Order-Unity ¹³C Nuclear Polarization of [1-¹³C]Pyruvate in Seconds and the Interplay of Water and SABRE Enhancement

Abstract

Signal Amplification By Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) is investigated to achieve rapid hyperpolarization of ¹³C₁ spins of [1-¹³C]pyruvate, using parahydrogen as the source of nuclear spin order. Pyruvate exchange with an iridium polarization transfer complex can be modulated via a sensitive interplay between temperature and co-ligation of DMSO and H₂O. Order-unity ¹³C (>50%) polarization of catalyst-bound [1-¹³C]pyruvate is achieved in less than 30 s by restricting the chemical exchange of [1-¹³C]pyruvate at lower temperatures. On the catalyst bound pyruvate, 39% polarization are measured using 1.4 T NMR spectrometer, and extrapolated to >50% at the end of build-up in situ. The highest measured polarization of a 30-mM pyruvate sample, including free and bound pyruvate is 13% when using 20 mM DMSO and 0.5 M water in CD₃OD. Efficient ¹³C polarization is also enabled by favorable relaxation dynamics in sub-microtesla magnetic fields, as indicated by fast polarization buildup rates compared to the T₁ spin-relaxation rates (e.g., ~0.2 s⁻¹ versus ~0.1 s⁻¹, respectively, for a 6 mM catalyst-[1-¹³C]pyruvate sample). Finally, the catalyst-bound hyperpolarized [1-¹³C]pyruvate can be released rapidly by cycling the temperature and/or by optimizing the amount of water, paving the way to future biomedical applications of hyperpolarized [1-¹³C]pyruvate produced via comparatively fast and simple SABRE-SHEATH-based approaches.

Graphical Abstract

Order unity ¹³C polarization for [1-¹³C]pyruvate was demonstrated for catalyst-bound species by SABRE-SHEATH, which becomes possible due to favorable ¹³C relaxation dynamics in a microtesla magnetic field. The magnetic field, temperature and co-solvents heavily modulate the attainable ¹³C polarization, providing an opportunity for optimization to deliver highly polarized [1-¹³C]pyruvate quickly and cheaply for biomedical and other applications.

Introduction

NMR hyperpolarization techniques transiently boost nuclear spin polarization (P) by several orders of magnitude.^{[1, 2]} The enhanced P can be stored on relatively long-lived ¹³C carriers of hyperpolarization, allowing them to be used as metabolic contrast agents with 4-6 orders of magnitude gain in NMR signal.^{[3, 4]}

[1-¹³C]pyruvate is the leading hyperpolarized (HP) contrast agent.^[5] It is currently under investigation in 14 clinical trials^[5] for spectroscopic imaging of aberrant metabolism of cancer^[4] and other diseases.^[6] Such real-time metabolic studies^[7] became possible due to the pioneering work of Ardenkjaer-Larsen and co-workers who established dissolution Dynamic Nuclear Polarization (d-DNP) technology (ca. 2003).^[8] Despite major success in the research domain,^[9] the production of HP [1-¹³C]pyruvate via d-DNP is slow (tens of minutes or longer) and expensive ($2M+ for a clinical device), representing a substantial barrier for clinical translation of this otherwise revolutionary contrast agent and molecular imaging modality. Metabolic MR scan with HP [1-¹³C]pyruvate takes less than a minute and uses no ionizing radiation.^[7] This is in sharp contrast to clinical [¹⁸F]fluorodeoxyglucose Positron Emission Tomography scan requiring ~2 hours and exposing subjects to 30-50 mSv radiation.

More efficient and cost-effective approaches for production of HP [1-¹³C]pyruvate are needed to enable its widespread clinical use. One recent development is the use of the Parahydrogen Induced Polarization (PHIP)^[10] technique by Reineri and co-workers, who pioneered the Side Arm Hydrogenation (SAH) variant of PHIP for production of HP [1-¹³C]pyruvate via SAH followed by magnetic field cycling (MFC) and de-esterification.^[11] Indeed, P_13C of up to 10% has been demonstrated for HP [1-¹³C]pyruvate using this approach^[12] for in vivo validation studies.^[13] The key limitation of this approach is the requirement for synthesis of unsaturated PHIP precursors.

Signal Amplification by Reversible Exchange (SABRE)^[14] is a PHIP variant that requires no hydrogenation—instead, SABRE relies on reversible exchange of parahydrogen (p-H₂) and the to-be-hyperpolarized substrate with a catalyst. Recently, after some success with indirect methods,^[15] Duckett and co-workers^{[16, 17]} and Goodson and co-workers^[18] independently demonstrated that SABRE can be employed for direct hyperpolarization of ¹³C carboxylate moieties—including [1-¹³C]pyruvate (P_13C~1.85%).^[16] [1-¹³C]pyruvate forms a complex with the standard catalyst used for SABRE, [IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD = cyclooctadiene)],^{[19, 20]} Scheme 1). The efficient hyperpolarization transfer from p-H₂-derived hydrides to the ¹³C nuclear spins of [1-¹³C]pyruvate becomes possible by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH).^{[21, 22]} In this technique, the use of microtesla magnetic fields during enables spontaneous polarization transfer from p-H₂-derived hydrides directly to the heteronucleus (i.e., anything else but protons) of to-be-hyperpolarized exchangeable substrate molecule, including ¹³C as shown in Figure 1a.^[21-23]

Scheme 1.

Formation of [IrCl(H)₂(DMSO)₂(IMes)] (2) and [Ir(H)₂(η²-[1-¹³C]pyruvate)(DMSO)(IMes)] (3) complexes following activation of [IrIMes(COD)Cl] (1) pre-catalyst. Complex 1 was synthesized previously^[24] according to Cowley and co-workers.^[19] The complexes 1, 2, 3a and 3b are as indicated by Duckett and co-workers.^[16]

Figure 1.

a) Chemistry of SABRE-SHEATH hyperpolarization exchange of dominant complexes 3a and 3b and corresponding ¹³C NMR spectrum from the HP composition (30 mM [1-¹³C]pyruvate, 20 mM DMSO, 6 mM pre-catalyst): the sample was hyperpolarized at 0 °C and transferred to a 1.4 T NMR spectrometer for detection in ~5 seconds. Note the appearance of the residual free HP [1-¹³C]pyruvate signal (most down-field resonance) originating from sample warm-up during sample transfer; faster transfer following SABRE under these conditions yields spectra that are more dominated by the bound 3b resonance (inset; see Figure 3 for more details). b) Corresponding thermal reference spectrum from neat [1-¹³C]acetic acid. c) Diagram of the gas manifold and key components within the PHIP hyperpolarizer experimental design to be described elsewhere.

In even more recent work describing low-cost 50% p-H₂ generators, we explored temperature dependence of SABRE-SHEATH polarization of pyruvate yielding ¹³C polarization of 1.2%.^[25]

Results and Discussion

Here, we demonstrate that SABRE-SHEATH can produce order-unity ¹³C polarization in [1-¹³C]pyruvate in seconds, Figure 1a. Figure 1a shows a spectrum of 6 mM Ir-[1-¹³C]pyruvate complex composed of both 3a and 3b with P_13C of 39% at the time of detection; a corresponding ¹³C reference spectrum from thermally polarized neat [1-¹³C]acetic acid is shown in Figure 1b. Most the ¹³C NMR measurements reported here were performed at the clinically relevant magnetic field of 1.4 T. Because the HP sample depolarizes during the 3-5-second long sample transfer (due to sample exposure in the Earth’s field and the 1.4 T field of the NMR spectrometer, see Supporting Information (SI) for details), the P_13C value at the time of production (i.e., at the moment of completion of the SABRE-SHEATH process) is estimated to be >50%. This polarization value represents a conservative estimate, because 3b is not the only complex present as seen from high-resolution ¹H NMR spectra of the hydride region (Figure S2), and our polarization calculations were based on the pre-catalyst 1 concentration, see SI. Therefore, we conclude that P_13C can approach order-unity values under ideal conditions of ¹³C SABRE-SHEATH polarization build-up. We note that here, we are focused on the ultimate limit of achievable ¹³C polarization, instead of bulk production of HP [1-¹³C]pyruvate.

By decreasing the temperature of ¹³C SABRE-SHEATH to 0 °C, where maximum P_13C is achieved, exchange is too slow to enable the appreciable ¹³C polarization build-up on the free species, Figure 1a. Only minute amounts of “free” HP [1-¹³C]pyruvate resonance (Figure 1a) is created during the ~3-5-second-long sample transfer from the shield (at 0 °C) to the bore of the 1.4 T NMR spectrometer (32 °C). The 3-5-second transfer period represents an experimental limitation required for reproducible detection of the NMR signal — indeed, in certain cases when faster transfer was used to reduce sample warm up, (inset of Figure 1a), the “free” HP pyruvate resonance is negligible. Therefore, we attribute virtually all of the detected HP pool to the catalyst-bound complexes 3a and 3b. The successive p-H₂ exchanges with 3b result in accumulating polarization of the complex.

Kinetically, the build-up to these high P_13C levels becomes possible because the ¹³C T₁ relaxation time of HP [1-¹³C]pyruvate in this complex at 0.30 μT is long: 9.5 s (i.e., the spin relaxation rate is ~0.1 s⁻¹) even at the relatively large catalyst concentration of 6 mM. In comparison, the ¹⁵N T₁ of HP [¹⁵N]pyridine is less than 2 s at 6 mM catalyst concentration.^[23] We rationalize these more favorable ¹³C relaxation dynamics in terms of the overall greater separation distance of the target ¹³C nucleus from the quadrupolar iridium catalyst center: i.e., the Ir-O-¹³C moiety versus the substantially shorter total bond distance (and by extension greater iridium-complex-mediated depolarization^[26]) with an Ir-¹⁵N moiety. As a result, the effective ¹³C polarization build-up constant, T_b = 4.9 s, corresponds to an effective build-up rate of ~0.2 s⁻¹—substantially faster (e.g., Figure 2c) than the corresponding spin relaxation rate (Figure 2d), allowing one to achieve order-unity ¹³C polarization.

Figure 2.

Total ¹³C polarization of ¹³C-1 (i.e., integrating over all bound and free resonances) in 30 mM [1-¹³C]pyruvate (pyruvate to DMSO ratio of 30:20) as a function of magnetic transfer field (a), temperature (b), p-H₂ bubbling duration (c), and in-shield decay (d). All experiments were performed at 7.7 atm of p-H₂ pressure at 70 sccm p-H₂ flow rate. Detailed mapping of other experimental parameters can be found in the SI. The data points marked with asterisks correspond to the cases when virtually all ¹³C HP signal originates from bound species 3a and 3b.

The reported SABRE-SHEATH experiments were performed using p-H₂ bubbling through the solution (Figure 1c), which can be quickly and conveniently interrupted and restarted at will—thereby allowing one to quickly map the experimental parameter space in a systematic manner, in addition to the studies of relaxation dynamics discussed above. Careful optimization of the magnetic field and temperature of ¹³C SABRE-SHEATH for [1-¹³C]pyruvate (Figures 2a,2b respectively) revealed the optimum polarization transfer field B_T of ~ 0.3 μT and the optimum polarization transfer temperature T_T of ~0 °C (P_13C values in Figure 2 were computed for total ¹³C polarization of 30 mM [1-¹³C]pyruvate to provide a fair comparison of the entire hyperpolarization pool, regardless of its origin: free, 3a, or 3b species).

Temperature has a profound effect on the exchange rates of [1-¹³C]pyruvate on complex 3b—Figure 3a shows a variable temperature SABRE-SHEATH experiment with monotonic disappearance of free HP resonance at lowered temperatures, because the ultra-slow exchange rate does not allow any appreciable release of HP species from complex 3b into the free state. When SABRE-SHEATH is performed at elevated temperatures (e.g., 22 °C, Figure 3a), [1-¹³C]pyruvate exchange with the polarization transfer complex is accelerated, leading to hyperpolarization of both free and bound 3b species in the expected pyruvate:pre-catalyst (~5:1) ratio. Even though the ¹³C polarization was nearly fully locked in the bound 3b state at 0 °C (Figure 3b, blue trace), the ¹³C polarization bolus can be rapidly released if the NMR tube containing the HP solution is rapidly warmed up after sample transfer from the shield, but before inserting the sample in the NMR detector. The effect of rapid release of HP pyruvate from 3b can potentially enable a wide range of new strategies to improve the efficiency of [1-¹³C]pyruvate polarization via SABRE-SHEATH.

Figure 3.

a) Stacked variable-temperature SABRE-SHEATH ¹³C spectra showing the interplay between HP complex 3b and “free” peaks as a function of temperature during hyperpolarization. b) Corresponding stacked plots of HP ¹³C spectra obtained after SABRE-SHEATH polarization at a fixed temperature of 0 °C, followed by rapid sample warm-up at different temperatures (~25-40 °C), illustrating the step-wise release of HP [1-¹³C]pyruvate during sample warm-up following hyperpolarization of slow-exchanging complex 3b at the lower temperature. All experiments were performed using the same sample of ~30 mM [1-¹³C]pyruvate, ~20 mM DMSO, and ~6 mM catalyst in CD₃OD. The stacked spectra presented are not to scale.

During the course of our experimental studies, we discovered that our p-H₂ generator (98.5% para-)^[27] was leaching residual moisture into our samples after repeated p-H₂ bubbling (on the time scale of hours), leading to reproducibility challenges. Although this experimental limitation was quickly remedied by thorough drying of the generator by H₂ purging at room temperature for 3 days, this observation prompted us to investigate the role of H₂O as a potential secondary co-ligand or substrate modifier in SABRE-SHEATH experiments. Indeed, systematic H₂O titrations (SI) revealed the dependence of P_13C not only on DMSO concentration, but also on water concentration. For example, Figure 4 shows a clear P_13C modulation of [1-¹³C]pyruvate by varying the concentration of H₂O added to the sample and variable-temperature SABRE-SHEATH experiments. As a result, the presence of H₂O in an optimal concentration (~0.5 M) was found to nearly double P_13C (compared to no water added), reaching ~13% at the optimum temperature (~8 °C)—a P_13C value that is ~7 times greater than the previously reported P_13C value of 1.85%.^[16] We speculate that water may potentially act either as a secondary co-ligand, or water molecule may interact with pyruvate and therefore alter [1-¹³C]pyruvate exchange dynamics of Ir-IMes hexacoordinate complex.

Figure 4.

a) total ¹³C polarization of the ¹³C-1 site (i.e., integrating over all bound and free resonances) in ~30 mM [1-¹³C]pyruvate in CD₃OD (pyruvate to DMSO ratio of 3:2) as a function of H₂O concentration and SABRE-SHEATH temperature. See SI for details; b) the ¹³C NMR spectrum of HP [1-¹³C]pyruvate with P_13C ~ 13% with SABRE-SHEATH at 8 °C and 0.5 M water content.

Although future systematic optimization studies are certainly warranted to further improve ¹³C polarization efficiency of HP [1-¹³C]pyruvate produced via SABRE-SHEATH, this report clearly demonstrates that order-unity ¹³C polarization is fundamentally attainable in this system. Moreover, the P_13C value of ~13% demonstrated here for the free substrate (Figure 4) is certainly already useable for in vivo studies^{[13, 28, 29]}; however, future in vivo feasibility studies will need to address remaining translational roadblocks by combining the present polarization techniques with ongoing efforts to achieve rapid SABRE catalyst removal^[30-32] and HP [1-¹³C]pyruvate reconstitution into biocompatible aqueous media of injectable solutions.^[29] Furthermore, recent studies utilizing pulsed-SABRE-SHEATH^[33] demonstrated that even higher polarization levels may be obtained that using conventional SABRE-SHEATH approach employed here. These so-called pulsed approaches benefit from the coherently driven hyperpolarization dynamics in signal amplification by reversible exchange^[34] and more favorable relaxation dynamics of heteronucleus during the pulse performed at tens of microtesla.^{[23, 26, 35, 36]} Indeed, very recent studies demonstrated that heteronuclear polarization can be boosted by a factor of 1.3 when employing pulsed-SABRE-SHEATH, a.k.a., alt-SABRE-SHEATH^{[37, 38]} Therefore, the future SABRE-SHEATH optimization studies of [1-¹³C]pyruvate may potentially benefit substantially from various pulsed variants. Finally, future robust production of HP [1-¹³C]pyruvate for biomedical applications (as well as optimization of experimental parameters reported here) using SABRE-SHEATH requires a dedicated purpose-built hyperpolarizer device. All results reported here were obtained using clinical-scale SABRE-SHEATH hyperpolarizer (Figure 1c shows the diagram of the gas manifold and key components within the PHIP hyperpolarizer), the experimental design of which will be described elsewhere similarly to our track record of sharing best laboratory practices of parahydrogen hyperpolarizers’ designs with the scientific community.^[39-41]

Conclusions

In conclusion, we have demonstrated that order-unity ¹³C polarization (P_13C>50%) of [1-¹³C]pyruvate is obtained during SABRE-SHEATH on the catalyst-bound complex 3b—the feasibility of high ¹³C polarization is also supported by the relaxation dynamics measurements. This is an important proof that highly polarized ¹³C-carboxylic acids (including key metabolites) can be produced by the SABRE technique. Second, we have shown that temperature modulates the efficiency of SABRE-SHEATH dramatically with optimum temperature of ~8 °C. Third, the presence of water in addition to DMSO co-ligand also modulates SABRE-SHEATH efficiency with the optimum water content found to be ~0.5 M. Taken together the presented results bode well for future biomedical use of [1-¹³C]pyruvate prepared via SABRE-SHEATH for future biomedical applications.