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Cite!
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A Practical Guide |
The method just described has been utilised in the methylation
analysis of polysaccharides from a variety of sources including
bacteria (capsular polysaccharides and lipopolysaccharides)
15
16
17
18
fungi 19
20
and algae 21
.
Oligosaccharides 22
23
, glycolipids 24
25
and
glycopeptides 26
27
have also been investigated. Polysachcarides containing uronic acids may be carboxyl-reduced before (carbodiimide - sodium borohydride 28 or after (lithium aluminium hydride 17 the permethylation step. If the reduction is performed with a deuterated reagent the neutral sugar obtained is easily distinguished by MS (see section 5) from other neutral sugars present in the polysaccharides. This has been used in studies of bacterial and fungal polysaccharides. Some polysaccharides contain naturally methylated sugars. Thses units can be readily identified by GLC-MS of the derived alditol acetates obtained on hydrolysis of the polysaccharide and subsequent reduction-acetylation. Some examples of the use of this technique are the identification of 3-O-methyl-L-rhamnose in Klebsiella O group 10 lipopolysaccharides 29 and Rhodopseudomonas capsulata 30 , as well as 3-O-methyl-D- and L-xylose in lipopolysaccharides from Rhodopseudomonas viridis and Myxococcus fulvus 31 , respectively. Furthermore, the position of the naturally methylated sugar may be determined by methylation analysis. One example is the location of 3-O-methyl-L-rhamnose in the Klebsiella O group 10 lipopolysaccharide 29 . The permethylation was performed with trideuteriomethyl iodide in order to distinguish between the natural methoxyl groups and those introduced in the analytical procedure. In the subsequent GLC-MS analysis of the alditol acetates from the hydrolysate of the trideuteriomethylated polysaccharide it could be established that the naturally methylated sugar occupied only terminal positions in the polysaccharide chains since the O-methyl group was found only in the 3-O-methyl-2,4-di-O-trideuterio-methyl-L-rhamnose derivative. Reducing oligosaccharides should preferably be transformed into non-reducing (e.g. alditol) derivatives before methylation. This is to prevent any alkaline degradation and/or presence of different forms during the methylation which might complicate results. It should also be noted that permethylated oligosaccharide glycosides 32 and alditols 32 may be investigated as such by GLC-MS. Glycopeptides containing glycosyl-asparagine or glycosyl-hydroxy-proline linkages may be methylated by the Hakomori procedure but glycosyl-serine and glycosyl-threonine linkages are likely to be cleaved by these alkaline conditions and the liberated, reducing sugar residues further degraded. Direct methylation of these materials is therefore not recommended; they should first be subject to an alkaline borohydride degradation. 33 Methylation analysis is valuable for monitoring and evaluating various specific and non-specific degradations of polysaccharides, performed in order to determine the sequence of their sugar residues. Polysaccharide methodology 34 35 36 , including specific degradation methods 37 , has rescently been reviewed and the discussion of this subject will therefore be limited. Partial acid hydrolysis, followed by methylation analysis, of polysaccharides containing sugar residues which are highly susceptible to acid hydrolysis (e.g. furanosidic sugars and dideoxy sugars) in conjunction with the methylation analysis of the intact material gives information on the attachment site of these residues 15 38 . Likewise, methylated polysaccharides may be partially hydrolysed and remethylated using trideuteriomethyl iodide. This method yields similar but more detailed information on the position of the acid labile sugars 39 . The Smith degradation may conveniently be evaluated in this way 40 . In particular, the polyalcohol obtained on periodate oxidation - borohydride reduction may be mathylated before the mild hydrolysis step. Trideuteriomethylation after this treatment will give detailed information on the positions to which the oxidised residues were linked 18 41 . Methylation analysis has also been used to investigate the products obtained on uronic acid degradation 12 18 of polysaccharides and on oxidation-alkaline elimination of partially methylated polysaccharides 42 43 . The use of methylation analysis to monitor enzymic degradations of polysaccharides and glycoconjugates has yet only found limited application but offers obvious possibilities. In such experiments information could be obtained on the penultimate residue in the degraded material. Methylation analysis is also an important technique for the location of O-acyl groups in natural carbohydrates. In the method devised by Norrman-de Belder 44 , the substrate is first acetalated by treatment with methyl vinyl ether and then investigated by methylation analysis. The positions of O-methyl groups in the monomeric sugars reflect the substitution of O-acyl groups in the native material 15 39 . |