Diazirines as Potential Molecular Imaging Tags: Probing the Requirements for Efficient and Long-Lived SABRE-Induced Hyperpolarization

Abstract

Diazirines are an attractive class of potential molecular tags for magnetic resonance imaging owing to their biocompatibility and ease of incorporation into a large variety of molecules. As recently reported, ¹⁵N₂-diazirine can be hyperpolarized by the SABRE-SHEATH method, sustaining both singlet and magnetization states, thus offering a path to long-lived polarization storage. Herein, we show the generality of this approach by illustrating that the diazirine tag alone is sufficient for achieving excellent signal enhancements with long-lasting polarization. Our investigations reveal the critical role of Lewis basic additives, including water, on achieving SABRE-promoted hyperpolarization. The application of this strategy to a ¹⁵N₂-diazirine-containing choline derivative demonstrates the potential of ¹⁵N₂-diazirines as molecular imaging tags for biomedical applications.

Magnetic resonance imaging (MRI) is a powerful, non-invasive approach, based on nuclear magnetic resonance (NMR) spectroscopy, to visualize structure and function with high spatial and temporal resolution. Yet one of the most critical challenges of magnetic resonance (NMR and MRI),^[1] is poor nuclear polarization at thermal equilibrium associated with low sensitivity, especially for nuclei of low natural abundance such as ¹³C and ¹⁵N. Hyperpolarization induces non-equilibrium polarization, increasing fractional magnetization of target nuclei, and therefore raises detectable signal by several orders of magnitude.^[2] Hyperpolarized heteronuclei (e.g., ¹³C and ¹⁵N) often allow signal detection for extended time periods, due to their large relaxation time (T₁) compared to ¹H.^[3] Among different hyperpolarization techniques,^[4] Signal amplification by reversible exchange (SABRE) has been established as an experimentally simple and cost-effective method,^[5] which uses para-hydrogen^[6] as hyperpolarization source and iridium N-heterocyclic carbene complexes as catalysts.^[7] More recently, it has been shown to hyperpolarize ¹⁵N nuclei efficiently in a process termed SABRE in shield enables alignment transfer to (SABRE-SHEATH).^[8]

In the context of developing novel NMR and MRI strategies using hyperpolarized markers, we envisioned that ¹⁵N₂-diazirine motifs could serve as unique hyperpolarizable tags. First, the structure of the double ¹⁵N-labeled diazirines gives access to observable ¹⁵N singlets, which support long-lived states. Furthermore, ¹⁵N₂-diazirines have several attractive features, making them excellent candidates for imaging tags. For example, diazirines have been successfully incorporated into a large number of biologically relevant small molecules, metabolites, and biomolecules. They are small in size, therefore imparting minimal alteration to the original molecule’s properties,^[9] and are known to be biocompatible and stable under either acidic or basic conditions. Indeed, diazirines have been extensively applied as photoaffinity labelling tags for biochemical investigations.^[10] To explore the feasibility of a ¹⁵N₂-diazirine as a potential imaging tag, we have investigated the hyperpolarization of ¹⁵N₂-labeled diazirine 1 by SABRE-SHEATH (Figure 1).^[11] These studies established that ¹⁵N₂-diazirine 1 is capable of being hyperpolarized, achieving a 15000-fold signal enhancement (Ɛ) over thermal signal at the diazirine nitrogen atoms. Additionally, we found relaxation time constants of both ¹⁵N₂ magnetization (T₁) and singlet spin order (T_s) to be orders of magnitude larger than typical polarization decay time constants.^[11]

Figure 1.

¹⁵N₂-diazirines as potential molecular tags for MRI by SABRE-SHEATH hyperpolarization.

Herein, we report our investigations on the potential of diazirines to serve as molecular tags, which would provide enhanced signals with long lifetimes via SABRE-SHEATH. Several aspects had to first be assessed regarding diazirine hyperpolarization—a prerequisite for their ability to serve as general molecular tags. As the coordination of 1 to the iridium catalyst is essential for hyperpolarization by SABRE-SHEATH, we needed to clarify if either the nitrile or carboxylic acid group within 1 (Figure 1) were necessary for binding to iridium, thereby bringing the diazirine into the metalQs coordination sphere, or if the diazirine alone were sufficient. Additionally, we chose deuterated structure 1 in our previous studies with the hypothesis, based on related ¹³C-based hyperpolarization,^[12] that deuteration proximal to the diazirine functionality in 1 would help to extend the T₁ lifetime by reducing polarization loss. Yet the effects of deuteration within the molecule on the magnitude of hyperpolarization and its lifetime remained unclear. Finally, it would be desirable to establish the applicability of diazirines as molecular tags in biologically important molecules while retaining long-lasting hyperpolarization with large signal enhancements.

Our studies began with examining the structural features necessary for a diazirine-containing molecule to undergo SABRE-SHEATH hyperpolarization. To compare with the original structure of diazirine 1, a series of structural analogues 2–4 was prepared (Scheme 1). The absence of the Lewis basic carboxylic acid or nitrile group in 2–4 allowed us to probe the contribution of each functional group in successful diazirine hyperpolarization systematically. This would indicate whether the functional group might be involved in direct coordination to the catalyst (mode I) or if chelation were unnecessary, that is, the diazirine alone were sufficient to bind to Ir (mode II). Note that the hindered silyl ether of diazirine 4 was chosen to discourage, sterically and electronically, its coordination to the metal center. When subjected to standard SABRE-SHEATH conditions with pyridyl Ir precatalyst A (Scheme 1), cyanodiazirine 2, carboxylic acid-containing 3, and TBS-protected ether 4 are all efficiently hyperpolarized (Ɛ=7100, 3500, and 6400 at 8.45 T, respectively). Additionally, each substrate displays a T₁ of 3–4 minutes at 1 T. The relatively small differences in signal enhancement and T₁ among compounds 2–4 implies that while chelation might be possible in some cases (mode I), it is not required to achieve hyperpolarization. Therefore, coordination mode II is likely operative for diazirine hyperpolarization with complex A.^[13]

Scheme 1.

Hyperpolarization of ¹⁵N₂-diazirines 2–4. Standard conditions: diazirines 2–4 (12.5 mm) and Ir precatalyst A (125 μm) in [D₄]MeOH. [a] Signal enhancement measured at 8.45 T and calculated by comparison to a reference of neat ¹⁵N-acetonitrile. [b] T₁ measured at 1 T. Mes=2,4,6-trimethylphenyl; py=pyridyl; TBS=tert-butyldimethylsilyl.

During the SABRE-SHEATH hyperpolarization of diazirines 2–4, no hyperpolarization was observed for any substrate when the neutral Ir-chloride precatalyst B was employed under identical conditions to those with complex A (Table 1, entries 1, 5, and 9). Interestingly, the addition of exogenous Lewis bases restored effective hyperpolarization with complex B. For example, in the presence of pyridine, diazirines 2–4 were hyperpolarized with T₁ values of 2–4 min (entries 2, 6, and 10). Acetonitrile as an additive also enabled signal enhancements to be observed (entries 3, 7, and 11), although the volatility of this additive proved problematic in some instances (e.g., low signal enhancement and large T₁ error with silyl ether-containing diazirine 4, entry 11). It is noteworthy that diazirine 2, bearing a cyano group, is insufficient on its own to allow for SABRE hyperpolarization but exogenous nitrile, even though in lower concentration, is able to reverse this deficiency. The addition of D₂O as a Lewis base is also sufficient to bring about diazirine hyperpolarization (entries 4, 8, and 12); these conditions are similar to those originally reported for 1. Presumably the smaller steric presence of D₂O compared to [D₄]MeOH, the solvent, is the prime factor in facilitating SABRE-SHEATH in these instances even though the D₂O concentration is significantly lower. Signal enhancements and T₁ times are similar to those with pyridine or acetonitrile additives.

Table 1:

Hyperpolarization of diazirines 2–4 with Ir-chloride precatalyst B via the SABRE-SHEATH method.^[a]

Entry	Diazirine	Additive	Ɛ[e]	T1 [s] [f]
1		none	_[g]	n/a
2[b]		pyridine	3500	168 ± 18
3[c]		MeCN	3400	179 ± 16
4[d]		D2O	11200	176 ± 3
5		none	_[g]	n/a
6[b]		pyridine	6000	215 ± 22
7[c]		MeCN	3200	172 ± 17
8[d]		D2O	1900	234 ± 28
9		none	_[g]	n/a
10[b]		pyridine	16 000	141 ± 4
11 [c]		MeCN	1000	263 ± 83[h]
12[d]		D2O	2500	164 ± 25

^[a]Conditions for all experiments: diazirines 2–4 (12.5 mm) and Ir precatalyst B (125 μm) in [D₄]MeOH. See the Supporting Information for more details on the hyperpolarization method.

^[b]Pyridine (1.00 mm, 8 equiv with respect to B).

^[c]Acetonitrile (1.00 mm).

^[d]D₂O (925 mm).

^[e]Signal enhancement measured at 8.45 T and calculated by comparison to a reference of neat ¹⁵N-acetonitrile.

^[f]T₁ measured at 1 T. See the Supporting Information for measurements at other fields.

^[g]No hyperpolarized signal observed.

^[h]The low signal enhancement and volatility of acetonitrile contribute to the larger T₁ error. n/a=not applicable.

The fact that no signal enhancement was observed for any of the diazirines 2–4 with precatalyst B in the absence of a Lewis basic additive suggests that the diazirine moiety is either incapable of substituting the chloro ligand of B in the catalyst initiation step and/or of forming an active catalyst with three facially-coordinated diazirine ligands due to steric congestion (Figure 2, complex III). Attempts to substitute the chloride in complex B for a diazirine stoichiometrically, which would then lead to III upon para-hydrogen addition, failed to generate any observable reaction. We cannot rule out that III might be formed in small quantities in the presence of one of the Lewis basic additives; however, it is most likely that polarization transfer occurs from either complex IV or V.

Figure 2.

Possible coordination modes for SABRE-promoted hyperpolarization of diazirines.

We prepared a series of isotopomers of diazirine 2 to determine the effect of deuteration at the α-positions of the diazirine motif on hyperpolarization and T₁ relaxation times (Figure 3). We used Ir complex B in the presence of pyridine additive for a comparative study. Diazirines d₀-2, 2, and d₅-2 all provide comparable T₁ values of approximately three minutes at 1 T (Figure 3A), showing that deuterium incorporation does not strongly affect T₁ relaxation. The negligible effect of deuterium is consistent with small differences of T₁ between d₀-2 and d₅-2(Figure 3B).^[14]

Figure 3.

Effect of deuteration on T₁-relaxation rates and enhancements Ɛ of ¹⁵N₂-diazirine 2. A) Negligible effect of deuteration of diazirines 2 on T₁-relaxation rates at 1 T (within the experimental error). B) T₁-relaxation rate for ¹⁵N polarization of d₀−2 and d₅-2 as a function of the magnetic field. SABRE-SHEATH conditions: diazirine d₀-2, 2, or d₅-2 (12.5 mm) in [D₄]MeOH, Ir precatalyst B (125 μm), and pyridine (1.00 mm).

The study of the field dependence of T₁ relaxation rates is of special interest (Figure 3B) because for body-noise dominated hyperpolarized MRI, the signal-to-noise is roughly field independent whereas signal decay time constants are not.^[15] Thus, it is important for future applications and choice of optimal MRI field strength to understand at what magnetic field we observe longest hyperpolarization lifetimes. Mechanistically, SABRE-SHEATH involves coherent polarization transfer from p-H₂ to ¹⁵N at the region of Level Anti Crossings,^[16] which is most efficient at about 0.6 μT for the characteristic J_HH and J_NH couplings in iridium-diazirine complexes. Commercial MRI operates between 1 T and 4 T. It is therefore noteworthy that T₁ did not vary significantly over a large magnetic field range, falling significantly only at very high field strength (Figure 3B). The data indicate that magnetic fields of about 1 T and slightly below may be most advantageous for future applications, which is intriguing given the ongoing progress in low-field NMR^[17] and low-field MRI.^[18]

We have established the independence of the ¹⁵N₂-diazirine motif for SABRE-SHEATH hyperpolarization and its ability to deliver long-lasting polarization. To demonstrate the applicability of ¹⁵N₂-diazirines as molecular tags for biologically important molecules, we prepared ¹⁵N₂-diazirine-tagged choline derivative 5 (Figure 4). It has been shown that choline analogues, in which a methyl group is replaced with an alkyl chain of up to five carbon atoms in length, incorporate efficiently into phospholipids.^[19] Subjecting 5 to SABRE-SHEATH hyperpolarization with Ir precatalyst B in the presence of pyridine or D₂O as the Lewis base leads to greater than 2000-fold signal enhancement with T₁ of approximately three minutes. Compared to ¹⁵N-choline hyperpolarized by dynamic nuclear polarization (DNP),^[20] the effectiveness observed on hyperpolarized 5 by SABRE-SHEATH is comparable yet SABRE-SHEATH is operationally a simpler and more economical protocol. Particularly encouraging is the effective enhancement and long-lasting polarization observed in the presence of D₂O, presenting an important first step toward biomedical in vivo applications with this tag.

Figure 4.

Hyperpolarization of ¹⁵N₂-diazirine-tagged choline derivative 5. SABRE-SHEATH conditions: 5 (12.5 mm) and Ir precatalyst B (125 μm) in [D₄]MeOH with either pyridine (1.00 mm) or D₂O (925 mm). Signal enhancement measured at 8.45 T and calculated by comparison to a reference of neat ¹⁵N-acetonitrile. T₁ measured at 1 T. See the Supporting Information for more measurements at other fields. Ts=p-toluenesulfonyl.

In summary, we have established the independence of the ¹⁵N₂₋ diazirine group from other functional groups for SABRE-SHEATH hyperpolarization and its ability to support long-lasting hyperpolarization for signal enhancement, marking its potential as a molecular tag for NMR and MRI. Furthermore, the studies on different SABRE-SHEATH hyperpolarization conditions reveal a critical contribution of Lewis basic additives for generating active Ir catalysts for polarization transfer. These studies also provide some structural information regarding the likely coordination sphere at Ir in SABRE-SHEATH with diazirines. Finally, successful hyperpolarization of a ¹⁵N₂-diazirine-containing choline derivative demonstrates the applicability of ¹⁵N₂-diazirine tags within biologically relevant molecules. Future studies are directed towards incorporating ¹⁵N₂-diazirines into other biologically active molecules and their studies in molecular imaging.