A substantial barrier to progress in many areas of biomedical research is the lack of atomic-resolution structural and dynamic characterization of the biomolecule of interest in solution. The main obstacle is often the insufficient amount of available material or, alternatively, the need to keep the target molecule dilute to prevent its aggregation. The goal of this research is to develop novel tools to significantly increase the sensitivity of NMR spectroscopy in solution via laser-driven photo-chemically induced dynamic nuclear polarization (photo-CIDNP). The instrumentation involved in this work comprises high-power lasers (both continuous-wave and pulsed) coupled, via fiber optic, to a commercial 600 MHz NMR spectrometer equipped with a quadruple-resonance (HFCN) cryogenic probe. The advances enabled by this work will facilitate the tackling of important biological questions of medical significance by many investigators, and will enable high- resolution NMR analysis under mild, physiologically relevant conditions in dilute solution. The proposed research involves extensive method development in heteronuclear correlation photo-CIDNP NMR, including the generation of novel pulse sequences to enable 1, 2 and 3D photo-CIDNP sensitivity-enhanced data collection. Non-uniform sampling (NUS) will be implemented as part of the pulse sequence design and subsequent data reconstruction, for the higher-dimensionality experiments. Longitudinal equilibrium and non- equilibrium magnetization will be enhanced via the photo-CIDNP effect for a variety of spin-1/2 nuclei, primarily 1H, 15N, 13C and 19F. In the case of 13C and 19F, the particularly large hyperfine coupling of the transient radical cations of the biomolecules of interest (which is generated as part of photo-CIDNP) promise to yield extremely large enhancements. Preliminary data on 13C photo-CIDNP are extremely encouraging and support the feasibility of the proposed experiments. We will also develop novel photo-CIDNP photosensitizer dyes and matching experiments to use them. In addition, we will synergistically combine experiments with collaborative efforts involving ab initio electron density and hyperfine-coupling-constant calculations to identify and predict the best-performing dyes. Finally, we will develop pilot approaches employing double laser irradiation schemes, which involve the concerted use of a laser capable of exciting the photo-CIDNP dye in the visible range, and another laser exciting the molecule of interest in the ? AE?* transition range. The latter range corresponds to excitation of backbone and side-chain carbonyls of all amino acids, to render them more susceptible to transient oxidation and develop larger sensitivity-enhancement factors in the context of biomedically important polypeptides and proteins.

Public Health Relevance

The applicability of the photo-CIDNP approach proposed here extends to amino acids, polypeptides and proteins under mild and dilute-solution conditions. Hence, the proposed research has wide-ranging implications for a variety of biomedical investigations related to the analysis of dilute biomolecules and aggregation-prone species relevant to neurodegenerative disorders and other misfolding-based diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZGM1)
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Friedman, Fred K
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University of Wisconsin Madison
Schools of Arts and Sciences
United States
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Okuno, Yusuke; Cavagnero, Silvia (2018) Effect of heavy atoms on photochemically induced dynamic nuclear polarization in liquids. J Magn Reson 286:172-187
Okuno, Yusuke; Cavagnero, Silvia (2016) Fluorescein: A Photo-CIDNP Sensitizer Enabling Hypersensitive NMR Data Collection in Liquids at Low Micromolar Concentration. J Phys Chem B 120:715-23
Lee, Jung Ho; Okuno, Yusuke; Cavagnero, Silvia (2014) Sensitivity enhancement in solution NMR: emerging ideas and new frontiers. J Magn Reson 241:18-31
Lee, Jung Ho; Cavagnero, Silvia (2013) A novel tri-enzyme system in combination with laser-driven NMR enables efficient nuclear polarization of biomolecules in solution. J Phys Chem B 117:6069-81
Lee, Jung Ho; Sekhar, Ashok; Cavagnero, Silvia (2011) 1H-Detected 13C photo-CIDNP as a sensitivity enhancement tool in solution NMR. J Am Chem Soc 133:8062-5
Sekhar, Ashok; Cavagnero, Silvia (2009) 1H photo-CIDNP enhancements in heteronuclear correlation NMR spectroscopy. J Phys Chem B 113:8310-8
Sekhar, Ashok; Cavagnero, Silvia (2009) EPIC- and CHANCE-HSQC: two 15N-photo-CIDNP-enhanced pulse sequences for the sensitive detection of solvent-exposed tryptophan. J Magn Reson 200:207-13