Introduction

The study of transient intermediate states in chemical reactions has long proven difficult for chemists. NMR’s usefulness is limited in in this arena due to the low thermal polarization of atomic nuclei, which yields poor temporal resolution. In this study, we demonstrate that the NMR signal enhancement provided by hyperpolarized (HP) ¹³C NMR can be used to probe the dynamics of these intermediate states and lead to the quantification of reaction kinetics. HP 13C-bicarbonate, often used for pH imaging experiments, can be produced through the decarboxylation of HP [1-¹³C] pyruvate. However some aspects of this reaction are poorly understood; the proposed intermediate state, 2-hydroxyperoxy-2-hyroxypropranoate, has never been directly observed at room temperature, and a mysterious peak is often seen in spectra ~1.5ppm to the right of bicarbonate which has been previously assumed to be an impurity or secondary intermediate state [1]. Here we report the first observation of this reaction’s intermediate state at room temperature, as well as the characterization of the ‘mystery’ peak as peroxycarbonate, the product of a side reaction between CO₂ and the hydroperoxide ion, OOH^-.

Methods

All reactions were preformed by sequentially injecting reactants into a 20mm NMR tube placed in a 9.4T vertical bore magnet (Varian Inc). In the initial reaction analysis experiments, 28.6mg of [1-¹³C]-pyruvate was polarized to ~25% in a Hypersense DNP polarizer (Oxford Instruments) and quickly injected into the 20mm reaction tube where it was diluted to 6.7mM in 15mL of DI water with 100mM of tris, CHES, or CAPS buffer, depending on the target pH of the particular experiment. 80mM of H₂O₂ was injected into the reaction mixture <5 seconds after the HP pyruvate solution to initiate the reaction. Spectra were acquired for at least 100 seconds with a TR of 250ms and a flip angle of 10°. To help determine the identity of the compound producing the ‘mystery’ peak, this same experiment was repeated using [2-¹³C]-pyruvate. In order to confirm that this compound was indeed the product of a side reaction between CO₂ and the hydroperoxide ion, two additional reaction experiments were preformed. In the first experiment, directly polarized ¹³C-bicarbonate was mixed with H₂O₂ at elevated pH in the absence of pyruvate, the appearance of a peak at the same chemical shit as the ‘mystery’ peak would confirm that it is the result of a reaction between bicarbonate or CO₂ and H₂O₂ or OOH^-. In the second experiment, the decarboxylation reaction was repeated at elevated pH in the presence of >600 units/mL of carbonic anhydrase to assess the change in the ‘mystery’ compound dynamics when the produced CO₂ is rapidly converted to bicarbonate. All data was analyzed in custom Matlab software. A model of the full reaction dynamics was developed and implemented in Matlab.

Results

Using hyperpolarized ¹³C-NMR, we are able to acquire spectra of reactions in progress with temporal resolution as low as 100ms without significant loss in spectral resolution. As seen in Figure 1, 2, & 5, we have more than enough signal to directly quantify the dynamics of the intermediate state and we find it at the same chemical shift as previously reported in low temperature studies [2]. This constitutes the first direct observation of this intermediate state at room temperature. From the spectra of the decarboxylation reaction of [2-13C] pyruvate shown in Figure 3, we see all of the expected 2C peaks except for one that could represent the mystery compound. From this we conclude that the compound must contain just a single carbon atom. We theorized that this compound could be the peroxycarbonate ion, formed from the reaction of CO₂ and OOH^-. As shown in Figure 4, when H₂O₂ and NaOH are mixed with a solution of hyperpolarized ¹³C-bicarbonate and ¹³CO₂, peroxycarbonate is produced at the same chemical shift as the ‘mystery’ compound. When carbonic anhydrase was introduced into the decarboxylation reaction at elevated pH, the production of peroxycarbonate is hindered due to the rapid conversion of CO₂ to bicarbonate as shown in Figure 5. A model of the reaction was developed based on the results of these experiments, which provided reasonably good fits as seen in Figure 2.

Conclusions

We have used hyperpolarized ¹³C-NMR to thoroughly investigate and characterize the dynamics of the decarboxylation reaction of pyruvate via H₂O₂, a reaction often used for the production of high polarization _¹³C-bicarbonate. This study could serve as a template for using hyperpolarized ¹³C NMR to study the dynamics of innumerable other organic reactions with polarizable substrates, and demonstrates the wealth of information potentially available to chemists from such studies.

Acknowledgements

This work was supported by the National of Institutes of Health (NIH) R01 HL124986.