Influence of ¹³C Isotopic Labeling Location on Dynamic Nuclear Polarization of Acetate

Abstract

Dynamic nuclear polarization (DNP) via the dissolution method has alleviated the insensitivity problem in liquid-state nuclear magnetic resonance (NMR) spectroscopy by amplifying the signals by several thousand-fold. This NMR signal amplification process emanates from the microwave-mediated transfer of high electron spin alignment to the nuclear spins at high magnetic field and cryogenic temperature. Since the interplay between the electrons and nuclei is crucial, the chemical composition of a DNP sample such as the type of free radical used, glassing solvents, or the nature of the target nuclei can significantly affect the NMR signal enhancement levels that can be attained with DNP. Herein, we have investigated the influence of ¹³C isotopic labeling location on the DNP of a model ¹³C compound, sodium acetate, at 3.35 T and 1.4 K using the narrow electron spin resonance (ESR) linewidth free radical trityl OX063. Our results show that the carboxyl ¹³C spins yielded about twice the polarization produced in methyl ¹³C spins. Deuteration of the methyl ¹³C group, while proven beneficial in the liquid-state, did not produce an improvement in the ¹³C polarization level at cryogenic conditions. In fact, a slight reduction of the solid-state ¹³C polarization was observed when ²H spins are present in the methyl group. Furthermore, our data reveal that there is a close correlation between the solid-state ¹³C T₁ relaxation times of these samples and the relative ¹³C polarization levels. The overall results suggest the achievable solid-state polarization of ¹³C acetate is directly affected by the location of the ¹³C isotopic labeling via the possible interplay of nuclear relaxation leakage factor and cross-talks between nuclear Zeeman reservoirs in DNP.

TOC Graphic

1. Introduction

In vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) of nuclei with relatively low gyromagnetic ratio γ such as ¹³C is quite difficult and can be prohibitively time-consuming due to the inherently low Boltzmann thermal polarization of these nuclei at ambient conditions. One way to alleviate this NMR insensitivity issue is by employing a technique known as dynamic nuclear polarization (DNP)—a standard method used in preparing highly polarized protons and deuterons in the nuclear and particle physics community since 1960s. In DNP, the high electron spin alignment is transferred to the nuclear spins via microwave irradiation at cryogenic temperatures and high magnetic field, thus increasing the nuclear polarization and hence amplifying the NMR signal.¹ The NMR signal-enhancing capabilities of DNP was previously used primarily in nuclear scattering experiments at cryogenic temperatures until the invention of the dissolution DNP method in 2003.² This major breakthrough, pioneered by Ardenkjaer-Larsen and co-workers,² has extended the NMR signal amplification capability of DNP to the liquid-state in which the signals of low-γ nuclei such as ¹³C, ¹⁵N, ⁸⁹Y, etc.^2–6 are enhanced by several thousand-fold. Since its inception, dissolution DNP has become an emerging biomedical technique for in vitro and in vivo metabolic research using hyperpolarized ¹³C-enriched biomolecules. In particular, it has found practical applications in real-time metabolic assessment of healthy and diseased tissues with excellent sensitivity and high specificity afforded by hyperpolarized ¹³C NMR and MRI.^7–13

Since DNP involves the microwave-mediated interaction between electrons and nuclear spins, the choice of the sources of free electrons, typically provided by stable organic free radicals, can significantly affect the maximum achievable polarization level.^14–19 Furthermore, certain sample preparation methods such as the addition of trace amounts of lanthanides in the DNP sample can significantly boost the DNP-enhanced NMR signals.^5,20–23 Previous studies have shown that ¹³C enrichment²⁴ and deuteration^17,21,25 of the glassing matrix can expedite and also improve the DNP enhancement levels, respectively. All of these DNP sample optimization practices so far were additives or certain isotopic enrichment in the glassing matrix. In this work, we have investigated the effect of ¹³C isotopic enrichment location on the DNP of ¹³C substrate, in this case, ¹³C sodium acetate using the free radical trityl OX063 (see structure in Fig. 1) as the polarizing agent. Acetate is chosen for this work due to its availability, biochemical importance, and the usefulness of the carboxyl location as a model for DNP optimization of other important DNP substrates such as [1-¹³C] pyruvate. In particular, hyperpolarized ¹³C acetate has been used to probe myocardial,^26,27 hepatic,²⁸ and cerebral²⁹ metabolism in vivo.

Figure 1.

The free radical polarizing used in this work (left) and a representative ¹³C microwave DNP spectrum (right) of trityl OX063-doped [1-¹³C] acetate sample showing the optimum microwave irradiation frequencies P(+) and P(−) for DNP at 3.35 T and 1.4 K. All the other ¹³C isotopomers of acetate have the same locations of P(+) and P(−).

In this study, we have recorded and compared the relative ¹³C polarization levels achieved for carboxyl and methyl ¹³C spins of acetate samples with the following isotopic enrichments (see structures in Fig. 2): the carboxyl [1-¹³C], deuterated carboxyl [1-¹³C,d₃], methyl [2-¹³C], deuterated methyl [2-¹³C,d₃], doubly-labeled [1,2-¹³C₂], and doubly-labeled and deuterated [1,2-¹³C2, d₃] sodium acetate. While it is well-known in the liquid-state that carboxyl ¹³C spins have relatively longer T₁ relaxation time because of its relative isolation from the protons and that deuteration of the methyl ¹³C group would lead to longer ¹³C T₁ and higher liquid-state enhancement,^30–32 there is a dearth of comparative data of these ¹³C isotopomers in terms of solid-state ¹³C DNP polarization levels and T₁ relaxation times at cryogenic temperatures. The main goal of this study is to investigate the effect on DNP of these different ¹³C isotopic labeling locations of acetate in which intra-molecular forces between magnetically-active nuclei may play a significant role on the polarization transfer process at cryogenic temperature. In addition, this study also aims to provide details as to the effect of ²H enrichment of the methyl group, a practice which is proven to be beneficial in preserving the ¹³C polarization in the liquid-state, on the ¹³C DNP at cryogenic temperature from which the NMR signal amplification originates. Furthermore, the corresponding solid-state ¹³C T₁ relaxation times of the different ¹³C isotopic locations will also be measured and discussed in conjunction with the enhanced ¹³C polarization levels. With the plethora of ¹³C-labeled carboxylate compounds used as hyperpolarized ¹³C NMR and MRI agents,^7–9,30 the results of this study may provide insights with regards to the DNP efficiency of these ¹³C isotopomers of acetate or a carboxylate in general prior to the dissolution process.

Figure 2.

Structures and representative normalized hyperpolarized ¹³C NMR spectra of frozen solutions of sodium acetate with different isotopic labeling locations: (a) [1-¹³C], (b) [1-¹³C,d₃], (c) [2-¹³C], (d) [2-¹³C,d₃], (e) [1,2-¹³C₂], and (f) [1,2-¹³C₂,d₃]. These ¹³C NMR spectra were taken at 3.35 T and 1.4 K from 100 μL aliquots of 3 M ¹³C acetate samples in 1:1 v/v glycerol:water doped with 15 mM trityl OX063.

2. Experimental Section

Sample preparation:

The ¹³C-labeled acetate compounds (99% ¹³C enrichment), glassing solvents and free radical used in this study were obtained commercially and were used without further purification. The following amounts of ¹³C-enriched acetate compounds (Sigma-Aldrich, St. Louis, MO) were weighed out and prepared using Discovery semi-micro analytical balance (Ohaus, Parsippany, New Jersey): 24.9 mg [1-¹³C]sodium acetate (mw=83.03 g/mol), 24.9 mg [2-¹³C]sodium acetate (mw=83.03 g/mol), 25.8 mg [1-¹³C, d₃]sodium acetate (mw=86.04 g/mol), 25.8 mg [2-¹³C, d₃]sodium acetate (mw=86.04 g/mol), 25.2 mg [1,2-¹³C]sodium acetate, and 26.1 mg [1,2-¹³C,d₃]sodium acetate (mw=87.04 g/mol). 100 uL solutions were prepared in 1 mL microcentrifuge (Scientific USA, Ocala, FL) by mixing these compounds with 1:1 glycerol:water. 2.14 mg of trityl OX063 free radical (Oxford Instrument Biotools, MA) was added to each solution, then rapidly mixed and prepared using vortex mixer and microcentrifuge (ThermoFisher Scientific, WI). The final concentrations of ¹³C acetate and trityl OX063 in the solutions were 3 M and 15 mM, respectively. Fresh sample for each trial was prepared 10 hours prior to experiment and stored at −80° C in an ultra-low temperature freezer (Thermo Scientific, WI) to prevent degradation. All the procedures of sample preparation, storage, insertion, and acquisition were done in triplicate for each ¹³C acetate isotopomer.

Microwave frequency sweep.

The ¹³C microwave DNP spectrum, which determines the optimum microwave frequencies to polarize the samples, was done using a built-in NMR program in the HyperSense polarizer (Oxford Instruments, UK). 100 μL ¹³C acetate sample was placed in a PEEK cup and was quickly inserted into the polarizer which operates at 3.35 T and 1.4 K base temperature. The microwave power was set at 100 mW and the frequency was swept in the range 94.00–94.20 GHz with step interval of 5 MHz. The sample was irradiated for 3 minutes at each frequency step and the ¹³C NMR signal was automatically recorded for each frequency.

¹³C polarization buildups.

The ¹³C acetate samples were all irradiated at the positive polarization peak P(+) determined from the microwave frequency sweep. 100 μL aliquots of ¹³C acetate samples were placed in the polarizer at 3.35 T and 1.4 K. A ¹³C NMR spectrum was recorded every 5 minutes and plotted as a function of time. The ¹³C polarization buildup curves for each sample were done in triplicate. Since the same 100-µL volume was used for the doubly-labeled ¹³C samples ([1,2-¹³C]sodium acetate and [1,2-¹³C, d₃]sodium acetate), the polarization buildup intensities of these 2 samples were multiplied by ½ to be able to compare with the relative ¹³C DNP intensities of singly ¹³C-labeled samples ([1-¹³C]sodium acetate, [1-¹³C, d₃]sodium acetate, [2-¹³C]sodium acetate, and [2-¹³C, d₃] sodium acetate) with the sample ¹³C spin count. The relative ¹³C maximum DNP signals were determined (mean ± standard deviation) for each sample and compared in bar graph representations.

¹³C T₁ decay curves.

Each ¹³C acetate sample was polarized to its maximum ¹³C DNP intensity at 3.35 T and 1.4 K. Once the samples reached their maximum polarization, the microwave source was then turned off and the decay of hyperpolarized ¹³C NMR signal of the frozen sample at 3.35 T and 1.4 K was recorded every 20 minutes using a 2-degree RF pulse. To do this, an RG-58U RF cable from a Varian VNMR 400 MHz console (Agilent Technologies, Santa Clara, CA) was connected to the top-tuned NMR circuit box of the HyperSense polarizer. Depending on the ¹³C T₁, each T₁ decay curve from a ¹³C acetate sample can take 5–10 hours of NMR signal recording. The T₁ decay curves were then fitted using an equation that accounts ¹³C NMR signal decay due to T₁ relaxation and RF pulsing.⁵ The ¹³C T₁ values of these samples were extracted using the aforementioned equation.

3. Results and Discussion

The free radical trityl OX063 is very commonly used in dissolution DNP, and its electron spin resonance (ESR) linewidth is among the narrowest of all free radicals used in DNP.^18,34,35 This narrow linewidth allows for the DNP process to occur either via the solid effect and thermal mixing, depending on the nucleus being polarized.^24,34,36,37 The solid effect is dominant when the nuclear Larmor frequency is much larger than the linewidth of the free radical as is the case for the polarization of ¹H. In this mechanism, polarization is achieved through direct activation of zero or double quantum transitions by microwave irradiation at a frequency $\upsilon ={\upsilon }_s\pm {\upsilon }_I$ where ${\upsilon }_s$ and ${\upsilon }_I$ are the electron and nuclear Larmor frequencies, respectively. Thermal mixing, on the other hand, is dominant when the nuclear Larmor frequency is comparable to or less than the free radical ESR linewidth.³⁸ Even for a narrow linewidth radical such as trityl OX063, low γ nuclei meet this condition at the field considered in this work, so it is expected that ¹³C and ²H polarization will proceed by way of thermal mixing.^18,39,40 In this mechanism, spin systems are treated as thermodynamic heat reservoirs with an associated spin temperature.^38,41,42 In particular, reservoirs include the nuclear and electron Zeeman systems (NZS and EZS) and the electron dipolar system (EDS). Thermodynamically, polarization transfer proceeds when microwave irradiation near the electron resonance brings the three reservoirs into thermal contact and “cools” the NZS to a spin temperature value below thermal equilibrium.^1,43,44

The P(+) and P(−) polarization peaks in the microwave frequency sweep data in Fig. 1 denote the optimum microwave frequencies in which the trityl OX063-doped ¹³C acetate samples can be irradiated to achieve the maximum polarization enhancement. Conventionally, DNP at P(+) will result in more nuclear spins populating the lower Zeeman energy level and conversely, microwave irradiation at P(−) will cause more spins to populate the upper Zeeman energy state. This polarization gradient in the microwave frequency sweep can be qualitatively described by the Borghini or spin temperature model of DNP.⁴⁰ The ¹³C DNP samples in this study were irradiated at the P(+) microwave frequency. During DNP, all NMR-active nuclei in the DNP sample whose Larmor frequencies are comparable to the trityl ESR linewidth such as ¹³C, ²³Na, and ²H spins are expected to be in thermal contact with the trityl OX063 EDS. Protons, on the other hand, have a Larmor frequency that is much larger than the trityl OX063 ESR linewidth at 3.35 T and therefore ¹H spins are not in thermal contact with EDS as experimentally confirmed in a previous report.⁴⁵ We will revisit the pertinent discussion of this phenomenon later in the ¹³C DNP efficiency when ¹H spins are replaced with ²H spins in the methyl group of acetate.

Representative hyperpolarized solid-state ¹³C NMR spectra of the ¹³C isotopomers of sodium acetate at 3.35 T and 1.4 K are shown in Fig. 2. Since these frozen samples are under static or non-magic angle spinning (MAS) conditions, dipolar broadening is the most prominent feature visible in these ¹³C NMR spectra at cryogenic conditions. For instance, the ¹³C NMR spectrum of [1-¹³C] sodium acetate in Fig. 2a has a full-width at half-height value of around 9 kHz. Deuteration of the methyl group appears to have no significant effect on the solid-state ¹³C NMR spectrum of the carboxyl ¹³C spins of acetate as shown in Fig. 2b. Despite the large broadening, some of the NMR spectral features of these acetate samples with different ¹³C isotopic labeling locations are distinguishable. In particular, there is a slight difference in NMR resonance locations between the carboxyl ¹³C spins in Fig. 2a and methyl ¹³C spins in Fig. 2c. Furthermore, the NMR spectrum of methyl ¹³C spins in Fig. 2c has a slightly broadened shoulder ascribed to the coupling of methyl ¹³C spins with methyl protons. When protons are replaced with deuterons in the methyl group, the broadened shoulders seen previously in Fig. 2c disappear and the resulting NMR spectrum of deuterated methyl ¹³C spins in Fig. 2d resembles a symmetric single Gaussian or Lorentzian shape. On the other hand, the ¹³C NMR spectrum of the doubly-labeled [1,2-¹³C₂] acetate in Fig. 2e appears to be a superposition of the ¹³C NMR spectra of the carboxyl ¹³C spins in Fig. 2a and the methyl ¹³C spins in Fig. 2c. In a similar fashion, the NMR spectrum of doubly-labeled and deuterated [1,2-¹³C2,d₃] acetate displayed Fig. 2f is reminiscent of a combination of the ¹³C NMR spectra of [1-¹³C] acetate in Fig. 2a and [2-¹³C,d₃] acetate in Fig. 2d.

Next, we have investigated the effect of ¹³C isotopic labeling location in acetate on the efficiency of ¹³C DNP. Inspection of Fig. 3 reveals that not only do they strongly affect the NMR spectra, but the different ¹³C isotopic labels can significantly affect the achievable maximum ¹³C DNP levels. Relative ¹³C polarization buildup curves in Fig. 3a show that ¹³C labeling in the carboxyl location yielded about twice the ¹³C polarization obtained for the methyl or deuterated methyl ¹³C spins in acetate. Deuteration of the methyl group resulted in a slight but not statistically significant decrease in the solid-state polarization of the carboxyl ¹³C spins. In addition, the doubly-labeled [1,2-¹³C₂] and [1,2-¹³C₂,d₃] acetate samples yielded ¹³C polarization levels that are comparable with the methyl or deuterated methyl ¹³C spins in acetate. It should be noted that the ¹³C polarization buildup curves displayed in Fig. 3a were normalized to the same number of ¹³C spins for direct comparisons of relative solid-state ¹³C polarization levels. A bar graph of the relative maximum ¹³C polarizations that can be achieved from each isotopomer is shown in Fig. 3b. While it is expected that the ¹³C labeling in the carboxyl location can be polarized most efficiently due to its relative isolation from protons, it is quite surprising that the ¹³C polarization levels of the other samples are nearly equal. Based on dipolar relaxation arguments, it is also anticipated that methyl ¹³C spins would yield lower ¹³C polarization enhancement compared to ¹³C DNP of carboxyl ¹³C spins because of the direct coupling or proximity of methyl ¹³C to methyl protons which are a dominant source of fluctuating magnetic fields. As noted before, deuteration of the methyl ¹³C group is beneficial in the liquid-state because it increases ¹³C T₁ and thus higher liquid-state ¹³C NMR enhancements due to longer ¹³C polarization preservation time.^30–32 However, our solid-state ¹³C DNP results indicate that there is no improvement in the solid-state ¹³C polarization levels when the methyl group is deuterated for both the singly- and doubly- ¹³C labeled acetate samples. Although not statistically significant as shown in Fig. 3b, deuteration in the methyl location, in fact, causes a slight reduction in the average ¹³C polarization enhancement.H

Figure 3.

Relative solid-state ¹³C DNP signals of various isotopic labeling locations of sodium acetate taken at 3.35 T and 1.4 K: (a) Buildup curves of the relative ¹³C polarization as a function of microwave irradiation time. The error bars are standard deviations for trials done in triplicate. The down arrow indicates the direction of decreasing polarization level for the different isotopic labeling locations. (b) Maximum ¹³C DNP signals for the different isotopomers of sodium acetate taken from data in (a). The data for the doubly-labeled [1,2-¹³C₂] acetate and [1,2-¹³C₂,d₃] acetate samples were normalized according to the same ¹³C spin count for direct comparison with the maximum ¹³C DNP signals of the singly-labeled ¹³C acetate samples.

To explain these behavior regarding the effect of deuteration, we revisit the previous finding that the use of trityl OX063 as the polarizing agent, at least at 3.35 T and temperature close to 1 K, allows for the exclusion of ¹H spins from the polarization process via thermal mixing.⁴⁶ When ¹H is replaced with ²H, the NZS of ²H spins in the methyl groups is now brought into thermal contact with the EDS, thus increasing the total heat load for the EDS to cool, and thereby reducing the polarization enhancement.^21,25,38 A similar effect has been observed when ²H-enrichment is used in the glassing solvents where the ¹³C polarization is reduced significantly by 30–50%.^21,25 In the case of isotopic labeling of the substrate itself as in this work, ²H labeling in the methyl group resulted in similar or slightly lower ¹³C polarization compared to ¹³C DNP of non-deuterated methyl ¹³C spins, suggesting the extra ²H NZS heat load of the deuterons in the methyl group is not as significant as the DNP effect of ²H enrichment in the glassing matrix.^21,25 This is borne out by considering the relative number of hydrogen atoms in the two systems. In the glassing matrix, water and glycerol are approximately 55 M and 13 M respectively, far exceeding the 3 M concentration of acetate. As such, the reduction of ¹³C polarization induced by ²H labeling in the methyl group is small compared to that witnessed in deuteration of the glassing matrix. This is of great interest as it suggests that ²H labeling may be utilized to lengthen liquid-state ¹³C T₁ with only minor reductions in the solid-state ¹³C polarization enhancement.

Based on the high polarization of singly-labeled carboxyl ¹³C and the low polarization for other labels, it is suggested that polarization is strongly affected by relaxation interactions between nuclear spins which can be encompassed in a “leakage factor” f.^34,38 It should be noted that f varies with γ² of the nucleus in question, suggesting that ¹³C bonding to ¹H would lead to large f, while bonding to ²H or ¹³C would lead to comparatively small f.^1,46 From this, one possible scenario is that the ¹³C polarization of the methyl ¹³C spins is governed primarily by the leakage factor given the proximity with protons. When methyl ¹³C samples are deuterated, the leakage factor is reduced because of the weaker magnetic moment of ²H, but the heat load of the NZS is increased, ultimately resulting in slightly weaker polarization enhancement. In addition, our suggested explanation for the lower ¹³C polarization values of the doubly labeled [1,2-¹³C₂] and [1,2-¹³C₂,d₃] acetate samples is that the presence of both carboxyl and methyl ¹³C labels in acetate contribute to mutual sources of fluctuating magnetic fields via dipolar interaction, leading to shorter solid-state ¹³C T₁ values and thus lower ¹³C polarization levels.

In light of the suggested DNP explanations related to nuclear relaxation, we have performed ¹³C T₁ relaxation time measurements of these different ¹³C isotopomers of acetate at 3.35 T and 1.4 K. The ¹³C hyperpolarization decay curves of these different acetate samples along with the fits to the equation⁵ accounting for ¹³C T₁ relaxation and RF pulsing are displayed in Fig. 4a. We should note that for doubly labeled samples, the displayed T₁ is calculated by fitting a decay curve to the total signal obtained from both ¹³C peaks. In the supporting information, it is shown that the relaxation of the individual peaks mirror that of the convolution of the two spectra. The calculated average solid-state ¹³C T₁ values of these trityl OX063-doped ¹³C acetate isotopomers are displayed as a bar graph in Fig. 4b. It is apparent that the solid-state ¹³C T₁ values of the different acetate isotopomers shown in Fig. 4 closely correlate with their corresponding relative ¹³C polarization levels shown in Fig. 3. One prominent observation from Fig. 4 is that the carboxyl ¹³C T₁ relaxation value is about twice the ¹³C T₁ of the methyl ¹³C spins and the other isotopomers of acetate. These solid-state ¹³C T₁ results roughly scale with their solid-state ¹³C polarization values. Another important observation from Fig. 4 is that there seems to be a significant reduction of ¹³C T₁ when the methyl group is deuterated. This ¹³C T₁ reduction effect by deuteration is, as noted before, mirrored on a weaker scale by ¹³C polarization reduction for the deuterated versions of the acetate isotopomer shown in Fig. 3. The shortening of methyl ¹³C T₁ in the solid-state when ¹H (γ=42.6 MHz/T) is exchanged for ²H (γ=6.54 MHz/T) in acetate is somewhat unexpected considering that deuterons have weaker magnetic moments than protons, and therefore weaker sources of fluctuating magnetic fields for relaxation. As noted previously, the exact opposite occurs in the liquid-state in which ²H enrichment of the molecule leads to longer ¹³C T₁.⁴⁷ Though the cause of this counterintuitive result is not immediately clear, one possible explanation is that of heteronuclear “cross-talk” between hyperpolarized ¹³C and ²H spins. It has been found that there may be polarization exchange between ¹H and ²H in the presence of electrons, which is termed the heteronuclear cross effect, suggesting the possibility of a similar exchange between ¹³C and ²H.⁴⁸ This exchange causes the equalization of polarization enhancements of the two different spin species, suggesting its application to the present case. The solid-state relaxation time of deuterium is much shorter than that of ¹³C, so as ²H relaxes, these spins act as a “sink” for ¹³C polarization. That is, as ²H spins relax, polarization is pulled from the ¹³C spins, leading to slower ²H and faster ¹³C relaxation. Further studies are needed to confirm this or in general, pinpoint the exact cause of this effect on solid-state ¹³C T₁ by deuteration. Testing of the effect would require monitoring of the hyperpolarized ²H NMR signal during relaxation which is currently outside the capability of our hyperpolarizer. Nevertheless, our results here have indicated that the location of ¹³C isotopic labeling has a significant effect on the maximum achievable ¹³C polarization levels.

Figure 4.

Lifetimes of the solid-state ¹³C polarizations of hyperpolarized ¹³C isotopomers of sodium acetate at 3.35 T and 1.4 K: (a) Decay of the hyperpolarized ¹³C NMR intensity of each sample monitored by applying a 2-degree RF pulse every 20 minutes after the microwave source was turned off. The down arrow indicates the direction of decreasing relaxation times for the various ¹³C acetate isotopomers. The ¹³C DNP decay data were fitted to a single-exponential equation accounting for the DNP signal loss due T₁ decay and RF pulsing. (b) Comparative solid-state ¹³C T₁ values of the ¹³C-enriched acetate samples calculated from data in (a). The error bars are standard deviations for trials done in triplicate. The ¹³C T₁ values reported for the doubly-labeled [1,2-¹³C₂] sodium acetate and [1,2-¹³C₂,d₃] sodium acetate were done by integrating the areas of both the carbonyl and methyl ¹³C spins. See Supporting Information for separate T₁ analysis of the deconvoluted ¹³C NMR spectra of doubly-labeled ¹³C acetate samples.

4. Conclusion

It has been shown that the ¹³C isotopic labeling of acetate has a significant effect on ¹³C DNP efficiency when using the trityl OX063 free radical. When considering both the polarization enhancement and T₁, [1-¹³C] acetate has significant advantages over the other labelings studied due to its isolation from other nuclear magnetic spins. ¹³C labeling in the methyl group greatly reduced the efficiency of DNP, leading to lesser polarization enhancement and short T₁ due to the coupling to adjacent nuclear spins which increases the nuclear relaxation leakage factor. Contrary to liquid-state results, deuteration of the methyl group leads to smaller solid-state ¹³C polarization gain and faster relaxation due to possible increased NZS heat load and heteronuclear cross-talk between hyperpolarized ¹³C and ²H spins. Our results suggest that there is a close correlation between the maximum achievable solid-state ¹³C polarization values and their corresponding solid-state ¹³C T₁ relaxation times.

5. Supporting Information

In the supporting information, we show the deconvolution of ¹³C NMR spectra for the doubly labelled isotopomers of acetate. This allows for the determination of solid-state T₁ for each ¹³C location as well as the overall ¹³C signal of the molecule. For [1,2-¹³C₂] sodium acetate, the methyl ¹³C signal is dwarfed by the carboxyl, making an accurate assessment of the methyl T₁ difficult. In the case of [1,2-¹³C₂,d₃] sodium acetate, the peaks corresponding to the two locations are approximately equal in intensity, making the deconvolution more effective. This sample shows that there is “cross-talk” between the ¹³C spins in different locations, resulting in all ¹³C spins, regardless of location, to relax at approximately the same rate.