Facile Synthesis of Glycosulfopeptides and Related Bioconjugates

糖磺肽和相关生物结合物的简便合成

• 批准号: 8982359
• 负责人: RICHARD D CUMMINGS
• 金额: $ 60.8万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8982359
• 关键词: Affinity; Alkanesulfonates; Binding; Biological; Biological Assay; CCR5 gene; Carbohydrates; Cells; Cellular Structures; Chemicals; Chemistry; Complex; Disease; Epitopes; Exhibits; Factor VIII; Family member; Flow Cytometry; Generations; Genetic Techniques; Glycoconjugates; Human; Immune response; Inflammatory Response; Inorganic Sulfates; Integrins; Leukocytes; Libraries; Ligands; Ligation; Liposomes; Malignant Neoplasms; Membrane Glycoproteins; Microfluidics; Microspheres; Modeling; Molecular Genetics; Mus; Natural Immunity; P-Selectin; P-selectin ligand protein; Peptides; Phenylalanine; Play; Polysaccharides; Printing; Process; Role; Scheme; Selectins; Structure; Substrate Specificity; Surface; System; Therapeutic Agents; Thrombosis; Tyrosine; Unspecified or Sulfate Ion Sulfates; Ursidae Family; Variant; Vertebral column; analog; base; biomacromolecule; chemical genetics; chemokine; cold temperature; density; design; drug candidate; functional group; glycosylation; innovation; member; molecular assembly/self assembly; molecular recognition; mutant; novel; protein aminoacid sequence; public health relevance; sortase; sugar; tool; tyrosine O-sulfate

 DESCRIPTION (provided by applicant): "Glycosulfopeptides" (GSP) represent a novel type of complex glycoconjugate, which contain a recognition motif consisting of clustered tyrosine sulfates (Tyr-SO3H) and a core 2 O-glycan that bears a sialyl LewisX-containing hexasaccharide epitope (C2-O-sLeX). GSPs are present in PSGL-1, GPIba, endoglycan, VWF, Factor VIII, and CCR5, among many other biomolecules and play critical roles in innate immunity, thrombosis, and cancer. Despite the potential of sLeX-GSPs to serve as tools for biological studies and their promise as therapeutic agents, their utility has been limited by low yield and limited stability. Recent innovations by the investigative team in chemical and molecular genetic techniques have established an important new framework for the design of sLeX-GSPs and related multicomponent conjugates. Specifically, we intend to: (1) Define efficient schemes for scalable generation of GSPs containing tri-member recognition motifs (Tyr-SO3H, C2-O-sLeX, peptide backbone). Poor regio- and stereoselectivity and low yield continues to pose a significant challenge for the synthesis of C2-O-sLeX hexasaccharides and related variants. To overcome these limitations, we will design convergent glycosylation schemes in association with low temperature pre-activation, building blocks that require minimal protecting group manipulation, as well as one-pot schemes for glycans containing sLeX. To expand the diversity of novel GSP structures, new analogues will be generated in which tyrosine sulfates are replaced by sulfonates, Glu or Asp, and sulfated sugars incorporated in the glycan. (2) Identify orthogonal bioconjugation strategies for the construction of GSP bearing microarrays, as well as microspheres and liposomes as cellular `avatars'. Chemical and genetic orthogonal bioconjugation schemes will be investigated for the construction of microarrays containing printed GSPs, as well as GSP bearing microspheres and liposomes as cellular `avatars' to mimic cell-like presentation of ligands. In addition to standard bioorthogonal approaches, such as click chemistry and native chemical ligation, mutant sortase variants that exhibit orthogonal substrate specificity for peptide sequences LPETG, LAETG, and LPESG will be used to construct highly divergent GSP supramolecular systems. In the process, we will also define complementary approaches for the generation of GSP multimers and macrocycles. (3) Construct libraries of native and non-natural sLeX-bearing GSPs for the identification of highly potent selectin, integrin, and chemokine antagonists. We will generate microarrays, microspheres, and liposomes presenting GSPs modeled after human and murine PSGL-1, GPIba, endoglycan, and CCR5. Candidate high affinity selectin, integrin, and chemokine antagonists will be identified through a combination of microarray-based screens, Biacore analysis, flow cytometry, and microfluidic cell binding assays. GSP-derivatized microspheres and liposomes, as cellular `avatars', will define the density- and presentation-dependent features that influence molecular recognition.

 描述（由申请人提供）：“糖磺肽”（GSP）代表一种新型的复合糖缀合物，其含有由成簇的酪氨酸硫酸盐（Tyr-SO 3 H）组成的识别基序和带有含唾液酸化LewisX的六糖表位（C2-O-sLeX）的核心2 O-聚糖。GSP存在于PSGL-1、GPIba、内聚糖、VWF、因子VIII和CCR 5以及许多其他生物分子中，并且在先天免疫、血栓形成和癌症中起关键作用。尽管sLeX-GSPs有潜力作为生物学研究的工具，并有望作为治疗剂，但其实用性受到低产率和有限稳定性的限制。研究小组最近在化学和分子遗传技术方面的创新为sLeX-GSPs和相关多组分缀合物的设计建立了一个重要的新框架。具体而言，我们打算：（1）定义用于可规模化产生含有三成员识别基序（Tyr-SO 3 H、C2-O-sLeX、肽骨架）的GSP的有效方案。差的区域和立体选择性和低产率继续对C2-O-sLeX六糖和相关变体的合成构成重大挑战。为了克服这些限制，我们将设计与低温预活化相关的会聚糖基化方案，需要最小保护基团操作的构建模块，以及含有sLeX的聚糖的一锅法方案。为了扩大新型GSP结构的多样性，将产生新的类似物，其中酪氨酸硫酸盐被磺酸盐、Glu或Asp取代，并且硫酸化糖并入聚糖中。(2)确定用于构建GSP轴承微阵列以及微球和脂质体作为细胞“化身”的正交生物缀合策略。将研究化学和遗传正交生物缀合方案，用于构建含有印刷的GSP的微阵列，以及作为细胞“化身”的带有GSP的微球和脂质体，以模拟配体的细胞样呈递。除了标准的生物正交方法，如点击化学和天然化学连接之外，对肽序列LPETG、LAETG和LPESG表现出正交底物特异性的突变分选酶变体将用于构建高度发散的GSP超分子系统。在此过程中，我们还将定义生成GSP多聚体和大环化合物的补充方法。(3)构建天然和非天然sLeX-承载的GSPs文库，用于鉴定高效的选择素、整合素和趋化因子拮抗剂。我们将生成微阵列、微球和脂质体，这些微阵列、微球和脂质体呈现以人和鼠PSGL-1、GPIba、内聚糖和CCR 5为模型的GSP。候选的高亲和力选择素、整合素和趋化因子拮抗剂将通过基于微阵列的筛选、Biacore分析、流式细胞术和微流体细胞结合测定的组合来鉴定。作为细胞“化身”的GSP衍生微球和脂质体将限定影响分子识别的密度和呈递依赖性特征。