Water-Soluble Nanoparticle Receptors Supramolecularly Coded for Acidic Peptides

Article abstract of DOI:10.1002/chem.201703760

Sequence-specific recognition of peptides is of enormous importance to many chemical and biological applications, but has been difficult to achieve due to the minute differences in the side chains of amino acids. Acidic peptides are known to play importan

Full text of DOI:10.1002/chem.201703760

A Journal of
Accepted Article
Title: Water-Soluble Nanoparticle Receptors Supramolecularly Coded
for Acidic Peptides
Authors: Shixin Fa and Yan Zhao
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10.1002/chem.201703760
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Water-Soluble Nanoparticle Receptors Supramolecularly Coded
for Acidic Peptides
Shixin Fa and Yan Zhao*
Abstract: Sequence-specific recognition of peptides is of enormous
importance to many chemical and biological applications, but has
been difficult to achieve due to the minute differences in the side
chains of amino acids. Acidic peptides are known to play important
roles in cell growth and gene expression. In this work, we report
molecularly imprinted micelles coded with molecular recognition
information for the acidic and hydrophobic side chains of acidic
peptides. The imprinted receptors could distinguish acidic amino acids
from other polar and nonpolar amino acids, with dissociation
constants of tens of nanomolar for biologically active peptides
containing up to 18 amino acids.
hydrogen-bond-based molecular recognition, doing so for a
peptide chain with a complex combination of functionalities is a
completely different matter. When the binding takes place in water,
additional challenges exist because hydrogen bonds, arguably
the best tool available for selective molecular recognition, are
often ineffective in aqueous solution.[4]
In the last several decades, chemists have constructed
peptide receptors using many different materials including
macrocycles[1a, 1c] such as cyclodextrin[1b, 1d] and cucurbituril,[1h, 1j,
1l] amide oligomers[1a, 1f, 1g, 1i, 1k] and self-assembled nanocages.[1e]
Nonetheless, most of these receptors can only bind particular
types of peptides, usually fairly short ones. This is understandable.
Because numerous binding interactions need to work together to
achieve selective binding of a peptide even moderate in length,
the difficulty in the design and synthesis of its receptor increases
exponentially as the peptide gets longer, especially if the receptor
is molecular in nature.
Molecular imprinting is a distinctively different concept for
constructing guest-tailored receptors.[5] Instead of building
discrete molecular structures and then fitting them with guests,
researchers perform polymerization/cross-linking directly around
the guests (i.e., template molecules). Removal of the templates
leaves behind imprinted binding sites in the polymer matrix. The
technique has advanced greatly in the last decades and found
applications in separation, sensing, and enzymatic catalysis,
including in peptide recognition.[6] In addition to traditional
polymers and surfaces, imprinting could occur unimolecularly
within discrete structures such as dendrimers.[7]
One of the most difficult challenges in peptide recognition is
derived from the subtle structural difference of amino acids:
leucine and isoleucine differ by the position of a single methyl
group; aspartic acid and glutamic acid by the addition of one
methylene. Such minute differences demand an extremely high
level of precision in the molecular recognition. As a result, despite
decades of research, a general method for sequence-specific
binding of peptides remains elusive.[3, 8]
Introduction
Using 20 amino acids, nature constructs countless numbers of
peptides and proteins with diverse structures and functions.
These amino acids provide the functional “codes” that define the
hydrophobic, hydrophilic, acidic, and basic side chains at specific
positions along the peptide chain. The primary sequence is
important to the conformational preference of the peptide and its
secondary/tertiary structures under a given condition. Although
not a direct determinant of the final function, the primary “code” of
amino acids does contain all the information on the functional
groups of a protein, including those involved in the binding,
catalysis, or other molecular tasks the protein performs.
Chemists have long been interested in the molecular
recognition of peptides.[1] In addition to their fundamental
importance to supramolecular chemistry, these receptors have
practical applications. With many peptides serving as
neurotransmitters, neuromodulators, and hormones in biology,[2]
synthetic receptors could be used to alter or inhibit these
interactions. If effective strategies can be developed to recognize
peptides in a sequence-selective manner, it might be possible to
extend the method to recognize proteins by their characteristic
amino acids on the surface.[3]
We recently reported our first step toward a general method
for peptide recognition[9] via molecular imprinting within cross-
linked micelles.[10] However, since these receptors rely heavily on
hydrophobic interactions to operate, the method is expected to
work poorly for peptides rich in hydrophilic amino acids, excluding
a large number of biological peptides. Acidic peptides are known
to play important roles in cell growth and gene expression.[11]
Aspartic and glutamic acids are key to the aqueous solubility of
many proteins and play important roles in the catalysis of
numerous enzymes. In this work, we describe a method to encode
the receptors with molecular recognition information for acidic
groups on the side chain and at the C-terminus. The discovery
expands the supramolecular code of our molecularly imprinted
nanoparticles (MINPs) and significantly broadens the scope of
peptides to be recognized. Given the importance of peptide
recognition in many fundamental and applied research fields, we
In principle, to prepare a strong and selective receptor for a
peptide, all one needs to do is to create a “supramolecular code”
on the receptor, complementary to the amino acid “code” of the
peptide, in hydrophobicity, hydrogen bonds, and acidic/basic
functionalities. The challenge, however, lies in the technicalities.
If donor/acceptor motifs can be clearly identified and used in
Prof. Dr. Yan Zhao and Dr. Shixin Fa
Department of Chemistry
Iowa State University
Ames, IA 50011-3111, U.S.A.
Fax: (+1) 515-294-0105
E-mail: zhaoy@iastate.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201703760
Chemistry - A European Journal
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expect these easily synthesized, protein-sized, water-soluble
receptors for peptides to be very useful in biology and chemistry.
monomers (FMs) for the imprinting. Their design was based on
the ion-pairing interaction between carboxylic acid and
guanidinium or related cationic salts.[12] The two compounds differ
in hydrophobicity but have the same binding group. Their vinyl
group allows the compounds to be polymerized by radical
polymerization. To be useful in peptide recognition, the
thiouronium FM needs to be highly effective at binding carboxylic
acid but not overwhelming in strength to cause nonspecific
binding of acidic peptides. In addition, it needs to help the MINP
differentiate the two acidic amino acids, which can be very
challenging given their similarity.
Results and Discussion
Design and Synthesis
To recognize carboxylic acids on a peptide, we synthesized
thiouronium derivatives 1 and 2 (Scheme 1) as the functional
Scheme 1. Preparation of peptide-binding MINP with imbedded functional monomers (FMs).
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As shown in Scheme 1, the preparation of the MINP started
with solubilizing the peptide template within the micelle of cross-
linkable surfactant 3. Click-cross-linking of the micelle on the
surface using diazide 4 yielded surface-cross-linked micelle
(SCM).[13] The SCM had multiple residual alkyne groups because
of the 1:1.2 ratio between the tripropargylated cross-linkable
surfactant and the diazide cross-linker. Ligand 5 was installed by
another round of click reaction to cover the cross-linked micelle
with a layer of hydrophilic groups, allowing us to recover the final
MINPs by precipitation into acetone and washing with organic
solvents.[10] Right in the beginning, the micelles contained DVB,
DMPA (a photoinitiator), and functional monomer (FM) 1 or 2. UV
irradiation initiated free radical polymerization/cross-linking of the
micellar core, yielding the core-cross-linked SCM, with the
template still bound in the binding site and the FMs covalently
attached to the core. The template was removed by repeated
solvent washing, yielding MINP with the vacant binding site.
Usually, the entire preparation and purification were complete in
<2 days once the starting materials were available.
We did not attempt to control the pH of the solution during
MINP preparation because we did not want the micellization
process to be complicated by buffer molecules. The relatively high
pKa of S-alkylthiouronium (~9.8)[14] and low pKa of the acidic side
chains of peptides (~4) suggest that the hydrogen bond-
reinforced thiouronium–carboxylate salt bridge could dominate in
quite broad a range of pH near neutral.[15] The notion was
supported by our binding studies (vide infra).
Our first template was WDW. The tryptophan-aspartic acid-
tryptophan tripeptide has two hydrophobic residues and one
acidic one, making the total number of acids two including the C-
terminal one. The fluorescent W allowed us to study the binding
by both isothermal titration calorimetry (ITC) and fluorescence
titration. Its hydrophobicity enabled us to study the interplay
between hydrophobicity and the thiouronium–carboxylate salt
bridge in the imprinting and binding of the MINP.
O
OH
HN
NH
NH2
O
O
H
N
N
H
WDW
COOH
In general, ITC and fluorescence titrations showed excellent
agreement (Table 1). ITC has the advantage of affording the
number of binding sites per particle (N), in addition to other
thermodynamic binding parameters such as the binding constant
(Ka), enthalpy (∆H), and entropy (∆S).[17] However, since
fluorescence titration was faster and easier to perform, we used it
whenever it was possible (i.e., when the peptide contained
fluorescent tryptophan). Because the binding affinity changed
little (<10%) from water to 25 mM HEPES buffer (pH 7.4), with or
without FMs (entries 2 and 7), we performed the majority of the
bindings in water (we will come back to this point near the end of
the paper).
As shown in Table 1, MINP(WDW) prepared with the
template but without any FM bound the peptide with Ka = 15.3 ×
105 M-1 in water (entry 1). This binding constant reflects the
effectiveness of “hydrophobic encoding” of the imprinted receptor,
as polymerization/cross-linking around the indole side chain of
tryptophan is expected to create complementary pockets in the
nonpolar region of the cross-linked micelle.[9] Although the two
carboxylates of WDW were expected to form salt bridges with the
cross-linked ammonium surfactant,[10] we consider such
electrostatic interactions as background in the presence of
functional monomers such as 1 and 2, which are expected to form
specific hydrogen bond-reinforced ion pairs.
Synthesis and characterization of the materials followed
previously reported procedures[16] and the details are reported in
the Supporting Information. Generally, the reaction progress was
monitored by 1H NMR spectroscopy. Dynamic light scattering
(DLS) afforded the size and molecular weight of the MINP. The
nanoparticles were typically 4–5 nm in diameter, with an
estimated M.W. of 50,000–56,000. The size was confirmed by
transmission electron microscopy (Figure S1 in Supporting
Information).
Optimization of the MINP preparation
Table 1. Binding data for WDW by MINP(WDW).[a]
Ka
-∆G
entry
FM
surfactant
FM/template
N
∆H (kcal/mol)
T∆S (kcal/mol)
(× 105 M-1)
(kcal/mol)
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
6
6
7
7
0
15.2 ± 1.2 (15.7 ± 1.4)
(17.4 ± 0.1)[b]
0.80 ± 0.03
8.4
8.5
8.9
8.8
9.2
9.4
9.4
9.2
9.1
8.2
9.4
8.4
9.3
-76.7 ± 3.3
-68.3
--
2
0
--
--
3
3:1
1:1
2:1
3:1
3:1
4:1
5:1
0
33.4 ± 1.5 (34.3 ± 1.8)
28.8 ± 2.9 (28.8 ± 1.2)
54.4 ± 5.6 (53.6 ± 4.9)
82.7 ± 4.9 (80.8 ± 7.2)
(76.5 ± 0.5)[b]
1.08 ± 0.01
-59.8 ± 0.4
-50.9
-47.7
-66.0
-110.4
--
4
0.77 ± 0.01
-56.5 ± 1.2
5
0.80 ± 0.01
-75.2 ± 1.1
6
0.83 ± 0.02
-119.8 ± 3.3
7
--
--
8
57.9 ± 3.8 (58.7 ± 3.3)
45.4 ± 2.3 (39.1 ± 1.0)
(10.1 ± 1.6)
1.26 ± 0.01
-101.6 ± 0.8
-92.4
-94.8
--
9
1.15 ± 0.01
-103.9 ± 0.8
10
11
12
13
--
--
--
--
--
--
--
--
3:1
0
(76.7 ± 1.9)
--
(14.3 ± 3.4)
--
3:1
(63.3 ± 6.0)
--
[a] The titrations were performed in Millipore water unless indicated otherwise. The Ka values in the parentheses were determined by fluorescence titration and
those without the parentheses by ITC. [b] The binding was measured in 25 mM HEPES buffer (pH 7.4).
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solvation demands the peptide to stay near the surface of the
micelle during imprinting and binding. Quite likely, such a
configuration would not allow the relatively short hydrophobic side
chains (even that of tryptophan) to penetrate deep into the
hydrophobic core when an amide layer exists in the MINP.
One of the most important properties of the MINP is its
controllable number of binding sites.[10] Usually, we keep the
surfactant/template ratio to 50:1. With each MINP containing ~50
(cross-linked) surfactants, the ratio gives one binding site per
nanoparticle on average. The number was confirmed by the ITC
studies, as shown in Table 1. The 1:1 binding stoichiometry was
also evident from the Job plots, for the MINP prepared with or
without FM 2 (Figure 1). Our previous work shows that, by
changing the surfactant/template ratio, the number of binding
sites can be easily controlled.[10]
The binding constant above was somewhat weaker than
what was obtained for WGWG by MINP(WGWG) under similar
conditions (Ka = 56.2 × 105 M-1).[9] Unless the last glycine (G)
contributes significantly to the binding of WGWG, the weaker
binding in WDW (by its own MINP) seems to suggest that the
aspartic acid negatively impacts the imprinting/binding of the
peptide in the absence of specific FMs. Ionic groups such as
carboxylate have a strong tendency to stay on the surface of the
micelle, to be solvated by water. Hydrophobic binding between
the tryptophans and the MINP, on the other hand, is stronger
when the binding pocket is relatively deep in the hydrophobic core
[18]. Although we could not pin down the exact reason for the
weakened binding of WDW (in the absence of FMs), the above
two requirements do seem to contradict each other. (We will come
back to this point again later in this paper.)
FM 1 and 2 both enhanced the binding, indicating that the
hydrogen bond-reinforced thiouronium–carboxylate salt bridges
were effective. At the same FM/template ratio (3:1), the more
hydrophobic 2 strengthened the binding more than the less
hydrophobic 1 (entries 3 and 6). We attributed the difference to
their different incorporation into the micelle: being cationic, both 1
and 2 are repelled by the cationic micelle. The more hydrophobic
2 is anticipated to have a stronger tendency to stay in the
hydrophobic microenvironment of the micelle, whereas 1 may
migrate into the aqueous phase due to its water solubility. For the
imprinting to work, the FM clearly needs to stay with the template
within the micelle during polymerization.
Our data also show that the optimal FM/template ratio was
3:1 for FM 2 (Table 1, entries 4–9). Since WDW has two
carboxylates in the structure, the ratio suggests a 50% excess of
the thiouronium was needed for the best result. As will be shown
by additional studies, this ratio was fairly constant over different
templates (vide infra).
Figure 1. Job’s plots for WDW with (a) MINP(WDW) prepared (a) without FM
(a) and with 3 equiv FM 2 (b). The total concentration of MINP and the guest
was 10 µM. χ = [Host]/{[Host] + [Guest]}.
Effects of hydrophobicity and the number of salt bridges
We recently reported amide-containing cross-linkable
surfactants 6 and 7.[18] The hydrogen-bonding capabilities of the
amide were found to enhance the imprinting and binding of
hydrogen bond-containing templates significantly, including
oligosaccharides.[16f] However, with or without the thiouronium FM,
surfactant 6 or 7 afforded MINPs with weaker binding than those
prepared with 1 (entries 10–13). Thus, even if hydrogen bonds
could be formed between the cross-linked surfactants and the
peptide template, they brought no net benefit.
The two tryptophans afforded significant hydrophobic
driving force to the binding (Table 1, entry 1).[19] FM 2 was able to
increase the binding constant by 5 times (compare entries 1 and
6). This is good news to us but how about other peptides? Is the
salt bridge equally effective for peptides with different
hydrophobicity?
Table 2 addresses these questions. All the peptides have
two carboxylic acids, one from the aspartic acid side chain and
the other from the carboxy terminal. The number of tryptophan
increased from 0 to 1 to 2 from GDG to GDW to WDW. Note that
the positions of the carboxylic acids stay the same within the
series.
O
O
O
O
O
O
Cl
Cl
N
N
N
N
2
H
H
7
6
We were pleased to see that even the most hydrophilic
peptide (GDG) showed significant binding toward its MINP, with
Ka = 0.53 × 105 M-1 in water (entry 1). Since this peptide does not
have any significant hydrophobicity, the 6.4 kcal/mol binding free
energy (-∆G) is quite impressive. The “background” electrostatic
interactions between the oppositely charged MINP and template,
the hydrogen bonding interactions formed between the peptide
and the MINP (e.g., with triazole on the cross-linked surfactant or
hydroxyl groups on the cross-linker or surface ligand), and any
desolvation during the binding probably all contributed to the
binding.
The behavior of the amide-containing MINPs might be
puzzling, given that they enhanced the imprinting and binding of
18]
many templates with hydrogen-bonding functionalities.[16f,
However, our previous work also showed that location of the
hydrogen-bonding group in the template is highly important to the
performance of such MINPs [18]. The templates that benefit most
from the amide generally need to have their hydrophobic group
penetrate through the amide layer, reaching the hydrophobic core
of the micelle in order to maximize the hydrophobic interactions.
In addition to many hydrogen-bonding groups in the main chain,
a
peptide has ionic ammonium/carboxylate groups. Their
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Table 2. Effects of peptide hydrophobicity on imprinting and binding using FM 2.[a]
Ka
-∆G
entry
template
FM/template
Krel
N
∆H (kcal/mol)
T∆S (kcal/mol)
(× 105 M-1)
(kcal/mol)
1
2
3
4
5
6
GDG
GDG
GDW
GDW
WDW
WDW
0:1
3:1
0:1
3:1
0:1
3:1
0.53 ± 0.1
4.6 ± 0.3
6.4 ± 0.7
19.8 ± 1.0
15.2 ± 1.2
82.7 ± 4.9
0.69 ± 0.36
1.03 ± 0.02
0.83 ± 0.03
1.02 ± 0.06
0.80 ± 0.03
0.83 ± 0.02
6.4
7.7
7.9
8.6
8.4
9.4
-42.2 ± 5.4
-20.1 ± 0.9
-96.5 ± 5.0
-88.6 ± 2.0
-76.7 ± 3.3
-119.8 ± 3.3
-35.8
-12.4
-88.6
-80.0
-68.3
-110.4
8.7
3.1
5.4
[a] The Ka values were determined by fluorescence titrations in Millipore water. Krel is the binding constant of the MINP with the thiouronium FM relative to that
without.
The addition of the first tryptophan increased the binding by
1.5 kcal/mol (entry 3) and the second addition by 0.5 kcal/mol
(entry 5). Note that these numbers represent the binding between
each peptide and its own MINP, not different peptides to the same
receptor. The energy difference, thus, reflects the effectiveness of
the imprinting, as well as that of the binding.
Importantly, in all three peptides, the thiouronium FM clearly
enhanced the binding, shown by the Krel value, i.e., the binding
constant of the MINP with the thiouronium FM relative to that
without. Meanwhile, as the number of hydrophobic residues
increased, the enthalpic driving force became stronger but were
offset by an increasingly larger (unfavorable) entropic term (Table
2). Thus, all the bindings were enthalpically driven, including
those shown in Table 1.
We then studied the effect of the (hydrogen bond-
reinforced) salt bridge on the imprinting and binding (Table 3). In
this series, we kept the number (= 2) and the position of the
tryptophan the same and increased the number of aspartic acid
from 1 to 3. As a result, the salt bridges that could be potentially
formed between the peptide and its MINP increased from 2 to 4.
support that, without proper binding groups for the carboxylates,
their strong solvation by water makes the peptide stay closer to
the surface of the micelle and interferes with the hydrophobic
imprinting that requires the hydrophobic residues to move deeper
into the micellar core.
Encouragingly, Krel increased steadily, from 5.1 for WDW to
10.2 for WDWD, and to 14.5 for WDWDD at the same
FM/carboxylate ratio (1.5:1). As shown by entries 4–6, the 50%
excess FM per carboxylate was optimal for the imprinting of
WDWD, the same as found before.
The results so far clearly demonstrated the effectiveness of
the thiouronium–carboxylate salt bridge in the supramolecular
coding of the MINP receptors: they worked for both hydrophilic
peptides and hydrophobic ones, and continued to enhance the
binding as the number of salt bridges increased.
In Table 4, we varied both the hydrophobicity and the
number of salt bridges. In these examples, we kept the
FM/carboxylate ratio at 1.5:1, the optimal number confirmed by
two studies shown above.
Table 4. Interplay of hydrophobicity and salt bridges.[a]
Table 3. Effects of the number of salt bridges on imprinting and binding using
FM 2.[a]
# of salt
bridge[b]
0
Ka
-ΔG
(kcal/mol)
8.94
Entry template
FM
Krel
(×105 M-1)
Ka
-ΔG
(kcal/mol
8.45
1
2
3
4
5
6
7
8
WNW
WNW
WWW
WWW
WDW
WDW
WEW
WEW
none
2
35.7 ± 5.1
Entry
template FM/template
Krel
(×105 M-1)
1
0
1
0
2
0
2
59.1 ± 5.9
123.5 ± 9.9
159.6 ± 6.5
15.7 ± 1.4
80.8 ± 7.2
16.2 ± 1.5
92.7 ± 9.2
1.7
1.3
5.1
5.7
9.24
9.67
9.83
8.45
9.42
8.47
9.50
1
2
3
4
5
6
7
8
WDW
WDW
0:1
3:1
15.7 ± 1.4
none
2
80.8 ± 7.2
11.4 ± 0.8
78.8 ± 12.7
116.5 ± 17.2
94.7 ± 8.1
9.3 ± 0.8
5.1
9.42
8.26
9.41
9.64
9.52
8.14
9.73
WDWD
WDWD
WDWD
WDWD
WDWDD
WDWDD
0:1
none
2
3:1
6.9
10.2
8.3
4.5:1
6:1
none
2
0:1
[a] The Ka values were determined by fluorescence titrations in Millipore water.
When FM 2 was used, the FM/carboxylate ratio was kept 1.5:1 in all cases. Krel
is the binding constant of the MINP with the thiouronium FM relative to that
without. [b] The salt bridge refers to the hydrogen bond-reinforced thiouronium–
carboxylate salt bridge.
6:1
134.8 ± 10.5
14.5
[a] The Ka values were determined by fluorescence titrations in Millipore water.
Krel is the binding constant of the MINP with the thiouronium FM relative to that
without.
Our data indicate that, as the number of carboxylate
increases, the binding became weaker for the MINP prepared
without FM (Table 3, entries 1, 3, and 7). Although the effect was
moderate, the trend corroborated with our earlier results to
For the two very hydrophobic peptides (WNW and WWW),
the hydrophobic imprinting apparently was highly efficient,
affording nearly 9 kcal/mol in binding free energy without any FMs.
With one thiouronium–carboxylate salt bridge (at the carboxy
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terminal), the enhancement (Krel) was expectedly low, only 1.3–
1.7 (Table 4, entries 2 and 4). The data in entries 5 and 6 were
the same as in Tables 1–3, and are listed here for comparison
purposes. As shown by entries 7 and 8, the thiouronium FM also
worked well for peptides containing glutamic acid (E), enhancing
the binding constant by a similar degree as for aspartic acid (D).
interference of hydrophobic binding by the ionic carboxylates, the
stronger binding of WNW may not seem as strange. Essentially,
the imprinting and binding of WDW have to deal with the
conflictory requirements of the ionic and hydrophobic side chains
for optimal interactions, as discussed earlier. Once the aspartic
acid is replaced with asparagine, the dilemma no longer exists.
Being extremely similar in structure to the template (i.e., WDW),
WNW should be able to occupy the binding site created after
WDW. Not only so, its tryptophans can now optimize the
hydrophobic interactions with the binding pockets, without the
constraint set by the aspartic acid ionic side chain—a feature
beneficial to the binding.
When MINP(WDW) was encoded with the carboxylate-
binding thiouronium groups, the binding affinity improved as
shown by our earlier data, as well as Table S1. Importantly, the
binding selectivity was maintained in most cases (Figure 2). Most
notably, WNW, which showed the unusually stronger binding than
the template itself toward the MINP prepared without FM, now
behaved normally by displaying a weaker binding than the
templating peptide. Thus, once the aspartic acid side chain was
made to participate in the specific carboxylate–thiouronium salt
bridge, the residue becomes an important contributor to the
binding selectivity (as well as to binding affinity).
Binding selectivity of small peptides
Molecular imprinting has the benefit of supramolecularly
encoding the receptor with molecular recognition information for
the entire peptide template, main chain and side chains at the
same time. Since the hydrophobic residues on a peptide give us
excellent sequence-selectivity according,[9] we focused on several
closely related amino acids in this study, knowing that the
hydrophobic residues will continue to impart selectivity to the
MINP receptor.
Figure 2 compares the binding affinity of different peptides
toward the MINP prepared from WDW, normalized to the binding
affinity of the template itself. We included data for MINP(WDW)
prepared with and without FM 2. In the absence of FM, replacing
one of the tryptophans with glycine (in GDW) or removing it (in
DW) weakened the binding significantly. Switching the position(s)
of the tryptophan (in DWW or WWD), as well as adding a
tryptophan (in WWW) weakened the binding even more. All of
these results are fully consistent with the rules found in the
hydrophobic imprinting.[9] Thus, although hydrophobic effect is
nonspecific by itself, imprinted hydrophobic binding pockets with
specific shape and size can be highly discriminating in their
binding.
Table
5 shows the (absolute) binding constants of
MINP(WDW) and MINP(WEW) for peptide WDW and WEW. The
comparison shows the binding selectivity of the MINPs for the two
subtly different acidic amino acids. In all cases, the MINP always
preferred its own template over the minutely different competitor,
indicative of effective molecular imprinting. Encouragingly, both
the MINPs prepared with and without FM 2 displayed moderate
selectively: with the cross-reactivity (i.e., Krel) ranging from 0.54 to
0.72 for the non-matching peptide. Although the selectivity might
have improved slightly with MINP(WDW) functionalized with the
thiouronium binding group (entry 6), the improvement was rather
limited. The binding studies also demonstrated that the
thiouronium FM strengthened the binding, without comprising the
binding selectivity.
Table 5. Differentiation of aspartic acid (D) and glutamic acid (E).[a]
Ent
ry
templat
e
Ka
Guest
FM
Krel
(×105 M-1)
1
WDW
WDW
WEW
WEW
WDW
WDW
WEW
WEW
WDW
none
none
none
none
2
15.7 ± 1.4
2
3
4
5
6
7
8
WDW
WEW
WEW
WDW
WDW
WEW
WEW
10.7 ± 0.7
16.2 ± 1.5
11.7 ± 1.2
80.8 ± 7.2
44.0 ± 4.2
92.7 ± 9.2
66.1 ± 5.1
0.68
0.72
0.54
0.71
Figure 2. Binding selectivity of MINP(WDW) prepared without (blue bars) and
with FM 2 (red bars). The binding data are reported in Table S1 in the Supporting
Information. Krel is the binding affinity of different peptides toward the MINP
prepared from WDW, normalized to the binding affinity of the template itself.
2
2
For the binding of MINP without FM, the most unusual result
was that WNW showed stronger binding for MINP(WDW) than the
templating peptide WDW (Figure 2). This would be normally
interpreted as failure in molecular imprinting. However,
asparagine (N) and aspartic acid (D) differ subtly: the two have
the same number of carbons and both contain a carbonyl group,
except one (carbonyl) from a primary amide and the other form a
carboxylic acid. If we consider our earlier evidence for the
2
[a] The Ka values were determined by fluorescence titrations in Millipore water.
Krel is the binding constant of the MINP with the thiouronium FM relative to that
without. The FM/carboxylate ratio was kept 1.5:1 in all cases.
Imprinting and binding of biological peptides
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Having confirmed that thiouronium FM 2 was able to
effectively encode the MINP receptor for aspartic acid and
glutamic acid, we moved on to study a number of biological
peptides bearing acidic side chains (Figure 3). The 1:1.5
carboxylate/FM 2 was maintained in all cases.
For the simplest DGEA, a tetrapeptide in collagen critical to
the binding of the alpha 2 beta 1 integrin receptor,[20] FM2
enhanced the binding impressively, by nearly 50 times. As we
have observed previously for the hydrophobic peptides, the
benefit of the molecular imprinting is that, the longer the peptide,
the more hydrophobic and now also acidic residues would
contribute to the binding and the stronger the binding will be.
Impressively, for HIFF-1 alpha, a peptide made of 18 amino acids
and the longest we have imprinted so far, a dissociation constant
of 62 nM was obtained.
Our model peptide was DYKDDDDK, the FLAG-tag
frequently used for protein purification and for studying proteins in
living cells.[21] Its abundant acidic and basic groups suggest that
its binding could be quite sensitive to solution pHs. As shown in
Table 6, its binding constant was 20.4 × 105 M-1 in water, without
any pH adjustment. The binding constant dropped approximately
by half in 10 mM HEPES buffer. At first appearance, there
seemed to be a quite significant pH effect. However, when the pH
was changed to 6.0 and 9.0 using two other buffers, the binding
constant stayed nearly the same, suggesting otherwise.
Suspecting that the sulfonate and carboxylate groups of the these
buffer molecules might have competed with the carboxylate
groups of the peptide for the thiouronium binding groups, we
performed the binding in Tris-HCl buffer at both pH 7.4 and 9.0.
Indeed, in the absence of competing binding functionalities, the
binding became stronger, similar to that in water.
O
O
H
:
:
2
2
Without FM
With FM
H2N
N
N
N
COOH
Table 6. Effects of pH and buffer on the binding of DYKDDDDK.[a]
-1
-1
=
±
± x
µ
M
Ka
0.04) 105 M
Ka
0.6) 105 M
H
H
x
=
(0.14
µ
(6.7
O
HOOC
Ka
=
=
Kd 71
M
Kd 1.5
COOH
Entry
buffer
pH
(×105 M-1)
DGEA
1
2
3
4
5
6
none
unadjusted
20.4 ± 0.7
CONH2
N
COOH
10 mM HEPES
7.4
6.0
9.0
7.4
9.0
11.7 ± 0.6
OH
:
2
With FM
O
O
O
O
O
H
H
N
H
H
-1
±1.29) 106 M
nM
10 mM MES
H2N
N
N
N
N
=
x
Ka
(8.37
11.2 ± 0.5
12.6 ± 1.0
N
N
N
COOH
H
O
H
H
H
H
=
Kd 120
O
O
O
HOOC
10 mM Bicine
10 mM Tris-HCl
10 mM Tris-HCl
COOH
COOH
c-Myc
Peptide
NH2
25.9 ± 2.0
24.2 ± 3.1
OH
NH2
HOOC
:
2
COOH
With FM
O
O
O
H
O
[a] The Ka values were determined by ITC. The FM/carboxylate ratio was kept
H
H
-1
=
Ka
±0.07) 106 M
x
H2N
HOOC
N
N
N
(2.04
N
N
N
N
COOH
1.5:1 in all cases.
H
H
H
H
=
nM
Kd 490
O
O
O
COOH
COOH
DYKDDDDK
NH2
Thus, the binding of DYKDDDDK showed little dependence
on solution pH (6–9), despite its many ionizable groups (Table 6).
This feature makes the binding more predictable in different
environments and could be quite useful in biological applications
of the MINPs. Cationic micelles are known to be slightly basic on
their surface due to their electrical potential, even when the bulk
solution is neutral .[22] MINP, being a polycation, should do the
same.[23] Since the acidic side chains of aspartic acid and glutamic
acids have a pKa of ~4, they should be readily deprotonated near
the surface of the MINP during the binding. This could be the
possible reason for the lack of pH effect at 6–9 for the MINP
binding.
CONH2
O
COOH
COOH
O
O
H
H
N
H
H
N
HOOC
N
N
N
N
H
N
H
N
H
O
O
O
O
O
O
HOOC
:
With FM 2
S
HN
HN
O
-1
OH Ka
0.13) 107 M
=
± x
(1.62
nM
S
=
Kd 62
O
O
H
O
H
N
O
H
N
N
N
N
N
H
H2N
N
N
H
H
H
O
O
O
O
COOH
COOH
COOH
HIF-1
alpha (556-574)
Figure 3. Structures of biological peptides studied and the binding constants
(Ka) and dissociation constants (Kd) obtained.
Conclusions
pH effect on the binding of biological peptides
Many imprinted polymers have been used for peptide recognition,
ranging from materials useful for peptide extraction and
separation to lightly cross-linked nanogels with improved
biocompatibility.[6] In comparison to these materials, our cross-
We performed the majority of the binding studies in Millipore
water because our initial studies showed that the binding of WDW
by its MINP showed little change (<10%) from water to HEPES
buffer (pH 7.4), with or without FMs (Table 1, entries 2 and 7).
Negligible pH effects in MINP binding has also been observed
previously for other peptides (e.g., WWGG).[9] Nonetheless, the
biological acidic peptides contain many ionizable groups. It is
important to understand how the MINP receptors behave under
different pH conditions.
linked
micelles
generate
protein-sized
water-soluble
nanoparticles with a controllable number of binding sites. This
work has expanded the “supramolecular codes” of our imprinted
micellar receptors significantly. Thiouronium FM 2 turned out
highly effective in the molecular imprinting of peptides containing
acidic side chains. In the presence of the FM (typically used at 1.5
equiv to the carboxylic acids), both hydrophobic residues and the
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T. Halbert, A. R. Urbach, J. Am. Chem. Soc. 2009, 131, 2408-2415; i) S.
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acidic side chains contribute strongly to the binding of the
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gets longer.
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for example, was bound more strongly by its MINP than WNW (by
its own) in the presence of FM 2; in the absence of the FM, the
opposite trend was observed.
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Conflict of interest
The authors declare no conflict of interest.
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Keywords: peptide binding • molecular recognition • receptor •
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Entry for the Table of Contents
Layout 1:
FULL PAPER
We describe a general method to
construct synthetic receptors with high
sequence specificity for the acidic and
hydrophobic residues of peptides. The
receptors could bind biological
peptides with tens of nanomolar of
binding affinity and were prepared
straightforwardly through molecular
imprinting in less than 2 days.
Shixin Fa, Yan Zhao*
Page No. – Page No.
Water-Soluble Nanoparticle
Receptors Supramolecularly Coded
for Acidic Peptides
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