Solid-Phase Fragment Coupling Synthesis of Granulocyte Colony-Stimulating Factor

描述(申请人提供)：粒细胞集落刺激因子(G-CSF)是一种重要的造血生长因子，属于一大类生物相关的糖蛋白细胞因子。G-CSF有两种治疗形式，临床上用于治疗化疗所致的中性粒细胞减少症。这些形式之间的主要区别是苏氨酸-133上有或没有O-连接的碳水化合物。通过化学合成方法制备这两种治疗形式的既定平台可以解决人们对G-CSF糖基化重要性的担忧。此外，这一研究战略的进一步发展将允许以模块方式获得更有效的G-CSF变种。出于这些原因，糖蛋白G-CSF被认为是化学合成的目标。 开发有效的合成方法来制备这种治疗性糖蛋白是我们实验室正在进行的目标。这一建议需要对我们最近描述的固相片段偶联(SPFC)方法的反应范围和局限性进行调查。该方法可用于C端多肽硫酸和N端多肽固相偶联聚合制备大分子多肽和糖多肽。SPFC技术的应用将加快靶向糖蛋白G-CSF的合成。为了推进这一方法，将解决以下合成挑战：(1)C-末端硫代酸(+15-MERS)的高效制备和(2)异腈介导的C-末端硫酸与树脂结合的N-末端多肽的片段偶联以获得大的多肽(+30-MERS)。在这一进展之后，SPFC技术的实用性将在与G-CSF合成相关的靶向多肽片段的背景下进行考察。这些靶向多肽将通过化学连接技术组装起来，以制备均一的糖基化和非糖基化形式的G-CSF。

DESCRIPTION (provided by applicant): Granulocyte colony-stimulating factor (G-CSF) is an important hematopoietic growth factor belonging to a larger class of biologically relevant glycoprotein cytokines. Two therapeutic forms of G-CSF are used clinically for the treatment of neutropenia induced by chemotherapy. The principal difference between these forms is the presence or absence of an O-linked carbohydrate at threonine-133. An established platform to prepare both therapeutic forms by means of chemical synthesis could address concerns with regard to the importance of G-CSF glycosylation. Additionally, further development of this research strategy would allow for modular access to more efficacious variants of G-CSF. For these reasons, glycoprotein G-CSF is considered a target for chemical synthesis. The development of efficient synthetic methods for the preparation of such therapeutic glycoproteins is an ongoing goal in our laboratory. This proposal entails the investigation of reaction scope and limitations with regard to our recently described solid-phase fragment coupling (SPFC) method. This method will be useful for the convergent preparation of large polypeptides and glycopeptides by the coupling of C-terminal polypeptide thioacids and N-terminal polypeptides on solid-phase. The application of SPFC technology would expedite the synthesis of target glycoprotein, G-CSF. To advance this method, the following synthetic challenges will be addressed: (1) the efficient preparation of C-terminal thioacids (+15-mers) and (2) the isonitrile mediated fragment coupling of C-terminal thioacids with resin-bound N-terminal polypeptides to access large polypeptides (+30-mers). Following this advance, the utility of SPFC technology will be examined in the context of target polypeptide fragments relevant to G-CSF synthesis. These targeted polypeptides will be assembled by chemical ligation techniques to prepare homogenously glycosylated and non-glycosylated forms of G-CSF.