Saccharide assembly

E. The Use of Cell-free Systems in Studying Saccharide Assembly Some Problems... 

These hazards are probably more serious in the study of saccharide assembly than in the synthesis of polypeptide. The absolute requirement for a message in the form of a linear polynucleotide, to specify the particular amino acyl transfers that can occur, largely prevents the possibility of inappropriate synthesis of polypeptide, and powerful techniques exist for identifying and characterising the labelled products of such synthesis. While the true rates of various steps in this process may be distorted in a cell-free system, the sequence of events and the mechanisms of each step are demonstrably almost unaltered and are accessible to detailed analysis. With oligo- and poly-saccharides there can be difficulties and these must be clearly appreciated in evaluating much of the work to be described below and in the chapters that follow. 

Eukaryotic cells have vastly more elaborate systems of membranes than have bacteria. Because these membranes can fold back and come into close apposition to each other, forming a reticulum, many of the limitations of the bacterial systems of saccharide assembly could be avoided. Indeed, eukaryotes have a far more elaborate biochemistry and molecular biology of carbohydrates (see Chapters 4 and 5). Nevertheless, the bacterial systems have been astonishingly successful. 

Both of the first two types of saccharide assembly are sequential, so that saccharides always grow and branch from the reducing terminal towards the... 

In what follows, the experimental evidence for and against these concepts of saccharide assembly will be evaluated. 

The role of soluble, cytoplasmic enzymes in saccharide assembly seems chiefly to be in the phosphorylation of sugars and the interconversion of sugar phosphates. The enzymes of sugar nucleotide interconversion are generally membrane-associated and must, in most cases, be firmly membrane-bound. They are, therefore, likely to be integral membrane proteins and must, for the most part, be constituents of the endoplasmic reticulum. 

The reality of the glycosyltransferase system will, of course, be much more complex in particular the association of enzyme and modulator subunits would have to be controlled in order to select for appropriate patterns of saccharide assembly and that, in turn, would need some genetic information. Hence, even allowing for economies of this type, the eukaryotic cell still must have a considerable genetic commitment to the specificity of glycosyltrans-ferases alone. 

Scheme 3 The first example of armed/disarmed saccharide assembly...

With remarkable accuracy, Democritus in the fifth century B.C. set the stage for modem chemistry. His atomic theory of matter, which he formulated without experimental verification, still stands, more or less intact, and encapsulates the profound truth that nature s stunning wealth boils down to atoms and molecules. As science uncovers the mysteries of the world around us, we stand ever more in awe of nature s ingenious molecular designs and biological systems nucleic acids, saccharides, proteins, and secondary metabolites are four classes of wondrous molecules that nature synthesizes with remarkable ease, and uses with admirable precision in the assembly and function of living systems. 

In 2006, Milosavljevic and co-workers64 reported a study of the complete 4H and 13C NMR assignment of a new triterpenoid saponin, leucantho-side-A (13), from Cephalaria leucantha L. In the course of determining the structure and assigning the spectra, the authors made extensive use of the normal ensemble of 2D NMR experiments in use for the characterization of natural product structures HSQC, HMBC, DQF-COSY, TOCSY, and NOESY. The authors supplemented the aforementioned list of experiments with 2D /-resolved, DINE-(Double INEPT-Edited)-HSQC, and INADEQUATE spectra. The authors made no mention of the use of the connectivity information derived from the 1,1-ADEQUATE spectrum in the assembly of the triterpene nucleus of the molecule but reported extensive tabulations of the 1,1-ADEQUATE correlations that were used to sequence and assign the saccharide resonances of the tri- and di-saccharide sub-units, 14 and 15, respectively, linked to the triterpene nucleus. 

Our original approach to polysaccharide C-13 n.m.r. spectral analysis consisted of making a minimum number of hypotheses about expected structure-to-spectra relationships (8). By then comparing spectra to known structure for a series of D-glucans, we attempted to establish the validity of these hypotheses and to establish how diverse a structural difference could be accommodated The hypotheses were as follows. Firstly, that each polymer could be considered as an assembly of independent saccharide monomers. Secondly, that these hypothetical saccharide monomers would be 0 alkylated (0 -methylated) in the same positions as the actual saccharide linked residues (it had previously been established that 0-methylation of any a-D-glucopyranosyl carbon atom position resulted in a down-field displacement of vlO p.p.m. for the associated resonance). Thirdly, that each differently substituted residue would have a completely different set of chemical shift values for each carbon atom position (different from the unsubstituted saccharide) but that only the carbon atom positions involved in inter-saccharide linkages would have A6 greater that 1 p.p.m. And, fourthly, that the hypothetical 0-alkylated residues would contribute resonances to the total spectrum proportional to their mole ratio in the polymers. 

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