| Summary: | We have developed a novel chemoenzymatic strategy to GDP-azidodeoxymannoses which differs from the natural substrates of mannosyltransferases of the Leloir type due to the presence of an azide group instead of hydroxyl functionality on the mannopyranose ring. We first chemically synthesized 2-, 3-, 4- and 6-azidodeoxy-α-D-mannose-1-phosphates, and then tested them as substrates for a recombinant GDP-Mannose Pyrophosphorylase from <i>Salmonela enterica </i>produced as polyhistidine-tagged fusion protein in <i>E. coli. </i>This enzymatic step yielded all the corresponding GDP-azidodeoxymannoses in 41%, 52%, 55% and 63% yields respectively, which were characterized by high resolution MS, <sup>1</sup>H- <sup>13</sup>C and <sup>31</sup>P NMR. Furthermore, we have optimized a high throughput colourimetric screen (via malachite green dye) which allows for indirect monitoring of this enzymatic reaction, and could be applied in future studies of enzymatic directed evolution to improve the efficiency of unnatural products formation. The GDP-azidodeoxymannoses so formed were tested as substrates for the soluble fragment of an α-1,2-Mannosyltransferase (Kre2p/Mnt1p) from <i>S. cerevisiae </i>which we produced in <i>E. coli. </i>Our preliminary data demonstrated that GDP-4-azidodeoxymannose was the most efficient donor substrate when transferred to a mannose α-methyl glycoside acceptor, as monitored by electrospray ionization mass spectrometry, and we foresee that the other isomers may find similar applications on further enzymes. Consequently, GDP-azidodeoxymannoses may serve as valuable tools for glycobiological studies using bioorthogonal azide chemistry.
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