Glycolipids and glycoproteins are involved in a variety of molecular interactions which are crucial to eukaryotic cellular function. The enzymatic mechinery required for the initial assembly of the majority of cell surface carbohydrates is located in the membrane of the endoplasmic reticulum (ER). This glycan-assembly apparatus consists of a wide spectrum of proteins (mainly glycosyltransferases (GTs)) involved in the construction of N-glycans and glycosylphosphatidylinositol (GPI) anchors. These ER proteins are typically membrane-bound, highly labile and, unlike their counterparts in the Golgi, not generally amenable to conventional protein purification; with very few exceptions only partial purifications have been achieved. Photoaffinity labeling provides an alternative to conventional chromatographic methods in which enzymatic activity must be retained. In photoaffinity labeling, a radiolabeled substrate analog which contains a functional group that can be photoactivated to a highly reactive species upon irradiation with ultraviolet light and thus form covalent bonds with nearby molecules (i.e., proteins) is used. Therefore, one needs enzymatic activity only once to form the non-covalently bound analog-enzyme complex, and following UV-irradiation, the radiolabel is covalently incorporated into the protein. Purification of the protein using conventional chromatographic techniques can then be perfoirmed using the photo-incorporated radiolabel as a marker for the protein. We have developed synthesis schemes for two classes of photoaffinity probes. The structures of the probes are based on structural motifs present in glycosyltransferase substrates which are essential for enzymatic recognition of substrate. In the case of nucleotide-sugar utilizing GTs, the nucleotide itself acts as a competitive inhibitor to enzymatic acitivity, whereas the sugar or sugar-phosphates do not. This demonstrates specific recognition of the nucleotide by the enzyme. Therefore, we have initially based the structure of our probes on uridine-nucleotides. The two classes of probes are 1) 5-azido-UTP and 5-azido-UMP and 2) P3-(4-azidoanilido)-uridine 5?-triphosphate. The synthesis scheme for the first class of probes is an alternative but improved method to existing procedures for synthesis of this class. The advantage of this procedure is ease of separation of reaction products (using chromatographic methods, silica gel and anion exchange) due to the hydrophobicity of the benzoyl-blocking group of the starting material (2?, 3?-dibenzoyluridine). The synthesis of P3-(4-azidoanilido)-uridine 5?-triphosphate is based on analogy to an existing procedure for the guanosine derivative. The uridine derivative synthesis has not been reported previously in the literature. Our motive for utilizing the NMRFAM is to provide routine 1D spectra (1H, 13C, 15N and 31P) for structural characterization of synthetic intermediates and products of the photoaffinity probes described above. The samples would be submitted for service spectroscopy on the DMX 400 MHz spectrometer.
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