New trityl radicals for studies of biopolymers by NMR and EPR spectroscopies

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Nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies are commonly used for investigation of biopolymers. NMR can provide high-resolution structures but is an inherently insensitive technique, which limits its applications. However, the rapidly emerging field of dynamic nuclear polarization (DNP) has led to an increased sensitivity of NMR, of up to several hundred-fold, by transferring spin polarization from electrons to nuclear spins. The main goal of this proposal is the preparation of new triarylmethyl (trityl)-based mono- as well as biradicals for DNP-NMR. A spirocyclohexanolyl-derived trityl radical will address shortcomings of limited water-solubility of trityls and of their electron relaxation rates. In addition to addressing water-solubility of trityls for biological applications with monoradicals, new biradicals will also be prepared, in particular nitroxide-trityl radicals. Moreover, two new classes of trityl-based radicals will be prepared, trityl-trityl biradicals and 1,3-bisdiphenylene-2-phenylallyl (BDPA)-trityl biradicals. These narrow-line biradicals will be prepared for increased polarization of several low-gamma nuclei (13C, 15N and 29Si). Since trityl radicals possess high stability under reducing conditions, some of the new radicals will be used for in-cell DNP-NMR. Maleimide- and iodoacetamide-derived trityl radicals will be prepared for site-directed spin labeling of proteins and the latter will also be applied for nucleic acids, for studies by EPR spectroscopy. Trityl radicals will also be attached to a fluorescently-labeled, cell-penetrating peptide for cellular delivery. Such peptide conjugates will enable visualization of the cellular location of the trityl radicals by fluorescence microscopy. Selected trityl radicals will also be used for state-of-the-art pulsed DNP-NMR experiments. The proposed radicals have the potential to significantly impact the field of DNP-NMR, as well as EPR.