Effects of Deuteration of ¹³C-Enriched Phospholactate on Efficiency of Parahydrogen-Induced Polarization by Magnetic Field Cycling

Abstract

We report herein a large-scale (>10 g) synthesis of isotopically enriched 1-¹³C-phosphoenolpyruvate and 1-¹³C-phosphoenolpyruvate-d₂ for application in hyperpolarized imaging technology. The 1-¹³C-phosphoenolpyruvate-d₂ was synthesized with 57% overall yield (over two steps), and >98% ²H isotopic purity, representing an improvement over the previous report. The same outcome was achieved for 1-¹³C-phosphoenolpyruvate. These two unsaturated compounds with C=C bonds were employed for parahydrogen-induced polarization via pairwise parahydrogen addition in aqueous medium. We find that deuteration of 1-¹³C-phosphoenolpyruvate resulted in overall increase of ¹H T₁ of nascent hyperpolarized protons (4.30 ± 0.04 s versus 2.06 ± 0.01 s) and ¹H polarization (~2.5% versus ~0.7%) of the resulting hyperpolarized 1-¹³C-phospholactate. The nuclear spin polarization of nascent parahydrogen-derived protons was transferred to 1-¹³C nucleus via magnetic field cycling procedure. The proton T₁ increase in hyperpolarized deuterated 1-¹³C-phospholactate yielded approximately 30% better ¹³C polarization compared to non-deuterated hyperpolarized 1-¹³C-phospholactate. Analysis of T₁ relaxation revealed that deuteration of 1-¹³C-phospholactate may have resulted in approximately 3-fold worse H→¹³C polarization transfer efficiency via magnetic field cycling. Since magnetic field cycling is a key polarization transfer step in the Side-Arm Hydrogenation approach, the presented findings may guide more rationale design of contrast agents using parahydrogen polarization of a broad range of ¹³C hyperpolarized contrast agents for molecular imaging employing ¹³C MRI. The hyperpolarized 1-¹³C-phospholactate-d₂ is of biomedical imaging relevance because it undergoes in vivo dephosphorylation and becomes ¹³C hyperpolarized lactate, which as we show can be detected in the brain using ¹³C hyperpolarized MRI; an implication for future imaging of neurodegenerative diseases and dementia.

Graphical Abstract

INTRODUCTION

Parahydrogen Induced Polarization (PHIP)^1–2 is a fast and efficient hyperpolarization technique.^3–8 When parahydrogen pairwise addition is performed to C=C or C≡C unsaturated chemical bond, the symmetry of nascent parahydrogen-derived protons can be broken due to their magnetic inequivalence in the product of this chemical reaction.^9–10 High-levels of polarization can be retained by the nascent protons.^11–15

Because proton sites typically retain hyperpolarized (HP) state for a short period of time due to relatively short exponential decay time constants (e.g. spin-lattice relaxation time constant T₁) on the order of a few seconds,¹⁶ the PHIP studies demonstrated a significant advantage of transferring parahydrogen-derived polarization to ¹³C^{13–15, 17} and ¹⁵N sites with longer T₁ values on the order of a minute or more (Scheme 1a).¹⁸ The resulting ¹³C hyperpolarized (HP) compounds have been successfully employed as HP ¹³C contrast agents to probe metabolism and biochemical functions in vivo as long as sufficiently high concentration and high polarization level can be achieved.^{15, 19–29}

Scheme 1.

a) Schematic of Parahydrogen Induced Polarization (PHIP) Process with polarization transfer to heteronuclei.⁵ (b) Schematic of PHIP hyperpolarization of 1-¹³C-phospholactate-d₂ (PLAC-d₂) by pairwise p-H₂ addition to unsaturated precursor (1-¹³C-phosphoenolpyruvate-d₂). Magnetic field cycling (MFC) procedure is employed in the second step for polarization transfer from p-H₂-derived protons to ¹³C nucleus.

Two key approaches have been developed for polarization transfer from nascent parahydrogen-derived protons. In the first approach, radio-frequency (RF) pulses are employed, taking advantage of direct spin-spin couplings between the two protons and the ¹³C nucleus.^{14, 30–39} In the second approach, a magnetic field cycling (MFC) procedure is performed in weak magnetic fields (from nano-Tesla to tens of micro-Tesla) for spontaneous polarization transfer via the network of spin-spin couplings between parahydrogen-derived protons and the ¹³C nucleus.^{14–15, 20, 30, 40–41} The key advantage of the second approach is the possibility of longer-range polarization transfer over up to 5 chemical bonds away.⁴² The other advantage of MFC is a significantly less demanding hardware operations compared to RF-based methods: MFC requires a magnetic shield,^{20, 40} whereas RF-based methods require a reasonably homogeneous magnet, RF coils, transmitters and other related hardware.^{19, 24, 28, 34, 43–44} As a result, Side Arm Hydrogenation (SAH) approach has been developed,^{42, 45–48} where parahydrogen (p-H₂) pairwise addition is performed into an ester moiety, and polarization is transferred to ¹³C-carboxyl site, which has long T₁. Recently, PHIP-SAH has been demonstrated for efficient hyperpolarization of ¹³C-acetate and ¹³C-pyruvate moieties.^49–50 HP ¹³C-acetate and ¹³C-pyruvate have been shown to report on metabolic activities in cancers, liver disease, and brain.^51–64 Furthermore, PHIP-SAH HP 1-¹³C-pyruvate has been shown recently to probe altered heart metabolism in vivo.⁶⁵

Despite the recent progress with PHIP-SAH relying on MFC, ¹³C hyperpolarization levels demonstrated to date are lower than those reported by dissolution Dynamic Nuclear Polarization (d-DNP; ¹³C polarization, P_13C up to 60%⁶⁶) and by PHIP relying of RF pulses (P_13C up to 59%^{14, 67}).

The motivation of this work is to study the effect of deuteration on ¹³C hyperpolarization relying on MFC procedure with the goal of improving the ¹³C hyperpolarization yields. Here, we employed HP 1-¹³C-phospholactate (PLAC) to study the effect of deuteration on the hyperpolarization via MFC, because HP PLAC can be converted to HP ¹³C-lactate in vivo in vasculature,⁶⁸ and its imaging feasibility has already been shown.²⁸ To achieve this goal, we have prepared deuterated and non-deuterated forms of 1-¹³C-phosphoenolpyruvate via improved synthetic procedure.⁶⁹ This unsaturated precursor is required for p-H₂ pairwise addition, which leads to formation of HP 1-¹³C-phospholactate (Scheme 1b).

METHODS

General Considerations.

All glassware (round bottom flasks, magnetic stir bar, addition funnel, glass funnel and measuring cylinder) were oven dried.

Potassium 1-¹³C-phosphoenolpyruvate-d₂ (PEP-d₂) synthesis.

1-¹³C-pyruvic-d₄ acid (1) (10 g, 110 mmol, 1 eq) was added to a tared, oven-dried round bottom flask (previously flushed with Argon), immediately followed by the addition of carbon tetrachloride (40 mL). Then, the flask was equipped with an addition funnel for dispensing the mixture of bromine (~5.7 mL, 111 mmol, 1 eq) and carbon tetrachloride (~4 mL) into a stirring solution dropwise under the presence of argon over the course of 3 h at 4 °C (Figure S9). Upon bromine color disappearance (after 3 hours), the resulting mixture was evaporated in vacuo (0–2 mbar) with the co-solvent of methylene chloride (40 mL) and the process was repeated 3 times (3×40 mL of CH₂Cl₂ in total) until the solid material was dried completely to provide crude product 2 (16.7 g, 0.1 mol, 90% yield). The prepared crude material was used in the next step without purification, Scheme 2.

Scheme 2.

Synthesis of 1-¹³C-phosphoenolpyruvate-d₂ (PEP-d₂).

A solution of above-mentioned crude bromopyruvic acid ( 16.7 g, 0.1 mol) in anhydrous THF (50 mL) was added dropwise to a dried and argon-flushed round bottom flask (250-mL size) containing trimethyl phosphite, P(OMe)₃, (~1.1 eq., 14.8 mL, 125 mmol) and anhydrous THF (50 mL) via an additional funnel at a rate that the reaction solution temperature never exceeded 45 °C. The round-bottom flask was equipped with magnetic stirrer, thermometer and addition funnel attached via a Claisen adapter (see Figure S10 for details). The mixture was stirred for 1h before the solvents were evaporated under the reduced pressure for 3 h. D₂O (80 mL) was added to the viscous residue until it was dissolved, and it was left stirring at RT overnight. Next, the mixture was transferred to an ice-chilled beaker and the pH was adjusted to approximately 2.8 using deuterated KOH (10 g KOH, 20 mL D₂O). Then, activated charcoal (0.5 g) was added to the solution, filtered, and the filtrate was evaporated under reduced pressure to provide solid 3, which was re-dissolved in D₂O (100 mL) in the presence of activated charcoal (~0.5 g), filtered and the filtrate was evaporated under reduced pressure. The resulting solid was purified by recrystallization using water (~40 mL) and ethanol (~300 mL) to afford white crystal needles (13.1 g, 0.06 mol, 57% yield over two steps).

The produced material (1-¹³C-phosphoenolpyruvate-d₂, 3) was characterized by high-resolution NMR spectroscopy (Figures S5–S7) and by high-resolution mass spectrometry (HR-MS) (Figure S4).

¹³C NMR (100 MHz, D₂O): 166.4. ³¹P NMR (162 MHz, D₂O): −5.5. Proton characterization is not useful because of the deuteration.

HR-MS was performed by direct liquid infusion using an Orbitrap mass spectrometer (Thermo-Finnigan, San Jose, CA) equipped an Ion-Max source housing and a standard electrospray (ESI) ionization probe in negative ion mode at a resolving power of 60,000 (at m/z 400). ¹²C₂¹³C₁H₂D₂O₆P⁻ (M-H-): 169.99101; found 169.99079 (−1.3 ppm). The product ¹³C isotopic purity was >99% (determined by the isotopic purity of the starting material), ²H isotopic purity was >98% (determined from HR-MS and by NMR data).

Potassium 1-¹³C-phosphoenolpyruvate (PEP) synthesis followed the same procedure as that for 1-¹³C-phosphoenolpyruvate-d₂ described above with the exception that (i) D₂O was not employed (H₂O was used instead), and (ii) the starting material was 1-¹³C-pyruvic acid (Isotec-Sigma-Aldrich, 677175). The overall synthesis is summarized in Scheme 3. The overall yield was similar to that for 1-¹³C-phosphoenolpyruvate-d₂ reported above. The product ¹³C isotopic purity was 99% in accord with the starting material enrichment employed in this synthesis: 1-¹³C-pyruvic acid (Isotec-Sigma-Alrich, 677175). NMR spectroscopic characterization of the obtained product is provided in Figures S1–S3.

Scheme 3.

Synthesis of 1-¹³C-phosphoenolpyruvate (PEP).

¹H NMR (400 MHz, D₂O): 5.43 (d.t., 1H, J = 10 Hz and 2.2 Hz), 5.77 (d.t., 1H, J = 3.5 Hz and 2.2 Hz). ¹³C NMR (100 MHz, D₂O): 166.4 (d, J = 7 Hz). ³¹P NMR (162 MHz, D₂O): −5.5.

NMR hyperpolarization.

Commercially available bis(norbornadiene)rhodium(I) tetrafluoroborate ([Rh(NBD)₂]BF₄, NBD = norbornadiene, Strem 45–0230), 1,4-bis[(phenyl-3-propanesulfonate)phosphine]butane (Sigma-Aldrich 717347) and ultra-pure hydrogen (H₂) (>99.999%) were used as received. The overall scheme of experimental setup is presented in Figure 1. Hydrogen gas was enriched with p-H₂ up to 66–85% para-state using a home-made parahydrogen generator. p-H₂ gas flow rate was regulated with mass flow controller (SmartTrak 50, Sierra Instruments, Monterey, CA). The previously described procedure³² was used for the preparation of homogeneous catalyst solution in D₂O (~5.3 mM) followed by addition of PEP or PEP-d₂ to obtain approximately 30 mM substrate concentration. Medium-wall 5 mm NMR tubes (Wilmad glass P/N 503-PS-9) tightly connected with ¼ in. outer diameter PTFE tubes were filled with 0.5 mL of resultant solution under argon atmosphere and pressurized with p-H₂ up to 70 psig. The samples were preheated either up to 60 °C using NMR spectrometer temperature control unit (in case of PASADENA^9–10 experiments) or up to ~70–80 °C using hot water (in case of ALTADENA⁷⁰ and MFC experiments). The p-H₂ gas was bubbled at 140 standard cubic centimeters per minute (sccm) flow rate and 70 psig pressure. In PASADENA experiments, the sample was located inside the probe of 9.4 T Bruker NMR spectrometer during p-H₂ gas bubbling, while in ALTADENA and MFC experiments p-H₂ gas was bubbled while the sample was located in the Earth’s magnetic field. After termination of p-H₂ bubbling the sample was either transferred directly to the NMR probe (in ALTADENA experiments) or placed inside the mu-metal magnetic shield described in detail in Ref. # ⁴¹ in experiments employing MFC polarization transfer. The magnetic field inside the shield was adjusted using additional solenoids placed inside the mu-metal shield (Note the mu-metal shield provides an isolation of approximately 1,200 according to the manufacturer’s specification; therefore, the use of the shield in the Earth’s magnetic field results in the minimum residual magnetic field of approximately 40 nT). Then the sample was slowly (~1 s) pulled out of the shield and placed inside the NMR probe for detection. PHIP spectra were acquired as pseudo 2D sets consisting of 64 ¹H NMR spectra in order to avoid delays between placing the sample into the probe and starting of the acquisition. The acquisition of these pseudo 2D sets was always started before termination of p-H₂ bubbling, and the first spectra containing signal were used for presentation here. In PASADENA experiments NMR spectra were acquired using 45° RF excitation pulse, while in ALTADENA and MFC experiments 90° RF pulse was used. Note that in all experiments the temperature of the sample inside the NMR probe was set to 60° C using NMR spectrometer temperature control unit. The sample transfer time in and out of the shield was approximately 2 seconds, and the sample transfer time into 9.4 T NMR spectrometer was ~5–6 seconds.

Figure 1.

Diagram of experimental setup for ¹³C hyperpolarization and NMR spectroscopic detection of 1-¹³C-phospholactate (PLAC) or 1-¹³C-phospholactate-d₂ (PLAC-d₂). The safety valve indicated as ⊘ was set to 70 psig. Adopted with permission from ref. ⁴⁹, Copyright (2018) American Chemical Society https://pubs.acs.org/doi/abs/10.1021%2Facsomega.8b00983

PLAC-d₂ hyperpolarization for in vivo experiments was performed as described previously.^{28, 69} The resulting mixture contained approximately 25 mM of HP PLAC-d₂ with %P_13C ~ 5%.

Calculation of NMR Signal Enhancement and Nuclear Spin Polarization.

¹H and ¹³C NMR signal enhancement factors (ε_1H and ε_13C, respectively) were calculated using NMR signals of thermally polarized 1-¹³C-phospholactate molecules as a reference, according to formulas:

$${\text{ε}}_{1\text{H}}=\frac{{\text{I}}_{1\text{H}-\text{HP}}}{{\text{I}}_{1\text{H}-\text{PLac}}}\times {\text{N}}_{1\text{H}-\text{PLac}}$$

$${\text{ε}}_{13\text{C}}=\frac{{\text{I}}_{13\text{C}-\text{HP}}}{{\text{I}}_{13\text{C}-\text{PLac}}}$$

where I_1H-HP, I_1H-PLac, I_13C-HP and I_13C-PLac are, respectively, ¹H and ¹³C NMR signal intensities of HP and thermally polarized PLAC after hydrogenation reaction (I_1H-HP and I_1H-PLac are signals of methyl group), N_1H-PLac is the number of protons in the methyl group of corresponding PLAC molecule (N_1H-PLac = 3 for non-deuterated PLAC and N_1H-PLac = 1 for PLAC-d₂).

Nuclear spin polarizations P_1H and P_13C were calculated using the formula

$$P=\text{ε}\times P_0$$

where P₀ is the equilibrium nuclear spin polarization of ¹H or ¹³C at the 9.4 T magnetic field and 333 K temperature (P₀ = 2.9×10^–3% for ¹H and P₀ = 7.2×10^–4% for ¹³C). Because experiments were performed with broadly varied p-H₂ fraction (66–85%), the observed polarizations were also recalculated to the highest utilized p-H₂ fraction (85%) for the sake of comparison using the formula⁴

$$P_{85\%}=P\times \frac{4\times 0.85-1}{4{\text{χ}}_{\text{p}}-1}$$

where P_85% is polarization recalculated to 85% p-H₂ fraction and χ_p is the p-H₂ fraction employed in particular experiment.

RESULTS AND DISCUSSION

Synthesis of potassium 1-¹³C-phosphoenolpyruvate-d₂ (PEP-d₂).

The overall yield (over two steps) was 57%, which represents an improvement over the previously reported 43% yield.⁶⁹ The isotopic ¹³C enrichment was ~99%. The deuterium isotopic purity was >98%, which is significantly better than the previously reported procedure, which reported ~80% deuterium isotopic purity.⁶⁹ The synthesis was repeated several times (N>3), and the yields and material purity were reproducible for both PEP-d₂ and PEP. All reported improvements can be primarily attributed to a better source of the starting material: 1-¹³C-pyruvic-d₄ acid (Isotec-Sigma-Aldrich) versus previously employed 1-¹³C-pyruvic acid (Isotec-Sigma-Aldrich 677175), which was deuterated in the first step. We note that the overall yield is consistent with the previous reports for preparation of unlabeled phosphoenolpyruvate,⁷¹ although the prior reports with the exception of Ref. # ⁶⁹ did not perform deuteration of phosphoenolpyruvate. The overall purity (~99%) of the synthesized PEP-d₂ was assessed by ³¹P NMR spectroscopy (Figure S6). It compares favorably to our previous report for PEP-d₂ of overall purity of ~96.3% (Figure S5 from Ref. # ⁶⁹).

¹H PHIP Hyperpolarization.

¹H hyperpolarization of 1-¹³C-phospholactate and 1-¹³C-phospholactate-d₂ via PASADENA and ALTADENA protocols was investigated (see Figure 2a and Figure 3a). It was found that PASADENA protocol (hydrogenation in the high magnetic field) was significantly more efficient than ALTADENA protocol (hydrogenation in the Earth’s magnetic field with subsequent transfer of the sample to the high field for detection). For example, for non-deuterated 1-¹³C-phospholactate ε_1H was ~240 in PASADENA experiment presented in Figure 2b–c, whereas in ALTADENA experiments presented in Figure 3b–c ε_1H was only ~17. Similar results were obtained for 1-¹³C-phospholactate-d₂: ε_1H in PASADENA experiment presented in Figure 2d–e was ~3 times greater than in ALTADENA experiments presented in Figure 3d–e (~640 vs. ~220). Importantly, it was found that deuteration of the PEP allows obtaining significantly higher ¹H polarization of the resultant phospholactate (P_1H ~2.5% for phospholactate-d₂ vs. P_1H ~0.71% for non-deuterated phospholactate at 85% p-H₂ fraction and in the case of PASADENA experimental protocol, see Table S1).

Figure 2.

(a) Scheme of phosphoenolpyruvate hydrogenation with p-H₂ producing HP 1-¹³C-phospholactate with optional deuterium labeling (denoted as Z = H or D). (b-c) ¹H NMR spectra acquired in PASADENA hyperpolarization of non-deuterated 1-¹³C-phospholactate (b) immediately after termination of p-H₂ bubbling and (c) after relaxation of hyperpolarization. (d-e) ¹H NMR spectra acquired in PASADENA hyperpolarization of 1-¹³C-phospholactate-d₂ (d) immediately termination of p-H₂ bubbling and (e) after relaxation of hyperpolarization. Note that spectra (c) and (e) are multiplied by a factor of 16. p-H₂ was bubbled for ~5 s at 140 sccm flow rate and 70 psig total pressure. H_A and H_B are the two spin-correlated p-H₂ derived protons.

Figure 3.

(a) Scheme of phosphoenolpyruvate hydrogenation with p-H₂ producing HP 1-¹³C-phospholactate with optional deuterium labeling (denoted as Z = H or D). (b-c) ¹H NMR spectra acquired in ALTADENA hyperpolarization of non-deuterated 1-¹³C-phospholactate (b) immediately after the placement of the sample inside the NMR probe and (c) after relaxation of hyperpolarization. (d-e) ¹H NMR spectra acquired in ALTADENA hyperpolarization of 1-¹³C-phospholactate-d₂ (d) immediately after the placement of the sample inside the NMR probe and (e) after relaxation of hyperpolarization. p-H₂ was bubbled for ~5 s at 140 sccm flow rate and 70 psig total pressure. H_A and H_B are the two spin-correlated p-H₂ derived protons.

Polarization transfer to ¹³C nucleus via Magnetic Field Cycling (MFC).

MFC experiments were performed for the transfer of polarization to ¹³C nuclei of 1-¹³C-phospholactate molecules. First, magnetic field profile of ¹³C polarization was obtained at the optimal in terms of resulting ¹H polarization duration of p-H₂ bubbling (Figure 4). It was found that the optimal magnetic field for MFC experiments is ~0.05 μT. The maximum ¹³C polarization for PLAC-d₂ was ~1.3 times higher than that for non-deuterated PLAC (P_13C ~0.10% (ε_13C ~ 104) or for phospholactate-d₂ vs. P_13C ~0.078% (ε_13C ~ 85) for non-deuterated 1-¹³C-phospholactate at 85% p-H₂ fraction, see Figure 5a (the schematic of the process), Figure 5b (the ¹³C spectrum of HP 1-¹³C-phospholactate), Figure 5c (the ¹³C spectrum of thermally polarized 1-¹³C-phospholactate after HP state decay seen in Figure 5b), Figure 5d (the ¹³C spectrum of HP 1-¹³C-phospholactate-d₂), Figure 5e (the ¹³C spectrum of thermally polarized 1-¹³C-phospholactate-d₂ after HP state decay seen in Figure 5d) and Table S2).

Figure 4.

Dependence of ¹³C polarization of 1-¹³C-phospholactate (black squares) and 1-¹³C-phospholactate-d₂ (red circles) on magnetic field (produced by the solenoid) employed in MFC experiments. ¹³C polarizations correspond to 85% p-H₂ fraction. Duration of p-H₂ bubbling was ~5 s. Note the mu-metal shield provides an isolation by approximately 1,200-fold according to the manufacturer’s specifications; therefore, the use of the shield in the Earth’s magnetic field results in the minimum residual magnetic field of approximately 40 nT, which corresponds to the zero mark on x-axis. The addition of the magnetic field (which was carefully calibrated by the gaussmeter and then attenuated by the resistor banks⁷²) via the solenoid (see Methods for details) adds or subtracts the magnetic field from the residual value (40 nT)—therefore, the maximum observed at 40–50 nT in this plot is not surprising, because it likely corresponds to the null point, where the residual field is compensated by the induced field of the solenoid.

Figure 5.

(a) Scheme of 1-¹³C-phosphoenolpyruvate hydrogenation with p-H₂ and polarization transfer via MFC producing ¹³C HP phospholactate with optional deuterium labeling (denoted as Z = H or D). (b-c) ¹H NMR spectra acquired in MFC experiments for ¹³C hyperpolarization of non-deuterated 1-¹³C-phospholactate (b) immediately after termination of p-H₂ bubbling and (c) after relaxation of hyperpolarization. (d-e) ¹H NMR spectra acquired in MFC experiments for ¹³C hyperpolarization of 1-¹³C-phospholactate-d₂ (d) immediately termination of p-H₂ bubbling and (e) after relaxation of hyperpolarization. Note that spectra (c) and (e) are multiplied by a factor of 8. p-H₂ was bubbled for ~5 s at 140 sccm flow rate and 70 psig total pressure. H_A and H_B are the two spin-correlated p-H₂ derived protons.

Spin-lattice T₁ relaxation results of hyperpolarized ¹H and ¹³C sites and its influence on the levels of obtained polarization.

The first advantage of the use of deuterated precursors for production of HP biomolecules is the reduction of the number of proton spins, which reduces the complexity of the spin system,^{69, 73} and it was found helpful for improving polarization transfer efficiency by methods relying on RF-based polarization transfer. ^{22–23, 69} The second advantage of deuteration is the usually observed increase of hyperpolarization lifetime.^{22–23, 69, 74–75} Indeed, ¹H T₁ measurements at 9.4 T magnetic field yielded T₁ = 2.06 ± 0.01 s for non-deuterated PLAC and T₁ = 4.30 ± 0.04 s for PLAC-d₂, that is ~2 times difference. In contrast, ¹³C T₁ measurements at the same field resulted in T₁ = 16.26 ± 0.04 s for non-deuterated PLAC and 12.98 ± 0.09 s for PLAC-d₂. We expect ¹H and ¹³C T₁ values to be similar at high (9.4 T) and low (the Earth’s and micro-Tesla) magnetic fields based on recently published relaxation study of metronidazole.⁷²

First, and foremost, because ¹H T₁ values are significantly lower than the sample transfer time (ca. 5–6 s), significant polarization losses occur during the sample transfer. Not surprisingly, PASADENA signal enhancements (when the sample was reacted inside the NMR spectrometer, and the sample transfer was not needed) were significantly greater than those obtained using ALTEDENA approach. The effect is more pronounced for non-deuterated HP PLAC, because it has more than 2-fold lower T₁ of HP protons than that in PLAC-d₂. Therefore, the fact that the resulting PASADENA ¹H signal enhancement for PLAC (ε_1H ~240) was significantly lower than that for PLAC-d₂ (ε_1H ~640) can be explained by greater depolarization of nascent HP protons during 5-second-long p-H₂ bubbling. Moreover, the additional time interval of ~5–6 seconds required for sample transfer caused disproportionately greater polarization losses in ALTADENA hyperpolarization of PLAC (ε_1H ~17) versus PLAC-d₂ (ε_1H ~220). Thus, generally the higher ¹H signal enhancements observed for PLAC-d₂ versus those detected in PLAC can be explained by longer lifetime of ¹H polarization.

It should be noted that in MFC experiments the lifetime of ¹H polarization is important, because hyperpolarization is initially generated on protons while the unsaturated precursor is hydrogenated in the Earth’s magnetic field. Thus, the relaxation of hyperpolarization before the step of polarization transfer to ¹³C nuclei may have significant implications on the resulting ¹³C polarization. To this extent, the maximum obtained ¹³C signal enhancement levels in PLAC (ε_13C ~ 85) and PLAC-d₂ (ε_13C ~ 104) are unexpected, because since PLAC-d₂ has significantly longer ¹H T₁ values, one would expect ε_13C to be several-fold greater in PLAC-d₂ vs. PLAC. Because ¹³C T₁ values are significantly greater than the sample transfer time (and quite similar for both PLAC and PLAC-d₂), the ¹³C polarization losses due to T₁ relaxation after polarization transfer are likely relatively small for both substrates (< 40%). When the observed PASADENA ¹H polarization levels are employed for computing the efficiency of polarization transfer from nascent protons to ¹³C, the resulting efficiencies are relatively low: 0.078%/0.71% or ~11% for PLAC and 0.1%/2.5% or ~4% for PLAC-d₂. While arguably some ¹H polarization losses have occurred during ~2-second-long sample manipulation delay (between cessation of p-H₂ bubbling and sample insertion in the magnetic shield), and they have caused the apparent reduction of polarization transfer efficiency, PLAC was likely penalized disproportionately more by these relaxation losses, because PLAC ¹H T₁ is more than a factor of two lower than that of PLAC-d₂ (and therefore reducing the apparent value of the polarization transfer efficiency more in PLAC vs. PLAC-d₂). To summarize, the polarization transfer efficiency from nascent p-H₂ protons to ¹³C nucleus by the magnetic field cycling process appears to be nearly threefold worse in the deuterated PLAC-d₂ compared to non-deuterated PLAC. We note that if the analysis of polarization losses due to T₁ relaxation is not taken into account, one may arrive to a conclusion that deuteration is helpful for the MFC efficiency, because the maximum obtained ¹³C signal enhancements were better (by a factor of ~1.3) for PLAC-d₂ versus PLAC. The latter observation is likely caused by longer proton T₁ values for PLAC-d₂ versus those for PLAC. A potential explanation of the worse polarization transfer efficiency in deuterated PLAC-d₂ is polarization transfer from p-H₂-derived protons to deuterons.⁷⁶ However, we note that the experimental results presented here had the following limitations. First and foremost, the reproducibility of the data was significantly impacted by a very short T₁ values of proton sites and relatively short T₁ values of ¹³C sites. As a result, a small variation (e.g. ~0.5–1 second for PLAC with proton T₁ of ~2 s) in the duration of the sample manipulation (especially during field cycling and hydrogenation, when the decay of polarization is governed by proton T₁ values) would have a substantial overall effect on the observed signal and polarization values, and would have significantly impacted shot-to-shot reproducibility of our data. Second, the duration of the field cycling was manually controlled, and it is estimated to be in the range between 0.5 and 1.5 seconds. This variability would also inevitably have impacted the data reproducibility for the magnetic field plots shown in Figure 4. Third, the exact values for T₁ in the low and nano-Tesla magnetic field regimes have not been determined here, but may have a significant effect on the overall polarization transfer. The last but not the least, it is very surprising that it is the deuterated sample that shows more significant polarization fluctuations with small changes in the low-field value—note that over the entire range of fields explored, the ²H Zeeman precession rate is << 1 Hz, so for a low-field residence time of < 1s, the ²H precession angle is quite small. It is therefore somewhat puzzling how the exact value of the field could affect the outcome so much. The latter question can clearly benefit from in-depth theoretical investigations and simulations, which are certainly warranted. Furthermore, it is entirely possible that the optimization of the field cycling procedure (i.e. the timing and the amplitude of the field sweep), may significantly impact the polarization transfer efficiency in deuterated versus non-deuterated compounds. Despite the above-mentioned limitations, to the best of our knowledge, this work is the first report highlighting that deuteration of the substrate maybe detrimental to polarization transfer from parahydrogen-derived protons to ¹³C nucleus in the context of magnetic field cycling methods for polarization transfer.

Outlook for contrast agent development by MFC.

The efficient relaxation processes can be a significant practical limitation for the use of HP contrast agents. In the current study, the ¹H polarization losses due to efficient relaxation during ~5 s long reaction time have caused significant reduction of polarization of nascent protons. Moreover, only a small fraction of material was converted during ~5-second-long reaction time (less than 20%, Figure S8). Both of these issues can be potentially remedied through use of high-pressure PHIP hyperpolarizers, where PHIP precursors can be reacted on the time scale significantly shorter than ¹H T₁.^{19, 24, 28–29, 34, 77–78} From the practical perspective, the use of PHIP moieties with long T₁ values will be beneficial to maximize the level of polarization through minimization of relaxation losses. Such moieties may include allyl side arm,^49–50 and others.⁴¹ While PHIP precursor deuteration is helpful in the context of RF-based methods,^{13–14, 23, 67, 69} the data presented here points that deuteration (while helpful to increase proton T₁ values) may be detrimental to the efficiency of polarization transfer via magnetic field cycling approach.²⁰

Feasibility of brain imaging with HP PLAC-d₂.

5XFAD mice were maintained at Vanderbilt University under standard conditions, in a 12-h light/dark cycle and with free access to food and water, as we described in the past.⁷⁹ The 5XFAD mice over express both mutant human APP and PS1, correlating with high burden and accelerated accumulation of the Aβ. A colony of 5XFAD transgenic mice obtained from Jackson Laboratories was maintained by crossing 5XFAD mice with a wild-type (wt) C57BL/6J strain. The mice were genotyped by a standard polymerase chain reaction using DNA isolated from tail tips with the following primers: PSEN1 forward, 5’–TCATGACTATCCTCCTGGTGG-3’ and reverse, 5’-CGTTATAGGTTTTAAACACTTCCCC-3’. For APP, forward, 5’ -AGGACTGACCACTCGACCAG-3’ and reverse, 5’-CGGGGGTCTAGTTCTGCAT-3’. We also genotyped mice for the presence of retinal degeneration Pde6brd1 mutation using forward, 5’-AAGCTAGCTGCAGTAACGCCATTT-3’ and reverse, 5’-ACCTGCATGTGAACCCAGTATTCTATC-3’. After polymerase chain reaction amplification, the DNA product of each reaction was analyzed by size fractionation through a 1% agarose gel; with Pde6b mutant = 560bp, APP transgene = 377bp and PSEN1 transgene = 608bp. The 5XFAD mice were maintained as heterozygous. Animal experiments were conducted per the guidelines established by Vanderbilt University’s Institutional Animal Care and Use Committee. At the end of the study, animals were euthanized by cervical dislocation after sedation with isoflurane. Clinical signs were used to verify euthanasia, including heartbeats and reflection to toe-pinching. Further, if animals showed signs of illness (weight loss, food withdrawal, or infection) they were sacrificed before the endpoints. All experimental procedures in this study were approved by the Vanderbilt University IACUC panel.

Mouse tail-vein catheter.

An in-house generated catheter was comprised of detached insulin needle cannula (30G) inserted into a catheter (Micro-Renathane Implantation Tubing), of which, the other end was inserted with a needle (28G) hub for filling heparin (1%)-based saline to prevent blood clotting. Intravenous (IV) insertion of the catheter on a ketamine/xylazine (0.15 mg/g/0.01 mg/g)-induced mouse was started with the insertion of the needle at either side of the lateral veins. Appropriate IV catheter preparation was evidenced and confirmed only when copious amount of blood appeared in the catheter (Figure 6a). Then, the animal was aligned on the MRI holder equipped with designated surface RF coils before MRI (Figure 6b). The holder containing the animal and ¹³C surface RF coils was placed inside a volume ¹H RF coil. HP molecular probe injection was performed via the catheter while the animal was appropriately aligned inside the magnet with heartbeat and body temperature monitoring.

Figure 6.

(a) The photograph of an anesthetized animal with tail vein catheter installed. (b) The photograph of the same animal with the ¹³C surface RF coil placed over the animal head. (c) 2D proton anatomical MRI image with slice selection via RF pulse sequences. (d) 2D ¹³C projection MRI obtained after injection of HP PLAC-d₂; the RF-pulse sequence slice selection was not employed to maximize signal-to-noise ratio (SNR). (e) Non-localized ¹³C NMR spectroscopy recorded immediately after tail-vein injection of the HP PLAC-d₂. ¹³C region selectivity over the brain in displays (d) and (e) was achieved through the use of the surface RF coil (shown in display (b)), which has detection primarily over the brain region. All images and spectra are recorded using Varian 4.7 T small-animal MRI scanner.

Animal imaging study.

Because the overall ¹³C polarization levels achieved using MFC were relatively low, the in vivo experiments proceeded with high-pressure hyperpolarizer with polarization transfer implemented with the use of RF pulses. The RF pulse sequence developed by Goldman and co-workers¹³ was employed as described previously.²⁸

The level of hyperpolarization of HP PLAC-d₂ was checked in situ of the hyperpolarizer as described previously.^{28, 69} The HP PLAC-d₂ solution was ejected in a plastic syringe and buffered for a pH with a solution of phosphate buffer. Approximately 0.2 mL of resulting HP liquid was injected via tail vein, and non-localized MR spectroscopy was recorded using approximately 15° RF excitation pulse. The MRS recorded every ~3 seconds (Figure 6e) shows initial contrast agent delivery to the brain, which is observed as initial signal growth followed by the decay of HP signal. We note that the PLAC undergoes fast dephosphorylation in vivo and becomes HP 1-¹³C-lactate as discussed previously,^{29, 68} although ¹³C chemical shift of HP PLAC-d₂ and HP ¹³C-lactate-d₂ are indistinguishable, because the chemical shift difference of ~0.3 ppm⁶⁸ could not be resolved in vivo. Only one ¹³C HP NMR resonance was detected. The overall HP dynamics of PLAC-d₂ is similar to that reported for mice previously.²⁸

2D HP ¹³C imaging was performed using gradient-echo sequence (GRE, under GEMS name on Varian platform) without slice selection. The slice selection is effectively achieved through the use of the surface RF coil, which has a limited excitation range immediately under the RF coil (see Figure 6b). The 2D ¹³C image with the highest intensity is shown in Figure 6d corresponding to the maximum determined by the MRS (in Figure 6e). Comparison with ¹H anatomical imaging recorded using the volume RF coil (Figure 6c) shows that the HP signal indeed originates from the brain rather than other parts of mouse head.

The pilot data presented in Figure 6 shows a promise of HP PLAC-d₂ for molecular imaging of brain metabolism in a manner similar to that using HP compounds produced by d-DNP.⁵⁷ The use of more advanced RF pulse sequence is certainly warranted to improve spectral, temporal and spatial resolution of HP images.^80–81

CONCLUSION

To conclude, we have reported on large–scale (>10 g) synthesis of PEP-d₂ and PEP, which serves as precursors for parahydrogen induced polarization of PLAC-d₂ and PLAC, respectively with ~57% yield and >98% deuterium purity (for PEP-d₂), which represents an improvement over the previous report. The deuteration enhances T₁ of nascent HP protons by approximately 2-fold, which is a clear advantage, because it minimizes T₁-associated-polarization losses during hyperpolarization procedure. At the same time, the deuteration of PEP leads to approximately 3 times worse ¹H→¹³C polarization transfer efficiency via magnetic field cycling, which is a disadvantage in the context of developing HP contrast agents for biomedical imaging applications, where high polarization level is required. These findings will be helpful for more rationale design of HP molecular precursors for development of HP contrast agents by PHIP especially in the context of SAH approach. The pilot use of HP PLAC-d₂ (produced using RF-based approach to enhance %P_13C to ~ 5%) is demonstrated for in vivo spectroscopy and imaging of brain metabolism in mouse model of Alzheimer’s disease.