Synthese van cyclische polymeren en karakterisering van hun Diffusive Motion in de Smelt staat bij de Single Molecule niveau
Summary
Een protocol voor de synthese en karakterisering van diffuse beweging van cyclische polymeren op de single molecule-niveau wordt gepresenteerd.
Abstract
We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.
Introduction
Cyclic polymers are unique in that they do not have chain ends. They often exhibit unusual behaviors which is distinct from their linear counterpart, including increased thermal stability of polymer micelles by a linear-to-cyclic conversion,1,2 and spatial organization of DNA in bacterial cells by a loop formation.3 Topological interactions between the cyclic chains are believed to be the critical factor for such unusual behaviors.4,5 Therefore, characterizing the motion and relaxation of cyclic polymers under entangled conditions has been an important research topic in polymer science for decades.6
Cyclic polymer dynamics has been investigated using both synthetic and naturally occurring molecules by means of ensemble averaged experimental methods such as nuclear magnetic resonance (NMR), light scattering, and viscosity measurements.7-9 However, these studies often suffer from impurity molecules in the samples.10 Furthermore, spatiotemporal heterogeneities of the motion of individual molecules caused by inherent structural heterogeneity of entangled polymers are often hidden behind the ensemble averaging in these studies. In order to characterize molecular level dynamics of cyclic polymers, a synthesis method that provides high purity cyclic polymers and an experimental and analysis methods that allow for quantitative characterization of molecular motion at the single molecule level have to be developed. Here, we will show a method to synthesize high-purity cyclic and dicyclic poly(THF)s that incorporate a fluorophore unit using an electrostatic self-assembly and covalent fixation (ESA-CF) process11-13 and a method to analyze the motion of the individual fluorophore-incorporated polymer chains using a combination of mean-squared displacement (MSD) and cumulative distribution function (CDF) analyses.
A proper data processing has been shown to be essential for the accurate characterization of the diffusive motion. With an adequate MSD and CDF analyses, a multiple-mode diffusion of the cyclic and dicyclic polymers in the melt and semi-dilute solution of the linear polymer chains has been revealed,14-16 suggesting the significant effects of the topological states of the polymers on the diffusive motion of the chains under entangled conditions.17 While the experimental and analytical approaches to characterize the motion of the cyclic polymers are described in this protocol, the same method can be used to quantitatively characterize the diffusive motion in many other heterogeneous systems. The approach would be especially suitable when multiple diffusion components existing in the samples are to be analyzed.
Protocol
Representative Results
Discussion
De 4-armige en 8 gevormde polymeren werden bereid via de ESR-CF protocol (figuur 1), die een kritische stap voor de synthese. 12,24 monofunctionele en bifunctionele lineaire poly (THF) s N -phenylpiperidinium eindgroepen bereid volgens de voorgaande procedure. 11 de ionenwisselende werd uitgevoerd door opnieuw neerslaan van een acetonoplossing van een polymeer precursor met triflaat tegenanionen uitgevoerd in een waterige oplossing die een overmaat carboxylaat.
<p clas…Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research No. 22750122 (S.H.), No. 26288099 (T.Y.), and No. 23350050 (Y.T.) of the Japan Society for the Promotion of Science. S.H. is grateful for The Kurata Memorial Hitachi Science and Technology Foundation. The research reported in this publication was supported by the King Abdullah University of Science and Technology (S.H.).
Materials
| Materials | |||
| THF | Godo | ||
| Wakosil C-300 | Wako Pure Chemical Industries | ||
| Acetone | Godo | ||
| Toluene | Godo | ||
| n-Hexane | Godo | ||
| CHCl3 | Kanto Chemical | ||
| Bio-Beads S-X1 | Bio-Rad | ||
| Methyl triflate | Nacalai Tesque | ||
| Triflic anhydride | Nacalai Tesque | ||
| Potassium Hydroxide | Wako Pure Chemical Industries | ||
| Ethanol | Wako Pure Chemical Industries | ||
| Poly(tetrahydrofuran) | Aldrich | ||
| Chloroform | Wako Pure Chemical Industries | ||
| Immersion oil | Cargille | Type 37 / Type A | |
| Equipment | |||
| 2-Neck 100-mL round-bottom flask | |||
| Flask | |||
| Beaker | |||
| Funnel | |||
| Filter paper | Whatman | ||
| Reflux condenser | |||
| Syringe | |||
| Water bath | |||
| Magnetic stirrer | |||
| Rotary evaporator | |||
| Microscope cover slips (24 x 24 mm, No. 1) | Matsunami Glass | CO22241 | |
| Staining jar | AS ONE Corporation | 1-7934-01 | |
| Ultrasonic cleaner | VWR International | 142-0047 | |
| Inverted microscope | Olympus | IX71 | |
| Ar-Kr ion laser | Coherent | Innova 70C | |
| Berek compensator | Newport | 5540 | |
| Excitation filter | Semrock | LL01-488-12.5 | |
| Dichloric mirror | Omega optical | 500DRLP | |
| Emission filter | Semrock | BLP01-488R-25 | |
| Lens and mirror | Thorlabs | ||
| EM-CCD camera | Andor Technology | iXon | |
| Objective lens (x100, N.A. = 1.3) | Olympus | UPLFLN 100XOP | |
| Objective heater | Bioptechs | ||
| Preparative GPC | Japan Analytical Industry | LC-908 |
Referencias
- Honda, S., Yamamoto, T., Tezuka, Y. Topology-Directed Control on Thermal Stability: Micelles Formed from Linear and Cyclized Amphiphilic Block Copolymers. J. Am. Chem. Soc. 132 (30), 10251-10253 (2010).
- Honda, S., Yamamoto, T., Tezuka, Y. Tuneable enhancement of the salt and thermal stability of polymeric micelles by cyclized amphiphiles. Nat. Commun. 4, (2013).
- Jun, S., Mulder, B. Entropy-driven spatial organization of highly confined polymers: Lessons for the bacterial chromosome. Proc. Natl. Acad. Sci. U. S. A. 103 (33), 12388-12393 (2006).
- McLeish, T. Polymers without beginning or end. Science. 297 (5589), 2005-2006 (2002).
- McLeish, T. Polymer dynamics: Floored by the rings. Nat. Mater. 7 (12), 933-935 (2008).
- Roovers, J., Tezuka, Y. . Topological Polymer Chemistry: Progress of Cyclic Polymers in Syntheses, Properties and Functions. , 137-156 (2013).
- Klein, J. Evidence for reptation in an entangled polymer melt. Nature. 271 (5641), 143-145 (1978).
- Leger, L., Hervet, H., Rondelez, F. Reptation in entangled polymer-solutions by forced rayleigh light-scattering. Macromolecules. 14 (6), 1732-1738 (1981).
- von Meerwall, E. D., Amis, E. J., Ferry, J. D. Self-diffusion in solutions of polystyrene in tetrahydrofuran – comparison of concentration dependences of the diffusion-coefficients of polymers, and a ternary probe component. Macromolecules. 18 (2), 260-266 (1985).
- Kapnistos, M., et al. Unexpected power-law stress relaxation of entangled ring polymers. Nat. Mater. 7 (12), 997-1002 (2008).
- Adachi, K., Takasugi, H., Tezuka, Y. Telechelics having unstrained cyclic ammonium salt groups for electrostatic polymer self-assembly and ring-emitting covalent fixation. Macromolecules. 39 (17), 5585-5588 (2006).
- Oike, H., Imaizumi, H., Mouri, T., Yoshioka, Y., Uchibori, A., Tezuka, Y. Designing unusual polymer topologies by electrostatic self-assembly and covalent fixation. J. Am. Chem. Soc. 122 (40), 9592-9599 (2000).
- Yamamoto, T., Tezuka, Y. Topological polymer chemistry: a cyclic approach toward novel polymer properties and functions. Polym. Chem. 2 (9), 1930-1941 (2011).
- Habuchi, S., Fujiwara, S., Yamamoto, T., Tezuka, Y. Single-molecule imaging reveals topological isomer-dependent diffusion by 4-armed star and dicyclic 8-shaped polymers. Polym. Chem. 6 (22), 4109-4115 (2015).
- Habuchi, S., Fujiwara, S., Yamamoto, T., Vacha, M., Tezuka, Y. Single-Molecule Study on Polymer Diffusion in a Melt State: Effect of Chain Topology. Anal. Chem. 85 (15), 7369-7376 (2013).
- Habuchi, S., Satoh, N., Yamamoto, T., Tezuka, Y., Vacha, M. Multimode Diffusion of Ring Polymer Molecules Revealed by a Single-Molecule Study. Angew. Chem. Int. Ed. 49 (8), 1418-1421 (2010).
- Habuchi, S., Tezuka, Y. . Topological Polymer Chemistry: Progress of Cyclic Polymers in Syntheses, Properties and Functions. , 265-290 (2013).
- Fernandez, P., Bayona, J. M. Use of off-line gel-remeation chromatography normal-phase liquid-chromatography fro the determination of polycyclic aromatic-compounds in environmental-samples and standard reference materials (air particulate matter and marine sediment). J. Chromatogr. 625 (2), 141-149 (1992).
- Biesenberger, J. A., Tan, M., Duvdevan, I., Maurer, T. Recycle gel permeation chromatography. 1. recycle principle and design. J. Polym. Sci. Pol. Lett. 9 (5), 353 (1971).
- Kusumi, A., Sako, Y., Yamamoto, M. Confined lateral diffusion of membrane-receptors as studied by single-particle tracking (nanovid microscopy) – effects of calcium-induced differentiation in cultured epithelial-cells. Biophys. J. 65 (5), 2021-2040 (1993).
- Schutz, G. J., Schindler, H., Schmidt, T. Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys. J. 73 (2), 1073-1080 (1997).
- Vrljic, M., Nishimura, S. Y., Brasselet, S., Moerner, W. E., McConnell, H. M. Translational diffusion of individual class II MHC membrane proteins in cells. Biophys. J. 83 (5), 2681-2692 (2002).
- Margineanu, A., et al. Photophysics of a water-soluble rylene dye: Comparison with other fluorescent molecules for biological applications. J. Phys. Chem. B. 108 (32), 12242-12251 (2004).
- Tezuka, Y., Oike, H. Self-assembly and covalent fixation for topological polymer chemistry. Macromol. Rapid Commun. 22 (13), 1017-1029 (2001).
- Deres, A., et al. The Origin of Heterogeneity of Polymer Dynamics near the Glass Temperature As Probed by Defocused Imaging. Macromolecules. 44 (24), 9703-9709 (2011).
- Flier, B. M. I., et al. Heterogeneous Diffusion in Thin Polymer Films As Observed by High-Temperature Single-Molecule Fluorescence Microscopy. J. Am. Chem. Soc. 134 (1), 480-488 (2012).
- Habuchi, S., Oba, T., Vacha, M. Multi-beam single-molecule defocused fluorescence imaging reveals local anisotropic nature of polymer thin films. Phys. Chem. Chem. Phys. 13 (15), 6970-6976 (2011).
- Zettl, U., et al. Self-Diffusion and Cooperative Diffusion in Semidilute Polymer Solutions As Measured by Fluorescence Correlation Spectroscopy. Macromolecules. 42 (24), 9537-9547 (2009).
- Kirstein, J., Platschek, B., Jung, C., Brown, R., Bein, T., Brauchle, C. Exploration of nanostructured channel systems with single-molecule probes. Nat. Mater. 6 (4), 303-310 (2007).
