State-of-the-art accounts of hyperpolarized 15N-labeled molecular imaging probes for magnetic resonance spectroscopy and imaging

Hyperpolarized isotope-labeled agents have significantly advanced nuclear magnetic resonance spectroscopy and imaging (MRS/MRI) of physicochemical activities at molecular levels. An emerging advance in this area is exciting developments of 15N-labeled hyperpolarized MR agents to enable acquisition of highly valuable information that was previously inaccessible and expand the applications of MRS/MRI beyond commonly studied 13C nuclei. This review will present recent developments of these hyperpolarized 15N-labeled molecular imaging probes, ranging from endogenous and drug molecules, and chemical sensors, to various 15N-tagged biomolecules. Through these examples, this review will provide insights into the target selection and probe design rationale and inherent challenges of HP imaging in hopes of facilitating future developments of 15N-based biomedical imaging agents and their applications.


Magnetic resonance spectroscopy and imaginggeneral information and limitations
Magnetic resonance spectroscopy and imaging (MRS/MRI) are powerful non-invasive molecular imaging modalities that provide biochemical and anatomical information about the human body. MR imaging mainly concerns the generation of anatomical images translated into spatial maps to distinguish healthy tissues from diseased areas. 1 MR spectroscopy, performed along with MRI, analyzes chemical processes and metabolic contents of the scanned tissue. MRS offers qualitative and quantitative assessments of various MR-active nuclei (i.e., 1 H, 13 C, 15 N, and 31 P) in metabolites using chemical shi assignments in the NMR spectra. 2,3 Therefore, MRI and MRS have been routinely used in research and clinical practices as imperative diagnostic techniques that offer valuable biochemical and anatomical information. Despite these advancements, magnetic resonance spectroscopic technologies suffer from low sensitivity and clinical MRS/MRI are restricted to the most abundant proton ( 1 H) resonances as the signal source.
All MR scans are evolved from nuclear magnetic resonance (NMR) and the imaging sensitivity mainly relies on the abundance and polarization levels of nuclear spins. As thermal polarization levels of nuclear spins are small, traditional 1 H-MRI detects highly abundant 1 H signals in the form of water and fat to provide sufficient sensitivity. Yet scanning other MRactive nuclei found in biomolecules is challenging due to their low natural abundances. For example, carbon and nitrogen are among the most common elements found in the structures of biomolecules. The natural abundance for 13 C and 15 N is only 1.1% and 0.37%, respectively, in comparison to 99.99% for 1 H (Table 1). 4 Other factors related to the MR signal intensity are the gyromagnetic ratio (g) and concentration of the nuclei of interest. The g value directly correlates with the NMR signal sensitivity; for instance, 13 C has a low gyromagnetic ratio, which is less than 1/4 of g( 1 H), and therefore has a lower relative sensitivity to 1 H. Furthermore, the ultra-low g of 15 N (1/10 of g( 1 H)) translates into a signicant decrease in sensitivity.
In addition to the low g values, the sensitivity of isotopeenriched metabolites may suffer from the low concentration (sub-millimolar) of the interrogated metabolic species in vivo. 5 Accordingly, it is challenging to observe these isotope signals, especially those of 15 N nuclei, from biomolecules in vivo with the conventional MRS/MRI. Yet, several hyperpolarization techniques have emerged to tackle the challenge of MR sensitivity.

Hyperpolarization technique and current methods
The sensitivity of MR correlates with nuclear-spin polarization. The NMR signal intensity is governed by the population difference between two nuclear spin states, also referred to as the polarization level. The polarization is affected by the gyromagnetic ratio (g) and the magnetic eld strength (B 0 ). At thermal equilibrium, polarization levels of MR-active nuclei are low (only 10 À6 to 10 À4 ), which is the reason for the low sensitivity of MRI/MRS. 6 The hyperpolarization (HP) technique addresses the sensitivity problem and has revolutionized the eld of MR spectroscopy and imaging. Hyperpolarization articially induces a nonequilibrium polarization of nuclear spins for a period of time (Fig. 1). The HP technique can enhance signal sensitivity by several orders of magnitude by increasing the spin state population difference. 6 The dramatic signal enhancements allow real-time detection of both introduced hyperpolarized imaging agents and their metabolic products. Thus, HP-MR scans of isotope-labeled probes provide unparalleled ability to monitor complex biological processes through advantageous features of the NMR spectroscopy combined with its high structural specicity, non-invasiveness, and quantitative analysis.
Among several available hyperpolarization methods, two techniques have been used mainly for polarizing non-gaseous isotopes. The rst technique is dynamic nuclear polarization (DNP), which is currently the most clinically advanced method that has been used for in vivo hyperpolarization studies. 5,7 DNP relies on polarization transfer from electrons to the nuclei of interest dissolved in glass-forming solvents via microwave irradiation at low temperatures (1-2 K) for approximately 1-3 hours. 8 Aer polarization build-up, the frozen pellet containing a hyperpolarized imaging agent is quickly dissolved with hot water (hence dissolution-DNP, d-DNP), generating a hyperpolarized solution ready for in vivo imaging. d-DNP is the most established polarization method used for preclinical and clinical imaging, as according to its principle, any molecule of interest can be hyperpolarized in water.
The second hyperpolarization method uses para-hydrogen as the polarization transfer source. 9,10 For example, para-hydrogeninduced polarization (PHIP) can be achieved by catalytic hydrogenation of para-H 2 across unsaturated bonds (e.g., alkene or alkyne) located near the MR-active isotope. Thus, the reduction of the unsaturated bond with a concomitant break of para-H 2 symmetry enables polarization transfer from 1 H to nearby 13 C or 15 N nuclei via networks of J-coupling. 11 The PHIP method is not generally applicable as d-DNP as the substrate needs to have an unsaturated bond. Alternatively, para-H 2 can be used to deliver polarization transfer by signal amplication by reversible exchange (SABRE) through reversible binding to a metal catalyst from both para-H 2 and the substrate. [12][13][14] Thus, SABRE can hyperpolarize a broader scope of substrates than the traditional PHIP method that relies on the irreversible hydrogenation reaction. The detailed mechanisms of these hyperpolarization techniques have been described in several review papers. [15][16][17] Overall, these developments in polarization techniques have signicantly advanced simple proof-of-concept ideas of hyperpolarized MRS/MRI into clinical applications.

Development of hyperpolarized MRI/MRS agents: considering factors and current progress
Hyperpolarized imaging studies rely on molecular probes, which are isotope-enriched chemical agents used to visualize, characterize, and quantify biological processes. 18,19 These probes can be ne-tuned to characterize a specic molecular or cellular process of interest for diagnostic or therapeutic applications. Developing an effective hyperpolarized molecular imaging probe is challenging, particularly in addressing several important considerations that are specic to hyperpolarized imaging. First, the labeled nuclei should have a long longitudinal relaxation time, denoted as T 1 . The MR signal detection window strictly depends on the T 1 value, which represents approximately 1/3 of polarization decay back to the thermal equilibrium of the spin population. Therefore, great efforts have been devoted to extending the polarized state in the hyperpolarization process and identifying isotope-labeled functional groups and centers with long T 1 lifetimes. For example, 13 C centers without directly attached protons, such as 13 C centers in carbonyl groups, benet from the decreased dipolar relaxation and commonly have longer T 1 values. 20 In addition to the dipolar contribution, the magnetic eld strength also affects the T 1 value. The magnetic eld strength, commonly measured in tesla (T), correlates with the signal-tonoise ratioa stronger magnetic eld yields stronger signals over background noise and consequently, provides a better image. Routinely used clinical MRI scanners have eld strengths of 1.5 and 3.0 T, while research MRI and laboratory NMR spectrometers commonly have eld strengths of 7.0, 11.7, and 14.1 T. Generally, the T 1 has an inverse correlation with the magnetic eld, so the higher elds result in shorter T 1 values. Second, the design of HP-MR probes should consider the difference in the chemical shi between the probe and its reaction product (i.e., metabolite). A larger chemical shi difference in the NMR spectra will provide more distinguishable peak identication and quantication, especially in lower magnetic elds (for example, typically 5-40 ppm for 13 C). 21 In the current eld of HP imaging, 13 C tracers are the most explored for studying metabolic processes, largely because carbon serves as a backbone for nearly all organic biomolecules. Several comprehensive review papers delineate hyperpolarized 13 C probes exploited for preclinical and clinical research, [20][21][22][23][24][25][26][27][28] which is beyond the scope of this review paper. The success in hyperpolarized 13 C imaging has validated the applicability of HP MRI/MRS technology in clinical settings for monitoring disease progression and therapy response. At the same time, 13 C-labeled agents oen manifest short polarization lifetimes, with T 1 values of only tens of seconds, presenting a limitation for imaging slower biological processes beyond rapid metabolic systems.
Compared to 13 C-based probes, hyperpolarized 15 N agents have proved to offer much longer T 1 lifetimes and are well suited for sensor designs. 29 This review will present current accounts of hyperpolarized 15 N-labeled biomolecular probes studied in the literature, the advantages and challenges associated with 15 N-probes, and how 15 N-agents can provide unique directions in the eld of hyperpolarized imaging. Nitrogen atoms are present ubiquitously in bioorganic molecules, and in principle, any nitrogen center can be isotopeenriched with 15 N nuclei. 30 As 15 N has a gyromagnetic ratio lower than those of 1 H and 13 C, the 15 N-NMR signal suffers from poorer sensitivity. However, the reduced interaction of 15 N with an external magnetic eld allows longer polarization lifetimes of 15 N centers in the order of several minutes. Such long polarization lifetimes expand the imaging window of the hyperpolarized species and dramatically broaden the potential applications in biomedical imaging beyond rapid metabolism tracing restricted by the shorter T 1 lifetime of 13 C metabolites.

Hyperpolarized 15 N probes
Besides potentially long hyperpolarized lifetimes of the 15 N nucleus, 15 N-NMR has a wider range of chemical shis. This warrants a greater sensitivity of 15 N chemical shi to its environment. The development of non-1 H-based MRI and MRS agents has been partially motivated by the difficulty in deconvoluting many metabolite resonances in the narrow chemical shi range of $10 ppm of the 1 H spectrum. In comparison, peaks corresponding to 13 C metabolites of interest occur over a much wider range of approximately 200 ppm. A wider range of chemical shis provides hyperpolarized 13 C-based probes with greater qualitative analysis capability to trace complex biochemical processes. In this respect, the 15 N spectrum provides an even more comprehensive range up to 900 ppm, 29 thus providing hyperpolarized 15 N probes with an even higher detection accuracy and an extended scope of chemical complexity. These favorable features of hyperpolarized 15 Nprobes offer promising biomedical and clinical imaging applications.
This review presents the up-to-date progress in the development of various 15 N agents for hyperpolarized bioimaging. The HP 15 N agents reported so far are classied into three main categories in this review: (1) 15 N-enriched endogenous molecules and drugs, (2) 15 N sensors designed for specic physiological parameters, and (3) biomolecules labeled with 15 N molecular tags. The discussion on these probes generally covers the design principles, considerations, and imaging performances in each molecular probe category.

15 N-Enriched endogenous molecules and drugs
Isotope enrichment is the most straightforward approach in designing HP agents, including 15 N-labeled endogenous metabolites and drug molecules. Ideally, HP agents should have low toxicity and high cellular uptake for in vivo imaging. Considering that the imaging agents are typically hyperpolarized ex vivo and injected intravenously into animals, high concentrations of HP agents (generally 10-100 mM) are oen needed, taking into account the dilution in the blood, to produce sufficiently detectable NMR signals in vivo. The cytotoxicity proles of endogenous metabolites and drug molecules are readily available, which expedited the in vivo applications of 15  is an endogenous molecule involved in phospholipid metabolism. Elevated metabolism of choline to phosphocholine (PCho) catalyzed by choline kinases is a known characteristic of cancer, making choline an ideal biomarker for tumor imaging. 31,32 None of the carbon centers in the natural choline molecule (CH 3 ) 3 N + CH 2 CH 2 OH would retain a long T 1 lifetime if 13 C-enriched. However, the quaternary amine is suitable for 15 N-enrichment to achieve long-lasting polarization, beneting from the absence of proton-based dipole relaxation. 15 N-Enriched choline has been hyperpolarized and studied to monitor in vitro choline metabolism to 15 N-PCho for the rst time by Gabellieri et al. (Fig. 2A). 33 The non-basic and symmetrical environment of the quaternary 15 N center in choline led to exceptionally long T 1 values of 285 AE 12 s in water and 120 AE 10 s in blood at 37 C in a magnetic eld of 11.7 T. A reduction of T 1 in the blood is a documented phenomenon and can result from the increased relaxation caused by the viscosity of blood, presence of red blood cells, and hydrogen-bonding with biomolecules. This study monitored the in vitro enzymatic conversion of hyperpolarized 15 N-Cho to 15 N-PCho, with a maximum buildup of 15 N-PCho observed at 114 s. The initial rate of 15 N-PCho buildup was estimated to be 1.45 mM min À1 using choline kinase (2 mM). Such kinetics information obtained using the plotted hyperpolarization signal over time is vital for estimating substrate buildup rates and enzyme activity. Most encouragingly, the 15 N signal remained aer 10 min, substantially exceeding the longevity compared to deuterated 13 C-choline with a T 1 of $30 s (11.8 T). 34 On the other hand, the 15 15 N-Cho build-up in a rat brain. This in vivo study has established the feasibility of detecting hyperpolarized 15 N signals in the animal model for the rst time. 35 Injection of 15 Ncholine infusate at $90 mM was tolerated without severe toxicity, although previous work has reported that MRI of choline was problematic due to its toxicity at high doses. 36 As shown in Fig. 2C, hyperpolarized 15 N-Cho provided a T 1 of 126 AE 15 s in vivo (9.4 T) and the 15 N-Cho signals were detectable well over 100 s, possibly over 300 s based on the T 1 value. However, the choline kinase activity was not observed in the animal model, possibly owing to the decreased sensitivity of the 15 N signal in vivo and slow Cho uptake.
Promising potential of hyperpolarized choline in diverse applications has also attracted efforts to improve hyperpolarization efficiency and lifetimes of 15 N-Cho, for example, by a deuteration strategy (Fig. 3). A study by Sarkar et al. showed that naturally abundant choline-d 9 with deuterated methyl groups showed a T 1 ( 15 N) of 390 AE 110 s, and 15 N-Cho showed a T 1 of 189 AE 2 s (both in D 2 O at 7 T). 37 Note that compared to 285 AE 12 s (11.7 T) in Gabellieri et al., 33 the shorter relaxation of 15 N-Cho shown in Sarkar's study was due to the addition of free radicals used for d-DNP hyperpolarization. In another study by Kumagai et al., the fully deuterated 15 N-choline-d 13 showed a T 1 of 580 AE 10 s (9.4 T). 38 Similarly, a 15 N-choline analog has been hyperpolarized using PHIP in aqueous media, achieving a T 1 of 348 AE 10 s and 494 AE 13 s for protonated and deuterated substrates, respectively (9.4 T). 39 Longer relaxation times of hyperpolarized 15 N signals in these deuterated choline analogs were rationalized by the reduced dipolar relaxation pathway with every neighboring proton (spin ¼ 1 2 ) replaced with deuterium (spin ¼ 1). These studies also demonstrate that the deuteration of nearby protons can increase the polarization lifetime of 15 N ammonium centers up to 3-fold.
2.2.2. Permethylated, perdeuterated 15 N-amino acids. 15 N-Enriched derivatives of amino acids, another type of endogenous molecules, have been studied extensively for long-lasting hyperpolarized perfusion imaging. Specically, the 15 N-  enriched amino acids can perform as tracers to study renal functions, such as ltration rates and tubular properties that are vital for diagnosing metabolic disorders.
Chiavazza et al. have rst prepared permethylated, perdeuterated derivatives of glutamine, glutamate, and lysine. 40 The design of perdeuterated glutamine compounds was based on the long relaxation time previously observed for deuterated ammonium centers that benet from the reduced dipolar interaction with neighboring protons. The a-glutamine-15 N, prepared by 15 N-enrichment of a-amine ( 15 NH 2 ) in naturally occurring amino acids, had a T 1 value of merely 8 s (14.1 T). In comparison, perdeuteromethylation of a-15 N-amine in amino acids as a strategic approach dramatically increased the T 1 values up to 220-250 s (Fig. 4A).
Durst et al. applied the amino acid derivative (CD 3 ) 3 15 N + Gln to compare the hyperpolarized imaging performances of 15 Nprobes and 13 C-urea in HP-MRI perfusion studies ( Fig. 4B and C). 41 The signal from the 15 N-glutamine analog was localized to the kidney area and detectable for more than 5 minutes. In contrast, the 13 C signal from [ 13 C, 15 N 2 ]urea was delocalized around the tissue and disappeared within 90 s (Fig. 4B). In practice, the hyperpolarized signal of 15 N had a lower SNR than that of 13 C, as the SNR correlates with the gyromagnetic ratio (Fig. 4C). However, this was offset by the slow signal decay of the 15 N-glutamine analog in the order of several minutes.
These permethylated, perdeuterated amino acid analogs had long signal retention and showed minimal toxicity in an animal model, meeting the requirements for in vivo imaging applications of hyperpolarized probes. The high T 1 values and strong localization properties make perdeuteromethylated 15 N probes promising candidates for perfusion imaging. These examples also reinforce design principles to increase T 1 ( 15 N) by reducing the dipolar interaction with neighboring protons and installing a symmetrical environment of the 15 N nucleus.
2.2.3. 15 N-Carnitine. The ideal HP properties of quaternary 15 N centers are further illustrated by 15 N-labeled L-carnitine, 42 an endogenous metabolite involved in acetyl-coenzyme A and fatty acid metabolism (Fig. 5A). The T 1 times of L-15 N-carnitined 9 were determined to be 210 s in water and 160 s in vivo (4.7 T) (Fig. 5B). Furthermore, the MR spectroscopic imaging of HP 15 N-carnitine in the rat abdomen three minutes aer injection showed 15 N signals localized in the liver and kidney area, proving the feasibility of imaging the biodistribution of an 15 Nagent for an extended period (Fig. 5C-E). However, no downstream 15 N-acetyl-carnitine metabolites were detected in this study due to magnetic isolation of the 15 N-quaternary atom.
The 15 N-labeled choline, amino acids, and carnitine studies show several benets of simple isotope-enrichment of endogenous molecules, such as ease of synthesis, high aqueous solubility, and low cytotoxicity. Nonetheless, 15 N-labeled endogenous molecules do not present detectable chemical reactions and thus cannot capture real-time physicochemical activities. Discovery of imaging agents that undergo an enzymatic or chemical reaction with signicant 15 N chemical shi differences will provide even greater analytical appeal in terms of structure determination and quantication.
2.2.4. 15 N-Azidothymidine (AZT). 15 N-Enrichment of nitrogen-containing drug molecules may offer the capability of monitoring the drug's location and metabolism by HP imaging. A good example of this category is azidothymidine (AZT), an azide-containing antiviral drug that prevents reverse transcriptase from forming viral DNA. 43 45 In this study, the singly labeled 1-15 N center was affected by the scalar relaxation with neighboring 14 N (I ¼ 1), leading to unmeasurable T 1 at 1 T. These studies exemplify the synthesis of 15 N-labeled drug molecules with the potential to monitor drug activities.
2.2.5. 15 N-Nicotinamide and 15 N-dalfampridine. Hyperpolarized 15 N-heterocycles have been explored as potential drug contrast agents. Nicotinamide, also known as vitamin B3 amide, is a drug that is used for the treatment of M. tuberculosis, HIV and cancer. 46  The feasibility of hyperpolarized 15 N-drug imaging is yet to be conrmed with in vivo studies. In addition to hyperpolarization efficiency, factors such as drug metabolism rate, cellular uptake, and cytotoxicity of the probe of interest need to be scrutinized to meet the criteria for preclinical applications.  15 N-Heteroatom bases as pH sensors. Imbalanced intracellular pH is closely related to pathological processes and is a hallmark for diseases such as cancer. 52 Developing pH sensors for effective cancer diagnosis has attracted continuous interest, including isotope-labeled hyperpolarized pH sensors. Several 13 C-pH sensors have been developed, such as [1-13 C]bicarbonate 53,54 and 13 C 2 -zymonic acid, 8 allowing for pH detection via the proton exchange of 13 C-carboxylic acids.  Alternatively, 15 N-based pH sensors have been developed for direct 15 N-protonation-based chemical shi imaging because sp 2 -hydridized, aromatic nitrogen centers can be protonated near physiological pH and cause signicant electronic changes in the 15 N atom.
Jiang et al. rst illustrated hyperpolarized 15 N-pyridine and pyridine derivatives as potential pH sensors ( Fig. 7A and B). 55 15 N-Pyridine demonstrated pH-sensitive chemical shi changes up to 90 ppm at a pH range of 2.1-8.5. Sharper chemical shi changes were observed in pH near a 15 N-pyridine pK a of 5.17, and the pH sensitivity was further altered by adding substituents to the pyridine derivatives. Yet the 15 N-pyridines suffered from a short hyperpolarization lifetime, with a T 1 value of 41 s for non-protonated 15 N-pyridine (pH 8.4) that decreased to 11 s in plasma (9.4 T). The reduced T 1 is due to an added relaxation pathway from proton exchange between the 15 N atom (H-15 N + ) and water.
A study by Shchepin et al. examined 15 N 2 -imidazole as a pH sensor by SABRE-SHEATH hyperpolarization ( Fig. 7C and D). 56 15 N 2 -Imidazole, with a pK a of $7.0, showed higher sensitivity near physiological pH than that of 15  These studies use isotope-enriched substrates because of the low natural abundance of 15 N (0.37%). Notably, high levels of 15 N polarization of naturally abundant substrates (i.e., pyridine, metronidazole and acetonitrile) up to P 15N ¼ 51% have been achieved using SABRE hyperpolarization in the presence of amines as coligands of the SABRE catalyst. Such a study will allow simple and efficient hyperpolarization of nitrogencontaining pH sensors and relevant biomolecules for 15 N-MRI. 58 2.3.2. 15 N-TMPA for detection of ROS and enzyme activity. Unlike the above-mentioned 15 N-based pH sensors designed with an all-in-one 15 N-sensing and signaling unit, the probes can be designed with a separate sensing unit and a signal unit. In this alternative design, the sensing unit surveys a biological system of interest while a remote 15 (Fig. 8B and D). Such a small chemical shi difference corresponds to a 15 N frequency of only 60 Hz at 9.4 T and even smaller at clinically relevant magnetic elds, with 19 Hz at 3 T and 9.7 Hz at 1.5 T, which would be insufficient for signal distinction. These results suggest that a 15 N chemical shi change of larger than 1.5 ppm is needed to distinguish the peaks for accurate analysis of the signals.
The excitingly long T 1 values in these 15 N-based probes signicantly broaden the HP imaging possibilities for in vivo characterization of slower biochemical reactions, such as enzymatic reactions, redox activities, and cellular signaling pathways, which would be otherwise challenging with a short signal lifetime of HP 13 C-probes.
2.3.3. 15 N-Metronidazole and 15 N-nimorazole as hypoxia sensors. 15 N-Labeled probes for hypoxia sensing have been developed as an imaging model of the tumor microenvironment. Hypoxia, a condition with inadequate oxygen supply in tissues, is a common feature in solid tumors and a diagnostic marker for therapy-resistant tumors. 60 Thus, non-invasive and reliable hyperpolarized hypoxia sensors offer valuable tools for cancer diagnosis and predicting therapy efficacy.
Nitroimidazoles have been widely used as hypoxia markers through immunohistochemistry and PET imaging. Under hypoxic conditions, the nitro group of these nitroimidazole compounds is expected to undergo sequential bioreduction to form nitroso, hydroxylamine, and amine derivatives (Fig. 9A). These hypoxia-based reactions can potentially provide signicant 15 N chemical shi changes and make 15 N-nitroimidazoles suitable candidates for MRS/MRI probes. So far, two types of 15 N-labeled nitroimidazoles have been investigated as hypoxia sensors.
Metronidazole is an FDA-approved nitroimidazole-type antibiotic drug. It can be administered safely at high doses, which well suits the use of hyperpolarized solution at high concentrations for HP-MR studies. Efficient hyperpolarization of naturally abundant metronidazole 61 as well as 15 N-enriched [ 15 N 3 ]-metronidazole 62 has been demonstrated using SABRE--SHEATH. In the work by Shchepin et al., all three 15 N sites had high polarizations of $16% and long polarization lifetimes (Fig. 9B). 62 Among the three 15 N centers, 15 NO 2 had an extraordinarily long T 1 value of 9.7 min (1.4 T), and the two aromatic 15 N-1 and 15 N-3 centers in the imidazole ring had T 1 values of 3.1 and 3.8 min, respectively. Nimorazole is another imidazole-based radiosensitizer drug for head and neck cancer. [ 15 N 3 ]-Nimorazole has also been studied as a potential HP sensor for tumor hypoxia. Salnikov et al. reported hyperpolarized [ 15 N 3 ]-nimorazole as a potential theranostic agent for dual therapy and imaging of tumor hypoxia (Fig. 9C). 63 Hyperpolarization of [ 15 N 3 ]-nimorazole using SABRE-SHEATH provided long T 1 lifetimes, especially for 15 NO 2 (5.9 min, 1.4 T). Such remarkably long-lasting polarizations open opportunities for hyperpolarized hypoxia MR imaging for over tens of minutes.
Although neither of these two studies have reported metabolic imaging of 15 N-nitroimidazoles, the ab initio calculations  revealed that the sequential hypoxic reduction processes shown in Fig. 9A were expected to provide signicant 15 N chemical shi differences, with nearly 800 ppm difference for the 15 Nnitro center. 63 Such a dynamic chemical shi range of the 15 Nsites bodes well for future in vivo imaging of nitroimidazole metabolism. One challenge is that the 15 NH 2 metabolite from hypoxic reduction would deliver a short T 1 because of the proton-coupled relaxation pathway. A possible alternative readout to monitor hypoxia is the other two sp 2 -15 N atoms that may also lead to chemical shi changes upon 15 NO 2 reduction.
While in vivo imaging has not been demonstrated in these studies, the 2D 15 N MRI visualization of [ 15 N 3 ]-metronidazole 64 and [ 15 N 3 ]-nimorazole 63 displayed high spatial and temporal resolution (Fig. 9D), highlighting the prospects of highresolution 15 N-imaging.
2.3.4. Coordination-based detection of Ca 2+ and Zn 2+ metal ions. MR probes have also been designed for sensing biologically important metal ions. Free metal ions, such as calcium and zinc, participate in essential cellular ionic signaling cascades and oxidative balance. The importance of metal ion homeostasis suggests the promise of in vivo metal ion concentrations as diagnostic markers for analyzing diseases associated with metal ion imbalance. So far, hyperpolarized sensors for metal ions have been developed by designing chelators that can coordinate to metal ions to induce electron localization and chemical shi changes.
[ 15 N, d 9 ]-TMPA has been studied as a potential sensor for calcium ions (Ca 2+ ). Calcium ions are ubiquitous signaling molecules that control various cellular functions, and abnormal Ca 2+ concentrations are responsible for several pathological processes. 65 The design of [ 15 N, d 9 ]-TMPA used (CD 3 ) 3 15 N + as the signaling unit and triacetic acid branches as the Ca 2+ chelator. Unfortunately, small 15 N chemical shi changes up to 1.5 ppm were inadequate for unambiguous Ca 2+ detection (Fig. 10A). 59 To address this limitation, another Ca 2+ sensor 15 (Fig. 10B). 15 N-Labeled sensors for Zn 2+ metals have also been reported.
Elevated cellular Zn 2+ levels are highly toxic and linked to cancer and neurodegenerative disorders. Imaging labile zinc ions as biomarkers presents a promising approach for diagnosing these diseases. 67 (Fig. 10D). These studies show versatile 15 N design principles through metal-ligand coordination-based chemical shi changes for

15 N-Molecular tags and biomolecules
An alternative to isotope enrichment, an attractive new strategy in designing HP 15 N-labeled agents, is to install biocompatible and long-lasting polarized 15 N-molecular tags onto biologically relevant molecules. Such a molecular tagging strategy can potentially introduce a 15 N-signaling moiety into any target of interest. In the probes mentioned above, long-lived 15 N signals rely on permethylated 15 N-ammonium or 15 N-heterocycles as the common 15 N-centers. In comparison, the 15 N-molecular tags can constitute various nitrogen-containing functional groups that are non-proton bound and symmetrical, selected for optimal polarization efficiency and long-lived polarization states.
2.4.1. 15 N 2 -Diazirine tags. 15 N 2 -Diazirines are one of the rst 15 N-based molecular tags explored for HP MRS/MRI. 70 Structurally, 15 N 2 -diazirines are three-membered rings containing a nitrogen-nitrogen double bond (Fig. 11). Diazirines have desirable physicochemical properties for a molecular tag, including small size, biocompatibility and stability under physiological conditions, and minimal effects on the physicochemical properties of biomolecules. 71,72 Particularly attractive for HP-MR detection, 15 N 2 -diazirines have a unique symmetrical molecular structure that stores polarization for an extended period through a singlet state. The singlet state (T s ) has a zero magnetic moment, so the symmetry has to be broken to be NMR-detectable. This also means that the singlet spin order is immune to many relaxation mechanisms and polarization is long-lived. In particular, SABRE-SHEATH hyperpolarization of 15 N 2 -diazirine-labeled compounds had a long singlet relaxation of T s ¼ 23 min. 70 Furthermore, several 15 N 2 -diazirine-labeled biomolecules have been hyperpolarized by SABRE-SHEATH 73 and d-DNP 74 methods. Examples include the 15 N 2 -diazirine tagged analogs of amino acids, glucose, and drug molecules. Hyperpolarization by d-DNP showed that all provided T 1 values in the 3-4 min range (1 T) (Fig. 11). The study showed the considerable inuence of the solubility of the 15 N-tagged molecules on their hyperpolarization efficiencies. High solubility of the hyperpolarized probes in the aqueous glassing solvent (at least 100 mM) is crucial for effective hyperpolarization of non-polar endogenous or drug molecules for practical applications.
2.4.2. 15 N 3 -Azide tags. Azides, unique linear species containing three nitrogen atoms, have been known as bioorthogonal reactive partners and possess desired features for a molecular tag. 75,76 15 N-Azides have been demonstrated as another class of 15 N-molecular tags for hyperpolarized imaging by Bae et al. 45 Triply labeled 15 N 3 -azides have been incorporated into choline, glucose, and tyrosine analogs for investigation. Hyperpolarization of all these 15 N 3 -tagged molecules by d-DNP demonstrated long lifetimes up to 9.8 min (1 T) (Fig. 12). The terminal nitrogen, 15 Ng, retained the longest HP signal, followed by 15 Nb and 15 Na, in which the long T 1 corresponds to increased distance from the nearest protons. The 15 N 3 -azide tag is especially interesting as three distinct 15 N signals can be monitored simultaneously. Additionally, the extended imaging time window opens possibilities for 15 N 3 -azide bioconjugation reaction in vivo (i.e., azide-alkyne cycloaddition) for hyperpolarized secondary labelling.
The 15 N-tagging strategy demonstrated in 15 N-azide and 15 Ndiazirine compounds will broaden the application of HP 15 N imaging beyond nitrogen centers in heteroarenes and permethylated amines. Moreover, these 15 N-tag motifs can be readily introduced into a broad range of biomolecules, allowing for preparing a variety of hyperpolarized imaging probes with a long polarization lifetime. Of note, in some examples where the 15 N-tags are generally installed several bonds away from the metabolic sites, signicant 15 N chemical shi changes may not be observed upon metabolism. Nevertheless, the 15 N-azide and 15 N-diazirine-tagged molecules are of great interest for future studies on their applications in monitoring cellular uptake and accumulation.

Summary and outlook
This review provides the current state of development of HP 15 Nprobes, including their hyperpolarization performances in relation to design principles. As an emerging molecular imaging technique, hyperpolarized 15 N MRS/MRI shows promising potential for biomedical applications. Several 15 Nlabeled endogenous and de novo molecular probes delivered long hyperpolarization lifetimes in the order of several minutes. Such substantial hyperpolarization lifetimes allow an extended imaging period to capture slower biochemical reactions that are useful for disease diagnosis. At the same time, long T 1 lifetimes of HP 15 N agents can compensate for the low sensitivity issues, as shown in the MRS/MRI scans of 15 N-amino acids and 15 Ncarnitine acquired over several minutes.
Despite recent progress and increased interest in 15 N-based imaging in the past decade, hyperpolarized 15 N MR has not gained widespread use to enter the preclinical stage. As re-ected in the analysis of currently studied HP 15 N-probes in this review, advancing 15 N MRS/MRI into a practical imaging tool requires advancements in multiple aspects such as new probe design, extensive animal imaging studies, and improved MR technology.
Fundamental considerations for the design of novel 15 Nprobes include the factors of T 1 lifetime, chemical shi differences, and toxicity. First, the discussion on the reported 15 N probes in this review reveals that the 15 N signal lifetime can be greatly extended by the probe design to reduce dipole-relaxation pathways (i.e., deuteration of neighboring protons). Compared to commonly observed 13 C carbonyl centers, the 15 N centers of the HP probes in the literature have greater structural diversity, such as quaternary amine, diazirine, and azides. All these 15 N centers warrant a long T 1 lifetime. So far, most studies have presented polarization lifetimes at high B 0 (7-11.7 T). Future work on demonstrating T 1 in clinically relevant magnetic elds (1-3 T) will be important to accurately predict the performances of HP 15 N probes in in vivo imaging. Second, accurate measurement of chemical reactions would require signicant chemical shi differences. A serviceable chemical shi difference needed for HP imaging is affected by the magnetic eld, polarization levels, and spectral resolution. Finally, the probe candidates must be biocompatible and non-toxic in living systems. The cytotoxicity proling is critical for exogenous 15 Nmolecular agents, especially at high concentrations (mM range). The current exogenous 15 N-probes solely demonstrate spectroscopic analysis, and only endogenous compounds (i.e., 15 N-choline) have advanced to in vivo MRI studies.
Extensive characterization of 15 N-labeled agents must be performed to understand the potential use of hyperpolarized 15 N imaging in clinical studies. Cellular experiments of 15 Nlabeled HP-NMR agents can provide information on the membrane permeability of probes and cellular reaction kinetics. Additionally, in vivo imaging should be conducted to validate the hyperpolarization measurements and sensitivity threshold of the 15 N probes. So far, most studies have demonstrated MRS experiments. The conjunction of MRS with MRI in small animal model imaging is desirable, which will provide not only pharmacokinetic data to quantify the rate of substrate buildup and metabolite conversion but also anatomical distribution of 15 N signals for accurate and quantitative analysis in preclinical studies.
Developing hyperpolarized 15 N imaging for preclinical studies requires addressing several technical challenges of MR scanners' technical challenges. For instance, 15 N imaging requires dedicated 15 N radiofrequency coils, which are not widely available in conventional MR scanners. 69 Parallel efforts in improving pulse sequences and multichannel coils may be crucial. Advances in hyperpolarization techniques can increase polarization efficiency and address the sensitivity issues associated with 15 N imaging.
Overall, the insights into the chemical and physical properties of 15 N-molecular probes gained through the up-to-date examples will assist in more effective designs for future hyperpolarized 15 N-based probes. Along with the advancement in MRI/MRS techniques, emerging next-generation probes are expected to foster hyperpolarized 15 N-sensors as widespread molecular imaging technology in the future.

Author contributions
Both H. P. and Q. W. contribute to the writing of this manuscript.

Conflicts of interest
There are no conicts to declare.