After 2 days at room temperature, TLC (80:20 CHCl3, CH3OH) indicated the disappearance of the starting 4-hydroxy TEMPO. bound conformations and binding motifs of substrates include trNOE spectroscopy and STD spectroscopy. Use of trNOEs has been extensively reviewed and there are a number of applications to carbohydrates bound to proteins.13 trNOEs are acquired using standard 2D NOE sequences to monitor correlations among ligand resonances and provide distance constraints between proximate pairs of protons. Use in cases where ligands are rapidly exchanging between bound and free forms is particularly advantageous because excess ligand (10C30 X) can be used to improve sensitivity while NOEs from the bound state still dominate the observed average. This weighted average occurs because of the linear dependence of magnetization transfer rates on correlation times for large systems. STD spectroscopy is of more recent vintage and is used to identify binding epitopes on the surfaces of ligands. Spectra are collected with and without saturation of regions of the spectrum containing only protein LY-2584702 hydrochloride resonances. In the presence of excess ligand, differences between the spectra primarily show resonances belonging to ligand protons in close proximity to protein protons. STD depends on the efficient spin diffusion of magnetization among protons within large proteins and the transfer of this magnetization from protein to ligand protons in a 1/r6 dependent fashion. The third method used for investigation of ligand geometry is intended to retrieve information on the relative placement of the two ligands in the binding pocket. In principle, this could be determined using inter-ligand NOEs from the trNOE experiments, but these data are limited by the requirement of very close approach and reduction of signal due to partial occupations of each site. An alternate approach uses perturbation of spin relaxation by the presence of an unpaired electron on one of the ligands. Relaxation of spins on the other ligand is enhanced with the same 1/r6 distance dependence as an NOE, LY-2584702 hydrochloride but because the magnetic moment of an electron is on the order of 1000 times that of a proton, the distance range is larger. The unpaired electron in our case was introduced by replacing the GlcNAc in UDP-GlcNAc with a nitroxide containing TEMPO moiety. This compound is a novel addition to a set of tools that LY-2584702 hydrochloride might be used to investigate properties of a number of glycosyltransferases. Use of TEMPO analogues to retrieve structural constraints is well established in systems where the nitroxides are covalently attached to a protein and amide protons of the protein are observed; here distances between protons and nitroxide as large as 20 ? have LY-2584702 hydrochloride been characterized.14 In our case we detect relaxation effects by introducing spin relaxation delays in the preparation period of HBEGF 1H-13C HSQC experiment of rapidly exchanging ligands. The 2D HSQC experiment provides the resolution needed to individually monitor effects on a large number of sites on the acceptor in the presence of the TEMPO analogue of the sugar donor. Modeling of all data collected provides a picture of bound ligand geometry that constrains the active site of a structurally uncharacterized protein. In principle, the geometry could be used to begin to rationally design inhibitors that could modulate activity of an enzyme whose activity can regulate the invasiveness and metastatic potential of malignant cells. Results Binding Epitopes from Saturation Transfer Difference Saturation transfer difference spectroscopy was used to determine the binding epitopes of the acceptor,.