S26: Mass spectrometry characterization for chemoenzymatic glycoprotein synthesis

Chemoenzymatic synthesis is a powerful technique utilized to perform more efficient synthesis, and in some cases products unobtainable by classic chemical methods.  Numerous analytical techniques are commonly used to characterize the final product, however, process development and optimization can also benefit from such technologies.  As an example, the current project applies mass spectrometry for the reaction optimization and characterization of a chemoenzymatic modification of bovine ribonuclease B (RNase B).  A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through Endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation.  Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision induced dissociation (CID) mass spectrum.  Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry.  LC-MSⁿ using four sequential rounds of CID verified that the original glycosylation site had been reorganized to contain the new glycan.  The techniques described herein are not limited to this analyte or glycan, and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations.