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.

Spectral 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-H2) and to-be-hyperpolarized substrate molecules on a catalyst[21−23]—in solutions or in “neat” liquids.[24] Polarization of target nuclear spins (e.g., 1H,[21]15N,[25,26] or 31P[27]) occurs spontaneously when the applied static magnetic field BT is “matched” to the corresponding spin–spin couplings between the nascent para-H2 hydride pair and the target nuclei (Figure a). Homonuclear (i.e., 1H) SABRE was demonstrated first[21] using BT in the mT range; the approach was later extended to heteronuclei (e.g., 15N, 31P, etc.) via SABRE in shield enabling alignment transfer to heteronuclei (SABRE-SHEATH[25]) utilizing BT 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% P15N (corresponding to >30 000-fold 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-H2 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 15N sites can be HP with relaxation time constants ranging from 1 min[26] to 10 min.[30] Furthermore, compared to 13C enrichment of leading 13C HP contrast agents (e.g., pyruvate-1-13C[31,32]), spin labeling with 15N uses relatively straightforward chemistry replacing N-sites in N-heterocycles with 15N.[26,33]

Figure 1

(a) Generalized scheme of SABRE and SABRE-SHEATH hyperpolarization processes. (b) Chemical structure of the activated Ir-IMes hexacoordinate complex after activation with H2. The complex undergoes fast exchange with para-H2 and free imidazole-15N2, which enables spontaneous polarization transfer from para-H2 (in the form of Ir-hydrides) to 15N nuclei of imidazole-15N2 in μT magnetic fields.[25,26]

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.[20,31,34,35] While several translational challenges of conventional SABRE have been addressed recently, i.e., demonstration of SABRE in aqueous media,[36−38] and implementation of heterogeneous SABRE catalysts,[39,40] most SABRE-hyperpolarized compounds studied to date have limited biological relevance (although nicotinamide,[21] pyrazinamide, and isoniazid[41] have been demonstrated). Recently, 15N 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 15N isotropic chemical shifts, which are >90 ppm for the protonated and deprotonated states of the 15N-heterocycles.[42] As a result, 15N isotropic chemical shifts of 15N-hyperpolarized probes may be ideal reporters of in vivo pH. This approach has two key advantages compared to the current HP 13C-bicarbonate pH sensing approach.[15] First, in vivo 15N T1 is significantly longer than that for 13C (e.g., ∼10 s for 13C bicarbonate[15]). Second, pH sensing using bicarbonate requires measurement and detection of both 13C bicarbonate and its exchanging partner 13CO2 via spectroscopic imaging (MRSI)—a demanding approach with respect to SNR, because the relative signal ratio of 13C bicarbonate and 13CO2 peaks must be measured with good precision, whereas this approach only requires accurate measurement of 15N 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 pKa 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 pKa ∼ 6.6. However, 2,6-lutidine is not readily amenable to SABRE-SHEATH hyperpolarization.[24] The pKa 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 15N HP pH sensing. Indeed, proton binding induces easily measured 15N chemical shifts of ∼30 ppm (Figure ).[45−47] Note that both 15N 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 15N sites are equivalent and have the same chemical shift. As a result, imidazole-15N2 is an excellent delivery vehicle, because its two 15N sites carry twice the hyperpolarization payload of (single-site) pyridine derivatives.

Figure 2

(a) Molecular diagram of imidazole-15N2 protonation; note that the effective molecular symmetry in unprotonated (due to fast proton hopping between two 15N sites) and protonated states results in the same 15N chemical shift of both sites. (b) Determination of imidazole-15N2 pKa using isotropic 15N chemical shift in aqueous solutions. (c) Selected (thermally-polarized) 15N spectra of imidazole in water used for pKa determination. (d) 15N NMR spectrum of HP imidazole-15N2 (∼0.1 M) in methanol:water (∼1:1) produced via SABRE-SHEATH (BT < 0.1 μT, [catalyst] ∼ 4 mM); note the inset spectrum showing the other HP enlarged resonances: the large changes (i.e., ≥10 ppm) of a 15N chemical shift of these species are caused by the imidazole position in the hexacoordinate complex (e.g., equatorial vs axial position, Figure b), binding state (e.g., free vs catalyst-bound states, Figure b),[25,26] and protonation states.[42] (e) 15N spectrum of a 15N signal reference. (f,g) SABRE-SHEATH optimization of magnetic transfer field BT and temperature, respectively. All NMR spectra are recorded using a 400 MHz Bruker NMR spectrometer.

Here, 15N-SABRE-SHEATH hyperpolarization of imidazole-15N2 is demonstrated. Figure b shows the exchange process of imidazole-15N2 and para-H2 gas on the activated Ir-IMes hexacoordinate complex of the most potent SABRE hyperpolarization catalyst to date.[23] As shown in Figure , 15N signal enhancement ε15N of ∼2000-fold is detected on each of the two 15N sites in a methanol:aqueous (pH ∼ 12) buffer (∼1:1) solution of ∼0.1 M substrate utilizing only 50% para-H2 gas and the hyperpolarization setup described previously.[26] Note the broad appearance of the HP NMR line in pure methanol-d4 (Figure S3) owing to intermediate proton chemical exchange between the two 15N sites described above; the 15N NMR line is no longer broadened in aqueous solution (Figure d). The additional 15N HP resonances (seen as narrow lines) are due to the presence of catalyst-bound 15N imidazoles (Figure d inset and Figure S3a)—which have different pKa values, protonation states, and proton exchange rates. If 100% para-H2 would have been utilized (vs ∼50% para-H2 utilized here), the enhancement would be effectively tripled to ε15N ∼ 6000-fold, corresponding to P15N ∼ 2%. Temperature and BT(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 g) was found optimal for 15N SABRE-SHEATH in the aqueous medium (Figure f,g).

These results represent the highest payload (defined as the product of 15N concentration and polarization) for any SABRE-hyperpolarized compounds with the exception of 15N-nicotinamide (50 mM and P15N ∼ 11% at ∼100% para-H2 limit), which was achieved in pure methanol-d4 using preactivation with pyridine,[33] whereas here, 15N SABRE-SHEATH was performed in an aqueous medium, which is known to provide lower enhancements due to lower para-H2 solubility.[37] A potential solution is a further significant increase of para-H2 pressure (compared to ∼6.5 atm used here), which could potentially enable significantly larger polarization levels,[24,26] e.g., P15N ∼ 10% or more. 15N T1 of imidazole-15N2 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 T1 increase to 86 ± 2 s (Figure S2) indicating that the in vivo T1 (with the absence of both alcohol and exchangeable catalyst) could potentially exceed 1 min.[49] The 15N 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-15N2 in aqueous media was performed at several different pH values (below and above the pKa, Figure ) demonstrating that (i) 15N chemical shift of the HP probe indeed changes by ∼30 ppm, and (ii) the 15N 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 15N shift.

Figure 3

15N NMR spectra of imidazole-15N2 hyperpolarized via SABRE-SHEATH at various pH values (below and above pKa) in aqueous solutions containing <50% methanol. Note a minor shift of ∼2 ppm between resonances shown in Figure c (not color matched) and here due to temperature difference of ∼40 °C.

Conventional 1H-SABRE of methanol-d4 solution yielded εH ∼ 50–100-fold (Figure S3e), i.e., values lower than the corresponding 15N enhancements (Figure S3a)—in agreement with previous 15N SABRE-SHEATH studies of 15N-pyridine.[26] Moreover, Figure S3d also shows in situ (or “high-field”) SABRE 1H NMR spectroscopy of imidazole-15N2 recorded inside a 9.4 T spectrometer (the spectrum was recorded approximately 2 s after para-H2 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] and (ii) upfield 1H signals from intermediate hydride species formed transiently during the catalyst activation process.[37] Taken together, the 15N SABRE-SHEATH and 1H SABRE results indicate that imidazole-15N2 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-15N2, 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]15N SABRE-SHEATH allows preparation of HP imidazole-15N2 (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 15N-imidazole moieties for structural and functional studies of peptides and proteins.[51,52]