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15N Hyperpolarization of Imidazole-15N2 for Magnetic Resonance pH Sensing via SABRE-SHEATH

15N nuclear spins of imidazole-15N2 were hyperpolarized using NMR signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH). A 15N NMR signal enhancement of ∼2000-fold at 9.4 T is reported using parahydrogen gas (∼50% para-) and ∼0.1 M imidazole-15N2 in methanol:aqueous buffer (∼1:1). Proton binding to a 15N site of imidazole occurs at physiological pH (pKa ∼ 7.0), and the binding event changes the 15N isotropic chemical shift by ∼30 ppm. These properties are ideal for in vivo pH sensing. Additionally, imidazoles have low toxicity and are readily incorporated into a wide range of biomolecules. 15N-Imidazole SABRE-SHEATH hyperpolarization potentially enables pH sensing on scales ranging from peptide and protein molecules to living organisms.

S pectral sensing or imaging of local pH variances in vivo has been of long-standing interest for characterizing a host of pathological conditions, including various cancers. 1−6 For example, a variety of MR-based approaches using both exogenous and endogenous agents (e.g., refs 6−17) have been investigated as less invasive alternatives to using microelectrode probes. 6 However, sensitivity presents a significant challenge to otherwise powerful MR-based methods due to the typically low concentrations of probe molecules compared to water in vivo.
One way to combat such MR sensitivity limitations is hyperpolarization. NMR hyperpolarization techniques significantly enhance nuclear spin polarization (P), resulting in large gains in NMR signal. 18−20 One such approach is signal amplification by reversible exchange (SABRE), a technique that relies on exchange of parahydrogen (para-H 2 ) and to-behyperpolarized substrate molecules on a catalyst 21−23 in solutions or in "neat" liquids. 24 Polarization of target nuclear spins (e.g., 1 H, 21 15 N, 25,26 or 31 P 27 ) occurs spontaneously when the applied static magnetic field B T is "matched" to the corresponding spin−spin couplings between the nascent para-H 2 hydride pair and the target nuclei ( Figure 1a). Homonuclear (i.e., 1 H) SABRE was demonstrated first 21 using B T in the mT range; the approach was later extended to heteronuclei (e.g., 15 N, 31 P, etc.) via SABRE in shield enabling alignment transfer to heteronuclei (SABRE-SHEATH 25 ) utilizing B T static fields in the μT range. Alternatives to spontaneous SABRE or SABRE-SHEATH include radiofrequency irradiation targeting level anti-crossings (LAC) 28 and low-irradiation generation of high tesla-SABRE (LIGHT-SABRE). 29 These RF-based approaches are attractive because they yield hyperpolarization directly in the magnet where detection takes place. However, the spontaneous/static-field approaches currently yield larger polarization levels, up to 10% P 15N (corresponding to >30 000fold signal enhancement at 9.4 T). A key advantage of all SABRE hyperpolarization methods is their fast polarization buildupachieving high P levels in only a few seconds. Moreover, spontaneous SABRE and SABRE-SHEATH are not instrumentally demanding and only require access to readily produced para-H 2 and a weak static magnetic field. Furthermore, SABRE-SHEATH addresses a critical challenge faced by all hyperpolarization techniques: Upon injection of hyperpolarized (HP) material into a system of interest, signals usually decay rapidly, with decay constants on the order of seconds up to a minute. However, with SABRE-SHEATH, long-lived 15 N sites can be HP with relaxation time constants ranging from 1 min 26 to 10 min. 30 Furthermore, compared to 13 C enrichment of leading 13 C HP contrast agents (e.g., pyruvate-1- 13 C 31,32 ), spin labeling with 15 N uses relatively straightforward chemistry replacing N-sites in N-heterocycles with 15 N. 26,33 The development of all hyperpolarization techniques has largely been driven by their use in biomedicine to image organ function and probe metabolic processes in vivo. 20 15 N heterocycles have been shown to be potent for pH imaging. 42 In this case, hyperpolarization was performed with the well-established yet expensive dissolution-DNP (dynamic nuclear polarization) 43 modality and pH sensing was achieved by detecting changes in 15 N isotropic chemical shifts, which are >90 ppm for the protonated and deprotonated states of the 15 N-heterocycles. 42 As a result, 15 N isotropic chemical shifts of 15 N-hyperpolarized probes may be ideal reporters of in vivo pH. This approach has two key advantages compared to the current HP 13 C-bicarbonate pH sensing approach. 15 First, in vivo 15 N T 1 is significantly longer than that for 13 C (e.g., ∼10 s for 13 C bicarbonate 15 ). Second, pH sensing using bicarbonate requires measurement and detection of both 13 C bicarbonate and its exchanging partner 13 CO 2 via spectroscopic imaging (MRSI)a demanding approach with respect to SNR, because the relative signal ratio of 13 C bicarbonate and 13 CO 2 peaks must be measured with good precision, whereas this approach only requires accurate measurement of 15 N frequency, which can be performed with relatively low SNR.
A key challenge for in vivo pH sensing is a relatively narrow pH range for the extracellular compartments for most conditions of interest, requiring that a given pH probe provide a wide dynamic range of signal response over a relatively narrow range of pH values (i.e., ∼1.5 pH units). As a result, the pH sensor must have a pK a close to physiological pH of ∼7. Initial studies of six-membered N-heterocycles (see Supporting Information Figure S1 and ref 42) identified only one somewhat suitable candidate: 2,6-lutidine, 42 with pK a ∼ 6.6. However, 2,6-lutidine is not readily amenable to SABRE-SHEATH hyperpolarization. 24 The pK a of imidazole is ∼7.0a property that has already been exploited for in vivo tumor pH imaging via proton detection without hyperpolarization. 6,44 Therefore, imidazole nitrogen-15 sites are excellent candidates for 15 N HP pH sensing. Indeed, proton binding induces easily measured 15 N chemical shifts of ∼30 ppm ( Figure 2). 45−47 Note that both 15 N sites have the same chemical shift in the deprotonated form because of fast proton hopping between these two sites in aqueous media. 45−47 In the protonated form, both 15 N sites are equivalent and have the same chemical shift. As a result, imidazole-15 N 2 is an excellent delivery vehicle, because its two 15 N sites carry twice the hyperpolarization payload of (single-site) pyridine derivatives.
Here, 15 N-SABRE-SHEATH hyperpolarization of imidazole-15 N 2 is demonstrated. Figure 2b shows the exchange process of imidazole-15 N 2 and para-H 2 gas on the activated Ir-IMes hexacoordinate complex of the most potent SABRE hyperpolarization catalyst to date. 23 As shown in Figure 2, 15 N signal enhancement ε 15N of ∼2000-fold is detected on each of the two 15 N sites in a methanol:aqueous (pH ∼ 12) buffer (∼1:1) solution of ∼0.1 M substrate utilizing only 50% para-H 2 gas and the hyperpolarization setup described previously. 26 Note the broad appearance of the HP NMR line in pure methanol-d 4 ( Figure S3) owing to intermediate proton chemical exchange between the two 15 N sites described above; the 15 N NMR line is no longer broadened in aqueous solution (Figure 2d). The additional 15 N HP resonances (seen as narrow lines) are due to the presence of catalyst-bound 15 N imidazoles (Figure 2d inset and Figure S3a)which have different pK a values, protonation states, and proton exchange rates. If 100% para-H 2 would have been utilized (vs ∼50% para-H 2 utilized here), the enhancement would be effectively tripled to ε 15N ∼ 6000-fold, corresponding to P 15N ∼ 2%. Temperature and B T 48 of the SABRE-SHEATH procedure were optimized to achieve the largest enhancements under our conditions. We note that unusually (for SABRE) high temperature (>340 K, Figure 2g) The complex undergoes fast exchange with para-H 2 and free imidazole-15 N 2 , which enables spontaneous polarization transfer from para-H 2 (in the form of Ir-hydrides) to 15 N nuclei of imidazole-15 N 2 in μT magnetic fields. 25,26 was found optimal for 15N SABRE-SHEATH in the aqueous medium (Figure 2f,g).
These results represent the highest payload (defined as the product of 15 N concentration and polarization) for any SABRE-hyperpolarized compounds with the exception of 15 Nnicotinamide (50 mM and P 15N ∼ 11% at ∼100% para-H 2 limit), which was achieved in pure methanol-d 4 using preactivation with pyridine, 33 whereas here, 15 N SABRE-SHEATH was performed in an aqueous medium, which is known to provide lower enhancements due to lower para-H 2 solubility. 37 A potential solution is a further significant increase of para-H 2 pressure (compared to ∼6.5 atm used here), which could potentially enable significantly larger polarization levels, 24,26 e.g., P 15N ∼ 10% or more. 15 N T 1 of imidazole-15 N 2 in methanol:aqueous (pH ∼ 12) buffer (∼1:1) solution in the presence of SABRE catalyst was 24 ± 1 s at 9.4 T, whereas further reduction of methanol fraction (to an estimated value of <10% by volume) resulted in a T 1 increase to 86 ± 2 s ( Figure  S2) indicating that the in vivo T 1 (with the absence of both alcohol and exchangeable catalyst) could potentially exceed 1 min. 49 The 15 N hyperpolarization lifetime could also be further enhanced via long-lived spin states and the use of lower magnetic fields. 30 Motivated by potential biomedical translation, SABRE-SHEATH hyperpolarization of imidazole-15 N 2 in aqueous media was performed at several different pH values (below and above the pK a , Figure 3) demonstrating that (i) 15 N chemical shift of the HP probe indeed changes by ∼30 ppm, and (ii) the 15 N NMR resonances are sufficiently narrow to discriminate minute changes in pH in the physiologically relevant range. Therefore, this HP molecular probe can potentially enable in vivo pH sensing with an estimated ∼15 ppm range covering pH range 6.5 to 7.5, and it should provide resolution of 0.1 unit of pH per 1.5 ppm of 15 N shift.
Conventional 1 H-SABRE of methanol-d 4 solution yielded ε H ∼ 50−100-fold ( Figure S3e), i.e., values lower than the corresponding 15 N enhancements ( Figure S3a)in agreement with previous 15 N SABRE-SHEATH studies of 15 N-pyridine. 26 Moreover, Figure S3d also shows in situ (or "high-field") SABRE 1 H NMR spectroscopy of imidazole-15 N 2 recorded inside a 9.4 T spectrometer (the spectrum was recorded approximately 2 s after para-H 2 bubbling (conducted at 9.4 T) was stoppednote (i) the partial SABRE signal enhancement of one of the imidazole protons, manifested as the signal with negative (emissive) phaseconsistent with the previously described "high-field" SABRE effect; 50    imidazole-15 N 2 reversible exchange (and SABRE in general) have the same key features as the most-studied SABRE substrate, pyridine.
While d-DNP could in principle be employed for hyperpolarization of imidazole-15 N 2 , it is an instrumentationally demanding and expensive hyperpolarization technique, and DNP hyperpolarization processes for this class of compound typically require ∼2 h of polarization build-up. 42 15 N SABRE-SHEATH allows preparation of HP imidazole-15 N 2 (and potentially other imidazole-based biomolecules) in less than a minute using a very simple experimental setup, paving the way to pH sensing (imaging and localized spectroscopy) in vivo. Furthermore, in combination with recent demonstrations of SABRE in aqueous media 36−38 and in "neat" liquids, 24 the presented work potentially enables the hyperpolarization of 15 N-imidazole moieties for structural and functional studies of peptides and proteins. 51,52 ■ ASSOCIATED CONTENT

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00231.