| Résumé | Novel Aspect: Novel HILIC-MS approach for the selective detection of sugar-nucleotides synthesized in vivo by an engineered E. coli strain. Introduction: Natural product glycorandomization is a chemoenzymatic strategy used for the synthesis of glycosylated secondary metabolites that may have novel therapeutic potential. The process of natural product glycorandomization involves three promiscuous enzymes to activate (anomeric sugar kinases and nucleotidlyltransferases) and attach (glycosyltransferases) a repertoire of unique sugar libraries to various complex natural product aglycons. Previously, sugar phosphates produced in vivo by an engineered E. coli strain was successfully determined by CE-ESMS. We report here a hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) approach for sugar-nucleotides produced by the in vivo bioconversion. Methods: HILIC-MS was performed using an Agilent 1100 Series LC system coupled to a 4000 QTRAP mass spectrometer (AB/MDS Sciex) equipped with a TurboV source for electrospray ionization. HILIC separations were performed on a TSK-gel Amide-80 column (4.6 x 250 mm, Tosoh Biosciences) using mobile phases based on acetonitrile and ammonium acetate. Selective detection of the sugar-nucleotides was achieved using MRM highlighing the transition from the parent ions to the characteristic nucleotide fragment ions. Preliminary Results: The HILIC-MS parameters were optimized using a mixture of sugar-nucleotide standards consisting of TDP-D-glucose, UDP-a-D-glucose, ADP-glucose, GDP-D-a-mannose and CMP-ß-L-sialic acid. A detection limit in the low fmol level was achieved and the method was shown to be linear from 50 pg to 500 ng. Application of the method for the determination of natural and unnatural TDP-linked sugar-nucleotides synthesized in vitro revealed over half of the enzyme reactions to be successful. The analysis of cell lysates from the engineered E. coli strain revealed intracellular sugar-nucleotides corresponding to the specific sugar substrates, such as glucose, 2-deoxy-glucose and 6-azido-glucose, which had been fed to the cells. These data confirmed that the in vivo glycorandomization process was occurring. The preliminary data also indicated differences in the degree of in vivo bioconversion of the different sugar substrates and additional experiments will be performed to more accurately assess the ability of the engineered E. coli strain to generate natural and unnatural sugar nucleotide libraries. HILIC-MS combined with MRM provided a rapid and selective screening method for the detection of sugar-nucleotides synthesized by both in vitro and in vivo glycorandomization. |
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