Efficient Hyperpolarization of U-13 C-Glucose Using Narrow-Line UV-Generated Labile Free Radicals

Abstract

Free radicals generated by UV-light irradiation of a frozen solution containing a fraction of pyruvic acid (PA) have demonstrated their dissolution dynamic nuclear polarization (dDNP) potential, providing up to 30 % [1-¹³ C]PA liquid-state polarization. Moreover, their labile nature has proven to pave a way to nuclear polarization storage and transport. Herein, differently from the case of PA, the issue of providing dDNP UV-radical precursors (trimethylpyruvic acid and its methyl-deuterated form) not involved in any metabolic pathway was investigated. The ¹³ C dDNP performance was evaluated for hyperpolarization of [U-¹³ C₆ ,1,2,3,4,5,6,6-d₇ ]-d-glucose. The generated UV-radicals proved to be versatile and highly efficient polarizing agents, providing, after dissolution and transfer (10 s), a ¹³ C liquid-state polarization of up to 32 %.

Keywords: NMR spectroscopy; glucose; hyperpolarization; radicals.

Figure 1

Free radicals generated by UV-irradiation of PA and structures of TriPA and d9-TriPA.

Figure 2

X-band ESR measurement (black line) and ESR spectrum fit (dashed red line) after 5 min UV-light irradiation at 77 K of a single 4.0±0.2 µl bead of a frozen solution containing PA:GW55 1:9 (v/v) (A); [1-¹³C]PA:GW55 1:9 (v/v) (B); [2-¹³C]PA:GW55 1:9 (v/v) (C); TriPA:GW55 1:9 (v/v) (D). GW55 is an abbreviation for the solvent composition glycerol:water 50:50 (v/v). In the insert the structural formula of the radical precursor is shown.

Figure 3

Radical generation at 77 K as a function of UV-irradiation time for frozen mixtures of 0.7 M TriPA (red curve), d₉-TriPA (black curve) and PA (blue curve) with 2 M [U-¹³C,d₇]-D-glucose dissolved in GW55. Measurements were performed on a single 4.0±0.2 µL frozen bead immersed in liquid nitrogen (A). Room temperature UV-vis absorption measurements for the same sample solutions (left y-scale); the light blue coloured area represents the spectral irradiance (right y-scale) of the UV-source (Dymax, BlueWave 75) at maximum power (19 W/cm²) used to photo-generate the radicals (B).

Figure 4

The longitudinal detected (LOD) ESR spectrum (grey curve) and the ¹³C DNP microwaves sweep without modulation (blue curve) and with modulation (red curve) are reported for TriPA_DNP-sample (panel A) and d₉-TriPA_DNP-sample (panel B). All measurements were performed at 6.7 T and 1.1 K. Samples were UV-irradiated in liquid nitrogen for 300 s.

Figure 5

The ¹³C polarization solid-state build-up (left panel) and liquid-state relaxation (right panel) are showed for TriPA_DNP-sample, d₉-TriPA_DNP-sample and Trityl_DNP-sample. Solid-state measurements were performed at 6.7 T and 1.1 K by mw irradiation at 188.19 GHz (20 MHz amplitude modulation and 1 kHz frequency modulation) for TriPA_DNP-sample and d₉-TriPA_DNP-sample; Trityl_DNP-sample was polarized at the 187.94 GHz (no microwaves modulation) corresponding to the optimum for Trityl (see Supporting Information). Liquid-state measurements were acquired on 9.4 T high resolution magnet equipped with a 5 mm NMR probe kept at 40 °C. The transfer of the HP solution from the polarizer to the high resolution magnet took 10 s. In the inset the HP [U-¹³C,d₇]-D-glucose NMR spectrum corresponding to the first point of d₉-TriPA_DNP-sample signal decay is reported (C2-C5 DNP enhancement = 41000).

Figure 6

The ESR signal as a function of temperature is reported for TriPA_DNP-sample (panel A) and d₉-TriPA_DNP-sample (panel B). In both cases, the ESR signal suddenly disappeared above 190 K. The two samples became diamagnetic when still frozen.

Figure 7

UV-TriPA hyperpolarised [U-¹³C,d₇]glucose conversion in live prostate carcinoma cells (PC-3). A build-up of the glycolysis derived metabolite lactate could be followed over 30 s. Intermediates in glycolysis are identified by ¹³C-¹³C coupling constants due to the uniformly labelled substrate and indicated by black bars in a sum of 30 spectra: Lactate (LAC), Glutamate carbon #1 and #5 (GLU-C1 and C5), 3-phophoglucerate (3PG), unspecified amino acid (AA). Remaining radical precursor is identified outside ppm area of interest as singlets (TriPA). The signal denoted * is likely to originate from the precursor.