Facile Synthesis of Glycosulfopeptides and Related Bioconjugates

 描述(由申请人提供)：“糖磺肽”(GSP)是一种新型的复合糖共轭化合物，它包含一个识别基序，该基序由簇状酪氨酸硫酸盐(Tyr-SO3H)和一个核心的O-糖链组成，该基序带有唾液酸路易斯X-六糖表位(C2-O-SLeX)。GSP存在于PSGL-1、GPIba、内聚糖、VWF、因子VIII和CCR5等许多生物分子中，在先天性免疫、血栓形成和癌症中发挥关键作用。尽管SLeX-GSP具有作为生物学研究工具的潜力和作为治疗剂的前景，但它们的用途一直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设计SLeX-GSP和相关的多组分连接物建立了一个重要的新框架。具体地说，我们打算：(1)定义高效的方案，用于可扩展地生成包含三成员识别基序(Tyr-SO3H，C2-O-SLeX，肽骨架)的GSP。较差的区域和立体选择性以及较低的产率仍然是合成C2-O-SLeX六糖及其相关变体的一个重大挑战。为了克服这些限制，我们将设计与低温预活化相结合的收敛糖基化方案，需要最少保护基团操作的构建块，以及包含SLeX的糖链的一锅方案。为了扩大新的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，并在多糖中加入硫酸糖。(2)确定用于构建携带GSP的微阵列以及作为细胞“Avatars”的微球和脂质体的正交生物结合策略。将研究化学和遗传的正交生物结合方案，以构建含有印刷的GSP的微阵列，以及含有GSP的微球和脂质体作为细胞的“Avatars”，以模拟配体的细胞样呈现。除了标准的生物正交法，如点击化学和天然化学连接，对肽序列LPETG、LAETG和LPESG表现出正交底物专一性的突变的索酸酶变体将被用于构建高度不同的GSP超分子系统。在这一过程中，我们还将确定产生普惠制多聚体和大循环的补充方法。(3)构建天然和非天然携带SLeX的GSP文库，用于鉴定高效的选择素、整合素和趋化因子拮抗剂。我们将产生微阵列、微球和脂质体，呈现仿照人和小鼠PSGL-1、GPIba、内聚糖和CCR5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、Biaccore分析、流式细胞术和微流控细胞结合分析相结合的方法进行鉴定。GSP衍生的微球和脂质体，如细胞的“Avatars”，将定义影响分子识别的密度和呈现相关特征。

 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.