Rapid 13C Hyperpolarization of the TCA Cycle Intermediate α-Ketoglutarate via SABRE-SHEATH

Abstract

α-Ketoglutarate is a key biomolecule involved in a number of metabolic pathways─most notably the TCA cycle. Abnormal α-ketoglutarate metabolism has also been linked with cancer. Here, isotopic labeling was employed to synthesize [1-¹³C,5-¹²C,D₄]α-ketoglutarate with the future goal of utilizing its [1-¹³C]-hyperpolarized state for real-time metabolic imaging of α-ketoglutarate analytes and its downstream metabolites in vivo. The signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH) hyperpolarization technique was used to create 9.7% [1-¹³C] polarization in 1 minute in this isotopologue. The efficient ¹³C hyperpolarization, which utilizes parahydrogen as the source of nuclear spin order, is also supported by favorable relaxation dynamics at 0.4 μT field (the optimal polarization transfer field): the exponential ¹³C polarization buildup constant T_b is 11.0 ± 0.4 s whereas the ¹³C polarization decay constant T₁ is 18.5 ± 0.7 s. An even higher ¹³C polarization value of 17.3% was achieved using natural-abundance α-ketoglutarate disodium salt, with overall similar relaxation dynamics at 0.4 μT field, indicating that substrate deuteration leads only to a slight increase (∼1.2-fold) in the relaxation rates for ¹³C nuclei separated by three chemical bonds. Instead, the gain in polarization (natural abundance versus [1-¹³C]-labeled) is rationalized through the smaller heat capacity of the "spin bath" comprising available ¹³C spins that must be hyperpolarized by the same number of parahydrogen present in each sample, in line with previous ¹⁵N SABRE-SHEATH studies. Remarkably, the C-2 carbon was not hyperpolarized in both α-ketoglutarate isotopologues studied; this observation is in sharp contrast with previously reported SABRE-SHEATH pyruvate studies, indicating that the catalyst-binding dynamics of C-2 in α-ketoglutarate differ from that in pyruvate. We also demonstrate that ¹³C spectroscopic characterization of α-ketoglutarate and pyruvate analytes can be performed at natural ¹³C abundance with an estimated detection limit of 80 micromolar concentration × *%P_13C. All in all, the fundamental studies reported here enable a wide range of research communities with a new hyperpolarized contrast agent potentially useful for metabolic imaging of brain function, cancer, and other metabolically challenging diseases.

Figure 1.

a) Schematic of SABRE-SHEATH hyperpolarization process and relevant polarization transfer path of [1-¹³C,5-¹²C,D₄]α-KG. b) ¹³C spectrum of HP [1-¹³C,5-¹²C,D₄]α-KG; carbon positions are labeled by blue numerals. c) Representative stacked variable-temperature ¹³C spectra of 5 mM [1-¹³C,5-¹²C,D₄]α-KG demonstrating the interplay between HP complex 3b and the “free” peak as a function of temperature during the SABRE-SHEATH hyperpolarization process; d) corresponding ¹³C spectrum of thermally polarized neat [1-¹³C]acetic acid employed as signal reference for computation of signal enhancements. e) Build-up and decay of total ¹³C polarization of ¹³C-1 (i.e., integrating over all bound and free resonances) in [1-¹³C,5-¹²C,D₄]α-KG at B_T=0.42 μT; f) corresponding ¹³C-1 T₁ relaxation curves at the Earth’s and the clinically-relevant 1.4 T field of the benchtop spectrometer. Total ¹³C polarization of ¹³C-1 in [1-¹³C,5-¹²C,D₄]α-KG as a function of temperature (g) and magnetic transfer field (h). All experiments are performed at 1.4 T using a SpinSolve NMR spectrometer in CD₃OD at T_T=+10 °C (unless otherwise noted).

Figure 2.

a) Schematic of SABRE-SHEATH hyperpolarization process and relevant polarization transfer path of natural-abundance α-KG; carbon positions are labeled by blue numerals. b) Representative HP ¹³C spectrum of 5.6 mM natural-abundance α-KG obtained by performing SABRE-SHEATH at +10 °C in CD₃OD at 1.4 T; c) corresponding ¹³C spectrum of thermally polarized neat [1-¹³C]acetic acid; d) Total (bound+free) ¹³C polarization build-up and decay at B_T=0.42 μT and T_T=+10 °C; e) Total (bound+free) ¹³C polarization decay at the Earth’s field and 1.4 T. Total ¹³C polarization of ¹³C-1 in natural abundance α-KG as a function of temperature (f) and magnetic transfer field (g). All experiments are performed at 1.4 T using a SpinSolve NMR spectrometer in CD₃OD.

Figure 3.

a) Schematic of SABRE-SHEATH hyperpolarization process and relevant polarization transfer path of natural-abundance sodium pyruvate; carbon positions are labeled by blue numerals. b) Representative HP ¹³C spectrum of 8.6 mM natural-abundance sodium pyruvate obtained by performing SABRE-SHEATH at +10 °C in CD₃OD at 1.4 T; c) corresponding ¹³C spectrum of thermally polarized neat [1-¹³C]acetic acid; d) Total (bound+free) ¹³C polarization build-up and decay at B_T=0.42 μT and T_T=+10 °C; e) Total (bound+free) ¹³C polarization decay at the Earth’s field and 1.4 T. Total ¹³C polarization of ¹³C-1 in sodium pyruvate as a function of temperature (f) and magnetic transfer field (g). All experiments are performed at 1.4 T using a SpinSolve NMR spectrometer in CD₃OD.

Scheme 1.

Formation of [IrCl(H)₂(DMSO)₂(IMes)] (2) and [Ir(H)₂(η2-substrate)(DMSO)(IMes)] (3) complexes following activation of [IrIMes(COD)Cl] (1) pre-catalyst. Catalyst 1 was prepared previously according to Cowley et al. The species 1, 2, 3a and 3b are as indicated by Iali et al. for pyruvate variants. R = CH₃ for pyruvate and R = CH₂-CH₂-COO⁻ for α-KG.

Scheme 2.

Overall synthetic schematic of [1-¹³C,5-¹²C,D₄]α-KG. See Supporting Information (SI) for details of experimental procedure and characterization details.