Synthesis and Purification of PGN Precursors

Large scale synthesis and purification of the PGN precursors lipid I and lipid II were performed as previously described (Ling et al., 2015 (link)). UDP-N-acetyl-muramic acid pentapeptide (UDP-MurNAc-pp) was purified according to the protocol elaborated by Kohlrausch and Höltje (Kohlrausch and Höltje, 1991 (link)). Undecaprenyl phosphate (C₅₅P) and undecaprenyl diphosphate (C₅₅PP) were purchased from Larodan Fine Chemicals AB (Malmö, Sweden). The phospholipids 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and 1′,3′-bis[1,2-distearoyl-sn-glycero-3-phospho]-glycerol (DOCL, cardiolipin) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The concentration of purified PGN and wall teichoic acid precursors was quantified on the basis of their phosphate content as described (Rouser et al., 1970 (link)).

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Shukla R., Peoples A.J., Ludwig K.C., Maity S., Derks M.G., de Benedetti S., Krueger A.M., Vermeulen B.J., Lavore F., Honorato R.V., Grein F., Bonvin A., Kubitscheck U., Breukink E., Achorn C., Nitti A., Schwalen C.J., Spoering A.L., Ling L.L., Hughes D., Lelli M., Roos W.H., Lewis K., Schneider T, & Weingarth M. (2023). A new antibiotic from an uncultured bacterium binds to an immutable target. bioRxiv.

Publication Preprint 2023

Undecaprenyl phosphate (C55P) 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1′,3′-bis[1,2-distearoyl-sn-glycero-3-phospho]-glycerol (DOCL, cardiolipin)

Alabaster Cardiolipin Dopc Dopg Glycerol Lipid i Lipid ii Lipids Phosphate Phospholipids Sn glycero 3 phosphocholine Synthesis Teichoic acid Udp n acetyl muramic acid pentapeptide Undecaprenyl diphosphate Undecaprenyl phosphate

Corresponding Organization : University Hospital Bonn

Other organizations : University of Groningen, German Center for Infection Research, Novartis (United States), University of Florence, Interuniversity Consortium for Magnetic Resonance, Northeastern University

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Variable analysis

independent variables

Large scale synthesis and purification of the PGN precursors lipid I and lipid II
Purification of UDP-N-acetyl-muramic acid pentapeptide (UDP-MurNAc-pp)
Purchase of undecaprenyl phosphate (C55P) and undecaprenyl diphosphate (C55PP)
Purchase of the phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and 1',3'-bis[1,2-distearoyl-sn-glycero-3-phospho]-glycerol (DOCL, cardiolipin)

dependent variables

Concentration of purified PGN and wall teichoic acid precursors, quantified on the basis of their phosphate content

control variables

Protocols for large scale synthesis and purification of PGN precursors lipid I and lipid II, as previously described
Protocol for purification of UDP-N-acetyl-muramic acid pentapeptide (UDP-MurNAc-pp), as elaborated by Kohlrausch and Höltje

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