PROJECT SUMMARY Bacterial cells surround themselves with a peptidoglycan (PG) cell wall, an essential structure that resists changes in osmotic pressure and other environmental insults. The goal of this proposal is to develop a PG fragment library that captures the natural diversity to facilitate the proper innate and adaptive immunity biological studies. PG fragments are used by the innate immune system to correctly recognize and respond to the presence of bacteria. We hypothesize that PG diversity naturally present across the multitude of bacterial species is essential for generating the correct immune response for host defense. Study of these important PG fragments has been hampered by the lack of reproducible, high purity compounds. Currently, researchers are limited to few carbohydrate probes, such as MDP, and even fewer larger fragments. We propose to functionalize three carbohydrate cores: monosaccharide, disaccharide and peptide dimers, with select amino acids which are highly represented across a variety of bacterial species. The synthetic workflow is modular, builds off our expertise in producing highly pure PG fragments and permits for modification with chemical biology probes at multiple pinpoints on the library members. A large PG fragment library of this type has not been produced before. To overcome this challenge we propose to use common intermediates for all three carbohydrate cores. We aim to use this library to interrogate innate immune responses elicited in macrophages. In Aim One, a large-scale, modular synthetic effort will be undertaken to produce the >400 member library. For Aim Two the disaccharide and peptide linked dimer family members will be employed in an unbiased genome wide transcriptome analysis to unravel gene expression signatures that define the responses to PG classes in bone marrow derived macrophages. We will synthesize the chemical biology probes to investigate receptors and signaling partners. Aim Three will probe the innate immune response of the PG through the production of a PG-microarray. This array will fix the PG derivatives in multiple orientations permitting the sweeping of potential receptors across chemical space. The arrays will be used to assess the substrate binding preferences of a variety of innate immune receptors indicated in PG recognition. This library will accurately capture the PG fragment diversity, providing powerful probes for the proposed immunological assays and new tools for the microbial and immunological communities.