Using hyperpolarised ¹⁵ N derived synthons to create hyperpharmaceuticals through SABRE Field of the invention The present invention relates to a method for the production of a ¹⁵ N-hyperpolarised agent and its subsequent assessment via NMR or MRI. More particularly, the present invention provides a method for magnetic resonance studies of a sample containing MRI and NMR active ¹⁵ N in a variety of species where PHIP is used to provide the initial hyperpolarisation. The initial hyperpolarisation transfer described herein is generally based on the SABRE effect. The present invention also provides an agent for magnetic resonance studies; the use of hyperpolarised ¹⁵ N species; a method of hyperpolarising a substance; a method to detect the chemical transformation of that substance through NMR or MRI apparatus. Background of the invention Positron emission tomography (PET) is a very sensitive technique that uses gamma cameras to image changes in metabolic processes, blood flow and agent absorption in the body. It takes long-lived radionuclides, output from a cyclotron, and embeds them into suitable receptors to create the radiopharmaceuticals that convey the diagnostic response. Unfortunately, this process can be complex and costly. Magnetic resonance imaging (MRI) is another powerful diagnostic method, but inherent low sensitivity means routine clinical measurements probe highly abundant water. Consequently, there has been a great deal of excitement in the clinical community associated with a method called hyperpolarisation that gives MRI the sensitivity needed to visualise changes in metabolic flux by detecting biomolecules that encode disease. The most clinically developed method in this area currently involves the use of dynamic nuclear polarisation (d-DNP). ^1-2 One alternative method to create hyperpolarisation is parahydrogen (p-H ₂ ) induced polarisation (PHIP), which despite being discovered in the 1980’s, is only now receiving worldwide attention. Two recent significant PHIP advances that utilise p-H ₂ are Signal Amplification by Reversible Exchange (SABRE) ³ and p-H ₂ induced polarisation with side-arm hydrolysis (PHIP-SAH). ⁴ As p-H ₂ can be prepared to a level of 50% purity by simply cooling H ₂ gas by liquid nitrogen, ⁵ one could imagine the wide spread future use of this sensitisation approach. The expectation is that hyperpolarised pyruvate could transform the clinical diagnosis of diseases such as cancer by tracking pyruvate metabolism and consequently the potential health benefits of this approach are significant. Pyruvate is in fact the end-product of glycolysis, it is derived from additional sources in the cellular cytoplasm, and is ultimately destined for transport into mitochondria as a fuel input in the citric acid cycle. In mitochondria, pyruvate drives ATP production by oxidative phosphorylation and multiple biosynthetic pathways intersecting the citric acid cycle. Mitochondrial pyruvate metabolism is regulated by many enzymes, including pyruvate dehydrogenase, and pyruvate carboxylase. Mutations in any of the genes encoding for proteins regulating pyruvate metabolism may lead to disease. Aberrant pyruvate metabolism plays an especially prominent role in cancer, heart failure, and neurodegeneration. Because most major diseases involve aberrant metabolism, understanding and exploiting pyruvate carbon flux may yield novel treatments that enhance human health. Therefore, pyruvate is a key molecule critical for numerous aspects of eukaryotic and human metabolism. Its ¹³ C-hyperpolarisation has been harnessed successfully in the creation of an MRI method to assess abnormalities in pyruvate metabolism that link to cancer. Unfortunately, it is difficult to see how DNP can be easily scaled up to a widely available technique due to the initial equipment cost, the amount of liquid helium it uses and the small number of samples that such devices can be output daily. While routine NMR and MRI techniques mainly detect ¹ H responses, the next most probed heteronucleus, ¹³ C, is 6400 times harder to detect than ¹ H. This is due to ¹³ C’s 1% abundance and small gyromagnetic ratio ( ^) which means the Zeeman splitting yielding the resonance frequency is four times smaller than ¹ H. Consequently, the macroscopic nuclear magnetisation detected that results from the normally very small nuclear spin-orientation population imbalance is minute. Hyperpolarisation enlarges this imbalance to create bigger signals and it is for this reason that the DNP hyperpolarisation of pyruvate is needed when it is used as a marker to probe for cancer by MRI. The most common isotope of nitrogen is ¹⁴ N, which has a nuclear spin quantum number of 1 and this makes it unsuitable for most NMR and MRI studies. Less utilised ¹⁵ N has a highly informative 1350 ppm chemical shift range and long T _1, ⁶ but its 0.36% abundant and ^ which is 10 times smaller than ¹ H combine to make it 277,000 times harder to detect than ¹ H; it can, however, be sourced cheaply in reactive materials like ¹⁵ NH ₄ Cl. Due to the poor signal levels, high concentration samples in conjunction with extensive signal averaging is needed for studies at natural abundance. ¹⁵ N NMR though is vital to the examination of proteins and its inefficient detection has driven many of the developments in NMR over the last three decades. We also note that establishing the structures of poly-nitrogen containing compounds that feature widely in drugs is often very hard to achieve due to their sparsity of hydrogen and carbon atoms. Furthermore, the in vivo imaging of such agents is expected to be diagnostic. US Patent No. 9,658,300 describes creating hyperpolarised samples of target molecular species through spin transfer from hyperpolarised xenon atoms or other source isotopes. Reversible nanoscale solid state contact is achieved between the hyperpolarised xenon atoms and molecules of a target species. The hyperpolarised target species can then be introduced into a subject of a nuclear magnetic resonance (NMR) experiment. US Patent application No. 2011/0050228 describes an agent for magnetic resonance studies comprising hyperpolarised ¹⁵ N labelled N ₂ O in solution or liquid ¹⁵ N—N ₂ O. We have now found that it is possible to turn PHIP into a versatile tool for the preparation of a family of long-lived and highly Magnetic Resonance (MR) visible precursors containing ¹⁵ N, akin to the radionuclides of PET. These reactive synthons are then rapidly embedded into important molecular reporters to illustrate the creation of the hyperpharmaceutical. This can be achieved by harnessing species like reactive nitrite (NO ₂ -), nitrate (NO ₃ -), ammonia (NH ₃ ), amines, hydroxylamine (NH ₂ OH), hydrazine (N ₂ H ₄ ), azide (N ₃ -), isocyanate (NCO-/RNCO) cyanate (OCN-/ROCN), thiocyanate (SCN-) and nitrosonium (NO ⁺ ) which alongside nitrate, dinitrogen and nitrous oxide (N ₂ O) can be transformed into useful biological probes. These species reflect some of the key chemicals involved in the life sustaining nitrogen cycle. In fact, many useful hyperpolarised materials could be prepared from this small pool of starting materials and these are reflective of many pharmaceuticals. Summary of the Invention This success in hyperpolarisation outcome represents an important breakthrough in enabling SABRE for biochemical analysis as it not only dramatically widens the range of agents it works with but improves diagnostic potential more generally. ^29-32 The success in sensitising the ¹ H, ¹⁵ N, ¹⁹ F and ¹³ C NMR profiles of these and other materials more generally marks therefore a significant breakthrough in hyperpolarisation which has implications for both NMR and particularly MRI. Thus, according to a first aspect of the invention there is provided a method for the magnetic resonance studies of a sample using an MRI active agent’s ¹⁵ N form in solution or liquid comprising: hyperpolarising a ¹⁵ N-form using SABRE; delivering the hyperpolarised ¹⁵ N form to a predetermined region of a sample; applying a magnetic field to the sample; exciting the predetermined region of the sample with an excitation pulse suitable for exciting an NMR signal from ¹⁵ N in the applied magnetic field; and acquiring magnetic resonance data associated with ¹⁵ N from the sample. Generally in the method of the present invention various PHIP approaches are used to simply hyperpolarise the MRI or NMR active ¹⁵ N forms. This means an imbalance is created in one of the two possible nuclear spin orientations (+½ or -½) of ¹⁵ N that is maintained for 10’s of minutes if placed in an appropriate magnetic field. ⁶ Furthermore, the nitrite ion, usually as sodium nitrite, finds widespread use in the chemical industries, due to its oxidizing properties and role in organic transformations. ²⁷ Hyperpolarised ¹⁵ N NMR can aid in this process as a further outcome. In this regard, nitrite, such as sodium nitrite, can be used to diazotize many precursors and reflects an important step in many named reactions such Sandmeyer, Pschorr, Gomberg– Bachmann, Balz–Schiemann, Meerwein arylation, Kikukawa–Matsuda, Suzuki- coupling, Heck reaction, Stille cross-coupling. The products of these reactions then find applications as fine chemicals, dyes and pigments, or pharmaceuticals (e.g. prosulfuron, thiazolidinedione derivatives (pioglitazone), O-anisamide and Calpain I inhibitors). Hence, using such protocols provides a transformative approach to improve drug analysis and synthesis. The azide anion is an excellent nucleophile that readily forms organic azides such as the anti-retroviral AZT. This functionality can be readily reduced to create amines, and through the Curtius rearrangement carbamates. Copper catalysed azide-alkyne cycloadditions or click reactions are also important. Consequently, azide represents an important precursor to agrochemicals, pharmaceuticals and natural products such as Avapro, Diova and Tamiflu. Normally labelled materials can be examined, and as an example, nitrite ions can be probed by ¹⁵ N NMR in solution and the solid state ⁷ and used to study chemodenitrification in humic substances ⁸ and nitric oxide release from copper (II) sites as examples. ⁹ By measuring at 9.4 T, 100% nuclear spin polarisation will improve the ¹⁵ N signal strength by 310,000 times its usual level. The sensitivity of PHIP has been used widely in the study of organic and inorganic chemistry and it has made the detection of previously hidden intermediates possible. ^10-13 To date, there is a single report of Na ¹⁵ NO ₂ hyperpolarised by dissolution dynamic nuclear polarisation (d-DNP). ¹⁴ Therefore, the aim of the present invention is to illustrate this hyperpharmaceutical concept by establishing that chemical reactivity can be tracked, and the diagnostic fingerprinting of materials can be performed, thereby dramatically expanding chemical diversity in the field of hyperpolarisation, including the simple creation of very long-lived singlet spin order.

The process may begin with nitrite (NO ₂ - ), a reagent that is formed during the nitrification of ammonia by nitrosomas. ¹⁹ Whilst mammals do not absorb nitrites directly, plants use it to form essential nitrogen containing molecules such as amino acids and the further aerobic oxidation of nitrite leads to nitrate. Nitrite is used a food additive for cured meats ²⁰ and approximately 7% of our ingested nitrite comes this source while the remainder come from nitrate in saliva and stomach through the enterosalivary pathway. ^21-22 Furthermore, although nitrites are non-carcinogenic, their ability to form nitrosamines can lead to toxicity ²³ as examined by the research community and mainstream media. ^24-25 The action of metmyoglobin production by nitrite is, however, beneficial in the treatment of cyanide poisoning and sodium nitrite remains as one of primary antidotes for acute intoxication. ²⁶

In a particular aspect of the invention the source of ¹⁵ N may comprise one or more of nitrite (NO ₂ -), ammonia (NH3), ammonium (NH ₄ ⁺ ), hydroxylamine (NH ₂ OH), hydrazine (N ₂ H ₄ ), nitrosonium (NO ⁺ ), , dinitrogen (N ₂ ), nitrate (NO ₃ -), azide (N ₃ -), isocyanate (NCO-/RNCO), cyanate (OCN-/ROCN), thiocyanate (SCN-), amine (RNH ₂ etc.) and nitrous oxide (N ₂ O).

If the source of ¹⁵ N contains protons its ² H-counterparts are also included (including combinations of protons and ² H-counterparts) such as ammonia (ND ₃ ND ₂ H and

NDH ₂ ), ammonium (ND ₄ ⁺ , ND ₃ H ⁺ , ND ₂ H ₂ ⁺ and ND ₃ H ⁺ ), hydroxylamine (NH ₂ OD,

NHDOD, NHDOH, ND ₂ OD and ND ₂ OH), amine (RNHD, RND ₂ , R'RND where R/R` is a suitable group like alkyl or aryl), hydrazine (N ₂ D ₄ , N ₂ D ₃ H, N ₂ D ₂ H ₂ and N ₂ DH ₃ ) etc. The presence of nitrosamines as impurities has led to recent drug recalls (valsartan and ranitidine). Hence understanding more clearly pathways to their formation may affect drug manufacture. The method of the present invention provides a route to the detection and quantification of such impurities during synthesis or in the formulation through SABRE. Nitrite is an ambidentate ligand that can bind via the N- or O- atoms to form nitro or nitrito complexes respectively ^28-29 with Ni ^30-33 and Pt ^34-36 examples being the most prevalent. As SABRE works through reversible binding of the agent to become hyperpolarised to a metal complex, we hypothesised that polarisation of NO ₂ - via this route might be possible. ^37-40 There are a few examples of ionic species such as sodium pyruvate, ^41-42 sodium acetate ⁴³ and naicin ⁴⁴ that undergo SABRE. A scalar coupling network is required to exist between the target analyte and p-H ₂ derived protons in the catalyst. ^{45- 48} Hence a η ¹ -NO ₂ (N-nitro) form, where any hydride- ¹⁵ N coupling will be preferred over the η ¹ -ONO (O-nitrito) and η ² -O–N–O (O,O-bidentate) linkage isomers. Theoretical descriptions of SABRE are provided by Barskiy and others ^49,50,51 that account for the magnetisation transfer conditions needed. ⁵² They are achieved at low magnetic field, typically 6 mT for ¹ H, or through r.f. excitation at high field ⁵³ and drive the sensitisation of ¹ H, ¹³ C, ¹⁵ N, ¹⁹ F, ³¹ P and ²⁹ Si (etc.) ^{41-43, 54-64} nuclei provided the catalyst lifetime matches spin-state evolution times. Typically, when an iridium N-heterocyclic carbene (NHC) catalyst is used, NMR signal strengths that are many orders of magnitude higher than that which would be obtained at thermal equilibrium can be achieved. ^65-66,67 Warren in particular stands- out for his early work on ¹⁵ N ⁶⁸ called ¹⁵ N-SABRE here, but 79% polarisation has been reported more recently ⁶⁹ . Tessari has developed analytical science applications ⁷⁰ and other catalyst types have been reported. ⁷¹ Hyperpolarised long-lived singlet states ⁷² , as pioneered by Levitt ⁷³ , have also been created and detected 15 mins. after their formation by ourselves. ⁷² However, field dependent ¹⁵ N relaxation times can themselves exceed 10 minutes. ^74-75 These properties are harnessed here to extend utility of the novel SABRE hyperpolarised products. Thus, one particular benefit is that the hyperpolarised state, if stored in an optimal magnetic field means it may last of several 1000 seconds, which turns these agents into useful synthetic tools like radio labelled precursors for PET, but without the toxicity issues. By reference to NO ₂ - we have established that the reaction shown below takes place with [IrCl(COD)(IMes)] and H ₂ .

Scheme 1: Reaction of [IrCl(COD)(IMes)] with Na ¹⁵ NO ₂ in the presence of hydrogen and pyridine These complexes, depending on the identity of A then enable the SABRE hyperpolarisation of ¹⁵ NO ₂ - in a process whose outcome efficiency is controlled by the magnetic field experienced by the sample during catalysis. The diagram below demonstrates how this approach leads to a versatile range of feedstocks that allow hyperpharmaceuticals to be created for use in patient diagnostics, synthetic screening, kinetic modelling and/or agent quantification. The harnessing of the broad ranging reactivity through nitrogen derived synthons make the realisation of hyperpharmaceuticals possible. For use in living systems, these must be associated with biocompatibility, long-lived magnetisation and high polarisation levels; urea, N ₂ O, N ₂ and glutamate reflecting suitable examples of clinical MRI potential. Similarly, the nitrogen derived synthons may also be useful in the preparation of nitrogen containing PET agents, and the like. Additionally, the long magnetic state lifetimes and diverse reactivity shown by demonstrate that a diverse portfolio of reaction products can be created that become readily visible to NMR and MRI. For this diverse portfolio of hyperpolarised reaction products we have coined the term “hyperpharmaceuticals”, where synthesis, akin to the production of radiopharmaceuticals, is used to create a versatile range of highly visible agents that may be selected according to their relevance for the future diagnose of disease, since ¹⁵ N is a cheap and easily sourced NMR/ MRI label. Thus, according to this aspect of the invention there is also provided a hyperpharmaceutical agent wherein said agent includes a hyperpolarised or hyperpolarisable ¹⁵ N moiety. According to this aspect of the invention there is provided a hyperpharmaceutical agent wherein said agent is created through a reaction involving a ¹⁵ N containing hyperpolarised agent and wherein said agent allows the embedding of hyperpolarised nuclei in the hyperpharmaceutical agent reaction product. There is further provided a method of creating a hyperpharmaceutical agent, said method comprising creating a ¹⁵ N containing hyperpolarised agent and wherein the resulting ¹⁵ N hyperpolarisation is shared with the hyperpharmaceutical agent through spin coupling driven propagation within the hyperpharmaceutical agent. In the method of creating a hyperpharmaceutical agent said method comprises maximising its utility by creating a (i) more diagnostic or (ii) longer lived or (iii) stronger response. The effect of polarisation transfer field on the SABRE polarisation of Na ¹⁵ NO ₂ in the presence of pyridine is illustrated in Figure 1 and harnessing such effects represents a way to optimise the delivered hyperpolarisation. According to a particular aspect of the invention the step of hyperpolarisation includes the use of a SABRE hyperpolarisation catalyst. The hyperpolarisation may be achieved by polarisation transfer after, spin refrigeration, DNP, para-hydrogen induced polarisation (PHIP), SABRE or from a suitable molecule in a singlet state. However, in one particular aspect of the invention the hyperpolarisation is introduced by SABRE and thus, the transfer catalyst is a magnetisation transfer catalyst, especially a SABRE magnetisation transfer catalyst. There are a number of ways to induce certain nuclei into a hyperpolarised state. The simplest way is to cool the material to very low temperatures in the presence of a magnetic field, which will favour population of the lower energy state in which the spins of the nuclei are aligned with the applied magnetic field. This method is suitable for the production of hyperpolarised monatomic gases such as xenon or helium-3. The polarisation levels of these nuclei have also been increased via the use of laser-based technologies. In SABRE, a catalyst reversibly binds p-H ₂ and the polarisable molecule to transfer dormant spin order from p-H ₂ into the substrate via a scalar-coupling framework to the target molecule. If there are two NMR active spins accepting polarisation, as exemplified by ¹³ C ₂ pyruvate, this will result in a singlet state in the polarisable molecule which will desirably be characterised by a long lifetime in a low magnetic field if there are two scalar coupled spin 1/2 nuclei present. Preferably, the resulting singlet state lifetime will be 20 seconds or more, preferably more than 20 seconds or more than 25 seconds or more than 30 seconds. The resulting singlet state lifetime may last one or more minutes. For the case of ¹⁵ N, this is exemplified by N ₂ O, N ₂ H ₄ , and R’ ₂ CN-NCR ₂ where R and R’ are suitable groups (viz aromatic, aliphatic etc.). When a SABRE type process is utilised as the method of hyperpolarisation, a SABRE hyperpolarisation catalyst (e.g. [IrCl(COD)IMes] or a ² H-labelled counterpart or a related catalyst may be used to optimise the process in a suitable solvent with the selected singlet state derived agent. A co-ligand may be added to promote SABRE for NO ₂ -, NO ₃ - (etc.). The co-ligand will be selected according to its binding properties, which must relate to those of NO ₂ -, NO ₃ - (etc.). Thus if the selected co-ligand binds too strongly it will suppress NO ₂ -, NO ₃ - (etc.) binding and be detrimental to SABRE. However, if it binds too weakly, the iridium catalyst will cease to have a suitable lifetime. The co-ligand is exemplified, but not limited to DMAP, DSMO, pyridine, NH ₃ and NH ₂ Ph (or their deuterated forms). H ₂ or parahydrogen (p-H ₂ ) gas may be the selected singlet state derived agent and after being added to the resulting system whilst agitating the system will activate the catalyst through a reaction whose speed may be enhanced by stirring, warming or shaking. Alternatively, the application of ultrasound may be used as a means of agitation. Hyperpolarisation transfer, by replacing the H ₂ gas with p-H ₂ may be performed to create a hyperpolarised transference complex whilst agitating the system as described herein. The addition of H ₂ or parahydrogen (p-H ₂ ) gas to the solvent may take place prior to the solvent system being agitated or may take place concurrent with agitation. Catalyst activation under parahydrogen may take place prior to the final hyperpolarisation transfer step or be part of the hyperpolarisation transfer step. The co-ligand (L) and the other ligands surrounding the catalyst may include ² H labels in order to make the hyperpolarisation transfer process more selective and or efficient. Hyperpolarisation will be transferred from parahydrogen into the polarisable target molecule in an optimised magnetic field to create a strongly hyperpolarised response. This may be subsequently converted into a singlet state across the spin-pair if desired. This conversion may occur spontaneously and optimised by selection of an appropriate magnetic field(s) for transfer or may be promoted by radio frequency excitation. It will be understood that a mixture of transfer catalysts may be included in the method of the invention to improve selectivity and allow mixtures to be examined. The magnetic field can be changed to focus or improve the efficiency of hyperpolarisation transfer. The type of magnetic states required in this process may be ultra-low magnetic fields, e.g. <<1G (<10 ^-6 T) which can spontaneously hyperpolarise the said singlet state. A change in magnetic field can be used to control which substrates in a mixture gain signal in order to introduce selectivity, while varying the field during transfer step to enhance the signal from all substrates. Hence it will be possible to use this magnetic field to optimally polarise the MR active nuclei in the target substrate rather than the ligand L. It will be understood that a mixture of target molecules may be included in the method of the invention. It will also be understood that in the conjugate acid forms, the protons on these agents can be removed by the addition of a base such as NaOH or Cs ₂ CO ₃ to form the corresponding anion in order to further optimise lone pair availability. This route can improve binding to the catalyst and thereby increase SABRE efficiency. The target molecule will then contain at least one –NH and may comprise an amine or amide moiety. Thus, a polarisable molecule containing at least one –NH may comprise, individually or in combination, a primary, secondary or tertiary amine, such as NH ₃ , NH ₂ OH, N ₂ H ₄ , NH ₂ Ph, NH ₂ CH ₂ Ph, NH ₂ CH ₂ HCH ₂ CH ₂ Ph and related amines; or an amide, such as NH ₂ COCH ₃ or NH ₂ CONH ₂ ; and the like. An amine or amide can be used to control the efficiency of hyperpolarisation transfer. For NO ₂ -, it is HNO ₂ , while for NO ₃ - it is HNO ₃ . Illustrative examples of target molecules which may be hyperpolarised via this route include, but shall not be limited to: (i) NR'R"R'" wherein R', R" and R'", which may be the same or different, each represents H, alkyl _C1-20 , aryl, base pair, etc. and combined in structures like glutamine, glutamate and GABA; (ii) NR'R"COR'" wherein R', R" and R'", which may be the same or different, each represents R', R" or R'" = H, CH ₃ , alkyl, aryl, vinyl, or any combination exemplified by acetamide, urea, glutamine, glutamate, and the like; (iii) carbazides with general formula RR'N-NH(C=O)NH-NR''R''' wherein R, R', R" and R'" which may be the same or different, each represents R', R" or R'" = H, CH ₃ , alkyl, aryl, vinyl, or any combination. (iv) nitrogen containing platinum derived cancer drugs, which include, but shall not be limited to cisplatin, carboplatin, nedaplatin, oxaliplatin, triplatin, satraplatin; and the like; and (v) cancer drugs containing an acetamide group, which include, but shall not be limited to, taxanes such as paclitaxel, docetaxel, cabazitaxel; and the like. Generally in the method of the invention the step of hyperpolarisation includes the use of a SABRE hyperpolarisation catalyst. The SABRE hyperpolarisation catalyst will usually comprise a transition metal complex, for example comprising a metal atom selected from, but not limited to, Ru, Rh, Ir, W, Pd and Pt. In a particular aspect of the present invention, a SABRE hyperpolarisation catalyst may comprise an iridium-based catalyst whose key identity is controlled by the co-ligand. The nature of the catalyst may vary, but may, for example, take the form of a conventionally known hydrogenation catalyst. Thus, such catalysts may be homogeneous catalysts, for example, Wilkinson’s catalyst, or heterogeneous catalysts, such as Pd on carbon. Thus, such homogeneous catalysts may include, but shall not be limited to, rhodium based catalysts, such as Wilkinson’s catalyst and iridium based catalysts, such as Crabtree’s catalyst. Heterogeneous catalysts may comprises one or more platinum group metals, particularly platinum, palladium, rhodium and ruthenium, precious metal catalysts, such as silver or gold, or non-precious metal catalysts, such as those based on nickel, e.g. Raney nickel. Examples of preferred (SABRE) hyperpolarisation transfer precatalysts are thus described in International patent application No. PCT/GB2009/002860. Such catalysts include, for example, [IrCl(COD)(IMes)] and analogues thereof, (in which COD is cycloocta-1,5-diene). Alternatively, the SABRE hyperpolarisation catalyst may comprise a ² H-labelled counterpart of [IrCl(COD)(IMes)] or a catalyst optimised to work in the non-aqueous phase with the selected substrate. Alternatively, the SABRE hyperpolarisation catalyst may comprise of either of the two previous modifies in conjunction with a form like [IrL(COD)(IMes)]Cl which already contains L or a catalyst optimised to work in the non-aqueous phase with the selected substrate. Generally, an iridium magnetisation transfer catalyst will include iridium with at least one N-heterocyclic carbene (NHC) ligand or phosphine. Examples of such N-heterocyclic carbenes include, but shall not be limited to:

The transfer catalyst may be designed to produce an optimal lifetime and coupling framework for hyperpolarisation transfer under these conditions. It will be understood that a mixture of transfer catalysts may be included in the method of the invention. These species are often referred to as precatalysts because they are stable and become active during the catalytic process, in this case through their reaction with the small molecule substrate, the co-ligand L and H ₂ . The co-ligand may be selected from the group consisting of one or more of a sulfoxide, a thioester, a phosphine, an amine, CO, an isonitrile and a nitrogen heterocycle. A particular example of a co-ligand is one or more sulfoxides, although other co-ligands are contemplated herein. Examples of sulfoxide co-ligands include a wide range of sulfoxides, such as, alkylsulfoxides, including, but not limited to, dimethylsulfoxide, diethylsulfoxide, dibutylsulfoxide and methylethylsulfoxide; and arylsulfoxides, including, but not limited to, diphenylsulfoxide, dibenzysulfoxide, phenylmethylsulfoxide, phenylethylsulfoxide, phenylvinyl sulfoxide and dimesityl sulfoxide (1,3,5-trimethyl-2-(2,4,6-trimethylphenyl)sulfinylbenzene); depending on the identity of the target analyte (illustrated above for pyruvate), the polarisation transfer mechanism, and the desire to create singlet polarisation in a suitable spin pair or Zeeman polarisation, the need for biocompatibility, the identity of the NHC and the choice of solvent. The co-ligand will generally be bound to a SABRE catalyst. A variety of solvents may be used in preparing the fluid required for the method of the present invention. Such solvents will generally be organic solvents, e.g. a non- aqueous solvent; and may comprise polar, non-polar solvents, non-protic and protic solvents. Such solvents include, but shall not be limited to H ₂ O, CH ₃ OH, CH ₃ CH ₂ OH, CH ₂ OH, CH ₂ Cl ₂ , CHCl ₃ , THF, DMF, nitromethane, alkanes and aromatic hydrocarbons, such as benzene or toluene; the deuterated counterparts of any of the aforementioned solvents. Selection of an appropriate solvent may be used to control one or more of the steps herein defined in the method of the invention. According to a further aspect of the invention a biphasic element may be introduced into the solvent in order to separate the hyperpolarised target molecule from the transfer catalyst. When SABRE hyperpolarisation is used, a SABRE hyperpolarisation catalyst (e.g. [Ir(Cl(COD)(IMes)] or a ² H-labelled counterpart or one containing L or a catalyst optimised to work in the polar phase with the selected singlet state derived substrate). When the agent to be hyperpolarised is an anion, like NO ₂ - or NO ₃ - a phase transfer promoter like a crown ether, aza-crown ether, thia-crown ether, cyclodextrin, cryptophane or cryptand may be used in conjunction with a cation like Na ⁺ or K ⁺ to improve solubility. When a mixed solvent system is used a solvent phase-separation promoter e.g. NaCl or NaO ₂ CCH ₃ or NaOH or NaHCO ₃ or Na ₂ CO ₃ or ethanol, at a suitable concentration may be added to the system. The concentration of the phase-separation promoter may be an amount suitable to: (i) achieve physiological conditions; (ii) vary the solutions pH to achieve optimal SABRE; (iii) optimise organic phase extraction; and/or (iv) optimise the speed of phase-separation. Any known phase-separation promoter may be used. Desirably such a phase- separation promoter will be suitable for in vivo use and therefore should be suitable to achieve physiological conditions. In addition, the phase-separation promoter should be suitable to withstand variations in pH which may be desirable to achieve optimal SABRE. Selection of the phase-separation promoter may also be desirable to optimise organic phase extraction; and/or to optimise the speed of phase-separation. Examples of phase-separation promoters include alkali metal salts, such as sodium or potassium salts; or alkaline earth metal salts, such as calcium. Alkali metal salts are preferred, such as NaCl, or NaO ₂ CCH ₃ , NaOH, NaHCO ₃ or Na ₂ (CO ₃ ). A further phase-separation promoter may comprise an alcohol such as ethanol. The amount of phase-separation promoters may vary depending, inter alia, upon the nature of the phase-separation promoters, the nature of hyperpolarisation target, etc. When the aim is to create a biocompatible system, NaCl or KCl may be used as a phase-separation promoter to produce a saline or saline-like solution. Therefore, the amount of the phase-separation promoter may vary depending upon, inter alia, the nature of the phase-separation promoter. Generally, the phase-separation promoter may be from about 0.33% w/v to about 9 % w/v. However, it will be understood by the person skilled in the art that more or less of the phase-separation promoter may be included, as required. The hyperpolarisation transfer may be performed with p-H ₂ to create a hyperpolarised target molecule whilst agitating the biphasic solvent as herein described. An appropriate amount of time may be allowed to enable the two solution phases to separate. Through the process described herein the NMR or MR response of the target molecule can be increased so that it is readily detectable in a high resolution or imaging experiment. The hyperpolarisation target molecule may reflect one or more of nitrite (NO ₂ -), ammonia (NH ₃ ), ammonium (NH ₄ ⁺ ), hydroxylamine (NH ₂ OH), hydrazine (N ₂ H ₄ ), nitrosonium (NO ⁺ ), dinitrogen (N ₂ ), nitrate (NO ₃ -), azide (N ₃ -), isocyanate (NCO- /RNCO), cyanate (OCN-/ROCN), thiocyanate (SCN-) and nitrous oxide (N ₂ O). If the source of ¹⁵ N contains protons its ² H-counterparts are also included such as ammonia (ND _3, ND ₂ H and NDH ₂ ), ammonium (ND ₄ ⁺ , ND ₃ H ⁺ , ND ₂ H ₂ ⁺ and ND ₃ H ⁺ ), hydroxylamine (NH ₂ OD, NHDOD, NHDOH, ND ₂ OD and ND ₂ OH), hydrazine (N ₂ D ₄ , N ₂ D ₃ H, N ₂ D ₂ H ₂ and N ₂ DH ₃ ) etc. It may also be the product of a reaction involving one of the above agents, which are now acting as synthons to a more complex molecule, or the hyperpharmaceutical. These will be prepared in a synthetic step(s) that individually or in combination proceed more rapidly than the ¹⁵ N T ₁ relaxes the created hyperpolarisation. The use of L (e.g. DMSO, diethylsulfoxide, (etc.)) can be used to control the efficiency of hyperpolarisation transfer in the first step. This is a result of the metal complexes reactivity, which can be optimised for specific solvent, cost, pressure of p- H ₂ and time of activation. The temperature can be changed to focus or improve the efficiency of hyperpolarisation transfer. We note, more than one target may be present. The transfer catalyst will usually comprise a transition metal complex, for example comprising a metal atom selected from, but not limited to, Ru, Rh, Ir, W, Pd and Pt. The transfer catalyst will usually comprise one or more ligands in addition to the ligand comprising the hyperpolarisable nuclei. These one or more other ligands may comprise organic or inorganic ligands and may be mono-, bi- or multidentate in nature. These one or more ligands may play a role in controlling the activity and stability of the metal centre. For example, the one or more ligands may comprise NHC ligands as herein described while the other ligand may be a sulfoxide. In one embodiment, the transfer catalyst comprises one or more phosphine/co-ligand combinations in addition to the ligand to be hyperpolarised. The transfer catalyst may be attached to a solid support, for example a polymer support. Attachment will usually be made through a ligand which links the metal centre to the support. Suitable linkers are known in the art. For example, the linker may comprise one or more in-chain atoms selected from C, O, N, S, P and Si. The linker may comprise a siloxane moiety for attachment to the support and/or a phosphine moiety for attachment to the metal of the complex. In embodiments, the linker is a group of the following formula: -O- Si(OMe) ₂ -(CH ₂ ) _n -P(Cy) ₂ -, wherein n is 0 upwards (e.g. 0, 1, 2, 3, 4, 5 or 6) and Cy is cyclohexyl. In a further embodiment, the NHC or phosphine and co-ligand are linked together and form what is known as a chelate. This can be achieved via appropriate substitutions and the NHC/phosphine and co-ligand. Both cis and trans spanning may be induced by changing the length of the spacer. In this case the pre-catalyst is preassembled to include the co-ligand L. For in vivo use an in-line UV probe may be used, if desired, to establish that the concentration of the catalyst is sufficiently for in vivo injection. This makes full use of the fact that the catalyst is no longer present and therefore unable to promote the relaxation of the agent, thereby maximising longevity of the resulting hyperpolarised signal. For systems where the catalyst concentration remains too high, a catalyst deactivator may be added after the polarisation transfer step, or the synthetic step(s). Examples of suitable catalyst deactivators include, but shall not be limited to a chelating ligand, such as, bipyridyl, EDTA and dimethylglyoxime. A catalyst deactivator can be added to facilitate catalyst release. An appropriate delivery device may be used to procure the hyperpolarised target molecule for detection by NMR or MRI which can facilitate some or all of the following: Using an appropriate delivery device to procure the hyperpolarised agent for detection by NMR or MRI which will facilitate some (all) of the following: (i) after an appropriate time removing a hyperpolarised sample from the aqueous phase or solvent; (ii) using UV monitoring to assess suitability immediately prior to sample removal or after sample removal; (iii) using pH monitoring to assess suitability immediately prior to sample removal or after sample removal; (iv) employing filtration to achieve sterility after sample removal; (v) injecting or transporting the sample into a target for subsequent detection by NMR or MRI, where the target might be a suitable sample tube, an animal or a human. In summary, we have illustrated a novel approach to hyperpolarise ¹⁵ N directly by the SABRE hyperpolarisation technique. In order to further optimise this process the sulfoxide ligand can be changed with suitable representative examples being diphenyl sulfoxide, dibutyl sulfoxide, dibenzyl sulfoxide, phenylmethyl sulfoxide, phenylethyl sulfoxide, phenylvinyl sulfoxide, dimesityl sulfoxide. In addition, the NHC can be varied according to the earlier figure in conjunction with the sulfoxide to further optimise this process for a given substrate. According to a further aspect of the invention there is provided an agent for magnetic resonance studies, the agent comprising SABRE hyperpolarised ¹⁵ N in solution or liquid ¹⁵ N. The source of ¹⁵ N may comprise one or more of nitrite (NO ₂ -), ammonia (NH ₃ ), hydroxylamine (NH ₂ OH), hydrazine (N ₂ H ₄ ), nitrosonium (NO ⁺ ), nitrate and dinitrogen. In a particular aspect of the invention the source of ¹⁵ N may comprise one or more of nitrite (NO ₂ -), ammonia (NH ₃ ), ammonium (NH ₄ ⁺ ), hydroxylamine (NH ₂ OH), hydrazine (N ₂ H ₄ ), nitrosonium (NO ⁺ ), dinitrogen (N ₂ ), nitrate (NO ₃ -), azide (N ₃ -), isocyanate (NCO-/RNCO), cyanate (OCN-/ROCN), thiocyanate (SCN-) and nitrous oxide (N ₂ O). If the source of ¹⁵ N contains protons its ² H-counterparts are also included such as ammonia (ND _3, ND ₂ H and NDH ₂ ), ammonium (ND ₄ ⁺ , ND ₃ H ⁺ , ND ₂ H ₂ ⁺ and ND ₃ H ⁺ ), hydroxylamine (NH ₂ OD, NHDOD, NHDOH, ND ₂ OD and ND ₂ OH), hydrazine (N ₂ D ₄ , N ₂ D ₃ H, N ₂ D ₂ H ₂ and N ₂ DH ₃ ) etc. The solution may comprise at least one selected from water, blood, oxygenated blood, deoxygenated blood, plasma, fat or oil. In a preferred aspect of the invention the imaging medium comprises a solution of a target molecule in a saline solution of a hyperpolarised target molecule. According to a further aspect of the invention there is provided the use of SABRE hyperpolarised ¹⁵ N in solution or liquid as an agent in the methods as herein described. In particular there is provided the use of hyperpolarised ¹⁵ N wherein the source of ¹⁵ N comprises one or more of nitrite (NO ₂ -), ammonia (NH ₃ ), ammonium (NH ₄ ⁺ ), hydroxylamine (NH ₂ OH), hydrazine (N ₂ H ₄ ), nitrosonium (NO ⁺ ), nitrate, dinitrogen, nitrate (NO ₃ -), azide (N ₃ -), isocyanate (NCO-/RNCO), cyanate (OCN-/ROCN), thiocyanate (SCN-) and nitrous oxide (N ₂ O) of their reaction products. According to a further aspect of the invention there is provided a method of hyperpolarising a substance, the method comprising: liquefying the SABRE hyperpolarised ¹⁵ N agent; dissolving the substance to be hyperpolarised in said liquefied ¹⁵ N agent; and removing said ¹⁵ N that is now converted to a product. According to a further aspect of the invention there is provided an MRI apparatus comprising: means to apply a magnetic field to a SABRE hyperpolarised sample; means to apply RF radiation to the sample; and means to detect an NMR signal arising from the sample due to the application of the magnetic field and RF radiation, wherein the means to apply a magnetic field and the means to apply RF radiation are configured to excite an NMR signal from ¹⁵ N; and wherein the means to detect an NMR signal are configured to detect an NMR signal from ¹⁵ N. The invention will now be illustrated by way of example only and with reference to the accompanying drawings, in which: Figure 1 illustrates the effect of polarisation transfer field on the SABRE polarisation of Na ¹⁵ NO ₂ in the presence of pyridine; Figure 2 illustrates the effect of co-ligand, A-H, on the SABRE polarisation level found for Na ¹⁵ NO ₂ after reaction of [IrCl(COD)(IMes)] with Na ¹⁵ NO ₂ (25 eq.) and the co-ligand (4 eq.) in methanol-d ₄ at 298 K. The ¹⁵ N signal enhancements for Na ¹⁵ NO ₂ result after polarisation transfer under 3 bar p-H ₂ in a −3.5 mG field; Figure 3 illustrates the effect of NHC identity on the ¹⁵ N NMR signal enhancement level for Na ¹⁵ NO ₂ based on using the precatalyst [IrCl(COD)(NHC)] (5 mM), DMAP- d ₂ (6 eq.) and Na ¹⁵ NO ₂ (25 eq.) in methanol-d ₄ with polarisation transfer at −3.5 mG under 3 bar p-H ₂ ; Figure 4 illustrates the conversion of nitrite into a diazonium cation and N ₂ as revealed by hyperpolarised ¹⁵ N NMR thereby illustrating the hyperpharmaceutical concept. This starts with hyperpolarised ¹⁵ NO ₂ -. A hyperpolarised response for ¹⁵ N ₂ is noteworthy, alongside the efficient sharing of polarisation into both of the ¹⁵ N coupled spins in the product even though only one is initially polarised; Figure 5 illustrates utilizing the reactivity of Na ¹⁵ NO ₂ towards aniline- ¹⁵ N to enable the tracking of diazotization with the product reacting with mono- ¹⁵ N labelled sodium azide to subsequently form of two isotopomers of phenyl azide via a normally invisible cyclic intermediate; Figure 6 illustrates the utilization of hyperpolarized ¹⁵ N NMR spectroscopy to probe: a) copper free click chemistry of hyperpolarised PhN ₃ , formed as in Figure 5; b) two routes to the formation of hyperpolarized N _2(g) ; c) Quaternization of an amine with DCl(aq) and the corresponding imine formation with benzaldehyde; d) Amidation, sulfonamidation, imine formation utilizing benzylamine- ¹⁵ N; e) the copper free-click chemistry with 1- ¹⁵ N-NaN ₃ with an alkyne to form a triazole; Figure 7 illustrates that ¹⁵ N NMR measurements allow the time course of a reaction to be quantified, in this case, the conversion of nitrite into a diazonium cation; Figure 8 illustrates the demonstration of NO ₃ ^{- 15} N-hyperpolarisation under SABRE. Top trace showing a thermally polarised reference spectrum of Na ¹⁵ NO ₃ recorded using 512 signal averages with the bottom showing the SABRE hyperpolarized spectrum recorded in a single scan; and Figure 9 illustrates the demonstration of ¹⁵ N-hyperpolarisation aqueous bolus preparation under SABRE exemplified by NO ₂ - (a) Picture of sample used in analysis; (b) demonstration of ¹⁵ N NO ₂ - hyperpolarisation in aqueous phase achieved using phase transfer catalysis via high-resolution 7 T spectrum; (c) two spatially resolved ¹⁵ N-images showing the response for the upper aqueous phase at different spatial resolution; (d) time course map showing decay of signal as a function time after polarisation transfer; (e) thermally polarised comparison to demonstrate need for hyperpolarised response. Experimental Procedures Materials All of the experimental procedures associated with this work were carried out under nitrogen using standard Schlenk techniques. The solvents used were dried using an Innovative Technology anhydrous solvent system, or distilled from an appropriate drying agent under nitrogen. The catalyst precursor ([Ir(IMes)(COD)Cl] (1) employed in this work was synthesized by established procedures according to literature methods. Deuterated chloroform (CDCl ₃ ), deuterated water (D ₂ O), deuterated ethanol (EtOD) and pyrazine (2) were purchased from Sigma Aldrich and used as supplied. Example 1 Demonstration that an active SABRE catalyst forms with Na ¹⁵ NO ₂ For successful SABRE transfer to occur, the formation of a complex exhibiting spin- spin couplings between the bound substrate and p-H ₂ derived hydride nuclei is required. Classically, this involves the reaction of a precatalyst (most commonly [IrCl(COD)(IMes)] (1) (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazolylidene)), with an excess of the selected substrate under a H ₂ atmosphere. Complexes of type [Ir(H) ₂ (IMes)(sub) ₃ ]Cl, when the substrate is a neutral N-heterocycle such as pyridine, meet this requirement. Consequently, our initial efforts targeted the synthesis of an active SABRE catalyst with bound NO ₂ ⁻ . When Na ¹⁵ NO ₂ (1 eq.) was added to a solution of [IrCl(COD)(IMes)] (1, 5 mM) in methanol-d ₄ the complete conversion to [Ir( ¹⁵ NO ₂ )(COD)(IMes)] (2) at 298 K (Scheme 1) is observed. This change was readily evident as the ¹⁵ N signal for free Na ¹⁵ NO ₂ at δ _N 611.8 moved to δ _N 490.7 at 254 K. When complex 2 is exposed to a 3 bar pressure of H ₂ at 254 K, the oxidative addition of hydrogen takes place to form [Ir(H) ₂ ( ¹⁵ NO ₂ )(COD)(IMes)] (3). This complex exhibits ¹ H NMR resonances for its hydride ligands at δ _H −18.69 (hydride trans to ¹⁵ NO ₂ ⁻ , ² J _HN = 23.0 Hz and ² J _HH = -3.6 Hz) and δ _H −14.01 (hydride trans to COD, ² J _HH = 3.6 Hz). Additionally, the signal for the bound ¹⁵ NO ₂ ⁻ ligand appears at δ _N 376.6. Hence there is a strong hydride- ¹⁵ N coupling in this material commensurate with SABRE. Subsequently, this sample was warmed to 298 K for 20 minutes. This led to the formation of multiple hydride containing products; some of which display PHIP on exposure to p-H ₂ . Pleasingly, a hyperpolarised signal for free Na ¹⁵ NO2 is observed in the ¹⁵ N NMR spectrum after SABRE transfer at −5 mG. Whilst the ¹⁵ N signal enhancement was just 134-fold, it confirms that the reversible binding of NO ₂ ⁻ took place. However, when this sample was left at room temperature for >2 hours SABRE activity is lost. Hence, we sought to create alternative catalysts that would both improve the ¹⁵ N signal enhancement level and be suitable for repeated measurement over long periods. Co-ligands have been used to achieve stability in conjunction with weakly binding ligands, ^{40-41, 73} reduced spin dilution ^{42, 74-75} and hydride ligand chemical, rather than magnetic, inequivalence. ⁷⁶ We hypothesized that the addition of a suitable co-ligand with NaNO ₂ could therefore ameliorate low complex stability whilst maintaining NO ₂ ⁻ coordination and strong ¹⁵ N-hydride ligand couplings and set out to prove this. Thus, a sample containing [IrCl(COD)(IMes)] (1, 5 mM), Na ¹⁵ NO ₂ (5 eq) and pyridine (3 eq.) was interrogated by NMR spectroscopy. The initial formation of [Ir( ¹⁵ NO ₂ )(COD)(IMes)] (2) was indicated. Hence, nitrite outcompetes pyridine for Ir(COD)(IMes) ⁺ . Subsequently, exposing this sample to 3 bar H ₂ at 254 K led to the formation of neutral [Ir(H) ₂ (NO ₂ )(IMes)(η ¹ -COD)(pyridine)] (4 _A ) with diagnostic hydride resonances at δ _H −14.00 and −18.77 that share a ² J _HH coupling of −3.3 Hz; the latter resonance also exhibits a ² J _NH of 23.1 Hz. The bound resonance for NO ₂ ⁻ appears at δ _N 476.1. We note, that known [Ir(H) ₂ (IMes)(η ¹ -COD)(pyridine) ₂ ]Cl (5 _A ) forms alongside 4a in a 1:4 ratio. After warming the sample for 1 h at room temperature, further reaction, to form two additional hydride containing products, takes place. Of these, [Ir(H) ₂ ( ¹⁵ NO ₂ )(IMes)(pyridine) ₂ ] (6 _A ), with characteristic hydride peaks at δ _H −21.24 and −22.45, dominates. The former resonance exhibits a ² J _NH splitting of 28.3 Hz, and both show ² J _HH couplings of -7.5 Hz. The minor product proved to be Na[Ir(H) ₂ ( ¹⁵ NO ₂ ) ₂ (IMes)(pyridine)] (7 _A ). It yields hydride resonances at δ _H −22.02 ( ² J _NH = 28.5 Hz) and −23.01 with mutual ² J _HH splittings of -7.6 Hz.. Interestingly, no evidence for the formation of tris pyridine containing [Ir(H) ₂ (IMes)(pyridine) ₃ ]Cl, is observed. ⁷⁷ Unlike the complexes formed in the absence of pyridine, the pyridine derived NO ₂ ⁻ complexes 6 _A and 7 _A proved stable when left at room temperature for >24 hours and were therefore suitable for assessment of their SABRE performance. When the ratio of Na ¹⁵ NO ₂ to pyridine was set to 5:3, the ratio of 6 _A to 7 _A in solution proved to be 85:15. Addition of excess Na ¹⁵ NO ₂ (25 eq), whilst maintaining the pyridine concentration, only moderately shifted the equilibrium between 6 _A and 7 _A to 80:20 thereby confirming neutral [Ir(H) ₂ ( ¹⁵ NO ₂ )(IMes)(pyridine) ₂ ] (6 _A ) is the thermodynamic product. Example 2 SABRE assessment of [Ir(H) ₂ ( ¹⁵ NO ₂ )(IMes)(py) ₂ ] (6 _A ) and Na[Ir(H) ₂ ( ¹⁵ NO ₂ ) ₂ (IMes)(A)] (7 _A ) activity In order for effective SABRE, the lifetime of the active catalyst must match with the propagating couplings and a level anti-crossing condition be met. ^{43-44, 46} For 6 _A and 7 _A , the ² J _NH couplings have been measured to be 28.3 and 28.5 Hz and an optimum polarisation transfer field (PTF) on the order of mG will be needed. ^{54, 58-59} Therefore, to assess the SABRE performance of 6 _A and 7 _A , a series of shake and drop measurements were undertaken using a mu-shield ⁷⁸ to attenuate the earth’s field by a factor of 300. These involved first exposing an NMR tube equipped with a J. Youngs Tap containing a solution of [IrCl(COD)(IMes)] (1, 5 mM), Na ¹⁵ NO ₂ (5 eq.) and pyridine (4 eq.) in methanol-d ₄ (0.6 mL) to H ₂ (3 bar) for 1 h to form an 85 : 15 ratio of 6 _A to 7 _A in solution. Subsequently, the H ₂ atmosphere was replaced with p-H ₂ (3 bar) and the sample shaken for 10 seconds in the mu metal shield. After shaking, the sample was transferred into the 9.4 T detection field and a ¹⁵ N NMR spectrum recorded. Analysis of this NMR spectrum revealed that the free ¹⁵ N signal of Na ¹⁵ NO ₂ was now 878-fold larger than that of the corresponding thermally polarised NMR spectrum at 9.4 T; corresponding to a 0.29% ¹⁵ N polarisation level. SABRE transfer to the ¹⁵ N of unlabelled pyridine at this low field was also observed, and a 172-fold signal gain quantified for its resonance at δ _N 301. The ¹⁵ N NMR signals of the coordinated NO ₂ ⁻ ligands were also readily visible at δ _N 511.28 (J _HN = 28.3 Hz) for 6 _A and at δ _N 509.7 (J _HN = 28.3 Hz) for the NO ₂ ⁻ in the equatorial position and at δ _N 483.7 for the ligand in axial position of 7 _A . Repeating the experiment after polarisation transfer at 70 G and subsequently recording a ¹ H NMR spectrum revealed PHIP enhanced hydride resonances for 6A and 7A. SABRE hyperpolarisation was also quantified for the ¹ H resonances of free pyridine as ~230, 60 and 150-fold for its ortho, meta and para positions respectively. No evidence for a PHIP enhanced hydride resonance for [Ir(H)2(IMes)(py)3]Cl at δH −22.7 was observed. We conclude therefore that ¹⁵ NO2 ⁻ sensitisation is possible through the action of this co-ligand supported catalyst. Example 3 Effect of polarisation transfer field on level of ¹⁵ N NMR signal gain in Na ¹⁵ NO ₂ In order to improve the levels of signal gain a more precise polarisation transfer field needs to be used. To investigate this effect, a sample containing [IrCl(COD)(IMes)] (1, 5 mM), Na ¹⁵ NO ₂ (5 eq.) and pyridine (4 eq.) in methanol-d ₄ (0.6 mL) was exposed to p-H ₂ (3 bar) and polarisation transfer fields from +10 mG to −10 mG were deployed; these were created by a solenoid located within a mu-metal shield. A profile of the resulting SABRE enhanced resonance for Na ¹⁵ NO ₂ is presented in Figure 1. The highest signal enhancements were observed when the polarisation transfer field was nominally +5 mG or −3 mG with gains of 1948 and 2054-fold respectively. More precise probing of the polarisation transfer field around these maxima revealed that improvement could be achieved by using a −3.5 mG value. At this field a 2329-fold signal gain was quantified through subsequent measurement at 9.4 T which corresponds to a ¹⁵ N polarisation level of 0.77%. Example 4 Effect of co-ligand on SABRE catalysis In order to form 6 _A and 7 _A , ¹⁵ NO ₂ ⁻ must out-bind the stabilising co-ligand pyridine. This means that the SABRE processes should be sensitive to the identity of this co- ligand; this behaviour has recently been observed during the SABRE polarisation of sodium pyruvate bysulfoxides, ³⁹ although there are other examples. ^{41, 79-80} Additionally, isotopic labelling of these co-ligands has been shown to reduce the number of acceptor spins at the metal centre and modulate relaxation. ^{42, 81-82} Therefore, a range of co-ligands were examined in order to see if it was possible to improve the polarisation levels in free Na ¹⁵ NO ₂ , as detailed in Figure 2. In each case, samples containing [IrCl(COD)(IMes)], Na ¹⁵ NO ₂ (25 eq.) and the co- ligand (A-H, 4 eq.) were first exposed to 3 bar H ₂ at 298 K for 1 hour to form the corresponding complexes 6 and 7. Subsequently, the sample was exposed to 3 bar p- H ₂ , in a −3.5 mG field, prior to rapid insertion into the 9.4 T detection field. Under these conditions, pyridine (A) now yields a 2329-fold signal enhancement (0.77% ¹⁵ N polarisation). ¹⁵ N labelled pyridine (A- ¹⁵ N) was then examined, and this reduced the signal enhancement level for Na ¹⁵ NO2 to 2107-fold. This is likely to be due to the increase in spin dilution associated by increasing the proportion of spin-1/2 nuclei. The resonance for free ¹⁵ N-pyridine at δ _N 301 now exhibits a lower signal gain of 1558-fold. In contrast, the use of pyridine-d ₅ (A-d ₅ ) improves the SABRE hyperpolarisation for Na ¹⁵ NO ₂ as the new enhancement level is now 3007-fold. As expected, all the pyridine isotopologues yield complexes 6 to 7 in an 85:15 ratio. This ca. 30% improvement, when compared to the undeuterated ligand, is likely to be due to slower relaxation in the active catalyst. In order to further modulate the co-ligand, other pyridyl derivatives having differing steric and electronic properties were examined. 2,6-Lutidine (B), which until recently was inaccessible to SABRE, ^83-84 was chosen as its ortho methyl groups hinder binding to the metal centre, which might promote ligand loss. When B is employed in conjunction with Na ¹⁵ NO ₂ , an increase in SABRE polarisation level is overserved when compared to pyridine. Interestingly, the ratio of 6 _B to 7 _B is now 95 : 5, but slow activation means [Ir(H) ₂ (NO ₂ )(η ¹ -COD)(B)] (4 _B , c.f. scheme 1) is visible with hydride resonances at δ _H −18.78 ( ² J _NH = 22.6 Hz and ² J _HH = 6 Hz) and −14.18 ( ² J _HH = 6 Hz); at this stage, it is observed in a 1:1 ratio with 6 _B . Unfortunately, when this sample is left under a 3 bar atmosphere of H ₂ for 72 h, sample degradation and the formation of multiple hydride containing complexes is noted. Hence, B is unable to provide the long term stability needed to reliably probe its behaviour. The use of electron deficient methyl 4,6-d ₂ -nicotinate (C), which has been shown to exhibit ¹ H polarisation levels of ca. 60%, ^{42, 63} was found to decrease the signal enhancement of Na ¹⁵ NO ₂ to 1894. The formation of 7 _C is promoted by this change as the ratio of 6C to 7C became a 1:2. In contrast, electron rich dimethylamino pyridine (D, DMAP) forms 6 _D in a 17:1 ratio with 7 _D. Additionally, a significantly improved 10313-fold ¹⁵ N signal enhancement is observed for Na ¹⁵ NO ₂ which corresponds to the creation of a 3.4% ¹⁵ N polarisation level. Non-heterocyclic ligands can also be utilized for SABRE. As such, amine ligands have been shown to be able to form stable SABRE catalysts and are effective agents for SABRE-Relay polarisation transfer. ^{81-82, 85-87} When utilized as a co-ligand for the hyperpolarisation of Na ¹⁵ NO ₂ , benzylamine-d ₇ (E-d ₇ ) led to a ¹⁵ N signal gain of 2070- fold. The two hydride containing complexes 6 _E and 7 _E were formed under these conditions in a ca. 1 : 1 ratio with hydride resonances at δ _H −22.10 and −23.40 and δ _H −22.36 and −22.72 respectively. When aniline (F) was used as the co-ligand, a 3322- fold signals gain for Na ¹⁵ NO ₂ was quantified. In this sample, 6 _F now dominates. Similarly, sulfoxides have proven to be efficacious for the hyperpolarisation of sodium pyruvate and weakly coordinating substrates. ^{39-40, 79, 88} The co-ligand DMSO- d ₆ (G) gave a 6270-fold signal enhancement for Na ¹⁵ NO ₂ . Interestingly, whilst Na[Ir(H) ₂ ( ¹⁵ NO ₂ ) ₂ (IMes)(DMSO-d ₆ )] as 7 _G is now dominant in solution, a second isomer of (8 _G ), where the two ¹⁵ NO ₂ ligands lie cis to one another and trans to hydride is now observed. This complex gives rise to a single hydride resonance at δ _H −22.32 where J _NHcis + J _NHtrans is 27.6 Hz. The ¹⁵ NO ₂ resonance of 8 _G appears at δ _H 502.0. Isomer 6 _G is detected as a minor species, with the ratio of 6 _G : 7 _G : 8 _G in solution being ~1 : 9 : 5. Finally, acetonitrile ⁸⁹ gave a 2029-fold ¹⁵ N signal gain and the neutral complex [Ir(H) ₂ ( ¹⁵ NO ₂ )(IMes)(acetonitrile) ₂ ] (6 _H ), with hydride resonances at δ _H −22.66 ( ² J _NH = 26.7 Hz and ² J _HH = −7 Hz) and −21.77 ( ² J _HH = −7 Hz) was the only complex observed. Example 5 Identifying the optimum DMAP (D): Na ¹⁵ NO ₂ ratio The highest ¹⁵ N polarisation level for Na ¹⁵ NO ₂ was achieved with the DMAP co- ligand. Interestingly, this ligand yielded the highest concentration of isomer 6. We postulated that the concentration of 6 _D in solution could be further manipulated by changing the number of equivalents of D in relation to [IrCl(COD)(IMes)] (1) and Na ¹⁵ NO ₂ . Therefore, a series of samples were prepared with between 3 − 20 eq. of D relative to 1. After activation, they were exposed to 3 bar p-H ₂ whilst located in a −3.5 mG polarisation transfer field. The resulting signal enhancements at 9.4 T are shown in Figure 2. When 3 equivalents of D (with respected to iridium) is utilised, a 9086-fold signal enhancement is observed with the corresponding 6 _D : 7 _D ratio being 8:1. Increasing the concentration of DMAP to 4 equivalents improved the signal gain seen at 9.4 T to 11019-fold. The ratio of complex 6 _D : 7 _D also increased to 17 : 1. Further incremental increases in DMAP concentration, to 6, 8 and 10 eq., gave signal enhancements of 12036, 12079 and 11888-fold respectively. The ratio 6 _D : 7 _D was now 24 : 1 in all three samples. At higher loadings of D, the formation of [Ir(H) ₂ (IMes)(D) ₃ ]Cl is observed, as a single hydride resonance at δ _H −23.00. Clearly, this catalyst does not transfer hyperpolarisation to Na ¹⁵ NO ₂ and hinders the overall ¹⁵ N signal gain due to consumption of p-H ₂ . Therefore, we conclude that the optimum DMAP level lies between 6-10 equivalents with respect to iridium and this results in 6D being the dominant species in solution. Example 6 Synthesis and Utilization of DMAP-d ₂ for SABRE Deuteration of ligands within the active catalyst can provide a route to improved polarisation transfer due to reduced spin-dilution and an increased lifetime of the hyperpolarised state. ^{42, 75-76, 89-90} We postulated that deuteration of the ortho protons in D to give DMAP-d ₂ may lead to further improvements in the ¹⁵ N polarisation level in Na ¹⁵ NO2. Therefore, DMAP-d2 was synthesized via H/D exchange from DMAP in D ₂ O under microwave irradiation as reported in the literature. ⁹¹ Preparation of a sample containing [IrCl(COD)(IMes)] (5 mM), DMAP-d ₂ (6 eq.) and Na ¹⁵ NO ₂ (25 eq.) in methanol-d ₄ and exposure to 3 bar p-H ₂ at a polarisation transfer at −3.5 mG led to a signal gain of 13811 after interrogation at 9.4 T (4.56% ¹⁵ N polarisation level). The corresponding value with protio DMAP is 12036, and hence introducing the ² H can be concluded to be beneficial to the SABRE outcome. Interesting, no significant change in the T ₁ value of the hyperpolarised ¹⁵ NO ₂ ⁻ signal was seen and we conclude that the signal improvement results mainly from reduced spin-dilution around the active centre. Example 7 Effect of NHC identity on the efficiency of Na ¹⁵ NO ₂ polarisation Aiming to improve the polarisation outcome still further, a study of the effect of the NHC ligand was completed in conjunction with DMAP-d2. Previously, we have shown how manipulation of the steric and electronic properties of this ancillary ligand can result in improved ¹ H, ¹³ C and ¹⁵ N signal enhancements due to changes in the rate of ligand exchange. ⁶³ Having first noted that dissociation of the ¹⁵ NO ₂ ⁻ ligand bound in the equatorial plane is slower than that which would be predicted to be optimum, we sequentially increased the steric bulk of the NHC (quantified by the magnitude of %BurV ^92-93 ) to drive ligand exchange. On moving from the IMes ligand (%BurV 31.2) to SIMes (32.7) we saw a 14628-fold signal gain which is a slight improvement from the 13811-fold signal gain previously observed for IMes. IPr (33.6) and SIPr (35.7) both also led to increased signal enhancements of 15799 and 17149-fold. However, the best result was obtained for IPent which gave a 20337-fold signal gain and has the highest %BurV of 43.4. Next our focus turned to the electronic properties of the NHC ligand. As expected, electron deficient IMes ^Cl , which has chloro substituents on the imidazole ring, reduced the signal enhancement to just 5471. Introducing methyl groups on the imidazole ring showed minimal effect when compared to IMes (13336-fold vs. 13811- fold respectively). However, introduction of a single –NMe ₂ group increased the signal enhancement level to 16427-fold at 9.4 T. To combine the steric and electronic effects, we utilized the ligand IPr ^NMe2 , which has previously proven to be effective for Buchwald-Hartwing amination catalysis, ^94-95 to form the precatalyst [IrCl(COD)(IPr ^NMe2 )]. This catalyst gave the highest ¹⁵ N signal enhancement for Na ¹⁵ NO ₂ of 21967-fold at 9.4 T which is equivalent to 7.2% polarisation. These results are summarised in Figure 3. Example 8 Effect of Na ¹⁵ NO ₂ concentration on signal enhancement A series of samples were interrogated that contained varying concentration of Na ¹⁵ NO ₂ that differed from the standard 25 eq. that had been employed so far in this patent but utilized the optimum catalyst [IrCl(COD)(IPr ^NMe2 )] (5 mM) and DMAP–d ₂ . Reducing the substrate concentration to 10 eq. with respect to iridium increased the signal enhancement to 36629-fold. With just 4 eq. of Na ¹⁵ NO ₂ a signal enhancement of 62470-fold was quantified at 9.4 T which corresponds to 20.6% ¹⁵ N polarisation. Conversely increasing the substrate loading to 50 eq. reduced the signal gain to 10382 (3.17%), however, the final signal to noise level was improved. If the concentration of [IrCl(COD)(IPr ^NMe2 )] is reduced to 0.25 mM whilst maintaining 1:4 molar ratio with Na ¹⁵ NO ₂ the ¹⁵ N polarisation level further increases to 28.42%. This phenomenon is likely to be due to effectively increasing the excess of p-H ₂ relative to substrate and catalyst and has been observed previously. ^{56, 63} Example 9 Detection of unlabelled NaNO ₂ Given the high signal gains obtained for Na ¹⁵ NO ₂ , we wished to test the analogous SABRE hyperpolarization effect using NaNO ₂ where the ¹⁵ N label was present at natural abundance. When such a sample, containing 20 mM of NaNO ₂ , was examined with the [IrCl(COD)(IPr ^NMe2 )] (5 mM) and DMAP–d ₂ (6 eq,), a ¹⁵ N signal was easily seen whose signal enhancement was 115592-fold at 9.4 T; this corresponds to a 38.1% P _15N level. Example 10 Effect of 15-crown-5 and utilisation of biphasic catalysis Unfortunately, the ionic nature of NaNO ₂ acts to limit its solubility in the organic solvents typically employed for SABRE catalysis; it has a moderate solubility in methanol, however, it is sparingly soluble in other primary alcohols and insoluble in most apolar solvents. In an attempt to increase methanol-d ₄ solubility, the macrocycle 15-crown-5 was added, which has a high chelating affinity for Na ⁺ . ^96-97 SABRE transfer was therefore undertaken on a sample containing [IrCl(COD)(IMes)] (5 mM), Na ¹⁵ NO ₂ (25 eq.), DMAP (6 eq.) and 15-crown-5 (25 eq.) in methanol-d ₄ . This led to a ¹⁵ N signal enhancement of 12044-fold being obtained at 9.4 T. This corresponds to a ¹⁵ N polarisation level of 3.97%. It reflects a 10% improvement over the analogous measurement when 15-crown-5 is not present. Interestingly, the ratio of 6 _D to 7 _D was now 99 : 1 as opposed to 91 : 9 in the absence of 15-crown-5. Using the optimised catalyst and co-ligand ([IrCl(COD)(IPr ^NMe2 )] (5 mM), Na ¹⁵ NO ₂ (25 eq.), DMAP-d ₂ (6 eq.)) the effect of 15-crown-5 is less pronounced with the ¹⁵ N signal gain improving from 21967-fold to 23114-fold. When an NMR sample containing [IrCl(COD)(IMes)] (5 mM), Na ¹⁵ NO ₂ (25 eq.) and D (6 eq.) was prepared in dichloromethane-d ₂ (0.6 mL), the impact of insolubility of Na ¹⁵ NO ₂ was immediately evident. After sonication for 30 mins, the sample was exposed to H ₂ (3 bar). Interrogation by NMR spectroscopy revealed that the only hydride containing species present in solution was [Ir(H) ₂ Cl(IMes)(DMAP) ₂ ]. However, when an analogous sample was prepared containing 15-crown-5 in a 1 : 1 ratio with Na ¹⁵ NO ₂ , a different hydride containing complex formed. Its hydride resonances appear at ^ −22.66 ( ² J _HN = 27.5 Hz and ² J _HH = −7 Hz) and ^ −23.00 ( ² J _HH = −7 Hz) and match those of 6 _D . After SABRE transfer in a −3.5 mG field, a 3586-fold signal enhancement was observed at 9.4 T for the free ¹⁵ NO ₂ ⁻ resonance δ _N 618. Warming this sample to 308 K prior to polarisation transfer significantly improved the signal gain to 7248-fold and indicates that slow ligand exchange limits the polarisation level attained. However, warming further to 323 K yielded no further increase. Using the electron rich and sterically encumbered pre-catalyst [IrCl(COD)(IPr ^NMe2 )], also yielded improved polarisation transfer as an 8149-fold signal gain is seen at 9.4 T. Warming this sample further, however, had no benefit. Hence, we have demonstrated how significant polarisation levels for ¹⁵ NO ₂ ⁻ can be achieved in dichloromethane-d ₂ by the addition of 15-crown-5. Example 11 Producing hyperpolarised NO ₂ ⁻ in water For biological applications, it is desirable to produce hyperpolarised NO ₂ ⁻ in water. Unfortunately, 6 _A did not form when the analogous reaction was screened in this solvent. However, it could be obtained via its prior formation in methanol, solvent removal and replaced by D ₂ O. Preforming 6 _A in methanol-d ₄ prior to removing the solvent and replacing it with D ₂ O was also unsuccessful. One further other way to achieve this outcome is to use a biphasic ⁹⁸ dichloromethane-d ₂ approach which benefits from the fact the catalyst is not present in the aqueous layer. ^99-100 A sample containing [IrCl(COD)(IMes)] (5 mM), Na ¹⁵ NO ₂ (25 eq.) and DMAP (6 eq.) and 15- crown-5 (25 eq.) in dichloromethane-d ₂ (0.3 mL) was prepared and exposed to H ₂ (3 bar) to form the active catalyst. D ₂ O (0.3 mL) was then added under a nitrogen atmosphere. After SABRE transfer at −3.5 mG and phase separation, two hyperpolarised signals were seen in the result in the corresponding ¹⁵ N NMR spectrum for Na ¹⁵ NO ₂ at δ _N 618 and δ _N 609. These peaks had relative intensities of 1:70 and were assigned to Na ¹⁵ NO ₂ dissolved in the dichloromethane-d ₂ and D ₂ O phases respectively by comparison with data from independent solutions. The ¹⁵ N signal gain in the D ₂ O layer was estimated to be 4667-fold, assuming all of the Na ¹⁵ NO ₂ was present in it; this underestimates the real value. As 15-crown-5 can also play a role as a phase transfer catalyst, ¹⁰¹ a further 25 eq. was added to the sample and this proved to increase the signal enhancement level to 13794-fold. Further additions of 15-crown-5 did not improve on this, however, but warming the sample to 308 K resulted in a 26327-fold signal gain at 9.4 T. This is equivalent to an 8.69% polarisation level in Na ¹⁵ NO ₂ . Hence, we have created a simple route to hyperpolarised Na ¹⁵ NO ₂ in water. The level of signal gain compares favourably with the <1% reported with DNP. ¹² See figure 9. Example 12 Assessment of Na ¹⁵ NO ₂ relaxation rates The DNP hyperpolarised Na ¹⁵ NO ₂ is reported to have a T ₁ of 14.8 s in D ₂ O. ¹² We used a low-tip angle approach to assess the T ₁ of our SABRE polarised product at 9.4 T. It was found to be 16.45 s. This value was also probed using an automated hyperpolarisation device under reversible flow, after first conducting the SABRE process at −3.5 mG, prior to turning off the p-H ₂ supply and holding the sample in a defined magnetic field for a period of time, prior to transfer to 9.4 T to acquire a spectrum. Repeating this process for varying time points, enables the effective low field T ₁ value to be calculated. This was undertaken for samples stored in the mu- metal shield (ca. 300-fold shielding) and a -3.5 mT. The new T ₁ values were 14.9 s and 11.2 s respectively. These values suggest that there will be sufficient time to use ¹⁵ NO ₂ ⁻ synthetically to create other hyperpolarised products as 3 x T ₁ is available before the signal vanishes. Interestingly, as the T ₁ values for ¹⁵ N nuclei can dramatically be extended at appropriate magnetic fields reaction time of many minutes may be possible. Example 13 Conversion of hyperpolarised Na ¹⁵ NO ₂ to a diazonium via NO ⁺ The Sandmeyer reaction rapidly converts arylamines to an arylhalide via a diazonium salt intermediate. ¹⁰² Since the first reported example in 1884, ¹⁰³ it has become a mainstay of organic chemistry and many related reactions have been discovered. ¹⁰⁴ Classically, it utilizes either stoichiometric or catalytic amounts of copper halides, although, a number of metal free variants are known. ^105-107 The formation of the diazonium salt intermediate proceeds via nitrous acid addition, which is formed in situ from the reaction of NaNO ₂ and a strong acid. We sought to follow a diazotization reaction by SABRE hyperpolarised ¹⁵ N NMR spectroscopy. Therefore, we created hyperpolarised Na ¹⁵ NO ₂ using the previously optimized conditions ([IrCl(COD)(IMes)] (5 mM), DMAP (30 mM), Na ¹⁵ NO ₂ (125 mM) in methanol-d ₄ (0.6 mL)) prior to adding a solution of aniline (150 mM) and conc. HCl (100 ^L) in methanol-d ₄ (100 µL). The NMR tube was immediately transferred to the spectrometer and interrogated using a T ₁ corrected variable flip angle pulse sequence. It took between 3 and 5 s to start the measurements and hyperpolarised signals indicative of nitrous acid (H ¹⁵ NO ₂ , δ _N 563) and for phenyl diazonium chloride (δ _N 314) and ortho- ¹⁵ N ₂ (δ _N 309) were evident in these NMR spectra. Their identity was confirmed by their independent synthesis, and comparison to literature data. ¹⁰⁸ Over the course of 30 s, the signals for H ¹⁵ NO ₂ vanished. See Figure 4. Upon repeating this process using aniline- ¹⁵ N, reaction monitoring revealed the detection of hyperpolarised responses for both of the ¹⁵ N centres in the diazo product, at δ _N 315.0 and 232.6 which are consistent with values reported in the literature. ¹⁰⁹ This happens even though aniline itself was not hyperpolarised. Consequently, efficient polarisation transfer takes place during the reaction at low field within the coupled spin pair. As a control, we exposed a sample of phenyl diazonium chloride and the catalyst to p-H ₂ at −3.5 mG and noted no hyperpolarised ¹⁵ N resonances result. Consequently, all the hyperpolarised signals seen during this reaction originate from the initially hyperpolarised Na ¹⁵ NO ₂ . Example 14 Reactions of hyperpolarised ¹⁵ N ₂ -phenyl diazonium chloride and therefore the creation of hyperpharmaceuticals via NO ₂ - The phenyl diazonium chloride product proved to have hyperpolarised T1 values for the ¹⁵ N ₁ and ¹⁵ N ₂ nuclei of 29.4 and 39.2 s respectively at 9.4 T. Additionally, it proved to be relatively stable under these reactions conditions with only limited decomposition to hyperpolarised ¹⁵ N ₂ (g) (δ _N 309) being observed. This meant we could explore further the reactivity of the diazonium salt. It is known that such salts liberate N ₂ under photochemical or transition metal catalysed processes. ^110-111 Under our hyperpolarised regime, addition of CuI saw the rapid conversion of the phenyl diazonium chloride into N ₂ which is visible in solution through a signal at δ _N 308. It was also treated with NaN ₃ to form hyperpolarised phenyl azide. This material yields ¹⁵ N NMR resonances at δ _N 242.2 and 90.1 that share a common ² J _NN of 13.8 Hz. According to the literature, this reaction could proceed via a linear and/or a cyclic intermediate, which would deliver 5 and 3 distinct ¹⁵ N signals respectively. ^112-114 It interestingly we detect this transient species through signals at δ _N 356.8 and 298.2 ( ² JNN = 16.7 Hz) for the site connected to the C ₆ H ₅ ring which we assign to this product. Upon repeating this study with 1- ¹⁵ N NaN ₃ , these two signals gain further complexity and appear alongside one other at δ _N 387.3 (d, 17 Hz). These additional features are reflective of the two possible isotopologues that can result from ¹⁵ N ₁ -N ₃ - addition to form a cyclic intermediate, which place a Ph- ¹⁵ N next to two ¹⁵ N groups (a triplet at δ _N 298.6 with 17 Hz is seen for it alongside a doublet of 17 Hz at δ _N 356.9) or one (a doublet at δ _N 298.6 of 17 Hz is now seen, alongside a further triplet at 356.9 of 17 Hz and a doublet at δ _N 387.3 (d 17 Hz). Hence all three signals for this cyclic intermediate have been detected. We note its conversion to phenyl azide (Ph- ¹⁵ N= ¹⁵ N ⁺ = ¹⁵ N- and Ph- ¹⁵ N= ¹⁵ N ⁺ =N-) proceeds rapidly at 298 K and the signals for this product appear δ _N 90.3, 242.5 and 232 with apparent T ₁ values of 56, 192 and 101 s at 9.4 T, all respectively. See Figure 5 and 7. These long T ₁ values enable the creation of strongly hyperpolarized and reactive phenyl azide. When the reactive alkyne, (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9- ylmethanol, ¹²¹ is added to this sample as a third synthetic step, reaction to form the corresponding triazole occurs. Despite the corresponding ¹⁵ N signals for this product having sub 10 s T ₁ values, its formation is readily indicated in the associated hyperpolarized ¹⁵ N NMR measurements (δ _N 335.4 and 255.3 with a mutual ² J _NN 12.8 Hz). As copper-free click-chemistry is used widely for bioconjugation with nuclei acids we expect such measurements to help in the optimization of pharmaceutical preparations and/or in vivo detection. ^122-123 These data (see Figure 6) have clearly illustrated the successful examination of a multistep reaction as it proved possible to simultaneously see ¹⁵ N signals for the phenyl diazonium salt, the pentazole intermediate and phenyl azide or the pentazole intermediate, phenyl azide and the triazole. The detection of such species can be used in conjunction with the optimisation of a synthetic procedure or to create a hyperpharmaceutical. Example 15 Conversion of hyperpolarised Na ¹⁵ NO ₂ to ¹⁵ N ₂ through reaction with ¹⁵ NH ₄ Cl/HCl N ₂ gas spontaneously forms from the diazonium salts of primary amines. As ¹⁵ NH ₄ Cl is readily available we monitored its reaction with Na ¹⁵ NO ₂ and as expected see strong signals for ¹⁵ N ₂ in solution (see Figure 6). We have therefore detailed two approaches that ultimately are expected to lead to p-N ₂ ^{57, 115} . Example 16 Utilization of ¹⁵ N hyperpolarised azide, amines and ammonia as probes of reactivity and therefore the creation of hyperpharmaceuticals The SABRE hyperpolarisation of NH ₃ and amines, such as benzylamine, and their use in SABRE-Relay has been extensively reported. ^{81-82, 85-87} Additionally, ammonia and amines have been used as a co-ligand that leads to improved SABRE catalysis. ^{68, 116} The ¹⁵ N polarisation of benzylamine- ¹⁵ N (E- ¹⁵ N) is reported to be ca. 800-fold at 9.4 T. This involves [Ir(H) ₂ (IMes)(E- ¹⁵ N) ₃ ]Cl in dichloromethane-d ₂ solution. ⁸⁵ We restudied this process in order to improve the SABRE outcome and provide access to a further functional group to demonstrate hyperpolarised reactivity screening. Using the same conditions as previously reported ([IrCl(COD)(IMes)] (5 mM), benzylamine- ¹⁵ N (E- ¹⁵ N, 7 eq.)) we determined that optimal SABRE transfer occurs at −4 mG. At this field a 7751-fold signal enhancement was achieved at 9.4 T. As the rate of benzylamine dissociation from [Ir(H) ₂ (IMes)(E- ¹⁵ N) ₃ ]Cl in dichloromethane- d ₂ is slow ^{45, 86} we found that warming the sample to 308 K further improved the enhancement level to 11211-fold which corresponds to 3.7% ¹⁵ N polarisation; it has a 14 s T ₁ at 9.4 T in the absence of the catalyst, and 12.8 s when it is present. As amines show a wide range of reactivity, we exemplified the utilisation of hyperpolarised E- ¹⁵ N as a synthon for amidation, sulfamidation and imine formation. This resulted in the ¹⁵ N detection of the products shown in Figure 6. Their identity was verified by independent synthesis. Specifically, the addition of trifluoroacetic anhydride to hyperpolarised E- ¹⁵ N led to the formation and detection of N-benzyl-trifluoroacetamide- ¹⁵ N in the resulting ¹⁵ N NMR spectrum through a signal at δ _N 116.4. Similarly, triflic anhydride reacted to yield the analogous sulfonamide with a resonance at δ _N 88.6. Finally, addition of benzaldehyde to E- ¹⁵ N produced the imine condensation product as evident by a peak at δ _N 327.7. Ammonia is also widely used in synthetic chemistry and we sought to exemplify its hyperpolarised reactivity. Due to ¹⁵ NH _3(g) being expensive and difficult to handle, we pursued the use of an alternative ammonia source. By using a 1 : 1 mixture of ¹⁵ NH ₄ Cl/KO ^t Bu we were able to form the active SABRE catalyst [Ir(H) ₂ (IMes)( ¹⁵ ND ₃ ) ₃ ]Cl in methanol-d ₄ . After polarisation transfer at −4 mG, a 3268-fold ¹⁵ N signal gain was quantified. However, over the course of ca. 1 h, the signal enhancement diminishes when the SABRE process is repeated. In contrast, the use of ¹⁵ NH ₄ OH (available as a ¹⁵ N solution in H ₂ O) yielded the same active catalyst, but the sample is now stable >24 h. The ¹⁵ N signal enhancement is also slightly improved to 3765-fold. Changing the NHC ligand proved to have a modest effect on SABRE efficacy and warming the sample to 308 K now improved the signal gain to 4521-fold. However, dramatic improvements result with a co-ligand. Whilst the co- ligands DMSO-d ₆ , CD ₃ CN, NO ₂ ⁻ and DMAP were explored, pyridine-d ₅ proved to give the highest signal gain of 15145-fold (5.0% polarisation). The ¹⁵ N T ₁ value for ⁵ ND ₃ at 9.4 T proved to 37 s which means the wide over which a reaction can be examined is substantial. Protonation of ¹⁵ ND ₃ with DCl in D ₂ O led to the detection of hyperpolarised ¹⁵ ND ₄ ⁺ as a signal δ _N 15.93 with resolved ¹⁵ N-D scalar couplings, J _ND , of 10.8 Hz and a hyperpolarised T ₁ of 33.6 s. The SABRE hyperpolarization of the versatile synthon 1- ¹⁵ N NaN ₃ itself, using the co-ligand strategy with DMAP and 1 also proved successful. The reaction was found to proceed to form [Ir(H) ₂ (DMAP) ₂ (IMes)( ¹⁵ N=N=N)] which exhibits hydride signals at δ _H −23.1 ( ² J _HH = 8 Hz) and δ _H −25.0 ( ² J _HH = 8 Hz and ² J _NH = 8 HZ) alongside [Ir(H) ₂ (DMAP) ₃ (IMes)]Cl (δ _H −22.8). SABRE transfer at −3.5 mG yielded 3.2% hyperpolarization of the N ₃ ⁻ signal at ^ _N 95.7. A hyperpolarised solution of NaN ₃ was then reacted directly with (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol to form the triazole. Under these conditions, a single hyperpolarized ¹⁵ N response for the product was visible at ^ 321.3 as expected. The screen of this reagents reactivity has also been exemplified. We note its use in the formation of N ₂ in air bags, so using it as a simple solid state source of hyperpolarised N ₂ is feasible. Example 17 SABRE hyperpolarisation of Na ¹⁵ NO ₃ In contrast to nitrite, nitrate is usually non-coordinating, however, there are examples of it functioning as a weak monodentate or bidentate ligand. ^{27, 117-121} To further explore SABRE’s use as a tool to polarise materials featuring in the nitrogen cycle we explored the SABRE hyperpolarisation of Na ¹⁵ NO ₃ . As expected, in the absence of a co-ligand no active SABRE catalyst formed in the reaction between [IrCl(COD)(IMes)] and Na ¹⁵ NO ₃ under a H ₂ atmosphere (3 bar) in methanol-d ₄ . We screened a number of co-ligands (DMAP, 2-picoline, DMSO-d ₆ , DPSO or CD ₃ CN) and saw no evidence for the ¹⁵ N polarisation of Na ¹⁵ NO ₃ . In each case, the dominant hydride containing species in solution was [Ir(H) ₂ (IMes)(sub) ₃ ]Cl or [IrCl(H) ₂ (IMes)(sub) ₂ ]. However, when the ionic precatalyst [Ir(COD)(IMes)(pyridine)]BF ₄ was used with DMSO-d ₆ (2 eq.) a 547-fold signal enhancement for the ¹⁵ NO ₃ - signal at 9.4 T (0.18% ¹⁵ N polarisation) is observed. Example 18 Unexpected Reduction of Sodium Nitrate During the course of these investigations, a ¹⁵ N NMR signal appears at δ _N 511.28 (J _HN = 28.5 Hz) appears almost immediately when pyridine is used as a co-ligand. This matches the equatorial NO ₂ - resonance previously observed for [Ir(H) ₂ ( ¹⁵ NO ₂ )(IMes)(pyridine) ₂ ] (6 _A ). Relatively strong polarised signals for free and pyridine trans to hydride in [Ir(H) ₂ (IMes)(py) ₃ ]Cl ( δ _N 299.6 and 255.7 respectively) were also observed in these NMR spectra. As expected, the corresponding ¹ H NMR spectrum is dominated by the hydride signal of [Ir(H) ₂ (IMes)(py) ₃ ]Cl which appears at δ _H −22.7, although a weakly PHIP enhanced signal for 6 _A is visible in this spectrum at δ _H −21.49 (the peak at δ _H −22.71 cannot be observed due to overlap). No evidence for 7 _A was observed in either the ¹ H or ¹⁵ N NMR spectra which indicates that 6 _A is likely to be the kinetic product of this reaction. After waiting for 1 h, refreshing the sample with p-H ₂ and repeating the SABRE process, a polarised signal for free Na ¹⁵ NO ₂ (δ _N 611.9) could also be detected and the signal for 6 _A also increased in size. We therefore suspect that the reducing environment of this medium converts nitrate to nitrite in a metal catalysed reduction. To further probe this reduction, a sample containing [IrCl(COD)(IMes)] (20 mM), pyridine (3 eq.) and Na ¹⁵ NO3 (25 eq.) was exposed to 3 bar of H ₂ at 298 K for 24 h and the growth of the hydride ligand resonance for 6 _A at δ _N −21.49 was monitored by thermally polarised ¹ H NMR spectroscopy. The resulting integral data could be fitted to an exponential growth curve. After 24 h, and refreshing the H ₂ atmosphere, further conversion to 6 _A could again be seen which indicates that H ₂ is needed to drive this reaction.

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