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-hu...