Facile Synthesis Of Chiral Polymers With Defined Architecture Via Co-Operative Assembly Of Confined Templates
Herein is presented the synergistically self-assembled system as biomimetic polymerization media. This approach allows the facile synthesis of chiral amino acid-based polymers with high molecular weight and low dispersity inside of the bilayer of catanionic vesicles by using a conventional radical polymerization under moderate conditions. The amphoteric nature of synthetic amino acid-based poly-mers attract great interest due to their participation in chiral recognition, 1 sensitivity to external stimuli, and self-assembly into complex structures. Amino acid residues of these polymers can be in the main chain, as pendant groups, or as side-chain groups. Ring-opening polymerization of N -carboxy-α-amino acid anhydrides is commonly used for synthesis of polypeptides with amino acid residues in the main chain. Polymers with amino acids as side-chain groups with low molecular weights (MW) and low dispersity (Ð) values are most conveniently prepared using reversible deactivation radical polymerization (RDRP) techniques. However, extreme RDRP conditions, such as high-pressure or addition of metal as catalyst, can be used to prepare polymers with high MW and low Ð values (MW ~103 kg mol-1, Ð ~ 1. 10-1. 20). Different strategies employed in segregated envi-ronments include nanochannels of metal–organic framework, 17 molecular-scale stereoregular templates, and self-assembled media of dendritic polymers. These environ-ments produce increased radical lifetimes, but also decrease MWs and increase Ð values due to the formation of cylindri-cal polymers, crosslinking, or non-controlled propagation. By using synergistically assembled vesicles to conduct the polymerization inside of the individual bilayer these challeng-es can be overcome. During controlled polymerizations in the bilayer, vesicles remain colloidal and thermodynamically sta-ble as opposed to traditional polymerization techniques in a macro- or mini-emulsion, mainly due to the reduced possibility of bimolecular radical termination by isolation of growing polymeric radicals.
Herein is described a synergistically self-assembled system, wherein catanionic vesicles are used to template, compart-mentalize, and segregate confined monomer chains as they propagate via radical polymerization. The advantages of this type of bilayer-templated radical polymerization include elim-ination or substantial suppression of bimolecular termination due to the controlled propagation inside the bilayers of vesi-cles resulting in faster reaction rates compared to either solu-tion or bulk. As a result, free radical polymerization produc-es amino acid-based chiral polymers with high molecular weights (MW up to ~450 kg mol-1) and very low Ð values (≤1. 1).
Synergistic vesicle assembly
Amino acid-based methacrylate monomers were prepared from corresponding Boc-protected amino acids. 1H NMR spectra of all Boc-AA-HEMA monomers measured in acetone-d6 exhibit a carbamate resonance downfield shift as large as δ ~ 1. 2 ppm in compari-son to chloroform-d solutions. The main reason for this shift is the formation of intra- and intermolecular hydro-gen bonds between carbamate groups of Boc-AA-HEMA and/or amino group of monomer with the carbonyl group of acetone, similar to biological systems. NH protons are deshielded and due to changes in the field effects around the H, the chemical shifts comes into strong resonance downfield shifts of H-bonded groups. This effect is in part a conse-quence of the stronger H-bonding propensity of acidic pro-tons, and in part an inherent chemical shift effect. Additionally, the magnetic anisotropy of C-C double bonds have a deshielding region in the plane of the double bond. However, in methanol-d4 the carbamate resonance peak completely dis-appeared, probably due to hydrogen–deuterium exchange, strong hydrogen bonding and formation of complexes with methanol molecules, or dimers of monomers.
A mixture of hydrophobic monomers with surfactants (at 8:2 SDBS/CTAT ratio) immediately form stable vesicles with associated monomer inside the bilayer, with an average size of about 250±15 nm. A representative transmission electron microscopy (TEM) image showed spherical objects with moderate size distribution similar to previously reported monomer-loaded lipo-somes and surfactants-templated vesicles. 30-33 By comparison, DLS data of blank catanionic vesicles showed structures with average diameters of ~12 and 60 nm and a PDI of ~0. 2, with the majority of scattering arising from small particles and these catanionic vesicles were on the border of vesicles phase without additional aging. Differences in sizes between empty and loaded vesicles (with similar intensity of scattered light at applied temperatures) can be explained by rearrangement of vesicle bilayers in the presence of the hydro-phobic monomer, comparable to previous reports on the syn-ergistic formation of vesicles, where monomers play an active role in the bilayer assembly.
1H NMR analysis was used for the determination of com-partmentalization and association of monomers in vesicle bi-layers. Specifically, the original two single peaks of the vinyl protons in the monomer, are split into resolved downfield and upper field components by the incorporation of the monomer inside the bilayer. In agreement with previous reports, the sharp downfield component is caused by the vinyl protons in the outer monolayer, whereas, the broad upfield component is characteristic of the vinyl protons in the inner monolayer. Allocation of monomer molecules inside bilayer was defined using ratios of the integrated areas under the 1H-NMR peaks, and data were in good agreement with results previously shown for methacrylates distribution in vesicles measured by SANS and SAXS methods40 and de-pended on mobility and hydrophobicity of monomers37 and their ability to form intra- and inter-molecular complexes in-side the bilayer. It was found that the majority of monomers are associated with the hydrophobic tails of the bilayer (~1:1 for Boc-Ala-HEMA and increased with increasing of hydro-phobicity of monomers up to 1:6 for Boc-Leu-HEMA). Additionally, DLS and TEM data exhibit predominantly vesicles with a single population and narrow size distribution. In other words, the splitting of vinyl proton peaks were not because of vesicles with different sizes or different monomer/surfactant ratios. These results corroborate and expand data on synergis-tic assembly of vesicles in the present of methacrylic mono-mers. 30 Radical polymerization inside synergistic assembly. Homo-polymers with different hydrophobic side groups were syn-thesized. The chromatograms of the all P(Boc-AA-HEMA) polymers exhibit single sharp peaks indicating narrow chain length distribution. Additionally, it should be mentioned, that polymers form identical high MW even at low conversions. These results suggest fast and complete polymerization inside the bi-layer of an individual vesicle.
Upon heating, AIBN initiates polymerization of monomer inside the bilayer. TEM images of vesicles before and after polymerization of Boc-Ala-HEMA. (c) Size distribution of vesicles before (red line) and after (blue line) polymerization. DLS and TEM data revealed a decrease in vesicle diameter from ~220±30 nm (the synergistic assembly of vesicles with monomers, Figures 1b and 1c) to ~80±10 nm during polymer-ization of Boc-Ala-HEMA in the bilayer. Additionally, a decrease in the PDI of vesicles from ~0. 37 to ~0. 16 was also observed. In comparison with vesicles before polymerization, scattered light intensity of the solutions significantly increase during polymerization at all reaction temperatures. After polymerization, the resulting structures were colloidally stable for at least four months.
Addition of equivalent amount of methanol/acetone or meth-anol/acetone/hexane mixture and re-precipitation from acetone to hexane allowed the isolation of high molecular weight pol-ymer from low molecular weight surfactants. 1H NMR and 13C NMR spectra of the isolated polymer confirmed the newly generated polymers as P(Boc-AA-HEMA) homopolymers, with the corresponding SEC traces, for all P(Boc-AA-HEMA)). Molecular weights distribu-tion and shapes of the peak does not change during conver-sion of monomers in time, while retaining extremely low Ð values. In contrast to conventional micro-emulsion radical polymerization, there was no evidence of production of any polymers with ultrahigh molecular weight (~104 kg mol-1).
As expected for the monomers with more hindered side chains, SEC peaks shifted to longer retention time with a decrease of MW of polymers and insignificant increase of Ð values. In general, molecular weights and dispersity did not change with time of reaction or increasing concentration of AIBN, but did decrease with increased temperature of reac-tion. With reducing concentration of monomers Mn slightly reduced from ~430. 1 to ~330. 5 kg mol-1, but Ð values increased from 1. 05 to 1. 11, compared to reactions at regular concentration of monomer. However, Mn of the ho-mopolymer is similar and the Ð value is also very low with increased concentration of monomers. Given the near identical Mn values (with 20% reducing of Mn at low concentration of monomers) obtained at all three Boc-Ala-HEMA concentra-tions, it is apparent that homopolymer chains propagate to a critical chain length regardless of the monomer concentration. Additionally, monomers equally distributed in vesicles (almost identical Ð values of polymers in all cases and at all conversion rate). Polymerization entirely at 50 and 60 °C produces high molecular weights homopolymer with extremely low Ð values at all conversions, similarly to poly-mers prepared at 40 oC. With increasing conversion, molecular weights (and Ð values) of the homopolymer does not change significantly, while the vesicles’ size distribution also remains very narrow. Molecular weight of homopolymers remained the same and was around ~400. 1 kg mol-1 with Ð values ~1. 05. With increasing temperature up to 70 oC, polymers formed almost immediately, but molecular weight dropped to ~ 300. 1 kg mol-1 with increasing of Ð values to 1. 08. Hydrogen bonding behaviors of polymers were characterized by analyzing the solvent effect on its 1H NMR spectra. In the 1H NMR spectra, the peak of the carbamate NH group at ~5. 1 ppm in the monomer shifted to ~5. 5 ppm (measured in CDCl3, Figure S7) after polymerization. The main reason for shifting is formation of the hydrogen bond among the residues of amino acids in the polymer chain. Intra- and inter-strand hydrogen bonds in the polymer system are responsible for smaller changes in chemical shift of proton resonance and are expected in polymers relative to that of monomers. There are two types of intra-chain hydrogen bond-ing that can exist within the polymer chain of amino acid units: (a) C=O with the N-H of carbamates, and (b) ester C=O with the N–H of carbamate. 10 In polar solvents, the shielding effect of the bulky alkyl group contributes to the retention of the helical chain conformations and their ordered arrangement. In the 1H NMR spectra of polymers measured in non-polar as well as polar solvents, the peak of the carbamate N-H group at 5. 52 ppm shifted to 6. 30 ppm in polar solvents (acetone, and completely disappeared in methanol) and even to 7. 10 ppm in DMSO-d6, further con-firming hydrogen bond formation among the Boc-AA- resi-dues in the polymer chain.
The specific rotation of the polymers measured at room tem-perature in chloroform ranged from - 20 to + 22° and depend-ed on amino acid residues. It was found that specific optical rotation values changed perceptibly when comparing the monomer to the appropriate polymer, probably because of both the chiral residues of amino acids and a secondary helical conformation. Additionally, the specific rotation values of polymer solution changed in sign and/or magnitude after the solvent was replaced from chloroform to methanol, ace-tone and finally to DMSO. CD-spectra of the polymers measured in methanol showed two peaks at 205 and 235 nm also in chloroform it shows CD band at ~235 nm with a high molar ellipticity ([Q]). These peaks occur from the π−π* and n−π* transition of the carbonyl chromophore, because of the formation of a second-ary structure with strong Cotton effects, similar to literature reports for low molecular weight P(Boc-Leu-HEMA).
As it was shown previously for poly (N-propargyl carbamates and methyl esters) and low molecular weight poly Boc-AA-HEMA, this indicates a helical conformation stabilized by intra-molecular hydrogen bonding between the amino acid residues. In addition, with increasing of hydrophobic areas in the amino acid chain, hydrophobic interactions induced by the side groups contribute to helical conformation.
Deprotection of amino groups in polymers was accomplished at room temperature using TFA in DCM. Following dialysis against water and lyophilization, or simple precipitation in ether and re-precipitation from methanol to ether, all depro-tected homopolymers were obtained in >90% yields. All P(AA-HEMA) were found to have good methanol and water solubility. Disappearance of signals in the 1H NMR spectra at 1. 46 ppm suggests successful deprotection of polymers. Polarimetric and CD spectroscopy methods were used for estimation of conformations of all homopolymers in solution, and it was found that deprotected polymers demonstrate chiroptical properties equivalent to those of their Boc-protected counterparts.
In summary, radical polymerization of amino acid-based methacrylates inside the bilayer of catanionic vesicles yields chiral homopolymers with high MW and low Ð values via confinement of propagating chains. These optically active homopolymers show helical conformation. It was shown that intra- and inter-chain hydrogen bonds occurred in the polymer systems, and were involved in the stabilization of the helical structures. The ability to polymerize other conventional hy-drophobic monomers such as styrenes, substituted (meth)acrylates, and acrylamides could be one of the im-portant implication of these results. Additionally, with a little modification, reversible deactivated radical polymerization technique can be used to synthesize polymers with low disper-sity values and smaller molecular weights by using RAFT agents. In the case of branched polymers, it is possible to pre-pare polymers where active and polymerizable branches are parts of synergistic assembled bilayers and can be incorpo-rated into growing chains of polymer.
