Chemoenzymatic synthesis of glycosylated and sulfated CCR5 N-terminal peptide library

糖基化和硫酸化 CCR5 N 末端肽库的化学酶合成

• 批准号: 10360095
• 负责人: Hailiang Joshua Zhu
• 金额: $ 35.09万
• 依托单位: DORDT COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360095
• 关键词: Achievement; Amino Acids; Binding; Biological; Biomedical Research; CCR5 gene; Complex; Disease; Excision; Exposure to; Glycopeptides; Health; Immune response; Inflammatory; Libraries; Ligands; Malignant Neoplasms; Mass Spectrum Analysis; Methodology; Methods; Modification; N-terminal; Pathway interactions; Pattern; Peptide Library; Peptide Synthesis; Peptides; Phase; Play; Polysaccharides; Post-Translational Protein Processing; Process; Proteomics; Reaction; Research; Role; Site; Solid; Structure; Sulfate; Talents; Technology; Testing; Tyrosine; glycosylation; glycosyltransferase; human disease; novel; sugar; tool; tyrosine O-sulfate; undergraduate student

Co-localization of Tyrosine (Tyr) sulfation and O-glycosylation (CSOG) is an emerging global pattern of post-translational modifications (PTMs). CSOG is the major PTM pattern on the N- terminal peptide of CC chemokine receptor 5 (CCR5). CCR5 is involved in critical human diseases such as cancers and inflammatory diseases, and the N-terminal peptide of CCR5 is essential for the binding between CCR5 and its ligands. The access to sulfated and glycosylated N-terminal peptides of CCR5 (CCR5-SGNTPs) and any other peptides with CSOG is hindered by diversified patterns of glycosylation and sulfation, lability of sulfation, and complex sugar structures. This proposal aims to develop efficient chemoenzymatic methods for synthesizing glycopeptides containing O-glycans and Tyr sulfation. Therefore, we are aiming to efficiently synthesize CCR5-SGNTPs to build up the access to CCR5-SGNTPs and further provide new clues for related biomedical research. Lability of sulfate group during synthesis and complexity of sugar structures are the major obstacles to the achievement of the library of CCR5-SGNTPs. We provide a novel and efficient chemoenzymatic approach, merging two well- studied synthetic methodologies to overcome the obstacles. One of the two methodologies is solid-phase site-selective sulfation, and the other one is chemoenzymatic synthesis of glycopeptides. Site-selective sulfation with divergently protected Tyrs can efficiently build diversified sulfation patterns, and chemoenzymatic synthesis of glycopeptides can synthesize glycopeptides with complex sugar structures in high regio- and stereo-selectivity with high fidelity. Notably, the mild conditions (slightly basic) of glycosyltransferases catalyzed reactions are ideal for keeping the integrity of the labile sulfate group. Glycosylated-amino acids (GAAs) with core sugars and Fmoc-Tyrs with divergent protecting groups can be synthesized efficiently with current synthetic methodologies. Then the two classes of building blocks will be selectively incorporated into solid phase peptide synthesis (SPPS) to obtain protected sulfated and glycosylated peptides. After removal of protecting groups, glycosyltransferases will be used to extend the core sugars to generate larger and more complex glycopeptides following biosynthetic pathways. After binding study of the synthesized library, detailed information about how the two PTMs co-regulate binding processes will be obtained. Moreover, a peptide library with structure-defined CCR5-SGNTPs will provide standards for proteomics and glycomics research of CCR5-SGNTPs.

酪氨酸 (Tyr) 硫酸化和 O-糖基化 (CSOG) 的共定位是一个新兴的全球性研究 翻译后修饰（PTM）的模式。 CSOG 是 N-上的主要 PTM 模式 CC 趋化因子受体 5 (CCR5) 的末端肽。 CCR5 参与关键人类 癌症和炎症等疾病，CCR5的N端肽是 对于 CCR5 及其配体之间的结合至关重要。获得硫酸盐和 CCR5 的糖基化 N 端肽 (CCR5-SGNTP) 和任何其他带有 CSOG 的肽 受到糖基化和硫酸化的多样化模式、硫酸化的不稳定性的阻碍，并且 复杂的糖结构。该提案旨在开发有效的化学酶方法 合成含有O-聚糖和Tyr硫酸化的糖肽。因此，我们的目标是 有效合成 CCR5-SGNTP 以建立对 CCR5-SGNTP 的访问并进一步 为相关生物医学研究提供新线索。合成过程中硫酸基团的不稳定性 糖结构的复杂性是实现糖​​类库的主要障碍 CCR5-SGNTP。我们提供了一种新颖有效的化学酶方法，融合了两种良好的 研究合成方法来克服障碍。两种方法之一是 固相位点选择性硫酸化，另一种是化学酶法合成 糖肽。具有不同保护的 Tyrs 的位点选择性硫酸化可以有效地构建 多样化的硫酸化模式，化学酶法合成糖肽可以合成 具有复杂糖结构的糖肽，具有高区域和立体选择性，具有高 保真度。值得注意的是，糖基转移酶催化反应的温和条件（弱碱性） 是保持不稳定硫酸基团完整性的理想选择。糖基化氨基酸 (GAA) 可以高效合成具有核心糖和具有不同保护基团的 Fmoc-Tyrs 使用当前的合成方法。然后这两类构建块将被选择性地 纳入固相肽合成 (SPPS) 以获得受保护的硫酸化和 糖基化肽。去除保护基后，将使用糖基转移酶 扩展核心糖以产生更大和更复杂的糖肽 生物合成途径。对合成文库进行结合研究后，详细信息如下： 如何获得两个 PTM 共同调节结合过程。此外，肽库 具有结构定义的 CCR5-SGNTP 将为蛋白质组学和糖组学提供标准 CCR5-SGNTPs的研究。