Solid-State 17O NMR Spectroscopy of Fmoc-Protected Amino Acids

  • Author / Creator
    Klein, Brittney A.
  • Enhancing our understanding of the local structure and dynamics for different components within a biological system is key to unraveling their overall function. Nuclear magnetic resonance (NMR) spectroscopy has become an unparalleled technique for exploring both the structure and dynamics within these types of systems. Traditional NMR experiments have focused on investigating the hydrogen, carbon and nitrogen chemical environments comprising these systems which has provided us with invaluable information relating biological functions to physiological processes and has led to more targeted development of pharmaceuticals. However, oxygen, which is another essential element found in all biomolecules, has largely evaded incorporation into biomolecular NMR studies due to the only stable, NMR-active isotope of oxygen (17O) having comparatively unfavourable NMR properties. Past solid-state NMR (ssNMR) studies investigating 17O nuclei within biomolecules have faced significant hurdles trying to address the poor resolution and insensitivity associated with detection of this nucleus. Both these problems stem from the fact that 17O has a very low natural abundance (0.034%), a small magnetogyric ratio (γ= -5.774 MHz T-1) and is a quadrupolar nucleus (I = 5/2). To address these challenges a combination of isotopic enrichment, ultra-high magnetic fields, multidimensional and/or population transfer techniques have been used but implementing these solutions often translates to higher costs and/or longer experimental times. Despite these drawbacks, 17O NMR data has helped to address some of the more intricate issues surrounding the chemical bonding and structural environments in biological systems that are unable to be resolved using traditional methods.
    In this thesis, the 17O NMR parameters for the carboxylic acid moieties in a selection of N-α-fluoren-9-yl-methoxycarbonyl-O-t-butyl (Fmoc)-protected amino acids are investigated using a combination of isotopic enrichment and ultra-high magnetic fields for the first time. A “one-pot” multiple turnover reaction is utilized to introduce 17O labels into both the carbonyl (CO) and hydroxyl (COH) sites of Fmoc-L-isoleucine, Fmoc-L-tryptophan, Fmoc-L-proline, Fmoc-L-tyrosine and Fmoc-L-threonine. Both non-spinning and magic-angle spinning (MAS) 17O NMR spectra were acquired at 14.1 and 21.1 T and used to extract the relevant quadrupolar and chemical shift parameters. These experimental parameters are then compared with computed NMR parameters determined from density functional theory (DFT) computations. This work demonstrates key insights that can be gleamed from 17O NMR studies of small molecules including how 17O NMR spectroscopy can be utilized to monitor the protonation of labelled sites and highlights the importance of developing 17O NMR to enable the study of complex biological systems.

  • Subjects / Keywords
  • Graduation date
    Spring 2022
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.