Heterogeneous ¹H and ¹³C Parahydrogen-Induced Polarization of Acetate and Pyruvate Esters

Abstract

Magnetic resonance imaging of [1-¹³C]hyperpolarized carboxylates (most notably, [1-¹³C]pyruvate) allows one to visualize abnormal metabolism in tumors and other pathologies. Here we investigate the efficiency of ¹H and ¹³C hyperpolarization of acetate and pyruvate esters with ethyl, propyl and allyl alcoholic moieties using heterogeneous hydrogenation of corresponding vinyl, allyl and propargyl precursors in isotopically unlabeled and 1-¹³C-enriched forms with parahydrogen over Rh/TiO₂ catalysts in methanol-d₄ and in D₂O. The maximum obtained ¹H polarization was 0.6 ± 0.2% (for propyl acetate in CD₃OD), while the highest ¹³C polarization was 0.10 ± 0.03% (for ethyl acetate in CD₃OD). Hyperpolarization of acetate esters surpassed that of pyruvates, while esters with a triple carbon-carbon bond in unsaturated alcoholic moiety were less efficient as parahydrogen-induced polarization precursors than esters with a double bond. Among the compounds studied, the maximum ¹H and ¹³C NMR signal intensities were observed for propyl acetate. Ethyl acetate yielded slightly less intense NMR signals which were dramatically greater than those of other esters under study.

Graphical Abstract

Parahydrogen-induced polarization of acetate and pyruvate esters with ethyl, propyl and allyl alcoholic moieties was obtained using heterogeneous Rh/TiO₂ catalysts. ¹H and ¹³C polarizations of up to 0.6% and 0.10%, respectively, were demonstrated. Hyperpolarization of propyl and ethyl acetates was the most efficient among the esters under study.

Introduction

Hyperpolarization techniques enable dramatic increase in sensitivity of NMR spectroscopy and magnetic resonance imaging (MRI) techniques through the increase of nuclear spin polarization (P) by several orders of magnitude.^[1–6] For example, dissolution Dynamic Nuclear Polarization (d-DNP) technique^[7–10] provides nuclear spin polarization of tens % corresponding to NMR signal enhancement by 4–5 orders of magnitude at magnetic fields of several tesla. Compounds hyperpolarized by d-DNP have been successfully employed as molecular contrast agents for metabolic studies in vivo.^[11–13] For example, hyperpolarized (HP) [1-¹³C]pyruvate has demonstrated its utility as a molecular contrast agent for tumor diagnosis^[14,15] and monitoring of response to tumor treatment^[16,17] due to enhanced pyruvate metabolism in tumor tissues compared to normal tissues (Warburg effect^[18]). Moreover, HP [1-¹³C]pyruvate has been successfully employed in clinical trial MRI of prostate cancer.^[19,20] Other molecular contrast agents hyperpolarized by d-DNP are being considered, including carboxylates such as [1-¹³C]acetate^[21,22] and [1,4-¹³C₂]fumarate.^[23] [1-¹³C]acetate has attracted research interest as a potential contrast agent for metabolic investigations in brain,^[24,25] kidney,^[21,22] liver^[26] and muscle.^[27] HP [1,4-¹³C₂]fumarate showed the prospects for its application as a noninvasive marker of cellular necrosis in heart disease.^[23]

Despite the success of d-DNP, this technique has a significant drawback of highly expensive and complex hyperpolarization equipment, making its widespread use in the clinical setting challenging. Moreover, d-DNP technique requires relatively long ¹³C hyperpolarization times of ~1 h. The more affordable alternative is Parahydrogen-Induced Polarization (PHIP) technique which employs nuclear spin order of parahydrogen (p-H₂) molecule as a source of hyperpolarization.^[1,28,29] In PHIP, p-H₂ is added to an asymmetric unsaturated substrate in a pairwise manner, which means that two atoms from the same p-H₂ molecule end up in the same reaction product molecule. ¹H hyperpolarization in solution usually relaxes with spin-lattice relaxation time T₁ of several seconds. To prolong the hyperpolarization lifetime, polarization can be transferred from ¹H to heteronuclei such as ¹³C or ¹⁵N with T₁ values ranging from tens of seconds to tens of minutes.^[30–34] Transfer of hyperpolarization to heteronuclei can be carried out at a high magnetic field of NMR spectrometer using dedicated radiofrequency (RF) pulse sequences,^[35–40] or by magnetic field cycling (MFC) of the HP sample through microtesla magnetic fields where protons and heteronuclei of the hyperpolarized molecule come into level anti-crossing (LAC) regime.^[41–44]

A number of biologically relevant compounds have been directly hyperpolarized by PHIP technique so far, including succinate,^[45–48] phospholactate,^[48–51] and fumarate.^[52–54] Moreover, PHIP by means of side arm hydrogenation (PHIP-SAH), the approach developed by Reineri and co-workers,^[31] enables ¹³C polarization of carboxylates which cannot be hyperpolarized by PHIP directly due to the lack of corresponding unsaturated precursors. In PHIP-SAH the starting material is a ¹³C-labeled ester containing unsaturated alcoholic moiety which is hydrogenated with p-H₂. Next, polarization is transferred from ¹H to ¹³C nuclei of the reaction product either using RF pulse sequences^[40,55] or MFC.^[31,56,57] Subsequent cleavage of the formed ester via alkaline hydrolysis provides ¹³C HP carboxylate, e.g., [1-¹³C]pyruvate or [1-¹³C]acetate.

Most PHIP studies employ transition metal complexes as homogeneous hydrogenation catalysts to implement pairwise p-H₂ addition.^[58,59] These catalysts yield P of up to ~60% for the resultant HP reaction product.^[55] However, metal-based catalysts are toxic and thus should be separated from the HP compound before its administration to a subject. Reineri et al. demonstrated that the use of an immiscible with water organic solvent (e.g., chloroform) in PHIP-SAH experiments enables separation of a ¹³C HP carboxylate (which resides in aqueous phase after ester hydrolysis) and catalyst.^[31] An alternative approach is the use of heterogeneous (HET) hydrogenation catalysts which can be easily filtered out of the solution^[60,61] or can be used in a continuous vapor phase hydrogenation of an unsaturated ester with subsequent hydrolysis downstream.^[62] Moreover, HET-PHIP catalysts can be potentially recycled.^[63] Heterogeneous catalysts provide somewhat lower polarization levels compared to homogeneous catalysts due to lower contribution of pairwise hydrogen addition route to the overall hydrogenation mechanism.^[64–66] Nevertheless, the HET-PHIP catalyst development remains a hot area of research with the key goals to expand the scope of amenable substrates^[67–69] and improve polarization yields.^[66,70,71] In PHIP-SAH studies, heterogeneous Rh/TiO₂ catalyst allowed obtaining ¹³C polarization (P_13C) of 0.035% for ethyl [1-¹³C]acetate in organic solvents^[72] and P_13C = 0.011% in aqueous media.^[61] Ligand-capped Pd and Rh nanoparticles provided higher ¹³C polarization levels of up to 1.3% for ethyl [1-¹³C]acetate, but at the expense of lower chemical conversion of the reactant vinyl [1-¹³C]acetate to reaction product.^[33,73] Also heterogeneous PHIP-SAH approach was utilized to produce ¹³C HP amino acids with P_13C = 0.29% after side arm cleavage.^[74]

Recently, we presented an efficient synthetic approach for the preparation of 1-¹³C-labeled acetate and pyruvate esters containing vinyl, allyl and propargyl alcoholic moieties (Scheme 1),^[75] and investigated their use as unsaturated precursors for homogeneous PHIP-SAH studies.^[76] Here, we employed these compounds for systematic HET-PHIP-SAH investigations using Rh/TiO₂ catalysts for pairwise p-H₂ addition in methanol and in water aiming to compare the efficiency of HET-PHIP hyperpolarization of acetate and pyruvate esters with different alcoholic moieties.

Scheme 1.

Structures of unsaturated esters employed in this study for HET-PHIP-SAH experiments (shown here in isotopically unlabeled form).

Results and Discussion

General remarks.

HET-PHIP-SAH experiments were performed using Rh/TiO₂ catalysts (0.97 wt.% or 20 wt.% of Rh metal) in liquid phase with methanol-d₄ or D₂O as solvents. Rh/TiO₂ catalysts were chosen because they previously demonstrated the highest polarization levels among various monometallic supported metal catalysts.^[65,77,78] The following PHIP experiments were carried out: PASADENA^[29] (hydrogenation with p-H₂ at high magnetic field), ALTADENA^[79] (hydrogenation with p-H₂ at Earth’s magnetic field with subsequent transfer of the sample to the high field), and MFC^[41] (hydrogenation with p-H₂ at Earth’s magnetic field with subsequent transfer of the sample to magnetic shield and then to the high field) for polarization transfer to ¹³C nuclei. Since PHIP experiments were performed with a broadly varied p-H₂ fraction (from 58 to 89%), the observed polarizations were recalculated to the highest utilized p-H₂ fraction of 89% (see SI). Unless otherwise specified, all reported polarization values correspond to 89% p-H₂ fraction.

Hyperpolarization of ethyl acetate by HET-PHIP-SAH.

Heterogeneous liquid-phase hydrogenation of vinyl [1-¹³C]acetate with p-H₂ over Rh/TiO₂ catalysts to form HP ethyl [1-¹³C]acetate was previously investigated by some of us.^[61,72] Here we revisited this reaction to compare the obtained polarization levels of ethyl [1-¹³C]acetate with those of other esters under study at the same experimental conditions. To this end, 0.97 wt.% Rh/TiO₂ catalyst was tested in ALTADENA polarization of ethyl [1-¹³C]acetate in methanol-d₄ (Figure 1b,c) and in D₂O (Figure 1d,e), yielding ¹H polarizations (P_1H) of 0.5 ± 0.2% and 0.12 ± 0.04%, respectively. The obtained P_1H in methanol-d₄ was greater than that in the previous study by a factor of 17.^[72] The 2.5-fold increase of polarization levels should be expected based on the higher p-H₂ fraction employed here (89% vs. 50%). The additional ~7-fold increase is probably due to the fact that in this study we employed 0.97 wt.% Rh/TiO₂ catalyst instead of 23.2 wt.% Rh/TiO₂. Catalysts with higher metal loading provide greater chemical conversion of the reactant; however, the resultant polarization levels tend to be lower.^[72,80] Here, we obtained ~40% total conversion of the reactant, although ~16–30% of the reacted vinyl acetate formed ethylene, ethane and acetic acid due to C–O bond hydrogenolysis process (Table S1). Next, MFC experiments were performed for the transfer of polarization to ¹³C nuclei of ethyl [1-¹³C]acetate. The obtained P_13C were 0.10 ± 0.03% in methanol-d₄ (Figure 1g,h) and 0.07 ± 0.02% in D₂O (Figure 1i,j)—also several times greater than in the previous studies.^[61,72]

Figure 1.

(a) Reaction scheme of hydrogenation of vinyl [1-¹³C]acetate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst. (b) ALTADENA ¹H NMR spectrum of HP ethyl [1-¹³C]acetate in CD₃OD and (c) corresponding thermal ¹H NMR spectrum (multiplied by a factor of 8). ε_1H = 168, P_1H = 0.49% (0.51% at 89% p-H₂ fraction). (d) ALTADENA ¹H NMR spectrum of HP ethyl [1-¹³C]acetate in D₂O and (e) corresponding thermal ¹H NMR spectrum. ε_1H = 35, P_1H = 0.10% (0.12% at 89% p-H₂ fraction). (f) Scheme of polarization transfer from ¹H to ¹³C nuclei in ethyl [1-¹³C]acetate. (g) ¹³C NMR spectrum of HP ethyl [1-¹³C]acetate in CD₃OD and (h) corresponding thermal ¹³C NMR spectrum (multiplied by a factor of 16). ε_13C = 124, P_13C = 0.09% (0.10% at 89% p-H₂ fraction). (i) ¹³C NMR spectrum of HP ethyl [1-¹³C]acetate in D₂O and (j) corresponding thermal ¹³C NMR spectrum (multiplied by a factor of 2, acquired with 4 signal accumulations). ε_13C = 86, P_13C = 0.06% (0.07% at 89% p-H₂ fraction).

Hyperpolarization of propyl acetate by HET-PHIP-SAH.

Next, allyl acetate was employed as a PHIP precursor to HP propyl acetate. In PASADENA experiments with 0.97 wt.% Rh/TiO₂ catalyst in methanol-d₄ P_1H = 0.3 ± 0.1% was obtained (Figure S1b,c), while ALTADENA experiments under the same conditions yielded P_1H = 0.6 ± 0.2% (Figure 2b,c). In D₂O, 0.97 wt.% Rh/TiO₂ catalyst provided lower ¹H polarization levels of 0.25 ± 0.08% and 0.28 ± 0.08% in PASADENA and ALTADENA experiments, respectively (Figure S1d,e and Figure 2d,e). 9.79 wt.% Rh/TiO₂ catalyst in D₂O provided PASADENA P_1H = 0.09% at slightly lower conversion level than in the case of 0.97 wt.% catalyst (Table S2 and Figure S2b,c). Next, we tested 20 wt.% Rh/TiO₂ catalyst for allyl acetate hydrogenation with p-H₂ in D₂O which allowed to increase conversion to 65%, although at the expense of significantly diminished polarization levels (P_1H = 0.02 ± 0.01% in PASADENA experiment, Figure S2d,e). Therefore, the use of the catalyst with higher Rh loading for hyperpolarization of propyl acetate seems impractical for future applications of HP compound. Hydrogenation of allyl acetate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst in methanol-d₄ with subsequent MFC allowed detecting ¹³C HP propyl acetate even at natural abundance of ¹³C nuclei (Figure 2g). Corresponding P_13C was estimated as 0.09 ± 0.03% using 0.74 M solution of vinyl [1-¹³C]acetate in methanol-d₄ as an external reference. Hydrogenation of allyl [1-¹³C]acetate over 0.97 wt.% Rh/TiO₂ in D₂O with MFC yielded P_13C = 0.03 ± 0.01% (Figure 2i,j). It should be noted that only ~60% of the reacted allyl acetate formed propyl acetate upon hydrogenation in both solvents, while the rest ~40% formed propylene, propane and CH₃COOH as a result of C–O bond hydrogenolysis (Table S2).

Figure 2.

(a) Reaction scheme of hydrogenation of allyl [1-¹³C]acetate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst. (b) ALTADENA ¹H NMR spectrum of unlabeled HP propyl acetate in CD₃OD and (c) corresponding thermal ¹H NMR spectrum (multiplied by a factor of 16). ε_1H = 167, P_1H = 0.49% (0.64% at 89% p-H₂ fraction). (d) ALTADENA ¹H NMR spectrum of HP propyl [1-¹³C]acetate in D₂O and (e) corresponding thermal ¹H NMR spectrum. ε_1H = 94, P_1H = 0.28% (0.28% at 89% p-H₂ fraction). (f) Scheme of polarization transfer from ¹H to ¹³C nuclei in propyl [1-¹³C]acetate. (g) ¹³C NMR spectrum of HP propyl acetate with natural abundance of ¹³C nuclei in CD₃OD (multiplied by a factor of 8). (h) Thermal ¹³C NMR spectrum of 0.74 M solution of vinyl [1-¹³C]acetate in CD₃OD used as a reference. ε_13C = 72, P_13C = 0.05% (0.09% at 89% p-H₂ fraction). (i) ¹³C NMR spectrum of HP propyl [1-¹³C]acetate in D₂O and (j) corresponding thermal ¹³C NMR spectrum (multiplied by a factor of 4). ε_13C = 44, P_13C = 0.03% (0.03% at 89% p-H₂ fraction).

Hyperpolarization of allyl acetate by HET-PHIP-SAH.

When propargyl acetate was hydrogenated with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst in methanol-d₄, formation of both allyl and propyl acetates was observed (Table S3). Also, C–O bond hydrogenolysis took place leading to formation of propylene, propane and acetic acid. As a result of lower conversion of the reactant (~17%) and selectivity of its hydrogenation to allyl acetate (~40%), only ~7% of the propargyl acetate was converted to allyl acetate after 10–20 s of H₂ bubbling through the solution (Table 1). PASADENA and ALTADENA P_1H were 0.16 ± 0.05% and 0.22 ± 0.07%, respectively (Figure S3b,c and Figure S4b,c). When propargyl [1-¹³C]acetate was hydrogenated with p-H₂ over 0.97 wt.% Rh/TiO₂ in methanol-d₄ with subsequent magnetic field cycling, P_13C of ca. 0.01% was observed for resulting allyl [1-¹³C]acetate (Figure S5). Taken together, HET-PHIP-SAH of allyl acetate in methanol-d₄ was significantly less efficient than that of ethyl and propyl acetates in terms of both polarization and conversion levels. In aqueous phase hydrogenation of propargyl acetate with p-H₂ over 0.97 wt.% Rh/TiO₂, only ~1% conversion of the reactant was obtained (Table S3). Nevertheless, it was possible to detect ¹H PASADENA and ALTADENA polarization of allyl acetate with P_1H of ca. 0.6% and ca. 0.3%, respectively (Figure S3d,e and Figure S4d,e). Because the corresponding thermal ¹H NMR signals were almost at the noise level, these estimates are not particularly accurate. While 9.79 and 20 wt.% Rh/TiO₂ catalysts provided a higher conversion of ~5% in D₂O, the resultant ¹H PASADENA signals were lower in intensity due to lower P_1H = 0.14 ± 0.05% (Figure S6). All attempts to observe ¹³C polarization of allyl acetate in D₂O using 0.97 wt.% Rh/TiO₂ and MFC were unsuccessful despite the use of the ¹³C-enriched precursor.

Table 1.

The results of HET-PHIP-SAH experiments: conversion to hydrogenated esters (calculated as the product of total conversion of the reactant and selectivity to the hydrogenated ester), polarization levels (recalculated to 89% p-H₂ fraction for the sake of comparison) and relative intensities of NMR signals.

HP ester	Solvent	Conversion to ester, %	PASADENA P1H, %	ALTADENA P1H, %	P13C, %	PASADENA signal, a.u.	ALTADENA signal, a.u.	13C signal, a.u.
Ethyl acetate	CD3OD	27 ± 11	–[a]	0.5 ± 0.2	0.10 ± 0.03	–[a]	270	150
	D2O	35 ± 14	–[a]	0.12 ± 0.04	0.07 ± 0.02	–[a]	18	21
Propyl acetate	CD3OD	34 ± 14	0.3 ± 0.1	0.6 ± 0.2	0.09 ± 0.03	67	550	170
	D2O	20 ± 6	0.25 ± 0.08	0.28 ± 0.08	0.03 ± 0.01	22	58	25
Allyl acetate	CD3OD	7 ± 3	0.16 ± 0.05	0.22 ± 0.07	0.01	5.2	11	5.6
	D2O	1	0.6[b]	0.3[b]	–[c]	3.6	3.1	–[c]
Ethyl pyruvate	CD3OD	15 ± 6	0.11 ± 0.03	0.11 ± 0.03	–[c]	4.8	22	–[c]
	D2O	no reaction
Propyl pyruvate	CD3OD	7 ± 3	0.25 ± 0.08	0.3 ± 0.1	0.01	13	19	1.0
	D2O	2 ± 1	0.21 ± 0.07	0.3 ± 0.1	–[c]	5.0	3.4	–[c]
Allyl pyruvate	CD3OD	8 ± 4	0.06 ± 0.02	0.11 ± 0.03	–[c]	4.4	4.1	–[c]
	D2O[d]	1	0.1[b]	0.08[b]	–[c]	1.0	1.0	–[c]

^[a]Not studied.

^[b]These estimations are approximate because thermal NMR signals of the reaction product were too weak for reliable integration.

^[c]The ¹³C NMR signal was not detected.

^[d]The data were obtained using 20 wt.% Rh/TiO₂ catalyst (in contrast to 0.97 wt.% Rh/TiO₂ employed in other experiments reported in this table).

Hyperpolarization of ethyl pyruvate by HET-PHIP-SAH.

Next, heterogeneous PHIP-SAH of ethyl pyruvate was investigated. Hydrogenation of vinyl pyruvate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst in methanol-d₄ provided HP ethyl pyruvate with P_1H = 0.11 ± 0.03% in both PASADENA and ALTADENA experiments (Figure S7 and Figure S8). Note that in methanol the pyruvate esters are present in two forms (ketone and hemiketal) with the prevalence of the latter. ¹H chemical shifts of alcoholic moieties of vinyl and ethyl pyruvates are similar for both forms. Therefore, the estimated P_1H corresponds to the sum of both ketone and hemiketal forms. Conversion of vinyl pyruvate to ethyl pyruvate was ~15% (Table S4). Because of the lower conversion and polarization levels for ethyl pyruvate compared to those for allyl acetate, it was not possible to detect ¹³C hyperpolarization of ethyl pyruvate at natural abundance of ¹³C nuclei in MFC experiments (¹³C-enriched precursor was not available to us due to inefficient vinyl pyruvate synthesis^[75]). Aqueous phase hydrogenation of vinyl pyruvate was also unsuccessful—no reaction product was detected when either 0.97 or 20 wt.% Rh/TiO₂ was utilized.

Hyperpolarization of propyl pyruvate by HET-PHIP-SAH.

Hydrogenation of allyl pyruvate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst in methanol-d₄ under PASADENA conditions yielded HP propyl pyruvate with P_1H = 0.25 ± 0.08% (Figure S9). In ALTADENA experiment in methanol-d₄, P_1H = 0.3 ± 0.1% was observed (Figure 3b,c). However, the selectivity of allyl pyruvate hydrogenation to propyl pyruvate was only ~22%—the rest of reacted precursor molecules converted to propylene, propanol, propane and pyruvic acid as a result of C–O bond hydrogenolysis (Table S5). Hydrogenation of allyl pyruvate over 0.97 wt.% Rh/TiO₂ catalyst in D₂O gave similar ¹H polarization levels of 0.21 ± 0.07% and 0.3 ± 0.1% in PASADENA and ALTADENA experiments, respectively (Figure S10 and Figure 3e,f). However, total conversion of the reactant was only 8–10%—taking into account the similarly low selectivity to formation of propyl pyruvate, conversion of allyl pyruvate to the hydrogenated ester was only ~2%. In spite of a low amount of propyl pyruvate formed upon hydrogenation and modest P_1H, it was possible to detect ¹³C HP propyl pyruvate in MFC experiments in methanol-d₄ with P_13C = 0.01% (Figure S11). Note that propyl and allyl pyruvates were present in two forms in both methanol-d₄ and D₂O solvents, similar to ethyl and vinyl pyruvates. P_1H values were estimated for combination of both forms of propyl pyruvate which had similar chemical shifts, while P_13C was calculated using signal of hemiketal form only because it was not possible to detect thermal ¹³C NMR signal of ketone form of propyl pyruvate.

Figure 3.

(a) Reaction scheme of hydrogenation of allyl pyruvate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst in CD₃OD. (b) ALTADENA ¹H NMR spectrum of HP propyl pyruvate in CD₃OD and (c) corresponding thermal ¹H NMR spectrum. ε_1H = 52, P_1H = 0.15% (0.30% at 89% p-H₂ fraction). (d) Reaction scheme of hydrogenation of allyl pyruvate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst in D₂O. (e) ALTADENA ¹H NMR spectrum of HP propyl pyruvate in D₂O and (f) corresponding thermal ¹H NMR spectrum. ε_1H = 57, P_1H = 0.17% (0.31% at 89% p-H₂ fraction).

Hyperpolarization of allyl pyruvate by HET-PHIP-SAH.

Hydrogenation of propargyl pyruvate with p-H₂ over 0.97 wt.% Rh/TiO₂ catalyst in methanol-d₄ produced HP allyl pyruvate with PASADENA P_1H = 0.06 ± 0.02% (Figure S12) and ALTADENA P_1H = 0.11 ± 0.03% (Figure S13). These polarization values were estimated for combination of both ketone and hemiketal forms of allyl pyruvate present in methanolic solution. Similar to the case of allyl and vinyl pyruvates hydrogenation, significant amount of the reacted precursor produced propylene, propane and pyruvic acid due to C–O bond hydrogenolysis (Table S6). The total conversion of propargyl pyruvate was lower than in the case of allyl pyruvate hydrogenation (~15%). Considering low activity of the Rh/TiO₂ catalyst in production of allyl pyruvate from propargyl pyruvate and low ¹H polarization, it is not unexpected that ¹³C NMR signals of HP allyl pyruvate were not detected in MFC experiments even when ¹³C-enriched precursor was utilized. The 0.97 wt.% Rh/TiO₂ catalyst showed no activity in aqueous phase hydrogenation of propargyl pyruvate—even the NMR signal enhancement provided by the use of p-H₂ did not allow to detect allyl pyruvate. However, when 20 wt.% Rh/TiO₂ was employed, PASADENA and ALTADENA signals of HP allyl pyruvate were detected with P_1H of ca. 0.1% and ca. 0.08%, respectively (Figure S14 and Figure S15). Nevertheless, conversion of propargyl pyruvate was only ~1% (Table S6).

Efficiency of HET-PHIP-SAH of acetates and pyruvates.

The obtained ¹H and ¹³C polarization levels and NMR signal intensities of HP acetate and pyruvate esters are summarized in Table 1, along with conversions of the unsaturated precursors to the desired esters. The difference in PASADENA and ALTADENA polarizations is not unexpected due to different protocols of corresponding experiments. While in ALTADENA experiments significant part of polarization is lost because of relaxation during transfer of the sample from the Earth’s magnetic field to the NMR spectrometer, in PASADENA experiments the observed NMR signal is diminished due to anti-phase multiplet structure of PASADENA line.^[81] Three important trends can be deduced from the analysis of the data presented in Table 1. First of all, the efficiency of HET-PHIP-SAH approach for hyperpolarization of acetates significantly surpassed that of pyruvates as a result of the fact that in the former cases the polarization levels, conversions and selectivities to hydrogenated ester tend to be greater than those in the latter cases. The reasons behind these observations are not clear. One can speculate that the carbonyl group in pyruvate moiety may coordinate to the metal surface, thus facilitating hydrogenolysis of C–O bond which leads to lower selectivity towards hydrogenated esters. The bulkiness of pyruvate moiety may be a reason of lower catalytic activity in case of pyruvates. The ability of pyruvates to form hemiacetals and heminal diols can also have an important effect on the catalysts’ performance in hydrogenation of these substrates with p-H₂. Nevertheless, to the best of our knowledge this is the first demonstration of HET-PHIP hyperpolarization of pyruvate esters, and further catalyst optimization can make this approach more efficient. Second, the performance of Rh/TiO₂ catalysts in hydrogenation of propargyl esters with p-H₂ was inferior to that in case of hydrogenation of esters with a C=C bond. This is in agreement with results of gas-phase hydrogenation of propyne and propylene with p-H₂ over Rh/TiO₂ catalyst.^[82] In that study the intensities of ¹H NMR signals of HP propane were higher than those of HP propylene under otherwise similar reaction conditions. Therefore, Rh/TiO₂ catalyst is apparently more efficient in hydrogenation of double bonds with p-H₂ than in hydrogenated of triple bonds. Third, HET-PHIP-SAH in methanol-d₄ was significantly more efficient than in D₂O. This is not unexpected because hydrogen solubility in methanol is greater than in water, leading to higher conversion of the reactant. As a result of the aforementioned trends, the most intense ¹H and ¹³C NMR signals among HP esters under study were obtained for propyl acetate and ethyl acetate. This observation is in contrast with results of PHIP-SAH of the same six esters using cationic complex [Rh(NBD)(dppb)]BF₄ (NBD = norbornadiene, dppb = 1,4-bis(diphenylphosphino)butane) as homogeneous hydrogenation catalyst, where the highest hyperpolarization efficiency was demonstrated for allyl pyruvate produced by pairwise p-H₂ addition to propargyl pyruvate.^[76] In HET-PHIP with Rh/TiO₂ catalysts this ester yielded the least intense NMR signals; however, it is possible that other heterogeneous hydrogenation catalysts can be more efficient for hyperpolarization of this compound.

Conclusion

To conclude, we systematically investigated hyperpolarization of acetate and pyruvate esters with ethyl, propyl and allyl alcoholic moieties produced by heterogeneous hydrogenation of the corresponding unsaturated vinyl, allyl and propargyl precursors with p-H₂ over Rh/TiO₂ catalysts in methanol-d₄ and D₂O solvents. ¹H polarization levels of up to 0.6 ± 0.2% were demonstrated in ALTADENA experiments, while magnetic field cycling allowed to hyperpolarize ¹³C nuclei with polarization of up to 0.10 ± 0.03%. The hydrogenation of corresponding unsaturated precursors was accompanied by hydrogenolysis of the C–O bond leading to formation of alkene and alkane hydrocarbons from side arm alcoholic moiety and carboxylic acids from carboxylic moiety. As a result, the efficiency of conversion of unsaturated precursors to the hydrogenated esters was diminished. The most intense ¹H and ¹³C NMR signals in both methanol-d₄ and D₂O solvents were obtained for HP propyl acetate. Hyperpolarization of acetate esters was superior to that of pyruvates, while hydrogenation of the C=C bonds in vinyl and allyl moieties with p-H₂ was more efficient than hydrogenation of the C≡C bond in propargyl moiety. Further optimization of reaction conditions and catalyst nature may increase the efficiency of heterogeneous PHIP-SAH approach for the preparation of HP biocompatible compounds for possible MRI applications.

Experimental Section

Materials.

Commercially available methanol-d₄, D₂O (Sigma-Aldrich-Isotec) and ultra-high pure (UHP) hydrogen (>99.999%) were used as received. Unsaturated precursors (vinyl acetate, allyl acetate, propargyl acetate, vinyl pyruvate, allyl pyruvate and propargyl pyruvate, see Scheme 1) were synthesized according to previously reported procedures.^[75] These compounds were employed in both unlabeled and 1-¹³C-labeled (~98% ¹³C enrichment in the carboxyl group) forms, with the exception of vinyl esters (¹³C-enriched vinyl acetate and unlabeled vinyl pyruvate were used). The catalysts preparation procedure is described in the Supporting Information (SI).

PHIP experiments.

p-H₂ enrichment was achieved using custom-built ParaSun p-H₂ generator based on cryocooler module (SunPower, P/N 100490, CryoTel GT; a detailed description of the generator was published elsewhere^[75,83]). The generator was operated at 40–68 K, resulting in ca. 89–58% p-H₂ enrichment. For sample preparation, 20 mg of Rh/TiO₂ catalyst was placed at the bottom of a medium-wall 5 mm NMR tube (Wilmad glass P/N 503-PS-9) tightly connected with ¼ in. outer diameter PTFE tube. Next, 0.5 mL of 80 mM substrate solution in deuterated solvent was added.

The scheme of experimental setup is presented in Figure S16. The samples were pressurized up to 70 psig and preheated up to 55 °C (for CD₃OD samples) or to 65 °C (for D₂O samples) using NMR spectrometer temperature control unit. Hydrogen gas flow rate (140 standard cubic centimeters per minute, sccm) was regulated with a mass flow controller (SmartTrak 50, Sierra Instruments, Monterey, CA); duration of p-H₂ bubbling can be found in SI, Tables S1–S6. In PASADENA^[29] experiments the samples were located inside the probe of the NMR spectrometer. In ALTADENA^[79] experiments the samples were located at the Earth’s magnetic field during p-H₂ bubbling (in some experiments they were additionally heated to 80–85 °C in a beaker with hot water). After termination of p-H₂ flow, the samples were transferred to the NMR spectrometer for detection. MFC procedure was similar to ALTADENA but in this case the samples were placed inside the MuMETAL magnetic shield after cessation of p-H₂ flow, and then slowly (~1 s) pulled out of the shield and quickly placed inside the probe of the NMR spectrometer. The total sample transfer time in this case was ~8 s after the termination of H₂ gas bubbling. The magnetic field inside the shield was adjusted using additional solenoid placed inside the previously degaussed three-layered MuMETAL shield (Magnetic Shield Corp., Bensenville, IL, P/N ZG-206). This previously calibrated solenoid was powered by a direct current (DC) power supply (GW-Instek, GPR-30600), and the DC current was attenuated by a resistor bank (Global Specialties, RDB-10) to achieve the desired magnetic field inside the MuMETAL shield. The MuMETAL shield provides an isolation of approximately 1200 according to the manufacturer’s specification; therefore, the use of the shield in the Earth’s magnetic field results in the maximum residual magnetic field of ca. 40 nT. The magnetic fields employed for MFC are presented in Table S7.

NMR spectra were acquired on a 9.4 T Bruker NMR spectrometer using π/4 RF pulse for PASADENA experiments and π/2 RF pulse for ALTADENA and MFC experiments. The ¹H ALTADENA and ¹³C PHIP spectra were recorded as pseudo-two-dimensional (2D) sets consisting of 64 1D NMR spectra (acquisition time 1 s) to avoid delays between placing the sample into the probe and starting the acquisition. The acquisition of these pseudo 2D data sets was always initiated before the sample was placed inside the NMR probe.