【作者】 徐翔；

【导师】 郑楠；

【作者基本信息】 大连理工大学 ， 高分子材料， 2024， 博士

【摘要】 人工合成聚多肽作为天然蛋白质的类似物,不仅具有良好的生物相容性和独特的二级结构,而且通过引入各种非天然成分,可以实现结构和功能的多样性。相较于传统聚合物,功能聚多肽的二级结构带来的膜活性和可调控性使其在基因/药物递送、抗菌等方面得到了广泛应用,对聚多肽的化学结构和构象的合理设计可以赋予其独特的生物功能,在生物医用领域具有极大的应用价值和研究意义。聚多肽的二级结构受到多种因素影响,如氢键、极性和静电相互作用等,通过调控这些影响因素可以实现聚多肽的二级结构的稳定性和刺激响应性。聚合后修饰(Post-polymerization modification,PPM)策略是构建功能聚多肽的方法之一,其中,铜催化的叠氮-炔环加成(Azidealkyne cycloaddition,AAC)反应最为常见。因此,本文通过传统的AAC两组分反应和基于AAC拓展的多组分反应(Multicomponent reaction,MCR)对聚多肽进行PPM,合成了一系列结构多样、功能丰富的聚多肽材料,系统研究了侧链功能化聚多肽的二级结构,总结构效关系,并进一步拓展其在抗病毒领域、调控光敏剂活性、基因递送以及刺激响应型药物递送等方面的应用。主要研究内容如下:基于AAC反应构建了一系列阴离子聚多肽和高度硫酸化(>99%)的糖基聚多肽,并以严重急性呼吸综合症冠状病毒2(SARS-Co V-2)假病毒作为模型,系统研究了这些聚多肽和抗病毒活性之间的构效关系。结果显示α-螺旋构象和硫酸化糖起着关键作用。具有α-螺旋结构的硫酸化糖基聚多肽表现出优异的抗病毒活性,其抑制效果高达85%,半数抑制浓度为0.71μg/m L。刚性侧链结构和适度的分子量也有助于防止病毒进入宿主细胞。此外,这些硫酸化糖基聚多肽还能有效防止肠病毒感染,其抑制效率高达86%。基于AAC反应设计合成了带有四苯基卟啉(Tetraphenylporphyrin,TPP)侧链的阳离子聚多肽,系统研究了聚多肽二级结构在克服疏水光敏剂(Photosensitizers,PS)的聚集诱导猝灭(Aggregation-caused quenching,ACQ)效应方面的作用。通过全原子分子动力学模拟和实验结果的结合,证明了α-螺旋聚多肽骨架通过抑制TPP单元之间的π-π堆积相互作用,显著提高了荧光量子产率和活性氧(Reactive oxygen species,ROS)产率。α-螺旋构象也提高了聚多肽的穿膜能力、细胞摄取水平和细胞内的ROS产率。同时,在H22荷瘤小鼠体内也展现出了增强的光动力治疗(Photodynamic therapy,PDT)效率。在AAC的基础上,提出了“叠氮、炔、亲电试剂”的MCR策略作为聚多肽的PPM策略,构建了一类以1,4,5-全取代三氮唑(1,4,5-Fully substituted triazole,FT)为连接基团的阳离子聚多肽。该策略避免了传统AAC引入的1,4-二取代三唑(1,4-disubstituted triazole,DT)连接基上的氢键作用,使得聚多肽在广泛的pH范围内保持稳定的α-螺旋结构。这种α-螺旋聚多肽具有优异的穿膜活性、基因包封效率和低毒性,能够作为基因载体实现体外基因转染。FT结构上的反应性基团(烯烃)还为后续的功能化提供了修饰位点,进一步通过“巯基-烯”反应引入羟基提高了阳离子聚多肽的抗血清能力,促进了具有多个官能团的功能性聚多肽的开发,还为设计广谱pH范围下具有稳定α-螺旋结构的聚多肽提供了新的途径。在“叠氮、炔、亲电试剂”的MCR策略的基础上,发展了一类“磺酰基叠氮、炔、胺”的MCR策略对聚多肽进行PPM,在聚多肽侧链上引入了功能性的连接基团N-磺酰脒(N-sulfonymidine,SAi)。系统研究了SAi结构在响应pH变化时调控聚多肽从无规卷曲到α-螺旋的构象转变的能力。通过调控侧链SAi的结构,SAi-聚多肽能够精确响应肿瘤微酸环境并发生构象变化。在生理条件下(pH=7.4),由于SAi基团去质子化引起的静电相互作用使得聚多肽呈现无规卷曲构象。而在肿瘤细胞外(pH=6.8),SAi的质子化对电荷的中和诱导了α-螺旋构象的恢复,促进了细胞内摄取过程。同时,细胞内环境的进一步酸化加速了药物释放。在H22荷瘤小鼠模型中证实了SAi-聚多肽的良好的生物安全性和作为药物载体增强肿瘤化疗的能力。这种连接基团的未知功能的探索不仅丰富了二级结构的调控策略,还为pH响应型材料的设计提供了新的见解。更多还原

【Abstract】 Artificially synthesized polypeptides,as analogs of natural proteins,not only exhibit excellent biocompatibility and unique secondary structures but also achieve structural and functional diversity through the introduction of various non-natural components.In comparison to traditional polymers,the membrane activity and controllability brought about by the secondary structure make functional polypeptides widely applied in gene/drug delivery,antimicrobial activities,and more.The rational design of the chemical structure and conformation of polypeptides can impart them with unique biological functions,rendering them highly valuable and significant in the field of biomedical applications.The secondary structure of polypeptides is influenced by various factors such as hydrogen bonds,polarity,and electrostatic interactions,providing insights into regulating the stability of polypeptides’ secondary structures and designing stimuli-responsive materials based on polypeptides.Postpolymerization modification(PPM)strategie is one of the methods to construct functional polypeptides,with copper-catalyzed azide-alkyne cycloaddition(AAC)reactions being the most common.Therefore,this paper employs traditional AAC two-component reactions and AAC-based multicomponent reactions(MCR)for the PPM of polypeptides,synthesizing a series of structurally diverse and functionally rich polypeptide materials.The study systematically investigates the secondary structure of side-chain-functionalized polypeptides,summarizes structure-function relationships,and further explores their applications in the fields of antiviral activity,regulation of photosensitizer activity,gene delivery,and stimuli-responsive drug delivery.The main research contents are as follows:A series of anionic polypeptides and highly sulfated(>99%)glycosylated polypeptides were constructed based on AAC reactions.Using the severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)pseudovirus as a model,the study systematically investigates the structure-activity relationship between these polypeptides and antiviral activity.Results show that the α-helical conformation and sulfation of glycosylated polypeptides play a crucial role.Glycosylated polypeptides with an α-helical structure exhibit excellent antiviral activity,with an inhibition efficiency of up to 85%,and a half-maximal inhibitory concentration of 0.71μg/m L.Other structural characteristics,including a rigid chain structure and moderate molecular weight,also help prevent virus entry into host cells.These sulfated glycosylated polypeptides also prevent enterovirus infection with an inhibition efficiency of up to 86%.Cationic polypeptides with side chains containing tetraphenylporphyrin(TPP)were designed and synthesized based on AAC reactions.The study systematically investigates the role of polypeptides’ secondary structure in overcoming the aggregation-induced quenching(ACQ)effect of hydrophobic photosensitizers(PS).Through a combination of all-atom molecular dynamics simulations and experimental results,it is demonstrated that the α-helical polypeptide backbone significantly enhances fluorescence quantum yield and reactive oxygen species(ROS)production by inhibiting π-π stacking interactions between TPP units.The α-helical conformation also enhances the transmembrane ability,cellular uptake levels,and intracellular ROS production of polypeptides.Moreover,enhanced photodynamic therapy(PDT)efficiency is demonstrated in H22 tumor-bearing mice.Based on AAC,a multicomponent reaction(MCR)strategy involving "azide,alkyne,electrophilic reagent" is proposed as a PPM strategy for polypeptides.A class of cationic polypeptides with 1,4,5-fully substituted triazole(FT)as a linking group is constructed.This strategy avoids the hydrogen bonding on the 1,4-disubstituted triazole(DT)connecting group introduced by traditional AAC,allowing polypeptides to maintain a stable α-helical structure over a wide pH range.These α-helical polypeptides exhibit excellent transmembrane activity,gene encapsulation efficiency,and low toxicity,making them suitable as gene carriers for in vitro gene transfection.The reactive group(alkene)on the FT structure also provides additional modification sites for subsequent functionalization.The introduction of thiol-alkene groups on FT also enhances the serum resistance of cationic polypeptides and promotes the development of functional polypeptides with multiple functional groups.This approach also provides a new pathway for designing polypeptides with a stable α-helical structure over a broad pH range.Based on the MCR strategy of "azide,alkyne,electrophilic reagent," a novel MCR strategy involving "sulfonyl azide,alkyne,amine" is developed for PPM of polypeptides,introducing the functional connecting group N-sulfonymidine(SAi)into the side chain of polypeptides.The study systematically investigates the ability of SAi structure to regulate the conformational transition of polypeptides from random coils to α-helices in response to pH changes.By modulating the structure of side-chain SAi,SAi-polypeptides can precisely respond to the acidic microenvironment of tumors and undergo conformational changes.Under physiological conditions(pH = 7.4),electrostatic interactions induced by SAi deprotonation cause polypeptides to adopt random-coil conformations.However,in the extracellular environment of tumor cells(pH = 6.8),protonation of SAi neutralizes the charge,inducing the recovery ofα-helical conformation,promoting cellular uptake.Simultaneously,further acidification of the intracellular environment accelerates drug release.The study confirms the excellent biocompatibility of SAi-polypeptides and their ability to enhance tumor chemotherapy as drug carriers in an H22 tumor-bearing mouse model.The exploration of the unknown functions of this connecting group not only enriches the regulation strategies of secondary structures but also provides new insights into the design of pH-responsive materials.更多还原