The secondary cell wall polysaccharide (SCWP) of Bacillus anthracis plays a key role in the organization of the cell envelope. Through non-covalent interactions, it anchors S-layer proteins (SLP) and S-layer associated proteins (BSL) to the cell surface. Proper S-Layer assembly was shown to be important for virulence and various cell maintenance functions. Specific functionalities on the non-reducing SCWP trisaccharide unit including a 4,6-O-pyruvyl ketal, a free amine, and O-acetylation are thought to play a role in SLP and BSL binding. Herein, the synthesis of a library of compounds based on the non-reducing unit of B. anthracis SCWP is described. The compounds were utilized in microarray and ELISA experiments to probe the importance of various structural features for binding against a panel of mCherry fusion proteins constructed from the S-Layer homology binding domains of various SLP and BSL proteins. A number of synthetic challenges were addressed including the development of synthetic procedures and glycosylation conditions for the unique pyruvylated donors. The SCWP structural features vital for SLP and BSL binding were successfully identified. PatB1 is a putative O-acetyltransferase believed to play a role in the 3-O-acetylation of the penultimate beta-GlcNAc on the B anthracis SCWP. In order to characterize PatB1 specificity and specific activity in the context of its natural environment, a panel of oligosaccharides based on the trisaccharide repeating unit was synthesized with galactose substitutions at biologically relevant positions. A common trisaccharide intermediate was synthesized with levulinoyl, fluorenylmethyloxycarbonate, and 2-naphthylmethyl protecting groups installed at key branching points. The orthogonal protecting groups were regioselectively removed allowing for site specific installation of galactose. A trisaccharide based on the non-reducing SCWP unit was also synthesized to determine if the 4,6-O-pyruvyl ketal and/or free amine can affect PatB1 activity and specificity. It was found that the presence of galactosylation did not alter specificity, but led to a decrease in specific activity. Furthermore, it was found that the trisaccharide bearing the 4,6-O-pyruvyl ketal and free amine showed a significant increase in specific activity demonstrating that these features are likely utilized in vivo as PatB1 binding epitopes and help to facilitate 3-O-acetylation of the penultimate beta-GlcNAc.