ReviewDerivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry☆,☆☆
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].
Section snippets
Permethylation
Permethylation of carbohydrates is the most widely used technique for reducing polarity and for increasing thermal stability for analysis by techniques such as combined gas chromatography/mass spectrometry (GC/MS) and fast-atom bombardment (FAB) MS. Permethylation is used today with the newer mass spectral techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) MS for improving detection limits and providing linkage information from mass spectral
Derivatization of the reducing terminal
The single reducing terminus of carbohydrates has been exploited in many ways to attach groups such as fluorescent labels or charged moieties in order to render the molecules amenable to various analytical techniques. Predominant among these is the attachment of chromophores or fluorophores, mainly for detection following high-performance liquid chromatography (HPLC).
Derivatives of sugars with naturally-occurring amino groups
The presence of amino groups in sugars, often at the 2-position as, for example, following deacylation of acetylamino-sugars, provides the opportunity to use other chemistries as well as carbonyl condensation reactions to prepare derivatives. Acylation, as for acetylation (above) is possibly the most commonly used method but other methods are available.
Derivatives for stabilization of sialic acids in MALDI MS
Sialic acids are notoriously unstable under MALDI MS conditions and readily eliminate the sialic acid moiety. This reaction is the result of the labile acidic proton and thus, blocking this reaction by salt formation or derivatization of the acid stabilizes the compounds. Methyl ester formation is a common technique for achieving this stabilization and was introduced by Powell and Harvey for N-glycans and gangliosides in 1996 [283]. Reaction was achieved by conversion of the acid to its sodium
1,2-Diamino-4,5-methylenedioxybenzene (DMB) derivatives of sialic acids
The α-keto-carboxy group of sialic acids has been reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB, 132, Scheme 27) in dilute acetic acid to form fluorescent (λexc: 373 nm, λem: 448 nm) quinoxaline derivatives 133 [294] that have proved very useful for separations of different sialic acids by HPLC, ESI [295] and MALDI [296] MS. Although sialic acids are known to migrate under certain conditions, the reaction did not appear to be susceptible to this problem. The reagent is sold in kit form
Derivatives of asparaginyl carbohydrates
N-glycans are sometimes examined as asparaginyl derivatives after hydrolysis of the protein chain of their parent glycoproteins with pronase. Asparagine contains both carboxy and amino groups that can be targets for further derivatization to impart chromophores or fluorophores. Eight fluorescence reagents, (4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F, 134, Scheme 28), NBD-F (119), dansylchloride (DNS-Cl, 135), 2,3-naphthalenedialdehyde (NDA, 136), 1-pyrenesulfonyl
Conclusions
It is not possible, in a review of this size, to cover all aspects of carbohydrate derivatization but it is hoped that examples of most of the major methods are covered. Although a large number of techniques have been described, only a few may be regarded as standard in this area. Many of the older methods for monosaccharides have given way to newer methods that are more appropriate for the new instrumental techniques that have been introduced over the last two decades. However, it should be
Acknowledgements
I thank Professor Raymond A. Dwek, Director of the Oxford Glycobiology Institute for his help and encouragement.
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This paper is part of the special issue “Enhancement of Analysis by Analytical Derivatization”, Jack Rosenfeld (Guest Editor).
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This journal does not include paper titles in the References section. This author regards this information as an important aspect of the review and, consequently, full bibliographical data will be found in “Supplementary data”.