Conformational Selection as the Mechanism of Guest Binding in a Flexible Supramolecular Host

Article abstract of DOI:10.1021/jacs.7b03812

This study offers a detailed mechanistic investigation of host-guest encapsulation behavior in a new enzyme-mimetic metal-ligand host and provides the first observation of a conformational selection mechanism (as opposed to induced fit) in a supramolecula

Full text of DOI:10.1021/jacs.7b03812

Article
pubs.acs.org/JACS
Conformational Selection as the Mechanism of Guest Binding in a
Flexible Supramolecular Host
Cynthia M. Hong,† David M. Kaphan,† Robert G. Bergman,* Kenneth N. Raymond,*
and F. Dean Toste*
Chemical Sciences Division, Lawrence Berkeley National Laboratory and Department of Chemistry, University of California,
Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: This study offers a detailed mechanistic investigation of host−guest encapsulation behavior in a new enzyme−
mimetic metal−ligand host and provides the first observation of a conformational selection mechanism (as opposed to induced
fit) in a supramolecular system. The Ga4L4 host described features a C3-symmetric ligand motif with meta-substituted phenyl
spacers, which enables the host to initially self-assemble into an S4-symmetric structure and then subsequently isomerize to a T-
1
symmetric tetrahedron for better accommodation of a sufficiently large guest. Selective inversion recovery H NMR studies
provide structural insights into the self-exchange behaviors of the host and the guest individually in this dynamic system. Kinetic
analysis of the encapsulation−isomerization event revealed that increasing the concentration of guest inhibits the rate of host−
guest relaxation, a key distinguishing feature of conformational selection. A comprehensive study of this simple enzyme mimic
provides insight into analogous behavior in biophysics and enzymology and aims to inform the design of efficient self-assembled
microenvironment catalysts.
Until recently, conformational selection was considered by
some to be an uncommon mechanism, with few definitive
examples reported in the literature.5 However, single molecule
fluorescence6,7 and NMR relaxation8,9 experiments in the past
decade have resulted in the accumulation of significant evidence
in support of conformational selection as a competing
mechanism for protein−ligand interactions.1,10 While indirect
evidence for conformational selection can be garnered by
techniques including crystallographic analysis, selective muta-
tion of receptors, and in silico molecular dynamics, the most
compelling evidence to distinguish the induced fit and
conformational selection mechanisms arises from the kinetic
profile of the binding event. Specifically, the rate of approach to
the binding equilibrium shows saturation kinetics in ligand
concentration for induced fit and inverse dependence on ligand
concentration for the conformational selection mechanism
(Figure 2). Deciphering the mechanism of protein−ligand
recognition in the context of these two general mechanisms has
INTRODUCTION
■
The understanding of protein−ligand molecular recognition is
of paramount importance to the study of enzymatic catalysis
and allosteric regulation of cell signaling as well as to the design
of more efficient drugs.1 The high levels of specificity observed
in enzyme catalysis and protein−ligand binding have been
classically accepted to proceed predominantly by the “induced
fit” hypothesis, introduced in 1958 (also referred to as the
Koshland−Nemethy−Filmer model), rather than a concerted
binding model.2,3 The induced fit model stipulates that a
protein encounters its ligand in an inactive state, and an
optimum fit is achieved only after structural rearrangement is
induced by the interaction with the ligand (Figure 1). Seven
years later, an alternative mechanism for ligand recognition was
proposed, referred to as “conformational selection” or the
Monod−Wyman−Changeux model.4 This mechanistic hypoth-
esis postulates that the enzyme exists in solution as a dynamic
set of Boltzmann-populated conformations, and the ligand
selectively interacts with the active conformation of the protein
to form the ligated complex and subsequently shifts the
equilibrium distribution of protein conformations.
Received: April 15, 2017
Published: June 5, 2017
© 2017 American Chemical Society
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independent of ligand concentration, while the reverse reaction
is inhibited by the ligand. The resulting rate constant describing
the approach to equilibrium under the conformational selection
mechanism shows overall partial inverse order in ligand
concentration. While counterintuitive, this prediction has
been experimentally validated in the molecular biology
literature.12,13,15,16
The symbiotic relationship between molecular biology and
supramolecular chemistry suggests that similar kinetics
scenarios might arise in dynamic synthetic systems. Simple
biomimetic supramolecular assemblies have served as model
systems to provide insight into the underlying stereoelectronic
interactions that drive molecular recognition and enzymatic
reactivity in biological systems, and in turn, lessons from
biology have contributed to the design of synthetic receptors
and catalysts. Self-assembled nanovessels, including hydrogen
bond and metal−ligand based assemblies, have been partic-
ularly fruitful as models for the study of molecular recognition
and microenvironment catalysis.17−21
The Raymond, Toste, and Bergman collaboration has
extensively studied the host−guest chemistry and catalytic
behavior of K12Ga4L6 tetrahedral assembly 1, which is
composed of six biscatecholate ligands bridging four homo-
chiral (ΔΔΔΔ or ΛΛΛΛ) pseudo-octahedral gallium(III)
atoms (Figure 3A).22 Assembly 1 features a range of enzyme-
Figure 1. Induced fit and conformational selection mechanisms of
substrate binding.
since become a very active topic of study in biophysics and
enzymology.1,4
The mechanisms for induced fit and conformational selection
each consist of two consecutive reversible reactions, where
guest binding precedes isomerization in induced fit binding and
isomerization precedes guest binding in conformational
selection. A complete description of their respective rate laws
is beyond simple analysis and can be found in the literature.11
However, under the “rapid equilibrium approximation”, a
kinetic scenario that often prevails, expressions for the binding
rate constant (kobs) can be derived (Figure 2, eqs 1−3).
Specifically, the rapid equilibrium approximation makes the
simplifying assumption that the ligand-binding step is fast and
reversible on the time scale of the conformational change,
allowing the consecutive equilibria to be treated independently
(see the Supporting Information).5,12,13
For an induced fit mechanism, the rate law for relaxation of
inactive enzyme (Ei) to the active enzyme (Ea) bound to
substrate (L) can be expressed by applying a pseudoequilibrium
approximation to the guest binding step. This enables an
expression for the concentration of intermediate EiL in terms of
[Ei·total] and [L]. The concentration of [EiL] can then be used
to express the overall rate constant as the sum of the forward
and the backward rate constants for conformational change.14
The rate law for an induced fit mechanism shows saturation
kinetics in ligand concentration in the forward direction, while
the reverse reaction is independent of ligand. This results in
overall saturation kinetics with respect to ligand concentration.
In contrast, a similar analysis applied to the mechanism for
conformational selection reveals that the forward reaction is
Figure 3. (A) Tetrahedral M4L6 supramolecular host 1, (B) previously
reported M4L4 host 2, and isomeric host 3.
Figure 2. Observed rate constants (kobs) for an approach to substrate binding equilibrium under concerted, induced fit, and conformational selection
mechanisms (left) and qualitative comparison of observed rate constants as a function of ligand concentration for induced fit (blue dots),
conformational selection (orange dots), and concerted binding (red squares) mechanisms (right).
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mimetic behaviors including hydrophobic encapsulation of
neutral and anionic guests,23,24 Michaelis−Menten-type mech-
anisms for a range of catalytic applications with rate
accelerations (kcat/kuncat) of up to 1.9 × 107 fold,25−27 unusual
product selectivity reminiscent of enzymatic catalysis,28−30 and
even protein-like amide H−D exchange behavior.31 While
assembly 1 has proven a fruitful supramolecular enzyme mimic,
it is inherently limited with respect to the steric and electronic
properties of the catalyst active site. While structural analogues
of 1 have been reported,32,33 the rapid diversification of novel
M4L6 assemblies is significantly impaired by the fact that
tetrahedra of M4L6 stoichiometry are entropically disfavored
relative to the M2L3 helicate. Only the carefully tuned geometry
of the internal spacer in the bis-bidentate ligand of 1 prevents
fragmentation to the helicate, and subtle variations from this
structure often leads to the incomplete self-assembly of the
tetrahedron or selective formation of the M2L3 helicate.34
In contrast, M4L4 tetrahedra assembled from C3-symmetric
tris-bidentate ligands are less likely to suffer from this entropic
complication, given that the ligands are sufficiently rigid. The
Raymond group has previously reported the synthesis of M4L4
assembly 2 (Figure 3B); however, the scope of host−guest
chemistry observed with 2 was prohibitively limited in
comparison with assembly 1.35 We hypothesized that the
disparity in encapsulation between these hosts may be
attributed to a difference in the innate flexibility or “breath-
ability” of their cavity structures. In much the same way that a
highly specific protein−ligand interaction requires that the
protein undergo a configurational change to accommodate and
conform to the structure of its ligand (vide supra), so too
should supramolecular hosts conform to complement their
guest molecules for optimal binding.
host reorganization supports the conclusion that a conforma-
tional selection mechanism of molecular recognition is
operative, the first such observation in the context of synthetic
supramolecular enzyme mimics.
RESULTS AND DISCUSSION
■
With ligand 4 in hand, the synthesis of host 3 was attempted
under the established self-assembly conditions. Ligand 4 and
Ga(acac)3 in equimolar quantities were suspended in degassed
methanol-d4 followed by the addition of 3 equiv of potassium
hydroxide as a methanolic solution. This gave rise to
spontaneous self-assembly to form a single species, assigned
as host 3, which could be isolated by precipitation with acetone.
1
However, the H NMR spectrum of the resulting assembly
revealed the presence of 24 unique resonances, which is
inconsistent with the eight unique chemical environments of a
M4L4 host of T-symmetry (Figure 4, top). This low-symmetry
In assembly 1, the amide substitution on the central
naphthalene linker is offset from the center of the ligand,
which allows the walls of the cavity to expand and contract
through rotation about the amide−aryl nitrogen−carbon bond.
This is evidenced by the crystallographic observation that the
encapsulated guest inside 1 can influence the cavity volume
within from at least 253 to 435 Å3.36 In contrast, the amide−
aryl bonds within the ligand of assembly 2 are oriented radially
from the origin of C3-symmetry, and as a result, any structural
accommodation of guest molecules must occur predominantly
by enthalpically costly bond distension. We therefore
hypothesized that the introduction of rotational flexibility to
the M4L4 structural manifold might reintroduce the “breath-
ability” required for efficient guest encapsulation while opening
the opportunity for host diversification by maintaining the
benefits of entropic stability.
In accordance with this hypothesis, tetrahedral assembly 3
was selected as a synthetic target by self-assembly of ligand 4
(Figure 3). Host 3 is isomeric to assembly 2, differing only in
the meta-substitution pattern of the trianiline-linker that
generates the C3-symmetry of ligand 4 (as opposed to para-
substitution in the ligands of host 2). This meta-substitution
pattern introduces 60° of curvature in each arm of the ligand
and offsets the metal binding moiety from the center of the
ligand. This was expected to grant assembly 3 the necessary
flexibility to conform to guest molecules without incurring a
significant enthalpic penalty. We discuss here the dynamic
structural consequences of this simple isomeric substitution,
including the observation of a guest-induced structural
reorganization upon encapsulation of an appropriately large
guest molecule. Mechanistic investigation of the guest-induced
1
Figure 4. H NMR spectrum of host 3 immediately after assembly
(top) and after the addition of approximately 1.5 equiv of
tetraethylphosphonium iodide (bottom).
species was persistent, even upon heating in methanol for
several days (60 °C). Furthermore, the addition of potential
guest cation tetraethylphosphonium resulted in a rapid
reduction in the number of proton resonances from 24 to 8,
along with the appearance of two new resonances around −0.5
and −1.5 ppm (Figure 4, bottom). These resonances and their
integrations were indicative of a molecule of tetraethylphos-
phonium encapsulated within host 3, with 1H NMR resonances
shifted upfield due to close contact with the aromatic ring
currents of the assembly walls. These observations are
consistent with formation of T-symmetric M4L4 inclusion
+
complex PEt4 ⊂ 3; however, the structure of the initially
formed low symmetry species remained unclear.
Characterization of Assembly 3. Three potential
structures might explain the species observed from host 3 in
the absence of a guest. First, the increased flexibility of ligand 4
might alleviate the mechanical coupling that enforces
homochirality on the Ga(III) centers in assemblies 1 and 2;
if this is the case, then assembly 3 might adopt the S4-
symmetric, mixed metal-chirality isomer (ΛΛΔΔ) in the
absence of guest. Second, it is also plausible that a reduction
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in overall symmetry of host 3 may arise from a collapse of the
flexible ligand walls while maintaining homochirality of the
Ga(III) metal centers. This conformational variation would
reduce the host to D2 symmetry and might arise from pinching
two pairs of vertices together. Lastly, it is also conceivable that
if the ligand 4 is sufficiently flexible, an M2L2 structure of D2
symmetry could initially form under self-assembly conditions.
This would require that the addition of a guest initiates a rapid
rupture and dimerization of two complexes to form the
helicate isomerizations.43,44 The first Bailar twist from S4-
symmetric 3 would generate a C3-symmetric intermediate, and
a second Bailar twist would then provide either the T-
symmetric 3 or a second isomeric S4-symmetric host 3 (Figure
5).
+
observed inclusion complex PEt4 ⊂ T-3.
Diffusion-ordered NMR spectroscopy (DOSY) and electro-
spray mass spectrometry (ESI-MS) were employed to differ-
entiate the predicted M4L4 host 3 from this potential M2L2
structure. It was anticipated that a low-symmetry M4L4 host
would have a similar hydrodynamic radius and therefore rate of
+
diffusion compared to those of the inclusion complex PEt4
⊂
T-3. In contrast, an M2L2 complex would have a significantly
smaller hydrodynamic radius and thus diffuse much more
quickly. DOSY NMR revealed a diffusion coefficient of 1.52 ×
Figure 5. Stepwise Bailar twist mechanism for ΔΔΛΛ-3 degenerate
isomerization.
10−5 cm2/s for low symmetry host 3, while the complex PEt4
+
⊂ T-3 had a diffusion coefficient of 1.96 × 10−5 cm2/s. The
proximity of these values was a strong indication that the M4L4
stoichiometry is preserved between two forms of 3. ESI-MS
measurements of the low symmetry host 3 corroborated this
conclusion, as signals consistent with an M4L4 host were
observed.37 These data together supported the conclusion that
the host 3 adopts a low symmetry structure in the absence of
guest.
An S4-symmetric ΛΛΔΔ conformation of host 3 may form a
“collapsed” assembly, reducing the internal cavity volume and,
by extension, reducing the entropic penalty incurred by
trapping solvent molecules inside. Desymmetrization of the
assembly from T to S4 symmetry would be consistent with the
3-fold increase in unique proton resonances upon isomer-
ization. While the majority of M4L4 and M4L6 tetrahedra exhibit
T symmetry due to ligand-enforced mechanical coupling,
examples of M4L6 assemblies have been reported to exist as
near-statistical mixtures of T- (ΔΔΔΔ/ΛΛΛΛ), C3- (ΛΔΔΔ/
ΔΛΛΛ), and S4-symmetric (ΛΛΔΔ) structures.38−42 To the
best of our knowledge, no M4L4 or M4L6 assembly has been
reported in which the S4-symmetric ΛΛΔΔ isomer is observed
exclusively.
It should be noted that the plausibility of an empty T-
symmetric host 3 as an intermediate in the self-exchange of S4-3
depends on the relative energetic barriers for the S4-C3 Bailar
twist and the C3-T Bailar twist. Because these relative barriers
could not be probed, analysis of the kinetics parameters of self-
exchange do not necessarily directly inform the barrier of S4- to
T-symmetric host isomerization involved in guest binding.
However, as these processes are mechanistically related, these
self-exchange parameters are taken to shed a qualitative light on
the kinetic parameters of this process.
The degenerate isomerization of 3 was initially established by
the observation of 1H NMR peak coalescence at elevated
temperatures. In D2O, the 24 observed resonances of 3
coalesced into eight broad peaks at approximately 90 °C,
implying the observation of the time-averaged T-symmetric
conformation from rapid S4 isomerization (see the Supporting
Information). However, line-broadening effects and complica-
tions with the temperature dependence of isotropic chemical
shifts prevented an accurate determination of kinetic activation
parameters from this method.
To avoid these issues, the technique of selective inversion
recovery (SIR) 1H NMR was employed to measure the
isomerization kinetics of the low symmetry host 3 (Figure 6).
SIR experiments provide an excellent method of measuring
rates of slow-exchange (rates of 0.01 s−1 to 100 s−1) in resolved
systems at equilibrium.45 A typical experiment proceeds by
selective inversion of the spin population for a unique
resonance, effectively labeling those nuclei via magnetization.
This is followed by a variable delay time, during which the
chemical-exchange process causes a measurable magnetization
transfer of the spin-labeled nuclei between the exchange-related
chemical environments, followed by the acquisition of a 1D
spectrum. This process is repeated for a series of increasing
delay times to generate a profile of magnetization transfer over
time. The chemical exchange between the inverted signal and
the exchanging signal results in signal attenuation in the
exchanging resonance and accelerated relaxation in the inverted
signal. By modeling these rates in conjunction with
independently measured T1 relaxation times, the rate of
chemical exchange can be extracted.
Similarly, a D2-symmetric conformation of host 3 arising
from a pair of pinched vertices and collapsed ligands would also
reduce the internal cavity volume. If indeed the D2-symmetric
structure was an energetic minimum for host 3, the
discrimination of these structures with 1H NMR would require
that their barrier of interconversion be significantly higher than
would be expected for a process invoking simple conforma-
tional exchange of ligand 4 within host 3.
Conformational Fluxionality of Host 3. To interrogate
the configurational changes that give rise to the reduction of
overall symmetry in 3, the dynamics of host isomerization were
studied. Because the C3-symmetry of ligand 4 is broken upon
self-assembly into host 3, the unique chemical environments
created by this desymmetrization were expected to be related
by conformational exchange of each unique ligand resonance in
the cases of both S4- and D2-symmetric proposed structures of
host 3. If the low symmetry of host 3 is a consequence of mixed
Ga(III) chirality (ΔΔΛΛ), then the exchange process that
interconverts the desymmetrized protons was anticipated to
proceed via stepwise Bailar twists by analogy to studies in the
literature on the mechanisms of Ga(III) triscatecholate and
Although SIR NMR experiments are typically performed on
exchange reactions between two chemically distinct environ-
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Figure 6. (A) Total magnetization (difference in integration at equilibrium and various mixing times) for the selectively inverted proton resonance
(7.50 ppm) and two resonances related by chemical exchange (8.38 and 6.97 ppm) for a representative SIR experiment. (B) 1H NMR spectrum of 3
indicating the resonance selectively inverted (blue circles) and the resonances attenuated as a result of chemical exchange (red and green circles).
(C) Normalized integral as a function of mixing time for the three resonances related by chemical exchange. (D) Eyring plot for the degenerate
isomerization of ΔΔΛΛ-3, generated by SIR experiments at various temperatures.
Figure 7. (A) Induced fit and conformational selection mechanism for encapsulation of ammonium guests by cluster 3. (B) (Left) Kinetic profile for
+
+
approach to equilibrium of tetraethylammonium encapsulation in methanol at 8 °C (blue circles are NEt4 ⊂ T-3, red squares are NEt4 ⊂ S4-3).
(Right) Natural log plot displaying first-order kinetics for approach to equilibrium. (C) Rate dependence of the approach to encapsulation
equilibrium on the concentration of tetraethylammonium ion concentration (top) and tetrapropylammonium ion concentration (bottom) for cluster
3.
ments, the data collection and analysis were extended to
evaluate the 3-fold symmetric exchange of ligand resonances
upon isomerization of host 3. Inversion of the singlet appearing
temperatures from 33 to 53 °C to extract the kinetic parameters
of activation by Eyring analysis (Figure 6D). This analysis
afforded an enthalpy of activation (ΔH⧧) of 12.7(3) kcal/mol
and an entropy of activation (ΔS⧧) of −17.4(5) cal/mol*K,
corresponding to a free energy of activation (ΔG⧧) of 17.8(3)
kcal/mol at 298 K.46,47
1
in the H NMR spectrum of 3 at 7.50 ppm resulted in the
attenuation of signal intensity for the resonances at 8.38 and
6.97 ppm in a manner characteristic of symmetric 3-fold
exchange. The rates of this process were measured at
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1
These data show that the host isomerization event is
significantly entropically disfavored, indicating an ordered
transition state. These observations are consistent with the
expected parameters for a consecutive Bailar twist mechanism
for the degenerate isomerization of S4-symmetric 3 (ΔΔΛΛ).
In comparison, the isomerization of a mononuclear Ga(III)
triscatecholate proceeds with an enthalpy of activation (ΔH⧧)
of 11.0 kcal/mol, an entropy of activation (ΔS⧧) of −11.4 cal/
mol*K, and a free energy of activation (ΔG⧧) of 14.4 kcal/mol
at 298 K.43 The activation parameters for these two processes
are very similar, and the slight increase of both the entropy and
enthalpy of activation for isomerization of 3 is attributed to the
effect of minor mechanical coupling by the polymacrocyclic
framework of the host.48 Having established this species as the
S4-symmetric M4L4 3 of mixed chirality, the generality of this
phenomenon was further interrogated and established by the
synthesis of a new assembly bearing similar design features, in
addition to a survey of encapsulation behavior for neutral guests
(see the Supporting Information).
Evaluation of the Conformational Selection Mecha-
nism for Guest Binding. To understand the mechanism by
which the addition of a guest effects the isomerization of host 3
from the S4-symmetric conformation to the T-symmetric
isomer, the rate dependence of the approach to the
encapsulation equilibrium with respect to guest concentration
was studied. A number of supramolecular systems have been
shown to undergo configurational changes in order to
accommodate and conform to their respective guest
molecules.49−58 This phenomenon is often referred to
imprecisely as “induced fit” binding in reference to the overall
shift in host conformation, rather than the mechanism by which
this conformational change takes place. To the best of our
knowledge, no mechanistic studies have been performed on
synthetic hosts to differentiate between an induced fit and
conformational selection binding mechanism. By analogy to
enzymatic substrate binding, supramolecular assembly 3 might
display either induced fit or conformational selection behavior
upon binding of ammonium guests (Figure 7A).
It was possible to observe the approach to encapsulation
equilibrium by cooling a methanolic solution of empty host 3 in
an NMR tube to −78 °C, followed by addition of the guest at
low temperature and then allowing the resulting mixture to
warm in a precooled NMR spectrometer. At 8 °C, the approach
to equilibrium occurred on an appropriate time scale for
kinetics study, and this relaxation was observed to follow
pseudo-first order kinetics (Figure 7B).
The rate of approach to encapsulation equilibrium (kobs) for
both tetraethylammonium and tetrapropylammonium guests
and host 3 was found to be inhibited by the concentration of
guest (Figure 7C). Guest inhibition of the encapsulation
relaxation rate is the classic kinetic signature of a conforma-
tional selection mechanism of molecular recognition (vide
supra). If the rapid equilibrium approximationguest exchange
is rapid compared to host isomerizationholds true, then the
rate constant for approach to equilibrium is shown in eq 4 on
the basis of the mechanism depicted in Figure 7A and by
analogy to the established analysis of enzymatic conformational
selection. The observation of inhibition with respect to the
concentration of ammonium is consistent with eq 4, supporting
the conformational selection mechanism of guest binding for
assembly 3.
kobs = k1 + k−1
k2
1 + [NR+4]
k−2
(4)
To evaluate the applicability of the rapid equilibrium
approximation for this system, the rate of guest self-exchange
for tetraethylammonium with the inclusion complex NEt4+ ⊂ 3
was evaluated by SIR NMR spectroscopy. Eyring analysis of
this system revealed that the barrier of guest self-exchange
(ΔG⧧ = 16.2(8) kcal/mol at 298 K) is dominated by entropic
contributions (ΔS⧧ = −46.3(30) cal/mol*K) with only a
modest enthalpic component (ΔH⧧ = 2.3(1) kcal/mol) (see
the Supporting Information). These observations are consistent
with the previous analysis of guest self-exchange in host 1,
where a significant entropic contribution to the barrier was
observed.59 The measured kinetic parameters for guest self-
exchange and degenerate host isomerization were extrapolated
to 8 °C as an indicator of whether the rapid equilibrium
approximation was valid. At this temperature, guest self-
exchange occurs with a barrier of 15.3(8) kcal/mol, as
compared to 17.6(3) kcal/mol for degenerate host isomer-
ization, implying that guest self-exchange occurs at a rate
greater than 60-fold faster than that of host isomerization. As
noted, the degenerate host isomerization is only an
approximation for the isomerization process for T-symmetric
guest binding; however, these processes are mechanistically
related, and their activation barriers should be similar. This
analysis supports the application of the rapid equilibrium
approximation in this system.
However, while the observed guest-inhibited relaxation rates
provide strong support for a conformational selection
mechanism for guest binding, this does not preclude the
possibility of fast, reversible guest association to the low-
symmetry host before equilibrium is reached. Indeed, there is
some support for the encapsulation of guest by the S4-
1
symmetric host, as evidenced by the large change in the H
NMR chemical shift of the ammonium ion in the presence of
S4-symmetric host (see the Supporting Information). As the
low-symmetry host is consumed, a concomitant decrease in the
magnitude of the ammonium ion chemical shift is observed.
This is consistent with the ammonium encapsulation or
external association by S4-3 that is fast and reversible on the
NMR measurement time scale.
In light of this evidence, the first guest association step of the
induced fit pathway is then kinetically viable, while the second
isomerization step is prohibited by the encapsulated guest.
Because the isomerization proceeds via stepwise Bailar twists,
we hypothesize that the C3-symmetric prismatic transition state
of the Bailar twist constricts the cavity volume sufficiently that
guest encapsulation inhibits the overall process, leading to
conformational selection.
Further evidence for guest association to the S4-symmetric
host is gathered from the interaction between 3 and
tetramethylammonium. Tetramethylammonium, presumably
due to decreased steric demand compared to its ethyl and
propyl-congeners, does not induce an increase in symmetry in
3. However, shifts in the 1H NMR spectrum indicate
association of the guest to the low-symmetry host. It was
hypothesized that if the ethyl- and propylammonium guests
were acting as inhibitors to the Bailar twist isomerization
mechanism, thus preventing the induced fit mechanism of guest
binding, then the addition of tetramethylammonium to S4-3
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would also inhibit the rate at which degenerate isomerization
occurred in the low symmetry host.
inhibition of initial rates would provide evidence for fast and
reversible association to S4-3 in addition to the conformational
selection pathway.
Repetition of the encapsulation kinetics experiments at
decreased temperature allowed accurate determination of the
pseudo-zero-order initial rate as a function of ammonium
concentration, which displayed clear inhibition by the
ammonium guest (Figure 9). This observation underscores
To evaluate this hypothesis, we again turned to SIR NMR to
study the rates of degenerate S4-3 isomerization in the presence
of the tetramethylammonium guest. Indeed, it was observed
that increasing concentrations of ammonium ion had an
inhibitory effect on the rate of degenerate self-exchange for S4-
3, further supporting the hypothesis that a guest molecule
disfavors the induced fit pathway by increasing the barrier to
the Bailar twist isomerization mechanism (Figure 8).60
Figure 9. Initial rates for approach to encapsulation equilibrium as a
function of the concentration of tetraethylammonium ion concen-
tration for host 3.61
Figure 8. . Rate of degenerate self-exchange of low-symmetry host 3 in
the presence of various concentrations of tetramethylammonium
guest.
the conclusion that a complete mechanistic picture for the
relaxation to guest binding equilibrium for host 3 involves guest
dissociation from S4-symmetric ΔΔΛΛ-3, followed by host
isomerization, and guest binding to T-symmetric homochiral 3.
Overall, this constitutes the first observation of a conforma-
tional selection mechanism in a synthetic host. Furthermore,
identification of the mechanism for host rearrangement as
stepwise Bailar twists provides a rationale for why conforma-
tional selection is preferred over induced fit binding.
Further evidence for reversible preassociation of tetraethy-
lammonium to low symmetry host 3 prior to isomerization can
also be inferred from evaluating the guest concentration
dependence on the pseudo-zero order initial rate of approach
to encapsulation equilibrium, as opposed to the first-order rate
constant, which describes the extended kinetic profile. As
previously described, the observed rate constant for a reversible
reaction is the sum of the forward and backward rate constants.
Inspection of eq 4 reveals that the forward rate constant
contribution (eq 4, first term) is independent of guest
concentration, while the reverse rate constant contributes the
guest inhibition to the overall rate constant (eq 4, second
term). If the conformational selection mechanism (Figure 7A)
is amended to include reversible guest binding for the low
symmetry host (k3/k−3), a new expression for kobs is derived, as
shown in eq 5.
CONCLUSIONS
■
Understanding the mechanism of binding for molecular
recognition processes is essential for the rational design of
novel host−guest systems, whether in the biological context of
drug design or in the context of synthetic supramolecular
sensors and catalysts. In this work, a previously unknown motif
in supramolecular metal−ligand self−assembly was identified
which gave rise to configurationally dynamic behavior upon
recognition of a tetraalkylammonium guest. This feature was
taken advantage of in order to study the mechanism by which
encapsulation of guests takes place. Specifically, the introduc-
tion of appropriate flexibility into the ligand structure of a self-
assembled tetrahedral M4L4 supramolecular cluster leads to a
thermodynamic preference for the ΔΔΛΛ-mixed metal center
chirality state of S4-symmetry. The addition of guest molecules
of sufficient steric bulk induces a structural rearrangement to
afford the homochiral host of overall T symmetry. It was found
that the rate at which this increase in symmetry occurs is
inversely correlated to the concentration of the guest molecule,
which is singularly consistent with a conformational selection
mechanism of molecular recogntion. This behavior is known to
be the operant mechanism of substrate binding for many
biological systems and reinforces the intimate relationship
1
1
kobs = k1
+ k1−
k3
k2
1 + [NR+4]
1 + [NR+4]
k−2
k−3
(5)
In this alternative expression of the observed rate constant,
both the forward and reverse reaction display inhibition with
respect to the concentration of the guest. The initial rate of the
reaction is dominated by contributions from the forward
direction (eq 4 and 5, first term) due to the lack of appreciable
product accumulation. It is therefore possible to discern these
mechanisms by assessing the effect of guest concentration in
the initial rate of the approach to encapsulation equilibrium. By
isolating the contribution from the forward reaction, an absence
of inhibition in the initial rates regime would indicate a simple
conformational selection mechanism, while guest-dependent
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J. Am. Chem. Soc. 2017, 139, 8013−8021

Journal of the American Chemical Society
Article
and reverse rate constants. Reversible bimolecular reactions simplify to
this form when the concentration of one of the components is
sufficiently large, which applies in this case due to the presumed excess
of substrate relative to enzyme.
(15) It is important to note that not all binding events that proceed
via conformational selection show ligand inhibition; when the rapid
equilibrium approximation does not hold, approximately first order or
saturation behavior can be observed. However, if ligand inhibition is
observed, the induced fit mechanism can be definitively ruled out in
favor of conformational selection. Finally, it is important to note that
many biological systems follow a much more complicated mechanism
that may incorporate elements of both conformational selection and
induced fit.
between synthetic and biological supramolecular systems. In
particular, this work highlights the useful role of synthetic
supramolecular analogs to biological systems and their
application as model systems for biology.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b03812.
■
S
Experimental details, synthetic procedures, kinetic rate
constant derivations, NMR and HRMS data, and other
data (PDF)
(16) Pozzi, N.; Vogt, A. D.; Gohara, D. W.; Di Cera, E. Curr. Opin.
Struct. Biol. 2012, 22 (4), 421.
(17) Yoshizawa, M.; Klosterman, J. K.; Fujita, M. Angew. Chem., Int.
Ed. 2009, 48 (19), 3418.
(18) Brown, C. J.; Toste, F. D.; Bergman, R. G.; Raymond, K. N.
Chem. Rev. 2015, 115 (9), 3012.
(19) Catti, L.; Zhang, Q.; Tiefenbacher, K. Synthesis 2016, 48 (3),
313.
AUTHOR INFORMATION
■
Corresponding Authors
*rbergman@berkeley.edu
*raymond@socrates.berkeley.edu
*fdtoste@berkeley.edu
(20) Wiester, M. J.; Ulmann, P. A.; Mirkin, C. A. Angew. Chem., Int.
Ed. 2011, 50 (1), 114.
(21) Raynal, M.; Ballester, P.; Vidal-Ferran, A.; van Leeuwen, P. W.
N. M. Chem. Soc. Rev. 2014, 43 (5), 1734.
(22) Caulder, D. L.; Powers, R. E.; Parac, T. N.; Raymond, K. N.
ORCID
F. Dean Toste: 0000-0001-8018-2198
Author Contributions
†C.M.H. and D.M.K. contributed equally.
Angew. Chem., Int. Ed. 1998, 37 (13−14), 1840.
(23) Brumaghim, J. L.; Michels, M.; Raymond, K. N. Eur. J. Org.
Chem. 2004, 2004 (22), 4552.
Notes
The authors declare no competing financial interest.
(24) Pluth, M. D.; Bergman, R. G.; Raymond, K. N. J. Am. Chem. Soc.
2007, 129 (37), 11459.
(25) Pluth, M. D.; Bergman, R. G.; Raymond, K. N. Science 2007, 316
ACKNOWLEDGMENTS
■
This research was supported by the Director, Office of Science,
Office of Basic Energy Sciences, and the Division of Chemical
Sciences, Geosciences, and Bioscience of the U.S. Department
of Energy at Lawrence Berkeley National Laboratory (Grant
No. DE-AC02-05CH11231). D.M.K. was supported by an NSF
GRFP (Grant No. DGE 1106400). We gratefully thank Dr. Rita
V. Nichiporuk for her expertise and guidance in electrospray
mass spectrometry of metal−ligand complexes, and we
especially thank Professor Alex Bain for fruitful discussions
regarding the use of selective inversion recovery (SIR) NMR in
systems of three-fold self-exchange.
(5821), 85.
(26) Hart-Cooper, W. M.; Clary, K. N.; Toste, F. D.; Bergman, R. G.;
Raymond, K. N. J. Am. Chem. Soc. 2012, 134 (43), 17873.
(27) Kaphan, D. M.; Levin, M. D.; Bergman, R. G.; Raymond, K. N.;
Toste, F. D. Science 2015, 350 (6265), 1235.
(28) Hastings, C. J.; Backlund, M. P.; Bergman, R. G.; Raymond, K.
N. Angew. Chem. 2011, 123 (45), 10758.
(29) Zhao, C.; Toste, F. D.; Raymond, K. N.; Bergman, R. G. J. Am.
Chem. Soc. 2014, 136 (41), 14409.
(30) Kaphan, D. M.; Toste, F. D.; Bergman, R. G.; Raymond, K. N. J.
Am. Chem. Soc. 2015, 137 (29), 9202.
(31) Hart-Cooper, W. M.; Sgarlata, C.; Perrin, C. L.; Toste, F. D.;
Bergman, R. G.; Raymond, K. N. Proc. Natl. Acad. Sci. U. S. A. 2015,
112 (50), 15303.
(32) Zhao, C.; Sun, Q.-F.; Hart-Cooper, W. M.; DiPasquale, A. G.;
Toste, F. D.; Bergman, R. G.; Raymond, K. N. J. Am. Chem. Soc. 2013,
135 (50), 18802.
(33) Hart-Cooper, W. M.; Zhao, C.; Triano, R. M.; Yaghoubi, P.;
Ozores, H. L.; Burford, K. N.; Toste, F. D.; Bergman, R. G.; Raymond,
K. N. Chem. Sci. 2015, 6 (2), 1383.
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