General Method for Peptide Recognition in Water through Bioinspired

Jun 14, 2019 - (40) All FMs have a styrenyl group as a hydrophobic anchor for their inclusion into the .... Although 10 seemed to be the best among th...
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Cite This: Chem. Mater. 2019, 31, 4889−4896

General Method for Peptide Recognition in Water through Bioinspired Complementarity Shixin Fa and Yan Zhao* Department of Chemistry, Iowa State University, Ames, Iowa 50011-3111, United States

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S Supporting Information *

ABSTRACT: A general method for peptide recognition has been elusive despite decades of research. Strong binding and selectivity among closely related peptides are necessary for biological applications but have been difficult to achieve with synthetic receptors. With inspiration from highly specific protein−protein and protein−ligand interactions, proteinsized, water-soluble imprinted nanoparticles were prepared via templated polymerization of peptides within cross-linked micelles. Combination of hydrophobic and polar interactions afforded micromolar to submicromolar binding affinities for selected tripeptides. A “golden pair” of functional monomers was identified to enhance both the affinity and selectivity of binding and enabled differentiation of subtly different sequences including singlepoint variation of lysine by arginine and insertion of a single glycine at the N- or C-terminus. Biological peptides (β-amyloid peptides) afforded even stronger binding (tens of nanomolar) due to a larger number of complementary interactions between the host and the guest, opening doors to a wide range of biological applications.

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INTRODUCTION Sequence-selective recognition of peptides has been a longsought-after goal in supramolecular and bioorganic chemistry.1−3 Motivated by their many potential applications, chemists in the last decades have prepared numerous synthetic receptors for peptides. Early works focused on peptides containing aromatic residues such as phenylalanine (F) and tryptophan (W), using various macrocycles4−7 and selfassembled nanocages. 8 More recent platforms include molecular tweezers and clips,9 cucurbiturils,10−12 pseudopeptidic cages,13,14 gold nanoparticles (NPs),15 and co-assembled amphiphiles.16 Peptides with acidic or basic side chains were also frequent targets because of the potentially strong salt bridges that can be formed.4,17−20 One challenge in peptide recognition is to achieve biologically competitive binding affinity. For a synthetic receptor using hydrogen bonds for binding, solvent competition from water tends to weaken the binding substantially. An exception is cucurbit[8]uril (Q8), which binds certain aromatic peptides (e.g., Tyr-Leu-Ala) with extraordinary tightness.11 Another challenge comes from complexity and size of the peptide guest. When a biological peptide with 10 or more residues needs to be recognized, because the guest contains too many supramolecular features, building a complementary receptor on a preorganized covalent framework is no longer practical through molecular synthesis. Molecular imprinting, on the other hand, offers a potential solution, by building binding sites directly around template molecules in a crosslinked polymeric network through covalent capture.21−26 Shea and co-workers, in particular, employed precipitation polymerization to obtain imprinted NPs for biological peptides.27 NPs imprinted against mellittin (the major component of the bee © 2019 American Chemical Society

venom) bound the toxin with a Kd of 7.3−25 pM and possessed excellent biocompatibility, allowing them to be used for removing the toxin from bloodstream of living mice.28 Despite the progress made so far, a general method is still missing to recognize peptides of different sizes, hydrophobic and hydrophilic alike. Biological receptors can easily distinguish highly similar sequences; most reported synthetic receptors only worked for specific types of peptides and had difficulty differentiating closely related structures. The need is especially urgent considering that protein−protein interactions are often bindings of a peptide epitope on one protein by another protein as the receptor. Readily available synthetic peptide receptors can be useful both in fundamental research and technological applications. In this work, we report water-soluble organic NP-based receptors for highly selective recognition of peptides in water. Through templated polymerization in cross-linked micelles with specifically designed functional monomers (FMs), we created complementary sites on the micelles for the hydrophobic, acidic, and basic side chains of peptides all at the same time. Our molecularly imprinted NPs (MINPs) readily differentiated closely related sequences including single-point variation of lysine by arginine and insertion of a single glycine at either the N- or C-terminus in tripeptides. The method is simple to perform and general, affording receptors for longer βamyloid peptides with tens of nanomolar binding affinities and opening doors to a wide range of biological applications. Received: April 23, 2019 Revised: June 13, 2019 Published: June 14, 2019 4889

DOI: 10.1021/acs.chemmater.9b01613 Chem. Mater. 2019, 31, 4889−4896

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Chemistry of Materials

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RESULTS AND DISCUSSION Design and Synthesis of Peptide-Binding MINP. Scheme 1 shows the general preparation of MINP.29 A

carboxylate-derived FM simultaneously for peptide recognition is obviously impossible due to their “self-destruction”. To overcome the above difficulties, we prepared a series of FMs 4−10 to target specific polar side chains of peptides. We envision that the functionalized MINPs then can bind all the hydrophobic, basic, and acidic groups at the same time, making them an excellent platform of “artificial antibody” for peptides.40 All FMs have a styrenyl group as a hydrophobic anchor for their inclusion into the micelle and polymerizable to be attached covalently to the MINP core. For FMs 4−7, the many hydrogen-bond receptors on the structure allow them to target functional groups rich in hydrogen-bond donors, for example, guanidinium of arginine and protonated N-amino group and side chain of lysine (Figure 1, bottom left).32 FMs

Scheme 1. Preparation of MINP with Covalently Attached FMs

template−FM complex is first solubilized with DVB and DMPA (a photoinitiator) in water by the micelle of crosslinkable surfactant 1. Cross-linking of the micelle with diazide 2 by the Cu(I)-catalyzed click reaction affords surface crosslinked micelles,30,31 which are functionalized by another round of click reaction with 3. UV irradiation leads to core crosslinking by free radical polymerization among the methacrylate of 1, vinyl of the FM, and DVB. Progress of reaction is monitored by 1H NMR spectroscopy, and the size/MW of MINP is determined by dynamic light scattering (DLS). The DLS size has been confirmed by transition mission microscopy.32,33 MINPs are typically ∼5 nm in diameter, with an estimated MW of 50 000−60 000. One equivalent of DVB to the surfactantthe highest amount solubilized by the micelleis typically used to ensure integrity of the imprinted binding site. A 50:1 surfactant/template ratio affords an average of one binding site per MINP because the NP contains ∼50 crosslinked surfactants. Different number of binding sites can be obtained with different surfactant/template ratios.29 Our design of peptide-binding MINP was inspired by naturally occurring protein−protein34 and protein−ligand interactions,35 both of which require combined hydrophobic and polar interactions for high binding strength and selectivity. Micellar imprinting is excellent at creating binding pockets for hydrophobic groups.29,36 However, recognition of polar side chains of peptides requires new strategies because polar binding interactions inevitably rely on hydrogen bonds, which face severe competition from solvent when the binding takes place in water. The most notable supramolecular handles of peptides are their acidic side chains (and the C-terminal carboxyl), the amino side chains of lysine (and the N-terminal amino), and the guanidinium of arginine. Cationic salts such as guanidinium or amidinium are well known to bind carboxylates,37−39 using a guanidinium-derived FM and a

Figure 1. Structures of FMs 4−10 and potential binding motifs of azacrown (bottom left) and thiouronium FMs (bottom right).

8−10 carry a cationic thiouronium, allowing them to interact with a carboxylate through a hydrogen bond-reinforced salt bridge (Figure 1, bottom right).33,41 Although hydrogen bonds are weak in water, they become more effective in the hydrophobic core of a micelle,42,43 allowing us potentially to overcome the difficult solvent competition during imprinting and binding. Not only so, when both the FM and the peptide template are solubilized by a micelle, their local concentrations are much higher than their bulk concentrationsa feature advantageous to the template-FM complexation during imprinting. Peptide Binding of Functionalized MINPs. Tripeptides WWD and KWW were our initial templates/guests to test the above strategy (Chart 1). With two large hydrophobic groups, these peptides are expected to have a strong hydrophobic driving force to enter their imprinted binding sites. We wanted to know whether any added polar interactions could contribute significantly under such a condition. We chose to include W also because its fluorescence makes it convenient to study the binding by fluorescence spectroscopy. Previously, both binding constants and stoichiometry obtained from fluorescence titrations were verified by isothermal titration calorimetry (ITC).36 4890

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Chemistry of Materials Chart 1. Structures of Tripeptides Studieda

binding energy of −ΔG = 8.68 kcal/mol. Inclusion of FM 4−7 in the MINP preparation all strengthened the binding, as indicated by the Krel values (entries 2−5). The increase in binding energy ranged from 0.13 to 0.36 kcal/mol. Although the enhancement was modest, it was significant, given that the FMs were designed to interact with a single N-terminal amino group over a very large background of 8.68 kcal/mol for the parent MINP. Another interesting finding is that the bicyclic azacrowns (6 and 7) outperformed the monocyclic ones (4 and 5) consistently, suggesting preorganization in the macrocycle was helpful to the molecular recognition in the micelle. The results strongly indicate that the azacrowns, assisted by the micelles, indeed were able to complex with the N-terminal ammonium in the overall aqueous solution. FMs 8−10 target the two carboxylate groups of WWD. The Krel values (2.64−3.49) were larger than those for the azacrowns (1.24−1.82) (compare entries 6−8 with 2−5). The corresponding change in binding energy ranged from 0.58 to 0.74 kcal/mol. Given the strong polarity of the aqueous solution (and the large hydrophobic background of the binding), the increase was significant. Although 10 seemed to be the best among the three thiouronium FMs, the difference was quite small, especially in comparison to 8. More revealing results were obtained when the FMs were used together to target the acidic and basic groups simultaneously. Entries 9−11 of Table 1 show that 10 continued to be the winner among the three thiouronium salts. Moreover, although the three thiouronium FMs showed small differences when used alone, their mixing with 4 spread them out: while 4 strengthened the binding of MINP prepared with 10 from 80.9 to 89.1 × 105 M−1, it weakened that with 8 from 74.7 to 53.7 × 105 M−1. We attributed the opposite effects of 4 on the coexisting FM to its ability to interact with the other FM and hinder both interactions with the template. Because 8 has abundant hydrogen-bond donors, it should compete with the amino (ammonium) group of WWD for 4 that is rich in hydrogen-bond acceptors. On the other hand, the thiouronium of 9 and 10 only have two NH bonds, thus difficult to interact with 4. Their benzyl or butyl groups also make them sterically encumbered to bind an azacrown, thus avoiding the “self-destruction” of 4 and 8. Because FM 10 was the best by itself or mixed with 4, we examined its mixture with 5−7. Encouragingly, the binding was strengthened in every case in comparison to MINP prepared with 10 alone (compare entries 11−14 with entry 8). The optimal stoichiometry of FMs was 1:1 for azacrown/ amine and 1.5/1 for thiouronium/carboxylate. Reducing the amount of either FM weakened the binding of MINP (entries 14−16). Increasing the FMs beyond the above ratios gave no improvement in binding (data not shown). Table S1 summarizes a similar study for KWW, a peptide with two amino groups and one carboxylate. Very similar trends were observed, that is, 4 and 8 were self-destructive, and 7 and 10 were the “golden pair”. The binding data could not differentiate 6 and 7 within our experimental error, but 6 was unstable during storage and was not used in further studies. Binding Selectivity of Functionalized MINPs. Although the FMs were beneficial to the binding affinity, their true power was shown in the selectivity of MINPs. Table 2 shows the binding of three closely related peptides, KDW, KKW, and KSW (Chart 1). Without any FMs, MINP prepared with KDW as the template bound the template with Ka = 1.35 × 105 M−1 in 25 mM HEPES buffer (entry 1). The more than one-order-

a Differences of structures are highlighted in red for peptides used to study cross-reactivity.

Table 1 shows the binding of WWD by various MINPs prepared with the same tripeptide template but different FMs. The template has a single amino group at the N-terminus. Entry 1 shows that, without FM, the binding by the parent MINP displayed an association constant of Ka = 23.2 × 105 M−1 in 25 mM HEPES buffer (pH 7.0), corresponding to a Table 1. Effects of FMs on the Binding of WWD by MINP(WWD) Determined By Fluorescence Titrationsa entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

FM1 1 1 1 1

equiv equiv equiv equiv

FM2

Ka (×105 M−1)

Krel

−ΔG (kcal/mol)

equiv equiv equiv equiv equiv equiv equiv equiv equiv equiv equiv

23.2 ± 1.4 28.7 ± 2.8 33.2 ± 1.5 41.3 ± 1.8 42.2 ± 0.6 74.7 ± 5.5 61.3 ± 0.8 80.9± 0.1 53.7± 3.1 71.1± 5.1 89.1± 2.7 92.8 ± 6.8 99.1 ± 9.3 102.5 ± 8.5 105.2 ± 9.1 80.8 ± 10.1

1.00 1.24 1.43 1.78 1.82 3.22 2.64 3.49 2.31 3.06 3.84 4.00 4.27 4.42 4.53 3.48

8.68 8.81 8.90 9.03 9.04 9.38 9.26 9.42 9.18 9.35 9.48 9.51 9.54 9.56 9.58 9.42

4 5 6 7

1 equiv 4 1 equiv 4 1 equiv 4 1 equiv 5 1 equiv 6 1 equiv 7 1.5 equiv 7 1 equiv 7

3 3 3 3 3 3 3 3 3 3 2

8 9 10 8 9 10 10 10 10 10 10

a The titrations were performed in HEPES buffer (25 mM, pH 7.0) at 298 K. Krel is the binding constant of a MINP prepared with a FM relative to that prepared without.

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should then cause a decrease in Ka instead of an increase. An earlier study of ours indicates that ionic carboxylates and hydrophobic side chains of a peptide prefer different locations of a micellesurface versus hydrophobic coreand often interfere with each other during imprinting.33 Gratifyingly, inclusion of 7 and 10 corrected the failure completely. MINP(KDW) prepared with the golden pair showed excellent selectivity, with the same D → K and D → S replacements bringing about >1 kcal/mol change in binding energy (entries 4−6). The binding constants weakened by 8− 11 times. Similar results were obtained when KKW was used as the template (entries 7−12). Interestingly, when KSW was used as the template, MINP(KSW) showed good selectivity without any FMs (Table 2, entries 13−15). If anything, inclusion of 7 and 10 decreased the selectivity for peptide KDW slightly, from a CRR of 0.24 to 0.40 (compare entries 15 and 17). Nonetheless, it should be pointed out that the golden pair always strengthened the binding of MINPs for its own template. If we examine the data closely, 7 and 10 strengthened the binding of templates while weakening the binding of nontemplates for both KDW and KKW (entries 1−12). These are highly desirable features for a receptor and precisely what we tried to achieve with an ideal combination of FMs. WDW has two tryptophans and one aspartic acid. WDWG and GWDW added a glycine at the C-terminus and the Nterminus, respectively (Chart 1). The three peptides thus all share the same WDW sequence except that the carboxylate is moved two atoms away to the right in WDWG and the amino group two atoms away to the left in GWDW. Binding selectivity of MINPs in these cases would be a good test for the importance of FMs in particular. Table 3 summarizes the selectivity results. In the absence of FMs, MINP(WDW) was able to distinguish the shifts of the carboxylate and amino group but with difficulty. The CRR value for WDWG and GWDW, for example, was 0.46 and 0.66, respectively. When the MINP was prepared with the golden pair 7 and 10, the glycine addition weakened the binding much more, with a CRR of 0.15 for WDWG and 0.22 for GWDW. These changes translate to a change of 0.90−1.12 kcal/mol in binding energy and are very significant for an addition of a single amino acid without any side chain. Not only did the FM pair enhance the selectivity, the binding constant for the templating peptide also increased from 15.7 to 96.6 × 105 M−1, equivalent to a strengthening of binding by 1.08 kcal/mol (entries 1 and 4). One particular challenge in peptide recognition is the differentiation of lysine and arginine. Because their cationic side chains can bind negative groups on cell membranes,

Table 2. Binding Selectivity of MINPs for KXW (X = D, K, and S)a entry

T

FM

guest

Ka (×105 M−1)

CRR

ΔΔG (kcal/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

KDW KDW KDW KDW KDW KDW KKW KKW KKW KKW KKW KKW KSW KSW KSW KSW KSW KSW

none none none 7 & 10 7 & 10 7 & 10 none none none 7 & 10 7 & 10 7 & 10 none none none 7 & 10 7 & 10 7 & 10

KDW KKW KSW KDW KKW KSW KKW KDW KSW KKW KDW KSW KSW KDW KKW KSW KDW KKW

1.35 0.99 2.90 9.60 0.85 1.20 1.86 0.98 2.83 6.70 1.23 2.00 4.60 1.09 1.29 10.4 4.15 2.78

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1 0.73 2.15 1 0.09 0.13 1 0.53 1.52 1 0.18 0.30 1 0.24 0.28 1 0.40 0.27

0.00 0.18 −0.45 0.00 1.44 1.23 0.00 0.38 −0.25 0.00 1.00 0.72 0.00 0.85 0.75 0.00 0.54 0.78

0.04 0.06 0.25 0.72 0.04 0.14 0.05 0.02 0.06 0.25 0.01 0.11 0.20 0.01 0.02 1.2 0.12 0.16

a The titrations were performed in HEPES buffer (25 mM, pH 7.0) at 298 K. For MINPs prepared with FMs, the following stoichiometry was used in the formulation: 1:1 for 7/amine and 1.5:1 for 10/ carboxylate. CRR is the cross-reactivity ratio, defined as the binding constant of a guest relative to that of the templating peptide for a particular MINP. ΔΔG = ΔG(guest) − ΔG(template).

of-magnitude weaker binding constant compared to that obtained for KWW (Table S1, entry 1) highlights the hydrophobic contribution of one tryptophan. Note that, in the absence of FMs, MINP(KDW) showed very poor selectivity. Replacement of the middle aspartic acid (D) with lysine (K) only weakened the binding slightly, showing a CRR (cross-reactivity ratio) of 0.73. In addition to the CRR values, we also included a column for ΔΔG, defined as the difference in binding energy between the template and a guest by the same MINP. According to the equation ΔΔG = ΔG(guest) − ΔG(template), a more selective MINP receptor will give a larger positive ΔΔG for its guests. Table 2 shows that ΔΔG was only 0.18 kcal/mol for KKW and even −0.45 kcal/mol for KSW (entries 2−3). In other words, MINP(KDW) bound KSW even more strongly than its own template, a classic case of imprinting failure. It is not entirely clear to us what caused the failure. One would expect the anionic carboxylate of aspartic acid to be imprinted well by the cationic cross-linked micelle of 1 through a salt bridge. Replacing the carboxylate with a hydroxyl in KSW Table 3. Effects of Glycine Insertion on the MINP Bindinga entry

Template

FM

guest

Ka (×105 M−1)

CRR

ΔΔG (kcal/mol)

1 2 3 4 5 6

WDW WDW WDW WDW WDW WDW

none none none 7 & 10 7 & 10 7 & 10

WDW WDWG GWDW WDW WDWG GWDW

15.7 ± 1.4 7.2 ± 0.6 10.4 ± 0.7 96.6 ± 8.6 14.7 ± 0.6 21.1± 2.2

1 0.46 0.66 1 0.15 0.22

0.00 0.46 0.24 0.00 1.12 0.90

a The titrations were performed in HEPES buffer (25 mM, pH 7.0) at 298 K. For MINPs prepared with FMs, the following stoichiometry was used in the formulation: 1:1 for 7/amine and 1.5:1 for 10/carboxylate. CRR is the cross-reactivity ratio, defined as the binding constant of a guest relative to that of the templating peptide for a particular MINP. ΔΔG = ΔG(guest) − ΔG(template).

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Chemistry of Materials Table 4. Differentiation of Lysine and Arginine by MINPa entry

T

FM

guest

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

KWW KWW KWW KWW KWW KWW KWW KWW RWW RWW RWW RWW RWW RWW RWW RWW RWW RWW

none none 4 4 4 & 10 4 & 10 7 & 10 7 & 10 none none 4 4 4 & 10 4 & 10 7 & 10 7 & 10 4 & 7 & 10 4 & 7 & 10

KWW RWW KWW RWW KWW RWW KWW RWW RWW KWW RWW KWW RWW KWW RWW KWW RWW KWW

Ka (×105 M−1)

CRR

ΔΔG (kcal/mol)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.00 0.51 1.00 0.93 1.00 0.65 1.00 0.20 1.00 1.11 1.00 0.69 1.00 0.32 1.00 0.97 1.00 0.63

0.00 0.40 0.00 0.04 0.00 0.26 0.00 0.95 0.00 −0.06 0.00 0.22 0.00 0.68 0.00 0.02 0.00 0.28

20.7 10.5 36.7 34.1 58.1 37.5 77.4 15.5 14.7 16.3 43.1 29.6 62.9 20.1 41.5 40.3 57.5 36.1

1.7 0.5 2.4 2.1 1.0 2.4 3.8 0.9 1.0 1.2 3.8 2.1 0.6 0.3 3.3 1.5 1.5 0.1

a The titrations were performed in HEPES buffer (25 mM, pH 7.0) at 298 K. For MINPs prepared with FMs, the following stoichiometry was used in the formulation: 1:1 for 7/amine, 1:1 for 4/arginine, and 1.5:1 for 10/carboxylate. CRR is the cross-reactivity ratio, defined as the binding constant of a guest relative to that of the templating peptide for a particular MINP. ΔΔG = ΔG(guest) − ΔG(template).

Table 5. Binding of β-Amyloid Peptides by MINPs Determined by ITCa entry 1 2 3 4 5 6 7 8

Template β-amyloid (1−11) (DAEFRHDSGYE) β-amyloid (11−22) (EVHHQKLVFFAE) β-amyloid (18−28) (VFFAEDVGSNK) β-amyloid (25−35) (GSNKGAIIGLM)

FM/template

Ka (×105 M−1)

Krel

none 4 & 7 & 10 none 7 & 10 none 7 & 10 none 7 & 10

26.0 92.2 155 322 252 415 134 190

± ± ± ± ± ± ± ±

1.00 3.55 1.00 2.08 1.00 1.65 1.00 1.42

1.6 9.8 32 45 46 60 16 31

−ΔG (kcal/mol)

N 1.31 1.01 0.84 0.83 0.80 0.89 0.84 0.85

± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

8.6 9.5 9.9 10.3 10.0 10.4 9.6 10.0

ΔH (kcal/mol) −60.2 −94.5 −108.3 −113.2 −66.9 −109.1 −86.2 −115.5

± ± ± ± ± ± ± ±

0.5 1.1 2.1 1.1 1.0 1.1 1.0 1.6

TΔS (kcal/mol) −51.6 −85.0 −98.4 −102.9 −56.9 −98.7 −76.6 −105.5

a

The titrations were performed in HEPES buffer (25 mM, pH 7.0) at 298 K. For MINPs prepared with FMs, the following stoichiometry was used in the formulation: 1:1 for 7/amine, 1:1 for 4/arginine, and 1.5:1 for 10/carboxylate. Krel is the binding constant of a MINP prepared with a FM relative to that prepared without.

lysine- and arginine-rich peptides often have antimicrobial44,45 and cell-penetrating properties.46−50 To distinguish lysine and arginine, we prepared MINPs for KWW and RWW with different FMs. The difference between the two peptides is subtle considering the very similar cationic side chain and the two large, identical tryptophans present (Chart 1). Without any FMs, we achieved a 2:1 selectivity for KWW and RWWnot a trivial task given the similarity of the two guests (Table 4, entries 1 and 2). FM 4, although enhanced the binding for KWW, lowered the selectivity (entries 3 and 4). Adding FM 10 strengthened the binding further but did not give any significant improvement in the selectivity (entries 5 and 6). The golden pair 7 and 10, once again, demonstrated their unified power. Together, they strengthened the binding of KWW from Ka = 20.7 to 77.4 × 105 M−1, equivalent to an increase of binding energy of 0.78 kcal/mol (Table 4, entries 1 and 7). Meanwhile, the increase of binding for RWW was much smaller, resulting in an increase of binding selectivity (CRR = 0.20 and ΔΔG = 0.95 kcal/mol (entry 8). When RWW was used as the template, MINPs without FMs showed a reversed selectivity for KWW, another imprinting failure (entry 10). FM 4 strengthened the overall binding and

improved the selectivity somewhat (entries 11 and 12). Notably, 7 and 10 no longer was the best pair for the arginine-containing peptide, while another couple, 4 and 10, shined in both binding affinity and selectivity (entries 13−16). With an open structure and abundance of hydrogen-bond acceptors, FM 4 apparently had a strong preference for the guanidinium side chain, whether alone or in combination with 10. Because RWW contains both a guanidinium and an amino group in the structure, we also tried all three FMs in combination4 for the guanidinium group, 7 for the ammonium group, and 10 for the carboxylate. The binding data suggest that we have reached the point of diminishing returns with the trio, at least in this case (compare entries 17− 18 and 13−14). Imprinting and Binding of Biological Peptides. Unlike receptors prepared from step-by-step total synthesis, MINPs for larger biological peptides were made as easily as those for small peptides.51 Table 5 shows the binding properties of MINPs for four β-amyloid peptides. We chose these peptides because the full-length peptide was cleaved from the amyloid precursor protein and contains approximately 40−42 aminoacid residues.52 Amino acids 25−35, in particular, contribute 4893

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Chemistry of Materials greatly to the aggregation of the peptide,53 which forms fibril structures that eventually turns into the plaque found in the brains of Alzheimer’s patients. Synthetic materials that can bind and remove these peptides in a competitive aqueous environment thus have important biological implications.

Table 5 shows that all bindings including those by MINPs prepared without FMs were enthalpically driven, with unfavorable entropic changes. Classical hydrophobic interactions are considered entropically driven, at least at low temperatures.54 In the past, we have obtained enthalpically driven binding even multiple hydrophobic groups were present in the guest.55 This is most likely due to the complexity of the overall binding mechanism. For example, thermodynamic details of hydrophobic interactions can be different depending on the (aliphatic/aromatic) nature of the guests and the size/ shape of the hydrophobic surfaces.56−62 When electrostatic interactions are involved, as in the current peptide bindings, they can be either enthalpically or entropically favored.63−67

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CONCLUSIONS We now indeed have a general method to recognize peptides of different sizes, hydrophobic and hydrophilic alike in their natural solvent (water). MINPs bridge difficult-to-synthesize molecular receptors with less well-defined MIPs by having a nanodimension, water solubility, and a tunable number of binding sites. They are prepared in a one-pot reaction in less than two days without any special technique once the crosslinkable surfactant, FMs, and cross-linkers are available. Purification requires nothing other than precipitation (into acetone) and washing (with organic solvents). The same method can be applied to small tripeptides as well as biological peptides with over ten amino acid residues. Micellar imprinting tends to have extraordinary imprinting effects. Even for very hydrophobic peptides such as WKW that are expected to have strong nonspecific hydrophobic interactions, the imprinting factor was very high, ∼40 for the parent MINP and >100 for MINP prepared with 2 equiv FM 4 (Figure S67). This work shows that, with appropriate FMs, MINPs can have excellent binding affinity and selectivity for peptides, making them potentially useful for inhibition of important biological interactions. Our study yields several important learnings (1) Aromatic side chains such as tryptophan and phenylalanine provide a significant hydrophobic driving force for MINP binding. As shown in Table 3, a single tryptophan in tripeptides (together with the less welldefined background interactions) afforded micromolar binding affinity without any FMs. (2) Whereas hydrophilic peptides with nonionic side chains can be imprinted well without any FMs, appropriate FMs are needed if the target peptides are rich in ionic groups. The golden pair 7 and 10 generally is able to boost both the binding affinity and selectivity of the MINP, even for peptides with substantial hydrophobicity. (3) FM 10, due to its reduced number of NH bonds and alkyl substituents, can be used with 4 or 7 to target both acidic and basic groups simultaneously. FM 4 is particularly suitable for the guanidinium of arginine. FM 7 is selective for amino (ammonium) groups at the N-terminus and on lysine. FM 10 targets the carboxylic acids on the C-terminus, aspartic acid (D), and glutamic acid (E). The optimal ratio for the MINP preparation is 4/R = 1:1, 7/amino = 1:1, and 10/carboxylate = 1.5:1. (4) Choice of FMs ultimately depends on the nature of the peptide to be recognized and also on the demand of application for binding affinity and selectivity. The

The binding data (Table 5), obtained from ITC, showed that the FMs helped the binding in all cases. Among the four peptides, β-amyloid (1−11) is the most hydrophilic, containing four acidic amino acids and an arginine. The only notable hydrophobic group is phenylalanine. Without any FMs, its MINP gave an impressive binding constant of Ka = 26.0 × 105 M−1 or a submicromolar binding affinity in 25 mM HEPES buffer (pH 7.0). MINPs contain hydrogen-bonding groups including triazole, hydroxyl, and ester. Although these “background” interactions cannot be defined as precisely as the complementary hydrophobic pockets or specific FM−template interactions, they are expected to be optimized by imprinting for the peptide backbone and side chains. For the β-amyloid peptide (1−11) with 11 amino acid residues, these background interactions were quite strong. Inclusion of 4, 7, and 10 increased the binding by 3.55 times (ΔΔG = 0.9 kcal/mol), affording a dissociation constant of Kd = 108 nM. As the peptides became more hydrophobic and carried fewer acidic/basic groups, the FMs were no longer necessary for high binding affinity. For β-amyloid peptides (11−22), (18−28), and (25−35), Kd ranged from 40 to 74 nM for MINPs prepared without FMs, and 24−53 nM with the appropriate FMs. Thus, as long as selectivity is not a concern, the parent MINPs are sufficient for hydrophobic peptides. 4894

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Chemistry of Materials

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benefit of molecular imprinting is that both the peptide backbone and the side chains will be imprinted simultaneously and, as our data has shown, the background interactions can be substantial for a long peptide. For simplicity, a hydrophobic peptide or a largely polar nonionic peptide can be recognized by MINPs without any special FMs. Combination of FMs works best when closely related hydrophilic peptides need to be distinguished or when additional enhancement in binding affinity is needed.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.9b01613.

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Experimental details, fluorescence and ITC titration curves, and additional data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yan Zhao: 0000-0003-1215-2565 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Institute of General Medical Sciences of the National Institutes of Health (R01GM113883) for financial support of the research.

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REFERENCES

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