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

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

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

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

Over 20% ¹⁵N Hyperpolarization in Under One Minute for Metronidazole, an Antibiotic and Hypoxia Probe

Direct NMR hyperpolarization of naturally abundant ¹⁵N sites in metro­nid­azole is demonstrated using SABRE-SHEATH (Signal Amplification by Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei). In only a few tens of seconds, nuclear spin polarization <i>P</i>_¹⁵N of up to ∼24% is achieved using parahydrogen with 80% <i>para</i> fraction corresponding to <i>P</i>_¹⁵N ≈ 32% if ∼100% parahydrogen were employed (which would translate to a signal enhancement of ∼0.1-million-fold at 9.4 T). In addition to this demonstration on the directly binding ¹⁵N site (using <i>J</i>²_H‑¹⁵N), we also hyperpolarized more distant ¹⁵N sites in metro­nid­azole using longer-range spin–spin couplings (<i>J</i>⁴_H‑¹⁵N and <i>J</i>⁵_H‑¹⁵N). Taken together, these results significantly expand the range of molecular structures and sites amenable to hyperpolarization via low-cost parahydrogen-based methods. In particular, hyperpolarized nitro­imid­azole and its derivatives have powerful potential applications such as direct <i>in vivo</i> imaging of mechanisms of action or hypoxia sensing

NMR SLIC Sensing of Hydrogenation Reactions Using Parahydrogen in Low Magnetic Fields

Parahydrogen-induced polarization (PHIP) is an NMR hyperpolarization technique that increases nuclear spin polarization by orders of magnitude, and it is particularly well-suited to study hydrogenation reactions. However, the use of high-field NMR spectroscopy is not always possible, especially in the context of potential industrial-scale reactor applications. On the other hand, the direct low-field NMR detection of reaction products with enhanced nuclear spin polarization is challenging due to near complete signal cancellation from nascent parahydrogen protons. We show that hydrogenation products prepared by PHIP can be irradiated with weak (on the order of spin–spin couplings of a few hertz) alternating magnetic field (called Spin-Lock Induced Crossing or SLIC) and consequently efficiently detected at low magnetic field (e.g., 0.05 T used here) using examples of several types of organic molecules containing a vinyl moiety. The detected hyperpolarized signals from several reaction products at tens of millimolar concentrations were enhanced by 10000-fold, producing NMR signals an order of magnitude greater than the background signal from protonated solvents

Facile Removal of Homogeneous SABRE Catalysts for Purifying Hyperpolarized Metronidazole, a Potential Hypoxia Sensor

Here, we report a simple and effective method to remove IrIMes homogeneous polarization transfer catalysts from solutions where NMR signal amplification by reversible exchange (SABRE) has been performed, while leaving intact the substrate’s hyperpolarized state. Following microtesla SABRE hyperpolarization of ¹⁵N spins in metronidazole, addition of SiO₂ microparticles functionalized with 3-mercaptopropyl or 2-mercaptoethyl ethyl sulfide moieties provides removal of the catalyst from solution well within the hyperpolarization decay time at 0.3 T (<i>T</i>₁ > 3 min) and enabling transfer to 9.4 T for detection of enhanced ¹⁵N signals in the absence of catalyst within the NMR detection region. Successful catalyst removal from solution is supported by the inability to “rehyperpolarize” ¹⁵N spins in subsequent attempts, as well as by ¹H NMR and inductively coupled plasma mass spectrometry. Record-high ¹⁵N nuclear polarization of up to ∼34% was achieved, corresponding to >100 000-fold enhancement at 9.4 T (or >320,000-fold enhancement at 3.0 T), and approximately 5/6th of the ¹⁵N hyperpolarization is retained after ∼20 s long purification procedure. Taken together, these results help pave the way for future studies, involving in vivo molecular imaging using agents hyperpolarized via rapid and inexpensive parahydrogen-based methods

¹⁵N Hyperpolarization by Reversible Exchange Using SABRE-SHEATH

NMR signal amplification by reversible exchange (SABRE) is a NMR hyperpolarization technique that enables nuclear spin polarization enhancement of molecules via concurrent chemical exchange of a target substrate and parahydrogen (the source of spin order) on an iridium catalyst. Recently, we demonstrated that conducting SABRE in microtesla fields provided by a magnetic shield enables up to 10% ¹⁵N-polarization (Theis, T.; et al. <i>J. Am. Chem. Soc.</i> <b>2015</b>, <i>137</i>, 1404). Hyperpolarization on ¹⁵N (and heteronuclei in general) may be advantageous because of the long-lived nature of the hyperpolarization on ¹⁵N relative to the short-lived hyperpolarization of protons conventionally hyperpolarized by SABRE, in addition to wider chemical shift dispersion and absence of background signal. Here we show that these unprecedented polarization levels enable ¹⁵N magnetic resonance imaging. We also present a theoretical model for the hyperpolarization transfer to heteronuclei, and detail key parameters that should be optimized for efficient ¹⁵N-hyperpolarization. The effects of parahydrogen pressure, flow rate, sample temperature, catalyst-to-substrate ratio, relaxation time (<i>T</i>₁), and reversible oxygen quenching are studied on a test system of ¹⁵N-pyridine in methanol-<i>d</i>₄. Moreover, we demonstrate the first proof-of-principle ¹³C-hyperpolarization using this method. This simple hyperpolarization scheme only requires access to parahydrogen and a magnetic shield, and it provides large enough signal gains to enable one of the first ¹⁵N images (2 × 2 mm² resolution). Importantly, this method enables hyperpolarization of molecular sites with NMR <i>T</i>₁ relaxation times suitable for biomedical imaging and spectroscopy

Rational Design of Novel Pyridinol-Fused Ring Acetaminophen Analogues

Acetaminophen (ApAP) is an electron donor capable of reducing radicals generated by redox cycling of hemeproteins. It acts on the prostaglandin H synthases (cyclooxygenases; COXs) to reduce the protoporphyrin radical cation in the peroxidase site of the enzyme, thus preventing the intramolecular electron transfer that generates the Tyr385 radical required for abstraction of a hydrogen from arachidonic acid to initiate prostaglandin synthesis. Unrelated to this pharmacological action, metabolism of ApAP by CYPs yields an iminoquinone electrophile that is responsible for the hepatotoxicity, which results from high doses of the drug. We synthesized novel heterocyclic phenols predicted to be electron donors. Two of these inhibited the oxygenation of arachidonic acid by PGHS-1 and myoglobin and also were shown to be more metabolically stable and exhibited less direct cytotoxicity than acetaminophen. They are leading candidates for studies to determine whether they are free of the metabolism-based hepatotoxicity produced by acetaminophen

Long-Lived ¹³C₂ Nuclear Spin States Hyperpolarized by Parahydrogen in Reversible Exchange at Microtesla Fields

Parahydrogen is an inexpensive and readily available source of hyperpolarization used to enhance magnetic resonance signals by up to four orders of magnitude above thermal signals obtained at ∼10 T. A significant challenge for applications is fast signal decay after hyperpolarization. Here we use parahydrogen-based polarization transfer catalysis at microtesla fields (first introduced as SABRE-SHEATH) to hyperpolarize ¹³C₂ spin pairs and find decay time constants of 12 s for magnetization at 0.3 mT, which are extended to 2 min at that same field, when long-lived singlet states are hyperpolarized instead. Enhancements over thermal at 8.5 T are between 30 and 170 fold (0.02 to 0.12% polarization). We control the spin dynamics of polarization transfer by choice of microtesla field, allowing for deliberate hyperpolarization of either magnetization or long-lived singlet states. Density functional theory calculations and experimental evidence identify two energetically close mechanisms for polarization transfer: First, a model that involves direct binding of the ¹³C₂ pair to the polarization transfer catalyst and, second, a model transferring polarization through auxiliary protons in substrates