Regular ArticleOne-Day Enzymatic Synthesis and Purification of UDP–N- [1-14C]Acetyl-Glucosamine☆

Add time:09/07/2019    Source:sciencedirect.com

UDP–GlcN[1-14C]Ac was synthesized in a single enzymatic reaction from [1-14C]acetate and commercially available precursors on both a microcurie (micromole) and a millicurie (millimole) scale. The reaction was catalyzed by the action of acetyl coenzyme A synthetase, inorganic pyrophosphatase, and the bifunctionalEscherichia coliGlmU protein. Within 2 h 86 to 94% reaction is attained, and it approaches 99% completion overnight. GlmU protein was prepared in the form of a fusion suitable for nickel chelate affinity chromatography. Several methods were developed for rapid purification of UDP–GlcN[1-14C]Ac: an HPLC method handled micromole (microcurie) loads. Alternatively, ion exchange chromatography over DOWEX AG1 X-2 using a batch elution procedure was compatible with millimole (millicurie) amounts of radiolabel and yielded both chemically and radiochemically homogeneous UDP–GlcN[1-14C]Ac. These methods allow laboratories to quickly produce and purify microcurie to millicurie quantities ofN-acetyl-labeled UDP–GlcNAc by a choice of methods from relatively inexpensive precursors.

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