BDPA: An Efficient Polarizing Agent for Fast Dissolution Dynamic Nuclear Polarization NMR

Fast dissolution-dynamic nuclear polarization (DNP)^[1] is the latest and most successful attempt to solve the problem of low sensitivity in liquid-state nuclear magnetic resonance (NMR). The most prominent applications of this technology are in vitro and in vivo NMR spectroscopy and imaging with hyperpolarized ¹³C which has been used to study biochemical/metabolic activities in various tissues (heart, liver, tumors) in real time.^[2–4] Non-biological applications of this technique include the monitoring of transient chemical kinetic reactions and fast sample characterization via one- and two-dimensional high resolution NMR spectroscopy.^[5,6]

The choice of free radicals is critical in DNP. Mounting evidence^[7–9] show that higher nuclear polarization levels can be achieved when radicals with narrower EPR linewidths D are used. Currently, the water soluble trityl-based stable free radicals (Figure 1) are the most frequently used polarizing agents in ¹³C and ¹⁵N dissolution DNP because their EPR linewidth is among the narrowest (D = 60–70 MHz).^[9] Although the physical principles of DNP have been described in detail,^[10] the chemical ramifications of the nature of the radical are not well understood yet. This research was initiated to explore other stable radicals as DNP polarizing agents. Here we demonstrate that the well-known 1,3-bisdiphenylene-2-phenylallyl (BDPA) radical may offer a viable alternative to trityl radicals in dissolution DNP. BDPA (Figure 1), first synthesized by Koelsch in 1957,^[11] is one of the most stable carbon-centered free radicals. It has a comparable D to that of trityl radicals and has been used in polarized target experiments and solid-state DNP.^[12–14] However, this radical has never been tried in dissolution DNP, most likely because it is practically insoluble in water and therefore, cannot be used in conventional water-based glassing mixtures such as a glycerol-water matrix. Our preliminary experiments have shown that BDPA is reasonably soluble (over 100 mM) in sulfolane, a dipolar aprotic solvent that can dissolve a wide range of compounds. Sulfolane (tetramethylene sulfone) is a crystalline solid at room temperature (m.p. = 27.5 °C); however, it forms a glass at low temperature when mixed with other solvents. To demonstrate the general applicability of BDPA as a DNP polarizing agent, we have polarized a number of compounds in the presence of this radical using the HyperSense commercial polarizer. The DNP experiments were performed as previously described.^[1,2]

Figure 1.

Structure of free radical polarizing agents discussed in this work.

The data in Table 1 show that a variety of spin I= ½ nuclei with a gyromagnetic ratio ranging from γ = −2.086 MHz/T (⁸⁹Y) to 17.24 MHz/T (³¹P) and an I=1 nucleus ⁶Li (γ = 6.2655 MHz/T) could be polarized with an efficiency comparable to or higher than what has been achieved with trityl radicals. The glassing mixtures usually consisted of sulfolane-diethylene glycol monobenzyl ether mixtures (Supporting Information). Samples containing water-soluble compounds were dissolved with 4 mL water after DNP. The use of water for dissolution offered a very simple way to remove the BDPA radical from the hyperpolarized sample: the radical naturally precipitated out from the solution and could be completely removed by a simple and rapid filtration (Supporting Information). The dissolution of hydrophobic substrates was carried out with methanol, in which the BDPA remained in solution.

Table 1.

Liquid-state NMR enhancements (N=3) measured 8 s after dissolution of various compounds polarized with BDPA (40 mM) and trityl OX063 (15 mM) in sulfolane-based glassing matrices (Supporting Information). The liquid-state T₁ values and NMR enhancements were measured at 9.4 T and 298 K in unfiltered dissolution liquids.

Nucleus/Compound	Dissolution solvent	T1 (s)	Enhancement BDPA (OX063)
13C-pyruvic acida,c	water	42	14,000 (12,000)
13C-ureaa	water	46	10,010 (10,160)
13C-maleic acida	water	30	8,250
13C-benzenea	methanol	33	8,840
13C-tetramethylalleneb	methanol	59	9,120
15N-cholinea	water	215	8,000 (8,560)
15N-pyridinea	water	40	4,920
15N-nitrobenzenea	methanol	100	7,880
31P-triMe phosphateb	water	20	5,550
29Si-tetraethylsilaneb	methanol	88	6,330
6LiCla	water	273	6,480
89Y(acac)3b	methanol	287	3,740
89Y(OTf)3b	waterd	460	7,420

^aenriched

^bnatural abundance

^cthe dissolution liquid consisted of 15 mM NaHCO₃

^dthe dissolution liquid consisted of 10-fold excess of diethylenetriaminepentaacetic acid solution (pH 7.8).

Due to its biological importance and favourably long longitudinal relaxation time T₁, hyperpolarized [1–¹³C] pyruvic acid is frequently used to study metabolism in heart, liver, kidney, and tumors.^[2–4] Here we demonstrate that BDPA in sulfolane is capable of producing large NMR enhancements of ¹³C pyruvic acid samples. The ¹³C DNP microwave spectra of [1-¹³C]pyruvic acid recorded in sulfolane in the presence BDPA and trityl OX063 almost overlap with a separation of 60–70 MHz between the positive and negative polarization peaks (Figure 2a). This close similarity of microwave ¹³C DNP spectra is the result of the comparable EPR linewidths of the two radicals, indicating the same dominant DNP mechanism (thermal mixing). The optimal concentration of BDPA in pyruvic acid-sulfolane (1:1) samples that would afford maximum polarization in approximately 1 hour of DNP irradiation time was 40 mM (Figures S1 and S2, Supporting Information). Figure 2b shows comparable solid-state ¹³C polarization P of [1-¹³C]pyruvic acid:sulfolane samples doped with the optimum concentrations of trityl OX063 (15 mM)^[15] [P=12 %] and BDPA (40 mM) [P=14 %]. We note that DNP of neat [1-¹³C]pyruvic acid (self-glassing) doped with 15 mM trityl OX063 in the HyperSense yielded P=15 % while TEMPO-doped (40 mM) ¹³C pyruvate samples in glycerol:water matrix produced lower polarization (P=5.5 %) (Supporting Information).

Figure 2.

a) Microwave DNP spectra of 1:1 [1-¹³C]pyruvic acid:sulfolane doped with BDPA (40 mM) (●) and trityl OX063 (15 mM) (○). The up and down arrows denote the positive [P(+)=94.06 GHz)] and negative [P(−)=94.13 GHz] polarization peaks, respectively. b) Polarization buildup curves of ¹³C pyruvic acid doped with BDPA(●) and OX063 (○) taken at P(+). c) Liquid-state T₁ decay of ¹³C pyruvic acid samples: BDPA filtered (□), unfiltered (▲) and OX063 (○).

The average liquid-state NMR enhancement ε of BDPA-doped pyruvic acid:sulfolane sample was about 14,000 [P=11 %] times the thermal signal at 298 K in a 9.4 T magnet whereas the same sample doped with trityl yielded ε =12,000 [P=9.7 %]. The slight polarization decrease from solid-state to liquid-state is attributed mainly to T₁ decay and field gradients experienced by the sample during dissolution transfer^[16] (in our case, the tranfer time is 8 s). Upon dissolution, the BDPA separated out and could be removed from the hyperpolarized pyruvate solution by filtration (Figures S4 and S5, Supporting Information), while the trityl radical stayed in solution. The absence of BDPA radical in the dissolution liquid after filtration, an important consideration for the production of injectable liquid samples for in vivo experiments, was verified by UV-vis spectrophotometry (Figure S6, Supporting Information). The filtered and unfiltered dissolution liquids of samples polarized with BDPA had practically identical T₁ values while the ¹³C T₁ value was slightly shorter in the sample polarized with the trityl radical due to the paramagnetic effect of the dissolved radical (Figure 2c).

We managed to polarize several other water soluble compounds (maleic acid, choline, pyridine, trimethyl phosphate, and LiCl) to achieve high liquid-state NMR enhancements of ¹³C, ¹⁵N, ³¹P and ⁶Li nuclei (Table 1) which further demonstrate that BDPA works well with water-soluble substrates.

As one would expect, BDPA in sulfolane is extremely well suited for the hyperpolarization of water-insoluble aromatic and aliphatic hydrocarbons as well as other hydrophobic compounds (Table 1). While usually ¹³C-enriched substrates are used in in vivo hyperpolarized experiments, unlabeled compounds can also be polarized to produce high signal enhancement as demonstrated by the ¹³C DNP-NMR spectrum of nonlabeled tetramethylallene (Figure 3).

Figure 3.

Hyperpolarized (1 scan) and thermal (64 scans) ¹³C NMR spectra of tetramethylallene after dissolution with methanol at 9.4 T and 298 K. The C4,5,6,7 NMR signal at 21 ppm (Figure S7, Supporting Information) is not shown here because of the lower intensity due to short T₁.

In summary, we have shown that carbon-centered stable free radical BDPA is an efficient polarizing agent in dissolution DNP experiments not only for hydrophobic compounds but also for a number of hydrophilic substrates. Large signal enhancements of ¹³C, ¹⁵N, ³¹P, ⁶Li, ²⁹Si, and ⁸⁹Y nuclei have been achieved with BDPA using sulfolane-based glassing matrices. After the DNP of water-soluble compounds such as [1-¹³C]pyruvic acid or ¹⁵N-choline, when water is used as the dissolution solvent, the hydrophobic BDPA radical can conveniently be removed from the hyperpolarized solution by a simple and rapid mechanical filtration process. In addition, water-soluble derivatives of BDPA^[17] may allow the use of water-containing glassing mixtures, thus widening the range of compounds that could be polarized with BDPA-based radicals.