Detection of pyridine derivatives by SABRE hyperpolarization at zero field

Over the past decade, zero- and ultralow-field (ZULF) NMR has emerged as a complementary method to conventional high-field NMR, enabling direct probing of spin-spin interactions at zero field where the Zeeman interaction and chemical shift vanish and signals are governed purely by J-coupling. The resulting information-rich J-spectra feature narrow lines, allowing high-resolution spectroscopy without the need for cryogenic cooling, facilitating portable, low-cost spectrometers. Because zero-field measurements lack an external field, samples must be pre-polarized. Thermal pre-polarization is universal but yields low sensitivity, whereas hyperpolarization, particularly para-hydrogen-induced polarization (PHIP) and its non-hydrogenative variant SABRE, can deliver substantial signal enhancement under ZULF conditions. SABRE uses a reversible interaction among an iridium catalyst, substrate (e.g., pyridine), and para-hydrogen to transfer spin order from pH2 to the substrate via the catalyst’s J-coupling network, releasing the molecule unchanged but highly polarized. Zero-field NMR requires heteronuclear J-coupling, so the low natural abundance of spin-1/2 nuclei (e.g., 13C, 15N, 29Si) limits signal from unlabeled samples; SABRE can mitigate this by efficiently polarizing 1H, 13C, 15N, 19F, 31P, 29Si, and others. Pyridine and derivatives are effective SABRE targets and are relevant to pharmaceuticals (e.g., nicotinic acid and nicotinamide). Prior ZULF NMR studies demonstrated chemical identification and hyperpolarization separately, but not combined for naturally abundant analytes. This work investigates naturally abundant 15N (0.36%) pyridine derivatives under ZULF with SABRE, demonstrating that derivatives differing by substituent type or position yield distinct zero-field spectra. The authors develop a long-term stable measurement system using a nitrogen vapor condenser, enabling detection at millimolar levels and in-situ monitoring of catalyst activation, and validate observations with numerical simulations, paving the way for chemical identification of unlabeled compounds using zero-field NMR.

The paper reviews: (1) ZULF NMR advantages and prior demonstrations of high-resolution J-spectroscopy and applications without strong fields; (2) the need for pre-polarization at zero field and limitations of thermal pre-polarization; (3) hyperpolarization methods with emphasis on PHIP and SABRE as well-suited to ZULF; (4) prior SABRE studies achieving high polarization for pyridine and derivatives and their biomedical relevance (e.g., nicotinamide/vitamin B3), including prior conventional-field NMR/MRI uses; (5) earlier ZULF demonstrations of chemical identification and parahydrogen enhancement, noting the gap in combining ZULF detection with SABRE on naturally abundant analytes. This context motivates the present study of SABRE-hyperpolarized, unlabeled pyridine derivatives for zero-field chemical fingerprinting.

Samples: For catalyst activation and stability monitoring, a 300 µL methanol solution containing 70 mM [15N]-pyridine (CAS 34322-45-7) and 2 mM Crabtree’s catalyst [Ir(COD)(PCy3)(Py)]PF6 (CAS 64536-78-3) was used. For naturally abundant derivatives, 25 mM [IrCl(COD)(IMes)] (air-stable SABRE precatalyst) was combined with 2 M solutions of the target pyridine derivative (pyridine, 3,5-dichloropyridine, 3-methoxypyridine, 4-methoxypyridine, 3-methylpyridine, 4-methylpyridine) in 500 µL methanol. Due to low solubility of nicotinamide, a 1 M nicotinamide solution was prepared with 20 µL DMSO and 480 µL methanol plus 25 mM precatalyst. All chemicals from Sigma-Aldrich were used without further purification. Para-hydrogen production and SABRE procedure: Para-hydrogen (44% pH2) was generated by passing H2 through Fe(III) oxide at 77 K (home-built para-hydrogen generator) into an aluminum cylinder. Precatalyst activation was first performed in Earth’s field by bubbling pH2 for a few minutes until the solution became transparent. For some experiments, full activation used a 10 min purge at 300 sccm before measurement. For SABRE at zero field, pH2 was bubbled through a capillary immersed in the sample for 6–15 s at ~80 sccm and 5 bar. After bubbling ceased, a DC magnetic-field pulse corresponding to a π/2 rotation for 1H was applied, followed by signal acquisition. Long-term stability: A home-built nitrogen vapor condenser cooled the top of the 5 mm NMR tube (≈0 °C) while the bottom of the sample was warmed to ~40 °C. This recondensed solvent carried by dry gas, enabling long-term measurements (>7.5 h for 0.5 mL). Zero-field NMR spectrometer: Detection used two QuSpin QZFM optically pumped magnetometers situated near the sample in a 3D-printed coil frame inside a compact three-layer mu-metal shield (Twinleaf MS1F) with ferrite inner layer. Absolute zero field (<0.1 nT) was achieved with shim coils; separate coils applied DC pulses. The NMR tube passed through a solenoid (not used here) and connected to the bubbling system with solenoid valves. Signals were digitized at 2000 Sa/s via an NI USB-6002 DAQ, which also orchestrated timing (valves, pulses) using LabVIEW. Data processing and simulations: Magnetometer signals were de-trended via polynomial fitting; the first 35 ms (saturated by the pulse) were removed and reconstructed by autoregressive backprediction. Exponential apodization improved SNR (10 s acquisition); spectra from both sensors were Fourier transformed, phase-corrected, and combined. Simulations computed zero-field spectra by numerical diagonalization of the zero-field Hamiltonian and calculation of magnetization along the OPM-sensitive axis using the Spintrum library. The initial density matrix after the pulse was set proportional to nuclear polarization along the sensitive direction. Literature J-couplings (Supplementary Tables 1–4) were used; single-exponential line broadening matched experiments. Experimental design choices: Initial efficiency studies used Crabtree’s catalyst; naturally abundant measurements used [IrCl(COD)(IMes)] for higher polarization transfer to pyridine derivatives. The condenser enabled consistent signal averaging over tens of minutes without solvent loss.

- Zero-field NMR spectra were acquired for SABRE-hyperpolarized, naturally abundant pyridine derivatives (pyridine, 3,5-dichloropyridine, 3-/4-methoxypyridine, 3-/4-methylpyridine, nicotinamide) without isotopic labeling, relying on 0.36% natural abundance of 15N. - Dominant peaks across derivatives appear at ~3/2·2JNH ≈ 15 Hz (e.g., 15.4–15.7 Hz), reflecting the strongest 2J coupling between 15N and two ortho 1H on the ring. - Substitution patterns uniquely modify spectra via weaker heteronuclear and homonuclear couplings, enabling chemical discrimination: • 3,5-dichloropyridine shows a single narrowed line at 15.7 Hz due to substitution of two hydrogens with self-decoupled 35/37Cl, reducing weak JNH interactions. • Nicotinamide exhibits additional features and lower SNR (limited solubility), and is the first biomolecule (vitamin B3) hyperpolarized via SABRE and detected at zero field. • Methyl-substituted derivatives show broadened main peaks due to additional methyl 1H couplings; methoxy-substituted derivatives show comparatively narrower lines due to weaker long-range couplings. - No peaks from natural-abundance 13C isotopomers were observed, attributed to reduced 13C hyperpolarization efficiency in the presence of quadrupolar 14N and complex splitting into many weak lines. - Catalyst activation dynamics were monitored in situ at zero field: signal intensity increases with pH2 exposure time; fully activated samples (10 min, 300 sccm purge) yielded roughly 2× larger amplitude and highly reproducible signals (≈2.3% standard deviation over hundreds of transients). - A nitrogen vapor condenser enabled long-term, stable measurements (0.5 mL sample measured >7.5 h at ≈0 °C condenser temperature), preventing solvent loss and enabling robust averaging. - Numerical simulations using literature J-couplings accurately reproduced experimental line positions and relative amplitudes for all derivatives, validating the spin models. - Sensitivity: Identification was demonstrated with samples containing 1–2 M unlabeled derivatives (3.6–7.2 mM 15N-active molecules) in fewer than 200 transients; the stabilized setup supports identification of naturally abundant hyperpolarized molecules down to ~1 mM levels as claimed.

Combining SABRE hyperpolarization with zero-field detection addresses the sensitivity challenge inherent to ZULF NMR’s reliance on heteronuclear couplings of low-abundance nuclei. The study shows that unlabeled pyridine derivatives, differing by substituent type or position, produce distinct zero-field J-spectra due to variations in coupling topologies and constants, enabling chemical fingerprinting without external fields. Monitoring catalyst activation directly via the zero-field signal provides a practical route to optimize polarization transfer, while the nitrogen vapor condenser ensures sample integrity over long averaging times critical for low natural-abundance detection. Observations such as line narrowing upon chlorine substitution, broadening from methyl groups, and the lack of 13C features are consistent with coupling-network arguments and prior hyperpolarization behavior in systems with quadrupolar nuclei. The validated simulations confirm the interpretability and predictability of zero-field spectra for these systems. Altogether, the results demonstrate that SABRE-enhanced ZULF NMR can support chemical identification of unlabeled compounds at low concentration, with potential for extension to biorelevant molecules (e.g., nicotinamide) and imaging applications.

The work demonstrates, for the first time, zero-field NMR detection and chemical identification of naturally abundant pyridine derivatives hyperpolarized by SABRE, with spectral uniqueness arising from substituent identity and position. A robust experimental system with a nitrogen vapor condenser enabled long-term, reproducible measurements and in-situ monitoring of catalyst activation, improving sensitivity and stability. Simulations faithfully reproduced experimental spectra, validating the theoretical approach. Future directions include implementing in-situ SABRE-RELAY at zero field to extend hyperpolarization to a broader class of molecules without labeling, advancing ZULF NMR as a specific, sensitive, portable, and cost-effective modality for chemical/biochemical analysis and potentially for fundamental physics investigations and ultralow-field imaging.

- Dependence on the low natural abundance of 15N limits signal fraction; current demonstrations used relatively high substrate concentrations (1–2 M, corresponding to 3.6–7.2 mM 15N-active molecules) and signal averaging. - Nicotinamide’s low solubility in methanol reduced SNR and required DMSO co-solvent; solubility constraints may restrict analyte scope or require solvent optimization. - Some zero-field line splittings could not be resolved, manifesting as broadened peaks (e.g., methyl-bearing derivatives), potentially complicating spectral assignment at lower SNR. - Absence of detectable natural-abundance 13C features in the presence of 14N reflects limited polarization transfer and spectral complexity for multi-heteronuclear systems. - The approach requires specialized infrastructure (pH2 generator, iridium catalysts, controlled bubbling, zero-field shielding, OPM detection) and careful control of evaporation (addressed here by a vapor condenser).