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15N-Azides as practical and effective tags for developing long-lived hyperpolarized agents

Azide moieties, unique linear species containing three nitrogen atoms, represent an attractive class of molecular tag for hyperpolarized magnetic resonance imaging (HP-MRI). Here we demonstrate (15N)3-azide-containing molecules exhibit long-lasting hyperpolarization lifetimes up to 9.8 min at 1 T with remarkably high polarization levels up to 11.6% in water, thus establishing (15N)3-azide as a powerful spin storage for hyperpolarization. A single (15N)-labeled azide has also been examined as an effective alternative tag with long-lived hyperpolarization. A variety of biologically important molecules are studied in this work, including choline, glucose, amino acid, and drug derivatives, demonstrating great potential of 15N-labeled azides as universal hyperpolarized tags for nuclear magnetic resonance imaging applications.

The following procedure was adapted from a previously reported procedure. 2 To a 2neck 50-mL round-bottom flask, sodium metal (220 mg, 9.6 mmol, 1.25 equiv) was added portion-wise to ethanol (10 mL) over an ice-water bath. The mixture was stirred at room temperature until all solid material was completely consumed (about 1.5 hours).
Meanwhile, in a separate 25-mL pear-shaped flask, sodium nitrite-15 N (915 mg, 13.8 mmol, 1.8 equiv) was dissolved in 2 mL water. This solution was cooled with an ice-water bath and 3-methyl-1-butanol (1.50 mL, 13.8 mmol, 1.8 equiv) was added. Next, 4 M H2SO4 (1.73 mL, 6.9 mmol, 0.9 equiv) was added dropwise over 5 minutes, and the reaction was stirred vigorously over the icewater bath for 30 minutes. Upon addition, the reaction turned blue-green and slowly became colorless. Afterwards, the bilayer mixture was transferred to a 25-mL separatory funnel and the bottom layer was removed. The top layer (neat, crude isopentyl nitrite-15 N S1) was washed with 5 mL saturated sodium chloride solution. While the top layer remained in the separatory funnel, a small amount (~100 mg) sodium sulfate was directly added to the funnel to dry the crude material, which was subsequently drawn from the separatory funnel directly using a syringe. About 1.5 mL of the light yellow, crude S1 was collected.
To the previous sodium/ethanol mixture (once all solid material was completely consumed), hydrazine-15 N2 monohydrate and the crude S1 were added subsequently at room temperature. Following addition, a white solid began to precipitate. The reaction was heated at 60 °C for 4 hours, after which the reaction mixture was cooled using an ice-water bath. The reaction was filtered, and the filter cake was washed with ethanol (20 mL). The solid was dried under vacuum overnight to yield sodium azide-15 N3 as a fine, white powder (473.7 mg, 6.96 mmol, 91%).

2-(Azido-15 N3)-2-deoxy-D-glucose
To a 20-mL scintillation vial chilled over an ice-water bath, methanol (1.0 mL) and sodium metal (11 mg, 0.48 mmol, 1.1 equiv) were added in succession. This mixture was stirred over the ice-water bath until all solids were fully dissolved (~10 minutes), after which a solution of S4 (166 mg, 0.44 mmol, 1.0 equiv) in methanol (1.0 mL) was added. The reaction was warmed to room temperature and stirred at room temperature for 2 hours, during which the colorless solution became a light-yellow suspension. The reaction was quenched by adding acidic ion-exchange resin (450 mg of Dowex® 50WX8 resin, hydrogen form, 200-400 mesh) and stirring for 5 minutes at room temperature. This mixture was filtered and washed with additional methanol (~10 mL). The filtrate was concentrated in vacuo and subjected to column chromatography (0.5% methanol-ethyl acetate) to yield 2 as a sticky, yellow oil (34.9 mg, 0.17 mmol, 38% The spectral characterization of S4 and 2 prepared by the above procedures were in accord with unlabeled versions reported previously. 3

2-[2-(Azido-15 N3)acetylamino]-2-deoxy-D-glucose
To a 20-mL scintillation vial, S6-15 N3 (142.9 mg, 0.33 mmol, 1.0 equiv) was dissolved in methanol (2.0 mL) and tetrahydrofuran (0.5 mL), and the solution was chilled over an ice-water bath. A solution of lithium hydroxide monohydrate (20.8 mg, 0.50 mmol, 1.5 equiv) in water (0.50 mL) was added dropwise, and the reaction was stirred over the ice-water bath for 2 hours. Subsequently, the reaction was quenched by adding acidic ion-exchange resin (300 mg of Dowex® 50WX8 resin, hydrogen form, 200-400 mesh) and stirring for an additional 15 minutes over the ice-water bath. This mixture was filtered and washed with methanol (~10 mL), and the filtrate was concentrated in vacuo. The crude material was then dissolved in a small amount of methanol (~0.5 mL), triturated with dichloromethane (10 mL), filtered, and washed with additional dichloromethane (10 mL) to yield 3 as a fine, white solid (61.6 mg, 0.23 mmol, 70%).    The spectral characterization of S6 and 8 prepared by the above procedures were in accord with unlabeled versions reported previously. 4

O-2-(Azido-15 N3)ethyl-L-tyrosine
To a 2-dram vial, S8 (79.8 mg, 0.22 mmol, 1.0 equiv) was dissolved in methanol (0.50 mL). A solution of lithium hydroxide monohydrate (13.7 mg, 0.33 mmol, 1.5 equiv) in water (0.25 mL) was added dropwise, and the colorless solution turned light yellow. The reaction was stirred at room temperature for 1 hour, after which 2 M hydrochloric acid (162 μL, 0.34 mmol, 1.5 equiv) was added dropwise, and the reaction became white and cloudy. Additional water (1 mL) was added and the mixture was extracted with ethyl acetate (3 mL × 5). The organic layers were combined, dried with sodium sulfate, and concentrated in vacuo. The crude material was transferred to a 25-mL round-bottom flask and dissolved in dichloromethane (2 mL). After chilling the solution in an ice-water bath, trifluoroacetic acid (166 μL, 2.2 mmol, 10 equiv) was added dropwise, then the reaction was warmed to room temperature. The reaction was stirred at room temperature for 1 hour, after which it was concentrated in vacuo. Diethyl ether (5 mL) was added to the crude material, causing formation of a white precipitate. This suspension was chilled in an ice-water bath for 30 minutes, after which it was filtered and washed with additional diethyl ether (~10 mL). The solid was transferred to a 1-dram vial, dissolved in water (1 mL), and then concentrated hydrochloric acid (190 μL, 2.2 mmol, 10 equiv) was added dropwise. The solution was stirred for 1 hour, after which it was frozen by placing the vial in a -80°C freezer overnight. The frozen pellet was lyophilized, yielding 4 as a fluffy, white solid (29.4 mg, 0.10 mmol, 47%). Note that substantial formation of S10 has been observed with substitution reactions of Otosylserine derivatives, including azidation. 5 To conserve the labeled sodium azide reagent, excess amino acid precursor was used sacrificially in our procedure. The spectral characterization of S9, S10, and 5 prepared by the above procedures were in accord with unlabeled versions reported previously. [5][6] 2,3'-anhydro-5'-O-(4-methoxybenzoyl)thymidine (S11). S11 was synthesized following a reported procedure 7 to afford the desired product as a white solid (0.577g, 1.61 mmol, 81%). Rf = 0.14 (5% methanol-dichloromethane);  -15 N3)-3′-deoxy-5′-O-(4-methoxybenzoyl)thymidine (S12-15 N3).

S14
To a 25-mL round-bottom flask, S11 (287 mg, 0.80 mmol, 1.0 equiv) and sodium azide-15 N3 (82 mg, 1.2 mmol, 1.5 equiv) were suspended in DMF (20 mL). The reaction was heated to 125 ºC and stirred for 8 hours. The resulting orange solution was then cooled to room temperature, poured into H2O (15 mL) and was added 0.2 M HCl (4 mL). The aqueous layer was extracted with EtOAc (10 mL × 3). The combined organic layers were washed with water (10 mL) followed by saturated sodium chloride solution (10 mL), dried with sodium sulfate, and filtered. The filtrate was concentrated in vacuo to yield S12-15 N3 as a foamy solid. The crude material was used directly for the next step without purification. (6).

3′-(Azido-15 N3)-3′-deoxythymidine
To a 25-mL round-bottom flask was added crude S12-15 N3 and methanol (4 mL). A solution of NaOMe in methanol (1 M, 0.92 mmol, 0.92 mL, 1.15 equiv) was added dropwise, and the resulting mixture was stirred at room temperature for 2 hours. Water (6 mL) was added to this mixture, and then the methanol was evaporated in vacuo. The aqueous layer was washed with diethyl ether (10 mL × 2). Acidic ion-exchange resin (~500 mg of Dowex® 50WX8 resin, hydrogen form, 200-400 mesh) was added to the aqueous later, and the mixture was stirred at room temperature for 15 minutes, resulting in a milky, off-white solution. The resin was filtered, washed with water (20 mL), and the filtrate was frozen at -80 ºC. The frozen pellet was lyophilized to afford 6 as a fluffy white solid (43 mg, 0.162 mmol, 20%).

NMR Spectroscopic Data Acquisition and Analysis
For T1 measurements of 15 N3-azide compounds hyperpolarized by d-DNP, a series of 15  In the current procedures, the radicals were not removed in the dissolution step of d-DNP experiments before NMR detection. There was no removal of the catalyst in SABRE experiments before NMR detection. Note that the presence of either the radical or the catalyst does present a potential concern with respect to toxicity for in vivo applications or reducing T1 relaxation lifetime. On the positive note, several strategies regarding on removing the radicals for DNP experiments or removing catalysts in SBARE experiments have been reported, such as by the precipitation and filtration during the dissolution process, 9 the use of functionalized SiO2 microparticles, 10-11 and other methods. 12-16 S18

Dependence of 14 N-Mediated Scalar Relaxation on the Applied Magnetic Field
Since 14 N (I = 1) is a quadrupolar nucleus, scalar and dipolar relaxation mechanisms both contribute to 15 N (I = 1/2) relaxation lifetimes within azides 7-9. In contrast, in the absence of 14 N nuclei, the scalar relaxation mechanism is significantly less pronounced for azides 1-6. 18 The contribution of the 14 N-mediated scalar relaxation (second kind; : " () ), which is dependent on the applied magnetic field, can be expressed as follows, 19,20 : During sample transfer from DNP polarizer (B = 3.35 T) to NMR spectrometer (B = 1 T), the magnetic field of the transfer path is < 10 G. As a result, polarization is lost during this process caused by scalar coupling of the 15 N to the fast-relaxing quadrupolar 14 N within azides 7-9. 20,21 Furthermore, scalar relaxation scales with J-coupling. The different J14N-15N coupling among ( 15 N)( 14 N)2-azides 7-9 leads to varied 14 N-mediated scalar relaxation contribution, resulting in differences of the observed polarization for the compounds.
The 15 N T1 lifetimes were measured at magnetic field of 1 T. The similar 15 N T1 lifetimes for azides 7-9 and azides 1-6 suggest that 14 N-mediated scalar relaxation is not a dominant contribution to 15 N T1 lifetime at magnetic field of 1 T.

Theoretical Derivation of Resonance Conditions that Create Hyperpolarized 15 N Magnetization
In a SABRE experiment, a polarization transfer catalyst was used to establish contact between the polarization source, para-H2 and the target nuclei. As shown in Scheme S1, the catalyst has an octahedral coordination environment for iridium. The para-H2 derived hydrides and an 15 N3-azide occupy the catalyst equatorial position and act as exchanging ligands. A DMSO (dimethyl sulfoxide) and an IMes ([1,3-bis(2,4,6-trimethylphenyl)-imidazoyl] occupy the catalyst axial position and act as non-exchanging ligands. In the catalyst equatorial plane, polarization is transferred from hydrides to the 15 N nuclei through the J-coupling network across the iridium center. The reversible exchange of hydrides and the substrate results in the continuous hyperpolarization buildup on the 15 N nuclei if fresh para-H2 was continuously supplied. Scheme S1. Schematic representation of generation of hyperpolarized 15 N3-azide. IMes represents [1,3-bis(2,4,6trimethylphenyl)-imidazoyl].
The polarization transfer occurs in a AA'BMX spin system, containing five NMR active nuclei (two 1 H nuclei and three 15 N nuclei). A simplified sketch is shown in Scheme S2. Scheme S2. Spin system for polarization transfer. In the depicted AA'BMX spin system, polarization is transferred from para-H2 derived hydrides to 15 N3-azide. Note that 15 N (3, 4, 5) corresponds to 15 N (γ, β, α) in the main text. It is assumed that the polarization transfer process occurs at a magnetic field that is sufficiently low so that the frequency of both hydrides is equivalent to one frequency vH and that the frequency of nitrogens is equivalent to one frequency vN.
To understand the resonance conditions for polarization transfer driven in the spin system, the Hamiltonian was derived, The complete Hamiltonian (Eq. S4) represented in the basis introduced in Eq. S5 leads to a 32×32 matrix. The Hamiltonian can drive the hyperpolarization from the hydrides' singlet state (M ! 5 state) to other 15 N states (. 0 -, . ' states for the 15 N (3,4) spin pair or the 7 -, Rstates for the 15 N (5) spin) so that hyperpolarized 15 N magnetization can be created.
Initially, the parahydrogen derived singlet on the hydride pair is populated and all other states have nearly zero population (Eq. S6): After deriving the resonance conditions, we obtain the magnetic field All such subsets of the state combinations from the Hamiltonian matrix were identified as listed below: After deriving the resonance conditions, we obtain the magnetic field _ = ± > ", + > +* ± (> "4 + > ,4 ) − (> +4 + > *4 ) ∓ (> "+ + > "* + > ,+ + > ,* ) 4 The driving force of hyperpolarization transfer derived from the subsets of the state combinations shown above (Eq. S10 and Eq. S12) are ± (NO. M14) According to Eq. S14, hyperpolarization is transferred by depleting the hydride singlet and affecting population in the |7 > and |R > states. The driving force of the hyperpolarization transfer is the off-diagonal element '8 45 08 85 ,√, . Since other resonance conditions that deplete the hydride singlet and affect the |7 > and |R > population also have the potential to create hyperpolarized 15 N magnetization on the 15 N (5) spin, we identified all such subsets of the state combinations from the Hamiltonian matrix as listed below, After deriving the resonance conditions, we obtain the magnetic fields for Eq. S15 and Eq. S16, respectively, The driving force of hyperpolarization transfer derived from the subsets of the state combinations shown above (Eq. S15 and Eq. S16) are ±( μT (calculated by Eq. S17), ~ 0.9 μT and ~ 0.1 μT (calculated by Eq. S18).
While the SABRE-SHEATH polarizations are relatively low, the dramatic advantages of this method (including reduced cost and convenience) suggest these are promising targets. In the simplest cases of SABRE polarization (such as 15 N-pyridine) level anticrossing conditions are traditionally used to calculate an "optimal" field in the microtesla regime; in this case, the complex coupling network makes such calculations quite problematic. Some ongoing work (unpublished) shown that highly nontuitive field conditions are capable of enhancing these signals. Figure S1. a)-f) Hyperpolarized 15 N spectra of 15 N3-azides (1-6) and J-coupling analysis. The spectra were measured by d-DNP at magnetic field of 1 T with 10 o small flip angle pulses. The spectra acquired at t = 0 are shown. Note that line broadening (lb) = 0.1 Hz was applied to spectra shown in a), b), c) and f). lb = 0.2 Hz and 0.4 Hz were applied to spectra shown in d) and e), respectively. Figure S2. a)-c) Hyperpolarized 15 N spectra of ( 15 N)( 14 N)2-azides (7-9), respectively. The spectra were measured by d-DNP at magnetic field of 1 T with 10 o small flip angle pulses. The spectra acquired at t = 0 are shown for 15 Nγ sites. Note that line broadening (lb) = 1 Hz was applied to all spectra. The 15 N spectra are T2-broadened due to the fast quadrupolar relaxation of the directly bonded 14 N nuclei. Signal to noise ratio too low for the detection at 15 Nα site.