A bacterial
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D-glucopyranosyl-1-phosphate thymidylyltransferase was found to couple four hexofuranosyl-1-phosphates, as well as apentofuranosyl-1-phosphate, with deoxythymidine 5'-triphosphate, providing access to furanosyl nucleotides. The enzymatic reaction mixtureswere analyzed by electrospray ionization mass spectrometry and NMR spectroscopy to determine the anomeric stereochemistry of furanosylnucleotide products. This is the first demonstration of a nucleotidylyltransferase discriminating between diastereomeric mixtures of sugar-1-phosphates to produce stereopure, biologically relevant furanosyl nucleotides.