Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

Abstract

Carbohydrates display a large diversity of structures and their analysis presents many obstacles as the result of properties such as isomeric diversity, existence of branched structures and the lack of chromophores or fluorophores. Consequently, many analytical approaches depend on the application of chemical modifications such as hydrolysis or derivative formation. This review covers various aspects of derivatization that are used for such approaches as improving thermal stability and volatility for gas-phase analyses, introduction of fluorophores for optical detectors, introduction of charge for mass spectral analyses and attachment of bioaffinity tags for bioactivity studies. Reducing carbohydrates contain, in addition to multiple hydroxyl groups, several other sites for derivatization such as the single anomeric site that has been used in numerous methods for attaching various property-enhancing tags. Other sites are restricted to specific carbohydrates but include carboxy groups in sialic acids and amino groups in glycosylamines. All of these groups have been the targets of derivatization and this review attempts to summarise the main methods used for these various functional groups.

Introduction

Carbohydrates comprise one of the largest group of compounds found in nature and range in size from simple monosaccharides to very large molecules with molecular weights exceeding 1 mDa. Their analysis is challenging for several reasons: many compounds are isomeric, monosaccharides can adopt different structures (ring opened or closed, different ring sizes and conformations), structures of oligosaccharides are often branched, unlike the other common biopolymers (proteins and nucleic acids) and most carbohydrates lack chromophores or fluorophores, a property that makes detection difficult. But it is perhaps the large number of isomers that discourages investigators from analysing these compounds. For example, it has been estimated that there are 1.05 × 10¹² possible isomers for a reducing hexasaccharide [1] which, in terms of the average size of a carbohydrate molecule, is quite small. However, in reality, the situation simplifies considerably when it is realised that, in nature, these compounds are synthesised by very specific enzymatic reactions and within many types of carbohydrate only a very few, often only one, of the possible isomers actually exists. Nevertheless, even in these cases, structural determination can still present major problems.

Nuclear magnetic resonance (NMR) spectrometry is possibly the most powerful technique for structural analysis and has been applied extensively in the analysis of carbohydrates. However, in many cases, such as the analysis of N- and O-linked glycans from glycoproteins, there is seldom enough material available and the preferred techniques are chromatography and mass spectrometry (MS). Derivatization plays a major role in these analyses, both for the analysis of the intact molecules and for products obtained from various methods of depolymerization. For example, lipophilicity can be increased for studies by gas-phase techniques by derivatizing all hydroxy groups and, in addition, the unique reducing group in reducing sugars provides a convenient attachment point for fluorescent tags. This review will attempt to outline the major uses of derivatization in this field but, because of the enormous amount of reported work, by no means claims to be comprehensive. For other earlier reviews on carbohydrate derivatization, see [2], [3], [4], [5], [6], [7], [8], [9], [10] and the more general book on derivatization for mass spectrometry by Zaikin and Halket [11].