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-糖基化的共定位（CSOG）是一种新兴的全球性的 翻译后修饰（PTMs）。CSOG是N-S上的主要PTM模式， CC趋化因子受体5（CCR 5）末端肽。CCR 5参与了关键的人类 疾病如癌症和炎性疾病，并且CCR 5的N-末端肽是 CCR 5与其配体结合所必需的。获得硫酸盐和 CCR 5的糖基化N-末端肽（CCR 5-SGNTPs）和具有CSOG的任何其它肽 受到糖基化和硫酸化的多样化模式、硫酸化的不稳定性和 复杂的糖结构。该提案旨在开发有效的化学酶促方法， 合成含有O-聚糖的糖肽和Tyr硫酸化。因此，我们的目标是 有效地合成CCR 5-SGNTPs以建立对CCR 5-SGNTPs的获取，并进一步 为相关的生物医学研究提供了新的线索。合成过程中硫酸根基团的不稳定性， 糖结构的复杂性是实现 CCR5-SGNTPs。我们提供了一种新的和有效的化学酶的方法，合并两个良好的- 研究综合方法来克服障碍。两种方法之一是 固相定点硫酸化，另一种是化学酶促合成 糖肽用不同保护的Tyr进行位点选择性硫酸化可以有效地建立 多样化的硫酸化模式，和糖肽的化学酶促合成可以合成 具有复杂糖结构的糖肽具有高区域和立体选择性， 忠诚值得注意的是，糖基转移酶催化反应的温和条件（微碱性） 是保持不稳定硫酸根完整性的理想选择。糖基化氨基酸（GAs） 具有不同保护基的Fmoc-Tyr可以有效地合成 与目前的合成方法。然后，这两类构建块将被选择性地 结合到固相肽合成（SPPS）中以获得保护的硫酸化的和 糖基化肽除去保护基后，糖基转移酶将用于 延伸核心糖以产生更大和更复杂的糖肽， 生物合成途径在合成文库的结合研究之后，关于 将获得两个PTM如何共同调节结合过程。此外， 具有结构定义的CCR 5-SGNTPs将为蛋白质组学和糖组学提供标准 CCR 5-SGNTPs的研究。