ternary complexes. In
contrast, it is not possible to regulate the activity of AChE in
this manner. The relatively open active and peripheral sites of
AChE bind to both two-faced inhibitors 4 and 5 and their CB[7]
complexes (e.g., CB[7]•4 and CB[7]•5).
Beyond the system specific results described above, our
results allow us to speculate on the reasons why Nature evolved
allostery, rather than the strategy described in this paper
involving direct competition between receptors for a common
target, as a method to control enzymatic catalysis. In allosteric
enzymes, a small molecule binds to a site remote from the
enzyme active site resulting in decreased (allosteric inhibitor)
or increased (allosteric activator) enzymatic activity. In contrast,
the direct competition between receptors (e.g., BCA and CB[7])
for a two-faced inhibitor (e.g., 1 or 2) is only capable of
decreasing enzymatic activity relative to its natural rate because
our strategy relies on occupation of the enzyme active site.
Second, because allosteric activator or inhibitor concentration
can be varied independently of substrate concentration in
allosteric systems it is not necessary for the affinity of the
allosteric activator (or inhibitor) toward the enzyme to exceed
that of the enzyme•substrate interaction. As we have seen for
the BCA case study presented above, the requirement that the
Ka of the two-faced inhibitor toward the synthetic host exceeds
that toward the enzyme may result in slow kinetics of on-off
switching (e.g., CB[7]•1 has a half-life of hours). Lastly, in
contrast to BCA whose active site is highly structured and buried
deeply, the active site of many enzymes are located close to
their surface in sterically less demanding environments. Since
the direct competition between receptors relies on the fact that
the CB[7]•two-faced inhibitor complex is a poor enzyme
inhibitor due to the increased steric bulk of the complex this
strategy is limited to enzymes like BCA with deep active sites.
In contrast, allosteric control operates efficiently regardless of
the location of the active site. For living systems, which operate
based on complex networks of interactions, it is clearly
Figure 7. Plot of the initial slope of a plot of absorbance versus time for
the hydrolysis of 6 (521 µM) by AChE (0.084 units) versus concentration
of CB[7].
have been made by Nau and co-workers for the Trypsin
catalyzed hydrolysis of peptides containing CB[7] binding
sites.37
Origin of the Difference in the Behavior of Two
Enzymes
What factors result in the very different behavior observed
for BCA and AChE in the presence of two-faced inhibitors 1-5
and CB[7] (Scheme 6 versus Scheme 7)? One obvious difference
between BCA and AChE is the size and location of their active
and peripheral binding sites. Bovine carbonic anhydrase has an
unusually deep (∼15 Å) and narrow conical binding site.38
Sulfonamide inhibitors bind to the Zn2+ cofactor that is located
at the bottom of the cleft. This Zn2+•inhibitor coordination
interaction is highly directional and the majority of the strength
of the BCA•inhibitor complex originates from this interaction.29
On the other hand AChE is not a metalloprotease. An analysis
of the crystal structure of AChE form Torpedo California shows
that the active site of the enzyme is present in a ∼20 Å deep
gorge that widens out near the base. At the rim of the gorge,
(∼14 Å from the surface) AChE also has a peripheral binding
site.39 The binding of ligands to these sites are driven by
nondirectional forces like hydrophobic effects, π-π interactions,
cation-π interactions, and electrostatic interactions.40 For ex-
ample, in the crystal structure of tacrine•torpedo acetylcho-
linesterase, the acridine is stacked against the indole of Trp-84.
Compounds 4 and 5 show mixed inhibition behavior indicating
that they bind to the peripheral site. In contrast to the active
site of BCA that is well-structured and interacts with its
inhibitors by directional interactions, the active and peripheral
sites of AChE are flexible and can accommodate a variety of
ligands (inhibitors) of different shapes and sizes. CB[7] is able
(37) Hennig, A.; Ghale, G.; Nau, W. M. Chem. Commun. 2007, 1614–
1616.
(38) Hakansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol.
1992, 227, 1192–1204. Saito, R.; Sato, T.; Ikai, A.; Tanaka, N. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 2004, D60, 792–795.
(39) Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker,
L.; Silman, I. Science 1991, 253, 872–9.
(40) Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.; Hirth,
C.; Axelsen, P. H.; Silman, I.; Sussman, J. L. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 9031–9035.
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Ka determinations contained CB[7] and an excess of the two
competitive guests. H NMR spectra were acquired with a delay
advantageous to employ allosteric small molecules to control
catalytic processes because they are capable of participating at
multiple points in the network resulting in feedback and
regulation.
1
time of five times the longest T1 of the guest to ensure that
systematic errors due to differences in relaxation times were
eliminated. All spectra were referenced relative to residual HOD
at 4.79 ppm.
UV-vis Experiments. UV-vis spectra were recorded on a Cary
100-Bio UV-visible spectrophotometer using 1 cm path length
cells. The temperature was held constant at 25.0 °C using a RTE
bath/circulator containing a microprocessor controller. The kinetic
assays were performed using the literature procedures.22-24 The
relative rates of reaction were determined from the initial slope of
plots of absorbance versus time.
Fluorescence Spectroscopy. Solutions for fluorescence titrations
were prepared in 20 mM NaH2PO4 buffer, pH 7.3. All spectra were
measured on a Hitachi F-4500 fluorescence spectrophotometer with
excitation and emission band passes set at 5 nm. The temperature
was maintained at 25.0 °C using a Neslab RTE-111 bath/circulator.
An excitation wavelength of 290 nm was used to excite BCA. The
change of fluorescence at 460 nm was measured by integrating the
area (from 400 to 500 nm) under each spectrum. The change in
area under the emission spectra versus [11] was used to determine
the Ka value of 11 by nonlinear least-squares analysis fitting to a
1:1 binding model using Associate 1.6.42
What then are the future prospects for the use of the strategy
described here involving the direct competition between syn-
thetic CB[n]-type and biological receptors for a common two-
faced inhibitor described here? The largest advantage is
selectivity. Because the binding sites of synthetic receptors (e.g.,
CB[n]) are built from synthetic (e.g., nonpeptidic) building
blocks our expectation is that they will display affinity and
selectivity profiles that are distinct from their natural counter-
parts. Therefore, building on the initial steps described in this
paper, we expect that it will be possible to use CB[n]-type
receptors in concert with two-faced guests to compete with and
thereby control the activity of specific portions of the interaction
networks of complex biological systems. A second major
advantage is the growing technology available to synthetically
modify CB[n]-type receptors.2,41 For example, CB[n]-type
receptors embedded inside a dendrimer core should be bulky
enough to sequester two-faced inhibitors even from enzymes
with shallow active sites thereby greatly expanding the ap-
plicability of the system described herein. Furthermore, with
synthetic modification comes the ability to attach groups tailored
for targeting and imaging applications. For all these reasons,
we envision that CB[n]-type receptors will become valued tools
that allow scientists to interface with and exert control over
complex biological systems.
Acknowledgment. We thank the National Science Foundation
(CHE-0615049 and CHE-0914745) for financial support of this
work.
Supporting Information Available: Synthetic procedures and
1
characterization data; H and 13C NMR spectra for all new
Experimental Section
1
compounds; details of the kinetic assays; H NMR spectra for
Materials. Carbonic anhydrase (Sigma C3934); acetylcholinest-
erase (Sigma C3389); compounds 6, 7, and 9-12; and ꢀ-CD were
obtained from commercial sources. The synthesis and characteriza-
tion of 1-5 are described in the Supporting Information.
competitive binding experiments; Scatchard plots for BCA•1
and BCA•2; Measurement of dissociation rate constants of 1 -
3, CB[7]•1, CB[7]•2, and 11; Lineweaver-Burk plots for
AChE•4 and AChE•5. This material is available free of charge
via the Internet at http://pubs.acs.org.
1H NMR Competition Experiments. 1H NMR competition
experiments were performed on a 400 MHz NMR spectrometer as
described previously.11 The temperature was maintained at 298 (
0.5 K with a temperature control module that had been calibrated
using the separation of the resonances of methanol. Samples for
JA910915K
(42) Peterson, B. R. Ph.D. Thesis; University of California:Los Angeles,
1994.
(41) Isaacs, L. Chem. Commun. 2009, 619–629.
9
4454 J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010