Sequence-Specific, Nanomolar Peptide Binding via Cucurbit[8]uril-Induced Folding and Inclusion of Neighboring Side Chains

Article abstract of DOI:10.1021/jacs.5b00718

(Chemical Equation Presented). This paper describes the molecular recognition of the tripeptide Tyr-Leu-Ala by the synthetic receptor cucurbit[8]uril (Q8) in aqueous buffer with nanomolar affinity and exceptional specificity. This combination of characteristics, which also applies to antibodies, is desirable for applications in biochemistry and biotechnology but has eluded supramolecular chemists for decades. Building on prior knowledge that Q8 binds to peptides with N-terminal aromatic residues, a library screen of 105 peptides was designed to test the effects of residues adjacent to N-terminal Trp, Phe, or Tyr. The screen used tetramethylbenzobis(imidazolium) (MBBI) as a fluorescent indicator and resulted in the unexpected discovery that MBBI can serve not only as a turn-off sensor via the simultaneous inclusion of a Trp residue but also as a turn-on sensor via the competitive displacement of MBBI upon binding of Phe- or Tyr-terminated peptides. The unusual fluorescence response of the Tyr series prompted further investigation by ¹H NMR spectroscopy, electrospray ionization mass spectrometry, and isothermal titration calorimetry. From these studies, a novel binding motif was discovered in which only 1 equiv of peptide binds to Q8, and the side chains of both the N-terminal Tyr residue and its immediate neighbor bind within the Q8 cavity. For the peptide Tyr-Leu-Ala, the equilibrium dissociation constant value is 7.2 nM, whereas that of its sequence isomer Tyr-Ala-Leu is 34 μM. The high stability, recyclability, and low cost of Q8 combined with the straightforward incorporation of Tyr-Leu-Ala into recombinant proteins should make this system attractive for the development of biological applications.

Full text of DOI:10.1021/jacs.5b00718

Article
pubs.acs.org/JACS
Sequence-Specific, Nanomolar Peptide Binding via Cucurbit[8]uril-
Induced Folding and Inclusion of Neighboring Side Chains
Lauren C. Smith,† David G. Leach,† Brittney E. Blaylock, Omar A. Ali, and Adam R. Urbach*
Department of Chemistry, Trinity University, San Antonio, Texas 78212, United States
S
* Supporting Information
ABSTRACT: This paper describes the molecular recognition of the tripeptide Tyr-Leu-Ala by the synthetic receptor
cucurbit[8]uril (Q8) in aqueous buffer with nanomolar affinity and exceptional specificity. This combination of characteristics,
which also applies to antibodies, is desirable for applications in biochemistry and biotechnology but has eluded supramolecular
chemists for decades. Building on prior knowledge that Q8 binds to peptides with N-terminal aromatic residues, a library screen
of 105 peptides was designed to test the effects of residues adjacent to N-terminal Trp, Phe, or Tyr. The screen used
tetramethylbenzobis(imidazolium) (MBBI) as a fluorescent indicator and resulted in the unexpected discovery that MBBI can
serve not only as a turn-off sensor via the simultaneous inclusion of a Trp residue but also as a turn-on sensor via the competitive
displacement of MBBI upon binding of Phe- or Tyr-terminated peptides. The unusual fluorescence response of the Tyr series
1
prompted further investigation by H NMR spectroscopy, electrospray ionization mass spectrometry, and isothermal titration
calorimetry. From these studies, a novel binding motif was discovered in which only 1 equiv of peptide binds to Q8, and the side
chains of both the N-terminal Tyr residue and its immediate neighbor bind within the Q8 cavity. For the peptide Tyr-Leu-Ala, the
equilibrium dissociation constant value is 7.2 nM, whereas that of its sequence isomer Tyr-Ala-Leu is 34 μM. The high stability,
recyclability, and low cost of Q8 combined with the straightforward incorporation of Tyr-Leu-Ala into recombinant proteins
should make this system attractive for the development of biological applications.
Q8 (Figure 1) is derived from the cucurbit[n]uril (Qn)
family of organic macrocycles,10 which have earned distinction
for their capacity to encapsulate a wide array of organic
molecules in primarily aqueous media with Kd values that can
extend to attomolar levels.11−13 Numerous Qn homo-
logues,14,15 derivatives,16−20 and acyclic congeners21 have
extended the family to myriad structures and applications.22
Q8 and its homologue cucurbit[7]uril (Q7) have been shown
to bind amino acids, peptides, and proteins, with a preference
for the aromatic residues tryptophan (Trp), phenylalanine
(Phe), and tyrosine (Tyr), especially when they are located at
the N-terminal position in the polypeptide chain.6 An N-
terminal aromatic residue is recognized in the same fashion as
many other Qn-based guests, which is by inclusion of the
hydrophobic group (the side chain) within the nonpolar Qn
cavity and stabilization of the cationic group (the N-terminal
INTRODUCTION
■
The discovery and development of synthetic compounds that
can recognize specific peptides and proteins underpins the
effort to interface with living systems. One of the principal
technological successes in this area includes monoclonal
antibodies, which bind their peptide targets with equilibrium
dissociation constant (Kd) values in the nanomolar range and
with exquisite specificity.1 Supramolecular chemists have
developed a wide range of synthetic receptors for peptides in
aqueous solution,2−9 but most have Kd values in the
micromolar to millimolar range. Compared with antibodies,
synthetic receptors offer the advantages of being more
economical to produce, more stable, more scalable, and more
reliable. These advantages, however, will only become relevant
for biological applications if the receptor can recognize a
peptide in aqueous solution at nanomolar concentrations with
at least 1000-fold sequence specificity, as described here for the
synthetic receptor cucurbit[8]uril (Q8).
Received: January 21, 2015
© XXXX American Chemical Society
A
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fluorescence, higher chemical and thermal stability, and greater
synthetic tunability. Q8 binds to MBBI with a Kd value of 1.2
μM, and the Q8·MBBI complex binds to Trp-containing
peptides with affinities and sequence specificities that are
remarkably similar to those of the Q8·MV complex. The
simultaneous binding of MBBI and a second guest within the
Q8 cavity quenches the intrinsic fluorescence of MBBI, and
thus, the Q8·MBBI complex can be used to sense the binding
of both fluorescent and nonfluorescent guests.
The peptide library was screened for binding to Q8·MBBI by
measuring the change in the fluorescence intensity of Q8·MBBI
upon the addition of each peptide in 10 mM sodium phosphate
solution (pH 7.0) at room temperature. A comparison of the 35
peptides containing an N-terminal Trp residue (Figure S52 in
the Supporting Information) revealed that the fluorescence
intensity of Q8·MBBI generally decreased upon binding to the
Trp-containing peptides, as expected.27 Compared with that of
Q8·MBBI, the fluorescence intensities decreased by 4% to 57%,
and in the singular case of Trp-Phe-Ala, the intensity increased
by 4%. The decrease in fluorescence intensity indicates the
simultaneous inclusion of MBBI and the indole side chain of
Trp within the cavity of Q8, as observed previously.27 For the
35 peptides containing an N-terminal Phe residue (Figure S51),
the fluorescence intensity increased for all of the peptides,
covering the range 0.45% to 24%. This unexpected increase
may be explained in light of two previously reported
observations: (1) the fluorescence intensity of free MBBI is
higher than that of the Q8·MBBI complex;27 and (2) peptides
containing an N-terminal Phe residue are known to bind to Q8
in a 2:1 peptide:Q8 stoichiometry.28 With these observations in
mind, we hypothesized that the increase in fluorescence
intensity is due to displacement of MBBI by the peptides.
The most curious result of this experiment, however,
involved the 35 peptides with an N-terminal Tyr residue
(Figure 2). In this series, the influence of the peptide on the
Figure 1. Chemical formulas and peptide sequences used in this study.
ammonium) by the carbonyl oxygens that surround the two
constricted entrances to the cavity. Q8 is large enough to
include two aromatic guests,23,24 and the earliest report of
peptide recognition by the Qn family25 involved a ternary
complex of Q8 with methyl viologen (MV) and the indole side
chain of a Trp-containing peptide. In this system, Q8 first binds
to MV, and the Q8·MV complex then binds to the indole side
chain of a Trp-containing peptide. Specificity for Trp at the N-
terminal position is gained by interaction with the N-terminal
ammonium group when it is part of the Trp residue and thus
proximal to the indole side chain.
In order to explore the scope and limitations of the affinity
and selectivity in the Q8·MV·Trp system, we studied the effects
of residues adjacent to Trp and found little, if any, influence on
binding to Q8·MV.26 That study, however, was limited to Trp-
containing peptides because of the need for a fluorophore. We
have shown that MV can be replaced by tetramethylbenzobis-
(imidazolium) (MBBI) while still maintaining the molecular
recognition properties for Trp-containing peptides.27 The
intrinsic fluorescence of MBBI broadens the scope of possible
analytes to include nonfluorescent guests, thus enabling the
exploration of a larger peptide sequence space, the first report
of which is presented here.
RESULTS AND DISCUSSION
■
Design and Screen of a Peptide Library. A peptide
library (Figure 1) was designed to examine the effects of the
amino acid sequence on the binding of Q8 to peptides
containing an aromatic residue at the N-terminus. All of the
compounds were tripeptides comprising natural, unmodified
side chains. The N-terminal residue was Trp, Phe, or Tyr. The
residue immediately adjacent to the N-terminal residue (Var1)
or two residues away (Var2) was varied among 18 genetically
encoded amino acids while the other position in the tripeptide
was held constant with an alanine (Ala) residue. Trp and
cysteine (Cys) were not included at the variable positions
because of complications introduced by having a second Trp
binding site and the fact that Cys residues form disulfide bonds
under ambient conditions. This design yielded a library of 105
peptides (leaving out replicates when Var1 = Var2 = Ala). The
library was synthesized by parallel solid-phase synthesis on Rink
amide Lantern resins, and thus, all of the C-termini were
primary amides.
Figure 2. Fluorescence relative to the Q8·MBBI complex for mixtures
of Q8·MBBI with each of 35 tripeptides of sequence Tyr-Var1-Ala and
Tyr-Ala-Var2. Samples contained 200 μM peptide and 40 μM Q8·
MBBI at room temperature in 10 mM sodium phosphate (pH 7.0).
Values are averages of at least three experiments. Error bars represent
standard deviations.
observed fluorescence varied significantly and was highly
sequence-dependent. The fluorescence intensity decreased for
19 peptides (0.21% to 6.9%) and increased for 16 peptides
(0.39% to 21%). In several cases (Var = Leu, Phe, Tyr, Met,
Thr, Ser, and Asn), the fluorescence intensity increased for the
peptide Tyr-Var1-Ala but decreased for the peptide Tyr-Ala-
Central to the assay design is the fluorophore MBBI,27 which
was shown previously to have molecular recognition properties
similar to those of MV but with the advantages of intrinsic
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Var2. This remarkable result suggests that there may be a
sequence-dependent change in binding mode for these
peptides. The largest observed difference (23%) was observed
for Var = Leu (i.e., Tyr-Leu-Ala vs Tyr-Ala-Leu). These two
sequence isomers were therefore chosen for in-depth study to
further explore this phenomenon.
A detailed investigation of the binding of Tyr-terminated
peptides was carried out using 1H NMR spectroscopy in
deuterium oxide at 25 °C. Binding of a guest or a portion of a
guest within the cavity of a cucurbit[n]uril is known to induce
an upfield perturbation of the chemical shifts of the
corresponding signals.29 As shown previously27 and again
here (Figure 4a,b), this phenomenon is observed for MBBI in
the presence of Q8. Upon addition of 1 equiv of Tyr-Leu-Ala to
the Q8·MBBI complex (1:1:1 mixture; Figure 4c), the chemical
shifts of both MBBI signals return to those of free MBBI,
whereas all of the signals corresponding to the Tyr and Leu
residues move upfield and those of the Ala residue move
downfield. On the basis of these results, and in corroboration of
the fluorescence data, we conclude that the peptide
competitively displaces MBBI from the Q8 cavity. Moreover,
it is clear that both the Tyr and Leu side chains are bound
within the cavity of Q8 and that the Ala side chain is located
near the Q8 portal. In the absence of MBBI, the 1:1 mixture of
Tyr-Leu-Ala and Q8 (Figure 4d) shows peptide signals
identical to those of the 1:1:1 mixture of Tyr-Leu-Ala, MBBI,
and Q8. This result shows that MBBI does not influence the
binding mode of Tyr-Leu-Ala with Q8. The 1:2 mixture of Q8
and Tyr-Leu-Ala (Figure 4e) has a spectrum identical to that of
the Q8·Tyr-Leu-Ala complex with the addition of a second set
of resonances corresponding to the free peptide (Figure 4f).
These spectra confirm that Tyr-Leu-Ala binds to Q8 in a 1:1
stoichiometry and provide evidence that the kinetics of
exchange between the bound and unbound states is slow on
the NMR time scale, thus suggesting a low dissociation rate and
a high binding affinity. The presence of the 1:1 peptide·Q8
complex was confirmed by ESI-MS (Figure S2).
In-Depth Study of Tyr-Leu-Ala and Tyr-Ala-Leu. In the
library screen described above, the peptides were synthesized in
parallel on an 8 μmol scale and used without further
purification (see the Supporting Information for experimental
details). For the in-depth studies described here, Tyr-Leu-Ala
and Tyr-Ala-Leu were synthesized individually on a 37 μmol
scale, purified by reversed-phase HPLC, and characterized by
1H NMR spectroscopy and electrospray ionization mass
spectrometry (ESI-MS) (Figures S1, S3, S19, and S25). The
library screen showed that the fluorescence intensity of Q8·
MBBI increased in the presence of Tyr-Leu-Ala but decreased
in the presence of Tyr-Ala-Leu. In that experiment, however,
the emission intensity data were collected at only a single
wavelength (340 nm). In order to probe any additional spectral
effects, full emission spectra were acquired for Q8·MBBI in the
absence and in the presence of Tyr-Leu-Ala or Tyr-Ala-Leu in
10 mM sodium phosphate (pH 7.0) at room temperature
(Figure 3). Interestingly, the fluorescence spectrum of the
The slow exchange kinetics observed for the Q8·Tyr-Leu-Ala
complex facilitated the acquisition of a rotating-frame nuclear
Overhauser effect spectroscopy (ROESY) spectrum (Figure
S24) in order to gain more information on the conformation of
the peptide in the complex. Nine nuclear Overhauser effects
(NOEs) between the side chains of Tyr and Leu residues
(inter-residue) and 17 NOEs between the peptide and Q8
(intermolecular) were observed (see Table S1 in the
Supporting Information). On the basis of these data, a
semiempirical computational model was constructed that is
consistent with the NMR data (Figure 5). The model shows
the side chains of Tyr and Leu residues bound deeply within
the Q8 cavity. It also suggests electrostatic stabilization of the
complex via five putative hydrogen bonds between peptide NH
+
and NH3 groups and Q8 carbonyl oxygens.
In contrast to Tyr-Leu-Ala, studies of the sequence isomer
Tyr-Ala-Leu revealed substantially different results. In the H
Figure 3. Fluorescence emission spectra (λex = 297 nm) acquired at
room temperature for mixtures containing 40 μM MBBI, 40 μM Q8·
MBBI, and 40 μM Q8·MBBI in the presence of 200 μM peptide, all in
10 mM sodium phosphate (pH 7.0).
1
NMR spectrum of Tyr-Ala-Leu combined with Q8 and MBBI
(Figure S28), the MBBI peaks broaden, but their chemical
shifts do not return to those of free MBBI. The Tyr peaks shift
upfield and broaden substantially, and the Ala and Leu peaks
broaden slightly, but their chemical shifts do not change
appreciably. These observations corroborate the fluorescence
data and demonstrate that MBBI and Tyr are bound
simultaneously within the Q8 cavity. When Tyr-Ala-Leu is
combined with Q8 (Figure S26), the Tyr peaks are perturbed
upfield significantly, and they broaden but not as much as when
MBBI is present. The Ala and Leu peaks broaden slightly, and
their chemical shifts are not perturbed significantly. In the 2:1
Tyr-Ala-Leu:Q8 mixture (Figure S27), the peaks observed in
the 1:1 mixture broaden much further, and no new set of peaks
was observed. These results suggest that the chemical exchange
mixture containing Q8, MBBI, and Tyr-Leu-Ala is essentially
identical to that of free MBBI (also shown). This result is
consistent with the displacement of MBBI from the cavity of
Q8 upon binding of Tyr-Leu-Ala. The fluorescence spectrum of
the mixture containing Q8, MBBI, and Tyr-Ala-Leu, however,
shows a decrease in fluorescence intensity compared with that
of Q8·MBBI, which is consistent with prior work showing the
quenching of Q8·MBBI fluorescence upon the inclusion of the
side chain of Trp. Therefore, we hypothesized that a portion of
Tyr-Ala-Leu was bound within the cavity of the Q8·MBBI
complex, and further study was undertaken to explore the mode
of binding in more detail.
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Figure 4. 1H NMR spectra (500 MHz) acquired at 25 °C for samples containing (a) MBBI; (b) a 1:1 mixture of Q8 and MBBI; (c) a 1:1:1 mixture
of Q8, MBBI, and Tyr-Leu-Ala; (d) a 1:1 mixture of Q8 and Tyr-Leu-Ala; (e) a 1:2 mixture of Q8 and Tyr-Leu-Ala; and (f) Tyr-Leu-Ala. Samples
were approximately 0.5 mM in unbuffered deuterium oxide.
only Tyr binds within the Q8 cavity. The 1:1 Q8·Tyr-Ala-Leu
complex was confirmed by ESI-MS (Figure S4).
Extension of the Tyr-Var Motif. The contrasting results
for Tyr-Leu-Ala and Tyr-Ala-Leu described above suggest that
the Tyr and Leu residues need to be neighbors in order for the
peptide to bind in a 1:1 complex with Q8 with the inclusion of
both side chains. It was not clear, however, whether the Tyr-
Leu binding site needs to be located at the N-terminus. To
address this question, the sequence isomer Ala-Tyr-Leu was
1
synthesized, purified, and characterized by H NMR spectros-
copy and ESI-MS (Figures S5 and S29). The NMR spectrum of
Ala-Tyr-Leu in combination with Q8 (Figure S30) reveals the
upfield perturbation of chemical shifts for Tyr and Leu side
chains and thus their inclusion within the Q8 cavity. At
substoichiometric ratios of Q8 to Ala-Tyr-Leu, two sets of
peptide resonances are observed (free and bound), thus
revealing a 1:1 complex and slow exchange on the NMR
time scale (data not shown). The 1:1 Ala-Tyr-Leu·Q8 complex
was observed by ESI-MS (Figure S6). Therefore, the Tyr-Leu
binding site does not need to be located at the N-terminus.
Having established the unique nature of the Tyr-Leu binding
site, we then wondered whether this motif could extend beyond
Var = Leu. In the fluorescence screen described above, Var =
Leu was chosen for further study because Tyr-Leu-Ala led to an
increase in fluorescence and Tyr-Ala-Leu caused a decrease in
fluorescence, and the difference between their changes in
fluorescence intensity was the largest (23%). Among the other
peptides for which this pattern was observed, large differences
were also observed for Var = Phe (21%) and Tyr (18%).
Therefore, the peptides Tyr-Phe-Ala, Tyr-Ala-Phe, Tyr-Tyr-Ala,
Figure 5. Semiempirical model of the Q8·Tyr-Leu-Ala complex. The
model was built in Maestro by manually docking the peptide to fit the
and Tyr-Ala-Tyr were individually synthesized, purified, and
side chains of Tyr and Leu residues inside the Q8 cavity and then
1
characterized by H NMR spectroscopy and ESI-MS (Figures
S9, S11, S13, S15, S37, S39, S41, and S43) in order to probe
subjecting the model to a geometry minimization in the OPLS 2005
molecular mechanics force field with implicit solvent using upper-
bound distance restraints generated from the nine inter-residue (Tyr-
to-Leu) NOEs observed in the ROESY spectrum (Figure S24).
1
the possible extension of this binding motif. The H NMR
spectra of Tyr-Phe-Ala and Tyr-Tyr-Ala show upfield
perturbation of both aromatic side chains upon binding to
Q8, indicating simultaneous inclusion within the host (Figures
S38 and S42). In addition, the 2:1 peptide:Q8 mixtures show a
set of bound peptide resonances and a set of unbound peptide
resonances (data not shown), and thus, the kinetics of exchange
is slow on the NMR time scale and the complexes have a
peptide:Q8 stoichiometry of 1:1. In both cases, a 1:1
kinetics are intermediate on the NMR time scale and are
consistent with low-affinity binding. Unfortunately, the lack of
spectral resolution limits the depth of the NMR analysis. It is
clear, however, that only the peaks corresponding to the side
chain of Tyr experience a significant upfield perturbation in
chemical shift in the presence of Q8, and thus, we conclude that
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Table 1. Thermodynamic Data for Binding to Cucurbit[8]uril
a
b
a
c
peptide
Kd (μM)
ΔG (kcal/mol)
ΔH (kcal/mol)
−TΔS (kcal/mol)
Tyr-Leu-Ala
Tyr-Ala-Leu
Ala-Tyr-Leu
Tyr-Lys-Ala
Tyr-Tyr-Ala
Tyr-Phe-Ala
0.0072 0.0003
34. 7.
−11.2 0.1
−6.2 0.2
−7.6 0.2
−9.2 0.2
−8.5 0.1
−9.0 0.2
−15.5 0.4
−9.5 0.1
−14.1 0.5
−15.5 0.1
−16.1 0.2
−16.1 0.2
4.3 0.2
3.4 0.1
6.5 0.1
6.3 0.2
7.7 0.2
7.2 0.1
3.1 0.5
0.20 0.03
0.70 0.06
0.29 0.04
a
b
Mean values measured from at least two ITC experiments at 27 °C in 10 mM sodium phosphate (pH 7.0). Gibbs free energy values calculated
from Kd values. Standard deviations for the ΔG values were calculated as the relative errors observed in the Kd values because of their relationship by
c
a natural logarithm. Entropic contributions to ΔG were calculated from the Kd and ΔH values, with error propagated from those of Kd and ΔH.
peptide:Q8 stoichiometry was corroborated by ESI-MS
(Figures S10 and S14). Similar to Tyr-Ala-Leu, the H NMR
Gly versus Gly-Phe by Q7 (23000-fold).34 In their study and
others,28,35 the specificity for the N-terminal Phe is thought to
derive from the location of the aromatic residue relative to the
charged groups at each terminus, whereas in the current study,
the specificity of Tyr-Leu-Ala versus Tyr-Ala-Leu is thought to
derive from inclusion of the second side chain within the Q8
cavity. This hypothesis is supported by the enthalpy and
entropy data (Table 1). The binding of both Tyr-Leu-Ala and
Tyr-Ala-Leu is enthalpically driven and entropically unfavor-
able. The 5.0 kcal/mol difference in the free energy of
complexation is due mostly to enthalpy (6.0 kcal/mol), whereas
the entropic contributions to the binding energy are similar to
within 1 kcal/mol. This observation suggests that compared
with the Tyr-Ala-Leu·Q8 complex, the Tyr-Leu-Ala·Q8
complex is stabilized by the additional van der Waals
interactions of the Leu side chain with the Tyr side chain
and the walls of the Q8 cavity. Desolvation of the isobutyl
group may contribute to the entropic stabilization but would be
offset by the immobilization of the side chain within the tightly
packed cavity. The low affinity of the Tyr-Ala-Leu complex was
shared by the other two Tyr-Ala-Var2 peptides (Tyr-Ala-Tyr
and Tyr-Ala-Phe), whose ITC data proved difficult to fit (data
not shown).
1
spectra of Tyr-Ala-Tyr and Tyr-Ala-Phe also show extensive
signal broadening in the presence of Q8, thus complicating
further analysis (Figures S40 and S44). On the basis of these
data, we conclude that the Tyr-Var motif extends beyond Var =
Leu to include Var = Phe and Tyr.
The Tyr-Var·Q8 complex described here represents a novel
binding motif in which the host accommodates the inclusion of
two neighboring amino acid residues within its cavity and binds
in a 1:1 stoichiometry. Because of the tight fit, the peptide has a
very limited range of possible conformations, and thus, Q8
serves as a template for the induced folding of the peptide.
Although the inclusion of two amino acid residues within the
cavity of a synthetic receptor and the induced folding of a
peptide by a synthetic receptor are not entirely unique in the
literature, they are rare, especially in aqueous media. In
particular, work by Fujita and co-workers has demonstrated the
sequence-specific recognition of multiple amino acids and the
folding of peptides into α-helix and β-turn conformations by
self-assembled coordination cages, with several complexes
exhibiting micromolar affinity in aqueous solution.8,30−32
Thermodynamic Characterization of Peptide Binding.
Thermodynamic constants for binding of peptides to Q8 were
determined by isothermal titration calorimetry (ITC) (Table 1
and Figures S45−S50). As expected, all of the complexes were
observed to bind in a 1:1 peptide:Q8 ratio (the “n” values were
within 10% of 1.0). We were surprised to observe that Tyr-Leu-
Ala binds Q8 with a Kd value of 7.2 nM.33 To the best of our
knowledge, this is the strongest reported affinity of a synthetic
receptor for an unmodified peptide in aqueous solution. This
result is further highlighted by the fact that the buffer contains
10 mM sodium, which binds to the host with a Kd value of 1
mM10 and thus diminishes the observed binding affinity by a
factor of 10; therefore, we estimate the Kd of the Tyr-Leu-Ala·
Q8 complex to be subnanomolar in pure water. The next-
highest affinity reported in the literature is the binding of Phe-
Gly by Q7 in pure water (Kd = 33 nM), as determined by Kim,
Inoue, and co-workers.34 The strong affinity of Tyr-Leu-Ala is
consistent with the slow exchange kinetics observed in the
NMR data. The peptide Ala-Tyr-Leu showed a 430-fold loss in
affinity compared with Tyr-Leu-Ala. Therefore, although the
Tyr-Leu sequence can be bound by Q8 at both N-terminal and
non-N-terminal positions, there is substantial specificity for N-
terminal Tyr-Leu.
The Kd values for Tyr-Phe-Ala and Tyr-Tyr-Ala were 0.29
and 0.70 μM, respectively. These submicromolar affinities are
strong but significantly weaker than that of Tyr-Leu-Ala.
Looking back at the fluorescence data from the initial library
screen shows that Tyr-Phe-Ala (14%) and Tyr-Tyr-Ala (13%)
exhibited a lower extent of fluorescence enhancement than Tyr-
Leu-Ala (19%) upon binding to Q8. This observation is
consistent with a model in which the extent of fluorescence
enhancement is proportional to the affinity of the peptide and
thus the extent of MBBI displacement at a given concentration.
Those data show Var1 = Lys (i.e., Tyr-Lys-Ala) to be the
peptide that induces the greatest extent of fluorescence
enhancement upon binding to Q8 (21%), so we synthesized
1
and purified Tyr-Lys-Ala and characterized it by H NMR
spectroscopy and ESI-MS (Figures S7 and S31). As expected,
Tyr-Lys-Ala interacted with Q8 in a similar manner as for other
1
Tyr-Var peptides as determined by H NMR and ESI-MS
analyses (Figures S8 and S32−S34). A semiempirical model
(Figure S37) based on ROESY data (Figure S36) is consistent
with the inclusion of both side chains and an additional
interaction of the side-chain ammonium group with the
opposite carbonyl portal of Q8. Tyr-Lys-Ala binds Q8 with a
Kd value of 0.20 μM, which is stronger than that of Tyr-Tyr-Ala
and Tyr-Phe-Ala but weaker than that of Tyr-Leu-Ala. Although
we expected Tyr-Lys-Ala to bind a little stronger than Tyr-Leu-
Ala on the basis of the fluorescence screen, the observed
discrepancy is within the error of the fluorescence screen.
The sequence isomer Tyr-Ala-Leu binds Q8 with a Kd value
of 34 μM, which is 4700-fold weaker than its sequence isomer
Tyr-Leu-Ala. This is exceptionally high sequence specificity for
a synthetic receptor in aqueous solution, second only to that
reported by Kim, Inoue, and co-workers for the binding of Phe-
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Phenylalanine-Terminated Peptides. The library screen
for phenylalanine-terminated peptides (Figure S51) showed
that the fluorescence intensity of the Q8·MBBI complex was
consistently enhanced upon addition of peptide. As discussed
above, we thought that this result was due to the binding of 2
equiv of peptide to Q8 because we had observed this result
previously for Phe-Gly-Gly.28 In light of the results reported
here on tyrosine-terminated peptides, however, we revised our
thinking on how Phe-terminated peptides may displace MBBI.
Given the similarity between the structures of Phe and Tyr, we
predicted that Phe-Leu-Ala, for example, should bind Q8 in a
similar fashion as Tyr-Leu-Ala.
To test this prediction, we synthesized and characterized
Phe-Leu-Ala and Phe-Ala-Leu and studied their interaction with
Q8 by ITC, NMR, and ESI-MS (Figures S54−S68). Prior work
has shown Phe-terminated peptides to bind to Qn hosts with
higher affinity than Tyr-terminated peptides,6 presumably
because of the greater hydrophobicity of Phe. The semi-
empirical model of the Q8·Tyr-Leu-Ala complex (Figure 5)
shows the tyrosine hydroxyl group making no direct contact
with peptide or Q8, and it was therefore reasonable to predict
even stronger binding for Phe-Leu-Ala. The ITC experiments
for binding of Phe-Leu-Ala to Q8 reveal a 1:1 Q8:peptide
micromolar affinity (Kd) values for these peptides. Remarkably,
Tyr-Leu-Ala bound to Q8 with a Kd value of 7.2 nM in 10 mM
sodium phosphate buffer, which is, to the best of our
knowledge, the highest reported affinity for a synthetic receptor
binding to an unmodified peptide in aqueous solution. Moving
the Leu residue further away (e.g., Tyr-Ala-Leu) markedly
diminished the binding affinity (4300-fold), showing excep-
tionally high sequence specificity. In the Tyr-Ala-Leu·Q8
complex, only the Tyr residue was bound within the Q8
cavity, allowing a second equivalent of peptide or MBBI to bind
simultaneously. This result is important because it brings us
significantly closer to the long-standing goal of developing a
synthetic receptor that can bind peptides in aqueous media
with sufficiently high affinity and specificity as to make them,
like antibodies, practical for applications in biochemistry and
biotechnology. Q8 binding to Tyr-Leu-Ala is not a general
approach to peptide recognition, but Q8 has the advantages of
being small, highly stable, recyclable,36 and many orders less
expensive than a monoclonal antibody. Moreover, this peptide
sequence is minimal and can be readily engineered into
recombinant proteins for use as an affinity tag for biological
applications.37−39
ASSOCIATED CONTENT
stoichiometry and an average Kd value of 0.43
0.12 μM,
■
which is significantly weaker than that of the Tyr analogue. This
is consistent with our prediction that Phe-Leu-Ala should bind
Q8 in a similar fashion as Tyr-Leu-Ala, but it was surprising that
the affinity is weaker. The ESI-MS data confirm the presence of
1:1 and 2:1 peptide:Q8 complexes. The NMR data confirm the
inclusion of Phe and Leu side chains within the Q8 cavity and
thus a similar mode of binding as Tyr-Leu-Ala.
In contrast to the results for Tyr-containing peptides,
however, the ITC, NMR, and ESI-MS data for the sequence
isomer Phe-Ala-Leu reveal a 2:1 peptide:Q8 complex as the
dominant species. The ITC experiments show positive
cooperativity, as observed for Phe-Gly-Gly.28 The NMR
experiments show the 2:1 Phe-Ala-Leu:Q8 complex, even at
substoichiometric ratios (data not shown), and the inclusion of
only the Phe side chain within the Q8 cavity, which is also
consistent with prior work on Phe-Gly-Gly. These results
demonstrate that the increase in fluorescence intensity
observed in the library screen can be due to 1:1 or 2:1
peptide:Q8 binding, depending on the peptide sequence.
S
* Supporting Information
1
Experimental details and MS, H NMR, ITC, and fluorescence
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*aurbach@trinity.edu
■
Author Contributions
†L.C.S. and D.G.L. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge Prof. Christopher Bielawski for
providing the MBBI used in this study. This work was
supported in part by grants from the Welch Foundation (W-
1640 and W-0031), the National Science Foundation (CHE-
1309978, CHE-0748483, and CHE-0957839), the Henry
Dreyfus Teacher-Scholar Awards Program, and Trinity
University. O.A.A. was a Beckman Scholar. This paper is
dedicated to Prof. Peter Dervan on the occasion of his 70th
birthday.
CONCLUSIONS
■
This paper has explored the effects of sequence context on the
binding of the synthetic receptor cucurbit[8]uril (Q8) to
peptides containing an N-terminal aromatic residue. Using
MBBI as a fluorescent indicator, we found that peptides
containing an N-terminal Trp bind to Q8 as a second guest,
causing a decrease in MBBI fluorescence, whereas Phe-
terminated peptides displace MBBI from the Q8 cavity, causing
an increase in fluorescence. This novel use of a fluorescent
indicator as both a turn-off and turn-on sensor based on two
different binding modes led to the discovery that the mode of
binding for Tyr- and Phe-terminated peptides depends on the
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