脂联素糖蛋白及其类似物的化学合成研究

Protein glycosylation is an important class of protein posttranslational modifications and plays key role in cell-cell interaction and signaling. Protein posttranslational glycosylation is characterized by its heterogeneity, wherein several glycoforms are usually generated with potentially different biological properties. In order to systematically explore the evolutionary and functional significance of such heterogeneity, homogeneous glycoproteins with the defined oligosaccharide structure are required. The difficulties and challenges to obtain the homogeneous entities in the isolation and purification of specific glycoforms from the heterogeneous mixture underpin the importance of the chemical synthesis approach. Thus, the enabling chemical synthesis in the provision of the homogeneous glycoprotein with the defined structure will be important for the study of to correlate the glycan structure with its functions. Unlike nucleic acid and protein research wherein chemical synthesis has become a routine tool to provide moieties of interest, synthesis of glycopeptides/glycoproteins is still a daunting task, requiring know-how expertise and extensive efforts. Development of new and effective methods and strategies towards the chemical synthesis of glycoproteins is the key to drive the chemical glycobiology research. The first objective of the proposed project is to develop novel methods and strategies towards glycopeptide and glycoprotein synthesis. Serine and threonine are very rich in both N- and O-linked glycoproteins. A chemical ligation capable of ligating peptide segments at serine and threonine sites will be advantageous for the glycoprotein synthesis. The second objective of the proposed project is to apply the serine/threonine mediated peptide ligation for the total synthesis of adiponectin. Adiponectin is an intriguing glycoprotein with four Glc-Gal disaccharides linked to 5-hydroxylysine residues. It is involved in obesity and breast cancer, with the promising applications as a drug candidate for the treatment of liver diseases, type II diabetes, hypertension and other obesity-related disorders. The adiponectin protein is mainly composed of a collagenous domain (glycopeptide) and a globular domain. The function of the collagenous domain and the glycan structure has not been well defined, as there are difficulties to obtain the collagenous domain by recombinant protein expression. In the proposed project, we aim to develop an efficient synthetic strategy towards the glycopeptide fragment (collagenous domain) of adiponectin using chemical ligation, and we plan to synthesize various adiponectin collagenous domain analogues with different glycosylation motifs, which will enable one to comprehensively investigate the structure-activity relationship in adiponectin molecule and identify a minimal structure capable of inducing the required pharmacological activities.

作为蛋白质翻译后修饰的重要类型，蛋白质糖基化与细胞间相互作用和信号转导等诸多过程密切相关。因其异质化的结构特征，从非合成途径获取具有均一糖基化模式的产物极为困难，使得化学合成糖肽/糖蛋白对其功能研究的开展具有重要意义。相比于核酸与多肽，糖蛋白的合成更为困难，亟待发展高效的新方法与策略。糖肽/糖蛋白富含丝氨酸/苏氨酸的特征，使选择该残基作为连接位点具有潜在的优势。本项目针对糖蛋白的结构特征，致力于发展基于非修饰丝氨酸/苏氨酸残基的化学选择性多肽连接新方法，并将其应用于脂联素蛋白糖基化胶原结构域的合成中。通过该结构域及具有不同糖基化程度和位点的类似物的合成，全面研究其构效关系，以寻找具有药理学活性的最小结构单元。

脂联素是一个主要由脂肪组织生产的糖蛋白。它是骨骼肌和肝脏等外周器官葡萄糖和脂质代谢的关键调节因子，能够增强全身胰岛素敏感性和能量稳态。脂联素水平下降与胰岛素抵抗、2型糖尿病、脂肪性肝炎、心血管疾病和某些类型的癌症的发病机制有关。在此背景下，补充脂联素可能是一种非常有益的方法，具有治疗代谢、癌症和心血管疾病的潜力。然而，基于脂联素的治疗目前还不可能，主要是因为很难获得全长糖基化的人脂联素。.在此背景下，本课题组开展了糖基化脂联素胶原结构域的化学合成工作。为从头合成糖基化脂联素胶原结构域，我们将其分成两个部分，其一，糖基化5-羟基赖氨酸的构建；其二，运用糖基化5-羟基赖氨酸砌块，通过丝氨酸/苏氨酸残基的新型多肽连接反应（Serine/Threonine Ligation，STL），构建糖基化脂联素胶原结构域和全长脂联素。.本研究首次实现了适合Fmoc固相合成的糖基化5-羟基赖氨酸砌块的大规模制备合成，对合成路线的验证过程中，开发了不受邻位高位阻保护基影响的糖苷化方法，实现了高选择性5-羟基赖氨酸糖苷化反应。进一步，我们发现苄基（Bn）保护基在糖肽合成中存在难以脱除的缺，因此，发展了对酸性更敏感的对甲基苄基（MBn）保护基保护的砌块，并成功将其应用在糖基化脂联素胶原结构域衍生物的合成中，为其他类型的糖肽的合成提供了有价值的参考。对糖基化脂联素胶原结构域衍生物的合成，拓展了STL的应用范围，预示着STL具备合成复杂的糖肽的能力，可为其他治疗性糖肽物质的合成提供方法学支持。对糖基化脂联素胶原结构域衍生物的合成，探索了糖基化位置和程度对脂联素活性的影响，经过筛查发现四糖基化产物WM656877101最接近全长脂联素的活性。最后，我们开发了全长脂联素合成路线，实现了不带糖基化修饰的全长脂联素的合成，为后续糖基化全长脂联素合成打下基础。