## 1 Introduction  
Lippolysaccharide (LPS) makes up approximately 30% of the outer membrane (Figure 1) of Gram-negative bacteria and is a multipurpose cellular component necessary for cellular motility, surface adhesion, and intermolecular interactions in a bacterial biofilm. LPS is also known for causing an immunological response in vivo, and is therefore, commonly referred to as an endotoxin [1, 2, 3]. LPS can also be utilized by bacteria to prevent the cell permeation of hydrophobic molecules, thereby serving as a bacterial antibiotic resistance mechanism [4, 5]. Furthermore, xenobiotics including Polymyxin B and metal ions, have been shown to trigger alterations of LPS structure to further fortify the outer membrane [6,7]. Additionally, previous studies have shown that removing genes involved in the inner core oligosaccharidesride biosynthesis, including HeptosyltransferaseI (HepI), results in increased bacterial antibiotic susceptibility and an expression of a deep-rough LPS morphology [8], which makes HepI a target of interest for Gram-negative antibacterial therapeutic design. As shown in Figure 2, HepI transfers a heptose moiety from ADP-L-_glycero_-d-_manno_-heptose (ADPH) to Kdo\({}_{2}\)-lipid A (KLA) in a nucleophilic substitution reaction. Kinetic characterization of the HepI reaction, which is enabled by detection of the ADP produced during the reaction via a UV-Vis coupled spectrophotometric assay with putative inhibitors, as well as biophysical characterization of HepI including circular dichroism spectroscopy, small-angle x-ray scattering, intrinsic tryptophan fluorescence, and isothermal titration calorimetry all require significant quantities of purified ADPH and KLA [9, 10, 11, 12]. In addition to their described use with HepI, ADPH and KLA have a variety of other applications in biological and biochemical investigations. ADPH and KLA have both been shown to stimulate an immune response in vivo [13,14]. A 2018 study done by [PERSON] et al. showed that organic extracts from _H. pylori_ were abundant in ADPH,  
Figure 1: Bacterial lipid bilayer with LPS transfer protein complex and the full LPS structure (inset). Figure 2: Reaction of HepI with ADPH and KLA showing the transfer of the heptose moiety (red). which was determined to be a pathogen-associated molecular pattern (PAMP) [15]. PAMPs are molecules found in a variety of infectious species that are detected by host cells and trigger immune pathways and inflammatory mediators. ADPH is also a substrate for HepII-IV [2,16] making it a vital compound for multiple steps of LPS biosynthesis. Previous work by [PERSON] et al. demonstrated the synthesis of a variety of ADPH analogs in an attempt to determine potential inhibitors for Heposyltransferase [17]. The first liganded crystal structure of HepI was published using a fluorine functionalized form of ADPH as it stabilized the compound for crystallization [18]. Dr. [PERSON] at the Institute of Organic Chemistry has published a library of work [15,19,20] pertaining to the synthesis of ADPH analogs and is interested in the recognition of Heptoses by surfactant proteins. KLA has also a wide variety of applications that are on the frontier of therapeutic design models. A review by [PERSON] et al. describes the application of KLA in drug development and its role as an endotoxin [21]. [PERSON] et al. explains how KLA exhibits equivalent immune stimulating effects to a fully formed LPS structure which is known to stimulate an aggressive immune response [22]. KLA is also a substrate for another enzyme known to modify KLA structures, _S. typhimurium_ Lipid A deacylase LpxR [23]. There are a notable variety of uses for ADPH and KLA, and therefore the need for proper protocol to successfully extract pure compound as an extremely cost effective and reliable option is critical to helping continue the projects described above. Here we have described, in excessive detail, the steps required to obtain these substrates as well as visual and analytical data for comparison.