A CALCULATION OF ALL POSSIBLE OLIGOSACCHARIDE ISOMERS BOTH BRANCHED AND LINEAR YIELDS 1.05X10(12) STRUCTURES FOR A REDUCING HEXASACCHARIDE - THE ISOMER-BARRIER TO DEVELOPMENT OF SINGLE-METHOD SACCHARIDE SEQUENCING OR SYNTHESIS SYSTEMS

The number of all possible linear and branched isomers of a hexasaccha ride was calculated and found to be > 1.05 x 10(12). This large number defines the Isomer Barrier, a persistent technological barrier to the development of a single analytical method for the absolute characteri zation of carbohydrates, regardless of sample quantity, Because of thi s isomer barrier, no single method can be employed to determine comple te oligosaccharide structure in 100 nmol amounts with the same assuran ce that can be achieved for 100 pmol amounts with single-procedure Edm an peptide or Sanger DNA sequencing methods. Difficulties in the devel opment of facile synthetic schemes for oligosaccharides are also expla ined by this large number, No current method of chemical or physical a nalysis has the resolution necessary to distinguish among 10(12) struc tures having the same mass. Therefore the 'characterization' of a midd le-weight oligosaccharide solely by NMR or mass spectrometry necessari ly contains a very large margin of error, Greater uncertainty accompan ies results performed solely by sequential enzyme degradation followed by gel-permeation chromatography or electrophoresis, as touted by som e commercial advertisements. Much of the literature which uses these s ingle methods to 'characterize' complex carbohydrates is, therefore, i n question, and journals should beware of publishing structural charac terizations unless the authors reveal all alternate possible structure s which could result from their analysis. Today, only a combination of quantitative sugar analysis, methylation linkage analysis, partial de gradation by enzymes or chemistry, and mass spectrometry can reduce th e number of possibilities to one, The present study yields a number of individual formulae and a master set of equations necessary for the d etermination of all possible reducing-end isomers for di- to octasacch arides, above which branching isomers generate astronomical numbers, l arger than Avogadro's number, Because hexasaccharides are generally am ong the largest biologically active, protein-recognized oligosaccharid e sequences, and also among the largest repeating units in polysacchar ides, the present calculation was limited to dp6. Despite this simplif ication, the number of possible structures calculated for reducing hex asaccharides comprised of D hexoses alone is > 10(12). Available micro chemistry for biologically active oligosaccharides requires between 10 and 100 nmol for a minimum necessary combination of wet chemistry/enz ymology/mass spectrometry employing partial degradation. The relativel y high limiting quantity for analysis of carbohydrates (compared with proteins and DNA) has remained static for 20 years, despite intense re search 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 an d Sanger DNA sequencing methods, It also reveals the barrier to facile synthetic methods for oligosaccharides comparable to those developed for peptide synthesis.