Hierarchical Assembly of Branched Supramolecular Polymers from

Sep 25, 2014 - †Key Centre for Polymers and Colloids, School of Chemistry, and ... *E-mail: [email protected]., *E-mail: s.perrier@warwick...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/Biomac

Hierarchical Assembly of Branched Supramolecular Polymers from (Cyclic Peptide)−Polymer Conjugates Ming Liang Koh,† Katrina A. Jolliffe,*,‡ and Sébastien Perrier*,†,§ †

Key Centre for Polymers and Colloids, School of Chemistry, and ‡School of Chemistry, University of Sydney, Building F11, Sydney, New South Wales 2006, Australia S Supporting Information *

ABSTRACT: We report the synthesis and assembly of (N-methylated cyclic peptide)−polymer conjugates for which the cyclic peptide is attached to either the α- or both α- and ω- end groups of a polymer. A combination of chromatographic, spectroscopic, and scattering techniques reveals that the assembly of the conjugates follows a two-level hierarchy, initially driven by H-bond formation between two N-methylated cyclic peptides, followed by unspecific, noncovalent aggregation of this peptide into small domains that behave as branching points and lead to the formation of branched supramolecular polymers.

■

INTRODUCTION Supramolecular polymers have unique properties that arise from the dynamic bonds that form their structures and further exploration in this field opens exciting opportunities of bottomup engineering to build highly functional materials.1,2 Materials based on noncovalent bonds can be designed to respond to external stimuli with applications from biosensors to targeted delivery vectors.3,4 Reversible bonds also allow for the deconstruction and reconstruction of the material with prospects in degradable, recyclable, processable, and selfhealing materials.5 The synthetic achievements in this field has grown remarkably from the earliest examples6,7 to the more recent developments of highly complex nanostructures8−10 owing to the development and exploration of associating motifs and hierarchical strategies. Among the many available motifs of molecular recognition, self-assembling cyclic peptides (CPs) comprised of even numbers of alternating D- and L-amino acid residues form part of a fascinating family of materials that can assemble via hydrogen bonded β-sheet stacks and lead to a unique class of supramolecular polymers.11−13 Containing Damino acids and of cyclical topology, these synthetic peptides are biologically relevant and proteolytically stable which have been used in a number of bioapplications such as antibacterial agents,14 ion sensors,15 and transmembrane channels.16 The conjugation of synthetic polymers with peptides has a number of advantages including the modification of nanostructures and functionalities in order to, for example, reduce toxicity, reduce enzyme degradation, or improve cell uptake.17 Recently, the functionality of self-assembling cyclic peptides have been dramatically expanded by conjugating polymers to these cyclic peptides in order to generate a wide range of functional peptide-core polymer-shell nanotubes,18−36 and have © XXXX American Chemical Society

been applied toward the enhanced delivery of anticancer agents.37,38 Due to the ease of synthesis and range of amino acids available, the cyclic peptides themselves are also excellent targets for adding functionality to these supramolecular polymers, to expand their properties and control their structure. For instance, the selective N-methylation of the cyclic peptide backbone provides an efficient route to controlling assembly on only one face of the peptide. This approach has previously been employed to limit the supramolecular polymerization of cyclic peptides, providing dimeric structures that have been used as model systems to understand the assembly into nanotubes.39−54 The attachment of N-methylated cyclic peptides to both α- and ω- end groups of a homotelechelic polymer provides a simple system capable of supramolecular polymerization via dimerization of these self-complementary end groups. Supramolecular structures generated by the association of polymeric chain ends are of great interest since the selfassembly of these ditopic macromolecules can result in the formation of dynamic polymeric structures with virtual molecular weights based on noncovalent interactions.55−57 This work shows that not only do the cyclic peptides provide complementary bases for the design of supramolecular polymers via directed H-bonds, but also they aggregate to form physical branching points, leading to the formation of a new family of branched supramolecular polymers. Equipment. Preparative reverse phase high-performance liquid chromatography (RP-HPLC) was performed on a Waters SunFire C18 5 μm 19 × 150 mm column at 7 mL/ Received: July 21, 2014 Revised: September 18, 2014

A

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

3300 (N-Hstr), 3120−2830 (C-Hstr), 1622 (COstr), 2095 (N3). r-Terminal alkyne RAFT agent 3 was prepared following similar protocol from our group.65 z,r-Termini dialkyne RAFT agent 4 was prepared by an extension to the protocol used to synthesize RAFT agent 3. 2Propanoic acid 3-propanoic acid trithiocarbonate (PAPATC, 1.00 g, 3.93 mmol), propargyl alcohol (0.98 g, 0.018 mol), and 4-(dimethylamino)pyridine (DMAP; 0.145 g, 1.19 mmol) was dissolved in DCM (5 mL) and cooled to −20 °C. N-(3(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDCl; 2.69 g, 0.0140 mol) was suspended in DCM (20 mL), cooled to −20 °C and then added dropwise to the main solution. The reaction was left to stir for 20 h at room temperature. The resulting solution was diluted with DCM, washed with HCl (1 M, 70 mL), NaOH (1 M, 70 mL), and water (70 mL), dried with anhydrous MgSO4, concentrated, and purified over a silica column with DCM as the eluent. The product was concentrated under vacuum to yield (propynyl 2propanoate)yl (propynyl 3-propanoate)yl trithiocarbonate (PPPPTC) 4 as a yellow oil: 0.595 g, 46% yield; 1H NMR (300 MHz, CDCl3) δ ppm: 4.83 (q, J = 7.4 Hz, 1H, R-CHCOO), 4.68−4.77 (m, 4H, Z-COO-CH2 + R-COO-CH2), 3.63 (t, J = 6.9 Hz, 2H, Z-S-CH2), 2.82 (t, J = 6.9 Hz, 2H, Z-CH2COO), 2.51 (dt, J = 3.6, 2.6 Hz, 2H, Z-CCH + R-CCH), 1.62 (d, J = 7.2 Hz, 3H, R-CH3); ESI-LCQ MS, m/z = 353 (M + Na)+. As a representative procedure of the polymerizations performed, the synthesis of poly(n-butyl acrylate) with an alkyne functionality at each end group 7 by RAFT polymerization is presented. PPPPTC 4 (0.071 g, 0.22 mmol), azobis(isobutyronitrile) (AIBN; 0.0038 g, 0.023 mmol), and n-butyl acrylate (BA; 1.00 g, 7.80 mmol) in dioxane (2 mL) was cooled in a cold water bath and purged with N2 for 20 min. The reaction was stirred at 70 °C under an atmosphere of N2 until 66% conversion determined by 1H NMR. The polymer was precipitated and washed with ice-cold water/MeOH (1:9) until 1 H NMR analysis showed no vinyl peaks due to BA monomer. PBA-dialkyne 7 was obtained as a yellow viscous liquid: Mn (1H NMR) = 3200 g·mol−1; 1H NMR (300 MHz, CDCl3) δ ppm: 4.62−4.88 (br m, 5H, PPPPTCR-COO-CH2 + PPPPTCZCOO-CH2 + pBA-S-CH), 3.86−4.22 (br m, 44H, 22 × pBACOO-CH2), 3.62 (br t, J = 6.9 Hz, 2H, PPPPTCZ-S-CH2), 2.81 (br t, J = 6.9 Hz, 2H, PPPPTCZ-CH2-COO), 1.06−2.61 (br m, 160H, PPPPTCR-CH-CH3 + 22 × pBA-CH2-CH2 + 22 × pBAbackbone-CH-CH2 + PPPPTCZ-CCH + PPPPTCR-C CH), 0.94 (t, J = 7.4 Hz, 66H, 22 × pBA-CH3); SEC-DRI (DMF + LiBr): Mn = 3200 g·mol−1, Đ = 1.14; ESI-FTICR and MALDI-ToF MS: (M + Na)+ observed; repeat unit m/z = 128, end group m/z = 330. As a representative procedure of the conjugation reactions, the preparation of ditopic conjugate 11 is presented. Microwave-assisted CuAAC was used to graft the polymer to the cyclic peptide under conditions similar to the synthesis of 9, 10, and 12 (except that peptide is in excess for 11). PBA22-dialkyne 7 (0.030 g, 0.0094 mmol, Mn = 3200 g·mol−1, Đ = 1.14), cyclic peptide 2 (0.021 g, 0.021 mmol), copper(II) sulfate (0.0094 g, 0.038 mmol), sodium ascorbate (0.074 g, 0.38 mmol), and DMF (2 mL) were transferred to a microwave vial. The reaction mixture was then placed in a microwave reactor, stirred, and irradiated at 100 °C for 15 min with a flow of N2 and delivering 200 W in the initial ramp. DMF was removed under vacuum and the product was redissolved in DCM and

min running a linear gradient eluent of water and acetonitrile (with 0.01% trifluoroacetic acid (TFA) in both solvents) with an inline Waters 2489 UV−visible detector. Microwave-assisted solid phase peptide synthesis was carried out on a CEM Liberty 1, automated microwave peptide synthesizer. Microwaveassisted copper(I) catalyzed azide−alkyne cycloaddition (CuAAC) reactions were performed using a CEM Discover SP microwave reactor using 5 mL borosilicate microwave vessels. Synthesis. A complete list of the synthetic methods can be found in the Supporting Information. Fmoc-L-Lys(N3)-OH was made from Fmoc-L-Lys-OH using a diazotransfer reagent,58,59 in a method described in literature.23 Fmoc-N-Me-L-Ala-OH was synthesized through an oxazolidinone intermediate60 described in the literature.61 Linear peptide 1 was synthesized by fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis62−64 using 2chlorotrityl chloride resin, Fmoc deprotection with piperidine/ N,N-dimethylformamide (DMF) (1:4 vol), amino acid coupling with HBTU/HATU, N,N-diisopropylethylamine (DIPEA), DMF, and cleaved from the resin with 1,1,1,3,3,3hexafluoroisopropanol (HFIP)/dichloromethane (DCM; 1:4 vol). Peptide 1 was also prepared using a microwave peptide synthesizer. Cyclic peptide 2 was made by head-to-tail coupling of linear peptide 1. H2N-L-Lys(N3)-D-Phe(-N-Me-L-Ala-D-Phe)3-OH 1 (0.30 g, 0.30 mmol) was dissolved in DMF (200 mL) and purged with nitrogen gas (N2) while cooled in an ice bath. HBTU (0.17 g, 0.44 mmol), HOBt (0.060 g, 0.44 mmol), and DIPEA (0.12 g, 0.90 mmol) were separately mixed with DMF (25 mL total) and sequentially added dropwise to the linear peptide solution. The solution was brought to room temperature and stirred for 48 h under an atmosphere of N2. The crude product was concentrated under reduced pressure, redissolved in acetonitrile and purified by preparative RPHPLC (60:40 to 0:100 (water/acetonitrile [0.01% TFA]) over 60 min, 7 mL/min, tR = 48 min) to obtain cyclo[-L-Lys(N3)-DPhe-(N-Me-L-Ala-D-Phe-)3] 2 as a white powder: 96.0 mg, 33% yield; 1H NMR (300 MHz, CDCl3) δ ppm: 8.34−8.86 (br m, NHdimer), 7.05−7.38 (br m, 20H, 20 × Phe-CHaromatic), 6.40− 6.71 (br m, NHunimer), 4.50−5.93 (br m, 8H, Lys(N3)-α-H + 4 × Phe-α-H + 3 × N-Me-Ala-α-H), 2.60−3.30 (br m, 19H, 4 × Phe-α-CH2 + 3 × N-Me-Ala-N−CH3 + Lys(N3)-CH2-N3), 0.35−1.55 (br m, 15H, 3 × N-Me-Ala-α-CH3 + Lys(N3)-αCH2-CH2-CH2); 1H NMR (300 MHz, THF-d8) δ ppm: 8.30− 8.90 (br m, NHdimer), 7.65−7.85 (br, NHLys), 7.02−7.38 (br m, 20H, 20 × Phe-CHaromatic), 4.20−6.00 (br m, NHunimer + 8H, NHunimer + Lys(N3)-α-H + 4 × Phe-α-H + 3 × N-Me-Ala-α-H), 2.55−3.30 (br m, 19H, 4 × Phe-α-CH2 + 3 × N-Me-Ala-NCH3 + Lys(N3)-CH2-N3), 0.60−1.45 (br m, 15H, 3 × N-MeAla-α-CH3 + Lys(N3)-α-CH2-CH2-CH2); 1H NMR (300 MHz, THF-d8 + LiBr (1:9 ([2]/[LiBr])) δ ppm: 9.71 (d, J = 9 Hz, 1H, NHPhe), 9.58 (d, J = 9 Hz, 1H, NHPhe), 9.51 (d, J = 9 Hz, 1H, NHPhe), 9.11 (d, J = 6 Hz, 1H, NHLys), 8.79 (d, J = 6 Hz, 1H, NHPhe), 7.71 (d, J = 6 Hz, 2H, 2 × Phe-α-CHaromatic), 6.95−7.50 (m, 18H, 18 × Phe-α-CHaromatic), 5.53 (q, J = 7 Hz, 1H, N-Me-Ala-α-H), 4.72−5.42 (m, 7H, Lys(N3)-α-H + 4 × Phe-α-H + 2 × N-Me-Ala-α-H), 3.10−3.50 (br m, 8H, Lys(N3)-CH2-N3 + 3 × Phe-α-CH2), 2.75−2.92 (br m, 2H, Phe-α-CH2), 2.40−2.65 (br m, 9H, 3 × N-Me-Ala-N-CH3), 0.80−1.60 (br m, 15H, Lys(N3)-α-CH2-CH2-CH2 + 3 × N-MeAla-α-CH3); HPLC-MS: 10:90 (H2O/CH3CN); ESI-LCQ MS, m/z = 999 (M + H)+, 1021 (M + Na)+; ATR-FTIR νmax cm−1: B

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

washed with EDTA (3 × 30 mL, 0.055 M, pH 8.5) and water (2 × 50 mL). Dried with anhydrous MgSO4 and concentrating under vacuum, (cyclic peptide)-polymer conjugate 11 was obtained as a film of brown liquid: 32.3 mg, 66% yield; 1H NMR (200 MHz, CDCl3) δ ppm: 8.30−8.85 (br m, NHdimer), 7.05−7.80 (br m, 42H, 40 × Phe-CH + 2 × triazole-CH), 6.65−6.85 (br m, NHunimer), 4.45−6.00 (br m, 21H, 6 × Ala-αH + 8 × Phe-α-H + 2 × Lys-α-H + pBA-S-CH + 2 × PPPPTCO-CH2-triazole), 3.60−4.45 (br m, 48H, 22 × pBA-O-CH2 + 2 × Lys-CH2-triazole), 2.60−3.60 (br m, 38H, 8 × Phe-CH2 + 6 × Ala-N-CH3 + PPPPTCz-S-CH2-CH2), 0.20−2.60 (br m, 254H, 22 × pBA-CH2-CH2-CH3 + 22 × pBAbackbone-CH-CH2 + PPPPTCR-CH-CH3 + 6 × Ala-α-CH3 + 2 × Lys-α-CH2-CH2CH2); ATR-FTIR νmax cm−1: 3304 (N-Hstr), 3050−2810 (CHstr), disappearance of 2095 (N3), 1728 (COp(BA)), 1630 (COCP‑str). In the SEC studies of 9, the sample was passed over a column of neutral alumina to remove copper as well as excess polymer. An alternative treatment to remove copper the column was generally used, shown below with 11 as a representative procedure. The crude product of conjugate 11 (10 mg) in DCM (1.0 mL) was added to I2 (0.062 g) in DCM (2.0 mL) and stirred for 20 h. The mixture was diluted with DCM and washed with an aqueous solution containing EDTA (0.055 M) and sodium thiosulfate pentahydrate (0.053 M; 2 × 30 mL, pH 8.5) followed by water (30 mL). The resulting yellow solution was dried with MgSO4 and concentrated under vacuum. Affording 11 as a yellow light-brown film: 10 mg; 11: 1 H NMR (300 MHz, CDCl3) δ ppm: 8.35−8.85 (br m, NHdimer), 6.85−7.80 (br m, 42H, 40 × Phe-CH + 2 × triazoleCH), 6.30−6.80 (br m, NHunimer), 4.45−6.00 (br m, 21H, 6 × Ala-α-H + 8 × Phe-α-H + 2 × Lys-α-H + pBA-S-CH + 2 × PPPPTC-O-CH2-triazole), 3.60−4.45 (br m, 48H, 22 × pBAO-CH2 + 2 × Lys-CH2-triazole), 2.60−3.55 (br m, 38H, 8 × Phe-CH2 + 6 × Ala-N-CH3 + PPPPTCz-S-CH2-CH2), 0.20− 2.60 (br m, 254H, 22 × pBA-CH2-CH2-CH3 + 22 × pBAbackbone-CH-CH2 + PPPPTCR-CH-CH3 + 6 × Ala-α-CH3 + 2 × Lys-α-CH2-CH2-CH2). ATR-FTIR νmax cm−1: 3304 (NHstr), 3120−2800 (C-Hstr), disappearance of 2095 (N3), 1728 (COp(BA)), 1630 (COCP‑str). Characterization. Size exclusion chromatography (SEC) was performed with an inline Shimadzu RID-10A Refractive Index Detector with tetrahydrofuran (THF) + hydroquinone (0.05%wt) and toluene (0.5%vol) as the flow rate marker at 40 °C and DMF + LiBr (0.1% wt) + hydroquinone (0.05% wt) and water (1.15% wt) as the flow rate marker at 50 °C on Polymer Laboratories (PL) 10 μm guard column with two PL Mixed-B columns and PolarGel 8 μm guard column with two PL PolarGel columns respectively at a flow rate of 1 and 0.5 mL/min. Samples were filtered through a PTFE filter (0.45 μm) prior to injection, injection volume 100 μL. Reported values are based on narrow polystyrene standards in the range of 1260−6035000 and 580−38640 Da. Fourier transform infrared spectroscopy (FTIR) using a midrange beam splitter with a single bounce diamond/KRS-5 ATR accessory and the system purged with N2; and a Bruker Alpha-E ATR-FTIR spectrometer with a ZnSe crystal. Dynamic light scattering (DLS) measurements were obtained using a Zetasizer Nano from Malvern Instruments with a 4 mW, 633 nm laser and a detector angle of 173°. The analysis of each sample is the result of 8 runs, with the correlation functions fitted by the nonnegative least-squares method. When temperatures varied, an equilibration time of at least 5 min was given to the samples. In

the monotopic additive experiments, samples were kept at 40 °C for at least 20 min for equilibration before acquisition. Static light scattering (SLS) experiments were done on a Brookhaven laser light scattering system. A 633 nm laser was used and the sample was kept at 25 °C throughout the experiment. Dark count rate was recorded, distilled and filtered toluene (n = 1.491, Rθ = 1.398 × 10−5) was used to calibrate the instrument and filtered THF (n = 1.409) was used as a blank prior to the measurements. Mw was obtained with a Berry plot using the Brookhaven Instruments Zimm Plot Software (BI-ZPW). Specific refractive index increment (dn/dc) = 0.057 for poly(n-butyl acrylate) in THF at λ = 633 nm at 25 °C66,67 was used as an approximation for the dn/dc of CuNP-free conjugate 9 and CuNP-free conjugate 11. CuNP-free conjugate 9 was analyzed at concentrations of 19.5, 9.7, 4.9, and 2.4 mg/ mL (5.9, 2.9, 1.5, 0.7 mM respectively) in filtered THF in a quartz cell at angles 15−115° taking 5 × 1 s intensity measurements at every 5° increment. CuNP-free conjugate 11 was analyzed at concentrations of 10.2, 5.1, and 2.6 mg/mL (2.0, 1.0, and 0.5 mM, respectively) in filtered THF in a quartz cell at angles 15−115° taking 5 × 1 s intensity measurements at every 5° increment. Converting angles to scattering vectors to plot SLS data as I−q plots was done using q = 4π·nTHF·sin(θ/ 2)/λ. Differential scanning calorimetry (DSC) was carried out on a Mettler Toledo DSC823e. NMeCP 2 (1.7 mg), pBA16alkyne 5 (5.0 mg), pBA30-alkyne 6 (4.1 mg), pBA22-dialkyne 7 (3.1 mg), pSty20-alkyne 8 (1.3 mg), NMeCP-pBA16 9 (3.8 mg), NMe CP-pBA30 10 (1.1 mg), diNMeCP-pBA22 11 (3.7 mg), and NMe CP-pSty20 12 (1.6 mg) were analyzed in an aluminum crucible with a pinhole against a reference crucible of the same setup. In the case of liquid conjugates (i.e., 9, 10, and 11), the sample was transferred using chloroform as a solvent, which was subsequently removed using a flow of N2 gas. At a rate of 10 °C/min, samples were heated to 100 °C, then 3× (cooled to −100 °C, isothermal for 10 min, heated to 100 °C). Small angle neutron scattering (SANS) measurements were performed on the NG3 beamline at the National Institute of Standards of Technology Center for Neutron Research in Gaithersburg, MD, U.S.A. Raw data was corrected for detector sensitivity, background, and empty cell scattering.68 Sample-to-detector distances of 1.33 and 8 m were used and the data combined for 0.0057 Å−1 ≤ q ≤ 0.43 Å−1. Hellma cells (with 2 mm path length) were used for data acquisition. Solvent SLDs were determined with literature values69 of scattering lengths and cross sections using the NCNR SLD calculator.70 Where removed, incoherent scattering was subtracted using the Porod Law (I(q)·q4 vs q4 plot).

■

RESULTS AND DISCUSSION

From the alt-D,L α-residue based CP compounds reported to date, cyclo[(-L-Phe-N-Me-D-Ala)4-] has one of the highest dimerization constants (Ka = 1260−2540 M−1 in CDCl3, 293 K)41−43 and has previously been modified to allow an investigation of photochromic supramolecular systems.71,72 We opted to synthesize a modified version of this structure, in which one of the N-methyl alanine residues was replaced by an azido-lysine residue to allow attachment of the resulting cyclic peptide 2 to a polymer with two alkyne end groups via a dual copper catalyzed azide−alkyne cycloaddition (CuAAC) reaction. Cyclic peptide 2 was prepared by the head-to-tail cyclization of the linear peptide precursor 1 (prepared on 2chlorotrityl chloride resin using standard solid phase peptide C

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Scheme 1. Synthetic Strategy for the Formation of the Monotopic (N-Methylated Cyclic Peptide)−Polymer Conjugates

constant (Ka) was determined to be 1700 M−1 in CDCl3 at concentrations of 0.005−0.007 M at 300 K. This is comparable to the Ka of the analogous cyclic peptide reported by Ghadiri et al. (Ka = 1260−2540 M−1),41−43 indicating that the removal of one of the N-methyl groups and the inclusion of the azide did not interfere with the dimerization process. Note that the broad peaks observed in the 1H NMR spectra of 2 in CDCl3 are due to the presence of multiple conformers, similar peaks are observed in THF-d8. However, the addition of LiBr, which is well-known to disrupt hydrogen bonding in peptide structures,74,75 to a solution of 2 in THF-d8 resulted in sharper peaks in the 1H NMR spectrum of the cyclic peptide (see Supporting Information, Figure S2), suggesting that complexation of Li+ ions results in a single peptide conformation.76−78 In addition, the signals attributable to the amide protons shifted downfield and were well resolved. Having demonstrated that 2 can hydrogen bond into dimers, we investigated the effect of tethering polymers to the cyclic

synthesis techniques) upon treatment with HBTU in dilute DMF solution (Scheme 1). Evidence for the ability of 2 to form β-sheet structures was obtained by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Signals at 1622 cm−1 (amide I) and 1217 cm−1 (amide III) are comparable with literature ranges of β-sheet structure. Absorption observed at 1680 cm−1 is indicative of antiparallel β-sheet structure. Furthermore, absorption at 3300 cm−1 (NH) can be correlated to a NH···O bond distance of 3.00 Å,73 which is comparable to other previously studied N-methylated cyclic peptides.13 The dimerization of 2 was investigated by 1H NMR spectroscopy in CDCl3 at 300 K where the spectra contained signals attributable to the amide protons of the hydrogen bonded dimeric (NHdimer) as well as the unimeric (NHunimer) species. The ratio of dimeric to unimeric species was observed to depend on the concentration of cyclic peptide (see Supporting Information, Figure S1) and the dimerization D

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 1. Size exclusion chromatogram of 2, 5, and 9 in THF + hydroquinone (0.05% wt). The shift in peak molecular weight (Mp) of 2 and 9, as well as the asymmetric tailing of the elution profile toward lower molecular weights, demonstrate the self-assembly into dimeric species in solution. Molecular weights have been calibrated with polystyrene standards with toluene (0.5% vol) used as a flow rate marker.

Figure 2. Size exclusion chromatogram of 2, 5, and 9 in DMF + LiBr (0.1%wt) + hydroquinone (0.05%wt). The “lack of shift” in peak molecular weight (Mp) of 2 and 9, as well as the symmetric tailing of the elution profile, shows the species are in a disassembled state. Molecular weights have been calibrated with polystyrene standards with water (1.15%wt) used as a flow rate marker.

cyclic peptides to polymers,23,24,27 to yield peptide polymer conjugates 9, 10, and 11, respectively (Scheme 1). The microwave-mediated CuAAC reaction also led to the formation of copper nanoparticles (CuNPs) of a diameter of about 100 nm and of narrow size distribution, presumably from the CuAAC catalyst, as evidenced by the surface plasmon resonance bands probed by UV/vis spectroscopy (Figure S4), the inorganic residue left after thermogravimetric analysis (Figure S5), TEM images (Figure S6), and DLS measurements (see Supporting Information). The formation of CuNPs from the reduction of Cu2+ upon microwave irradiation has been reported before, and recent work has suggested that welldefined particles such those obtained in our work require the presence of a surfactant to template their growth.85−87

peptide and the impact of this on self-assembly. A series of alkyne termini poly(n-butyl acrylate) of varying degrees of polymerization (DP) was synthesized by the polymerization of n-butyl acrylate (BA), mediated by alkyne functional RAFT agents 3 or 4; to yield 5 (Mn 2300 g·mol−1, DP 16, Đ = 1.15), 6 (Mn 4100 g·mol−1, DP 30, Đ = 1.16) and 7 (Mn = 3200 g· mol−1, DP 22, Đ = 1.14, Figure S3 for mass spectrum). RAFT polymerization79 is one of the most versatile techniques of polymerization to yield functional polymers with wellcontrolled molecular weight and high end-group fidelity.80−83 Polymers 5, 6, and 7 were conjugated to 2 using a microwaveassisted, copper-mediated azide−alkyne cycloaddition (CuAAC) reaction,84 which has previously been shown to be an efficient and high-yielding method for the attachment of E

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Considering our system, this suggests that the peptide polymer conjugates are surface active, a property that arises from the difference in polarity between the cyclic peptide end group and the polymer main chain, and promote the formation of CuNPs. The nanoparticles were removed by oxidation of Cu0 and Cu1+ to Cu2+ by the reduction of I2 to I−, followed by washing with EDTA, sodium thiosulfate, and water. The formation of dimers from the monotopic conjugates was investigated by focusing on conjugates 9 (DP 16) and 10 (DP 30). The formation of dimers from conjugate 9 was initially evidenced by 1H NMR spectroscopy, which showed a clear shift in signal for the NH protons involved in H-bonding (Figure S7). In addition, SEC analyses using THF as the eluent of 9 revealed an elution peak at about twice the peak molecular weight of the polymer, while analyses in DMF + LiBr (0.1%wt) eluent, which is expected to disrupt hydrogen bonds, shows a symmetrical elution profile close to that of the alkyne-bearing polymer, which we attribute to the disassembled conjugate (see Figures 1 and 2). Conjugate 10, on the other hand, was not observed to form dimers in THF by SEC (see Figure S8), thus, suggesting that the length of the conjugated polymeric chain can affect the assembly. ATR-FTIR spectroscopy also confirmed the presence of Hbonding (see Figure 3, full spectra Figure S9) of conjugate 9. A

Sample solutions were observed to be slightly opaque by visual inspection, thus conjugates 9 and 10 were also analyzed by light scattering in THF and CHCl3 solutions. To our surprise, dynamic light scattering (DLS) analyses of 9 at room temperature revealed large (Dh = 100−1000 nm) structures that disappear upon an increase in temperature and decrease in concentration, suggesting aggregation (Figures S10−S13). DLS analyses of conjugate 10 (after the removal of copper), however, did not reveal such structures (Figure S14), suggesting that the aggregation mechanism is also dependent on the length of the pBA chain. In order to investigate the impact of the polymer functionality, a conjugate based on the less polar poly(styrene) (pSty, 8; Mn 2400 g·mol−1, DP 20, Đ 1.15), 12, was synthesized following the procedure described above. SEC in THF eluent confirmed the formation of dimers (Figure S15), and DLS analyses in CHCl3 and THF (Figures S16−S21) reveal the formation of large structures (Figures S13 and S21; after copper is removed). Aggregation in large structures in solution seems therefore to be dependent on the formation of dimers and the polarity of the polymeric chain. The latter property is supported by the ill-defined CuNPs (Figure S22) that were generated by the conjugation of pSty, as compared to well-defined CuNPs (Figure S6) from the conjugation of pBA. This observation correlates well with the idea of conjugates possessing surface active properties which facilitate the formation of CuNPs. It therefore appears that peptide aggregation is to some extent driven by phase separation, induced by difference in polarity between the cyclic peptide and polymer components of the conjugate. We then turned our attention to ditopic conjugate 11, a homotelechelic polymer with α,ω-peptide termini, to be used as the base of a supramolecular polymer. Initial attempts to characterize assemblies of 11 by size exclusion chromatography (SEC) in THF and DMF + LiBr (0.1 wt %) resulted in only very weak signals corresponding to the elution time of 2 and the dialkyne polymer 7, suggesting that the self-assembled product of 11 is of very large size and is filtered by the guard column of the instrument, leaving only unreacted starting materials in the eluted sample. DLS analyses of the sample in THF revealed the presence of large (Dh = 100−1000 nm) structures, as observed for the other conjugates (Figures S23− S27). These structures were analyzed in THF by static light scattering (SLS), and a Berry plot90,91 was constructed to extrapolate a weight-average molecular weight (Mw) of (3 ± 1) × 107 g mol−1 for the assembly formed from 11 (see Figure S28). This calculated Mw (corresponding to a degree of polymerization of ca. 1400) is too large for an isodesmic supramolecular polymer with Ka ∼ 1700 M−1 (ca. DP 5 would be expected in a less H-bonding competitive solvent such as CDCl3 at 2 mM, according to the measured Ka by 1H NMR).92 DLS was utilized to further probe the nature of the structure obtained from 11 in THF and in CHCl3, by following the response with changes in concentration, solvent composition and heat. In an isodesmic assembly, decreasing concentration should lead to smaller aggregates as the probability for Hbonding groups to find each other is lowered. In our system, we observe no decrease in size upon dilution to 10, 1, and 0.05 mg/mL (2.0, 0.2, and 0.01 mM respectively) in both THF and CHCl3 (Figures S23−S24). Finally, 0, 1, 2, and 20 equiv of 2 and 9 were added to a solution of 11. The doping of monotopic species in supramolecular polymers have been found to greatly impact the physical properties of the system by acting as “chain stoppers”.93 Light scattering revealed that, even in the presence

Figure 3. ATR-FTIR spectra of 2 and 9 cast as a film from a chloroform solution. Labeled peaks: pBA CO stretch (1732 cm−1), antiparallel β-sheet (1682 cm−1), peptide CO stretch (Amide I, 1622 and 1628 cm−1), β-sheet/random-coil (Amide III, 1244 cm−1), β-sheet (Amide III, 1217 cm−1). These modes match the expectation from self-assembling cyclic peptides. See Figure S9 for the full spectra of all conjugates.

shift was observed in local absorption maximum to 1244 cm−1, in the region expected for a β-sheet structure, with a broad shoulder extending to 1290 cm−1, which is characteristic of the random coil structure of the polymer.88 Interestingly, the discrepancy with the β-sheet absorption of 2 (1217 cm−1) could signify the effect of a long chain covalently attached to βsheet sites. A closer look at the Amide I mode also shows a shift in the absorption maximum, between the peptide 2 (1622 cm−1) to the conjugate 9 (1628 cm−1), indicative of the peptides response to the polymeric environment.89 Furthermore, we also observe absorption in the range 1670−1695 cm−1, which is generally attributed to antiparallel β-sheet structures.88 F

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 4. Small angle scattering data of 9 (2 mM, 4 mM) and 11 (2 mM) in THF or THF-d8. Incoherent background scattering has been subtracted off SANS data and the intensity of the SLS data has been offset to match the corresponding SANS data.

layer of structure in the length scales of Ångstroms to nanometers. To investigate the origin of the small scale structure, we carried out SANS on the unconjugated N-methylated cyclic peptide 2 which showed scattering over the probed q-range. A uniform cylinder with fixed dimensions, based on the unit cell from crystallography11 to model a dimer (radius 5 Å, length 10.2 Å), was insufficient to describe the scattering observed (Figures S39−S41).68 Permitting changes to the dimensions and using simpler geometries (uniform spheres, uniform ellipsoids) provided better fits to the data when the overall volume of the shape was larger than that of a dimer (Figures S42−S44).68 These volumes trend upward with increasing concentration, and elongation is especially apparent in the 4 mM sample (Figure S44). This result suggests that Nmethylated cyclic peptides, and their dimers, are capable of assembling/aggregating in THF-d8 by other means than Hbond formation through stacking, and implicates that the endgroups of the conjugates are similarly able to associate. Collating our observations, we can conceive a picture of the new supramolecular structures we have designed (Scheme 2). As expected, H-bonding directs dimer formation via stacking of the cyclic peptides (from ATR-FTIR, NMR, and SEC). In THF, the ditopic species assemble and adopt a large (∼400 nm) and extended structure (from SLS and SANS) where the cyclic peptide end-groups segregate into local (∼1 nm) domains (from SANS) creating unspecific, noncovalent branch points. Such assembly seems to be driven by phase separation, induced by the difference in polarity between cyclic peptide and polymer within the conjugate. This second interaction between cyclic peptides lead to the formation of small peptide domains that act as physical branching points and lead to the formation of large branched supramolecular polymers. This observation is reminiscent of the accounts of lateral aggregation in the use of quadruple hydrogen bonding unit, ureidopyrimidinone,94 as end-groups.95−97 Sijbesma et al. has attributed the behavior to the combination of microphase separation and π−π stacking, which is moreover amplified by the introduction of urea or urethane groups in the conjugation process.97 Furthermore, in

of 20 molar excess of monotopic species, the structure remained intact, thus confirming the product is not obtained from the isodesmic association of 11 (Figures S29, S30; CuNPs were removed from 9 but kept in the samples of 11 as internal reference for size and intensity). Furthermore, analysis of 9 alone by SLS in THF and construction of a Berry plot revealed assemblies with Mw of (4 ± 1) × 107 g mol−1 (Figure S31), thus suggesting that the monotopic species also assemble in large structures. It is understandable that large supramolecular assemblies will alter (e.g., filtered at the guard column) when subjected to the conditions in SEC characterization, and given that the unimeric and dimeric form of 9 can be observed by SEC, it can be inferred that 11 has a higher propensity to form large structures than 9. In an attempt to understand the mechanism of formation of the large structures observed by light scattering, we turned our attention to small angle scattering techniques. When examining the SLS data of 9 and 11 using an I−q plot on a log−log scale (Figures 4 and S32), we observe a change in the intensity with angle, which suggests large structures that are an appreciable size compared to the wavelength of visible light. Notably a peak in scattered intensity at low q (instead of plateauing in approach to the Guinier regime) suggests particle−particle interactions that decrease with dilution but are still observed at concentrations that approach the detection limits (2 mg/mL). With interparticle interactions at these dilute conditions we conceive that the assemblage is probably a highly extended structure, which would explain the broad size distribution obtained from the DLS results. To probe the intermediate regime, small angle neutron scattering (SANS) was carried out on the conjugates to obtain intraparticle scattering information (i.e., form factor, see Figures S33−S38). First, a steep I α q−2 relationship (a power law of q−2 is typically associated with the geometry of disks or Gaussian polymer chains) was found at q < 0.01 Å for 9 and q < 0.03 Å for 11. This crudely matches the relationship found at the large angles of the SLS data (Figure 4) supporting the observation that the size of the structures formed is comparable to the wavelength of light. At q > 0.01 Å for 9 and q > 0.03 Å for 11, scattering indicates an additional G

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

In order to further evidence the formation of smaller domains from the aggregation of peptides within the polymer structure, we analyzed the conjugates in their dry state by differential scanning calorimetry (DSC). DSC traces (Figure 5) reveal two distinct phase transitions exist for 9 and 11, the first occurring around −50 °C, which is characteristic of the glass transition of pBA chains, and the second occurring around 50 °C. The second transition agrees with our light and neutron scattering characterization, and the formation of small domains, presumably driven by the aggregation of the cyclic peptide. Conjugate 10 and 12, on the other hand, only exhibit a transition corresponding to the polymeric chain, −50 °C (pBA) and 100 °C (pSty), respectively. The lack of the second transition in 10 coincides well with the lack of assembly and Hbonding observed from conjugate, likely due to the polymer shielding the peptide end groups. PSty is a solid where we would expect to see the transition due to the aggregation of the cyclic peptide; as a result, it is difficult to compare and interpret the DSC trace of 12.

Scheme 2. Self Assembly of Conjugate 11, Highlighting the Hierarchical Relationship where (a) is the Unimeric Form, (b) Dimerization by Directed H-Bonding, (c) Lateral Aggregation and Phase Separation of Cyclic Peptides, and (d) Resulting Structure when H-Bonding, Lateral Aggregation, and Phase Separation Occur

■

CONCLUSION In conclusion, we have shown that (N-methylated cyclic peptide)−polymer conjugates can assemble into large branched supramolecular polymers by a combination of H-bonds and physical aggregation of the cyclic peptides into small domains. The conjugates assemble and adopt a large (∼400 nm) and extended supramolecular branched structures, in which the cyclic peptides aggregate into small (∼1 nm) domains, creating physical branch points in the structure. Varying the length and chemical functionality of the attached synthetic polymer was found to impact the architecture of these novel branched supramolecular polymers, which structure forms an interesting new platform for the design of large supramolecular systems and organic gels.

the case of the monotopic species, the cyclic peptide aggregation into ∼1 nm domains leads to smaller assemblies consisting of a cyclic peptide core surrounded by a polymeric chain corona. Unsurprisingly, the polymer conjugate is a poor stabilizer in an organic solvent for such assemblies, which aggregate in much larger structures, as evidenced by the increase in scattering intensity upon increase in conjugate concentration.

■

ASSOCIATED CONTENT

S Supporting Information *

Experimental details and supplementary data, including NMR, MS, UV spectra, thermal gravimetry analysis, TEM, SEC, ATR-

Figure 5. DSC traces of peptides, polymers, and conjugates (all in bulk) presented in this manuscript. Heating at 10 °C/min. H

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(15) Motesharei, K.; Ghadiri, M. R. J. Am. Chem. Soc. 1997, 119, 11306−11312. (16) Ghadiri, M. R.; Granja, J. R.; Buehler, L. K. Nature 1994, 369, 301−304. (17) Vandermeulen, G. W. M.; Klok, H.-A. Macromol. Biosci. 2004, 4, 383−398. (18) Couet, J.; Jeyaprakash, J. D.; Samuel, S.; Kopyshev, A.; Santer, S.; Biesalski, M. Angew. Chem., Int. Ed. 2005, 44, 3297−3301. (19) ten Cate, M. G. J.; Severin, N.; Börner, H. G. Macromolecules 2006, 39, 7831−7838. (20) Couet, J.; Biesalski, M. Small 2008, 4, 1008−1016. (21) Loschonsky, S.; Couet, J.; Biesalski, M. Macromol. Rapid Commun. 2008, 29, 309−315. (22) Gokhale, R.; Couet, J.; Biesalski, M. Phys. Status Solidi A 2010, 207, 878−883. (23) Chapman, R.; Jolliffe, K. A.; Perrier, S. Aust. J. Chem. 2010, 63, 1169−1172. (24) Chapman, R.; Jolliffe, K. A.; Perrier, S. Polym. Chem. 2011, 2, 1956−1963. (25) Hourani, R.; Zhang, C.; van der Weegen, R.; Ruiz, L.; Li, C.; Keten, S.; Helms, B. A.; Xu, T. J. Am. Chem. Soc. 2011, 133, 15296− 15299. (26) Xu, T.; Zhao, N.; Ren, F.; Hourani, R.; Lee, M. T.; Shu, J. Y.; Mao, S.; Helms, B. A. ACS Nano 2011, 5, 1376−1384. (27) Poon, C. K.; Chapman, R.; Jolliffe, K. A.; Perrier, S. Polym. Chem. 2012, 3, 1820−1826. (28) Li, L. S.; Zhan, H.; Duan, P.; Liao, J.; Quan, J.; Hu, Y.; Chen, Z.; Zhu, J.; Liu, M.; Wu, Y.-D.; Deng, J. Adv. Funct. Mater. 2012, 22, 3051−3056. (29) Lin, Z.; Li, L.; Yang, Y.; Zhan, H.; Hu, Y.; Zhou, Z.; Zhu, J.; Wang, Q.; Deng, J. Org. Biomol. Chem. 2013, 11, 8443−8451. (30) Chapman, R.; Bouten, P. J. M.; Hoogenboom, R.; Jolliffe, K. A.; Perrier, S. Chem. Commun. 2013, 49, 6522−6524. (31) Chapman, R.; Jolliffe, K. A.; Perrier, S. Adv. Mater. 2013, 25, 1170−1172. (32) Chapman, R.; Koh, M. L.; Warr, G. G.; Jolliffe, K. A.; Perrier, S. Chem. Sci. 2013, 4, 2581−2589. (33) Chapman, R.; Warr, G. G.; Perrier, S.; Jolliffe, K. A. Chem.Eur. J. 2013, 19, 1955−1961. (34) Danial, M.; My-Nhi Tran, C.; Young, P. G.; Perrier, S.; Jolliffe, K. A. Nat. Commun. 2013, 4, DOI: 10.1038/ncomms3780. (35) Amorin, M.; Perez, A.; Barbera, J.; Ozores, H. L.; Serrano, J. L.; Granja, J. R.; Sierra, T. Chem. Commun. 2014, 50, 688−690. (36) Danial, M.; Tran, C. M. N.; Jolliffe, K. A.; Perrier, S. J. Am. Chem. Soc. 2014, 136, 8018−8026. (37) Wang, Y.; Yi, S.; Sun, L.; Huang, Y.; Lenaghan, S. C.; Zhang, M. J. Biomed. Nanotechnol. 2014, 10, 445−454. (38) Blunden, B. M.; Chapman, R.; Danial, M.; Lu, H.; Jolliffe, K. A.; Perrier, S.; Stenzel, M. H. Chem.Eur. J. 2014, DOI: 10.1002/ chem.201403130. (39) Saviano, M.; Zaccaro, L.; Lombardi, A.; Pedone, C.; Diblasio, B.; Sun, X. C.; Lorenzi, G. P. J. Inclusion Phenom. Mol. Recognit. Chem. 1994, 18, 27−36. (40) Sun, X. C.; Lorenzi, G. P. Helv. Chim. Acta 1994, 77, 1520− 1526. (41) Ghadiri, M. R.; Kobayashi, K.; Granja, J. R.; Chadha, R. K.; McRee, D. E. Angew. Chem., Int. Ed. 1995, 34, 93−95. (42) Kobayashi, K.; Granja, J. R.; Ghadiri, M. R. Angew. Chem., Int. Ed. 1995, 34, 95−98. (43) Clark, T. D.; Buriak, J. M.; Kobayashi, K.; Isler, M. P.; McRee, D. E.; Ghadiri, M. R. J. Am. Chem. Soc. 1998, 120, 8949−8962. (44) Bong, D. T.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 2163−2166. (45) Amorín, M.; Castedo, L.; Granja, J. R. J. Am. Chem. Soc. 2003, 125, 2844−2845. (46) Amorín, M.; Brea, R. J.; Castedo, L.; Granja, J. R. Org. Lett. 2005, 7, 4681−4684. (47) Amorín, M.; Castedo, L.; Granja, J. R. Chem.Eur. J. 2005, 11, 6543−6551.

FTIR, DLS, and SANS data of the conjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: kate.jolliff[email protected]. *E-mail: [email protected]. Present Address §

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom; Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, VIC 3052, Australia. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Paul FitzGerald and Gregory G. Warr for assistance and discussions on small angle scattering; Robert Chapman for providing polymers 5 and 6; Algi Serelis of DuluxGroup for providing PABTC and PAPATC; and En Hao Pan for TEM imaging. M.L.K. acknowledges the Australian Government for the provision of an Australian Postgraduate Award research scholarship. M.L.K. and S.P. acknowledge the Monash-Warwick Alliance for funding and S.P. acknowledges the Royal Society Wolfson Merit Award (WM130055). We acknowledge the Australian Research Council for funding part of this work (DP110101608) and the support of the National Institute of Standards and Technology, U.S. Department of Commerce, in providing the neutron research facilities used in this work. This work utilized facilities supported in part by the National Science Foundation under Agreement No. DMR0944772. The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy and Microanalysis Research Facility at the Australian Centre of Microscopy and Microanalysis, The University of Sydney.

■

REFERENCES

(1) Zelzer, M.; Ulijn, R. V. Chem. Soc. Rev. 2010, 39, 3351−3357. (2) Aida, T.; Meijer, E. W.; Stupp, S. I. Science 2012, 335, 813−817. (3) Cragg, P. J. Supramolecular Chemistry: From Biological Inspiration to Biomedical Applications; Springer: New York, 2010. (4) Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Angew. Chem., Int. Ed. 2009, 48, 872−897. (5) Wojtecki, R. J.; Meador, M. A.; Rowan, S. J. Nat. Mater. 2011, 10, 14−27. (6) Pedersen, C. J. J. Am. Chem. Soc. 1967, 89, 7017−7036. (7) Fouquey, C.; Lehn, J.-M.; Levelut, A.-M. Adv. Mater. 1990, 2, 254−257. (8) Ruff, Y.; Moyer, T.; Newcomb, C. J.; Demeler, B.; Stupp, S. I. J. Am. Chem. Soc. 2013, 135, 6211−6219. (9) O’Sullivan, M. C.; Sprafke, J. K.; Kondratuk, D. V.; Rinfray, C.; Claridge, T. D. W.; Saywell, A.; Blunt, M. O.; O’Shea, J. N.; Beton, P. H.; Malfois, M.; Anderson, H. L. Nature 2011, 469, 72−75. (10) Zhang, W.; Jin, W.; Fukushima, T.; Saeki, A.; Seki, S.; Aida, T. Science 2011, 334, 340−343. (11) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, N. Nature 1993, 366, 324−327. (12) Brea, R. J.; Reiriz, C.; Granja, J. R. Chem. Soc. Rev. 2010, 39, 1448−1456. (13) Chapman, R.; Danial, M.; Koh, M. L.; Jolliffe, K. A.; Perrier, S. Chem. Soc. Rev. 2012, 41, 6023−6041. (14) Fernandez-Lopez, S.; Kim, H. S.; Choi, E. C.; Delgado, M.; Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D. A.; Wilcoxen, K. M.; Ghadiri, M. R. Nature 2001, 412, 452−455. I

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(48) Brea, R. J.; Amorín, M.; Castedo, L.; Granja, J. R. Angew. Chem., Int. Ed. 2005, 44, 5710−5713. (49) Brea, R. J.; Pérez-Alvite, M. J.; Panciera, M.; Mosquera, M.; Castedo, L.; Granja, J. R. Chem.Asian J. 2011, 6, 110−121. (50) Hernández-Eguía, L. P.; Brea, R. J.; Castedo, L.; Ballester, P.; Granja, J. R. Chem.Eur. J. 2011, 17, 1220−1229. (51) Pérez-Alvite, M. J.; Mosquera, M.; Castedo, L.; Granja, J. R. Amino Acids 2011, 41, 621−628. (52) Guerra, A.; Brea, R. J.; Amorín, M.; Castedo, L.; Granja, J. R. Org. Biomol. Chem. 2012, 10, 8762−8766. (53) Panciera, M.; Amorín, M.; Castedo, L.; Granja, J. R. Chem. Eur. J. 2013, 19, 4826−4834. (54) Aragay, G.; Ventura, B.; Guerra, A.; Pintre, I.; Chiorboli, C.; García-Fandiño, R.; Flamigni, L.; Granja, J. R.; Ballester, P. Chem. Eur. J. 2014, 20, 3427−3438. (55) Binder, W. H.; Zirbs, R. Adv. Polym. Sci. 2007, 207, 1−78. (56) ten Brinke, G.; Ruokolainen, J.; Ikkala, O. Adv. Polym. Sci. 2007, 207, 113−117. (57) Folmer, B. J. B.; Sijbesma, R. P.; Versteegen, R. M.; van der Rijt, J. A. J.; Meijer, E. W. Adv. Mater. 2000, 12, 874−878. (58) Goddard-Borger, E. D.; Stick, R. V. Org. Lett. 2007, 9, 3797− 3800. (59) Goddard-Borger, E. D.; Stick, R. V. Org. Lett. 2011, 13, 2514− 2514. (60) Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S.; Arison, B. H. J. Org. Chem. 1983, 48, 77−81. (61) Zhang, S.; Govender, T.; Norstrom, T.; Arvidsson, P. I. J. Org. Chem. 2005, 70, 6918−6920. (62) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149−2154. (63) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161−214. (64) Moss, J. A. Current Protocols in Protein Science 2005, 18.17.11− 18.17.19. (65) Konkolewicz, D.; Gray-Weale, A.; Perrier, S. J. Am. Chem. Soc. 2009, 131, 18075−18077. (66) Robin, S.; Guerret, O.; Couturier, J.-L.; Gnanou, Y. Macromolecules 2002, 35, 2481−2486. (67) Robin, S.; Guerret, O.; Couturier, J.-L.; Pirri, R.; Gnanou, Y. Macromolecules 2002, 35, 3844−3848. (68) Kline, S. J. Appl. Crystallogr. 2006, 39, 895−900. (69) Sears, V. F. Neutron News 1992, 3, 26−37. (70) NCNR Scattering Length Density Calculator, http://www.ncnr. nist.gov/resources/sldcalc.html (accessed 2013). (71) Steinem, C.; Janshoff, A.; Vollmer, M. S.; Ghadiri, M. R. Langmuir 1999, 15, 3956−3964. (72) Vollmer, M. S.; Clark, T. D.; Steinem, C.; Ghadiri, M. R. Angew. Chem., Int. Ed. 1999, 38, 1598−1601. (73) Lautié, A.; Froment, F.; Novak, A. Spectrosc. Lett. 1976, 9, 289− 299. (74) Seebach, D.; Thaler, A.; Beck, A. K. Helv. Chim. Acta 1989, 72, 857−867. (75) Thaler, A.; Seebach, D.; Cardinaux, F. Helv. Chim. Acta 1991, 74, 617−627. (76) Kunz, C.; Jahreis, G.; Günther, R.; Berger, S.; Fischer, G.; Hofmann, H.-J. J. Pept. Sci. 2012, 18, 400−404. (77) Dugave, C.; Demange, L. Chem. Rev. 2003, 103, 2475−2532. (78) Boger, D. L.; Patane, M. A.; Zhou, J. C. J. Am. Chem. Soc. 1995, 117, 7357−7363. (79) Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecules 1998, 31, 5559−5562. (80) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2012, 65, 985−1076. (81) Perrier, S.; Takolpuckdee, P. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 5347−5393. (82) Semsarilar, M.; Perrier, S. Nat. Chem. 2010, 2, 811−820. (83) Barner-Kowollik, C.; Perrier, S. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 5715−5723.

(84) Appukkuttan, P.; Mehta, V. P.; Van der Eycken, E. V. Chem. Soc. Rev. 2010, 39, 1467−1477. (85) Pileni, M.-P. Nat. Mater. 2003, 2, 145−150. (86) Nakamura, T.; Tsukahara, Y.; Sakata, T.; Mori, H.; Kanbe, Y.; Bessho, H.; Wada, Y. Bull. Chem. Soc. Jpn. 2007, 80, 224−232. (87) Blosi, M.; Albonetti, S.; Dondi, M.; Martelli, C.; Baldi, G. J. Nanopart. Res. 2011, 13, 127−138. (88) Singh, B. R. Infrared Analysis of Peptides and Proteins: Principles and Applications; American Chemical Society: Washington, DC, 2000. (89) Kong, J.; Yu, S. Acta Biochim. Biophys. Sin. 2007, 39, 549−559. (90) Andersson, M.; Wittgren, B.; Wahlund, K.-G. Anal. Chem. 2003, 75, 4279−4291. (91) Berry, G. C. J. Chem. Phys. 1966, 44, 4550−4564. (92) Ciferri, A. J. Macromol. Sci., Part C 2003, 43, 271−322. (93) Bouteiller, L. Adv. Polym. Sci. 2007, 207, 79−112. (94) Sijbesma, R. P.; Beijer, F. H.; Brunsveld, L.; Folmer, B. J. B.; Hirschberg, J. H. K. K.; Lange, R. F. M.; Lowe, J. K. L.; Meijer, E. W. Science 1997, 278, 1601−1604. (95) Yamauchi, K.; Lizotte, J. R.; Hercules, D. M.; Vergne, M. J.; Long, T. E. J. Am. Chem. Soc. 2002, 124, 8599−8604. (96) Elkins, C. L.; Viswanathan, K.; Long, T. E. Macromolecules 2006, 39, 3132−3139. (97) Botterhuis, N. E.; van Beek, D. J. M.; van Gemert, G. M. L.; Bosman, A. W.; Sijbesma, R. P. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 3877−3885.

J

dx.doi.org/10.1021/bm501062d | Biomacromolecules XXXX, XXX, XXX−XXX