Metabolic Engineering with Bioorthogonal Chemical Reporters

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DESCRIPTION (provided by applicant): The broad objective of this project is to apply a technique developed in our lab termed metabolic oligosaccharide engineering to in vivo imaging of global changes in the glycome associated with embryonic development and cancer. The "glycome" is the totality of glycans that cells produce under specified conditions of time, space and environment. Changes in the glycome's composition and distribution are associated with embryogenesis and cancer progression. We seek to develop chemical tools for imaging the dynamic cell-surface glycome in living organisms. In the last granting period, we demonstrated that three important sectors of the glycome - sialylated glycans, mucin-type O-glycans and fucosylated glycans, can be metabolically labeled with azido analogs of their biosynthetic precursors. The azide served as a chemical reporter that was visualized by Staudinger ligation with phosphine probes. We performed non-invasive imaging of sialic acids in healthy mice by metabolic labeling with N-azidoacetylmannosamine (ManNAz) followed by sequential injection of biotinylated phosphine and fluorescent streptavidin conjugates. For direct labeling of azidosugars, we designed fluorescent phosphine probes with a variety of spectral properties. In order to improve the sensitivity and time resolution of glycan imaging, we developed a new bioorthogonal reaction with faster kinetics than the Staudinger ligation: the strain-promoted cycloaddition of azides and cyclooctynes ("Cu-free click chemistry"). We employed a difluorinated cyclooctyne (DIFO) to image spatiotemporal changes in the glycomes of live cells and developing zebrafish. In the next granting period we plan to build upon these discoveries with four specific aims. First, we will expand our analysis of glycomic transformations during zebrafish development (Aim 1). We will image new sectors of the glycome (e.g., sialylated glycans, fucosylated glycans, glycosaminoglycans and N-glycans) at various stages of development. In addition, we will perturb the expression of certain glycosyltransferases and monitor concomitant changes in the glycome by in vivo imaging. We will develop new cyclooctyne imaging reagents with improved pharmacokinetic and fluorogenic properties (Aim 2). With the use of new phosphine and cyclooctyne probes, we will image glycans in mouse tumor models (Aim 3). Finally, we will develop new bioorthogonal reactions to expand the scope of the chemical reporter method (Aim 4). PUBLIC HEALTH RELEVANCE: All human cells are coated with complex sugar molecules termed "glycans". Each type of cell in the human body has its own collection of these glycans coating the cell surface. When cells transform from an embryonic state to a mature state, or from a healthy state to a cancerous state, the collection of glycans changes its makeup. The goal of this project is to develop tools from the field of chemistry that can help researchers and physicians monitor the changes in cell- surface glycans inside the body using imaging techniques. These chemical tools could be useful for cancer detection and diagnosis.