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

项目概要 使用稳定的合成模拟物探索 O-乙酰唾液酸的生物学 术语“唾液酸”往往与 N-乙酰神经氨酸（Neu5Ac，通常称为 “娜娜”）。事实上，Neu5Ac 只是多样化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、其他脊椎动物和一些致病细菌中。 O-乙酰基 众所周知，修饰在许多生物和病理过程中发挥着关键作用，例如 免疫学、肿瘤学、病毒学和神经科学等。然而，尽管他们发现了几十年 以前，对这些修饰的研究因其不稳定性而受到极大阻碍。 O-乙酰基可以是 通过小的 pH 变化或通过酯酶很容易水解，并且唾液酸的 C-7 和 C-8 处的 O-乙酰基可以 有时甚至在生理条件下也会迁移到 C-9。迄今为止，还没有可靠的方法 系统地研究含有这些不稳定 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，将化学、生物和 计算专业知识以一种新的、系统的方式共同解决这个长期存在的问题。我们 假设用 N-乙酰基取代唾液酸上的 O-乙酰基是一种合适的策略，以提供 用于研究这些重要分子的稳定模拟物。为了原理证明，我们已经证明 实验和计算表明 5,9-二-N-乙酰神经氨酸 (Neu5Ac9NAc) 是 各种类型的研究中天然存在的 9-O-乙酰基-5-N-乙酰神经氨酸 (Neu5,9Ac2)。在当前 根据建议，我们将进一步研究该分子以及 N-乙酰神经氨酸 (Neu5Ac) 文库 在C-4、C-7或C-8处具有N-乙酰化，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 处，均代表已知存在的不稳定 O-乙酰化唾液酸的稳定模拟物 自然界中存在，但尚未得到充分探索。含有这些 N-乙酰基 Neu5Ac 衍生物的唾液酸苷将是 化学酶法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O-乙酰化唾液酸苷的结构比较 它们的 N-乙酰化对应物也将通过计算方法和核磁共振研究进行探索。这个 项目将设计和产生重要的方法来阐明基本机制和生物 唾液酸 O-乙酰化的后果，为许多以前棘手的问题打开了大门。这，在 反过来，将有助于开发针对传染性、恶性或免疫的潜在诊断和治疗方法 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，该方法可以 扩展到自然界中的其他 O-酰化唾液酸，例如 9-O-乳酰-Neu5Ac (Neu5Ac9Lt) 和 O- 非人类 N-羟乙酰神经氨酸 (Neu5Gc) 的乙酰化形式，可能会掺入人类体内 来自外源。

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.