Spin–Lattice Relaxation of Hyperpolarized Metronidazole in Signal Amplification by Reversible Exchange in Micro-Tesla Fields

Simultaneous reversible chemical exchange of <i>para</i>-hydrogen and to-be-hyperpolarized substrate on metal centers enables spontaneous transfer of spin order from <i>para</i>-hydrogen singlet to nuclear spins of the substrate. When performed at a sub-micro-tesla magnetic field, this technique of NMR signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH). SABRE-SHEATH has been shown to hyperpolarize nitrogen-15 sites of a wide range of biologically interesting molecules to a high polarization level (<i>P</i> > 20%) in 1 min. Here, we report on a systematic study of ¹H, ¹³C, and ¹⁵N spin–lattice relaxation (<i>T</i>₁) of metronidazole-¹³C₂-¹⁵N₂ in the SABRE-SHEATH hyperpolarization process. In the micro-tesla range, we find that all ¹H, ¹³C, and ¹⁵N spins studied share approximately the same <i>T</i>₁ values (ca. 4 s under the conditions studied) because of mixing of their Zeeman levels, which is consistent with the model of relayed SABRE-SHEATH effect. These <i>T</i>₁ values are significantly lower than those at a higher magnetic field (i.e. the Earth’s magnetic field and above), which exceed 3 min in some cases. Moreover, these relatively short <i>T</i>₁ values observed below 1 μT limit the polarization build-up process of SABRE-SHEATH, thereby limiting the maximum attainable ¹⁵N polarization. The relatively short <i>T</i>₁ values observed below 1 μT are primarily caused by intermolecular interactions with quadrupolar iridium centers or dihydride protons of the employed polarization transfer catalyst, whereas intramolecular spin–spin interactions with ¹⁴N quadrupolar centers have a significantly smaller contribution. The presented experimental results and their analysis will be beneficial for more rational design of SABRE-SHEATH (i) polarization transfer catalysts and (ii) hyperpolarized molecular probes in the context of biomedical imaging and other applications

Proton-Only Sensing of Hyperpolarized [1,2-¹³C₂]Pyruvate

Hyperpolarized MRI is emerging as a next-generation molecular imaging modality that can detect metabolic transformations in real time deep inside tissue and organs. 13C-hyperpolarized pyruvate is the leading hyperpolarized contrast agent that can probe cellular energetics in real time. Currently, hyperpolarized MRI requires specialized “multinuclear” MRI scanners that have the ability to excite and detect 13C signals. The objective of this work is the development of an approach that works on conventional (i.e., proton-only) MRI systems while taking advantage of long-lived 13C hyperpolarization. The long-lived singlet state of [1,2-13C2]pyruvate is hyperpolarized with parahydrogen in reversible exchange, and subsequently, the polarization is transferred from the 13C2 spin pair to the methyl protons of pyruvate for detection. This polarization transfer is accomplished with spin-lock induced crossing pulses that are only applied to the methyl protons yet access the hyperpolarization stored in the 13C2 singlet state. Theory and first experimental demonstrations are provided for our method, which obviates 13C excitation and detection for proton sensing of 13C-hyperpolarized pyruvate with an overall experimental-polarization transfer efficiency of ∼22% versus a theoretically predicted polarization transfer efficiency of 25%

Parahydrogen-Induced Polarization with a Rh-Based Monodentate Ligand in Water

Reported here is a water-soluble Rh­(I)-based catalyst for performing parahydrogen-induced polarization (PHIP). The [Rh­(I)­(norbornadiene)­(THP)₂]⁺[BF₄]⁻ catalyst utilizes the monodentate phosphine ligand tris­(hydroxymethyl)­phosphine (THP). The monodentate PHIP catalyst is less susceptible to oxygenation by air and the THP ligand is significantly less expensive than bidentate water-soluble PHIP ligands. In situ PHIP detection with this monodentate Rh­(I)-based catalyst in water yielded 12% ¹³C polarization for the parahydrogen addition product, 2-hydroxyethyl 1-¹³C-propionate-d_2,3,3 (HEP), with a ¹³C <i>T</i>₁ relaxation of 108 s at 0.0475 T. PHIP polarization yields were high, reflecting efficient hydrogenation even under conditions of high content of the oxidized phosphine form of the THP ligand

Efficient Synthesis of Nicotinamide-1-¹⁵N for Ultrafast NMR Hyperpolarization Using Parahydrogen

Nicotinamide (a vitamin B₃ amide) is one of the key vitamins as well as a drug for treatment of M. tuberculosis, HIV, cancer, and other diseases. Here, an improved Zincke reaction methodology is presented allowing for straightforward and scalable synthesis of nicotinamide-1-¹⁵N with an excellent isotopic purity (98%) and good yield (55%). ¹⁵N nuclear spin label in nicotinamide-1-¹⁵N can be NMR hyperpolarized in seconds using parahydrogen gas. NMR hyperpolarization using the process of temporary conjugation between parahydrogen and to-be-hyperpolarized biomolecule on hexacoordinate iridium complex via the Signal Amplification By Reversible Exchange (SABRE) method significantly increases detection sensitivity (e.g., >20 000-fold for nicotinamide-1-¹⁵N at 9.4 T) as has been shown by Theis T. et al. (<i>J. Am. Chem. Soc.</i> <b>2015</b>, <i>137</i>, 1404), and hyperpolarized in this fashion, nicotinamide-1-¹⁵N can be potentially used to probe metabolic processes in vivo in future studies. Moreover, the presented synthetic methodology utilizes mild reaction conditions, and therefore can also be potentially applied to synthesis of a wide range of ¹⁵N-enriched N-heterocycles that can be used as hyperpolarized contrast agents for future in vivo molecular imaging studies

Delivering Robust Proton-Only Sensing of Hyperpolarized [1,2-¹³C₂]‑Pyruvate Using Broad-Spectral-Range Nuclear Magnetic Resonance Pulse Sequences

Hyperpolarized [1-13C]pyruvate is the leading hyperpolarized injectable contrast agent and is currently under evaluation in clinical trials for molecular imaging of metabolic diseases, including cardiovascular disease and cancer. One aspect limiting broad scalability of the technique is that hyperpolarized 13C MRI requires specialized 13C hardware and software that are not generally available on clinical MRI scanners, which employ proton-only detection. Here, we present an approach that uses pulse sequences to transfer 13C hyperpolarization to methyl protons for detection of the 13C–13C pyruvate singlet, employing proton-only excitation and detection only. The new pulse sequences are robust to the B1 and B0 magnetic field inhomogeneities. The work focuses on singlet-to-magnetization (S2M) and rotor-synchronized (R) pulses, both relying on trains of hard pulses with broad spectral width coverage designed to effectively transform hyperpolarized 13C2-singlet hyperpolarization to 1H polarization on the CH3 group of [1,2-13C2]pyruvate. This approach may enable a broader adoption of hyperpolarized MRI as a molecular imaging technique

Parahydrogen Induced Polarization of 1-¹³C‑Phospholactate‑<i>d</i>₂ for Biomedical Imaging with >30,000,000-fold NMR Signal Enhancement in Water

The synthetic protocol for preparation of 1-¹³C-phosphoenolpyruvate-<i>d</i>₂, precursor for parahydrogen-induced polarization (PHIP) of 1-¹³C-phospholactate-<i>d</i>₂, is reported. ¹³C nuclear spin polarization of 1-¹³C-phospholactate-<i>d</i>₂ was increased by >30,000,000-fold (5.75 mT) in water. The reported ¹³C polarization level approaching unity (>15.6%), long lifetime of ¹³C hyperpolarized 1-¹³C-phospholactate-<i>d</i>₂ (58 ± 4 s versus 36 ± 2 s for nondeuterated form at 47.5 mT), and large production quantities (52 μmoles in 3 mL) in aqueous medium make this compound useful as a potential contrast agent for the molecular imaging of metabolism and other applications

Nanoscale Catalysts for NMR Signal Enhancement by Reversible Exchange

Two types of nanoscale catalysts were created to explore NMR signal enhancement via reversible exchange (SABRE) at the interface between heterogeneous and homogeneous conditions. Nanoparticle and polymer comb variants were synthesized by covalently tethering Ir-based organometallic catalysts to support materials composed of TiO₂/PMAA (poly­(methacrylic acid)) and PVP (polyvinylpyridine), respectively, and characterized by AAS, NMR, and DLS. Following parahydrogen (pH₂) gas delivery to mixtures containing one type of “nano-SABRE” catalyst particle, a target substrate, and ethanol, up to ∼(−)­40-fold and ∼(−)­7-fold ¹H NMR signal enhancements were observed for pyridine substrates using the nanoparticle and polymer comb catalysts, respectively, following transfer to high field (9.4 T). These enhancements appear to result from intact particles and not from any catalyst molecules leaching from their supports; unlike the case with homogeneous SABRE catalysts, high-field (<i>in situ</i>) SABRE effects were generally not observed with the nanoscale catalysts. The potential for separation and reuse of such catalyst particles is also demonstrated. Taken together, these results support the potential utility of rational design at molecular, mesoscopic, and macroscopic/engineering levels for improving SABRE and HET-SABRE (heterogeneous-SABRE) for applications varying from fundamental studies of catalysis to biomedical imaging

Open-Source Automated Parahydrogen Hyperpolarizer for Molecular Imaging Using ¹³C Metabolic Contrast Agents

An open-source hyperpolarizer producing ¹³C hyperpolarized contrast agents using parahydrogen induced polarization (PHIP) for biomedical and other applications is presented. This PHIP hyperpolarizer utilizes an Arduino microcontroller in conjunction with a readily modified graphical user interface written in the open-source processing software environment to completely control the PHIP hyperpolarization process including remotely triggering an NMR spectrometer for efficient production of payloads of hyperpolarized contrast agent and <i>in situ</i> quality assurance of the produced hyperpolarization. Key advantages of this hyperpolarizer include: (i) use of open-source software and hardware seamlessly allowing for replication and further improvement as well as readily customizable integration with other NMR spectrometers or MRI scanners (i.e., this is a multiplatform design), (ii) relatively low cost and robustness, and (iii) <i>in situ</i> detection capability and complete automation. The device performance is demonstrated by production of a dose (∼2–3 mL) of hyperpolarized ¹³C-succinate with %<i>P</i>_13C ∼ 28% and 30 mM concentration and ¹³C-phospholactate at %<i>P</i>_13C ∼ 15% and 25 mM concentration in aqueous medium. These contrast agents are used for ultrafast molecular imaging and spectroscopy at 4.7 and 0.0475 T. In particular, the conversion of hyperpolarized ¹³C-phospholactate to ¹³C-lactate <i>in vivo</i> is used here to demonstrate the feasibility of ultrafast multislice ¹³C MRI after tail vein injection of hyperpolarized ¹³C-phospholactate in mice

Spin Relays Enable Efficient Long-Range Heteronuclear Signal Amplification by Reversible Exchange

A systematic experimental study is reported on the polarization transfer to distant spins, which do not directly bind to the polarization transfer complexes employed in Signal Amplification By Reversible Exchange (SABRE) experiments. Both long-range transfer to protons and long-range transfer to heteronuclei, i.e., ¹³C and ¹⁵N, are examined. Selective destruction of hyperpolarization on ¹H, ¹³C, and ¹⁵N sites is employed, followed by their rehyperpolarization from neighboring spins within the molecules of interest (pyridine for ¹H studies and metronidazole-¹⁵<i>N</i>₂-¹³<i>C</i>₂ for ¹³C and ¹⁵N studies). We conclude that long-range sites can be efficiently hyperpolarized when a network of spin-1/2 nuclei enables relayed polarization transfer (i.e., via short-range interactions between sites). In the case of proton SABRE in the millitesla regime, a relay network consisting of protons only is sufficient. However, in case ¹³C and ¹⁵N are targeted (i.e., via SABRE in SHield Enables Alignment Transfer to Heteronuclei or SABRE-SHEATH experiment), the presence of a heteronuclear network (e.g., consisting of ¹⁵N) enables a relay mechanism that is significantly more efficient than the direct transfer of spin order from para-H₂-derived hydrides

Synthesis of Unsaturated Precursors for Parahydrogen-Induced Polarization and Molecular Imaging of 1-¹³C‑Acetates and 1-¹³C‑Pyruvates via Side Arm Hydrogenation

Hyperpolarized forms of 1-¹³C-acetates and 1-¹³C-pyruvates are used as diagnostic contrast agents for molecular imaging of many diseases and disorders. Here, we report the synthetic preparation of 1-¹³C isotopically enriched and pure from solvent acetates and pyruvates derivatized with unsaturated ester moiety. The reported unsaturated precursors can be employed for NMR hyperpolarization of 1-¹³C-acetates and 1-¹³C-pyruvates via parahydrogen-induced polarization (PHIP). In this PHIP variant, Side arm hydrogenation (SAH) of unsaturated ester moiety is followed by the polarization transfer from nascent parahydrogen protons to ¹³C nucleus via magnetic field cycling procedure to achieve hyperpolarization of ¹³C nuclear spins. This work reports the synthesis of PHIP-SAH precursors: vinyl 1-¹³C-acetate (55% yield), allyl 1-¹³C-acetate (70% yield), propargyl 1-¹³C-acetate (45% yield), allyl 1-¹³C-pyruvate (60% yield), and propargyl 1-¹³C-pyruvate (35% yield). Feasibility of PHIP-SAH ¹³C hyperpolarization was verified by ¹³C NMR spectroscopy: hyperpolarized allyl 1-¹³C-pyruvate was produced from propargyl 1-¹³C-pyruvate with ¹³C polarization of ∼3.2% in CD₃OD and ∼0.7% in D₂O. ¹³C magnetic resonance imaging is demonstrated with hyperpolarized 1-¹³C-pyruvate in aqueous medium