Over20% ¹⁵N Hyperpolarization in UnderOne Minute for Metronidazole, an Antibiotic and Hypoxia Probe

NMR hyperpolarization techniquesincrease nuclear spin polarization from thermal equilibrium levelsof 10^–6–10^–5 by ordersof magnitude—in some cases approaching 100%.¹⁻⁴ Adequately chosen nuclear spinsites can retain their hyperpolarized (HP) state for many minutes,decaying exponentially back to thermal equilibrium; when this decayis slow enough, HP substances can be used as exogenous contrast agentsto probe metabolism and function in vivo.^2,5,615N sites are particularlyinteresting because of their greater (e.g., compared to ¹³C) decay constants of up to 20 min.⁷⁻¹⁰ Moreover, ¹⁵N isotopic enrichmentis frequently performed using relatively straightforward and efficientchemistries and therefore could be significantly more cost-effectivethan production of ¹³C-enriched compounds such as 1-¹³C-pyruvate.^11,12 Currently, the rapidly growingfield of HP ¹³C molecular contrast agents is dominatedby dissolution Dynamic Nuclear Polarization (d-DNP), which typicallyachieves ¹³C polarization levels well above 10%.¹³ However, for hyperpolarization of ¹⁵N sites, the efficiency of d-DNP has been limited to a few percentand currently requires hours of polarization time. To date, ¹⁵N-choline (P_¹⁵N ≈ 3%in 2.5 h),¹⁴ pH sensors (P_¹⁵N ≤ 3% in 2 h),¹⁵ and a calcium sensor (P_¹⁵N < 2%)¹⁶ have been polarizedby d-DNP for potential use as injectable HP contrast agents.

Direct hyperpolarization of heteronuclei with NMR Signal AmplificationBy Reversible Exchange (SABRE¹⁷) is possiblevia SABRE-SHEATH (SABRE in SHield Enables Alignment Transfer to Heteronuclei).^18,19 Unlike d-DNP, SABRE-SHEATH^18,19 is scalable, providesefficient hyperpolarization in seconds, and uses very simple and inexpensivehardware.^7,2015N SABRE-SHEATH of several biomoleculeshas been demonstrated (including nicotinamide,^11,18 imidazole,²¹ and diazirines⁷), producing P_¹⁵N of up to 10% in ≤1 min. It is our goal to expand therange of biomedically useful compounds amenable to SABRE-SHEATH, whichmay enable unprecedented molecular imaging and spectroscopy experimentsthat would provide direct visualization of biochemical processes.

Despite the success of ¹⁵N SABRE-SHEATH, so far thisnew approach has been limited to hyperpolarization of ¹⁵N sites directly binding to the polarization-transfer catalyst. Thispathway exploits short-range two-bond spin–spin couplings (J²_H-¹⁵N) for polarizationtransfer from parahydrogen to ¹⁵N sites (Figure 1a) in a μT magnetic field(B_T).^18,19 Furthermore,sterically hindering groups (e.g., methyl groups) in the ortho-positionsof six-membered N-heterocycles significantly reduce the efficiencyof polarization transfer.²² These shortcomingslimit the use of ¹⁵N-SABRE-SHEATH.

Figure 1

(a) Schematic diagramof the SABRE process: coherent polarizationtransfer from parahydrogen-derived hydrides to ¹⁵N heteronuclei.(b) Molecular structure of metronidazole. (c) Schematicrepresentation of the AA′B spin system in the polarizationtransfer complex. Red circles represent naturally abundant ¹⁵N nuclei. (d) Most-probable metronidazole-bound structuresof the complex with relevant AA′B spin systems.

Here we overcome these limitations and demonstrate ¹⁵N SABRE-SHEATH on metronidazole, a member ofan importantgroup of antibiotics—the nitroimidazoles. Nitroimidazoleswere discovered in the 1950s as a class of antibiotics acting on awide range of anaerobic bacterial infections.²³ Anaerobic conditions protect the organism’s employment ofpyruvate synthase, which consequently acts irreversibly on the nitroimidazolemoiety—leading to selective drug uptake by anaerobic bacterialcells in the host.²³ The sensitivity ofnitroimidazole moieties to anaerobic conditions was laterexploited for hypoxia sensing in cancer. Despite a different mechanismof action²³ in mammalian anaerobic cancercells, the end result is the same: the chemical reaction of the nitromoiety leads to irreversible binding in hypoxic cells.^24,25 A number of nitroimidazole compounds were employed asmolecular contrast agents to probe hypoxia and upregulation of hypoxiainducible factor (HIF-1α) using Positron Emission Tomography(PET),²⁶ and Magnetic Resonance (MR) imagingand spectroscopy.²⁷ Hypoxia is a hallmarkof some aggressive forms of cancer,^28,29 and its imagingcan be used as a biomarker for cancer screening and monitoring responseto treatment.^26,30 The investigational use of fluoromisonidazole(FMISO),²⁶ SR4554,²⁷ and numerous other related nitroimidazole-based agentsfor hypoxia imaging has advanced well into clinical trials. Furthermore,the intravenous injection of up to 1.4 g/m² of nitroimidazolederivatives is well tolerated in patients,²⁷ which would certainly be sufficient for HP imaging.³¹

In the present SABRE-SHEATH hyperpolarization studies,we employedthe most efficient IrIMes polarization-transfer catalyst (using theestablished Ir catalyst precursor [IrCl(COD)(IMes)]; IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene;COD = cyclooctadiene)^32,33 and metronidazolewith ¹⁵N at natural abundance (∼0.364%). As thisabundance is low, the statistically most-abundant polarization-transferspecies contains only one ¹⁵N site. Therefore, the relevantspin system for polarization transfer contains only three spins: twomagnetically inequivalent protons and one ¹⁵N site (Figure 1c).^22,34 Because metronidazole contains three nitrogen sites,it can form three types of AA′B spin systems, respectivelyinvolving two-bond, four-bond, or five-bond spin–spin couplings(Figure 1d). The ¹⁵N NMR signal from nitrogens only two bonds away from thenascent parahydrogen pair was enhanced by ∼72 000-foldat 9.4 T (corresponding to P_¹⁵N ≈ 24%; see Supporting Information (SI) for additional experimental and analysis details) in 50 mMsolution of metronidazole in methanol-d₄ using ∼80% parahydrogen (Figure 2b); these results may be extrapolated toexpect P_¹⁵N ≈ 32% if∼100% parahydrogen were employed. Note that the ¹⁵N polarization was sampled ∼8 s after cessation of parahydrogenbubbling, and the measurement does not take into account polarizationdecay losses that have occurred during the sample transfer from thehyperpolarization setup into the bore of the 9.4 T NMR spectrometer.The P_¹⁵N value of ∼24%is the highest ¹⁵N hyperpolarization level achieved byany technique to date by at least several-fold, and it is demonstrateddespite the presence of one or two ortho substituents, which havepreviously prevented the observation of SABRE-SHEATH enhancement ofsix-membered N-heterocycles.²² This highlevel of P_¹⁵N is not far awayfrom the practical limit of 50% theoretically predicted in recentliterature.³⁴⁻³⁶ Furthermore, investigations using more concentratedsolutions (∼150 mM, Figure 2c, and ∼100 mM, Figure S5) additionally enabled detection of relatively efficient SABRE-SHEATHhyperpolarization of the other two ¹⁵N sites (more weaklycoupled, via J⁴_H-¹⁵N and J⁵_H-¹⁵N couplings). Although these other two sites exhibited significantlymore modest P_¹⁵N values of0.1–0.3% (i.e., “only” ∼1000-fold enhancements),it should be noted that neither the μT field nor the exchangerates governing the SABRE-SHEATH process were optimized for hyperpolarizationof these two remote ¹⁵N sites. The feasibility of ¹⁵N SABRE-SHEATH processes via long-range (up to J⁵_H-¹⁵N) spin–spincouplings (in a manner similar to homonuclear SABRE^17,32,37) enables ¹⁵N hyperpolarizationof a wider range of biomolecules via SABRE-SHEATH, because more ¹⁵N and potentially other heteronculear sites become amenableto SABRE-SHEATH.

Figure 2

(a) ¹⁵N NMR spectrum of thermally polarizedsignal reference,neat pyridine-¹⁵N (∼12.4 M). (b) ¹⁵Nspectrum of HP natural abundance 50 mM metronidazole at∼298 K. Note the appearance of NMR resonances as a doubletof quartets due to spin–spin coupling of ¹⁵N witharomatic and methyl protons (inset). (c) ¹⁵N spectrum ofHP natural abundance 150 mM metronidazole at ∼298K. (d) Optimization of HP metronidazole ¹⁵NNMR signal by varying the magnetic field in the shield (B_T). (e) Optimization of HP metronidazole ¹⁵N NMR signal by varying the temperature (estimated values)of the sample. (f) ¹⁵N T₁ signaldecay of HP metronidazole in methanol-d₄ at 9.4 T. All HP ¹⁵N spectra shown were obtainedusing 80% parahydrogen gas.

It should also be emphasized that ¹⁵N NMR spectrawithhigh signal-to-noise ratios (SNRs >200 and >100) were recordeddespitethe low natural abundance of ¹⁵N and despite using only80% parahydrogen (Figure 2) and 50% parahydrogen (see SI spectra),respectively. Moreover, this SNR level is more than sufficient foroptimization of experimental conditions, i.e., the μT magneticfield for polarization transfer B_T, temperature,and measurements of relaxation parameters (see Figure 2d–f). Furthermore, the reported ¹⁵N T₁ (36 ± 1 s) relaxationis dominated by ¹⁵N Chemical Shift Anisotropy (CSA) athigh magnetic field (e.g., 9.4 T used here), and T₁ is significantly extended (by several-fold) at the clinicallyrelevant fields of 1.5 T and below.⁷ Anexpected in vivo¹⁵N T₁ of 1–2 min¹⁴ may besufficient for hypoxia sensing, because nitroimidazole-basedcompounds reach the hypoxic region within the first minute after intravenousinjection.^38,39 Moreover, while other tissuesmay also absorb the injected hypoxia sensor,^27,38,39 the ¹⁵N chemical shifts of thereduced form of the nitroimidazole moiety may be sufficientlydifferent to provide a good mechanism of contrast of hypoxic regionsversus surrounding tissues—mitigating the requirement for clearanceof the background signal (the subject of future work).

In conclusion,record-level ¹⁵N hyperpolarization wasachieved via SABRE-SHEATH in seconds for a representative compoundfrom the nitroimidazole class of antibiotics/contrastagents. Substituted five-membered N-heterocycles can be efficientlyhyperpolarized via SABRE-SHEATH even in the presence of ortho substituents. ¹⁵N SABRE-SHEATH of distant nitrogen sites via long-range spin–spincouplings is demonstrated. Moreover, high levels of P_¹⁵N enable detection of HP ¹⁵N compoundseven at natural abundance and 50% parahydrogen (see SI for additional ¹⁵N spectra), which can be convenientlyutilized for screening of large libraries of compounds. Finally, theadvances presented here will likely be synergistically compatiblewith recent developments of heterogeneous SABRE^40,41 and SABRE in aqueous media,^20,42−45 key elements for future biomedical translation of this work intoanimal models and ultimately clinical trials.