Project Summary/Abstract This equipment supplement project aims to purchase a liquid chromatography coupled mass spectrometry (LCMS) instrument essential for the discovery, biosynthesis, and development of antifungal nucleoside antibiotics. The parent R01 aims to understand the biosynthesis of peptidyl nucleoside (PN) antifungal antibiotics active against multiple pathogenic fungi such as the causal agent of "Valley Fever" and to discover novel antifungal agents using this knowledge. For this study, one of the most significant technical challenges is the characterization of hydrophilic nucleoside compounds because these compounds do not bind to the conventional reverse-phase columns most frequently used for LCMS. To overcome this challenge, we propose the purchase of a biocompatible (metal-free) ultra-high-performance liquid chromatography (UHPLC) unit coupled with a quadrupole time of flight (QTOF) MS device. Biocompatible UHPLC will allow the use of various hydrophilic interaction chromatography (HILIC) columns for the analysis of polar metabolites. The QTOF MS detector will allow discovery-oriented (untargeted) analyses of complex metabolite mixtures. The proposed system will enable the purification of the analytes while monitoring MS chromatograms, which is impossible with other LCMS instruments available at Duke. Also, having an instrument dedicated to the preparation and analysis of hydrophilic compounds will significantly improve the turnaround time of our analyses from 2-3 weeks to within a day. Therefore, the purchase of the proposed LCMS will provide unique capabilities currently unavailable to us but essential for the successful characterization of nucleoside antibiotics proposed in the parent project. The proposed research is significant because it will provide a basis for the future chemoenzymatic preparation or genome mining discovery of novel nucleoside antifungals.