Enhancing NMR signals in liquids
Analyzing complex (bio)molecules using nuclear magnetic resonance (NMR) spectroscopy has often been challenging due to limitations in sensitivity. Now, a research team led by Marina Bennati at the Max Planck Institute (MPI) for Multidisciplinary Sciences and the University of Göttingen, in cooperation with Bruker BioSpin GmbH, has changed that: The researchers developed a new instrumental design that enables enhancing NMR signals of nuclei such as 13C or 19F in one- and two-dimensional NMR experiments. The research collaboration achieved signal enhancements up to two orders of magnitudes in small molecules, drugs, and metabolites. This paves the way for broader applications of enhanced liquid-state NMR.
NMR spectroscopy is a fundamental spectroscopic technique for the study of biological systems and materials, for molecular imaging, and for analytics of small molecules. Due to its ability to detect interactions at very low energy levels, it is non-invasive and suitable for applications in both animals and humans.
Despite its achievements, NMR faces a major challenge: One of its most severe limitations is the low sensitivity, resulting from the small interaction energies involved. At an NMR field of 9.4 Tesla, the spin polarization is about 32 parts per million (ppm) for proton (1H) and 8 ppm for 13-carbon (13C). The potential for improvement is therefore tremendous.
Dynamic nuclear polarization (DNP) is one way to enhance NMR signals. This technique uses microwave (MW) irradiation to transfer magnetization from a stable organic radical to nuclear spins of interest. DNP in liquids has been known since the early days of magnetic resonance through the so-called Overhauser effect. However, all previous attempts to implement liquid DNP in NMR have been hampered by a lack of NMR resolution, as well as by greatly reduced sample volumes and heating due to high-frequency MW absorption in liquids.
Marina Bennati and her team, together with colleagues at Bruker BioSpin, now realized a setup in which liquid DNP can be performed close to optimal NMR conditions (Nature Communications, July 13, 2024). This allows an unprecedented exploration of NMR signal enhancement for future implementation in high-resolution NMR.
The conceived setup is based on a commercial NMR device (Figure 1), here operating at 9.4 Tesla (400 MHz 1H resonance), which is additionally equipped with a tunable gyrotron MW source (ν ≈ 263.3±0.25 GHz) and a newly designed NMR probe to irradiate the liquid sample with MW. The design of the probe is patented (for reference see below).
The MW beam is spread on the liquid sample confined into a thin layer of variable thickness, that can be tuned on the order of the MW penetration depth for specific solvents. Electromagnetic field simulations were used to calculate and optimize the MW incident field on the sample. The cylindrical geometry of standard NMR sample tubes could be maintained, allowing for future implementation in existing NMR systems.
The performance of the experimental setup was demonstrated with a focus on 13C nuclei, which are crucial for structural NMR spectroscopy but are rather insensitive due to their low gyromagnetic ratio and the low 13C natural abundance (1.1 %). Although the polarization mechanism between the polarizer radical and 13C is site-specific, the method allows to exploit the power of multidimensional NMR spectroscopy, as the experiment can be repeated continuously under steady-state irradiation. For instance, a two-dimensional total correlation experiment (TOCSY) under DNP conditions of a metabolite (ethyl acetoacetate in a tautomeric equilibrium with ethyl 3-hydroxybut-2-enoate) showed cross peaks enhanced of about one order of magnitude. This translates into an up to a hundred times shorter spectral acquisition time.
Although the field of hyperpolarization re-emerged two decades ago, important multidimensional NMR experiments for determining chemical structures have not yet been implemented in conjunction with hyperpolarization. In other approaches, scientists have successfully introduced fast 2D NMR to circumvent the limited lifetime of hyperpolarized substrates. However, these methods face several challenges, either during the ex-situ hyperpolarization step or due to substrate specificity. The simplicity of in situ DNP will boost future research where the full potential of liquid NMR on multiple nuclei can be exploited. (mb)
Patent
Marquardsen, Thorsten; Bennati, Marina; Tkach, Igor; Levien, Marcel; Orlando, Tomas; Yang, Luming; Leveasly, Alisa; Wylde, Richard. DNP Probe Head for High Resolution, Liquid-State NMR. International publication number WO 2024/115698 A1 (6.6.2024)
