Sugar nucleotides procedures

A fermentation procedure has been described237,238 for large-scale production of the latter sugar nucleotide. Bakers yeast transforms guanosine 5 -phosphate into the D-mannosyl pyrophosphate ester in 45% yield when a mixture with D-glucose, potassium phosphate, and magnesium sulfate is incubated. 

The enzymatic preparation of the activated sugar nucleotide may also involve a cofactor regeneration system. An example of this is an economic one-pot procedure, in which N-acetylneuraminic acid (NeuAc) is generated in situ from IV-acetylmannosamine (ManNac) and pyruvate with sialic acid aldolase and then converted irreversibly to CMP-NeuAc ([14], see also Sec. III). 

Sugar nucleotides are generally extracted with ethanol or with dilute perchloric or trichloroacetic acids, so that the extracts are essentially free of protein. Ethanol may extract a considerable amount of lipid, while the acids tend to cause appreciable losses of sugar nucleotides by hydrolysis. Trichloroacetic acid can be extracted with ether, while perchloric acid is best removed as its potassium salt. All extractions must be performed in the cold and extraction with ethanol is possible well below 0°C, though it fails to denature all pyrophosphatases and this can lead to losses. Saukkonen (1964) has reviewed a number of procedures for extraction and there is undoubtedly still some room for improvements. 

The separation of sugar nucleotides from such extracts is by no means easy, especially since it has to be as rapid as possible in order to minimise hydrolysis losses. Adsorption onto a basic ion-exchange resin is commonly used as a first step. It can be performed as a column method, as a batch extraction or, on a micro-scale, on sheets of ion-exchange materials. Considerable losses may occur on the resin. Elution can be by one of several different procedures, of which washing with ammonium chloride is one of the more useful (Recondo, Dankert and Passeron, 1965), since the salt is somewhat volatile. Other systems used include displacement by the chlorides of calcium, sodium or lithium, or by ammonium formate buffer. Hydrolysis losses are particularly likely with the last. 

Because of the lability of sugar nucleotides, every effort should be made to minimise the number of procedures involved in their separation and characterisation and to make these as rapid and mild as possible. For this reason, thin layer procedures are greatly to be preferred. If initial cleaning-up and concentrating of extracts can be minimised or avoided, that too is advantageous. 

The same procedure was applied212 for the preparation of uridine 5 -(a-D-glucopyranosyl pyrophosphate-4"-t, or -5"-f. A cell-free extract of Phytophthora cinnamoni was used for the synthesis of the 6"-t derivative,213 as well as yeast pyrophosphorylase.2133 An enzyme from Salmonella typhimurium was found quite satisfactory214 for obtaining this and other labeled nucleotide sugars. ... 

Early reviews on the mass spectra of nucleotides, more particularly of mononucleotides, " covered trimethylsilyl and methyl derivatives of nucleotides. The silylation of nucleotides, for instance, takes place on the base, on the sugar hydroxyls, and on the free phosphate hydroxyls under mild (room temperature) and basic (pyridine) silylation conditions (BSTFA/TMCS). The fragmentation of derivatives obtained from both procedures has been well studied. Several typical fragments ([M — CHj]", [base -I- 2H], silylated and desilylated sugar, S-TMSOH, etc.), as well as a weak M+ ion have been recorded. The FD spectrum of an underivatized... 

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