Abstract/Summary

Sialic acids are a family of over 50 derivatives of neuraminic acid that fulfil functions in both the healthy and disease states of a number of species, including humans. They are usually found at the terminal position of glycans as part of glycoproteins or glycolipids located on cell surfaces or in cell secretions. The need for accurate and detailed analysis of this type of carbohydrate is mainly driven by studies into their mechanisms of action, which may differ from one derivative to the next. Furthermore, ongoing work suggests that sialic acid derivatives are cell, tissue and species specific and that cross-over between species could have adverse effects. To clarify the impact of sialic acid derivatives contained in biological samples, appropriate standards and efficient analytical methods are essential. Chapter 1 provides an overview of the current understanding of sialic acids and their place in biological systems. O-Acetylated sialic acid derivatives are the most abundant form found in Nature and are, therefore, of particular interest with regards to detection and characterisation. Chapter 2 outlines the synthesis of mono-O-acetylated sialic acid monosaccharides for use as analytical standards. All synthetic routes started from N-acetylneuraminic acid. Synthesis of the 4-0- and 9-0-acetylated derivatives was successful and routes towards the synthesis of the 2-0-, 7-0 - and 8-0-acetylated form were optimised. A synthetic pathway concerning the synthesis of N-glycolylneuraminic acid and its 0- acetylated derivatives was also explored. Due to the poor fluorescence properties of sialic acids, it is often beneficial to tag them with a fluorophore, particularly for greater ease of detection by high pressure liquid chromatography (HPLC). Unfortunately, the widely used 4,5-methylenedioxy-l,2-phenylenediamine dihydrochloride (DMB) exhibits several drawbacks, therefore, as outlined in Chapter 3, an alternative label to DMB was sought. Especially promising practical results were obtained by substituting l,2-diamino-4,5- dimethoxybenzene (DDB) for DMB, with DDB showing good fluorescence intensity and peak separation for the samples studied. Some theoretical work was also carried out in the form of computational studies on various dye structures. In Chapters 4 and 5, a high throughput analytical method incorporating a liquid handling robot, HPLC and mass spectrometry was applied to pharmaceutical and research grade erythropoiesis stimulating agents, follicle stimulating hormone and human chorionic gonadotrophin, leading to the detection of some previously undiscovered sialoglycan structures. Such techniques are of exceptional value in an industrial pharmaceutical setting, as they enable monitoring of glycosylation critical quality attributes, which are in turn scrutinised by regulating bodies.Overall, this work details the detection and characterisation of sialic acids and their derivatives in a number of biotherapeutics; efforts have also been made to improve the accuracy and rapidity of their analysis by synthesising standards, refining fluorophore tagging and streamlining processes.