类动物丝蛋白聚合物的合成、表征及其与蚕丝蛋白的共混
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摘要
本论文在二级结构水平上对动物丝(蚕丝、蜘蛛丝)蛋白进行了模拟物的合成，得到了一类结构明晰并具有与动物丝蛋白相类似二级结构的模拟聚合物，以用作研究成纤过程中丝蛋白分子链的折叠方式及其在三维空间的有序排列对所最终所得丝纤维力学性能影响的基本材料。
首先采用传统的液相多肽合成法合成了含有源于动物丝蛋白结晶区氨基酸序列(GlyAlaGlyAla)、(AlaGlyAlaGly)及(Ala)_4的四种功能性多肽单体M1～M4。然后在碱的催化作用下，将这些多肽单体和HDI在DMSO中进行聚合，得到四种新型类动物丝蛋白模拟聚合物P1～P4。所合成的模拟聚合物并不是蛋白质，而是短链多肽与非多肽片段的交替共聚物。
所合成的模拟聚合物难溶于通常溶剂(甚至DMSO)，但可以溶解在强酸溶剂(甲酸、二氯乙酸及三氟乙酸)和六氟异丙醇中；通过在二氯乙酸中25℃下的特性粘度测定而估算出聚合物的分子量在2～5万之间。由于模拟聚合物中所含的脲键存在着热不稳定性，所有聚合物的热分解温度都在220℃附近(低于天然动物丝蛋白分解温度)，并且所有聚合物样品在熔融之前已开始分解；另外只有分子量比较大的模拟聚合物P4有明显的玻璃化转变(T_g=107℃)。由此可见，模拟聚合物与动物丝蛋白在一级结构方面的差别导致了两者不同的热性能。
红外光谱、交叉极化／魔角旋转固态~(13)C核磁共振波谱及广角X射线衍射分析的结果表明：在所合成的多嵌段类动物丝蛋白模拟共聚物(P3、P4)中源于动物丝蛋白结晶区的多肽片段的分子链以β-折叠为主的构象状态存在(同时也存在着无
中英文摘要
规构象状态);这些以p一折叠状态为主的分子链聚集在一起形成类似于动物丝蛋
白的有序区域并因此而结晶，表现出和动物丝蛋白相类似的聚集态结构(二级结
构)。以上实验结果证实了本论文所提出的在二级结构水平上对动物丝蛋白进行
了模拟合成方法的可行性。
另外还进行了类动物丝蛋白聚合物I，3、P4和蚕丝蛋白的共混研究。分析结
果表明:在P3、P4中源于动物丝结晶区氨基酸序列的多肤片段一一一GlyAlaGlyAla
及(Ala)’和蚕丝蛋白分子链之间能形成很强的分子间氢键相互作用。纯蚕丝蛋白
膜和P4膜都处于以无规线团/a一螺旋为主.的构象状态，而共混后组分间产生的分
子间强相互作用诱导并促使丝素蛋白分子链中原处于无规线团/a一螺旋构象部分
向p一折叠构象转变，使整个共混膜处于无规线团/a一螺旋和p一折叠构象共同存在
的状态。而共混膜的结晶情况则和模拟聚合物中所含多肤片段的氨基酸序列相
关。另外在与蚕丝蛋白的共混体系中，P3、P4和蚕丝蛋白是基本相容的。这些
结论对今后要进行的类动物丝蛋白聚合物成纤条件研究及人工蚕丝、人工蜘蛛丝
的纺制都具有重要指导意义。
In this work, on the secondary structure level, a series of well-defined structure polymers which had the similar secondary structure with animal silk proteins (i.e. silkworm fibroin and spidroin) were synthesized by the condensed polymerization of the functional oligopeptides and diisocyanate. These silk-protein-like polymers could be used as the basic materials for understanding the effect of chain folding models of fibroin and their three-dimensional order which be formed in the spinning process on the mechanical properties of silk fibers.
Firstly, four functional oligopeptide monomers (M1~M4), containing the amino sequence [GlyAlaGlyAla, AlaGlyAlaGly, (Ala)4] which derived from the crystal region of silkworm (Bombyx mori) silk and spider dragline silk, were synthesized with the traditional liquid-phase peptide synthesis method. Then the well-defined structure multiblock polymers (P1~P4) were obtained from the polymerization of the functional oligopeptides and hexamethylene diisocyanate (HDI) with the base as catalyst. These simulated polymers were the alternating copolymers formed by oligopeptide segment and non-peptide segment instead of the pure protein.
The silk-protein-like polymers could be dissolved in hexafluroisopropanol and strong organic acid (e.g., formic acid, dichloroacetic acid and trifluroacetic acid), while they showed a poor solubility in common solvents. According to the intrinsic viscosity in dichloroacetic acid at 25, the molecular weight of polymers were estimated to be 20,000~50,000. Because of the thermal instability of the urea bond in
molecular chain, all synthetic polymers began to degrade around 220, and had no melting point. In addition, only P4 which had a higher molecular weight relative to the others showed an apparent glass transition at 107. Therefore, it's reasonable that the differences of thermal properties between the simulated polymers and animal silk protein were attributed to their different primary structures.
The FT-IR, CP/MAS Solid-state C-NMR and Wide Angle X-ray Diffraction measurements on P3 and P4 revealed that a major -sheet conformation as well as other conformations coexisted in the polymers. In solid-state P3 and P4, the molecular chain in p-sheet conformation spontaneously aggregated into some ordered regions, and then formed the crystals which be similar to that in natural silk. Namely the silk-protein like polymers had the similar solid-state structures with animal silk proteins. These results meant our design for the simulated synthesis of the silk-protein like polymers on the secondary structure level was feasible and successful.
In addition, the blends of the silk-protein like polymer (P3, P4) and silkworm fibroin were studied. The experimental results revealed that the P-sheet and random coil/a-helix conformation coexisted in the SF/P3 and SF/P4 blend films, while the predominant conformation in the pure P4 and fibroin film were random coil/a-helix. The intermolecular hydrogen-bond interaction, which be formed between the molecular chain of fibroin and the oligopeptide segments in the silk-protein like polymers, induced a partial random coil/a-helix conformation transfer to P-sheet conformation after blending, and some ordered regions were formed by the aggregation of the molecular chain in P-sheet conformation. The crystal properties of the SF/P3 and SF/P4 blend films were dependent on the amino acid sequence of the oligopeptide in P3 and P4. The cross-section morphology of the blend films indicated silkworm fibroin and these simulated polymers were miscible in their blend films. These conclusions would be important for searching
the spinning condition of the silk-protein like polymers as well as producing artificial fibers of animal silk protein.
首先采用传统的液相多肽合成法合成了含有源于动物丝蛋白结晶区氨基酸序列(GlyAlaGlyAla)、(AlaGlyAlaGly)及(Ala)_4的四种功能性多肽单体M1～M4。然后在碱的催化作用下，将这些多肽单体和HDI在DMSO中进行聚合，得到四种新型类动物丝蛋白模拟聚合物P1～P4。所合成的模拟聚合物并不是蛋白质，而是短链多肽与非多肽片段的交替共聚物。
所合成的模拟聚合物难溶于通常溶剂(甚至DMSO)，但可以溶解在强酸溶剂(甲酸、二氯乙酸及三氟乙酸)和六氟异丙醇中；通过在二氯乙酸中25℃下的特性粘度测定而估算出聚合物的分子量在2～5万之间。由于模拟聚合物中所含的脲键存在着热不稳定性，所有聚合物的热分解温度都在220℃附近(低于天然动物丝蛋白分解温度)，并且所有聚合物样品在熔融之前已开始分解；另外只有分子量比较大的模拟聚合物P4有明显的玻璃化转变(T_g=107℃)。由此可见，模拟聚合物与动物丝蛋白在一级结构方面的差别导致了两者不同的热性能。
红外光谱、交叉极化／魔角旋转固态~(13)C核磁共振波谱及广角X射线衍射分析的结果表明：在所合成的多嵌段类动物丝蛋白模拟共聚物(P3、P4)中源于动物丝蛋白结晶区的多肽片段的分子链以β-折叠为主的构象状态存在(同时也存在着无
中英文摘要
规构象状态);这些以p一折叠状态为主的分子链聚集在一起形成类似于动物丝蛋
白的有序区域并因此而结晶，表现出和动物丝蛋白相类似的聚集态结构(二级结
构)。以上实验结果证实了本论文所提出的在二级结构水平上对动物丝蛋白进行
了模拟合成方法的可行性。
另外还进行了类动物丝蛋白聚合物I，3、P4和蚕丝蛋白的共混研究。分析结
果表明:在P3、P4中源于动物丝结晶区氨基酸序列的多肤片段一一一GlyAlaGlyAla
及(Ala)’和蚕丝蛋白分子链之间能形成很强的分子间氢键相互作用。纯蚕丝蛋白
膜和P4膜都处于以无规线团/a一螺旋为主.的构象状态，而共混后组分间产生的分
子间强相互作用诱导并促使丝素蛋白分子链中原处于无规线团/a一螺旋构象部分
向p一折叠构象转变，使整个共混膜处于无规线团/a一螺旋和p一折叠构象共同存在
的状态。而共混膜的结晶情况则和模拟聚合物中所含多肤片段的氨基酸序列相
关。另外在与蚕丝蛋白的共混体系中，P3、P4和蚕丝蛋白是基本相容的。这些
结论对今后要进行的类动物丝蛋白聚合物成纤条件研究及人工蚕丝、人工蜘蛛丝
的纺制都具有重要指导意义。
In this work, on the secondary structure level, a series of well-defined structure polymers which had the similar secondary structure with animal silk proteins (i.e. silkworm fibroin and spidroin) were synthesized by the condensed polymerization of the functional oligopeptides and diisocyanate. These silk-protein-like polymers could be used as the basic materials for understanding the effect of chain folding models of fibroin and their three-dimensional order which be formed in the spinning process on the mechanical properties of silk fibers.
Firstly, four functional oligopeptide monomers (M1~M4), containing the amino sequence [GlyAlaGlyAla, AlaGlyAlaGly, (Ala)4] which derived from the crystal region of silkworm (Bombyx mori) silk and spider dragline silk, were synthesized with the traditional liquid-phase peptide synthesis method. Then the well-defined structure multiblock polymers (P1~P4) were obtained from the polymerization of the functional oligopeptides and hexamethylene diisocyanate (HDI) with the base as catalyst. These simulated polymers were the alternating copolymers formed by oligopeptide segment and non-peptide segment instead of the pure protein.
The silk-protein-like polymers could be dissolved in hexafluroisopropanol and strong organic acid (e.g., formic acid, dichloroacetic acid and trifluroacetic acid), while they showed a poor solubility in common solvents. According to the intrinsic viscosity in dichloroacetic acid at 25, the molecular weight of polymers were estimated to be 20,000~50,000. Because of the thermal instability of the urea bond in
molecular chain, all synthetic polymers began to degrade around 220, and had no melting point. In addition, only P4 which had a higher molecular weight relative to the others showed an apparent glass transition at 107. Therefore, it's reasonable that the differences of thermal properties between the simulated polymers and animal silk protein were attributed to their different primary structures.
The FT-IR, CP/MAS Solid-state C-NMR and Wide Angle X-ray Diffraction measurements on P3 and P4 revealed that a major -sheet conformation as well as other conformations coexisted in the polymers. In solid-state P3 and P4, the molecular chain in p-sheet conformation spontaneously aggregated into some ordered regions, and then formed the crystals which be similar to that in natural silk. Namely the silk-protein like polymers had the similar solid-state structures with animal silk proteins. These results meant our design for the simulated synthesis of the silk-protein like polymers on the secondary structure level was feasible and successful.
In addition, the blends of the silk-protein like polymer (P3, P4) and silkworm fibroin were studied. The experimental results revealed that the P-sheet and random coil/a-helix conformation coexisted in the SF/P3 and SF/P4 blend films, while the predominant conformation in the pure P4 and fibroin film were random coil/a-helix. The intermolecular hydrogen-bond interaction, which be formed between the molecular chain of fibroin and the oligopeptide segments in the silk-protein like polymers, induced a partial random coil/a-helix conformation transfer to P-sheet conformation after blending, and some ordered regions were formed by the aggregation of the molecular chain in P-sheet conformation. The crystal properties of the SF/P3 and SF/P4 blend films were dependent on the amino acid sequence of the oligopeptide in P3 and P4. The cross-section morphology of the blend films indicated silkworm fibroin and these simulated polymers were miscible in their blend films. These conclusions would be important for searching
the spinning condition of the silk-protein like polymers as well as producing artificial fibers of animal silk protein.
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