Directly Bound Deuterons Increase X‐Nuclei Hyperpolarization using Dynamic Nuclear Polarization

Abstract Deuterated 13C sites in sugars (D‐glucose and 2‐deoxy‐D‐glucose) showed 6.3‐to‐17.5‐fold higher solid‐state dynamic nuclear polarization (DNP) levels than their respective protonated sites at 3.35T. This effect was found to be unrelated to the protonation of the bath. Deuterated 15N in sites bound to exchangeable protons ([15N2]urea) showed a 1.3‐fold higher polarization than their respective protonated sites at the same magnetic field. This relatively smaller effect was attributed to incomplete deuteration of the 15N sites due to the solvent mixture. For a 15N site that is not bound to protons or deuterons ([15N]nitrate), deuteration of the bath did not affect the polarization level. These findings suggest a phenomenon related to DNP of X‐nuclei directly bound to deuteron(s) as opposed to proton(s). It appears that direct binding to deuterons increases the solid‐state DNP polarization level of X‐nuclei which are otherwise bound to protons.

Typical formulations for the hyperpolarization of the sugar analogs are described below and in Table S1.
The concentration of the sugar in the formulation was calculated by dividing the number of moles by the calculated volume of the solution.The latter was calculated according to the mass of H2O or D2O solution that was added, corrected for 1) the density of D2O (1.11 g/ml, for solutions in D2O), and 2) the solutions' volume increase upon the addition of the sugar, as described in Table S5.

Data analysis for polarization buildup
For 13 C polarization, the data were obtained using the polarizer's internal software.For 15 N polarization, each frequency domain spectrum was analyzed using a single Lorentzian line fitting and integration in Matlab (Mathworks, Natick, MA, USA).The polarization buildup time courses were fitted using the curve fitting option in Matlab, using the polarization buildup equation (Eq.1), where P(t) is the polarization level at each time point, Pmax is the maximal polarization level that could be reached, and Tb is the polarization buildup time constant.
Eq. 1  The first maxima of the sugar formulations used in the current study were found to be the same and this frequency was used for recording the polarization buildup time courses.
The data were normalized to the highest point of each profile.Data are presented as obtained from the polarizer's spectrometer, in magnitude mode.Further to obtaining higher polarization levels for the X-nuclei which were directly bound to deuterons, we wished to explore the potential mechanism underlying this observation.
A possible explanation has to do with prolongation of the solid-state T1, which would allow favorable buildup conditions.To this end, the experiments described in Figure 1B and Table 1 with 13 C-and deuterium-labeled-2DG analogs were reproduced on a second Hypersense polarizer at the University of Oxford.
A) A reproduction of the experiments shown in Figure 1B, with the same samples used for producing Figure 1B (n=3, for each sample, Formulations 2A and 2B).
B) At the end of each buildup duration, the MW irradiation was stopped, and the polarization level was monitored during its decay.
Mz was calculated from the Mxy data shown in this plot using the conversion: Mxy(t)=Mz(t)sin(), where , the flip angle for excitation, was 5 .The decay of Mz(t) data was then used to calculate the T1 of [ 13 C6,D8]2DG and [ 13 C6]2DG in solid-state.
The solid-state T1 of [ 13 C6,D8]2DG was found to be 1.8-fold longer than that of [ 13 C6]2DG (185.7±36.2 min and 101.7± 32.5 min, respectively).In this set of experiments the increase in maximal polarization level of the deuterated compound was 14.8-fold (more than in the previous set of experiments, Table 1) and the buildup time constant increased 1.2-fold (less than in the previous set of experiments, Table 1).These differences are likely due to slightly different temperatures of the sample during the DNP process across the two spin polarization systems, as it was previously shown that such sugar molecules' polarization is very sensitive to the temperature of the sample during the DNP process 6 .Nevertheless, the higher polarization level of the 13 C sites directly bound to deuterons was reproduced and the T1 in solid-state was indeed prolonged, providing a possible explanation for this observation.
Formulation #3A: [ 15 N2]urea in D2O:glycerol 70.9 mg of [ 15 N2]urea were combined with 186.6 mg of a 60:40 D2O:glycerol solution and 3.4 mg of OX063.The final concentrations in this formulation were 14.9 mM OX063 and 4.38 mol of [ 15 N2]urea per mg formulation.Formulation #3B: [ 15 N2]urea in H2O:glycerol 70.9 mg of [ 15 N2]urea were combined with 186.6 mg of a 60:40 H2O:glycerol solution and 3.4 mg of OX063.The final concentrations in this formulation were 14.1 mM OX063 and 4.38 mol of [ 15 N2]urea per mg formulation.Formulation #4A: sodium [ 15 N]nitrate in D2O:glycerol 210.4 mg of a D2O solution containing 7.5 M of [ 15 N]nitrate were combined with 64.6 mg of glycerol and 4.3 mg of OX063.The final concentrations in this formulation were 12.5 mM OX063 and 5.09 mol of [ 15 N]nitrate per mg formulation.Formulation # 4B: sodium [ 15 N]nitrate in H2O:glycerol 210.4 mg of a H2O solution containing 7.5 M of [ 15 N]nitrate was combined with 64.6 mg of glycerol and 4.3 mg of OX063.The final concentrations in this formulation were 11.5 mM OX063 and 5.65 mol of [ 15 N]nitrate per mg formulation.

Figure S1 .
Figure S1.MW frequency sweep profiles of deuterated and non-deuterated 13 C-labeled sugars in H2O and in D2O.

Figure S2 .
Figure S2.Individual time courses and curve fitting for polarization buildup of deuterated and non-deuterated 13 C-uniformly-labeled sugars.

Figure S3 .
Figure S3.Example intensity profiles of MW sweeps obtained with the formulations containing 15 N in D2O:glycerol.

Figure S5 .
Figure S5.Solid-state polarization buildup and decay of 13 C-labeled 2DG with or without deuteration.

Table S1 .
Formulations' components About 480-600 mg of each formulation were placed in the polarizer cup for monitoring MW irradiation profiles, and for recording the polarization buildup of [ 13 C6]2DG and [ 13 C6]Glc (formulations #1B and 2B).For recording the polarization buildup of [ 15 N]urea and sodium [ 15 N]nitrate approximately 110-120 mg and 190-200 mg of each formulation was used, respectively.

Table S2 .
Conditions and characteristics of deuterated and non-deuterated sugars' polarization.

Table S4 .
Chronological order of 3 C polarization buildup measurements for the sugar formulations.

Table S5 .
Volume increase in mixtures of glucose and water which were used in the formulations.

Table S6 .
The various conditions of protonation and deuteration of X-nuclei tested in this work.