Minimization of Synthetic Polymer Ligands for Specific Recognition and Neutralization of a Toxic Peptide

Article abstract of DOI:10.1021/jacs.5b05259

Synthetic polymer ligands (PLs) that recognize and neutralize specific biomacromolecules have attracted attention as stable substitutes for ligands such as antibodies and aptamers. PLs have been reported to strongly interact with target proteins and can be prepared by optimizing the combination and relative proportion of functional groups, by molecular imprinting polymerization, and/or by affinity purification. However, little has been reported about a strategy to prepare PLs capable of specifically recognizing a peptide from a group of targets with similar molecular weight and amino acid composition. In this study, we show that such PLs can be prepared by minimization of molecular weight and density of functional units. The resulting PLs recognize the target toxin exclusively and with 100-fold stronger affinity from a mixture of similar toxins. The target toxin is neutralized as a result. We believe that the minimization approach will become a valuable tool to prepare "plastic aptamers" with strong affinity for specific target peptides.

Full text of DOI:10.1021/jacs.5b05259

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Communication
Minimization of synthetic polymer ligands for specific
recognition and neutralization of a toxic peptide
Haejoo Lee, Yu Hoshino, Yusuke Wada, Yuka Arata, Atsushi Maruyama, and Yoshiko Miura
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 17 Aug 2015
Downloaded from http://pubs.acs.org on August 17, 2015
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Minimization of synthetic polymer ligands for specific recog-
nition and neutralization of a toxic peptide
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Haejoo Lee†, Yu Hoshino†*, Yusuke Wada†, Yuka Arata†, Atsushi Maruyama‡, Yoshiko Miura†
†Department of Chemical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
‡Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technolo-
gy, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan
Supporting Information Placeholder
ymerization of simple functional monomers, such as hydrophobic
N-tert-butylacrylamide (TBAm), negatively charged acrylic acid
(AAc), and aromatic n-phenyl acrylamide.6,7 Affinity can be en-
hanced further by optimizing the volume density of functional
groups7, molecular imprinting8, affinity purification9, and tuning
the flexibility and density of polymer chains10.
However, a strategy has not been described to prepare PLs capa-
ble of recognizing a specific peptide from a pool of targets with
similar molecular weight and amino acid composition. In this
study, we demonstrate one such approach, in which the molecular
weight of PLs and the relative proportion (density) of functional
units are minimized. Thus, we were able to generate multifunc-
tional PLs that specifically targeted and neutralized a peptide
toxin in a pool of similar peptides.
ABSTRACT: Synthetic polymer ligands (PLs) that recognize
and neutralize specific biomacromolecules have attracted atten-
tion as stable substitutes for ligands such as antibodies and ap-
tamers. PLs have been reported to strongly interact with target
proteins, and can be prepared by optimizing the combination and
relative proportion of functional groups, by molecular imprinting
polymerization, and/or by affinity purification. However, little has
been reported about a strategy to prepare PLs capable of specifi-
cally recognizing a peptide from a group of targets with similar
molecular weight and amino acid composition. In this study, we
show that such PLs can be prepared by minimization of molecular
weight and density of functional units. The resulting PLs recog-
nize the target toxin exclusively and with 100-fold stronger affini-
ty from a mixture of similar toxins. The target toxin is neutralized
as a result. We believe that the minimization approach will be-
come a valuable tool to prepare “plastic aptamers” with strong
affinity for specific target peptides.
Synthetic polymer ligands (PLs) that recognize and neutralize
target molecules have been evaluated as inexpensive and physico-
chemically stable substitutes for biomacromolecular ligands such
as antibodies and aptamers.1 Such PLs are prepared either by
modification of the polymer backbone with a number of small
ligands, and/or by copolymerizing a combination of a simple
functional monomers to accumulate a number of weak interac-
tions such as van der Waals, hydrophobic, electrostatic, hydrogen
bonding, and pi-pi stacking forces.
For instance, Schrader and colleagues used the first approach to
develop linear polyacrylamides functionalized with arginine re-
ceptors that target arginine-rich proteins.2 Following the same
strategy, Haddleton and colleagues prepared sequence-controlled
multi-block glycopolymers modified by carbohydrates to inhibit
the lectin DC-SIGN.3 Kiessling and her group also investigated
the effect of carbohydrate architecture in the ligand on the func-
tion and clustering of the lectin Con A.4
Using the second approach, Haag and coworkers developed a
dendritic polyglycerol modified with sulfate groups to target se-
lectin, even in vivo, by multipoint electrostatic interactions.5 Shea
and colleagues demonstrated that 3D nanoparticles (NPs) based
on p-N-isopropylacrylamide targeted a specific peptide and pro-
tein through a combination of electrostatic, hydrophobic and pi-
stacking interactions.6,7 These NPs are fabricated through copol-
Figure 1. (A) Amino acid sequence of melittin, magainin 1 and ponericin.
(B) Preparation of multifunctional PLs via RAFT living-radical polymeri-
zation.
Figure 1A shows the primary structure of melittin, the peptide of
interest, and of control peptides magainin 1 and ponericin. Melit-
tin is an α-helical hemolytic toxin in bee venom, and is well stud-
ied as a model target molecule for synthetic PLs. Melittin, with
MW 2846 Da, contains 50 % hydrophobic residues and six posi-
tive charges.11 Magainin 112 and ponericin13 were selected as con-
trol peptides, because they are also cell-lytic toxins, and have
characteristics similar to melittin in terms of molecular weight
(2409 Da and 2708 Da respectively), hydrophobicity (43% and
52% respectively), and number of positively charged amino acids
(six positive charges each).
We hypothesized that the specificity of PLs can be improved by
minimization of molecular weight. Thus, PL libraries containing
300-mer, 30-mer, and 15-mer monomers were synthesized by
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heat-initiated reversible addition-fragmentation chain-transfer
motif KRKR instead of KKKK, as in ponericin: Presumably, the
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(RAFT) polymerization, using benzylsulfanylthiocarbonylsulfanyl
propionic acid (BPA) as chain transfer agent and N-
isopropylacrylamide (NIPAm) as main monomer (Figure 1B).
Details of polymerization reactions and associated data are de-
scribed in S1.
two guanidium ions on the KRKR sequence enabled selective
affinity to melittin due to strong electrostatic interaction between
guanidium cation and carboxylate anion supported by two parallel
hydrogen bonds.2,15 In addition, melittin contains 5 or more hy-
drophobic amino acids right next to the KRKR sequence that
enable multipoint hydrophobic interactions simultaneously to the
electrostatic and hydrogen bonding interactions.6
To confirm the interaction between peptide and PLs, we used
isothermal titration calorimetry (ITC). A solution of 0.5 mM pep-
tide in PBS was titrated into 0.38 mg/mL synthetic PL. Titration
of magainin 1 into synthetic PLs and NP did not generate detecta-
ble changes in heat (S3), suggesting little interaction between the
molecules. In contrast, endothermic titration curves were observed
when NPs and 300-mer PLs were titrated with melittin and pon-
ericin. This result suggest that there is interaction between the
peptides and PLs which is entropically-driven presumably by
dissociation of water and/or counter ions from hydrophobic and/or
ionic functional groups on the peptides and PLs (S4). However,
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Figure 2. (A) Amount of peptides bound by solutions containing 1.9
mg/mL NP, 300-mer PL, and 30-mer PL from of a mixture of 0.1 mM
each of magainin 1, ponericin, and melittin in PBS. Each synthetic poly-
mer contained 20 mol% TBAm and 10 mol% AAc. Captured peptides
were analyzed by HPLC. (B) Apparent binding constant (Ka) between
peptides and synthetic PLs, as measured by ITC titration. Each 0.5 mM
peptide was titrated at 37 °C into 0.38 mg/mL synthetic polymers in PBS.
only melittin showed endothermic signal when 30-mer PLs was
titrated by each peptide (S3, S4), indicating that 30-mer PLs inter-
acted only to melittin as suggested by the competition filtration
assay.
Table 1. Hemolysis neutralization (%) by (A) 30-mer and (B) 15-mer PLs
containing with various ratios of functional units. PLs with negligible (<
10 %), moderate (20-80%), and almost complete (> 95%) neutralization
are highlighted in gray, yellow, and green respectively.
Because the target peptide includes hydrophobic and cationic
amino acids, PL libraries were prepared by controlling the relative
proportion (density) of functional monomers, such as hydrophobic
TBAm and negatively charged AAc, that could interact with the
peptide. The average number of functional units incorporated into
PLs was quantified by 1H NMR. We found that the relative pro-
portion of functional units incorporated into each PL was compa-
rable to the ratio of functional monomers in the feed (± 5%). The
polydispersity index of each PL was determined from GPC to be
1.1-1.4. For comparison, synthetic NPs were prepared as de-
scribed7 using the same density of functional units.
To investigate the effect of molecular weight on target specifici-
ty, 1.9 mg/mL NP, 300-, and 30-mer PLs, each containing 20
mol% TBAm and 10 mol% AAc, were incubated at 37 °C in PBS
(35 mM phosphate buffer pH 7.3, 150 mM NaCl) with a mixture
consisting of 0.1 mM each of magainin 1, ponericin and melittin.
It was confirmed that all PLs were soluble in the buffer (S2). The
amount of unbound peptide was quantified by HPLC after filtrat-
ing the PLs by a centrifugal filters (Milipore Co., Amicon Ultra-
0.5, 8,000 G, 37 °C, 30 min, NMWL; 10 kDa) (S2).
Figure 2A summarizes the amount of peptides bound by syn-
thetic PLs. Raw HPLC traces are collected in S2. Synthetic PLs
bound similar amounts of melittin regardless of molecular weight
because of multiple hydrophobic and electrostatic interactions. As
expected, NP and 300-mer PL complexed magainin 1 and pon-
ericin as well, because of the same interaction forces. However,
30-mer PL captured only a small amount of control toxins, indi-
cating that this PL recognizes melittin specifically.
Linear polymers, as well as polymer chains in NPs, can map on-
to target proteins and peptides to form high-affinity complex-
es.10,14 It has been reported that PLs with larger molecular weight
shows stronger affinity to melittin than the smaller one, because
the larger PLs has a higher degree of freedom in its structure and
more easily map onto melittin to form high affinity binding
sites.14 Our results in this study indicate that large PLs interacted
with all peptides, presumably because of the cumulative effects of
multipoint electrostatic and hydrophobic interactions along the
length of the flexible polymers. On the other hand, 30-mer PL has
limited length and surface area with which to generate such inter-
actions, even though it should be conformationally flexible
enough to do so. Nevertheless, 30-mer PL binds melittin strongly
because of specific features in the peptide sequence, including the
To compare affinity of PLs to each peptide, apparent binding
constants (Ka) were obtained by fitting the titration results to the
Langmuir binding model (Figure 2B). Here, we approximated that
all PLs has uniform molecular weight and structure and all bind-
ing event occurred in single site binding mode, although the mo-
lecular weight and structure of each PLs cannot be homogeneous.9
All synthetic PLs bind melittin with high apparent binding con-
stant around 5 x 10-5 M-1. On the other hand, affinity to ponericin
depended strongly on molecular weight: While NP bound pon-
ericin (2 x 10-5 M-1) with comparable affinity as melittin (4 x 10-5
M-1), affinity got weaker as PLs got smaller. Indeed, 30-mer PL
did not show significant affinity to ponericin. These data also
confirm the specific affinity of 30-mer PL to melittin.
The ability of PLs to neutralize melittin toxicity was investigated
by hemolysis neutralization assay (S5)7,14. A mixture of 1.8 ꢀM
melittin and red blood cells in PBS was incubated at 37 °C with
300 mg/mL 30- and 15-mer PLs. The amount of hemoglobin re-
leased from red blood cells was then measured after cells were
pelleted by centrifugation. Hemolysis neutralization activity in %
was calculated according to S6. Table 1 lists hemolysis neutrali-
zation by 30-mer and 15-mer PL. Longer PLs containing at least
20 % TBAm and 5 % AAc showed significant neutralization ac-
tivity. Those that contain at least 20 % TBAm and 10 % AAc
neutralized > 97 % of melittin toxicity. Interestingly, 15-mer PL
with the same density of functional units did not completely in-
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hibit melittin (58 %). However, 15-mer PL with 40 % TBAm and
Based on all data, we conclude that molecular weight must be
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20 % AAc achieved almost complete neutralization (100%). Note
that this PL has the same number (six TBAm and three AAc), but
twice the density, of TBAm and AAc as the neutralizing 30-mer
with 20% TBAm and 10 % AAc.
minimized to achieve target specificity in multifunctional PLs. In
addition, the density of functional units must also be minimized to
prevent nonspecific interactions. However, as observed for melit-
tin-binding PLs, a PL of minimal size must also contain a mini-
mum number of functional units.
These results demonstrate for the first time the ability to recog-
nize a specific target from a pool of similar peptides. We antici-
pate that this strategy of minimization will become a valuable
tool, besides molecular imprinting and affinity purification, to
generate inexpensive and physicochemically stable substitutes for
biomacromolecular ligands like RNA, DNA, and peptide ap-
tamers.
Based on this result, we conclude that there is a minimum num-
ber (not density) of functional units required to capture and neu-
tralize melittin: Multi-point electrostatic interaction between at
least three carboxylate anions on a polymer side chain and cations
on melittin supported by several hydrogen bonds to guanidium
groups and strong hydrophobic interaction given by at least six
tert-butyl groups on a PLs are both required to capture melittin.
This phenomenon is characteristic of low-molecular weight PLs.
In 300- and 1000-mer PLs14, as well as NPs7, the density (not
number) of incorporated functional units determine the affinity to
the target because all of those large PLs has a number of function-
al units which is far greater than that of melittin, thus PLs with
lower density can still form the multipoint interactions by map-
ping onto the sequence of melittin.14 However, for the small PLs,
such as 30-mer PLs, if density of AAc is lower than 10% and/or
density of TBAm is lower than 20%, the PLs cannot form such
multipoint binding structure because number of AAc and/or
TBAm on a polymer side chain is less than three and/or six re-
spectively.
To further characterize the influence of functional units on target
specificity, the binding properties of 30- and 15-mer PLs, which
showed almost complete melittin neutralization (neutralization >
97 %, green in table 1), were determined by the competition filtra-
tion assay using a mixture of target and control peptides (S7).
Results indicate that all of the 30-mers, regardless of composition,
captured similar amounts of melittin (Figure 3) as suggested by
the hemolysis neutralization assay. However, 30-mers consisting
of 20 % TBAm and 20 % AAc, and those containing 40 % TBAm
and 10 % AAc also captured magainin 1 and ponericin to a signif-
icant extent. These results indicate that target specificity in 30-
mer PLs decreases with increasing density of functional units.
Although, the control peptides does not have guanidium cations to
form the stable hydrogen-bonded salt bridges with PLs, PLs con-
taining more than 10 % AAc or more than 20 % TBAm can cap-
ture control peptides thorough multipoint electrostatic or hydro-
phobic interaction respectively. Consistent with this observation,
15-mer PL consisting of 40 % TBAm and 20 % AAc also cap-
tured control peptides. Because the high density functional groups
on the small PLs enabled multipoint electrostatic and hydrophobic
interaction with the positively charged and hydrophobic domains
on the control peptides even without drastic conformation change
expected only for large PLs. Taken together, these results indicate
that PLs, regardless of molecular weight, lose target specificity if
the density of functional units is not minimized.
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures and supporting data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
yhoshino@chem-eng.kyushu-u.ac.jp
ACKNOWLEDGMENT
We acknowledge financial support from MEXT (25107726),
JSPS (23750193), JST (AS2321466 and AS231Z01490D),
Ogasawara Foundation, Kakihara Foundation, and Kao Founda-
tion for Arts and Sciences.
REFERENCES
1) a) Fasting, C.; Schalley, C. A.; Weber, M.; Seitz, O.; Hecht, S.;
Koksch B.; Dernedde, J.; Graf, C.; Knapp, E.; Haag, R.. Angew.
Chem. Int. Ed. 2012, 51, 10472-10498
2) a) Koch, S. J.; Renner, C.; Xie X.; Schrader, T. Angew. Chem. Int.
Ed. 2006, 45, 6352-6355
b) Renner, C.; Piehler, J.; Schrader, T. J. Am. Chem. Soc. 2006, 128,
620-628
3) Zhang, Q.; Collins, J.; Anastasaki, A.; Wallis, R.; Mitchell, D. A.;
Becer, R.; Haddleton, D. M. Angew. Chem. Int. Ed. 2013, 52, 4435-
4439
4) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiesslin,
L. L. J. Am. Chem. Soc. 2002, 124, 14922-14933
5) a) Dernedde, J.; Alexandra Rausch, A.; Weinhart, M.; Enders, S.;
Tauber, R.; Licha, K.; Schirner, M.; Zügel, U.; Bonin, A. V.; Haag,
R. PNAS. 2010, 107, 19679–19684
b) Weinhart, M.; Groger, D.; Enders, S.; Riese, S. B.; Dernedde, J.;
Kainthan, R. K.; Brooks, D. E.; Haag, R. Macromol. Biosci. 2011,
11, 1088–1098
c) Weinhart, M.; Groger, D.; Enders, S.; Dernedde, J.; Haag, R. Bi-
omacronolecules 2011, 12, 2502–2511
6) a) Yoshimatsu, K.; Yamazaki, T.; Hoshino, Y.; Rose, P. E.; Epstein,
L. F.; Miranda, L.; Tagari, P.; Beierle, J. M.; Yonamine, Y.; Shea, K.
J. J. Am. Chem. Soc., 2014, 136, 1194–1197
b) Weisman, A.;Chen,A. C.; Hoshino, Y.; Zhang, H.; Shea, K. Biom-
acromolecules, 2014, 15, 3290–3295
7) Hoshino, Y.; Koide, H.; Furuya, K.; Haberaecher, W. W.; Lee, S.;
Kodama, T.; Kanazawa, H.; Oku, N.; Shea, K. J. Proc. Natl Acad.
Sci. USA, 2012, 109, 33–38
8) Hoshino, Y.; Kodama, T.; Okahata, Y.; Shea, K. J. J. Am. Chem. Soc.,
2008, 130, 15242–15243
9) Hoshino, Y.; Haberaecher, W. W.; Kodama, T.; Zeng, Z.; Okahata,
Y.; Shea. K. J. J. Am. Chem. Soc., 2010, 132, 13648–13650
10) Hoshino, Y.; Nakamoto, M.; Miura, Y. J. Am. Chem. Soc., 2012,
134, 15209–15212
Figure 3. Amount of peptides bound by 0.38 mg/mL synthetic polymer
ligands from of a pool consisting of 0.1 mM each of magainin 1, ponericin,
and melittin in PBS. Complexed peptides were analyzed by HPLC.
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Chem. 2001, 276, 17823-17829
11) Terwilliger, T. C.; Eisenberg, D. J. Biol. Chem. 1982, 257, 6016-
6022
14) Wada, Y.; Lee, H.; Hoshino, Y.; Kotani, S.; Shea, K. J.; Miura, Y. J.
Mater. Chem. B 2015, 3, 1706-1711
1
2
3
4
5
6
7
8
12) Zasloff, M. Proc. Natl Acad. Sci. USA, 1987, 84, 5449-5453
13) Orivel, J.; Redeker, V.; Le Caer, J. P.; Krier, F.; Revol-Junelles, A.
M.; Longeon, A.; Chaffotte, A.; Dejean, A.; Rossier, J. J. Biol.
15) Schmidtchen, F. P.; Berger, M. Chem. Rev. 1997, 97, 1609−1646
9
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