Exploring the biology of O-acetyl sialic acids using stable synthetic mimics

项目摘要 使用稳定的合成模拟物探索O-乙酰基唾液酸的生物学 术语“唾液酸”倾向于与N-乙酰神经氨酸（Neu 5Ac，通常称为“唾液酸”）同义使用。 “NANA”）。事实上，Neu 5Ac只是不同分子家族中最常见的成员。O-乙酰化 唾液酸广泛存在于人类、其它脊椎动物和一些致病细菌中。o-乙酰 众所周知，修饰在许多生物学和病理学过程中发挥关键作用， 如免疫学、肿瘤学、病毒学和神经科学。然而，尽管他们发现了几十年， 以前，这些修饰的研究由于它们的不稳定性而受到很大的阻碍。O-乙酰基可以是 容易通过小的pH变化或通过酯酶水解，并且唾液酸的C-7和C-8处的O-乙酰基可以 迁移到C-9，有时甚至在生理条件下。到目前为止，还没有可靠的方法来 系统地研究含有这些不稳定的O-乙酰基的唾液酸聚糖的细胞生物学或病理学。 该提案首次将三个实验室结合在一起，结合了化学，生物和化学。 计算专业知识，以一种新的和系统的方式共同解决这一长期存在的问题。我们 假设用N-乙酰基取代唾液酸上的O-乙酰基是提供 研究这些重要分子的稳定模拟物。为了证明原理，我们证明了 在实验和计算上，5，9-二-N-乙酰神经氨酸（Neu 5Ac 9 NAc）是 天然存在的9-O-乙酰基-5-N-乙酰神经氨酸（Neu 5，9Ac 2）在各种类型的研究中。在当前 建议我们将进一步研究这个分子，以及N-乙酰神经氨酸（Neu 5Ac）的库 在C-4、C-7或C-8处具有N-乙酰基，或在C-7和C-9处、在C-8和C-9处具有两个N-乙酰基的衍生物， 或在C-4和C-9，都代表已知存在的不稳定O-乙酰化唾液酸的稳定模拟物 但仍未被充分探索含有这些N-乙酰基Neu 5Ac衍生物的唾液酸糖苷将是 化学酶促合成并用作探针来研究各种唾液酸的配体特异性， 哺乳动物或微生物来源的结合蛋白。O-乙酰化唾液酸苷和 它们的N-乙酰化对应物也将通过计算方法和NMR研究来探索。这 该项目将设计和产生重要的方法，以阐明基本机制和生物 唾液酸O-乙酰化的后果，打开大门，许多以前棘手的问题。而这 反过来，将有助于开发潜在的诊断和治疗方法，感染，恶性或免疫 涉及这些常见但知之甚少的唾液酸形式的过程。从长远来看，这种方法可以 可以扩展到自然界中的其它O-酰化唾液酸，例如9-O-乳酰基-Neu 5Ac（Neu 5Ac 9 Lt）和O- 乙酰化形式的非人N-羟乙酰神经氨酸（Neu 5Gc），其可能掺入人体 外源性的。

Project Summary Exploring the biology of O-acetyl sialic acids using stable synthetic mimics The term “sialic acid” tends to be used synonymously with N-acetylneuraminic acid (Neu5Ac, often called “NANA”). In fact, Neu5Ac is just the most common member of a diverse family of molecules. O-Acetylated sialic acids are widespread in humans, other vertebrates, and some pathogenic bacteria. The O-acetyl modifications are well known to play key roles in many biological and pathological processes, in fields as diverse as immunology, oncology, virology and neuroscience. However, despite their discovery many decades ago, the study of these modifications has been greatly hampered by their instability. O-Acetyl groups can be hydrolyzed easily by small pH changes or by esterases, and O-acetyl groups at C-7 and C-8 of sialic acids can migrate to C-9, sometimes even under physiological conditions. To date, there is no reliable approach to systematically investigate the cell biology or pathology of sialoglycans containing these labile O-acetyl groups. This proposal brings together for the first time three labs with the combined chemical, biological, and computational expertise to jointly tackle this long-standing problem in a new and systematic way. We hypothesized that substituting O-acetyl on sialic acids by N-acetyl groups is a suitable strategy to provide stable mimics for investigating these important molecules. For proof of principle, we have shown experimentally and computationally that 5,9-di-N-acetylneuraminic acid (Neu5Ac9NAc) is a good mimic of naturally occurring 9-O-acetyl-5-N-acetylneuraminic acid (Neu5,9Ac2) in various types of studies. In the current proposal we will further investigate this molecule, as well as a library of N-acetylneuraminic acid (Neu5Ac) derivatives with N-acetylation at C-4, C-7, or C-8, or with two N-acetyl groups at C-7 and C-9, at C-8 and C-9, or at C-4 and C-9, all representing stable mimics of unstable O-acetylated sialic acids that are known to occur in nature, but have remained underexplored. Sialosides containing these N-acetyl Neu5Ac derivatives will be chemoenzymatically synthesized and used as probes to study the ligand specificity of various sialic acid- binding proteins of mammalian or microbial origin. The structural comparison of O-acetylated sialosides and their N-acetylated counterparts will also be explored by computational methods and by NMR studies. This project will design and generate important approaches to elucidate fundamental mechanisms and biological consequences of sialic acid O-acetylation, opening the door to many previously intractable questions. This, in turn, will help to develop potential diagnostic and therapeutic approaches for infectious, malignant or immune processes involving these common but poorly understood sialic acid forms. In the long run, the approach can be extended to other O-acylated sialic acids in nature such as 9-O-lactyl-Neu5Ac (Neu5Ac9Lt), and O- acetylated forms of non-human N-glycolylneuraminic acid (Neu5Gc) which might be incorporated into humans from exogenous sources.