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Exploiting Oligo(amido amine) Backbones for the Multivalent Presentation of Coiled-Coil Peptides Ulla I. M. Gerling-Driessen,† Nina Mujkic-Ninnemann,‡ Daniela Ponader,‡ Daniel Schöne,† Laura Hartmann,*,‡ and Beate Koksch*,† U. I. M. Gerling-Driessen, Dr., D. Schöne, and B. Koksch, Prof. Dr. †

Department of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany

D. Ponader, Dr., N. Mujkic-Ninnemann, and L. Hartmann, Prof. Dr. ‡

Max Planck Institute of Colloids and Interfaces, Research Campus Golm 14424 Potsdam, Germany S Supporting Information *

ABSTRACT: The investigation of coiled coil formation for one mono- and two divalent peptide−polymer conjugates is presented. Through the assembly of the full conjugates on solid support, monodisperse sequence-defined conjugates are obtained with defined positions and distances between the peptide side chains along the polymeric backbone. A heteromeric peptide design was chosen, where peptide K is attached to the polymer backbone, and coiled-coil formation is only expected through complexation with the complementary peptide E. Indeed, the monovalent peptide K-polymer conjugate displays rapid coiled-coil formation when mixed with the complementary peptide E sequence. The divalent systems show intramolecular homomeric coiled-coil formation on the polymer backbone despite the peptide design. Interestingly, this intramolecular assembly undergoes a conformational rearrangement by the addition of the complementary peptide E leading to the formation of heteromeric coiled coil−polymer aggregates. The polymer backbone acts as a template bringing the covalently bound peptide strands in close proximity to each other, increasing the local concentration and inducing the otherwise nonfavorable formation of intramolecular helical assemblies.

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INTRODUCTION

drug during transport and allow for release of the drug upon disassembly of the coiled coil, for example, at acidic pH.13,22 Recently, synthetic polymers presenting several coiled-coil motifs in the side chain along with an antibody fragment have been reported and applied to induce receptor cross-linking and thereby cell apoptosis.23 Overall, coiled-coil peptides have shown to allow for the supramolecular self-assembly of two or more (macro)molecules24 and thus have a great potential in building up complex, potentially functional structures similar to their natural analogues, the proteins. However, the influence of a polymeric backbone on the self-assembly of a coiled-coil sequence has only gained limited attention so far. On the one hand, it has been observed that polymer conjugation leads to an increase in thermal, chemical, and protease stability of the coiled-coil motif and that self-assembly seems to be undisturbed by the polymer, suggesting that straightforward combination of the two kinds of molecules (peptide and

In contrast to conventional polymers, the oligo(amido amine) backbone is synthesized via a building block approach using solid phase synthesis techniques yielding highly defined polymers with desired site-specific functionalization.1 The coiled coil is a protein folding motif that is often found in natural as well as novel synthetic peptides.2 Because of the wellunderstood relationship between the primary sequence of amino acids and the resulting three-dimensional structures as well as their controlled self-aggregation behavior, coiled-coil peptides have been widely applied ranging from fundamental studies on protein folding3−8 to their use in the biomaterial9−12 and biomedical field.13−15 Particular attention has been attracted by the combination of coiled-coil peptides and synthetic polymers.16 One example is the straightforward combination into a peptide-blockcopolymer, which can induce polymer gel formation upon self-aggregation of the coiled coil.17−20 Such noncovalent hydrogels are of particular interest in the areas of tissue culture and injectable cell scaffolds.21 Another approach uses the coiled-coil motif as linker between a hydrophilic polymer and a drug to obtain stabilization of the © 2015 American Chemical Society

Received: May 12, 2015 Revised: June 24, 2015 Published: June 26, 2015 2394

DOI: 10.1021/acs.biomac.5b00634 Biomacromolecules 2015, 16, 2394−2402

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Scheme 1. Schematic Presentation of Mono- and Divalent Coiled-Coil Peptide−Polymer Conjugates Studied and Their Assembly into Peptide−Polymer Complexes

within the previously assembled backbone; the first glycine residue was coupled to these secondary amine groups. Alloc cleavage: tetrakis(triphenylphosphine)palladium(0) (5.77 mg, 0.1 equiv) and N,N-dimethylbarbituric acid (39 mg, 5 equiv) were flushed under argon and dissolved in 4.5 mL of DCM under a gentle nitrogen stream for 4 min. The solution was transferred to the reaction vessel, which was shaken for 2 h. The whole procedure was repeated once before the resin was washed three times with DCM, three times with 0.2 M DIEA in DMF, and six times with DMF. As a linker between the backbone and the peptide K, two glycine residues were introduced. For coupling these two glycine residues (Gly2), a standard Fmoc peptide strategy was adopted. Fmoc-Gly-OH (119 mg, 8 equiv) was dissolved in DMF (1 mL) and mixed with O(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (150 mg, 7.9 equiv) in DMF (1 mL) and N,Ndiisopropylethylamine (DIPEA) (0.15 mL) under argon atmosphere. The mixture was transferred to the reaction vessel, which was shaken for 1 h; the resin was washed with DMF, and a second coupling was performed to ensure completion of the addition of the first glycine residue. Fmoc cleavage was performed by treatment with 25% piperidine in DMF for 10 min (three times) followed by washing the resin with DMF (10 times). The second glycine residue was also doubly coupled following the same procedure. EDS3ADS(Gly2)EDS3-7 (1). Reversed-phase high-performance liquid chromatography (RP-HPLC) (5 to 95% MeCN in H2O for 60 min) tR = 19.1 min. Electrospray ionization coupled with mass spectrometry (ESI-MS) for C89H146N18O31 [M + 2H]+ calcd. 981.5, found 981.8; [M + 3H]3+ calcd. 655.4, found 655.4; [M + Na]+ calcd. 985.0, found 985.1. EDS3ADS(Gly2)EDS3ADS(Gly2)-8 (2). RP-HPLC (5 to 95% MeCN in H2O for 60 min) tR = 24.1 min. ESI-MS for C116H175N23O37 [M + 3H]3+ calcd. 828.7, found 828.6; [M + 2H,1Na]3+ calcd. 836.0, found 836.2; [M + 3Na]3+ calcd. 850.7, found 850.5; [M + 4H]4+ calcd. 621.8, found 621.8. ADS(Gly2)EDS6ADS(Gly2)-8 (3). RP-HPLC (5 to 95% MeCN in H2O for 30 min) tR = 14.5 min. ESI-MS for C116H175N23O37 [M + 3H]3+ calcd. 828.7, found 828.7; [M + 2H,1Na]3+ calcd. 836.0, found 836.0; [M + 3Na]3+ calcd. 850.7, found 850.6; [M + 4H]4+ calcd. 621.8, found 621.7.

polymer) is possible.25,26 On the other hand, it is wellunderstood that the conjugation of a polymer to a peptide strongly influences its dynamic folding behavior through an increase in hydrodynamic radius and potential nonspecific interactions between the backbones of polymer and peptide, for example, through hydrophobic interactions. Here, we present a small set of highly defined polymer− coiled-coil peptide conjugates. Through the stepwise assembly of both the polymer backbone and the peptide, on a solid support, full control over the chemical structure is obtained, and thus effects like spacing between the peptide strands along the polymer backbone can be controlled and varied (Scheme 1).

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EXPERIMENTAL SECTION

Synthesis of Oligo(amido amine) Backbones. Solid Phase Polymer Synthesis. All solid-phase reactions were performed on a 0.05 mmol scale referring to the following general solid-phase protocols.1,27 Solid support Tentagel S RAM resin (loading 0.24 mmol/g) was used, which was swollen twice for 15 min in dichloromethane (DCM) before the initial Fmoc-deprotection was started. All building blocks were synthesized following previously established protocols.1,27 General Procedures. After the successful addition of the first building block, Fmoc deprotection was performed using 25% piperidine in dimethylformamide (DMF) for 10 min and checked by UV monitoring for the fluorenyl piperidine adduct at 301 nm. This step was repeated until the deprotection was complete. Subsequently, coupling of the second building block proceeded as described previously,1,27 followed again by Fmoc cleavage. Repetition of these two steps was performed until the full backbone was assembled on solid support. After cleavage of the Fmoc protecting group at the last building block of the backbone, the resulting terminal primary amine group was acetylated. For acetylation of the N-terminal site, 3 mL of Ac2O was added to the resin, and the reaction vessel was shaken for 5 min. Afterward, the resin was washed with DMF. The starting point for on-polymer peptide synthesis was Alloc deprotection, which was performed to release secondary amine groups 2395


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ESI-MS: [M + 5H]5+ calcd. 1359.5, found 1360.0; [M + 6H]6+ calcd. 1133.1, found 1133.5. Conjugate (6). RP-HPLC (5 to 70% ACN in H2O for 18 min) tR = 14.47 min. ESI-MS: [M + 5H]5+ calcd. 1359.5, found 1360.0; [M + 6H]6+ calcd. 1133.1, found 1133.5. Circular Dichroism (CD) Spectroscopy. Lyophilized peptide/ conjugate was dissolved in 10 mM phosphate buffer (pH 7.4), and the concentration was determined by UV spectroscopy at 320 nm (Abz) using a Varian Cary 50 spectrophotometer (Varian Medical Systems, Palo Alto, CA, USA) and poly(methyl methacrylate) (PMMA) cuvettes (10 mm path length, 1.5 mL, Plastibrand, VWR International GmbH, Darmstadt, Germany). Prior to analysis, a calibration curve with Abz-Gly-OH (Bachem GmbH, Weil am Rhein, Germany) in phosphate buffer (10 mM, pH 7.4) was recorded at different concentrations. Samples of 100 μM peptide concentrations were prepared for all peptides and polymer peptide conjugates. CD spectra were recorded with a Jasco J-810 spectropolarimeter at 20 °C. Quartz cells (1 mm path length) were used throughout. The spectra were the average of three scans obtained by collecting data from 240−190 nm at 0.5 nm intervals, 2 nm bandwidth, and 2 s response time. Spectra were background-corrected by subtracting the corresponding buffer spectra. The measured CD data in mdeg were converted into molar ellipticity per residue θM = (103 × mdeg cm2 dmol−1 residue−1). Thermal denaturation experiments of the divalent peptide−polymer conjugates (5) and (6) were performed in phosphate buffer (10 mM, pH 7.4) containing 3 M guanidine hydrochloride (GuHCl) on a Jasco J-810 spectropolarimeter equipped with a Jasco PTC-348WI Peltier thermostat. Melting curves were recorded using the signal at 222 nm applying a heating rate of 2 K min−1 from 5−100 °C. Each sample was prepared three times, and the melting curves were averaged. The helical content was calculated from the obtained CD spectra (Supporting Information). The relative percent helicity of peptides can be estimated by the mean residue ellipticity at 222 nm. We used the equation described by Baldwin28 and recently modified by Fairlie29 to calculate percent helicity for short peptides from the ratio [θ]222/ [θ]max, where [θ]max = (−44 000 + 250T)(1 − k/n)]. The [θ]max for 22-residue α-helices is calculated to be −31 909 for k = 4.0 and T = 20 °C. As discussed by Fairlie and Baldwin, the correct value of k (finite length correction) remains difficult to define for short peptides. Isothermal Titration Calorimetry (ITC). ITC measurements were performed for the heteromeric peptide−polymer complexes (7− 9) on a VP-ITC instrument (Microcal). All compounds were dissolved in 10 mM phosphate buffer, pH 7.4. Measurements were performed at 20 °C by titration of a 2- to 10-fold molar excess of compounds (4−6) to free peptide E. The concentration of peptide E was 50 μM in each case. The concentration of the monovalent peptide−polymer conjugate (4) was 500 μM. In case of the divalent peptide−polymer conjugates (5 and 6), the concentrations were varied from 100−250 μM. At least three repeats for each quantifiable binding titration were performed.

Synthesis of Peptide−Polymer Conjugates. The Glu-containing peptide (peptide E) of the heteromeric coiled coil, which was not coupled to the polymer, was synthesized according to standard Fmoc strategy on an Activo-P11 automated peptide synthesizer (Activotec, Cambridge, United Kingdom) using syringes with poly(tetrafluoroethylene) (PTFE)-frits. To enable photometric concentration determination, the peptides were equipped with anthranilic acid (Abz). A NovaSynTGA-resin (0.23 mmol/g, 0.1 mmol scale) was manually preloaded with the first amino acid Fmoc-Lys(Mtt)−OH. The Mtt protection group was removed under mild acidic conditions by treating the resin several times with a solution of 1% trifluoroacetic acid (TFA) in DCM. Subsequently, (Boc)-Abz was coupled to the side chain amino function of the first Lys residue using 10 eq 1-hydroxy-7azabenzotriazole (HOAt)/N,N′-diisopropylcarbodiimide (DIC) activation. After manual Fmoc deprotection on the first Lys, the remaining residues of the sequence were coupled using the synthesizer. Cterminal activation was carried out using 10 eq HOBt/DIC. Fmoc deprotection was achieved by treatment of the resin with 2% piperidine and 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (3 × 10 min). Peptides were cleaved from the resin by treatment with 2 mL of a solution containing triisopropylsilane (10%, w/v), water (1%, w/ v), and TFA (89%, w/v) purified by RP-HPLC using linear CH3CN/ H2O gradients containing 0.1% TFA and identified by ESI-time of flight (ToF) MS using an Agilent 6210 ESI-ToF LC−MS spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). RP-HPLC (5 to 70% ACN in H2O for 18 min) tR = 17.06 min. ESI-MS: [M + 3H]3+ calcd. 801.2, found 801.4; [M + 4H]4+ calcd. 601.1, found 601.3. Peptide K was synthesized on the particularly prepared polymer backbones (1−3), which were still attached to the solid support. For the monovalent peptide−polymer conjugate (4), the backbone (1), which contained an ADS building block at the center of the sequence, was used. Two glycine residues had been previously coupled as spacers to the ADS building blocks. After Fmoc deprotection using 2% piperidine and 2% DBU (3 × 10 min), the peptide sequence was grown using an Activo-P11 automated peptide synthesizer (Activotec, Cambridge, United Kingdom). C-terminal activation was carried out using 10 eq HOBt/DIC. Fmoc deprotection was achieved by treatment of the resin with 2% piperidine and 2% DBU (3 × 10 min). Abz was manually coupled to the N-terminus to enable photometric concentration determination of the conjugate. Conjugates were cleaved from the resin by treatment with 2 mL of a solution containing triisopropylsilane (10%, w/v), water (1%, w/v), and TFA (89%, w/v) purified by RP-HPLC using linear CH3CN/H2O gradients containing 0.1% TFA and identified by ESI-ToF MS using an Agilent 6210 ESI-ToF LC−MS spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). Conjugate (4). RP-HPLC (5 to 70% ACN in H2O for 18 min) tR = 14.17 min. ESI-MS: [M + 4H]4+ calcd. 1030.2, found 1030.4; [M + 5H]5+ calcd. 824.3, found 824.5. The two divalent peptide−polymer conjugates (5) and (6) were grown on the backbones (2) and (3), which also contained two glycine residues as spacers on the particular ADS building blocks. The peptides were simultaneously grown using 20 equiv of amino acid in an Activo-P11 automated peptide synthesizer (Activotec, Cambridge, United Kingdom). C-terminal activation was carried out using 20 equiv HOBt/DIC. Fmoc deprotection was achieved by treatment of the resin with 2% piperidine and 2% DBU (3 × 10 min). Abz was manually coupled to the N-termini to enable photometric concentration determination the conjugates. Conjugates were cleaved from the resin by treatment with 2 mL of a solution containing triisopropylsilane (10%, w/v), water (1%, w/v), and TFA (89%, w/ v) purified by RP-HPLC using linear CH3CN/H2O gradients containing 0.1% TFA and identified by ESI-ToF MS using an Agilent 6210 ESI-ToF LC−MS spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). Conjugate (5). RP-HPLC (5 to 70% ACN in H2O for 18 min) tR = 14.43 min.

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RESULTS AND DISCUSSION Synthesis of Oligo(amido amine) Scaffolds. The synthesis of the oligo(amido amine) backbone was performed following previously introduced solid phase polymer synthesis.1,27 Therefore, we applied the stepwise assembly of dimer building blocks introducing either a hydrophilic spacer unit (diethylenglycol-succinic acid dimer building block, EDS) or an Alloc protected secondary amine functionality (Alloc-ethylenetriamine-succinic acid dimer building block, ADS) following standard Fmoc coupling protocols. After assembly of the polymer backbone, the Alloc protecting group(s) was cleaved releasing a secondary amine group in the backbone as attachment point for the subsequent peptide synthesis. As a linker, we first coupled two glycine residues before the coiled coil peptide sequence was introduced. The nomenclature of the polymer−linker construct describes the sequence of building 2396


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Figure 1. (1a−d) Synthesis of polymer backbone on solid support followed by (2a,b) solid phase peptide synthesis on the polymer backbone and final cleavage of the polymer−peptide ligand. (1a) Stepwise coupling of three EDS building blocks is followed by (1b) introduction of a single ADS building block and (1c) again three EDS building blocks. Selective cleavage of the Alloc group (1d) releases a secondary amine used for the coupling of two glycine residues as linker for (2a) the consecutive synthesis of peptide sequence through solid phase peptide synthesis. (2b) The final product is isolated after cleavage from the resin.

core that represents the primary recognition motif of the coiled coil. Positions e and g carry the oppositely charged amino acids glutamic acid and lysine, which are responsible for secondary recognition through interhelical electrostatic interactions to further stabilize the coiled coil. Peptide K contains exclusively lysines in e and g positions, while peptide E contains only glutamic acids in these positions. The remaining positions b, c, and f are exposed to the solvent and occupied by alanine and lysine in both peptides. The oppositely charged residues in the e and g positions of peptide K and peptide E provide a coiled-

blocks with the side chain in brackets and the overall length of the polymer backbone without the side chains as a number at the end of the oligomer abbreviation, for example, EDS3ADS(Gly2)EDS3-7 (Figure 1). Design and Synthesis of the Peptide−Polymer Conjugates. The design of peptide E and peptide K is based on an α-helical coiled-coil folding motif (Figure 2A)30,31 and has been well described in literature before.32 Positions a are occupied by the hydrophobic amino acid isoleucine and positions d are filled with leucine, forming the hydrophobic 2397


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Figure 2. (A) Sequences and design of the parallel heteromeric coiled coil consisting of peptide E (blue) and peptide K (green).32,33 (B) CD spectra of single peptide components showing random-coil conformation (blue and green) and of the equimolar mixture of both components (black) showing the α-helical coiled-coil structure.

Figure 3. CD spectra of the peptide−polymer conjugates (4−6) and of the complexes (7−9) formed with peptide E.

Figure 4. CD spectra of the peptide−polymer conjugates (4−6) and of the complexes (7−9) formed with peptide E in presence and absence of 3 M GuHCl.

central and N-terminal position of the sequence, while backbone 3 contained the ADS-Gly2 building blocks in Cand N-terminal position. Thus, the two divalent systems (5 and 6) vary with regard to the backbone distance between the attached peptides stands. The complex formation was achieved by adding peptide E in solution to give the peptide−polymer complexes (7−9). An overview of the investigated peptide− polymer conjugates and the concept of complex formation with the complementary peptide E is visualized in Scheme 1. Aggregation Behavior of Peptide−Polymer Conjugates. The structure of the peptide−polymer conjugates was analyzed by CD spectroscopy. The monovalent peptide− polymer conjugate (4), containing peptide K linked to the polymer, showed a random-coil conformation (Figure 3A). After the addition of the complementary sequence (peptide E),

coil formation only in case of the heteromeric assembly of both peptides (Figure 2B), while each of the peptide variants is forming a random-coil structure in solution, which was proven by CD spectroscopy (Figure 2B). In case of the here studied peptide−polymer conjugates (4− 6) (Scheme 1), peptide K was attached to the particular polymer backbones (1−3) resulting in one monovalent system (4) and two divalent systems (5 and 6). Peptide K was stepwise grown on the polymer, which was connected to a solid support via the C-terminus, applying Fmoc-based solid phase peptide synthesis. For this purpose, the backbone contained ADS-building blocks containing two glycine residues as spacers at various positions in the polymer sequence. Backbone 1 contained ADS-Gly2 at a central position of the polymer sequence. Backbone 2 contained ADS-Gly2 building blocks in a 2398


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The fact that unfavorable homomeric coiled-coil formation occurs only in case of the divalent systems raised the question whether the polymer backbone performs a template effect that brings the peptide strands in close proximity to each other.38 The resulting high local concentration of the peptides on the polymer backbone might thus trigger the formation of homomeric assemblies. To confirm this hypothesis, we tested whether peptide K alone is able to adopt homomeric coiled coils at high concentrations. Peptide K was analyzed at various concentrations by CD spectroscopy (Figure 6A). The spectra revealed that peptide K forms random coils at concentrations smaller than 400 μM. At a peptide concentration of 800 μM, the formation of a helical structure was observed, which proved the ability of peptide K to form homomeric coiled coils. Further increase in the peptide concentration led to the formation of higher oligomeric helical structures (helical fibers), as indicated by a decreasing signal at 208 nm. In a control experiment, the structures of highly diluted samples of the divalent peptide−polymer conjugates (5 and 6) were investigated by CD spectroscopy (Figure 6B,C). At concentrations of only 5 μM, the divalent systems still showed helical structures, whereas peptide K shows random-coil structures up to a concentration of 100 μM. This result indicates that the homomeric coiled-coil formation results from a high local peptide concentration induced by the attachment to the polymer. The insensitivity of the helical signal to high dilution further indicates that the interaction in the formed assemblies is of intramolecular rather than of intermolecular nature. These results strengthen the assumption that the polymer-backbone performs a template effect in the divalent systems (5 and 6) that brings the peptide strands in close proximity and thereby creates conditions that allow for peptide K to adopt homomeric coiled-coil structures. The complex formation of the heteromeric assemblies (7−9) was further studied by ITC (Table 1). In all cases, the peptide− polymer conjugates (4−6) were titrated to a 50 μM solution of peptide E. As expected, for the monovalent heteromeric complex (7), we found a 1:1 interaction (N = 1) of peptide E to the peptide−polymer conjugate (4). In the case of the two divalent heteromeric complexes (8 and 9), interactions in a 1:2 ratio were observed (N = 0.5). The binding constant (K) of peptide E to the polymer-bound peptide K was found to be in the same range for all three systems. Thus, the multivalent presentation of the peptides does not significantly influence the binding affinity of the heteromeric coiled-coil assembly. Compared to the monovalent system, the two divalent systems show slight differences for the enthalpy and entropy values. This could be explained by coiled-coil formation occurring from two random-coiled peptides in case of the monovalent systems. In case of the divalent systems, a homomoeric coiled coil was already present, which disassembled in the presence of the complementary peptide sequence (peptide E) to form the heteromeric assemblies. The divalent systems, however, show only marginal differences in their thermodynamic parameters, indicating that the different backbone distances between the attached peptides (5 and 6) have no significant impact on the overall process of coiled-coil formation. This might be explained by the high flexibility of the polymer backbone, which presumably forms tightly packed coils in the aqueous environment and thus brings the covalently bound peptide strands in close proximity.

the formation of a helical coiled-coil structure was observed (Figure 3A). Interestingly, the two divalent peptide−polymer conjugates (5 and 6) showed helical spectra already in the absence of peptide E (Figure 3B,C), which can be explained by homomeric coiled-coil formation of peptide K on the polymer backbone. This behavior occurred for both divalent systems, although they differ in distance between the peptide strands. This observation was surprising because the formation of homomeric coiled coils is very unfavorable based on the peptide design.34−37 However, the addition of peptide E to the divalent peptide−polymer conjugates (5 and 6) resulted in helical structures for both systems (Figure 3B,C). To study whether the helical assemblies observed for the equimolar mixtures of the divalent systems (5 and 6) with peptide E were of homomeric or heteromeric nature, we treated the complexes with 3 M GuHCl (Figure 4). From the monovalent complex (7), we knew that the heteromeric coiled coil disassembles under these denaturating conditions (Figure 4A). The addition of 3 M GuHCl to the divalent complexes revealed a reduction of the helical content in both cases (Figure 4B,C), which proves that the assemblies were indeed the heteromeric complexes (8 and 9) of the divalent systems (5 and 6) with peptide E. The remaining helical content observed in the CD spectra resulted from the homomeric coiled coils of the two divalent systems (5 and 6) that are formed in situ upon the disassembly of the heteromeric species (8 and 9). The homomeric coiled coils formed by the dimeric peptide− polymer conjugates (5 and 6) were found to survive the addition of 3 M GuHCl as the CD spectra, which show helical structures of the same intensities in presence or absence of 3 M GuHCl (Figure 4B,C). In a further experiment, we tested whether both divalent peptide−polymer conjugates (5 and 6) differ in thermal stability. Melting curves for both divalent systems were recorded in the presence of 3 M GuHCl (Figure 5). The

Figure 5. Melting curves of the divalent peptide−polymer conjugates (5 and 6) in the presence of 3 M GuHCl.

divalent peptide−polymer conjugate (5) showed a higher melting point (54.4 °C) compared to divalent peptide− polymer conjugate (6), which has a melting point of 48.9 °C. Thus, it can be assumed that the shorter distance between the peptides on the polymer-backbone resulted in a stronger helical interaction in the divalent system (5). 2399


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Figure 6. CD spectra of peptide K and the divalent peptide−polymer conjugates (5 and 6) at different sample concentrations.

Table 1. Thermodynamic Data for the Multivalent Complexes (7−9) Extracted from ITC Measurements binding sites (N) K [M−1] ΔH [cal/mol] ΔS [cal/mol/deg]

monovalent system

divalent system 1

divalent system 2

1.000 ± 0.006 9.19 e6 ± 2.40 e6 −1.140 e4 ± 141.8 −7.02

0.464 ± 0.003 9.06 e6 ± 1.42 e6 −2.988 e4 ± 298.0 −70.1

0.525 ± 0.003 1.67 e7 ± 3.02 e6 −2.482 e4 ± 227.2 −51.6

Figure 8. Schematic representation of the investigated multivalent systems.

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CONCLUSION In the current study, three peptide−polymer conjugates were investigated regarding their assembly properties with the complementary peptide sequence that is added in solution. Three functionalized polymer-backbones were synthesized, and either one (monovalent) or two (divalent) peptide strands were covalently attached via an all-on-solid phase approach. Interaction with a complementary peptide sequence was studied. The divalent systems differ with regard to the distance between the peptides on the polymer. The polymer-bound peptide strands are one component of a heteromeric coiled-coil design that allows the formation of helical assemblies only for the combination of the two complementary peptide sequences. As expected, the monomeric system displays rapid coiled-coil formation when the two components are mixed. Surprisingly, the dimeric systems show homomeric coiled-coil formation, although such a folding is unfavorable based on the design of the heptad repeat sequence.34−37 The divalent system with the shorter distance between the peptides forms the stronger

homomeric assembly. However, the homomeric assemblies transform into heteromeric aggregates on addition of the complementary peptide with a 1:1 stoichiometry. The homomeric assembly was only found for the divalent conjugates (5 and 6) and thus can be explained by a template effect of the polymer,38 which brings the equally charged peptide strands in close proximity to each other and thus induces the formation of an otherwise unfavorable aggregation due to a high local peptide concentration. The behavior of the here-investigated systems can be summarized according to Figure 8. Ongoing studies extend on this concept by looking at higher-valent peptide−polymer conjugates as well as at the aggregation behavior of peptide−polymer conjugates that present both complementary strands on an oligomer backbone. Such conjugates are able to form highly regular network structures, which have potential application in biomaterials development. 2400


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ASSOCIATED CONTENT

S Supporting Information *

HPLC chromatograms of pure final compounds (4−6) and peptide E, ITC graphs of peptide−polymer conjugates (4−6) in the presence of peptide E. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00634.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Fax: +4930-838-55644. Present Address

L.H., Heinrich Heine University Düsseldorf, Institute for Organic Chemistry and Macromolecular Chemistry, Universitätsstr. 1, 40225 Düsseldorf, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We kindly thank the Collaborative Research Center 765 (SFB765/2-2014), the Deutsche Forschungsgemeinschaft (FG806-HA2686/3-2) for financial support. N.M.-N., D.P., and L.H. thank the Boehringer Ingelheim Foundation and Max Planck Society for their support.

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