Side-Chain Peptide-Synthetic Polymer Conjugates via Tandem “Ester

ARTICLE pubs.acs.org/Biomac

Side-Chain Peptide-Synthetic Polymer Conjugates via Tandem “Ester-Amide/Thiol
Ene” Post-Polymerization Modification of Poly(pentafluorophenyl methacrylate) Obtained Using ATRP Nikhil K. Singha,*,† Matthew I. Gibson,‡,§ Bishnu P. Koiry,† Maarten Danial,‡ and Harm-Anton Klok*,‡ †

Rubber Technology Centre, Indian Institute of Technology, Kharagpur, 721302, WB, India Ecole Polytechnique F
ed
erale de Lausanne (EPFL), Institut des Mat
eriaux and Institut des Sciences et Ing
enierie Chimiques, Laboratoire des Polymeres, B^atiment MXD, Station 12, CH-1015 Lausanne, Switzerland § Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom ‡


bS Supporting Information ABSTRACT: Herein the concept of tandem postpolymerization modification as a versatile route to synthesize well-defined, highly functionalized polymers is introduced. Poly(pentafluorophenyl methacrylate) obtained by atom transfer radical polymerization was first modified with allylamine, which displaces the active ester to give well-defined polymers with pendant alkene groups, which are difficult to obtain by direct (radical) polymerization of allylicfunctional monomers. The produced poly(allylmethacrylamide) was modified by a second postpolymerization modification reaction with a thiol-terminated peptide (CVPGVG) using AIBN as the radical source. NMR, IR, and SEC demonstrated successful conjugation onto the polymer to give a polymer
peptide hybrid material. This versatile strategy should extend the scope of controlled radical polymerization and “click”-type reactions

’ INTRODUCTION The synthesis of macromolecules with precise control over molecular weight, architecture, composition, and end groups is a key challenge in modern polymer chemistry. Traditional ionic polymerization methods are not compatible with most functional groups (i.e., alcohols, amines, thiols); therefore, the polymerization of functional monomers can only be achieved through the use of protection/deprotection strategies, which can result in inhomogeneous products and an increased synthetic burden. Modern controlled radical polymerization (CRP) methods display increased tolerance to functional groups and monomer types, allowing the direct polymerization of functional monomers without the need for protecting groups. This includes atomtransfer radical polymerization (ATRP),1,2 single electron transfer (SET) polymerization,3 reversible addition
fragmentation chain transfer (RAFT),4,5 and nitroxide mediated polymerization (NMP).6 Despite the versatility of these methods, it is still challenging to prepare libraries of functional (co)polymers by direct polymerization, where each constituent polymer has identical chain length and chain length distribution, limiting the opportunities to obtain quantitative structure
activity relationships. There are also some functional groups that are incompatible with CRP methods (such as thiols, alkenes, alkynes). Considering this, postpolymerization modification of reactive polymer precursors is an attractive route r 2011 American Chemical Society

toward functional polymers7 or polymer-coated surfaces.8 Whitesides et al. used free-radical polymerized poly(N-acryloxy-succinimide) (PNAS), which has pendant activated-ester side chains, as a reactive scaffold that could be modified with amino-functional sialic acid for evaluating the interactions of glycopolymers with viral surface proteins.9 Brocchini et al. demonstrated controlled radical polymerization of N-methacryloxysuccinimde (PNMAS) and used this to create polymer libraries to screen for DNA binding capacity.10 A norbornene-functionalized active ester was prepared by ROMP to obtain glycopolymers.11 Early examples of highly activated polymers were also described by Ferruti12 and Ringsdorf13 and several other examples of poly(activated esters) are known, as reviewed by Th
eato.14 Recently, RAFT polymerization was used to prepare well-defined poly(pentafluorophenyl methacrylate) (PPFMA) as a reactive precursor to obtain libraries of polymers by addition of functional amines, which also liberated an ω-terminal thiol group that is available for further orthogonal (bio)conjugations.15,16 Pentafluorophenyl esters are attractive because they show good hydrolytic stability and can be functionalized in near-quantitative Received: April 6, 2011 Revised: June 8, 2011 Published: July 07, 2011 2908

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Biomacromolecules yield with unhindered amines. A significant attraction of poly(activated esters) is their easy synthesis and the fact that they can be functionalized by using a variety of commercially available amines (or alcohols). Other postpolymerization modification reactions include aldehyde/amine (or hydrazide and hydroxyamines),17,18 Diels
Alder reactions,19 and nucleophilic displacement of halogens.20 In 2001, Sharpless introduced the concept of “click” chemistry. This broadly describes highly efficient coupling reactions that proceed to quantitative conversion without any side products or reactions, are highly orthogonal, and are preferably conducted in benign solvent media.21
23 The use of these reactions in polymer science to postmodify preformed polymers has been extensively investigated, with the [3 + 2] cycloaddition reaction between azides and alkynes24 or the reactions of thiols with alkenes by radical or Michael-type additions25 proving to be the most versatile. However, the main disadvantage of these strategies is that both alkenes and alkyne side chains are generally not compatible with controlled radical polymerizations because of their propensity to cross-link. Haddleton and coworkers have used poly(trimethylsilyl-propargyl methacrylate) to create “clickable” polymers, but this requires an additional deprotection step.24,26 Alternatively, Wooley et al. used the differential reactivity of styrenic dienes to prepare alkene-functionalized polymers by RAFT polymerization.27 The degree of conversion was precisely controlled to prevent secondary reactions of the pendant alkene. There are reports of preparation of polymers bearing pendant alkenyl functionality by using ring-opening polymerization of alkene or alkyne functional cyclic esters,28,29 N-Carboxyanhydrides,30,31 oxazolines,32 or selective anionic polymerization of butadiene.33 These pendant alkenyl functional groups were modified via thiol-ene reaction.32
34 However, preparation of these alkenyl-functionalized polymers needs very stringent reaction condition because of the ionic nature of polymerization, which is limited to only a few monomers. This manuscript describes the concept of tandem postpolymerization modification as an efficient route to “clickable” macromolecules. First, well-defined tailor-made PPFMA, a polymer bearing activated ester, was prepared via ATRP. This precursor polymer is then used as a reactive scaffold to introduce pendant alkene groups in the first postpolymerization modification step using allylamine. In this case, preparation of PPFMA via ATRP is an important step because RAFT-derived PPFMA15,35 would undergo several side reactions due to the formation of terminal
SH group from the RAFT terminal group during the modification reaction. Subsequently, this latent alkene-functional polymer is used as a reactive scaffold for biofunctionalization, which is demonstrated via the thiol
ene reaction using a thiol-functionalized elastin-derived peptide sequence.

’ EXPERIMENTAL SECTION Materials. Methacryloyl chloride, pentafluorophenol, triethyl amine (TEA), and allyl amine were purchased from Aldrich and were used as received. CuBr and CuCl (98%, Aldrich) were purified by washing with glacial acetic acid, followed by diethyl ether and then were dried under vacuum. Ethyl-2-bromoisobutyrate (EBiB) and N,N,N0 ,N00 ,N00 -penta methyl diethylene triamine (PMDETA) were purchased from Aldrich and were used as received. Pentafluorophenyl methacrylate was synthesized according to a literature protocol by the reaction of methacryloyl chloride with pentafluorophenol.15 The synthesis and characterization of peptide (CVPGVG) are described in the Supporting Information.

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Instrumentation and Analytical Methods. IH NMR spectra were acquired with a Bruker-Avance-400 spectrometer operating at 400 MHz using CDCl3 as solvent. Chemical shifts were reported relative to residual nondeuterated solvent. Thermogravimetric analysis (TGA) was carried out using a TGA Q50, TA Instruments USA, at a heating rate of 20 °C 3 min
1 under a nitrogen atmosphere. Infrared spectra were recorded on a Perkin-Elmer, Spectrum RX-L. Size exclusion chromatography (SEC) was performed on a Waters Alliance GPC V2000 system. Separation was carried out at 60 °C with TSK-Gel Alpha 3000 + 4000 columns using DMF containing 0.5 g 3 L
1 LiBr as the eluent and a flow rate of 0.6 mL 3 min
1. Molecular weights were determined relative to narrow polydispersity poly(methyl methacrylate) (PMMA) standards. Results were calculated using the Empower pro-multidetection GPC software (U-500). Mass spectra were obtained using a Perceptive Biosystems Voyager Elite MALDI-TOF mass spectrometer, equipped with a nitrogen laser (wavelength 337 nm). For MALDI-TOF sample preparation, the polymer was dissolved in THF (1 mg 3 mL
1) and mixed with sodium trifluoroacetate (1 mg 3 mL
1) and 2,5-dihydroxybenzoic acid (40 mg 3 mL
1) in the volume ratio of 1:10:10, respectively. This solution (0.5 μL) was spotted on the target plate and was allowed to dry at room temperature. All spectra were averaged over 128 laser shots. Synthesis. Atom Transfer Radical Polymerization (ATRP) of PFMA. In a typical ATRP, PFMA (0.50 g, 1.98 mmol) was taken in a dry Schlenk tube. Then, N,N,N0 ,N00 ,N00 pentamethyl diethylene triamine (PMDETA) (12.8 mg, 0.0743 mmol) and CuBr (7.11 mg, 0.05 mmol) were added to the Schlenk tube under N2 atm, and the tube was sealed with a silicone rubber septum. Nitrogen was purged through the tube for 10 min to remove oxygen. Subsequently, EBiB (9.67 mg, 0.050 mmol) was added to the tube under nitrogen purging; then, it was immersed in an oil bath already heated to 70 °C. After 45 min, the polymer was dissolved in THF and purified by passing through an alumina column. The final polymer was isolated under vacuum, and the conversion was determined gravimetrically. Isolated yield: 0.4 g, 80%. 1H NMR (400 MHz, CDCl3, δ): 0.9 (3H,
CH3 of EBiB), 1.2 to 1.8 (3H,
CH3), 2.0 to 2.6 (2H,
CH2
), 4.2 (2H,
OCH2
of EBiB). SEC (THF): Mn = 13 000 g 3 mol
1; Mw/Mn= 1.30. MADLI-TOF-MS: Mn = 7300 g 3 mol
1. FT-IR: 1776 cm
1 (>CdO of PPFMA) Synthesis of Poly(allylmethacrylamide) by Postpolymerization Modification of PPFMA. In a typical experiment, 200 mg (0.79 mmol) poly(pentafluorophenyl methacrylate) (PPFMA) was dissolved in 3 mL of dry DMF and was stirred to dissolution under nitrogen. Then, 3.3 mL of a 0.5 M solution of allylamine (containing 0.5 M triethylamine) in DMF was added via a syringe. The reaction mixture was stirred at 50 °C under nitrogen for 16 h; then, it was precipitated in diethylether. The precipitate was then redissolved in 2 mL of THF and precipitated in 35 mL of Milli-Q water and cooled to 5 °C for 1 h, and the white polymer product was isolated by centrifugation. Yield: 75 mg, 76% (assuming 100% conversion of PFMA groups). 1H NMR (400 MHz, CDCl3, δ): 0.9 (3H,
CH3), 2.0 (2H,
CH2
), 3.7 (2H,
CH2
of
NHCH2
), 5.1 and 5.8 (3H,
CHdCH2), 7.9 (1H, HN
). FT-IR: 1640 cm
1 (
NHCO
). Radical Addition of CVPGVG onto Poly(allylmethacrylamide). We dissolved 10 mg (0.08 mmol allyl groups) of poly(allylmethacrylamide), 4.0 mg (0.024 mmol) of AIBN, and 91 mg (0.16 mmol) of CVPGVG in 0.5 mL of dry DMF in Schlenk tube. This gave a molar ratio of [CdC]/[I]/[SH] of 1:0.3:2. The reaction mixture was degassed by three freeze
pump
thaw cycles finally filled with N2. The tube was placed in an oil bath thermostatted at 70 °C for 16 h. After this time, an additional portion of 4 mg (0.024 mmol) of AIBN dissolved in 0.1 mL of degassed DMF (three freeze
pump
thaw cycles) was added to the reaction vessel via syringe and again heated at 70 °C for a further 16 h. Following this time, the reaction mixture was diluted with 3 mL of MilliQ water and was dialyzed (MWCO 10 000 g 3 mol
1) against Milli-Q water for 3 days with regular changes of the solvent. The desired product 2909

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Scheme 1. Biofunctionalized Polymethacrylate via Post-Polymerization Modification

Table 1. ATRP of Pentafluorophenyl Methacrylate (PFMA) with Different Catalyst Systems and Comparison with Methylmethacrylatea Mn,theo (g 3 mol
1)c

Mn,SEC (g 3 mol
1)d

Mw/Mn (
)

80

8200

13 000

1.30

75

7500

9150

1.25

6700

9075

1.14

conversion (%)b

code (
)

monomer (
)

catalyst system (
)

1

PFMA

EBiB/CuBr/PMDETA

2

PFMA

EBiB/CuCl/PMDETA

3

MMA

EBiB/CuCl/PMDETA

65

Polymerizations conducted in bulk at 70 °C for 45 min. b Determined gravimetrically. [monomer]:[initiator]. d Determined by SEC in THF against PMMA standards. a

c

Determined from conversion and feed ratio

was obtained by freeze-drying as a white powder, isolated yield 20 mg, 41%. 1H NMR (400 MHz, CDCl3, δ): 0.8
1.4 (12H, m, CH3-iPr, 3H of
CH3 and 2H of
CH2-polymethacrylate part) 1.75
2.11 (8H, m, CH3
Ac + CH2
Pro + CH2
Pro + CH-iPr), 2.26 (1H, m, CH-iPr), 2.84 (2H, t, CH2
Pro), 3.68 (1H, m, CH-Cys), 3.82
3.93 (4H, 2xCH2-Gly), 4.1 (1H, d, CH-Val), 4.35
4.47 (3H, CH-Val + CH2
Cys + CH-Pro), 5.1 and 5.8 (3H,
CHdCH2), 3.7 (2H,
CH2
of
NHCH2
), 7.9 (1H, HN
). FT-IR: 1640 cm
1 (
NHCO
).

’ RESULTS AND DISCUSSION Polymerization of Pentafluorophenyl Methacrylate by ATRP. The synthetic pathway used in this investigation is shown

in Scheme 1. The first step required the synthesis of poly(pentafluorophenyl methacrylate) (PPFMA) by atom transfer radical polymerization (ATRP). ATRP was chosen rather than reversible addition
fragmentation chain transfer (RAFT) polymerization, as during the first postpolymerization modification step, using a primary amine, thiol groups would be generated because of cleavage of the RAFT end group.15 The presence of a thiol group would complicate the second postpolymerization modification reaction, which involves radical addition of a thiol onto an alkene. In contrast, ATRP generates a bromide (or other halogen) end group, which is not expected to interfere with any of the modification reactions. To the best of our knowledge, ATRP has not been explored to polymerize PFMA; therefore, a small screening of suitable catalyst systems was conducted. The results of the bulk-phase polymerization of PFMA (Table 1) indicated that ATRP of PFMA using ethyl-bromoisobutyrate (EBiB) as initiator and CuBr or CuCl as catalyst in combination with PMDETA ligand is faster than the ATRP of MMA. This is due to the presence of the pentafluorophenyl group in the PFMA, which activates the monomer, as has been observed by Hvilsted et al.; they reported higher reactivity of pentafluorostyrene than that of styrene during ATRP.36

Figure 1. Infrared spectrum of carbonyl region of poly(pentafluorophenyl methacrylate) (solid line) and following modification with allyl amine (dotted line).

ATRP of PFMA using EBiB as initiator and CuBr/PMDETA as catalyst at 70 °C was very fast, and the molecular weight of the resulting PPFMA was slightly higher than the theoretical molecular weight. Importantly, when CuCl was used as the catalyst, the molecular weight was controlled, and the polydispersity index was relatively narrow. This is due to the halogen exchange reaction between the initiator/macroinitiator and the catalyst, which leads to slow activation of the initiating species and faster deactivation by the copper catalyst.37 FTIR spectra (Figure 1) showed a single carbonyl peak at 1776 cm
1, demonstrating that no cleavage of the pentafluorophenyl ester had occurred, which would result in additional peaks in the carbonyl range. MALDI-TOF-MS indicated the presence of bromine end group and the integrity of the pentafluorophenyl esters, as has been previously demonstrated for PMMA.38,39 For example, a peak at m/z of 5260.60 in MALDI-MS spectrum is due to the chemical structure 2910

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EBiB-(PFMA)20,Na+. The results of the 1H NMR, FT-IR, SEC, and MALDI experiments indicate that ATRP of PFMA was controlled and that the activated ester side chains are not affected by the polymerization conditions. Postpolymerization Modification. Using the optimized polymerization protocols discussed above, well-defined PPFMA was synthesized that was suitable to explore for tandem Table 2. Sequential Post-Polymerization Modification of Poly(pentafluorophenyl methacrylate) (PPFMA) code Mn(SEC,THF) Mn(SEC,DMF) Mw/Mn Mn(NMR) (
) (g 3 mol
1) A

a

13000

(g 3 mol
1) 10200

allyl

peptide

content

content

(
) (g 3 mol
1)a (mol %)b (mol %)c 1.40

B

5400

1.52

6200

82

C

25800

1.40

17500

41

41

Calculation based on the Mn obtained from SEC analysis, followed by the degree of side-chain conversion, as determined by 1H NMR. b Repeat units bearing allylmethacrylate groups as a molar percentage the total number of repeat units. c Repeat units functionalized with the peptide expressed as molar percentage of the total number of repeat units of the polymer backbone.

postpolymerization modification (A in Table 2). The first postpolymerization modification step was the reaction of PPFMA with a three-fold molar excess (relative to active ester groups) of allylamine to introduce pendant alkene groups along the polymer backbone, producing poly(allylmethacrylamide), B. Figure 1 shows the FTIR spectrum of A before and after (B) reaction, indicating complete removal of the active ester group at 1776 cm
1 and formation of an amide at 1640 cm
1. A small peak at 1710 cm
1 indicates the formation of a small amount of methacrylic acid units presumably through competing hydrolysis reactions, which we have previously observed.15 Importantly, this side product cannot be detected when 19F NMR is used to assess conversion and was hence not used here. 1H NMR analysis (Figure 2B) indicated side-chain allyl functionality of 82%. SEC analysis also indicated a shift in molecular weight from 10 200 to 5400 g 3 mol
1 without any significant broadening of the distribution, which is essential if well-defined materials are to be obtained. The poly(allylmethacrylamide) was subsequently investigated for thiol
ene radical addition using the peptide CVPGVG, which was engineered to have a single cystine residue. VPGVG is the consensus repeat sequence of the elastomeric protein elastin, and previous studies have demonstrated that elastin side-chain polymers display lower critical solution

Figure 2. 1H NMR analysis of sequential postpolymerization modification. (A) Poly(pentafluorophenyl methacrylate), (B) poly(allyl methacrylamide) obtained by reaction of PPFMA with allylamine, and (C) peptide-grafted poly(allylmethacrylamide) obtained by thiol
ene radical addition of CVPGVG (represented by blue globe). Additional peaks corresponding to peptide are clearly visible. Full NMR spectrum of peptide is included in the Supporting Information. * Indicates residual solvent. 2911

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Figure 3. SEC chromatograms (in DMF) showing influence of postmodification on the elution behavior of polymers. (A) Poly(pentafluorophenyl methacrylate), (B) poly(allylmethacrylamide), and (C) Poly(allylmethacrylamide-graft CVPGVG).

temperature behavior and are thus an interesting class of stimuliresponsive polymers,40 which can undergo cooperative aggregation.41,42 Peptide-based polymers are important materials in different biomedical applications and biomimetic material research.43
45 Using AIBN as a source of radicals, CVPGVG was reacted with polymer B using a molar ratio of [CdC]: [AIBN]:[SH] of 1:0.3:2 in DMF with heating to 70 °C. After 16 h, a second portion of AIBN was added. Following precipitation in diethylether and exhaustive dialysis to remove unreacted peptide, polymer C was isolated by lypophilization. Polymer C was readily soluble in aqueous solution, unlike the precursor polymer B, indicating successful thiol
ene coupling. 1H NMR (Figure 2) showed additional peaks corresponding to the peptide. (1H NMR of pure peptide is included in the Supporting Information.) By comparing the residual allylic peaks to those of the polymer backbone, it was calculated that 41 mol % of repeat units were functionalized with the peptide, which is equal to 50 mol % of the allylmethacrylate units. Higher degrees of functionalization could not be achieved, suggesting that steric hindrance of the large peptide chains was the limiting factor. Schlaad et al. have previously shown that allyl-functional polyoxazolines can be quantitatively functionalized but only with relatively small thiols.32 SEC analysis (Table 2 and Figure 3) confirmed conjugation of the peptide to polymer B, with a shift in Mn from 5400 to 25 800 g 3 mol
1, which is equivalent to 36 peptides or 67% of available allyl groups being functionalized. This discrepancy between SEC and 1H NMR is probably due to the values from SEC being obtained relative to the structurally different PMMA and the comb-shaped nature of the peptide-grafted polymer. Intramolecular coupling of adjacent allyl groups cannot be ruled out,33 but the presence of residual allyl groups and broad agreement between NMR and SEC analysis for degree of functionalization clearly demonstrates successful grafting of the peptide onto the polymer chain without significant formation of side products. This facile, but versatile methodology will be applicable for the synthesis of a wide range of (bio) functionalized polymers and polymer libraries through highly efficient “click”-type reactions

’ CONCLUSIONS The concept of tandem postpolymerization modification of a polymethacrylate bearing an activated ester functionality is introduced in this manuscript. In this case, a well-defined precursor polymer, PPFMA, was first synthesized by ATRP through

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optimization of the catalytic system, which introduces benign end groups, which do not interfere with subsequent modifications. Postpolymerizaton modification of this polymer with allylamine resulted in a well-defined polymer with pendant allyl functionality, which cannot be obtained by direct polymerization of allyl-functional monomers due to cross-linking reactions. This poly(allylmethacryamide) was then investigated for the thiol
ene (“click” type) reaction with a cystine-terminated elastinderived peptide sequence CVPGVG. Using AIBN as a thermal radical source, CVPGVG could be grafted onto the polymer with 50% grafting density, as demonstrated by SEC and NMR. This report of the use of tandem postpolymerization modification to introduce “clickable” groups onto polymers derived by controlled radical polymerization will present the opportunity to synthesize increasingly complex (co)polymers for biotechnological applications, including “smart”/responsive materials, peptidic drug carriers, or as precursors for nanomedicines.

’ ASSOCIATED CONTENT

bS

Supporting Information. Information regarding the synthesis of peptide and its characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (N.K.S.); harm-anton.klok@epfl.ch (H.-A.K.).

’ ACKNOWLEDGMENT N.K.S. and H.A.K. are grateful to DST, New Delhi, India and EPFL, Switzerland for financial support under the Indo-Swiss collaborative research program (project no. INT/SWISS/RF & JUAF/2008). The EU is acknowledged for providing funding through the integrated project ‘‘Nanobiopharmaceutics,’’ NMP4CT2006-026723. We are thankful to Dr. Tuan Q. Nguyen and Mr. D. J. Haloi for analytical support. ’ REFERENCES (1) Matyjaszewski, K.; Xia, J. Chem. Rev. 2001, 101, 2921–2990. (2) Kamigaito, M.; Ando, T.; Sawamoto, M. Chem. Rev. 2001, 101, 3689–3745. (3) Rosen, B. M.; Percec, V. Chem. Rev. 2009, 109, 5069–5119. (4) Perrier, S.; Takolpuckdee, P. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 5347–5393. (5) Moad, G.; Rizzardo, E.; Thang, S. H. Polymer 2008, 49, 1079–1131. (6) Hawker, C. J.; Bosman, A. W.; Harth, E. Chem. Rev. 2001, 101, 3661–3688. (7) Gauthier, M. A.; Gibson, M. I.; Klok, H.-A. Angew. Chem., Int. Ed. 2009, 48, 48–58. (8) Barbey, R.; Klok, H.-A. Langmuir 2010, 26, 18219–18230. (9) Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Med. Chem. 1995, 38, 4179–4190. (10) Pedone, E.; Li, X.; Koseva, N.; Alpar, O.; Brocchini, S. J. Mater. Chem. 2003, 13, 2825–2837. (11) Strong, L. E.; Kiessling, L. L. J. Am. Chem. Soc. 1999, 121, 6193–6196. (12) Ferruti, P.; Bettelli, A.; A, F. Polymer 1972, 13, 462–464. (13) Batz, H.-G.; Franzmann, G.; Ringsdorf, H. Angew. Chem., Int. Ed. 1972, 11, 1103–1104. 2912

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