Direct enhancement of nitrogen-15 targets at high-field by fast ADAPT-SABRE

Graphical abstract


Introduction
Despite the huge success of NMR in a wide assortment of research fields ranging from structural material characterization to the imaging of internal human organs, it is still regarded to be underexploited based on its theoretical potential [1,2]. Most of the successes of NMR and MRI applications have been achieved utilizing the thermal level of nuclear spin polarization which is only of the order of 10 À5 at room temperature in a standard high field spectrometer [2]: only one spin in 30,000 contributes to the NMR signal for protons in a 9.4 T magnet. Improving this poor sensitivity would make NMR and MRI more widespread and cost-efficient. The solution to this challenge is offered by hyperpolarization methods that enhance the nuclear spin polarization by up to 5 orders of magnitude compared to standard thermal polarization [3]. This large sensitivity enhancement enables the completion of high-end MRI applications e.g. in vivo study of human cancer, which could otherwise not be performed due to sensitivity issues [4][5][6].
Within the class of hyperpolarization techniques, the Parahydrogen Induced Polarization (PHIP) method employs a highly ordered nuclear singlet, para-hydrogen (p-H 2 ) gas, to enhance poorly polarized substrate spins by several orders of magnitude [7,8]. An important variant of the PHIP technique, SABRE was introduced in 2009, that no longer requires active hydrogenation to hyperpolarize targeted molecules [9]. It relies on the temporary association of the substrate at a metal center where the associated J-coupling network ultimately enables the generation of hyperpolarized substrates (see Fig. 1a). SABRE provides a simple, fast and cost-efficient approach to hyperpolarize substrates in its original form and they can be re-polarized several times in quick succession, providing the opportunity to achieve continuous hyperpolarization [10]. The method has been successful in polarizing a large class of biologically relevant substrates where their 1 H, 13 C and 15 N nuclei [11][12][13][14] are senitised, and also in preparing long-lived forms that are detectable for up to 30 min [15][16][17][18][19]. Considering their biological relevance, hyperpolarizing spin-1/2 heteronuclei already show great importance for in vivo MRI studies [20,21].
The coherent spin mixing condition for heteronuclei in SABRE can be achieved by bubbling the solution at ultra-low magnetic field (typically 0.2-1.0 lT), as previously shown by Theis et al. [22]. However, this low-field technique requires field-cycling between low and high magnetic fields, a condition that is both technically demanding and unsuitable for immediate signal detection. Overcoming this challenge, Warren and co-workers proposed the LIGHT-SABRE approach to create a similar resonance condition at high field by applying an optimized spin-lock based RF pulse sequence [23]. Pravdivtsev  Here we show that, the recently published ADAPT (Alternating Delays Achieve Polarization Transfer) sequence [26] can be applied to transfer polarization from singlet hydrides to target 15 N nuclei in a repetitive fashion. It takes only few seconds to build up strong 15 N hyerpolarized signal whilst the transfer mechanism is fully compatible with LIGHT-SABRE [23], Level Anti-Crossing (LAC) [27] and PHIP spin-order transfer mechanisms [28].
This article is organized as follows: In Section 2 we briefly describe the ADAPT method and its optimizations by simulations to yield a SABRE perspective. In Section 3 we present experimental details and results. Finally, Section 4 contains conclusions and discussions.

ADAPT approach
We consider a system formed by three nuclear spins: two hydride 1 H (1 and 2 in Fig. 1a) and a 15 N (3 in Fig. 1a). The aim is to transfer nuclear spin polarization between the 1 H singlet spin population and 15 N longitudinal magnetization. 1 H chemical equivalence is assumed, and it is experimentally imposed by CW irradiation on the 1 H channel.
In a doubly rotating frame, if the 1 H nuclei are chemically equivalent, the J coupling terms form the coherent Hamiltonian: where x J ¼ 2pJ 12 ; x R ¼ 2pðJ 13 þ J 23 Þ and x D ¼ 2pðJ 13 À J 23 Þ. We use a basis formed by the direct product of the singlet-triplet basis for 1 H spins 1 and 2, and the eigenbasis for the I 3x operator of 15 N spin 3. In this basis, the Hamiltonian in Eq. (1) can be decomposed into the direct sum of four orthogonal subspaces as detailed in Ref. [26]. However, we restrict the ADAPT analysis to the two relevant orthogonal subspaces H a;b and H c;d containing the proton singlet state: ð2Þ ð3Þ where jai ¼ ÀðjS 0 ; ai À jS 0 ; biÞ; jbi ¼ ðjT 0 ; ai þ jT 0 ; biÞ; jci ¼ ÀðjT 0 ; ai ÀjT 0 ; biÞ and jdi ¼ ðjS 0 ; ai þ jS 0 ; biÞ. The out-of-diagonal elements À x D 4 in Eqs. (2) and (3) can be exploited to transfer polarization from jS 0 ihS 0 j to I 3x . One of such methods is the ADAPT sequence as shown in Fig. 1b. It performs the task via a number of RF pulses and delays that are recursively repeated. Small and/or large a tip angle pulses can be used, providing the experimentalist a further degree of freedom. The protocol includes a number of steps: (i) definition of the tip angle: for example a ¼ 30 , (ii) calculation of the conversion efficiency for a given range of delays D and loop numbers m as detailed in [26], (iii) choice of the optimal pair D opt and m opt . A 90 pulse at the end of the loops (see Fig. 1b), is inserted to transform I 3x into I 3z so as to retain magnetization on the substrate upon dissociation. For the present case, the J-coupling network is known: J 12 ¼ 8 Hz, J 13 À J 23 ¼ 25:5 Hz, and J 13 þ J 23 ¼ 25:5 Hz and a theoretical transfer of 93% is achieved by ADAPT 30 in about 40 ms with D ¼ 8 ms and m ¼ 5. In practice, multiple repetitions of this block are required to build up bulk longitudinal heteronuclear magnetization (see Fig. 5). To keep our studies simple, in this work we employ ADAPT 30 which experimentally we found to be optimal, however as predicted all of the combinations of a; D and m we tested produced a return [26].
ADAPT remains robust even in the case of uncertainty about the value of the heteronuclear J 23 (simulations not shown).

Initial state and trajectories
When the para-hydrogen and the substrate bind to the catalyst, the initial state can be represented as an overpopulation of the 1 H scalar singlet order. We can define a set of orthogonal operators I rs x ; I rs y ; I rs z with cyclic commutation relationships ½I rs x ; I rs y ¼ iI rs z , for each of the 2 Â 2 subspaces in Eqs. (2) and (3). The initial state and the observable I 3x 15 N-transverse magnetization are related to the following single transitions operators: From the set of Eq. (4), it is apparent that polarization transfer can be obtained, for example, when a sequence of events invert the sign of the operator I cd z while maintaining the sign of the operator I ab z . ADAPT achieves this by performing a p rotation in the subspace H c;d (see Fig. 2).

Analogy with the LIGHT-SABRE and the LAC approach
A LAC [27] occurs when (i) the energy level relative to a pair of states jmi and jni is equal E jmi ¼ E jni and (ii) there is a matrix element V mn ¼ hmjVjni for some operator V that splits them. Essentially, the LIGHT-SABRE and LAC methods share the same clever idea: a resonance condition between the spin populations of the     proportional to ffiffiffi 2 p x D [23]. To complete the analogy with the ADAPT method, it has also been noted by Theis et al. [23], that the effect of the CW irradiation is to produce a p pulse which transfers population between the hydrogen singlet population to the 15 N transverse magnetization: precisely the same transformation achieved by ADAPT in the subspace of Eq. (3)(see Fig. 2).

Experimental results and discussions
All the measurements were carried out with a 500 MHz Bruker Avance III spectrometer equipped with a broad-band (BBO) probe at 298 K. The sample was prepared in a 5 mm NMR tube by mixing 10 mM of [IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl) imidazole-2-ylidene, COD = cycloocta-1,5-diene) pre-catalyst and 50 mM of 15 N-ethylnicotinate [29] in 0.6 ml methanol-d 4 solution. A valve-controlled para-hydrogen flow PTFE (polytetrafluoroethylene) tube was immersed inside the NMR tube to bubble the solution with para-hydrogen originating from a para-hydrogen generator with 90% enrichment. To accurately assign the resonances of the concerned spins and measuring their coupling constants within the network, we first performed a standard in-magnet PHIP experiment by bubbling p-H 2 inside the spectrometer and recording a 1 H spectrum upon applying a 45°pulse on the proton channel. A large and transient antiphase spectrum is observed in the hydride region reflecting the hydrogenation product, resulting from reaction with p-H 2 . Both the hydrides (spin-1 and -2) show overlapping resonances at À22.66 ppm as shown in Fig. 3. The 2 J HH and cis-2 J NH coupling constants were simply measured from the hydride region of the spectrum. Whilst trans-2 J NH could not be observed it is expected to be negligible (<0.5 Hz) in these types of system [12,27].
Next we performed a standard SABRE-SHEATH experiment [22] by shaking the solution with p-H 2 inside a l-metal can and then quickly recording a 15 N spectra upon a 90°detection pulse. The purpose of this experiment was to find out the 15 N resonance frequencies for the different forms of the substrate; the substrate can be free from the catalyst and also bound to the catalyst. Two different forms of bound substrate exist here (equatorial and axial position). In our study, we observe 15 N free resonance at 303.22 ppm and equatorial-bound resonance at 256.60 ppm whilst the axialbound resonance remains undetected due to insufficient detectable polarization. The parameters obtained from these initial screening experiments were sufficient to optimize the ADAPT pulse sequence numerically (see Fig. 1b).  The high-field SABRE experiments were performed according to the experimental protocol depicted in Fig. 4. After a suitable relaxation delay, p-H 2 was bubbled through the solution for a duration of s b (typically 10 s) followed by a short waiting period (s w $ 1 s.) for the solution to settle. The optimized ADAPT pulse sequence immediately followed with a total duration of s rf (% D Â m Â n), selectively exciting the bound-15 N resonance without affecting the free resonance. The offset for 15 N channel was set on the bound-peak (256.60 ppm) whilst the band-width (BW) of the RF pulse was kept at 500 Hz (%10 ppm). After n number of repeated ADAPT blocks, a hard 90 pulse was applied to detect the magnetization. During the RF sequence, a continuous-wave (CW) pulse of 1 kHz band-width was applied in proton channel resonant with the hydride region to enforce chemical equivalence between the hydride protons. However, when performed without any CW pulse in proton channel, we observe polarization transfer, albeit with a 30-40% of less enhancement than earlier.
Using the numerically optimized parameters (see Section 2), the ADAPT sequence efficiently transfers singlet-order polarization of hydrides into longitudinal polarization of 15 N nuclei that are connected to the network. An optimum level of polarization can be realized for ADAPT 30 with D = 8 ms and m ¼ 5 in a total pulse duration of only 40 ms. Using these parameters, one ADAPT block achieves 196-fold enhancement factor () for the free 15 N target. The enhancement factor () of hyerpolarization was calculated as the ratio of the integral for the hyperpolarized signal and the integral for the thermal signal divided by the number of thermal scans. As SABRE is an exchangeable process, the polarization transfer rate is limited by the residence time of both the hydrides and the free substrate, which are in the range of 100-200 ms under standard SABRE conditions. As a result, hydrogenation with p-H 2 and therefore the polarization transfer does not occur simultaneously across the entire sample volume. For this reason, the ADAPT block has to be repeated multiple times to build up 15 N polarization. In practice, one block of ADAPT sequence transfers only a fraction of available polarization into 15 N magnetization. But as long as fresh p-H 2 keeps exchanging together with non-polarized substrates, we observe accumulation in 15 N magnetization by repeated application of the ADAPT block. Fig. 5 presents a gradual build-up in free 15 N magnetization with increasing numbers of ADAPT blocks. A maximum enhancement of 940-fold was achieved after 40 ADAPT blocks of total duration $1.6 s. Ultimately, spin relaxation together with p-H 2 consumption take over in the magnetization build-up process and starts decreasing with larger loop counts. The is also limited by several other factors e.g. RF inhomogeneity and imperfections caused by mixing and diffusion [30,31].
In Fig. 6 we show the success of the ADAPT-SABRE method in driving polarization from singlet-hydrides to bound 15 N nuclei of ethylnicotinate and enhancing the free 15 N response. The earlier parameters were used to achieve the 1-shot ADAPT-SABRE spectra in Fig. 6a. The corresponding thermal signal in Fig. 6b was acquired by averaging 400 transients with a recycle delay of 120 s (T 1 ( 15 N) = 21.5 AE 0.5 s), taking over 13.5 h. The bound peaks remain undetectable in the thermal measurements even after many scans signifying greater relative enhancements as predicted previously [25]. The lack of a thermal signal for the bound peaks can be attributed to their broad line shapes, they are visible after 2000 scans.

Conclusions
In summary, we have used ADAPT-SABRE to generate 15 N hyperpolarization at high magnetic field without the requirement of below-earth field sample mixing. The conversion is robust and faster than previously reported methods: it took only 1.6 s to reach nearly 3 orders of signal enhancement for a 15 N target. This method has several advantages over the low-field SABRE mechanism, e.g. constant field shuttling, unnecessary signal losses during transport. The presented scheme whilst being inherently simple, can be easily augmented to any SABRE active species and their 13 C, 19 F and 31 P nuclei. We are currently working on its further optimization via exchange rate, sample concentration and additive-dependence studies. In the future, we plan to examine how the dynamics of SABRE exchange can be harnessed to improve this ADAPT process. We believe it will be of particular importance in terms of achieving in vivo hyperpolarization, where sample transport poses a significant challenge to hyperpolarization based experiments.