We have been witnessing a rapid development in our understanding of the roles sugars play in biological processes. Sugars are vital components of all organisms - crucial to their structure and function. Uncovering their roles opens new avenues for research and applications in various medical, biochemical and biotechnological fields. These developments in glycobiology have created an urgent need for sugar building blocks such as rare sugars, including L-, azido-, and deoxy-sugars, and intermediates important for glycosylation reactions, including sugar-phosphates and activated sugars.
To address this need we have successfully synthesized a variety of rare sugars important for glycobiology. With the use of an engineered mannitol dehydrogenase, we have synthesized l-Ribose, l-Galactose, l-Fucose, and many other rare sugars. N-acetyl-d-glucosamine was also prepared from chitin by enzymatic degradation.
We have also created a library of kinases to produce a variety of sugar-1-phosphates (sugar-1Ps). This includes a thermostable kinase engineered to not only produce its native product, α-D-Galactose-1P, but many other sugar-1Ps at large scale with yields greater than 150 g/L. These include phosphates of l-Glucose, l-Arabinose, l-Xylose, 6-azido-6-deoxy-d-Galactose, d-fucose, 2-deoxy-d-Galactose, 6-amino-6-deoxy-d-Glucose and N-acetyl-d-Galactosamine. Enzyme reactions were also used for preparation of d-Fructose-1P, d-fructose-2,6-diphosphate, trehalose-6P.
Finally, activated sugars are important for production of oligosaccharides and glycosylation of small molecules and proteins. d-Galactose-1P, l-Glucose-1P and 2-deoxy-d-galactose-1P were synthesized and subsequently used in a nucleotidyltransferase reaction to prepare their respective activated sugar. We achieved almost 100% conversion of UTP (10 mM) and Gal-1P (10 mM) to UDP-α-D-Galactose in an overnight reaction. This enzyme also utilizes GDP and TTP to produce UDP, GDP- and TDP- l-Glucose, l-Glucose and 2-deoxy- l-Galactose.

F. Michael Racine

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