Detection of pyridine derivatives by SABRE hyperpolarization at zero field

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique, and its zero-field (ZULF) incarnation offers unique advantages. In ZULF NMR, the chemical shift vanishes, and NMR signals are solely determined by J-couplings, revealing detailed intramolecular interactions. This results in information-rich, chemically specific J-spectra with narrow lines, eliminating the need for cryogenic cooling and enabling the construction of portable, inexpensive spectrometers. However, ZULF NMR often suffers from low signal intensity due to the low natural abundance of certain nuclei like ¹⁵N. Hyperpolarization techniques, such as parahydrogen-induced polarization (PHIP), address this challenge by significantly enhancing NMR signal intensity. This study focuses on using SABRE, a non-hydrogenative PHIP method, to hyperpolarize naturally abundant pyridine derivatives and analyze their resulting ZULF NMR spectra. Pyridine and its derivatives are attractive targets due to their widespread occurrence in pharmaceuticals and their potential as MRI contrast agents. Although ZULF NMR and hyperpolarization methods have been individually demonstrated, their combined application for chemical identification with naturally abundant samples remained unexplored until this work. The aim is to demonstrate the ability to identify pyridine derivatives at natural isotopic abundance using ZULF NMR and SABRE hyperpolarization, and to develop an experimental setup capable of reliable, long-term measurements for weakly polarized samples.

Zero-field and ultralow-field NMR (ZULF NMR) has emerged as a complementary method to conventional high-field NMR, offering the unique advantage of directly probing spin-spin interactions through J-couplings. Previous research demonstrated high-resolution J-spectroscopy of aromatic compounds and the use of various hyperpolarization methods, such as PHIP, particularly SABRE, for signal enhancement in ZULF NMR. SABRE utilizes a reversible interaction between an iridium catalyst, a substrate (pyridine derivative), and parahydrogen (pH2) to transfer polarization from the pH2 to the substrate. Previous studies have demonstrated the potential of SABRE for hyperpolarizing pyridine and various derivatives, including those relevant to biomedicine, such as nicotinamide. The use of SABRE in conventional NMR and ZULF NMR for pyridine has been shown, but the combination of both for chemical identification of naturally abundant pyridine derivatives in zero field has been lacking. The literature shows various attempts to prolong the duration of SABRE hyperpolarization by techniques such as presaturation of the gas with methanol vapor; however, the approach in this paper uses a more efficient method using a vapor condenser to achieve the needed signal-to-noise ratio for hyperpolarized molecules at natural abundance.

The authors developed an experimental system employing a home-built nitrogen vapor condenser to maintain sample stability during prolonged measurements. This system uses a cold nitrogen vapor coolant to recondense solvent vapor, preventing evaporation and sample concentration changes. The bottom of the NMR tube is heated (~40°C) and the top cooled (near 0°C), creating a stable environment allowing for measurements lasting several hours. The study involved using two different iridium-based catalysts for SABRE: Crabtree's catalyst for initial investigations of polarization transfer efficiency, and the [IrCl(COD)(IMes)] precatalyst which provided higher polarization for natural abundance samples of pyridine derivatives. Samples were prepared by dissolving pyridine derivatives (pyridine, 3,5-dichloropyridine, nicotinamide, 3-methoxypyridine, 4-methoxypyridine, 3-methylpyridine, 4-methylpyridine) in methanol (with DMSO as a co-solvent for nicotinamide due to its low solubility). Para-hydrogen (pH2) was generated using an iron(III) oxide catalyst and bubbled through the samples. ZULF NMR spectra were acquired using an experimental setup featuring two optically-pumped magnetometers (OPMs) housed within a mu-metal magnetic shield to maintain zero-field conditions. DC magnetic field pulses were applied to initiate NMR signal acquisition. Data processing included baseline correction, signal saturation removal, and Fourier transformation. Numerical simulations were performed using the Spintrum library to complement the experimental results and verify the spectral assignments based on J-coupling constants from the literature.

The authors successfully demonstrated the ability to obtain and uniquely identify the ZULF NMR spectra of various pyridine derivatives at their natural isotopic abundance. They observed that the main peak position in zero-field spectra is determined by the strongest J-coupling between ¹⁵N and the two ortho-protons of the pyridine ring. The differences in spectral patterns are attributed to variations in spin topologies and J-coupling constants caused by the substituents and their positions. The spectra of 3,5-dichloropyridine shows a narrower line compared to pyridine due to the replacement of hydrogen atoms by chlorine atoms. The presence of substituents like amide, methoxy, and methyl groups also modifies the spectral features, providing unique fingerprints. Numerical simulations using the Spintrum library accurately reproduced the experimental spectra, supporting the interpretations of the spectral features. The study successfully detected the hyperpolarized zero-field spectrum of nicotinamide, a B3 vitamin, for the first time, at natural isotopic abundance, potentially enabling applications in ultralow-field imaging. The authors also observed no spectral peaks arising from ¹³C-isotopomers at natural abundance, which may be due to reduced hyperpolarization transfer efficiency in the presence of quadrupolar ¹⁴N nuclei and the complex spin system of ¹³C-¹⁵N isotopomers. This work demonstrates that chemical identification is feasible with samples containing 1-2 M pyridine derivatives at natural ¹⁵N abundance by measuring fewer than 200 transients, highlighting the potential of SABRE-enhanced ZULF NMR for detecting naturally abundant ¹⁵N compounds at millimolar concentration levels.

The findings demonstrate that SABRE hyperpolarization coupled with ZULF NMR is a viable technique for the identification of pyridine derivatives at natural isotopic abundance. The unique spectral signatures observed for each molecule indicate the potential of this approach for chemical analysis. The use of a nitrogen vapor condenser significantly enhances the robustness and practicality of the experimental method. The successful hyperpolarization and detection of nicotinamide opens possibilities for future applications of this approach in biomedicine. The absence of ¹³C signals from naturally abundant molecules suggests that hyperpolarization of certain nuclei might be more challenging in complex molecules. However, the ability to distinguish even subtle differences in the molecular structure opens up exciting possibilities for chemical analysis of complex mixtures. The method presented is compact, cost-effective, and offers high specificity, making it a promising technique for various applications.

This study successfully demonstrated the detection and unique identification of pyridine derivatives at their natural isotopic abundance using SABRE hyperpolarization and zero-field NMR. The development of a robust experimental setup with a vapor condenser enables long-term measurements crucial for detecting weakly polarized molecules. The results pave the way for future applications of zero-field NMR in chemical and biochemical analysis, particularly in detecting biomolecules at low concentrations without isotopic labeling. Future research may explore the application of in-situ SABRE-RELAY hyperpolarization to expand the range of detectable molecules, further improving the capabilities of zero-field NMR.

The study focuses solely on pyridine derivatives and their spectral analysis. The generalizability to other classes of molecules needs further investigation. The solubility of some compounds, particularly nicotinamide, limited the concentration used, potentially impacting the sensitivity. The signal-to-noise ratio in some spectra could be improved further to enhance the accuracy of spectral interpretation. The precise influence of the experimental conditions on the hyperpolarization efficiency requires further optimization.