Real-Time High-Sensitivity Reaction Monitoring of Important Nitrogen-Cycle Synthons by 15N Hyperpolarized Nuclear Magnetic Resonance.

Here, we show how signal amplification by reversible exchange hyperpolarization of a range of 15N-containing synthons can be used to enable studies of their reactivity by 15N nuclear magnetic resonance (NO2- (28% polarization), ND3 (3%), PhCH2NH2 (5%), NaN3 (3%), and NO3- (0.1%)). A range of iridium-based spin-polarization transfer catalysts are used, which for NO2- work optimally as an amino-derived carbene-containing complex with a DMAP-d2 coligand. We harness long 15N spin-order lifetimes to probe in situ reactivity out to 3 × T1. In the case of NO2- (T1 17.7 s at 9.4 T), we monitor PhNH2 diazotization in acidic solution. The resulting diazonium salt (15N-T1 38 s) forms within 30 s, and its subsequent reaction with NaN3 leads to the detection of hyperpolarized PhN3 (T1 192 s) in a second step via the formation of an identified cyclic pentazole intermediate. The role of PhN3 and NaN3 in copper-free click chemistry is exemplified for hyperpolarized triazole (T1 < 10 s) formation when they react with a strained alkyne. We also demonstrate simple routes to hyperpolarized N2 in addition to showing how utilization of 15N-polarized PhCH2NH2 enables the probing of amidation, sulfonamidation, and imine formation. Hyperpolarized ND3 is used to probe imine and ND4+ (T1 33.6 s) formation. Furthermore, for NO2-, we also demonstrate how the 15N-magnetic resonance imaging monitoring of biphasic catalysis confirms the successful preparation of an aqueous bolus of hyperpolarized 15NO2- in seconds with 8% polarization. Hence, we create a versatile tool to probe organic transformations that has significant relevance for the synthesis of future hyperpolarized pharmaceuticals.

A role for low concentration reaction intermediates in the signal amplification by reversible exchange process revealed by theory and experiment.

A route to monitor the involvement of less abundant species during the catalytic transfer of hyperpolarisation from parahydrogen into a substrate is detailed. It involves probing how the degree of hyperpolarisation transfer catalysis is affected by the magnetic field experienced by the catalyst during this process as a function of temperature. The resulting data allow the ready differentiation of the roles played by hard to detect and highly reactive complexes, such as [Ir(H)2(NHC)(substrate)2(methanol)]Cl, from dominant species such as [Ir(H)2(NHC)(substrate)3]Cl. The difference in behaviour results from changes in the interligand spin-spin coupling network within the active SABRE catalysts.

Delivering strong 1H nuclear hyperpolarization levels and long magnetic lifetimes through signal amplification by reversible exchange.

Hyperpolarization turns typically weak NMR and MRI responses into strong signals so that ordinarily impractical measurements become possible. The potential to revolutionize analytical NMR and clinical diagnosis through this approach reflect this area's most compelling outcomes. Methods to optimize the low-cost parahydrogen-based approach signal amplification by reversible exchange with studies on a series of biologically relevant nicotinamides and methyl nicotinates are detailed. These procedures involve specific 2H labeling in both the agent and catalyst and achieve polarization lifetimes of ca 2 min with 50% polarization in the case of methyl-4,6-d2 -nicotinate. Because a 1.5-T hospital scanner has an effective 1H polarization level of just 0.0005% this strategy should result in compressed detection times for chemically discerning measurements that probe disease. To demonstrate this technique's generality, we exemplify further studies on a range of pyridazine, pyrimidine, pyrazine, and isonicotinamide analogs that feature as building blocks in biochemistry and many disease-treating drugs.

Parahydrogen-Induced Hyperpolarization of Gases.

Imaging of gases is a major challenge for any modality including MRI. NMR and MRI signals are directly proportional to the nuclear spin density and the degree of alignment of nuclear spins with applied static magnetic field, which is called nuclear spin polarization. The level of nuclear spin polarization is typically very low, i.e., one hundred thousandth of the potential maximum at 1.5 T and a physiologically relevant temperature. As a result, MRI typically focusses on imaging highly concentrated tissue water. Hyperpolarization methods transiently increase nuclear spin polarizations up to unity, yielding corresponding gains in MRI signal level of several orders of magnitude that enable the 3D imaging of dilute biomolecules including gases. Parahydrogen-induced polarization is a fast, highly scalable, and low-cost hyperpolarization technique. The focus of this Minireview is to highlight selected advances in the field of parahydrogen-induced polarization for the production of hyperpolarized compounds, which can be potentially employed as inhalable contrast agents.

Catalyst-Substrate Effects on Biocompatible SABRE Hyperpolarization.

The hyperpolarization technique, Signal Amplification by Reversible Exchange (SABRE), has the potential to improve clinical diagnosis by making molecular magnetic resonance imaging in vivo a reality. Essential to this goal is the ability to produce a biocompatible bolus for administration. We seek here to determine how the identity of the catalyst and substrate affects the cytotoxicity by in vitro study, in addition to reporting how the use of biocompatible solvent mixtures influence the polarization transfer efficiency. By illustrating this across five catalysts and 8 substrates, we are able to identify routes to produce a bolus with minimal cytotoxic effects.

Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy.

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5s), therefore direct NO quantification is challenging. An indirect method - based on quantifying conversion of an l-Arg- to l-Cit-derivative by 1H NMR spectroscopy - is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a-j and 11a-c were better or equivalent substrates for the eNOS enzyme (NO2- production=19-46µM) than native l-Arg (NO2- production=25µM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by 1H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.

Harnessing polarisation transfer to indazole and imidazole through signal amplification by reversible exchange to improve their NMR detectability.

The signal amplification by reversible exchange (SABRE) approach has been used to hyperpolarise the substrates indazole and imidazole in the presence of the co-ligand acetonitrile through the action of the precataysts [IrCl(COD)(IMes)] and [IrCl(COD)(SIMes)]. 2 H-labelled forms of these catalysts were also examined. Our comparison of the two precatalysts [IrCl(COD)(IMes)] and [IrCl(COD)(SIMes)], coupled with 2 H labelling of the N-heterocyclic carbene and associated relaxation and polarisation field variation studies, demonstrates the critical and collective role these parameters play in controlling the efficiency of signal amplification by reversible exchange. Ultimately, with imidazole, a 700-fold1 H signal gain per proton is produced at 400 MHz, whilst for indazole, a 90-fold increase per proton is achieved. The co-ligand acetonitrile proved to optimally exhibit a 190-fold signal gain per proton in these measurements, with the associated studies revealing the importance the substrate plays in controlling this value. Copyright © 2017 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.

Characterisation and evaluation of paramagnetic fluorine labelled glycol chitosan conjugates for (19)F and (1)H magnetic resonance imaging.

Medium molecular weight glycol chitosan conjugates have been prepared, linked by an amide bond to paramagnetic Gd(III), Ho(III) and Dy(III) macrocyclic complexes in which a trifluoromethyl reporter group is located 6.5 Å from the paramagnetic centre. The faster relaxation of the observed nucleus allows modified pulse sequences to be used with shorter acquisition times. The polydisperse materials have been characterised by gel permeation chromatography, revealing an average molecular weight on the order of 13,800 (Gd), 14,600 (Dy) and 16,200 (Ho), consistent with the presence of 8.5, 9.5 and 13 complexes, respectively. The gadolinium conjugate was prepared for both a q = 1 monoamide tricarboxylate conjugate (r1p 11.2 mM(-1) s(-1), 310 K, 1.4 T) and a q = 0 triphosphinate system, and conventional contrast-enhanced proton MRI studies at 7 T were undertaken in mice bearing an HT-29 or an HCT-116 colorectal tumour xenograft (17 µmol/kg). Enhanced contrast was observed following injection in the tail vein in tumour tissue, with uptake also evident in the liver and kidney with a tumour-to-liver ratio of 2:1 at 13 min, and large amounts in the kidney and bladder consistent with predominant renal clearance. Parallel experiments observing the (19)F resonance in the holmium conjugate complex using a surface coil did not succeed owing to its high R2 value (750 Hz, 7 T). However, the fluorine signal in the dysprosium triphosphinate chitosan conjugate [R1/R2 = 0.6 and R1 = 145 Hz (7 T)] was sharper and could be observed in vivo at -65.7 ppm, following intravenous tail vein injection of a dose of 34 µmol/kg.

Iridium(III) hydrido N-heterocyclic carbene-phosphine complexes as catalysts in magnetization transfer reactions.

The hyperpolarization (HP) method signal amplification by reversible exchange (SABRE) uses para-hydrogen to sensitize substrate detection by NMR. The catalyst systems [Ir(H)2(IMes)(MeCN)2(R)]BF4 and [Ir(H)2(IMes)(py)2(R)]BF4 [py = pyridine; R = PCy3 or PPh3; IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene], which contain both an electron-donating N-heterocyclic carbene and a phosphine, are used here to catalyze SABRE. They react with acetonitrile and pyridine to produce [Ir(H)2(NCMe)(py)(IMes)(PPh3)]BF4 and [Ir(H)2(NCMe)(py)(IMes)(PCy3)]BF4, complexes that undergo ligand exchange on a time scale commensurate with observation of the SABRE effect, which is illustrated here by the observation of both pyridine and acetonitrile HP. In this study, the required symmetry breaking that underpins SABRE is provided for by the use of chemical inequivalence rather than the previously reported magnetic inequivalence. As a consequence, we show that the ligand sphere of the polarization transfer catalyst itself becomes hyperpolarized and hence that the high-sensitivity detection of a number of reaction intermediates is possible. These species include [Ir(H)2(NCMe)(py)(IMes)(PPh3)]BF4, [Ir(H)2(MeOH)(py)(IMes)(PPh3)]BF4, and [Ir(H)2(NCMe)(py)2(PPh3)]BF4. Studies are also described that employ the deuterium-labeled substrates CD3CN and C5D5N, and the labeled ligands P(C6D5)3 and IMes-d22, to demonstrate that dramatically improved levels of HP can be achieved as a consequence of reducing proton dilution and hence polarization wastage. By a combination of these studies with experiments in which the magnetic field experienced by the sample at the point of polarization transfer is varied, confirmation of the resonance assignments is achieved. Furthermore, when [Ir(H)2(pyridine-h5)(pyridine-d5)(IMes)(PPh3)]BF4 is examined, its hydride ligand signals are shown to become visible through para-hydrogen-induced polarization rather than SABRE.

Lower ligand denticity leading to improved thermodynamic and kinetic stability of the Gd3+ complex: the strange case of OBETA.

OBETA, OBETA, you bet: Thermodynamic and kinetic measurements show an apparent paradox. The stability of complexes of lanthanide trivalent ions is higher with the heptadentate ligand OBETA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) than with its octadentate homologue EGTA (2,2'-oxybis(ethylamine)-N,N,N',N'-tetraacetic acid). The unusual properties of Gd(OBETA)(-) (see structure), combined with the presence of two fast exchanging coordinated water molecules, candidates this complex as an MRI contrast agent.

Lanthanide(III) complexes with ligands derived from a cyclen framework containing pyridinecarboxylate pendants. The effect of steric hindrance on the hydration number.

Two new macrocyclic ligands, 6,6'-((1,4,7,10-tetraazacyclododecane-1,7-diyl)bis(methylene))dipicolinic acid (H2DODPA) and 6,6'-((4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diyl)bis(methylene))dipicolinic acid (H2Me-DODPA), designed for complexation of lanthanide ions in aqueous solution, have been synthesized and studied. The X-ray crystal structure of [Yb(DODPA)](PF6)·H2O shows that the metal ion is directly bound to the eight donor atoms of the ligand, which results in a square-antiprismatic coordination around the metal ion. The hydration numbers (q) obtained from luminescence lifetime measurements in aqueous solution of the Eu(III) and Tb(III) complexes indicate that the DODPA complexes contain one inner-sphere water molecule, while those of the methylated analogue H2Me-DODPA are q = 0. The structure of the complexes in solution has been investigated by 1H and 13C NMR spectroscopy, as well as by theoretical calculations performed at the density functional theory (DFT; mPWB95) level. The minimum energy conformation calculated for the Yb(III) complex [Λ(λλλλ)] is in good agreement with the experimental structure in solution, as demonstrated by the analysis of the Yb(III)-induced paramagnetic 1H shifts. The nuclear magnetic relaxation dispersion (NMRD) profiles recorded for [Gd(Me-DODPA)]+ are typical of a complex with q = 0, where the observed relaxivity can be accounted for by the outer-sphere mechanism. However, [Gd(DODPA)]+ shows NMRD profiles consistent with the presence of both inner- and outer-sphere contributions to relaxivity. A simultaneous fitting of the NMRD profiles and variable temperature 17O NMR chemical shifts and transversal relaxation rates provided the parameters governing the relaxivity in [Gd(DODPA)]+. The results show that this system is endowed with a relatively fast water exchange rate k(ex)(298) = 58 × 10(6) s(­1).

Responsive Mn(II) complexes for potential applications in diagnostic Magnetic Resonance Imaging.

The investigation of new Mn(II)-based MRI/Molecular Imaging probes responsive to the enzyme tyrosinase for potential diagnostic applications is herein described. The expression of the enzyme tyrosinase, an oxidoreductase, is up-regulated in melanoma cancer cells. Three novel ligands (L(1), L(2) and L(3)) were designed as modified acyclic polyaminocarboxylate chelates by introducing an l-tyrosine residue in place of an aminoacetate unit. The corresponding Mn(II) complexes were fully characterised by (1)H NMR relaxometric techniques in aqueous media. The responsive activity towards the expression of tyrosinase was then assessed by monitoring the (1)H 1/T(1) relaxivity changes during incubation experiments in buffered solutions containing tyrosinase at different concentrations and in B16F10 melanoma cell homogenate. New insight on the mechanism of action of these systems was gained by measuring the magnetic field dependence of the relaxivity and ESR spectra of the incubated solutions. The systems developed showed responsive activity to tyrosinase with a relaxation enhancement spanning from 50% (MnL(1)) to 350% (MnL(3)) which augurs well for the development of diagnostic probes to detect melanoma cancer.

Remarkable Levels of 15N Polarization Delivered through SABRE into Unlabeled Pyridine, Pyrazine, or Metronidazole Enable Single Scan NMR Quantification at the mM Level.

While many drugs and metabolites contain nitrogen, harnessing their diagnostic 15N NMR signature for their characterization is underutilized because of inherent detection difficulties. Here, we demonstrate how precise ultralow field signal amplification by reversible exchange (±0.2 mG) in conjunction parahydrogen and an iridium precatalyst of the form IrCl(COD)(NHC) with the coligand d9-benzylamine allows the naturally abundant 15N NMR signatures of pyridine, pyrazine, metronidazole, and acetonitrile to be readily detected at 9.4 T in single NMR observations through >50% 15N polarization levels. These signals allow for rapid and precise reagent quantification via a response that varies linearly over the 2-70 mM concentration range.

Relayed hyperpolarization from para-hydrogen improves the NMR detectability of alcohols.

The detection of alcohols by magnetic resonance techniques is important for their characterization and the monitoring of chemical change. Hyperpolarization processes can make previously inpractical measurements, such as the determination of low concentration intermediates, possible. Here, we investigate the SABRE-Relay method in order to define its key characteristics and improve the resulting 1H NMR signal gains which subsequently approach 103 per proton. We identify optimal amine proton transfer agents for SABRE-Relay and show how catalyst structure influences the outcome. The breadth of the method is revealed by expansion to more complex alcohols and the polarization of heteronuclei.

Harnessing asymmetric N-heterocyclic carbene ligands to optimise SABRE hyperpolarisation.

The catalytic signal amplification by reversible exchange process has become widely used for the hyperpolarisation of small molecules to improve their magnetic resonance detectability. It harnesses the latent polarisation of parahydrogen, and involves the formation of a labile metal complex that often contains an N-heterocyclic carbene (NHC) ligand (e.g. [Ir(H)2(NHC)(pyridine)3]Cl), which act as a polarisation transfer catalyst. Unfortunately, if the target molecule is too bulky, binding to the catalyst is poor and the hyperpolarisation yield is therefore low. We illustrate here the behaviour of a series of asymmetric NHC containing catalysts towards 3,4- and 3,5-lutidine in order to show how catalyst design can be used to dramatically improve the outcome of this catalytic process for sterically encumbered ligands.

Deactivation of signal amplification by reversible exchange catalysis, progress towards in vivo application.

The catalyst which is used in the signal amplification by reversible exchange (SABRE) process facilitates substrate hyperpolarisation while acting to speed up the rate of relaxation. Consequently, the lifetime over which the hyperpolarised contrast agent is visible is drastically reduced. We show that the addition of a chelating ligand, such as bipyridine, rapidly deactivates the SABRE catalyst thereby lengthening the agent's relaxation times and improving the potential of SABRE for diagnostic MRI.

Activation of a (cyclooctadiene) rhodium(I) complex supported by a chiral ferrocenyl phosphine thioether ligand for hydrogenation catalysis: a combined parahydrogen NMR and DFT study.

The reaction of [RhCl(P,S(t)Bu)(COD)] (1) or [Rh(P,S(t)Bu)(COD)]BF4 (2) where (P,S(t)Bu) is CpFe[η(5)-1,2-C5H3(PPh2)(CH2S(t)Bu)] with H2 in MeOH gives rise to COD hydrogenation and formation of a solvent-stabilized product. The formation of hydride species cannot be observed in view of a very rapid H/D exchange between H2 and the solvent. Introduction of pyridine or acetonitrile slows down this exchange process and allows observation of diastereometric dihydride complexes, [Rh(P,S(t)Bu)(H)2(L)2](+), the stereochemistry of which was fully elucidated. The hydride site exchange rates have been derived from EXSY NMR experiments and used, with assistance from DFT calculation, to elucidate the isomerization and site exchange mechanisms.

Solution properties of the Ln(III) complexes of a novel octadentate chelator with rigidified iminodiacetate arms.

A novel octadentate ligand derived from the core structure of EGTA (ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid) with two piperidine-cis-dicarboxylic acid moieties spaced by a triethylenedioxy chain (L1) has been synthesised with the aim to assess the effect of the structural rigidity on the thermodynamic and kinetic stabilities of its Ln(III) complexes. The stability constants of [Ln(L1)](-) complexes show a weaker binding affinity for Ln(3+) ions of L1 than EGTA. On the other hand, the selectivity of L1 for Ca(2+) over Mg(2+) is one of the highest ever reported. The kinetic inertness has been also investigated and faster rates of both acid catalysed and metal assisted decomplexation have been measured for [Gd(L1)](-) as compared to [Gd(EGTA)](-). The (1)H- and (13)C NMR spectra of the diamagnetic La(3+), Y(3+) and Lu(3+) complexes in aqueous media show pronounced stereochemical rigidity and the occurrence of a structural change across the lanthanide series. Finally, a complete (1)H and (17)O NMR relaxometric study indicates that [Gd(L1)](-) possesses a nine-coordinate ground state with one inner coordination sphere water molecule characterized by a fast rate of exchange ((298)k(ex) = 59 × 10(6) s(-1)).

Cleavable ß-cyclodextrin nanocapsules incorporating Gd(III)-chelates as bioresponsive MRI probes.

Perthiolated ß-cyclodextrin-based nanocapsules incorporating diaquo Gd(III)-complexes represent a promising new type of bioresponsive MRI contrast agent, showing a pronounced relaxivity change upon degradation in a reducing environment.

Mn(II) complexes of novel hexadentate AAZTA-like chelators: a solution thermodynamics and relaxometric study.

Three novel chelators based on the 6-amino-6-methylperhydro-1,4-diazepine scaffold and possessing three pendant N-acetic or N-α-methylacetic acid have been synthesised. The ligands contain six donor atoms for complexation of Mn(II) ions and thus potentially leave an additional site for coordination of a water molecule. The protonation constants of the ligands and the stability constants of their complexes formed with Mn(II) ion were determined by pH-potentiometric titrations in 0.15 M NaCl solution at 25 °C and compared to those of the parent AAZTA ligand (AAZTA = 6-amino-6-methylperhydro-1,4-diazepine tetraacetic acid). In spite of the similar value of the total basicity (Σlog K), the values of the stability constants of the Mn(II)AAZTA-like complexes are more than three orders of magnitude lower than that of MnAAZTA (log K(MnL) = 14.19). A detailed (1)H and (17)O NMR relaxometric study was carried out on the Mn(II) complexes in aqueous solution as a function of pH, temperature and magnetic field strength. The (1)H NMRD profiles of all the complexes show a similar shape, typical of low-molecular weight systems, but amplitudes that markedly differ to indicate a different degree of hydration. A similar behaviour is shown by the (17)O NMR transverse relaxation rates and chemical shift data as a function of temperature. The experimental data can be rationalised by considering the presence in solution of a mixture of two isomeric species differing in coordination number (7 and 6) and in the number (1 and 0) of bound water molecules. Whereas this type of coordination equilibrium has been previously reported for lanthanide(III) complexes, it is observed for the first time on Mn(II) chelates.