This is a competing renewal application of RO1 GM58867 (""""""""Metabolic Oligosaccharide Engineering""""""""). The long-term objective of this project is to develop metabolic oligosaccharide engineering as a tool for fundamental studies of glycan function, particularly with respect to human disease. In the last granting period we discovered that unnatural sugars bearing bioorthogonal functional groups, such as ketones and azides, are metabolized by cells and incorporated into cellular glycans. When displayed on the cell surface, the functionalized sugars can be modified by covalent reactions with exogenous reagents. We employed this new technique in numerous studies of glycobiology, including cell surface targeting as a function of glycan expression pattern. An integral component of the project was the development of bioorthogonal reactions that can be performed on modified glycans expressed on living cells. We developed a reaction of azides and phosphines, termed the Staudinger ligation, with such high selectivity that it can be executed on cultured cells without detriment to their physiology. We further demonstrated that the Staudinger ligation proceeds in living animals, permitting the covalent targeting of cell-surface azidosugars with phosphine probes in vivo. The first major objective of the next granting period is to explore applications of metabolic oligosaccharide engineering to tumor imaging and immunotherapy. This goal is reflected in Aims 1-4.
Aim 1 will define the scope and consequences of azidosugar metabolism in laboratory mice. This includes identifying the specific glycoconjugates that are labeled with azidosugars in vivo and comparative studies of azidosugar metabolism in normal and tumor tissues.
Aim 2 will apply azidosugar metabolism and the Staudinger ligation to noninvasive imaging of tumor glycosylation.
Aim 3 focuses on the development of caging strategies to enhance the selectivity of tumor labeling with azidosugars.
Aim 4 outlines a new approach to tumor vaccine therapy that, exploits unnatural sugars as neoantigens capable of breaking immune self-tolerance. The second major objective is to expand metabolic oligosaccharide engineering to encompass new carbohydrate pathways and new bioorthogonal chemistries. This goal is reflected in Aims 5 and 6.
Aim 5 will probe the metabolism of azido fucose analogs.
Aim 6 describes a modification to the Huisgen azide-alkyne cycloaddition that may render the reaction suitable for in vivo applications.
| Rodriguez-Rivera, Frances P; Zhou, Xiaoxue; Theriot, Julie A et al. (2018) Acute Modulation of Mycobacterial Cell Envelope Biogenesis by Front-Line Tuberculosis Drugs. Angew Chem Int Ed Engl 57:5267-5272 |
| Wan, Stephanie J; Sullivan, Aaron B; Shieh, Peyton et al. (2018) IL-1R and MyD88 Contribute to the Absence of a Bacterial Microbiome on the Healthy Murine Cornea. Front Microbiol 9:1117 |
| Tomlin, Frederick M; Gordon, Chelsea G; Han, Yisu et al. (2018) Site-specific incorporation of quadricyclane into a protein and photocleavage of the quadricyclane ligation adduct. Bioorg Med Chem 26:5280-5290 |
| Jolly, Amber L; Agarwal, Paresh; Metruccio, Matteo M E et al. (2017) Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding. FASEB J 31:2393-2404 |
| Rodriguez-Rivera, Frances P; Zhou, Xiaoxue; Theriot, Julie A et al. (2017) Visualization of mycobacterial membrane dynamics in live cells. J Am Chem Soc 139:3488-3495 |
| Zhu, Xuejun; Shieh, Peyton; Su, Michael et al. (2016) A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool. Chem Commun (Camb) 52:11239-42 |
| Kim, Justin; Bertozzi, Carolyn R (2015) A Bioorthogonal Reaction of N-Oxide and Boron Reagents. Angew Chem Int Ed Engl 54:15777-81 |
| Shieh, Peyton; Dien, Vivian T; Beahm, Brendan J et al. (2015) CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum. J Am Chem Soc 137:7145-51 |
| Beahm, Brendan J; Dehnert, Karen W; Derr, Nicolas L et al. (2014) A visualizable chain-terminating inhibitor of glycosaminoglycan biosynthesis in developing zebrafish. Angew Chem Int Ed Engl 53:3347-52 |
| Shieh, Peyton; Siegrist, M Sloan; Cullen, Andrew J et al. (2014) Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes. Proc Natl Acad Sci U S A 111:5456-61 |
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