Monosaccharides identification

N. K. Kochetkov and O. S. Chizhov, Application of mass spectrometry to methylated monosaccharide identification, Biochim. Biophys. Acta, 83 (1964) 134-136. 

Today (2000), GC/MS is still the method of choice for the determination of the monosaccharide composition of oligosaccharides and is widely used. The analysis starts with the hydrolysis or alcoholysis of the oligosaccharide to monosaccharides, which after derivatization (mostly by trimethylsilylation) are analysed by GC/MS. [221] Monosaccharide identification is based on comparison of retention time and fragmentation pattern with references. 

COLLECTION AND IDENTIFICATION OF BIOACTIVE ORGANIC COMPOUNDS OCCURRING IN RIVERS AND LAKES. ADSORPTION SELECTIVITY OF MONOSACCHARIDES ONTO HYDROUS METAL OXIDES... 

Ribose and a-glucose are monosaccharides that occur in nature. The carbon atoms are numbered for identification purposes. 

Constitutional studies have broadly followed the pattern of methyla-tion of the unsubstituted alcoholic groups in a carbohydrate, hydrolytic or oxidative disruption of the molecule and identification of the resulting methylated fragments. In this way methyl ethers have been invaluable in the determination of the ring structures of the monosaccharides and in the elucidation of the constitutions of the more complex saccharides. 

The partially methylated monosaccharides obtained on depolymerization of the permethylated sample are preferably analyzed as acetates by g.l.c.-m.s., as shown by Bjomdal and coworkers.41,42 The neutral sugars and the amino sugars obtained in acetolysis-acid hydrolysis are reduced, and acetylated for the analysis, and the amino-hexitol and the neuraminic acid residues are acetylated after methanolysis. Identification with the aid of g.l.c.-m.s. has been described for all of the common components of protein- and lipid-linked glycans and oligosaccharides from animal cells, namely, the neutral sugars,41-43 hexitols,44 hexosamines,29,43,45,46 aminohexitols,31,32 and neuraminic acids.33,34,47... 

Historically, techniques such as the formation of osazones and the demonstration of fermentation have contributed significantly to the separation and identification of carbohydrates. Observation of the characteristic crystalline structure and melting point of the osazone derivative, prepared by reaction of the monosaccharide with phenylhydrazine, was used in identification. This method is not completely specific, however, because the reaction involves both carbon atoms 1 and 2 with the result that the three hexoses, glucose, fructose and mannose (Figure 9.19), will yield identical osazones owing to their common enediol form. 

Identification of constitutive monosaccharides two-dimensional homonuclear NMR techniques such as DQF-COSY and TOCSY are used to assign chemical-shift values for all C-bonded protons in each individual monosaccharide (96). One-dimensional NMR spectra provide useful information about the chemical shifts and scalar couplings of such well-resolved signals as methyl groups for 6-deoxy monosaccharides (fucose, quinovose, and rhamnose) at 6 1.1-1.3 ppm. 

Although many analyses are performed on alditol acetates (see Section VII, p. 56), in order to avoid the formation of multiple peaks, such a reduction is not practical when the mixture contains ketoses, notably fructose. Such analyses are mainly encountered with medical samples and in the examination of sugars occurring free in Nature. Furthermore, the peak-area ratios may be used as a means of identification, to check on the completeness of trimethylsilylation,67,89 and, despite the complex chromatograms obtained from trimethyl-silyl derivatives, they have the merit of being rapidly formed.89 For all of these reasons, improvements in the separation of monosaccharides as their trimethylsilyl derivatives continue to be of considerable importance. 

Their characteristic optical rotatory dispersion or circular-dichroism curves, and their infrared spectra, rich in characteristic frequencies, may be useful. Paper chromatography permits preliminary identification of the glycosyl phosphate or monosaccharide resulting after degradation, and the specific enzymic reactions of these products are widely used to provide additional evidence. 

Enzymic reactions with D-galactose oxidase or L-fucose isomerase were used for identification of the monosaccharide isolated after degradation of the esters. 

Fig. 4.5.4 Identification of mutations in the transferrin protein by neuraminidase treatment. Unusual patterns in the IEF of serum transferrin might lead to pitfalls in CDG diagnostics. These varying patterns are often due to mutations of charged amino acids in the protein backbone of the transferrin molecule, which might lead, for example, to an accumulation of trisialo transferrin bands (lane 3, indicated by a question mark). Further investigations are carried out by cleaving off charged sialic acid monosaccharide moieties from transferrin-linked oligosaccharides by neuraminidase treatment, followed by IEF and transferrin antibody staining. In the case of protein mutations, additional bands below (lane 4) or above (not shown) the desialylated transferrin form appear...

The advantage of this approach to the identification of partially methylated monosaccharides involves the fact that no preparation of difficultly... 

However, two other applications of mass spectrometry to carbohydrate chemistry seem the most important and promising. These are the identification of partially methylated monosaccharides, and structural analysis of di- and oligo-saccharides (and penetration of this new technique into polysaccharide chemistry). 

As for the first application considered in Section III,2d, the method for identification of methyl ethers using trideuteriomethyl- derivatives has already been demonstrated to be valid for all of the major types of monosaccharides, and is now receiving practical application.98 We hope that the increasing availability of mass spectrometers will facilitate the introduction... 

Table IV, which briefly summarizes the material described in previous sub-sections, shows the total of the different monosaccharide components identified in bacterial polysaccharides, and our present knowledge about their activated forms. It may be seen that identification of the activated forms has been achieved for only approximately half of the monosaccharides known to be involved. The most striking gap in the information available is the lack of data about the activated forms of D-ribose-derived monosaccharides and of most of the D-fructose-derived aldoses having configurations other than gluco, galacto, and manno.

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