NMR Spectroscopy Unchained: Attaining the Highest Signal

Perspective Cite This: J. Phys. Chem. Lett. 2018, 9, 5481−5489

pubs.acs.org/JPCL

NMR Spectroscopy Unchained: Attaining the Highest Signal Enhancements in Dissolution Dynamic Nuclear Polarization Peter Niedbalski, Andhika Kiswandhi, Christopher Parish, Qing Wang, Fatemeh Khashami, and Lloyd Lumata*

J. Phys. Chem. Lett. 2018.9:5481-5489. Downloaded from pubs.acs.org by NEWCASTLE UNIV on 10/18/18. For personal use only.

Department of Physics, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States ABSTRACT: Dynamic nuclear polarization (DNP) via the dissolution method is one of the most successful methods for alleviating the inherently low Boltzmann-dictated sensitivity in nuclear magnetic resonance (NMR) spectroscopy. This emerging technology has already begun to positively impact chemical and metabolic research by providing the much-needed enhancement of the liquid-state NMR signals of insensitive nuclei such as 13C by several thousand-fold. In this Perspective, we present our viewpoints regarding the key elements needed to maximize the NMR signal enhancements in dissolution DNP, from the very core of the DNP process at cryogenic temperatures, DNP instrumental conditions, and chemical tuning in sample preparation to current developments in minimizing hyperpolarization losses during the dissolution transfer process. The optimization steps discussed herein could potentially provide important experimental and theoretical considerations in harnessing the best possible sensitivity gains in NMR spectroscopy as afforded by optimized dissolution DNP technology.

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here is no question that chemical research, from the fundamental to applied, has benefitted tremendously from the discovery and subsequent harnessing of the nondestructive analytical power of nuclear magnetic resonance (NMR) for spectroscopic and imaging (MRI) purposes. In spite of the multitude of uses and benefits of NMR, it suffers from a crippling downside, lack of signal sensitivity, which is prominently manifested in mass-limited samples and samples with low-receptivity nuclei.1,2 This unfortunate property, which sometimes requires prohibitively long acquisition times for NMR measurements, stems from the low Boltzmann-dictated nuclear polarization P, which for an ensemble of spin I = 1/2 nuclei can be as described as i γhB yz zz P = tanhjjj k 2kT {

In spite of the multitude of uses and benefits of NMR, it suffers from a crippling downside, lack of signal sensitivity, which is prominently manifested in masslimited samples and samples with low-receptivity nuclei. nuclei, cooling samples to improve Boltzmann polarization, increasing magnetic field strength to incredible heights, and using cryogenically cooled probes to minimize Johnson noise in signal detection.1 However, arguably the most significant development in terms of improving NMR signal strength has been dynamic nuclear polarization (DNP).1,2 In DNP, ensembles of nuclear spins are dynamically brought to a highly nonthermal equilibrium polarization state, greatly increasing their NMR signal for a time. There are a number of techniques that fall under the umbrella of hyperpolarization, including parahydrogen-induced polarization (PHIP), chemically induced dynamic nuclear polarization (CIDNP), and spin exchange optical pumping (SEOP).1 Traditionally, however, DNP refers to microwave-induced polarization transfer from free electrons to nearby nuclear spins.3 Generally, such polarization transfer falls into one of four categories: the Overhauser effect (OE), the solid effect (SE), the cross effect

(spin‐1/2 nuclei)

where γ is the nuclear gyromagnetic ratio (in MHz/T), h Planck’s constant, B the external magnetic field, k Boltzmann’s constant, and T the sample temperature.3 While the parameters B and T can be externally controlled via appropriate instrumentation, γ is something intrinsic or specific for an NMR active nuclear spin; thus, the low sensitivity problem in NMR is somewhat in its nature. Based on the Boltzmann equation, nuclear polarization at ambient NMR conditions for nuclei such as 13C spins remains well below a small fraction of 1% or on the order of only a few parts per million. To circumvent the low sensitivity problem in NMR, work has been performed along many avenues to improve NMR signal strength, including but not limited to using bulk samples of nuclear spins, isotopically enriching low-natural abundance © 2018 American Chemical Society

Received: May 31, 2018 Accepted: September 4, 2018 Published: September 4, 2018 5481

DOI: 10.1021/acs.jpclett.8b01687 J. Phys. Chem. Lett. 2018, 9, 5481−5489

The Journal of Physical Chemistry Letters

Perspective

Figure 1. Schematic diagram showing the spin transitions for the solid effect (SE) and cross effect (CE) and the heat-transfer mechanism for thermal mixing (TM).

(CE), or thermal mixing (TM).4 Of these four, OE was the initial discovery of DNP and is effective for polarizing metallic solids and liquid samples. This method exploits relaxation of electron−nucleus pairs along forbidden transitions to create nonequilibrium nuclear polarization states, thereby improving the NMR signal strength. In comparison to the other mechanisms of DNP (Figure 1), the Overhauser effect gained only relatively modest polarization enhancements.4 In the solid effect DNP mechanism, dipolar coupling between nuclei and nearby electron spins enables direct excitation of forbidden transitions by microwave irradiation at a frequency corresponding to ω = ωe ± ωN, thereby allowing for significant polarization enhancement of nuclei in the solid state.5 However, more common for the polarization of low-γ nuclei are the cross effect and thermal mixing, both of which invoke triple-spin flips.4,6,7 Thermal mixing is most often described in terms of thermodynamic reservoirs. Specifically, spin systems, including the electron Zeeman system, nuclear Zeeman system, and electron dipolar system are treated as thermodynamic reservoirs with an associated spin temperature.8 Microwave irradiation near the electron resonance brings the nuclear Zeeman system in contact with the electron systems, leading to dynamic cooling of the nuclear spins, which is equivalent to increasing polarization. Thermal mixing is dominant when the electron paramagnetic resonance (EPR) line width is broader than or comparable to the nuclear Larmor frequency and specifically requires high EPR line saturation. A special case of thermal mixing is the cross effect in which two electrons are coupled to a single nucleus. Moreover, the Larmor frequencies of the electrons are separated by nuclear Larmor frequency, i.e. ωN = ωe1 − ωe2. In practice, this corresponds to an electron paramagnetic resonance (EPR) line width that is inhomogeneously broadened such that its line width is greater than the nuclear resonance frequency.4,6 Microwave irradiation at one of the electron resonances leads to triple spin flips, which is a more effective method of polarizing spins than the double flips used in the solid effect. While the EPR line is completely saturated because of rapid spin diffusion across the EPR line in thermal mixing, the cross effect involves only partial saturation.6,7,9 These DNP mechanisms are capable of improving polarization several hundred-fold greater than thermal equilibrium. There are many benefits to this improvement as it pertains to basic research, but utilizing the highly improved polarization is hampered by the added requirements of high-resolution NMR in the solid state at the cryogenic temperatures necessitated by DNP. In 2003, Ardenkjær-Larsen and co-workers showed that the polarization gained through solid-state DNP could be harnessed in the liquid state through a rapid dissolution of a polarized sample.10 In this process known as dissolution DNP,

liquid-state samples are generated with polarization enhanced greater than 10 000-fold relative to thermal equilibrium signal. The dissolution DNP technology has unlocked numerous applications, most notably in vivo metabolic imaging using 13 C-labeled molecules and other target compounds with traditionally weak NMR nuclei.11,12 However, even as applications of dissolution DNP are in abundance, there are unanswered questions regarding the underlying physics and optimization procedures behind the process. Our research group has placed a strong emphasis on coming to a greater understanding of chemical, physical, and instrumentation methods by which polarization and the dissolution DNP process in general may be improved. We note that the bulk of our work has been in optimization of 13C polarization, but it is expected that the principles and procedures discussed are generalizable to other low-γ nuclei.

Because DNP involves transfer of polarization from paramagnetic centers to nuclei, the source of free electrons plays a central role in determining the overall efficiency of the DNP process. Because DNP involves transfer of polarization from paramagnetic centers to nuclei, the source of free electrons plays a central role in determining the overall efficiency of the DNP process.13 In dissolution DNP, the source of free electrons is most commonly trace amounts of stable, organic spin-1/2 free radicals that are added to samples of nuclear spins (Figure 2).11 The most heavily used radicals are nitroxide-based TEMPO radicals and carbon-centered trityls. TEMPO radicals have very broad EPR line widths and significant g-anisotropy, which reduces their efficiency for direct polarization of low-γ nuclei. Even so, it has been used with some degree of success to polarize 13C spins.14,15 Arguably the better use for TEMPO, though, is polarization of protons, as it polarizes 1H spins very efficiently. Perhaps the most significant use of TEMPO in dissolution DNP currently is for cross-polarization.16,17 In this process, TEMPO is used to hyperpolarize proton spins within a sample. Subsequently, NMR pulse sequences transfer the 1H polarization to the 13C or other low-γ nucleus used in the experiment. The advantages of this method are due to the properties of 1H nuclei: (1) the high γ of 1H ensures a high polarization due to compatibility of its nuclear Zeeman energy with the electron dipolar energy, and (2) 1H spins can be polarized and repolarized quickly. Cross-polarization has been found to yield extremely high 13C 5482



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effective for hyperpolarization of low-γ nuclei. The primary difference between the two is solubility. While a trityl radical such as OX063 is water-soluble, BDPA typically is hydrophobic and is soluble only in specialized solvents such as DMSO or sulfolane, so there is only a limited number of compounds that it can hyperpolarize. The one benefit that comes out in the hydrophobicity of BDPA is that it precipitates out after dissolution with water; thus, it can be easily and quickly removed from the hyperpolarized liquid with a commercially available syringe filter. Furthermore, BDPA derivatives soluble in water22 or pyruvic acid23 have been developed that could potentially increase its applicability in polarizing various substrates, though these have not yet seen extensive use in dissolution DNP especially in in vivo applications. Meanwhile, the organic free radicals DPPH24 and galvinoxyl25 also require specialized solvents in DNP sample preparation but were shown to provide decent signal enhancement numbers for 13C spins and other nuclei. DPPH and galvinoxyl have EPR line widths that are intermediate between that of trityl OX063 and TEMPO.24,25 Because the type of DNP mechanism involved for a particular nuclear species is intimately linked to the EPR line width of a polarizing agent,3,8 the availability of organic free radical polarizing agents with varying EPR line widths presents an opportunity to investigate the effects of chemical tuning of this EPR parameter on the DNP efficiency of various nuclei. In addition to these organic monoradicals, there has been a host of other paramagnetic centers used for DNP. For instance, the biradical BDPAesterTEMPO26 has been tested to be an effective polarizing agent for dissolution DNP with liquid-state NMR enhancements exceeding 50 000-fold at high field and room temperature. Meanwhile, trityl biradicals, along with other commonly used biradicals for solid-state DNP-NMR, gained only relatively modest enhancements because of their strong reduction effect on nuclear T1 relaxation.27 Other more novel polarization methods use paramagnetic defects in silicon nanoparticles or nanodiamond.28,29 Finally, perhaps the most promising novel radicals for dissolution are the nonpersistent, photoinduced radicals that are created through ultraviolet irradiation of pyruvic acid at low temperatures and immediately quenched upon dissolution.30 The quenching of photoinduced radical after dissolution translates to longer lifetime of the hyperpolarized nuclei in the liquid-state.

Figure 2. W-band ESR spectra, 13C microwave DNP sweeps, and structures of stable organic spin-1/2 free radicals (BDPA, trityl OX063, galvinoxyl, DPPH, and 4-oxo-TEMPO) that were tested and used as polarizing agents in dissolution DNP. The spread of the 13C microwave DNP sweep is proportional to the EPR spectral line width of the free radical.

polarizations in only a fraction of the time required by conventional methods.16,17 Cross-polarization currently remains somewhat on the fringe of dissolution DNP because of the specialized instrumentation required for the procedure, but it will likely become incorporated into mainstream DNP in the near future. In contrast to TEMPO, trityl radicals have extremely narrow EPR line widths, which makes them particularly effective for the direct polarization of low-γ nuclei like 13C and 15N.18 This is because the nuclear Zeeman energies of these low-γ nuclei match the electron dipolar energy of trityl; thus, thermal contact is established between the two energy reservoirs. Under the thermal mixing mechanism, microwave irradiation dynamically cools the electron dipolar system which in turn cools the nuclear Zeeman systems in thermal contact with it, resulting in enhanced nuclear polarization levels. Using trityl to directly polarize samples can yield solid-state 13C polarizations approaching 80% at high field such as 5 T and sub-Kelvin temperature.19 This high degree of efficiency has made trityl the most heavily used radical for dissolution DNP studies and has made it the standard by which other radicals are judged. Moreover, certain variants of trityl free radical such as AH111501 can be removed from solution post dissolution by lowering the pH of the solution significantly, which causes the radical to precipitate, whence it can be filtered out.11 Alternatively, the radical may be removed via chromatography.20 These methods of radical extraction make trityl a great polarizing agent for sterile in vivo studies, and in fact, a trityl radical has been used in the initial in vivo human 13C metabolic imaging studies using dissolution DNP.21 While trityl and TEMPO have seen the bulk of usage in dissolution DNP, there are a number of other free radicals that have been used with some success. Some of these include the stable organic free radicals BDPA, DPPH, and galvinoxyl.11 BDPA, like trityl, has a very narrow line width and is very

Because the NMR active nuclei in the glassing solvents may also participate in the microwavedriven DNP process, the composition of the glassing matrix could influence the maximum achievable DNP efficiency. Except for self-glassing 13C compounds such as neat pyruvic acid, another component of a DNP sample is the glassing matrix or solvent which ensures that the free radicals and target substrates are homogeneously dispersed in an amorphous state.11 Because the NMR active nuclei in the glassing solvents may also participate in the microwave-driven DNP process, the composition of the glassing matrix could influence the maximum achievable DNP efficiency. Our DNP results have shown that deuteration of the glassing matrix is beneficial for 5483



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13

C DNP samples doped with wide EPR line width free radicals such as TEMPO and DPPH because it can improve the 13C DNP signal by about a factor of 2.14,31,32 There is, however, a caveat: when doing 13C DNP using a narrow EPR line width free radical such as trityl OX063 or BDPA, our advice is to avoid using deuterated glassing solvent because it can reduce the 13C DNP signal by as much as a factor of 1/ 2.31,32 Such DNP behavior can be explained thermodynamically in terms of nuclear Zeeman heat loads.8,31 For samples doped with narrow EPR line width radicals such as trityl OX063, the electron dipolar system (EDS) is in thermal contact with 13C spins, but not 1H spins. In this case, glassing matrix deuteration implies that the EDS is in thermal contact with both 13C and 2H spins; thus, the total nuclear Zeeman heat load for the EDS to cool is increased, leading to lower 13C polarization.8,31,32 On the other hand, this practice is beneficial for 13C (γ = 10.7 MHz/T) samples doped with large EPR line widths such as TEMPO because EDS is already in thermal contact with the high-γ 1H spins; thus, replacement of 1H (γ = 42.6 MHz/T) spins by low-γ 2H (γ = 6.54 MHz/T) spins in the glassing matrix will lead to lower nuclear Zeeman heat load for EDS to cool, leading to higher 13C polarization.8,14,15,31,32 In other words, glassing matrix deuteration can be beneficial or detrimental to DNP efficiency, depending upon the type of polarizing agent used. These DNP effects involving glassing matrix deuteration are prominently observed in DNP at 3.35 T, but preliminary studies show that such effects become less evident in DNP at higher field, such as 5 T.32 As depicted in Figure 3, another simple optimization procedure that one can employ in DNP sample preparation to double or even triple the DNP signal is by adding trace amounts of paramagnetic agents to DNP samples containing trityl OX063.32−38 This DNP behavior was initially discovered with the addition of gadolinium contrast agents to DNP samples. DNP optimization studies from our research group have shown that this effect extends to a host of other paramagnetic additives; holmium, terbium, dysprosium, manganese, and superparamagnetic iron oxide nanoparticles (SPIONs) are all capable of improving 13C polarization to some degree (Figure 3).32−38 Importantly, there is some dependence on chelation of the paramagnetic species, as terbium and dysprosium chlorides led to no polarization improvement when used as additives, but the same ions chelated in a DOTA complex were quite successful.33 As shown in Figure 3, it appears that gadolinium complexes are by far the superior paramagnetic additives in terms of DNP signal improvement of samples in the frozen state. However, the story changes slightly upon dissolution of the DNP samples. Gadolinium has very strong relaxivity in the liquid state as compared to Tb3+-DOTA and Ho3+-DOTA.33,34 This leads to a greater relaxation of nuclei during the shuttling time from polarizer to spectrometer so that holmium and terbium-doped samples yield liquid-state polarization enhancement similar to that of gadolinium doped samples. A similar problem is present with iron oxide nanoparticles.37 Samples doped with this additive gain impressive solid-state polarization near that achieved by gadolinium-doped samples but relax extremely quickly in the liquid-state, which leads to relatively low liquidstate NMR enhancements. In this case, hyperpolarization losses due to shortening of liquid-state T1 relaxation time can be easily mitigated by in-line mechanical filtration of the largediameter nanoparticles.37

Figure 3. Influence of paramagnetic additives on 13C DNP: (a) Relative 13C solid-state DNP buildup curves at 3.35 T and 1.2 K for samples containing 3 M [1-13C] sodium acetate doped with 15 mM trityl OX063 and the indicated paramagnetic additive. The paramagnetic additives include lanthanide and transition metal complexes as well as superparamagnetic iron nanoparticle (SPION). Curves were normalized such that the control sample builds up to unity. (b) Comparative bar plot showing liquid-state 13C NMR signal enhancements obtained via DNP with various paramagnetic additives. These liquid-state NMR measurements were done at 9.4 T and 298 K approximately 8 s after dissolution of 13C DNP samples described in panel a.

Thus, it appears that electron T1 reduction of the polarizing agent by a paramagnetic additive is directly linked to the improvement seen in solid-phase 13C DNP signal. The DNP signal improvement due to paramagnetic additives is ascribed to the reduction of the free radical electron T1. Repeatedly, it has been shown that paramagnetic additives in the optimal concentration for DNP cause a nearly order of magnitude decrease in electron T1, specifically that of trityl OX063.18,33−37 It should be noted that not all paramagnetic additives can provide improvement in the DNP signal. In our study of 13C DNP with transition metal complexes R-NOTA (R = Mn2+, Cu2+, Co2+), Mn2+-NOTA doping led to about 2fold improvement of the 13C DNP signal at 3.35 T and 1.2 K, whereas Cu2+-NOTA and Co2+-NOTA doping yielded little to no improvement on solid-phase 13C DNP signal relative to the control sample under the same DNP conditions. Subsequent EPR measurements showed that Mn2+-NOTA doping led to the reduction of trityl OX063 electron T1, whereas the other two transition metal complexes yielded negligible effect in electron relaxation.35 Thus, it appears that electron T1 reduction of the polarizing agent by a paramagnetic additive is directly linked to the improvement seen in solid-phase 13C 5484



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situated in the cryostat. Superheated water or other solvents then can be injected into the sample cup to rapidly dissolve the sample, and thus a hyperpolarized liquid can be obtained. In dissolution DNP, the usable polarization for practical applications is the liquid-state or solution-phase polarization, which will always be less than that in the solid phase before dissolution because of the relaxation processes occurring during transit. It is obvious that a higher solid-state polarization will translate as a higher liquid-state polarization assuming no additional relaxation agents are used in gaining the higher polarization. There are several ways to further enhance the solid-state polarization, including using a hyperpolarizer with a higher magnetic field and a lower temperature,11,39 microwave frequency modulation,41 and crosspolarization.16,17 Additional DNP capabilities such as multiple sample polarization with a revolver-type sample holder allows one to make a series of dissolutions for rapid and sequential hyperpolarized magnetic resonance experiments.11 In an effort to improve polarization by increasing the operating magnetic field for DNP, our group has reported the construction and performance of a home-built dissolution hyperpolarizer operating at 6.4 T and a base temperature of 1.6 K,42 based mainly on the 4.6 T polarizer design reported previously.43 This design was slightly different from the original dissolution DNP system10 such that the cryostat is cryogenically separated from the superconducting magnet. We aimed to build a polarizer that can be easily constructed with basic machining tools and with relatively low cost. For these reasons, we opted to use modular home-built parts and a thin, smoothbore stainless-steel tubing as an oversized waveguide similar to a previously published design. We showed that even with this compromise, a 13C polarization slightly over 60% can be achieved in the frozen state of the DNP sample.42

DNP signal. This is backed up by recent theoretical work in DNP on the cross effect and thermal mixing, both of which predict improved polarization efficiency with faster free radical electron relaxation.6,7 It should be noted that the vast majority of studies involving paramagnetic additives to DNP samples have been conducted at 3.35 T. Our research group, along with others, has shown that improvements in 13C polarization efficiency are reduced but still present at higher magnetic field strengths.9,32,39 However, there is little data for this paramagnetic additive effect above 5 T,39 so further work is necessary to understand the possible polarization gains at higher magnetic field strength. Inclusion of paramagnetic additives to DNP samples has proven extremely effective for trityl OX063 but has seen only limited use for other free radicals, though some preliminary work shows that it may be at least somewhat less effective.38 Further work is needed to pinpoint the exact physical explanations regarding the apparent exclusivity of trityl free radical in terms of additional DNP improvements with inclusion of paramagnetic additives in the DNP samples. In addition to free radical and solvent considerations, the choice of target nucleus, and specifically the location of nuclear isotopes within the molecule, is essential to DNP optimization.40 Both the molecule’s ability to be polarized in the solid state and the T1 relaxation in the frozen state are vital considerations when choosing the location of an isotope within a molecule. We have shown using differently labeled variants of 13 C sodium acetate that, as would be expected, 13C spins isolated from 1H spins by more than one bond length yield the optimum performance both in terms of polarization and relaxation.40 The proximity of protons to the site of 13C isotopic labeling within a molecule has a detrimental effect to 13 C DNP efficiency. In other words, the immediate intramolecular environment could have a significant impact on the DNP performance of 13C spins located in different labeling sites.40 As understanding of the physical and chemical aspects of DNP optimization has progressed, so too has DNP instrumentation.11 These instrumental advances, driven mainly by the utility of hyperpolarized 13C magnetic resonance in metabolic research or diagnostic imaging, are geared toward improving the overall DNP efficiency by (i) maximizing polarization in the solid-phase at cryogenic temperature, (ii) increasing the speed the polarization process as well as sample throughput, and (iii) minimizing liquid-state hyperpolarization losses via improvement of the dissolution process and delivery.11 Although there are differences in some details and in added capabilities, the instrumental developments in this field generally follow the major design of the original dissolution DNP instrument first described by Ardenkjær-Larsen et al.10 Here, the major components of the dissolution DNP machine include a superconducting magnet, a liquid helium cryogenic system, a sweepable microwave source centered close to the EPR frequency at the field, a vacuum pump system to achieve a sample space temperature close to 1 K, in situ NMR circuit to monitor the growth and quantify the polarization, and a dissolution system. The original design has an integrated cryogenic system for both the magnet and the cryostat sample space, which was made by modifying the magnet such that the liquid helium used for cooling the sample can be drawn directly from the magnet. The dissolution was performed by using an injection wand which seals a cup containing a sample

With the rising cost of liquid helium, the use of a cryogen-free DNP system is a major advance that could make dissolution DNP operation cheaper in the long run. To mitigate the high cost of liquid helium consumption in routine DNP operations, our group has successfully constructed and demonstrated that a cryogen-free superconducting magnet system could be used to achieve relatively high 13C polarization (see Figure 4).44 With the rising cost of liquid helium, the use of a cryogen-free DNP system is a major advance that could make dissolution DNP operation cheaper in the long run. This cryogen-free DNP system that we reported has a variable magnetic field capability.44 This implies that, using this polarizer, it is possible to perform DNP at 180 GHz (6.4 T) at a temperature of 1.8 K and that after the desired polarization has been achieved, the microwave source turned off, then the field can be ramped to the different magnetic fields for the 13C T1 relaxation time measurement of the frozen polarized samples. Here we have shown for the first time that the solid phase 13C T1 of hyperpolarized 13C carboxylates such as [1-13C] pyruvate, [1-13C] acetate, and [1-13C] glycine becomes drastically longer at higher fields.45 For instance, the 13C T1 of [1-13C] sodium pyruvate in Figure 4 shows that at 1.8 K, T1 ranges from ∼5 min at B = 1 T to 5485



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taken not to induce heating on the surrounding area of the sample due to Eddy currents. For this reason, the microwave frequency modulation technique is regarded as more reliable. The theoretical work by Hovav et al. also shows that the frequency modulation also acts to enhance the spin diffusion, which is lowered when a lesser concentration of radicals is used.46 Therefore, this method allows for a lower concentration than optimal free radicals to be used, thus leading to lower liquid-state T1 reduction of hyperpolarized nuclei after dissolution. To harness the enhanced NMR signals in the frozen solid phase for chemical and biomedical studies, the next step is to dissolve the frozen hyperpolarized substrate at cryogenic temperature and quickly use it once the hyperpolarized liquid is at a physiologically tolerable temperature. Reduction of hyperpolarization losses during transfer of hyperpolarized liquid is one of the foremost challenges in dissolution DNP. For biomedical applications, ideally the substrate has to be injected or utilized within the time T1 from the moment the dissolution process is carried out. However, this condition is sometimes difficult to satisfy because of the distance between the hyperpolarizer magnet and the analyzer NMR magnet or MRI scanner, especially when the magnet is unshielded. One solution to this problem was reported by Milani and coworkers in the form of a magnetic tunnel.47 The main problem leading to the polarization loss was reported to be the sudden, nonadiabatic change in the magnetic field strength experienced by the substrate during transit as it travels from a high magnetic field to a low-field region. By sending hyperpolarized liquid through a magnetic tunnel, this problem is somewhat reduced for favorable molecules, specifically molecules with weak intrinsic relaxivity whose relaxation is driven by external paramagnetic species.47 The implementation of a magnetic tunnel has resulted in improvements of up to 1.5-fold in polarization. Reduction of dissolution liquid shuttling time is another solution to the problem of polarization loss due to T1 decay in dissolution transfer as presented by Hilty and Bowen.48 This group devised a DNP dissolution sytem with increased transfer speed while using a system of back-pressure regulation to minimize turbulence. This modified dissolution system was incorporated in the HyperSense commercial polarizer, and a dissolution transfer time of less than 1.2 s was achieved.48 This is significantly faster than the current commercial polarizers and has enabled detection and significant NMR signal improvements even for hyperpolarized nuclei with relatively short liquid-state T1 relaxation times.48 Although the actual polarization may remain the same, high dilutions of hyperpolarized liquids can become problematic for NMR or imaging (MRI) detection especially for in vivo applications or animal studies in which certain high concentration of hyperpolarized 13C solution is necessary for injection into the bloodstream. Thus, reduction of sample dilution is an important consideration for hyperpolarized magnetic resonance experiments. Preliminary solutions to the issue of high dilution factor in hyperpolarized liquids, which could typically go up to 100- to 200-fold, have been approached using chemical methods in dissolution. Peterson et al. reported on the use of a mixture of 75% volume of inert fluid (e.g., perfluorocarbon) plus 25% volume of water as the dissolution solvent in the commercial HyperSense polarizer.49 This protocol successfully reduced the injection volumes for 13 C-pyruvic acid and 13C-succinate by a factor of 4, making

Figure 4. Cryogen-free DNP at relatively higher field: (a) Representative hyperpolarized and thermal solid-phase 13C NMR signals at 6.4 T and 1.8 K. 13C P = 58% was recorded (adapted with permission from John Wiley & Sons, Ltd).44 (b) An example of a cryogen-free DNP system (adapted with permission from John Wiley & Sons, Ltd.)44 with variable magnetic field capability. (c) Field dependence of 13C T1 of hyperpolarized [1-13C] pyruvate at 1.8 K. 13 C T1 changes drastically from 300 s at 1 T to 160 000 s (44 h) at 8.9 T. Longer solid-state 13C T1 values may be tied up to the improved 13 C polarization levels observed at higher fields (adapted with permission from the American Chemical Society).45

about 44 h at B = 8.9 T.45 In DNP, there is a competition between the hyperpolarization process and depolarization process via nuclear relaxation. The drastically longer solid phase T1 of 13C spins at higher field, which means longer DNP preservation time, is probably one of the major reasons why 13 C DNP efficiency appears to improve with increasing operating field of the polarizer.

The drastically longer solid phase T1 of 13C spins at higher field, which means longer DNP preservation time, is probably one of the major reasons why 13C DNP efficiency appears to improve with increasing operating field of the polarizer. Another beneficial capability that can be added to the DNP instrumentation is the microwave frequency modulation technique,41 which is geared toward bringing more electron spins to participate in nuclear hyperpolarization. Conventionally, DNP is done by irradiation in a single optimum microwave frequency in which only a fraction of the free electrons’ spins participate in the DNP process because the field produced by the magnet is not completely homogeneous. Therefore, there is a distribution of electron and nuclear Larmor frequencies in the sample in the magnetic field. Because of this, modulation of the microwave frequency41,46 will help to increase the number of spins participating in DNP. Field modulation could be used rather than frequency modulation by using a modulation coil, but care must be 5486



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C magnetic resonance continues to be the flagship application of the dissolution DNP technology, significant interest is also growing in the DNP study of other low-γ nuclei such as 6Li, 15N, 89Y, 109Ag etc. for fundamental chemical research or potential practical applications.11 Many of these same optimization techniques are thought to be similarly effective for other low-γ nuclei, but these have not yet been fully or extensively explored. Significantly, there is greater room for DNP improvement with low-γ nuclei, as their thermal equilibrium polarization is very weak. While 13C liquid-state polarization enhancements of 10 000−50 000-fold in a 400 MHz NMR magnet have become somewhat commonplace with 13C compounds, reaching the same polarization percent would be equivalent to several hundred thousand-fold enhancements for ultralow-γ NMR nuclei like 89Y (γ = 2.09 MHz/T) or 109Ag (γ = 1.98 MHz/T) as illustrated in Figure 5.51,52 The hyperpolarized 109Ag NMR of a silver complex at millimolar concentration in Figure 5a has a liquid-state NMR signal enhancement of 7200-fold.51 This level of NMR signal enhancement for 109Ag is already impressive considering the excellent signal-to-noise ratio; however, if we calculate the actual polarization of this hyperpolarized signal, this corresponds to only 1.1%. The highest recorded liquid-state NMR signal enhancement for hyperpolarized 109Ag is ∼15 000-fold, which corresponds to 2.3% 109Ag polarization as denoted by a dashed arrow in Figure 5b.51 These 109Ag enhancement numbers are obviously more than enough for NMR characterization of Ag compounds, but the quest for higher enhancements could open up possible new applications. As a number of silver-based compounds have therapeutic properties, one can envision tracking a silver-based drug in vivo with highly enhanced 109Ag NMR sensitivity afforded by dissolution DNPa feat that has never been done before. The same can be said for 89Y-based complexes with long solution-phase T1, such as the pH sensor Y-DOTP. The highest 89Y NMR enhancement in the liquid state currently recorded at 9.4 T and 298 K is 65 000-fold.52 This is a high enhancement number, but it corresponds to only ∼10% 89Y polarization. In vivo pH imaging across tumors using hyperpolarized 89YDOTP53 could potentially materialize with higher NMR enhancement levels. There is, thus, enough scientific motivation and still wide room for improvement to maximize the DNP signals of low-γ nuclei with relatively long solutionphase T1 relaxation times. In summary, we have presented a portion of the work that our group and others have performed in the quest for optimal polarization enhancements in dissolution DNP of 13C and other potentially relevant nuclei. Attaining the highest liquidstate NMR signal enhancements for these nuclei may require a combination of chemical tuning or optimized sample preparation, improvement in the DNP instrumental conditions such as higher B/T ratio, RF manipulations such as crosspolarization and microwave frequency modulation, minimizing the loss in dissolution transit using accessories such as magnetic tunnels and fast passage systems, and using methods to reduce sample dilution. As dissolution DNP continues to expand its reach into numerous practical applications, especially in its translation to in vivo human metabolic imaging, the considerations discussed herein will allow researchers and clinicians to harness the best possible sensitivity gains in magnetic resonance experiments as afforded by optimized dissolution DNP technology. 13

them more suitable for injection into small animals and thus better detection in hyperpolarized 13C MRI experiments.49 This protocol worked because there was a rapid separation of water and the immiscible perfluorocarbon after dissolution. Furthermore, the aqueous solution contained the dissolved hyperpolarized 13C compound, which can easily be pipetted out and collected for injection. In a similar vein, Harris et al. presented a method that utilizes a mixture of aqueous and organic solvents such as water/toluene as the dissolution solvent.50 This dissolution mixture has a dual purpose: one solvent with lesser volume is meant to dissolve the target analyte to produce a highly concentrated hyperpolarized 13C liquid, while the solvent with larger volume is chosen to dissolve the copolarizing free radical as these two solvents naturally separate in the NMR tubes.50 In the final dissolution form, this protocol cleverly yields a highly concentrated hyperpolarized 13C solution with a longer liquid-state T1 because the free radical is naturally extracted out of the injectable solution.

Reduction of hyperpolarization losses during transfer of hyperpolarized liquid is one of the foremost challenges in dissolution DNP. Overall, since the inception of the dissolution DNP technology in 2003, significant advances were made in DNP instrumentation and optimization of the DNP signals, as driven mainly by the growing interest in using hyperpolarized 13C magnetic resonance for biomedical applications. Such instrumental and DNP signal optimization advances have resulted in research groups readily achieving 13C polarizations approaching 50%, with some groups reporting record-breaking solution-phase 13C polarizations approaching 70% at 7 T (Figure 5).39 While metabolic research using hyperpolarized

Figure 5. (a) Representative hyperpolarized and thermal 109Ag NMR signal of a Ag complex at 9.4 T and 298 K. The 109Ag DNP signal displayed has an NMR signal enhancement of 7200-fold, which corresponds to only ∼1.1% (adapted with permission from WileyVCH Verlag GmbH and Co. KGaA, Weinheim).51 (b) Plot of the expected NMR signal enhancements of representative low-γ nuclei (13C, 89Y, and 109Ag) at 9.4 T and 298 K vis-à-vis their corresponding liquid-state polarization values. The dashed arrows indicate the highest recorded liquid-state polarization values39,51,52 achieved to date via dissolution DNP of these representative nuclei. 5487



There is, thus, enough scientific motivation and still wide room for improvement to maximize the DNP signals of low-γ nuclei with relatively long solution-phase T1 relaxation times.

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ACKNOWLEDGMENTS

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REFERENCES

Perspective

The authors acknowledge support for this work by the UT Dallas startup research funding, the Welch Foundation Grant Number AT-1877, and the U.S. Department of Defense Grant Number W81XWH-17-1-0303. In addition, also thank our DNP collaborators from the National High Magnetic Field Laboratory in Florida and the neighboring University of Texas Southwestern Medical Center at Dallas.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lloyd Lumata: 0000-0002-3647-3753 Notes

The authors declare no competing financial interest. Biographies Peter Niedbalski earned his Ph.D. in Physics in the fall of 2017 under the supervision of Prof. Lloyd Lumata at UT Dallas where he worked on the mechanisms and optimization methods of dissolution DNP. He graduated Summa Cum Laude at Benedictine College in 2013 with a B.S. degree in Physics. He is currently working at Cincinnati Children’s Hospital Medical Center (CCHMC) as a postdoctoral fellow in the Center for Pulmonary Imaging Research (CPIR). Andhika Kiswandhi obtained his Ph.D. in Physics at the National High Magnetic Field Laboratory (NHMFL) at Florida State University in the Fall of 2014. He then worked at UT Dallas in 2015−2017 as a postdoc in Prof. Lumata’s lab wherein he led the construction of two home-built dissolution DNP systems. He is currently a postdoc at Department of Chemistry of Kyoto University with the support of the Japan Society for the Promotion of Science (JSPS) fellowship program. Christopher Parish joined the Lumata lab at UT Dallas as a Ph.D. candidate in 2013. He earned his B.S. in Physics and Mathematics at Midwestern State University in Wichita Falls, Texas. He is currently working on DNP optimization and the application of dissolution DNP in cancer metabolism studies. Qing Wang earned a M.S. in physics from the University of Massachusetts at Lowell in 2014. He is currently a graduate student at UTD working on a cryogen-free DNP system and the DNP of lowgamma nuclei in the Lumata lab. Fatemeh Khashami earned her B.S. Physics degree in Iran and is currently a graduate student in the UTD Department of Physics. She is working on metabolic studies of cancer cells using conventional and hyperpolarized 13C NMR spectroscopy. Lloyd Lumata obtained his B.S. Physics at Western Mindanao State University in the Philippines in 2002. He earned his Ph.D. in Physics at the NHMFL at Florida State University in 2008 under the tutelage of Prof. James Brooks and Dr. Arneil Reyes. In 2009−2014, he worked as a postdoctoral fellow at the Advanced Imaging Research Center (AIRC) at UT Southwestern Medical Center under the mentorship of Prof. Matthew Merritt and Prof. Zoltan Kovacs, as well as Prof. A. Dean Sherry and Prof. Craig Malloy. He then moved to UT Dallas in 2014 as an Assistant Professor in the Department of Physics where he currently leads a DNP research group. Research efforts in his group include DNP instrumentation and optimization and the application of this technology to cancer metabolism studies. 5488



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Nuclear Polarization by Superparamagnetic Iron Oxide Nanoparticle Doping. J. Phys. Chem. C 2017, 121, 19505−19511. (38) Lumata, L.; Merritt, M. E.; Malloy, C. R.; Sherry, A. D.; Kovacs, Z. Impact of Gd3+ on DNP of [1-13C]Pyruvate Doped with Trityl OX063, BDPA, or 4-Oxo-TEMPO. J. Phys. Chem. A 2012, 116, 5129−5138. (39) Yoshihara, H. A. I.; Can, E.; Karlsson, M.; Lerche, M. H.; Schwitter, J.; Comment, A. High-Field Dissolution Dynamic Nuclear Polarization of [1-13C]Pyruvic Acid. Phys. Chem. Chem. Phys. 2016, 18, 12409−12413. (40) Niedbalski, P.; Parish, C.; Kiswandhi, A.; Kovacs, Z.; Lumata, L. Influence of 13C Isotopic Labeling Location on Dynamic Nuclear Polarization of Acetate. J. Phys. Chem. A 2017, 121, 3227−3233. (41) Bornet, A.; Milani, J.; Vuichoud, B.; Perez Linde, A. J.; Bodenhausen, G.; Jannin, S. Microwave Frequency Modulation to Enhance Dissolution Dynamic Nuclear Polarization. Chem. Phys. Lett. 2014, 602, 63−67. (42) Kiswandhi, A.; Niedbalski, P.; Parish, C.; Ferguson, S.; Taylor, D.; McDonald, G.; Lumata, L. Construction and 13C Hyperpolarization Efficiency of a 180 GHz Dissolution Dynamic Nuclear Polarization System. Magn. Reson. Chem. 2017, 55, 828−836. (43) Lumata, L. L.; Martin, R.; Jindal, A. K.; Kovacs, Z.; Conradi, M. S.; Merritt, M. E. Development and Performance of a 129-GHz Dynamic Nuclear Polarizer in an Ultra-Wide Bore Superconducting Magnet. MAGMA 2015, 28, 195−205. (44) Kiswandhi, A.; Niedbalski, P.; Parish, C.; Wang, Q.; Lumata, L. Assembly and Performance of a 6.4 T Cryogen-Free Dynamic Nuclear Polarization System. Magn. Reson. Chem. 2017, 55, 846−852. (45) Niedbalski, P.; Wang, Q.; Parish, C.; Khashami, F.; Kiswandhi, A.; Lumata, L. Magnetic-Field-Dependent Lifetimes of Hyperpolarized 13C Spins at Cryogenic Temperature. J. Phys. Chem. B 2018, 122, 1898−1904. (46) Hovav, Y.; Feintuch, A.; Vega, S.; Goldfarb, D. Dynamic Nuclear Polarization Using Frequency Modulation at 3.34T. J. Magn. Reson. 2014, 238, 94−105. (47) Milani, J.; Vuichoud, B.; Bornet, A.; Miéville, P.; Mottier, R.; Jannin, S.; Bodenhausen, G. A Magnetic Tunnel to Shelter Hyperpolarized Fluids. Rev. Sci. Instrum. 2015, 86, 024101. (48) Bowen, S.; Hilty, C. Rapid Sample Injection for Hyperpolarized NMR Spectroscopy. Phys. Chem. Chem. Phys. 2010, 12, 5766−5770. (49) Peterson, E. T.; Gordon, J. W.; Erickson, M. G.; Fain, S. B.; Rowland, I. J. Dynamic Nuclear Polarization System Output Volume Reduction Using Inert Fluids. J. Magn. Reson. Imaging 2011, 33, 1003−1008. (50) Harris, T.; Bretschneider, C.; Frydman, L. Dissolution DNP NMR With Solvent Mixtures: Substrate Concentration and Radical Extraction. J. Magn. Reson. 2011, 211, 96−100. (51) Lumata, L.; Merritt, M. E.; Hashami, Z.; Ratnakar, S. J.; Kovacs, Z. Production and NMR Characterization of Hyperpolarized 107,109Ag Complexes. Angew. Chem., Int. Ed. 2012, 51, 525−527. (52) Lumata, L.; Jindal, A.; Merritt, M. E.; Malloy, C.; Sherry, A. D.; Kovacs, Z. DNP by Thermal Mixing Under Optimized Conditions Yields > 60,000-fold enhancement of 89Y NMR Signal. J. Am. Chem. Soc. 2011, 133, 8673−8680. (53) Jindal, A. K.; Merritt, M. E.; Suh, E. H.; Malloy, C. R.; Sherry, A. D.; Kovács, Z. Hyperpolarized 89Y Complexes as pH Sensitive NMR Probes. J. Am. Chem. Soc. 2010, 132, 1784−1785.

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NMR Spectroscopy Unchained: Attaining the Highest Signal

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