De l'usage des protons hyperpolarisés pour augmenter la sensibilité de la RMN

Nuclear Magnetic Resonance (NMR) has become an inescapable technique for spectroscopic identification. Its main advantage comes from the sensitivity of NMR active nuclei embedded in a molecule to their chemical environment. NMR is also used daily in medical imaging. Magnetic Resonance Imaging (MRI) is not only remarkably versatile, but has the precious advantage of being non-invasive; moreover, the range of radiofrequency used implies that MRI deposit a limited amount of energy in tissues under investigation. Nevertheless, compared to other spectroscopic methods, NMR suffers from a relative lack of sensitivity. Indeed as the NMR transitions are low in energy, the difference of populations between the levels involved, known as polarization, is extremely low. The NMR signal, which is directly proportional to this polarization, is thus many orders of magnitude inferior to the theoretical maximum at full polarization. Dynamic Nuclear Polarization (DNP) allows one to circumvent this disadvantage by transferring the high electron spin polarization to the nuclear spins. This transfer happens via microwave irradiation under optimized conditions. The method has been constantly developed since the âfifties. A substantial breakthrough was achieved in 2003 by Golman, Ardenkjaer-Larsen and their collaborators. They proposed to dissolve a sample that has been hyperpolarized at low temperature (at about 1 K) in order to inject it into an animal, or, in fine, into a human patient, and to follow its bio-distribution and eventual metabolic conversion by MRI. This technique, called Dissolution-DNP (D-DNP), allows a signal increase on the order of 10'000, opening the way to many new experimental possibilities. Research in Dissolution-DNP was largely oriented toward the optimization of 13C polarization because of its long lifetimes. In the course of this Thesis, an alternative way that takes advantage of the proton (1H) polarization will be explored. Protons have the advantage that they can be polarized to a higher levels in a shorter time compared to 13C. Unfortunately, once ejected from the polarizer, in solution and at room temperature, the high proton magnetization will be short-lived compared to 13C. Along the chapters of this Thesis, different approaches will be proposed to maximize the advantages of 1H polarization, while minimizing its inconveniences. It is possible to use a standard NMR technique, known as Cross-Polarization (CP), to transfer the abundant magnetization of hyperpolarized protons to other nuclei like 13C, using suitable radiofrequency pulse sequences. The advantages of the 1H polarization are exploited inside the polarizer, while the interesting properties of 13C are put to use after dissolution. Still, it is also possible to observe directly the proton signal after dissolution. This can be extremely interesting, especially in the context of drug screening for pharmaceutical research. Two examples of such methods will be described. Finally, the use of hyperpolarized 1H signals after dissolution can be greatly improved if their relaxation rates could be attenuated. A first way of doing this, consisting in removing the paramagnetic species by filtration, will be explored. The use of Long-Lived States (LLS) will also be presented

Cross Polarization for Dissolution Dynamic Nuclear Polarization Experiments at Readily Accessible Temperatures 1.2< T <4.2K

Cross polarization can provide significant enhancements with respect to direct polarization of low-γ nuclei such as 13C. Substantial gains in sample throughput (shorter polarization times) can be achieved by exploiting shorter build-up times τDNP(1H)<τDNP(13C). To polarize protons rather than low-γ nuclei, nitroxide radicals with broad ESR resonances such as TEMPO are more appropriate than Trityl and similar carbon-based radicals that have narrow lines. With TEMPO as polarizing agent, the main Dynamic Nuclear Polarization (DNP) mechanism is thermal mixing (TM). Cross polarization makes it possible to attain higher polarization levels at 2.2K than one can obtain with direct DNP of low-γ nuclei with TEMPO at 1.2K, thus avoiding complex cryogenic technolog

Microwave-gated dynamic nuclear polarization

Dissolution dynamic nuclear polarization (D-DNP) has become a method of choice to enhance signals in nuclear magnetic resonance (NMR). Recently, we have proposed to combine cross-polarization (CP) with D-DNP to provide high polarization P(C-13) in short build-up times. In this paper, we show that switching microwave irradiation off for a few hundreds of milliseconds prior to CP can significantly boost the efficiency. By implementing microwave gating, C-13 polarizations on sodium [1-C-13]acetate as high as 64% could be achieved with a polarization build-up time constant as short as 160 s. A polarization of P(C-13) = 78% could even be reached for [C-13]urea

Drug Screening Boosted by Hyperpolarized Long-Lived States in NMR

Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1(1H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak “spy ligands” can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput

Extending Timescales and Narrowing Linewidths in NMR

Among the different fields of research in nuclear magnetic resonance (NMR) which are currently investigated in the Laboratory of Biomolecular Magnetic Resonance (LRMB), two subjects that are closely related to each other are presented in this article. On the one hand, we show how to populate long-lived states (LLS) that have long lifetimes T_LLS which allow one to go beyond the usual limits imposed by the longitudinal relaxation time T_1. This makes it possible to extend NMR experiments to longer time-scales. As an application, we demonstrate the extension of the timescale of diffusion measurements by NMR spectroscopy. On the other hand, we review our work on long-lived coherences (LLC), a particular type of coherence between two spin states that oscillates with the frequency of the scalar coupling constant J_IS and decays with a time constant T_LLC. Again, this time constant T_LLC can be much longer than the transverse relaxation time T_2. By extending the coherence lifetimes, we can narrow the linewidths to an unprecedented extent. J-couplings and residual dipolar couplings (RDCs) in weakly-oriented phases can be measured with the highest precision

An automated system for fast transfer and injection of hyperpolarized solutions

Dissolution dynamic nuclear polarization (dDNP) has become a hyperpolarization method of choice for enhancing nuclear magnetic resonance (NMR) signals. Nuclear spins are polarized in solid frozen samples (in a so-called polarizer) that are subsequently dissolved and transferred to an NMR spectrometer for high sensitivity detection. One of the critical challenges of dDNP is that it requires both a fast transfer to limit nuclear spin relaxation losses as well as stability to guarantee high resolution (no bubbles nor turbulences). Here we describe the design, construction and performances of such a transfer and injection system, that features a 5 m/s speed and sub-Hz spectral resolution upon arrival at the detection spot. We demonstrate the use of such a system for inter-magnet distances of up to 10 m