本研究合成帶有正電荷之親水性雙嵌段聚胺基酸高分子，聚賴胺酸嵌段聚蘇胺酸(PLL-b-PLT)並且將其製備成水膠。利用聚賴胺酸具有無規則線圈、聚蘇胺酸具有折板之二級結構，探討聚胺基酸的比例對氫鍵作用力、水合力以及靜電排斥力的影響來研究形成水膠的形成機制；另外，藉由各種實驗分析PLL-b-PLT嵌段聚胺基酸分子間的排列。本實驗利用一級胺作為起始劑，對第一段胺基酸進行開環聚合，再由第一段末端的親核基對第二段胺基酸開環聚合，得到9種不同比例的嵌段聚胺基酸。針對各個比例的嵌段聚胺基酸進行成膠濃度測試，再利用IR以及CD光譜分析其二級結構，發現β-sheet結構的增加有助於臨界成膠濃度的降低，並且由實驗數據推論PLL鏈段上的正電荷亦會影響成膠濃度，代表水膠系統的成膠作用力由氫鍵作用力、水合力以及靜電排斥力達到平衡；另外，藉由SAXS以及TEM等儀器的分析發現在低濃度的高分子水溶液中，PLL-b-PLT自組裝形成奈米纖維(nanofibril)結構，並分析其mesh size，由XRD分析高分子鏈段排列，再利用SEM觀察水膠樣貌，推測水膠形成是藉由纖維纏繞(entanglement)而成的網絡。此外，利用流變儀測試水膠的強度、水膠回復情形，以及在不同溫度下的強度變化，再利用不同溫度的CD光譜交互比對，發現溫度的升高會造成β-sheet結構的增加以及水合能力的降低，以及水膠強度的增加。

In this study, we reported the hydrogel preparation by using coil-sheet diblock copolypeptide poly(L-lysine)-block-poly(L-threonine) (PLL-b-PLT) with different block ratios. FTIR and circular dichroism (CD) analyses confirmed that PLL and PLT segments adopted random coil and β-sheet conformations, respectively. The gelation of PLL-b-PLT was dictated the balance between hydrogen-bonding interaction, hydration, and charge repulsion. The formation of β-sheet conformation by PLT segments through hydrogen bond interactions resulted in the self-assembly of the PLL-b-PLT by XRD analyses. SAXS and TEM analyses revealed that the PLL-b-PLT formed fibrilar nanoassemblies. The mechanical properties of these PLL-b-PLT hydrogels were found to depend on polypeptides chain length and block ratio.

(1) Noller, H. F. The parable of the caveman and the ferrari: Protein synthesis and the RNA world. Philosophical Transactions of the Royal Society B: Biological Sciences 2017, 372.
(2) Nelson, D. L.; Nelson, D. L.; Lehninger, A. L.; Cox, M. M.: Lehninger principles of biochemistry; W.H. Freeman: New York, 2008.
(3) Creighton, T. E.: Proteins: structures and molecular properties; Macmillan, 1993.
(4) Meyers, R. A.: Molecular biology and biotechnology: a comprehensive desk reference; John Wiley & Sons, 1995.
(5) Schulz, G. E.; Schirmer, R. H.: Patterns of folding and association of polypeptide chains. In Principles of Protein Structure; Springer, 1979; pp 66-107.
(6) Kabsch, W.; Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen‐bonded and geometrical features. Biopolymers 1983, 22, 2577-2637.
(7) Carlsen, A.; Lecommandoux, S. Self-assembly of polypeptide-based block copolymer amphiphiles. Current Opinion in Colloid & Interface Science 2009, 14, 329-339.
(8) Branden, C. I.: Introduction to protein structure; Garland Science, 1999.
(9) Merrifield, R. Solid phase peptide synthesis. II. The synthesis of bradykinin. Journal of the American Chemical Society 1964, 86, 304-305.
(10) Cheng, J.; Deming, T. J.: Synthesis of polypeptides by ring-opening polymerization of α-amino acid N-carboxyanhydrides. In Peptide-based materials; Springer, 2011; pp 1-26.
(11) Leuchs, H. Ueber die Glycin‐carbonsäure. European Journal of Inorganic Chemistry 1906, 39, 857-861.
(12) Kricheldorf, H. R. Polypeptides and 100 Years of Chemistry of α‐Amino Acid N‐Carboxyanhydrides. Angewandte Chemie International Edition 2006, 45, 5752-5784.
(13) Huesmann, D.; Birke, A.; Klinker, K.; Türk, S.; Räder, H. J.; Barz, M. Revisiting secondary structures in NCA polymerization: Influences on the analysis of protected polylysines. Macromolecules 2014, 47, 928-936.
(14) Wong, S.; Kwon, Y. J. Facile synthesis of high‐molecular‐weight acid‐labile polypeptides using urethane derivatives. Journal of Polymer Science Part A: Polymer Chemistry 2015, 53, 280-286.
(15) Hehir, S.; Cameron, N. R. Recent advances in drug delivery systems based on polypeptides prepared from N‐carboxyanhydrides. Polymer International 2014, 63, 943-954.
(16) Hadjichristidis, N.; Iatrou, H.; Pitsikalis, M.; Sakellariou, G. Synthesis of well-defined polypeptide-based materials via the ring-opening polymerization of α-amino acid N-carboxyanhydrides. Chemical reviews 2009, 109, 5528-5578.
(17) He, X.; Fan, J.; Wooley, K. L. Stimuli‐Triggered Sol–Gel Transitions of Polypeptides Derived from α‐Amino Acid N‐Carboxyanhydride (NCA) Polymerizations. Chemistry–An Asian Journal 2015.
(18) Wei, Z.; Zhu, S.; Zhao, H. Brush macromolecules with thermo-sensitive coil backbones and pendant polypeptide side chains: synthesis, self-assembly and functionalization. Polymer Chemistry 2015, 6, 1316-1324.
(19) Ulkoski, D.; Meister, A.; Busse, K.; Kressler, J.; Scholz, C. Synthesis and structure formation of block copolymers of poly (ethylene glycol) with homopolymers and copolymers of l-glutamic acid γ-benzyl ester and l-leucine in water. Colloid and Polymer Science 2015, 293, 2147-2155.
(20) Wibowo, S. H.; Sulistio, A.; Wong, E. H.; Blencowe, A.; Qiao, G. G. Functional and Well‐Defined β‐Sheet‐Assembled Porous Spherical Shells by Surface‐Guided Peptide Formation. Advanced Functional Materials 2015, 25, 3147-3156.
(21) Lu, Y.; Ngo Ndjock Mbong, G.; Liu, P.; Chan, C.; Cai, Z.; Weinrich, D.; Boyle, A. J.; Reilly, R. M.; Winnik, M. A. Synthesis of polyglutamide-based metal-chelating polymers and their site-specific conjugation to trastuzumab for auger electron radioimmunotherapy. Biomacromolecules 2014, 15, 2027-2037.
(22) Daly, W. H.; Poché, D. The preparation of N-carboxyanhydrides of α-amino acids using bis(trichloromethyl)carbonate. Tetrahedron Letters 1988, 29, 5859-5862.
(23) Poché, D. S.; Moore, M. J.; Bowles, J. L. An Unconventional Method for Purifying the N-carboxyanhydride Derivatives of γ-alkyl-L-glutamates. Synthetic Communications 1999, 29, 843-854.
(24) Deming, T. J. Living polymerization of α-amino acid-N-carboxyanhydrides. Journal of Polymer Science Part A: Polymer Chemistry 2000, 38, 3011-3018.
(25) Chen, C.; Wu, D.; Fu, W.; Li, Z. Peptide hydrogels assembled from nonionic alkyl-polypeptide amphiphiles prepared by ring-opening polymerization. Biomacromolecules 2013, 14, 2494-2498.
(26) Peppas, N. A.; Khare, A. R. Preparation, structure and diffusional behavior of hydrogels in controlled release. Advanced Drug Delivery Reviews 1993, 11, 1-35.
(27) Buwalda, S. J.; Boere, K. W. M.; Dijkstra, P. J.; Feijen, J.; Vermonden, T.; Hennink, W. E. Hydrogels in a historical perspective: From simple networks to smart materials. Journal of Controlled Release 2014, 190, 254-273.
(28) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J. Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles. Nature 2002, 417, 424-428.
(29) Roorda, W. E.; BoddÉ, H. E.; de Boer, A. G.; Junginger, H. E. Synthetic hydrogels as drug delivery systems. Pharmaceutisch Weekblad 1986, 8, 165-189.
(30) Miyata, T.; Asami N Fau - Uragami, T.; Uragami, T. A reversibly antigen-responsive hydrogel.
(31) Wichterle, O.; Lim, D. Hydrophilic Gels for Biological Use. Nature 1960, 185, 117-118.
(32) Kopeĉek, J. Hydrogels: From soft contact lenses and implants to self-assembled nanomaterials. Journal of Polymer Science, Part A: Polymer Chemistry 2009, 47, 5929-5946.
(33) Choi, S. W.; Choi, S. Y.; Jeong, B.; Kim, S. W.; Lee, D. S. Thermoreversible gelation of poly (ethylene oxide) biodegradable polyester block copolymers. II. Journal of Polymer Science Part A Polymer Chemistry 1999, 37, 2207-2218.
(34) Jeong, B.; Lee, D. S.; Shon, J. I.; Bae, Y. H.; Kim, S. W. Thermoreversible gelation of poly (ethylene oxide) biodegradable polyester block copolymers. Journal of Polymer Science Part A: Polymer Chemistry 1999, 37, 751-760.
(35) Slager, J.; Domb, A. J. Biopolymer stereocomplexes. Advanced drug delivery reviews 2003, 55, 549-583.
(36) Chujo, Y.; Sada, K.; Saegusa, T. Cobalt (II) bipyridyl-branched polyoxazoline comolex as a thermally and redox reversible hydrogel. Macromolecules 1993, 26, 6320-6323.
(37) Chujo, Y.; Sada, K.; Saegusa, T. Iron (II) bipyridyl-branched polyoxazoline complex as a thermally reversible hydrogel. Macromolecules 1993, 26, 6315-6319.
(38) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Reversible hydrogels from self-assembling artificial proteins. Science 1998, 281, 389-392.
(39) Wang, C.; Stewart, R. J.; KopeČek, J. Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains. Nature 1999, 397, 417-420.
(40) Jing, P.; Rudra, J. S.; Herr, A. B.; Collier, J. H. Self-assembling peptide-polymer hydrogels designed from the coiled coil region of fibrin. Biomacromolecules 2008, 9, 2438.
(41) Zhang, X.-Z.; Wu, D.-Q.; Chu, C.-C. Synthesis, characterization and controlled drug release of thermosensitive IPN–PNIPAAm hydrogels. Biomaterials 2004, 25, 3793-3805.
(42) Salgado-Rodrıguez, R.; Licea-Claverıe, A.; Arndt, K. Random copolymers of N-isopropylacrylamide and methacrylic acid monomers with hydrophobic spacers: pH-tunable temperature sensitive materials. European polymer journal 2004, 40, 1931-1946.
(43) Ishihara, K.; Hamada, N.; Kato, S.; Shinohara, I. Photoinduced swelling control of amphiphilic azoaromatic polymer membrane. Journal of Polymer Science Part A: Polymer Chemistry 1984, 22, 121-128.
(44) Yu, X.; Chen, L.; Zhang, M.; Yi, T. Low-molecular-mass gels responding to ultrasound and mechanical stress: towards self-healing materials. Chemical Society Reviews 2014, 43, 5346-5371.
(45) Cravotto, G.; Cintas, P. Molecular self-assembly and patterning induced by sound waves. The case of gelation. Chemical Society Reviews 2009, 38, 2684-2697.
(46) Fan, J.; Zou, J.; He, X.; Zhang, F.; Zhang, S.; Raymond, J. E.; Wooley, K. L. Tunable mechano-responsive organogels by ring-opening copolymerizations of N-carboxyanhydrides. Chemical science 2014, 5, 141-150.
(47) Ostroha, J.; Pong, M.; Lowman, A.; Dan, N. Controlling the collapse/swelling transition in charged hydrogels. Biomaterials 2004, 25, 4345-4353.
(48) Estroff, L. A.; Hamilton, A. D. Water gelation by small organic molecules. Chemical reviews 2004, 104, 1201-1218.
(49) Adams, D. J.; Topham, P. D. Peptide conjugate hydrogelators. Soft Matter 2010, 6, 3707-3721.
(50) He, X.; Fan, J.; Wooley, K. L. A.-O. h. o. o. X. Stimuli-Triggered Sol-Gel Transitions of Polypeptides Derived from alpha-Amino Acid N-Carboxyanhydride (NCA) Polymerizations.
(51) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001, 294, 1684-1688.
(52) Zou, J.; Zhang, F.; Chen, Y.; Raymond, J. E.; Zhang, S.; Fan, J.; Zhu, J.; Li, A.; Seetho, K.; He, X. Responsive organogels formed by supramolecular self assembly of PEG-block-allyl-functionalized racemic polypeptides into β-sheet-driven polymeric ribbons. Soft Matter 2013, 9, 5951-5958.
(53) Park, M. H.; Joo, M. K.; Choi, B. G.; Jeong, B. Biodegradable thermogels. Accounts of chemical research 2011, 45, 424-433.
(54) Huang, J.; Hastings, C. L.; Duffy, G. P.; Kelly, H. M.; Raeburn, J.; Adams, D. J.; Heise, A. Supramolecular hydrogels with reverse thermal gelation properties from (oligo) tyrosine containing block copolymers. Biomacromolecules 2012, 14, 200-206.
(55) Hamley, I. W.; Daniel, C.; Mingvanish, W.; Mai, S.-M.; Booth, C.; Messe, L.; Ryan, A. J. From hard spheres to soft spheres: the effect of copolymer composition on the structure of micellar cubic phases formed by diblock copolymers in aqueous solution. Langmuir 2000, 16, 2508-2514.
(56) Chen, Y.; Pang, X. H.; Dong, C. M. Dual Stimuli‐Responsive Supramolecular Polypeptide‐Based Hydrogel and Reverse Micellar Hydrogel Mediated by Host–Guest Chemistry. Advanced Functional Materials 2010, 20, 579-586.
(57) Nakahata, M.; Takashima, Y.; Yamaguchi, H.; Harada, A. Redox-responsive self-healing materials formed from host–guest polymers. Nature communications 2011, 2, 511.
(58) He, X.; Fan, J.; Zhang, F.; Li, R.; Pollack, K. A.; Raymond, J. E.; Zou, J.; Wooley, K. L. Multi-responsive hydrogels derived from the self-assembly of tethered allyl-functionalized racemic oligopeptides. Journal of Materials Chemistry B 2014, 2, 8123-8130.
(59) Moon, H. J.; Choi, B. G.; Park, M. H.; Joo, M. K.; Jeong, B. Enzymatically degradable thermogelling poly (alanine-co-leucine)-poloxamer-poly (alanine-co-leucine). Biomacromolecules 2011, 12, 1234-1242.
(60) Benguigui, L.; Boué, F. Homogeneous and inhomogenous polyacrylamide gels as observed by small angle neutron scattering: A connection with elastic properties. The European Physical Journal B - Condensed Matter and Complex Systems 1999, 11, 439-444.
(61) Hirokawa, Y.; Okamoto, T.; Kimishima, K.; Jinnai, H.; Koizumi, S.; Aizawa, K.; Hashimoto, T. Sponge-like heterogeneous gels: hierarchical structures in poly (N-isopropylacrylamide) chemical gels as observed by combined scattering and confocal microscopy method. Macromolecules 2008, 41, 8210-8219.
(62) Horkay, F.; Basser, P. J.; Hecht, A.-M.; Geissler, E. Structural investigations of a neutralized polyelectrolyte gel and an associating neutral hydrogel. Polymer 2005, 46, 4242-4247.
(63) Singh, S. S.; Aswal, V.; Bohidar, H. Structural evolution of aging agar-gelatin co-hydrogels. Polymer 2009, 50, 5589-5597.
(64) Imran, A. B.; Esaki, K.; Gotoh, H.; Seki, T.; Ito, K.; Sakai, Y.; Takeoka, Y. Extremely stretchable thermosensitive hydrogels by introducing slide-ring polyrotaxane cross-linkers and ionic groups into the polymer network. Nature communications 2014, 5.
(65) Rösler, A.; Klok, H.-A.; Hamley, I. W.; Castelletto, V.; Mykhaylyk, O. O. Nanoscale structure of poly (ethylene glycol) Hybrid block copolymers containing amphiphilic β-strand peptide sequences. Biomacromolecules 2003, 4, 859-863.
(66) Moore, J. L.; Caprioli, R. M.; Skaar, E. P. Advanced mass spectrometry technologies for the study of microbial pathogenesis. Current opinion in microbiology 2014, 19, 45-51.
(67) Uzawa, T.; Nishimura, C.; Akiyama, S.; Ishimori, K.; Takahashi, S.; Dyson, H. J.; Wright, P. E. Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR. Proceedings of the National Academy of Sciences 2008, 105, 13859-13864.
(68) Forood, B.; Feliciano, E. J.; Nambiar, K. P. Stabilization of alpha-helical structures in short peptides via end capping. Proceedings of the National Academy of Sciences 1993, 90, 838-842.
(69) Gibson, M. I.; Cameron, N. R. Experimentally facile controlled polymerization of N‐carboxyanhydrides (NCAs), including O‐benzyl‐L‐threonine NCA. Journal of Polymer Science Part A: Polymer Chemistry 2009, 47, 2882-2891.
(70) Breedveld, V.; Nowak, A. P.; Sato, J.; Deming, T. J.; Pine, D. J. Rheology of block copolypeptide solutions: hydrogels with tunable properties. Macromolecules 2004, 37, 3943-3953.
(71) Sreerama, N.; Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Analytical biochemistry 2000, 287, 252-260.
(72) Brahms, S.; Brahms, J. Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. Journal of molecular biology 1980, 138, 149-178.
(73) Choi, Y. Y.; Jang, J. H.; Park, M. H.; Choi, B. G.; Chi, B.; Jeong, B. Block length affects secondary structure, nanoassembly and thermosensitivity of poly (ethylene glycol)-poly (L-alanine) block copolymers. Journal of Materials Chemistry 2010, 20, 3416-3421.
(74) Gibson, M. I.; Cameron, N. R. Organogelation of sheet–helix diblock copolypeptides. Angewandte Chemie International Edition 2008, 47, 5160-5162.
(75) Wong, M. S.; Cha, J. N.; Choi, K.-S.; Deming, T. J.; Stucky, G. D. Assembly of nanoparticles into hollow spheres using block copolypeptides. Nano Letters 2002, 2, 583-587.
(76) Jan, J.-S.; Lee, S.; Carr, C. S.; Shantz, D. F. Biomimetic synthesis of inorganic nanospheres. Chemistry of materials 2005, 17, 4310-4317.
(77) Hou, S.-S.; Hsu, Y.-Y.; Lin, J.-H.; Jan, J.-S. Alkyl-poly (l-threonine)/Cyclodextrin Supramolecular Hydrogels with Different Molecular Assemblies and Gel Properties. ACS Macro Letters 2016, 5, 1201-1205.
(78) Kikhney, A. G.; Svergun, D. I. A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Letters 2015, 589, 2570-2577.
(79) Ryan, A.; Hamley, I.; Bras, W.; Bates, F. Structure development in semicrystalline diblock copolymers crystallizing from the ordered melt. Macromolecules 1995, 28, 3860-3868.