Invited commentary: A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 × 10 structures for a reducing hexasaccharide: The Isomer Barrier to development of single-method saccharide sequencing or synthesis systems

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The number of all possible linear and branched isomers of a hexasaccharide was calculated and found to be >1.05 × 1012. This large number defines the Isomer Barrier, a persistent technological barrier to the development of a single analytical method for the absolute characterization of carbohydrates, regardless of sample quantity. Because of this isomer barrier, no single method can be employed to determine complete oligosaccharide structure in 100 nmol amounts with the same assurance that can be achieved for 100 pmol amounts with single-procedure Edman peptide or Sanger DNA sequencing methods. Difficulties in the development of facile synthetic schemes for oligosaccharides are also explained by this large number. No current method of chemical or physical analysis has the resolution necessary to distinguish among 1012 structures having the same mass. Therefore the 'characterization' of a middle-weight oligosaccharide solely by NMR or mass spectrometry necessarily contains a very large margin of error. Greater uncertainty accompanies results performed solely by sequential enzyme degradation followed by gel-permeation chromatography or electrophoresis, as touted by some commercial advertisements. Much of the literature which uses these single methods to 'characterize' complex carbohydrates is, therefore, in question, and journals should beware of publishing structural characterizations unless the authors reveal all alternate possible structures which could result from their analysis. Today, only a combination of quantitative sugar analysis, methylation linkage analysis, partial degradation by enzymes or chemistry, and mass spectrometry can reduce the number of possibilities to one. The present study yields a number of individual formulae and a master set of equations necessary for the determination of all possible reducing-end isomers for di- to octasaccharides, above which branching isomers generate astronomical numbers, larger than Avogadro's number. Because hexasaccharides are generally among the largest biologically active, protein-recognized oligosaccharide sequences, and also among the largest repeating units in polysaccharides, the present calculation was limited to dp6. Despite this simplification, the number of possible structures calculated for reducing hexasaccharides comprised of D hexoses alone is >1012. Available microchemistry for biologically active oligosaccharides requires between 10 and 100 nmol for a minimum necessary combination of wet chemistry/enzymology/mass spectrometry employing partial degradation. The relatively high limiting quantity for analysis of carbohydrates (compared with proteins and DNA) has remained static for 20 years, despite intense research activity. This calculation underscores the reason for the long-standing technology barrier for the development of a microchemistry in carbohydrate analysis comparable in sensitivity with Edman protein and Sanger DNA sequencing methods. It also reveals the barrier to facile synthetic methods for oligosaccharides comparable to those developed for peptide synthesis. © 1994 Oxford University Press.

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