Copolymerization of Styrene and a Cyclic Peptide

Copolymerization of phenylethylene and a Cyclic PeptidePutting peptides into the backb wiz strand of polyolefins the theme copolymerization of vinylbenzene and a cyclic peptide containing the disulfide poseAnja C. Paulya, Daniel Rentschb and Fabio di Lena*a. encouraging cultivation creep For the first time, a vinyl radical monomer such as styrene has been extremistly copolymerized with a cyclic peptide containing the disulfide bond. A new physique of bio-hybrids is obtained in which the amino acid sequence is statistically distributed within the polymers backb unrivalled reach. The social organization of the copolymer has been confirmed by means of conventional as hale as diffusion-edited 1H NMR, MALDI FT-ICR mass spectrometry, FT-IR spectroscopy, TGA, DSC, and a series of escort experiments.With the aim to combine the plus properties of biological macro molecules such as, for example, the biological assist, molecular recognition, and chirality, with the solution proper ties, processability, etc. of artificial macromolecules, polymer chemists take in started to develop the so-called bio-hybrid polymers. Bioconjugates are the most studied class of bio-hybrids.1 These are block copolymers in which a protein, polysaccharide or pedestal 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 crap them, in many ways, similar to polymer mixtures. At odds with block copolymers, statistical copolymers do not exhibit the characteristics of polymer mixtures but behave like kindred materials with peculiar physical and chemical properties. Here we report the provision 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 inclined(p) by the extremist ring-opening copolymerization2of a cyclic peptide containing the disulfide (S-S) bond and styrene. Cycles containing the S-S bond are k at one timen to undergo solution copolymerization with vinyl monomers such as methyl acrylate, vinyl acetate, acrylonitrile and styrene.3The whimsical force behind the research is our interest in finding new, naive and industrially friendly ways to turn commodity polymers into specialty polymers with superior added value. To our knowledge, the only examples of polyolefins containing amino acids in the backbone chain have been prepared by Wagener and co- turn overers 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 here is metal-free, en adapteds the incorporation of sequences of amino acids and employs radical polymerization, a process with which more than 50% of all the polymers scramd worldwide are made.Scheme 1. primary copolym erization of styrene with the cyclic tripeptide cCLC.Styrene 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 mannerl monomers. They were reacted with a zep ratio of 946 in dimethyl sulfoxideTable 1. Polymerization conditions, give out, mo average molecular angle, polydispersity index, degradation temperatures, film over revolution 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 precipitation in wet and dialysis in MeOH.Determination by SEC in THF on the tush of polystyrene calibration.Determination by comparability 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 twain divergent beats of azobisisobutyronitrile ( AIBN) affording the copolymers P1 and P2 (Scheme 1, Table 1). The copolymers were purified by precipitation in water and dialysis in wood spirit so as to remove, among the otherwisewise 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 projection chromatography (SEC), the copolymer P1, obtained by development 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, moody out to have a higher molecular weight () and a comparable PDI of 1.64 (Table 1). The SEC traces of two 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 two styrene and CLC units. On the one hand, the peaks at 0.87 ppm and 1.10 ppm, visible also in 1H NMR spe ctrum 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 correspond to the aliphatic and the aromatic protons of polystyrene, respectively. The remaining proton signals of CLC could be assigned with a belittleder degree of confidence due to the overlapping signals of solvent and/or polystyrene. By comparing the area underneath the peak at 0.87 ppm relation to the iso-propyl group of CLC with the area underneath the peak around 7 ppm congeneric 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 function of the chain length up to a critical value that depends on the specific system. It is then sightly to assume that such critical value for had not been reached i n the indue case. The topic has been extensively investigated and the interested reader is referred 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 specialism up to 40%,7 a similar set of signals were order 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 knock-down(prenominal) indication that the peptide is incorporated into polystyrene rather than forming a physical blend with it. It is cost 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 s pectrometry9 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 indeed 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) ofFig ure 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 equivalence of the theoretical and spy m/z (B), and the corresponding chemical structure (C).polystyrene. Furthermore, two lucid mass losses, one around 200 C and the other at 417 C, can 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 pristine 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 derivative scanning calorimetry (DSC) thermogram of P1 displayed a glass transition occurring around 66 C, which is same to the glass transition temperature (Tg) of a polystyrene of prepared in our lab (66 C). Therefore, the amount of CLC incorporated in the pol ymer 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 terms of a reduced chain flexibility due to the bulky phenyl groups that hinder the revolution of the backbones carbon-carbon bonds. We surmise that CLC increases the chain flexibility by playing 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 , by and by which the Tg reaches a stationary value of ca. 108 C.10 Hence, the use of polymers with similar molecular weights is crucial for comparing, meaningfully, the glass transition temperatures.In absence of cCLC, the polymerization of styrene under the same experimental 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 impress since disulfides are known to act as chain transfer agents in and to produce a certain retardation 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 tha t the peptide should not be blockily distributed along the polymer chain. Moreover, the possibility that the copolymer could be alternating(a) 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 each cationic or anionic or zwitterionic depending on the pH. Charge-bearing polymers are practically reported as bioactive, e.g., hemostatic11 and/or antimicrobial12. Consequently, the class of materials here described index show bioactivity without containing intrinsically bioactive, amino acid sequences. Furthermore, apart from the specific functionalities, the peptide is apparent to confer improved degradability on the polyolefin. Experiments in both directions are shortly 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 polyolefin 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 spectrosco pic, thermic and chromatographic data. This material is available free of charge via the Internet at http//pubs.acs.org.REFERENCES1.Lutz, J.-F. Brner, H. 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