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Derivatization of Hydroxyl Groups: Permethylation

Permethylation of carbohydrates is the most widely used technique for increasing thermal stability and reducing polarity. It is used extensively for analyses by FAB, MALDI, and combined gas chromatography/mass spectrometry (GC/ MS). Reduction in polarity increases sensitivity in MALDI analyses and allows small amounts of very large glycans to be examined [139]. Furthermore it stabilizes the sialic acids as discussed later. One of their main advantages over other alkylation and acylation techniques is the relatively small (14 mass units) mass increment attending derivatization, which is important because of the large number of hydroxyl groups in carbohydrates.

The synthetic method introduced by Hakomori in 1964 [140] has been widely adopted for permethylation of carbohydrates and consists in reacting the carbohydrate, in dimethyl sulfoxide (DMSO), with methyl iodide catalyzed by the methylsulfinyl carbanion, prepared from sodium hydride. This reaction replaces active hydrogen in hydroxyl, carboxy, and amino groups by a methyl group. In order to retain information on the occurrence of natural O-Me groups, [2W3]-methyl iodide can be used as the methylating reagent. The reaction requires considerable cleanup (reviewed by Levery in 1997 [141]), which can limit its usefulness for the analysis of small amounts of glycans. A simpler method that uses finely divided sodium hydroxide rather than the methylsulfinyl carbanion was introduced in 1984 by Ciucanu and Kerek [142] and has been widely adopted. A few problems have been reported but generally overcome by such modifications as adding a trace of water or using N,N- dimethylacetamide as the solvent [143]. Peeling reactions (base-catalyzed removal of monosaccharide residues from the reducing terminus) have been minimized by addition of acetic acid to the final reaction mixture and by keeping the mixture at 0 °C [144].

A recent modification that is useful for derivatization of very small amounts of carbohydrate is the use of small microspin columns or fused-silica capillaries (500 pm i.d.) packed with sodium hydroxide powder to which are added the analytes, mixed with methyl iodide in DMSO containing traces of water. Reactions were said to take less than one minute, and the procedure minimized oxidative degradation and peeling reactions and avoided the need of excessive cleanup. Picomole amounts of linear and branched, sialylated, and neutral gly- can samples were rapidly and efficiently permethylated by this approach [145, 146], and a high-throughput extension of the method utilizing spin columns packed with sodium hydroxide beads has recently been described [147]. A potential problem is the appearance in mass spectra of a series of ions 30 Da larger than those from the fully methylated carbohydrate. These “overmethylation” ions appear to arise following reaction of the glycan with iodomethyl methyl ether that is formed as a side reaction [148]. Various aspects of permethylation procedures have been described in reviews by Jay [149] and Levery [141].

A major application of the permethylation reaction is in the determination of glycan structure by fragmentation. Glycosidic cleavages leave OH groups at the original sites of glycan attachment, effectively labeling them [150-152]. Crossring cleavage reactions produce different fragments attached to the nonreducing terminus, depending on the position of the linked residues. Thus 2-linkages produce a 74 Da increment, 4-linkages an 88 Da increment, and 6-linkages a combination of 60 and 88 Da increments, but for 3-linkages, no related ions are created.

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