Rational design of [¹³C,D₁₄]tert-butylbenzene as a scaffold structure for designing long-lived hyperpolarized ¹³C probes

Abstract

Dynamic nuclear polarization (DNP) is a technique to polarize the nuclear spin population. As a result of the hyperpolarization, NMR sensitivity of the nuclei in molecules can be enhanced dramatically. Recent application of the hyperpolarization technique has led to advances in biochemical and molecular studies. A major problem is the short lifetime of the polarized nuclear spin state. Generally, in solution, the polarized nuclear spin state decays to a thermal spin equilibrium, resulting in loss of the enhanced NMR signal. This decay is correlated directly with the spin-lattice relaxation time T₁. Here we report [¹³C,D₁₄]tert-butylbenzene as a new scaffold structure for designing hyperpolarized ¹³C probes. Thanks to the minimized spin-lattice relaxation (T₁) pathways, its water-soluble derivative showed a remarkably long ¹³C T₁ value and long retention of the hyperpolarized spin state.

Graphical Abstract

Here we report [¹³C,D₁₄]tert-butylbenzene as a new scaffold structure for designing hyperpolarized ¹³C probes. Thanks to the minimized spin-lattice relaxation (T₁) pathways, its water-soluble derivative showed a remarkably long ¹³C T₁ value and long retention of the hyperpolarized spin state.

Dynamic nuclear polarization (DNP) is a technique to polarize the nuclear spin population.^[1–3] As a result of the hyperpolarization, NMR sensitivity of the nuclei in molecules can be enhanced dramatically. Recent application of the hyperpolarization technique has led to advances in biochemical and molecular studies.^[4] Hyperpolarized (HP) molecules have enabled highly sensitive and real-time monitoring of metabolic fluxes^[4,5] and chemical status^[6] in vivo. DNP has not only been applied to these biomedical studies, but also to molecular interaction analysis and chemical reaction monitoring.^[7] Such extensive research has demonstrated the high potential of HP-NMR techniques.

A major problem is the short lifetime of the polarized nuclear spin state. Generally, in solution, the polarized nuclear spin state decays to a thermal spin equilibrium, resulting in loss of the enhanced NMR signal. This decay is correlated directly with the spin-lattice relaxation time T₁. In solution, the T₁ value of ¹³C in small molecules is typically from a few to tens of seconds.⁴ This short lifetime has hampered the flexible design of HP chemical probes.

To overcome the decay problem and to generate a series of HP probes systematically, we have proposed a scaffold strategy.^[8–11] A representative scaffold structure is [¹⁵N,D]trimethylphenylammonium (TMPA), which comprises a long-lived HP signalling moiety (–¹⁵N(CD₃)₃) and an aromatic ring for facile chemical modifications (Figure 1). We have successfully demonstrated that a series of HP probes could be developed from the [¹⁵N,D]TMPA by introducing a molecular sensing unit R.^[9,10] However, in principle, the ¹⁵N NMR sensitivity is lower than that of ¹³C nuclei because of its smaller gyromagnetic ratio. Therefore, a ¹³C HP scaffold structure with a long T₁ value has been highly sought after.^[11]

Figure 1.

Schematic illustration of ¹⁵N and ¹³C scaffold structures reported in previous and this study, respectively.

Here, we report a ¹³C scaffold structure, [¹³C,D₁₄]tert-butylbenzene, that can be used to design long-lived HP ¹³C probes.

There are two approaches to achieving a long-lived HP spin state. One is to design a molecule with long T₁, because the HP spin state is directly correlated with T₁.^[4] The other is to use a nuclear singlet state with a magnetically equivalent spin −1/2 pair.^[12] Both approaches are attractive and have advanced long-lived hyperpolarization.^{[8–10,13–18]} In the present study, we adopt the more straightforward former approach.

T₁ relaxation is affected by several factors, such as dipole–dipole (DD) interactions, chemical shift anisotropy (CSA), spin–rotation, scalar coupling.^[19,20] For ¹³C small organic molecules, especially in high magnetic field, DD and CSA relaxations are dominant factors.^[4,6]

To minimize the CSA relaxation, sp³ carbon with symmetrical environment is preferable. The tert-butyl carbon has an sp³ hybridization. This carbon centre is also attractive for HP applications because it experiences only a small DD-induced T₁ relaxation because of the absence of neighbouring ¹H. Indeed, tert-butanol and 1,1-cyclopropane dimethanol have been shown to have long T₁ values and have been proposed as potential HPMRI probes.^[21,22]

We then designed [¹³C]tert-butylbenzene (1) as a potential ¹³C scaffold structure. Molecule 1 is composed of a ¹³C tert-butyl group attached to an aromatic ring for feasible chemical modification to incorporate functional groups such as sensing moieties. 1 was synthesized from [¹³C]benzoic acid in 2 steps. The ¹³C T₁ value of 1 was 76 ± 1 s (CD₃OD, 9.4 T, 25 °C). The ¹³C T₁ of [¹³C]benzoic acid, which was another candidate for a ¹³C scaffold utilizing a ¹³C carboxyl group as a HP unit, was 33.9 ± 0.3 s (CD₃OD, 9.4 T, 25 °C), suggesting an advantage in utilizing sp³ carbon as a long-lived HP unit.

To extend the ¹³C T₁ of 1 further, we then prepared [¹³C,D₁₄]tert-butylbenzene (1D, Figure 2), wherein all ¹H were replaced with ²H (D) to minimize ¹³C-¹H DD relaxation. This 1D was synthesized from [D₅]bromobenzene in 3 steps (Scheme 1). The ¹³C T₁ of 1D reached over 100 s (141 ± 3 s, CD₃OD, 9.4 T, 25 °C), which was 1.3 times longer than that of deuterated tert-butanol (106 s).

Figure 2.

T₁ of tert-butanol and tert-butylbenzene (1 and 1D). ¹³C T₁ were determined by using inversion recovery method (9.4 T, 25 °C, CD₃OD, tert-butanol = 1.0 M, [D₉]tert-butanol = 1.5 M, 1 = 0.05 M, 1D = 0.05 M)

Scheme 1.

Synthesis of [¹³C,D₁₄]tert-butylbenzene (1D).

T₁ relaxation of 1D was investigated further by decomposing it to each relaxation mechanism: ¹³C–¹H DD (T₁ ^CH-DD), CSA (T₁ ^CSA), dissolved oxygen (T₁ ^O2), and residual factors (T₁ ^res). Total T₁ can be described using these decomposed T₁ values as equation 1. These T₁ contributions were determined according to previous reports (experimental details, see: supporting information).^{[19,20,23–28]}

$$\frac{1}{T_1}=\frac{1}{T_1^{\mathit{\text{CH}}-\mathit{\text{DD}}}}+\frac{1}{T_1^{\mathit{\text{CSA}}}}+\frac{1}{T_1^{O2}}+\frac{1}{T_1^{\mathit{\text{res}}}}$$ (eq 1)

The estimated T₁ contributions in 1 and 1D are summarized in Figure 3. As shown in equation 1, a larger 1/T₁ ^X indicates the larger contribution of the relaxation mechanism X to shorten the apparent T₁. These data include three important implications. First, ¹³C–¹H DD relaxation of 1D is almost completely suppressed by full deuteration, while 1 suffers from non-negligible ¹³C–¹H DD relaxation (red bar). Second, the sp³ carbon of 1 and 1D experience low CSA relaxation, as designed. Third, as a result of sufficient minimization of ¹³C–¹H DD and CSA relaxation mechanisms, the relaxation from dissolved oxygen (T₁ ^O2) becomes the major contributor for T₁ relaxation (blue bar). In the case of 1D, T₁ ^O2 governs the majority of the apparent T₁ relaxation, resulting in the considerably longer T₁ value of 1D in degassed condition (541 ± 18 s, toluene-d8, 9.4 T, 25 °C). These data support the validity of our design and the potential of 1D as a long-lived HP scaffold.

Figure 3.

Stacked bar graph of 1/T₁ ^X of 1 and 1D (50 mM, toluene-d₈, 25 °C, 9.4 T). 1/T₁ ^X values were determined experimentally (see, supporting information).

Furthermore, we tried to achieve a T₁ value longer than 1D based on the understanding of T₁ contributions of 1D shown in Figure 3. Considering that the dissolved oxygen is a dominant relaxation factor of 1D, it was considered that a longer T₁ could be achieved by changing the solvent to water in which the oxygen concentration is lower than that in organic solvents.^[29] We then attempted to add the water solubility into 1D.

In order to increase the water solubility of 1D, sulfonated 1D (S1D, Figure 4a) was designed. Thanks to the rich chemical functionality of conjugated aromatic systems, a variety of substitutions can be introduced to the benzene ring of 1D. In this case, 1D was diversified by aromatic sulfonation with fuming sulphuric acid to produce S1D (Scheme S1) as an HP ¹³C probe with excellent water solubility (> 0.4 mol/L, r.t.).

Figure 4.

(a) Molecular design of S1D. T₁s in D₂O and 90% H₂O were determined by inversion recovery method at 50 mM S1D, 37 °C, 9.4 T. (b) ¹³C NMR spectra of the hyperpolarized probe S1D (2 mM in H₂O), stacked from 0 to 15 min (every 2 s, pulse angle 5°).

The probe S1D showed a remarkably long T₁ in water. The ¹³C T₁ exceeded 200 s in D₂O (209 ± 7 s, 9.4 T, 37 °C), which was much longer than that of the original (1D) in organic solvent despite the increased molecular weight. It was shortened in 90% H₂O, but still maintained a long T₁ (147 ± 1 s, 9.4 T, 37 °C). These values were longer than those for [D₉]tert-butanol (140 ± 7 in D₂O, 77± 1 s in H₂O, 9.4 T, 37 °C).

Finally, S1D was applied for a DNP-NMR study in water. S1D was hyperpolarized efficiently by dissolution DNP. The liquid state ¹³C NMR signal of the probe dissolved in water was enhanced by approximately 29,000-fold (%P_13C = 23.5%, T = 298 K, B₀ = 9.4 T) compared with the thermally polarized signal, allowing detection of ¹³C NMR signal by 1 scan. Figure 4b shows the time course stack of ¹³C NMR spectra of HP S1D. Because of the long T₁ value, S1D could retain a hyperpolarized spin state for a relatively long time, allowing observation of the HP ¹³C NMR signal for over 10 min under our experimental conditions. These results strongly indicate that this approach toward T₁ extension based on an analysis of the T₁ contribution is a useful approach to the design of HP probes.

In summary, we developed a new HP ¹³C scaffold [¹³C,D₁₄]tert-butylbenzene (1D). The significance of this scaffold can be summarized as follows. First, it satisfies the requirement of a long T₁. The 1D scaffold was designed by minimizing each relaxation mechanism to achieve longer T₁ value. Notably, S1D, developed from 1D, afforded a remarkably long ¹³C T₁ (209 s in D₂O and 147 s in 90%H₂O, non-degassed, 9.4 T, 37 °C). To our knowledge, this is the longest among the water-soluble ¹³C small organic molecules used for HP-NMR research. Second, it satisfies a facile functionalization of HP probes by feasible chemical modification. As we reported in the present and the previous papers, thanks to the rich organic chemistry of aromatic compounds, a variety of HP probes may potentially be developed using this ¹³C HP scaffold. Development of water soluble S1D from 1D scaffold is a good example of this versatility. Third is higher sensitivity. The present ¹³C scaffold is more sensitive than the previous ¹⁵N scaffold. Although further optimization may be necessary for practical applications, these advantages indicate the high potential of 1D as a new scaffold structure for designing a series of HP ¹³C MR probes.