Copolymerization of Styrene and a Cyclic Peptide

Copolymerization of styrene and a Cyclic PeptidePutting peptides into the backbone image of polyolefins the radical copolymerization of styrene and a cyclic peptide containing the disulfide bondAnja C. Paulya, Daniel Rentschb and Fabio di Lena*a.support Information rise For the first time, a vinyl monomer such as styrene has been radically copolymerized with a cyclic peptide containing the disulfide bond. A new class of bio-hybrids is obtained in which the amino point sequence is statistically distributed within the polymers backbone chain. The structure of the copolymer has been confirmed by manner of conventional as sanitary as diffusion-edited 1H NMR, MALDI FT-ICR mass spectrometry, FT-IR spectroscopy, TGA, DSC, and a series of control experiments.With the aim to combine the advantageous properties of biological macro groinecules such as, for example, the biological be given, molecular recognition, and chirality, with the solution properties, processability, etc. of man-ma de macromolecules, polymer chemists have started to develop the so-called bio-hybrid polymers. Bioconjugates ar the most studied class of bio-hybrids.1 These argon block copolymers in which a protein, polysaccharide or nucleotide is chemically linked to a synthetic polymer such as a polyolefin, polyether or polyester. In this type of structures, the constituent blocks maintain their individual properties, which make them, in many ways, similar to polymer mixtures. At betting odds with block copolymers, statistical copolymers do non exhibit the characteristics of polymer mixtures but behave like homogeneous materials with peculiar physical and chemical properties. Here we report the proviso of a new class of bio-hybrids in which, much like in statistical copolymers, an amino acid sequence is incorporated directly into the backbone chain of a polyolefin like polystyrene. The polymers are wide-awake by the radical ring-opening copolymerization2of a cyclic peptide containing the di sulfide (S-S) bond and styrene. Cycles containing the S-S bond are known to undergo radical copolymerization with vinyl monomers such as methyl acrylate, vinyl acetate, acrylonitrile and styrene.3The driving force behind the research is our interest in finding new, simple and industrially tender ways to turn commodity polymers into specialty polymers with high added value. To our knowledge, the just examples of polyolefins containing amino acids in the backbone chain have been prepared by Wagener and co-workers by means of acyclic diene metathesis (ADMET)4 polymerization of dienes containing a single amino acid residue conducted in the presence of a ruthenium carbene catalyst.5 The approach we describe present is metal-free, en fittings the internalisation of sequences of amino acids and employs radical polymerization, a process with which more than 50% of all the polymers produced worldwide are made.Scheme 1. Radical copolymerization of styrene with the cyclic tripeptide cCLC.S tyrene and the cyclic peptide S1,S3-cyclo(L-cysteinyl-L-leucyl-L-cysteine), from now on referred to as cCLC (or CLC when ring-opened), were chosen as model monomers. They were reacted with a molar ratio of 946 in dimethyl sulfoxideTable 1. polymerization conditions, yield, number average molecular load, polydispersity index, degradation temperatures, glass transition temperatures and CLC content of the copolymers.CopolymerP1P2Styrene/cCLC/AIBNa)94/6/5 molar ratio94/6/2 molar ratioYieldb)40 %43 %c)2,5005,400PDIc)1.791.64Tdeg1198C215CTdeg2417C419CTg66C54CCLC contentd)6 mol%mol%1M in DMSO.After haste in water and dialysis in MeOH.Determination by SEC in THF on the basis of polystyrene calibration.Determination by comparison of the integrated peaks in the 1H-NMR spectra of the isopropyl unit in CLC and the phenyl unit in polystyrene.(DMSO) at 70 C for 12h with two different amounts of azobisisobutyronitrile (AIBN) affording the copolymers P1 and P2 (Scheme 1, Table 1). The copolymers were purified by precipitation in water and dialysis in methanol so as to remove, among the other possible impurities, unreacted cCLC and/or cCLC-derived by-products. The overall yield was equal to 40% for P1 and 43% for P2. When analysed by means of size exclusion chromatography (SEC), the copolymer P1, obtained by using a higher amount of AIBN, resulted to have a number average molecular weight () of 2,500 and a polydispersity index (PDI) of 1.79. On the other hand, P2, synthesized by using a smaller amount of AIBN, dour out(p) to have a higher molecular weight () and a comparable PDI of 1.64 (Table 1). The SEC traces of both copolymers are shown in the Supporting Information (Figure S1).The signals in the 1H NMR spectra of P1 (Figure S2) and P2 (Figure 1A) could be assigned to both styrene and CLC units. On the one hand, the peaks at 0.87 ppm and 1.10 ppm, visible in addition in 1H NMR spectrum of unreacted cCLC (Figure S3), could be assigned to the iso-propyl residue of CLC. On the other hand, the two groups of peaks at 1.54 and 1.92 ppm, and at 6.55 and 7.05 ppm equalise to the aliphatic and the aromatic protons of polystyrene, respectively. The remaining proton signals of CLC could be assigned with a lower degree of confidence due to the overlapping signals of solvent and/or polystyrene. By analyse the area underneath the peak at 0.87 ppm relative to the iso-propyl group of CLC with the area underneath the peak around 7 ppm relative to the phenyl ring of styrene, it was calculated that the peptide makes up 6 mol% of copolymer P1 and 9 mol% of P2. A different degree of co-monomer incorporation is not odd if one considers that the composition, like other properties of a polymer, is hold out of the chain length up to a critical value that depends on the specific system. It is then reasonable to assume that such critical value for had not been reached in the present case. The topic has been extensively investigated and the interested reader is referre d to the literature for details.6 In the diffusion-edited mode, in which the 1H NMR spectra were recorded applying a flow-compensated double-stimulated-echo with a gradient might up to 40%,7 a similar set of signals were found for the styrene and CLC units (Figure 1B and S2). By exploiting the fact that the translational diffusion in solution is size-dependent, the diffusion-edited NMR is able to discriminate between signals relative to low and high molecular weight species.8 Since only the solvent signals disappeared, the NMR data are a strong distinction that the peptide is incorporated into polystyrene rather than forming a physical blend with it. It is worth noting that the diffusion-edited NMR is not quantitative and thus the molar composition of the copolymers could be determined only from the conventional 1H-NMR spectra. The analysis by MALDI FT-ICR mass spectrometry9 substantiates these conclusions. A mass distribution (Figure 2) that accurately matches that of monocharged polystyrene chains each containing one CLC moiety and AIBN-derived isobutyronitrile groups as both and -chain ends was hence obtained.Figure 1. 1H-NMR spectra of the copolymer P2 (A), 1H-diffusion edited 1H-NMR spectra of the copolymer P2 with gradient strength of 40% (B) in THF-d8, and the corresponding chemical structure (C).The results of all the other analytical techniques used to characterize the copolymers are in line with what found above. In the FT-IR spectra, for example, signals belonging to both styrene and amino acid moieties could be detected (Figure 3), which are (i) the bands at 1735 cm-1 (carboxylic group) and 1654 cm-1 (amide group) of CLC, which are also present in the FT-IR spectrum of unreacted cCLC and (ii) the signals of the aromatic carbon-carbon bonds (1492 and 1452 cm-1) and carbon-proton bond of the phenyl rings (736 and 696 cm-1) ofFigure 2. MALDI FT-ICR spectrum of the copolymer P2 in the positive mode (A), the magnification of the spectrum in the mass range 4600 5000 with the comparison of the hypothetic and observed m/z (B), and the corresponding chemical structure (C).polystyrene. Furthermore, two distinct mass losses, one around 200 C and the other at 417 C, lav be seen in the thermogravimetric (TGA) traces of the copolymers P1 and P2 (Table 1). By direct comparison with the TGA of the constituting materials, which show a mass loss at 208 C for unreacted cCLC and one at 418 C for original polystyrene, the two steps observed in the TGA of both copolymers could be assigned to the degradation of the CLC and styrene units, respectively (Figure S4). The differential scanning calorimetry (DSC) thermogram of P1 displayed a glass transition occurring around 66 C, which is identical to the glass transition temperature (Tg) of a polystyrene of prepared in our lab (66 C). Therefore, the amount of CLC incorporated in the polymer turned out to be too low to produce a measurable effect on the glass transition. In contrast, the amount of CLC in the copolymer P2 turned out to be sufficient to produce a change in the glass transition temperature, which was measured to be 54 C (Table 1). This is significantly lower than Tg of polystyrenes with (75 C) and (89 C) prepared in our lab. The DSC scans of the two copolymers P1 and P2 in comparison with polystyrenes with similar molecular weight are shown in Figure S5. The relatively high Tg of polystyrene is classically rationalized in wrong of a reduced chain flexibility due to the bulky phenyl groups that hinder the rotation of the backbones carbon-carbon bonds. We surmise that CLC increases the chain flexibility by acting as a spacer between the styrene units, which results in the lowering of the glass transition temperature. It is worth noting that the Tg and the of (atactic) polystyrene are positively correlated up to , after which the Tg reaches a stationary value of ca. 108 C.10 Hence, the use of polymers with similar molecular weights is essential for comparin g, meaningfully, the glass transition temperatures.In absence of cCLC, the polymerization of styrene under the same observational conditions afforded polymers with in 76% yield and in 73% yield for theFigure 3. FT-IR spectra of the cyclic tripeptide cCLC, the copolymer P2 and Polystyrene.lower and higher amounts of AIBN, respectively. In both cases, the molecular weights and reaction yields for pristine polystyrene were higher than those of the relative copolymers. This is not surprising since disulfides are known to act as chain transfer agents in and to produce a certain meanwhile effect on radical polymerization.3 When the polymerization was repeated omitting the styrene from the reaction mixture, no polymer was obtained. Hence, cCLC, like other cyclic disulfides,2 does not homopolymerize in the presence of a radical initiator. This control experiment suggests that the peptide should not be blockily distributed along the polymer chain. Moreover, the possibility that the copol ymer could be alternating is ruled out by the fact that the degree of peptide incorporation is well below 50 mol%. It is therefore reasonable to assume that both P1 and P2 are statistical copolymers of styrene and CLC.Peptides like cCLC are peculiar in that they bear unbound amine and carboxyl groups while being cyclic. This makes them and their copolymers either cationic or anionic or zwitterionic depending on the pH. Charge-bearing polymers are often describe as bioactive, e.g., hemostatic11 and/or antimicrobial12. Consequently, the class of materials here described might show bioactivity without containing intrinsically bioactive, amino acid sequences. Furthermore, apart from the specific functionalities, the peptide is likely to confer improve degradability on the polyolefin. Experiments in both directions are presently ongoing and will be the subject of another publication.In conclusion, we have shown that a peptide sequence can be incorporated into the backbone chain of a po lyolefin via radical polymerization. Styrene and a cyclic tripeptide containing the disulfide bond were chosen as model monomers. Although cyclic disulfides are known to ring-open via the homolytic cleavage of the S-S bond in the presence of certain radicals, the result reported in this work is not trivial since the efficiency of such a reaction depends significantly on the disulfide used. Investigations are presently underway in order to explore the monomer scope, in terms of both the olefin and the peptide, the bioactivity and degradability of the copolymers, as well as the possibility to extend the process to reversible-deactivation radical polymerizations13 such as ATRP14. The preparation of a whole new range of functional and degradable materials is anticipated.ASSOCIATED CONTENTSupporting InformationDetailed experimental procedures as well as spectroscopic, thermal and chromatographic data. This material is available free of charge via the Internet at http//pubs.acs.org.REFERE NCES1.Lutz, J.-F. Brner, H. 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