Application and methodology of dissolution dynamic nuclear polarization in physical, chemical and biological contexts

Highlights

• dDNP is a powerful tool for real-time monitoring of chemical reactions and biochemical interactions.

• DNP yields the potential of promising applications including metabolomics and protein structural dynamics.

• Higher-dimensional NMR and ultrafast methods coupled to d-DNP enable high-resolution studies of complex soft matter.

• Recent methodological developments hint towards the development of “transportable hyperpolarization”.

Abstract

Dissolution dynamic nuclear polarization (d-DNP) is a versatile method to enhance nuclear magnetic resonance (NMR) spectroscopy. It boosts signal intensities by four to five orders of magnitude thereby providing the potential to improve and enable a plethora of applications ranging from the real-time monitoring of chemical or biological processes to metabolomics and in-cell investigations. This perspectives article highlights possible avenues for developments and applications of d-DNP in biochemical and physicochemical studies. It outlines how chemists, biologists and physicists with various fields of interest can transform and employ d-DNP as a powerful characterization method for their research.

Introduction

Dissolution dynamic nuclear polarization (d-DNP) is a technique that aims at overcoming experimental limitations of solution state nuclear magnetic resonance (NMR) spectroscopy imposed by its intrinsically low sensitivity. Weak signal intensities in NMR are a major bottleneck that often impede rapid detection schemes by necessitating signal averaging or high (e.g., non-physiological) concentrations of target molecules. d-DNP [1] mitigates these constraints by providing hyperpolarized nuclear spin states, which feature dramatic signal intensity boosts by several orders of magnitude. It is probably the most versatile method among other established hyperpolarization techniques, such as para-hydrogen induced polarization (PHIP) [2], signal amplification by reversible exchange (SABRE) [3] or spin exchange or metastability exchange optical pumping (SEOP or MEOP) [4]. Several reviews describing these methods are already available [5].

d-DNP is based on the general concepts of DNP [6] making use of the high polarization of electron spins by transferring it to nuclear spins through microwave irradiation at low temperatures. The original idea behind d-DNP [1] consists in a fast dissolution of the sample after DNP, which preserves most of the hyperpolarization. This approach enables a plethora of solution-state applications in analytical NMR or magnetic resonance imaging (MRI). Here again several reviews are available in the literature [7], [8], [9], [10].

d-DNP provides the potential to boost signal intensities by three to four orders of magnitude thereby opening possibilities to access materials and properties that cannot be studied by conventional NMR [11], [12]. Hence, potential applications in various fields of research ranging from physical chemistry over nuclear physics to biochemistry are imaginable.

While one of the currently most prominent applications of d-DNP relies on the use of hyperpolarized biomolecular imaging tracers for in-vivo metabolic MRI [13], [14], this article targets the in-vitro analytical, physiochemical and biochemical applications and perspectives of d-DNP thereby trying to raise the attention of a multidisciplinary community. After a brief introduction, current uses in biochemical, chemical and physical contexts will be outlined and perspectives will be developed for future applications, such as real-time chemical reaction monitoring, investigation of biological substrates, metabolomics and nuclear spin physics of solids. Subsequently, based on the current state-of-the-art a perspective on future instrumentation and methods improvements will be developed in view of improving d-DNP to make it more efficient, general, or transportable.

d-DNP goes back to an invention by Ardenkjaer-Larsen, Golman and co-workers in 2003 [1], who combined DNP of frozen liquids at low-temperatures (see [15] and references therein for a detailed discussion of DNP mechanisms) with a subsequent dissolution and rapid transition to ambient temperatures to increase signal intensities in liquid state NMR spectroscopy. The combination of temperature jumps with DNP can spectacularly increase nuclear spin polarizations $P$_I (vide infra for a more detailed definition), yet such an experiment also requires peculiar instrumentation and workflows. Currently, a prototypical d-DNP experiment for hyperpolarized NMR consists of three principal steps:

• Dynamic nuclear polarization of a sample at a low temperature $T^{\mathrm{D}\mathrm{N}\mathrm{P}}\le$ 4.2 K within a dedicated DNP apparatus that maintains a static magnetic field 3.35 T $<B_0^{\mathrm{D}\mathrm{N}\mathrm{P}}<$ 11.8 T;

• rapid heating and dissolution of the sample to the liquid state prior to fast transfer to an NMR spectrometer operating at typical magnetic fields $B_0^{\mathrm{N}\mathrm{M}\mathrm{R}}>$ 7 T or dedicated low-field devices;

• NMR detection at close to ambient temperature $T^{\mathrm{N}\mathrm{M}\mathrm{R}}>$ 273 K.

The basic setup of such an experiment is sketched in Fig. 1, which depicts a DNP apparatus connected to a conventional NMR spectrometer via a dissolution and transfer system.

To give the reader an overview over the requirements of a d-DNP experiment, a brief description of steps (i), (ii) and (iii) is provided in the following:

(i) The experiment starts with the preparation of a sample that contains paramagnetic molecules that are often coined polarization agents (PAs). Stable radicals such as nitroxides or tri-aryl methyl compounds often serve as PAs. After freezing of the sample, partial saturation of the electron paramagnetic resonance (EPR) line of a PA by slightly off-resonant microwave irradiation will entail an increased nuclear spin polarization $P$ due to a transfer of polarization from the unpaired electrons of the PAs to the nuclei in their vicinity (the ratio of gyromagnetic ratios is γ(e⁻)/γ(¹H) ≈ 668). This process normally takes place in a dedicated DNP apparatus, as it requires probes with microwave irradiation and often NMR capabilities (see for example Refs. [1], [16], [17]). A more or less homogeneous distribution of PAs in the DNP samples usually leads to optimal hyperpolarization. Therefore, crystallization, which leads to uncontrolled local PA-enriched and -depleted phases needs to be avoided, if necessary enforced by addition of vitrification agents such as glycerol [18].

The sections “Sample Throughput” and “Transportable Hyperpolarization” provide a closer look at current developments concerning instrumentation and state-of-the-art PAs. For the moment it is sufficient to consider that microwave irradiation induces DNP by means of various mechanisms, most prominent, the cross effect and thermal mixing [19], [20]. The effectiveness of the different mechanisms depends on various factors such as the EPR line-shape, the concentration and the electron longitudinal relaxation time T_1e of the PA. In d-DNP applications, one typically operates at temperatures close to 1 K, where mixtures of several mechanisms may be operative. A detailed description of these processes and their relation to d-DNP is beyond the scope of this perspective article. The reader is referred to the seminal work by Abragam and Goldman [6] as well as to monographs [21], articles [15], [22], [23], [24], [25], [26] and reviews [5], [27]. Yet, for the following perspectives the reader should keep in mind that DNP of samples that contain millimolar quantities of PAs entails a transfer of polarization from a PA to nuclear spins in its vicinity causing a boost in NMR signal intensity by increasing the nuclear polarization ${\mathit{P}}_{\mathit{I}}.$ The resulting spin state is then denoted as “hyperpolarized”.

(ii) After the hyperpolarization procedure is complete, the sample is rapidly dissolved and diluted in hot heavy water and transferred to a conventional NMR spectrometer. The influence of the temperature jump during a d-DNP experiment can directly be deduced from Eq. (1):

$$P_{\mathrm{I}}^{\mathrm{D}\mathrm{N}\mathrm{P}}={\epsilon }^{\mathrm{D}\mathrm{N}\mathrm{P}}\times {\epsilon }^{\mathrm{j}\mathrm{u}\mathrm{m}\mathrm{p}}={\epsilon }^{\mathrm{D}\mathrm{N}\mathrm{P}}\times \frac{\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{h}\left(\frac{\hslash \gamma B_0^{\mathit{DNP}}}{2k_B{T}^{\mathrm{D}\mathrm{N}\mathrm{P}}}\right)}{\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{h}\left(\frac{\hslash \gamma B_0^{\mathit{NMR}}}{2k_B{T}^{\mathrm{N}\mathrm{M}\mathrm{R}}}\right)}{=\epsilon }^{\mathrm{D}\mathrm{N}\mathrm{P}}\times \frac{B_0^{\mathit{DNP}}{T}^{\mathrm{N}\mathrm{M}\mathrm{R}}}{B_0^{\mathit{NMR}}{T}^{\mathrm{D}\mathrm{N}\mathrm{P}}}$$

with ${\epsilon }^{\mathrm{D}\mathrm{N}\mathrm{P}}$ the polarization enhancement brought about by DNP, and $\hslash$ and $k_B$ the Planck and Boltzmann constants, respectively, and $\gamma$ the gyromagnetic ratio of the nuclear spin $I$. The lower the temperature $T^{\mathrm{D}\mathrm{N}\mathrm{P}}$ at which the DNP process takes place, the higher the thermal equilibrium polarization. The large signal enhancement in a d-DNP experiment can therefore be traced back to the multiplicative effects of DNP and the temperature jump.

The speed of the dissolution and transfer process is critical due to two major factors. Firstly, large fractions of hyperpolarization can be lost during the dissolution/heating process, if it proceeds to slowly. The target substance may pass through a regime in which nuclear spin relaxation is very effective (e.g., slow tumbling of the molecule during the dissolution and exacerbate paramagnetic relaxation) causing a rapid decay of the hyperpolarization. Secondly, the sample needs to be rapidly transferred from the DNP apparatus to the detection NMR device during a transfer time t^trans through the magnetic field B^trans. The latter might be uncontrolled or too low, which may cause enhanced losses of hyperpolarization via enhanced paramagnetic relaxation [28] or scalar relaxation of the first and second kind [29], [30]. Several recent developments try to mitigate this bottleneck of the d-DNP technology. For example, the use of magnetic tunnels [31] that maintain a stable magnetic field B^trans during the transfer or high-pressure transfer systems in which the sample is propelled within only 0.5 to 2 s (depending on the setup) from the DNP apparatus to the NMR spectrometer [32], [33].

Additionally, it has recently been proposed that the dissolution process can take place in the detection NMR spectrometer after the cold solid sample has been transferred and not as usual within the DNP apparatus before the transfer [34]. Several developments and perspectives in this regard will be discussed in more detail below in section “Solid transfer, liquid transfer”.

(iii) After the sample has been dissolved and transferred, NMR detection proceeds. The hyperpolarized spin states can be exploited in NMR experiments if their duration does not exceed the lifetime of the hyperpolarization. A single pulse experiment with a 90° detection pulse will yield maximum sensitivity in a single scan (see Fig. 2), but it is also very common to detect the hyperpolarized magnetization with a train of small angle detection pulses, especially when the experiment aims at monitoring kinetic or dynamic processes.

In contrast to conventional NMR, where the experiment may last indefinitely within spectrometer stability and schedule constraints, signal detection in a d-DNP experiment needs to be completed within a short time interval after the sample has been transferred (see Fig. 1). The hyperpolarization has a limited lifetime as it inevitably decays towards thermal equilibrium with the time constant of longitudinal nuclear relaxation T₁.

The limited lifetime of hyperpolarization and the irreversible nature of d-DNP imposes limitations in the application of conventional 2D and 3D experiments, which typically take longer than 2 min. Several approaches have been developed to resolve this dilemma. They will be discussed in more detail in the section “High-resolution d-DNP vs. high-resolution NMR” as well as “d-DNP and ultrafast NMR detection”. While, on the one hand, a d-DNP experiment is time-constrained, it is, on the other hand, boosted by signal enhancements ε^d-DNP of three to four orders of magnitude. ε^d-DNP is here understood as the ratio between nuclear spin polarization P^d-DNP of a hyperpolarized sample and corresponding polarization in thermal equilibrium P^TE. This ratio can reach over 1 000 in proton NMR and over 10 000 in NMR of heteronuclei [11], [12]. Hence, novel detection strategies for higher-dimensional or time-resolved NMR become conceivable.

Small angle detection pulses of typically 1−30° are sufficient to observe an intense signal although only a small share of the nuclear polarization is used for detection. The readout can therefore be repeated rapidly, e.g. every 100 ms, before the polarization has vanished. In contrast, conventional NMR typically employs longer waiting times between every detection together with a readout of the entire polarization by 90° pulses. These relations are visualized in Fig. 2.