Category Archives: Kinetics

Quantifying reaction kinetics of the non-enzymatic decarboxylation of pyruvate and production of peroxymonocarbonate with hyperpolarized 13C-NMR

Drachman, N., et al., Quantifying reaction kinetics of the non-enzymatic decarboxylation of pyruvate and production of peroxymonocarbonate with hyperpolarized 13C-NMR. Phys. Chem. Chem. Phys., 2017. 19(29): p. 19316-19325.

http://dx.doi.org/10.1039/C7CP02041D

The transient nature of intermediate states in chemical reactions has made their detailed investigation difficult. In this study, we demonstrate the utility of hyperpolarized 13C-NMR to directly observe and quantify the kinetics of the intermediate compound in the non-enzymatic decarboxylation of pyruvate via H2O2 with time resolutions of <1 s. Reactants were sequentially added to a reaction vessel within a 9.4 T NMR magnet while continuously acquiring spectra with a low flip angle, producing the first direct observation at room temperature of the previously proposed reaction intermediate, 2-hydroperoxy-2-hydroxypropanoate. We also performed a series of NMR experiments to determine the identity of a previously unidentified peak, which was found to be peroxymonocarbonate, the product of the side reaction between HCO3-/CO2 and H2O2/OOH-. Using the information obtained from these experiments, we developed a kinetic model which fully describes the mechanism of reaction and can be fit to experimental data to simultaneously determine multiple kinetic rate constants over several orders of magnitude. We also discuss the application of this reaction to the production of hyperpolarized bicarbonate for pH imaging experiments. This study presents a template for the use of hyperpolarized 13C-NMR to study the kinetics and reaction mechanisms of innumerable organic reactions which involve polarizable substrates.

Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water

Kim, J., M. Liu, and C. Hilty, Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water. The Journal of Physical Chemistry B, 2017. 121(27): p. 6492-6498.

http://dx.doi.org/10.1021/acs.jpcb.7b03052

Water–protein interactions play a central role in protein structure, dynamics, and function. These interactions, traditionally, have been studied using nuclear magnetic resonance (NMR) by measuring chemical exchange and nuclear Overhauser effect (NOE). Polarization transferred from hyperpolarized water can result in substantial transient signal enhancements of protein resonances due to these processes. Here, we use dissolution dynamic nuclear polarization and flow-NMR for measuring the pH dependence of transferred signals to the protein trypsin. A maximum enhancement of 20 is visible in the amide proton region of the spectrum at pH 6.0, and of 47 at pH 7.5. The aliphatic region is enhanced up to 2.3 times at pH 6.0 and up to 2.5 times at pH 7.5. The time dependence of these observed signals can be modeled quantitatively using rate equations incorporating chemical exchange to amide sites and, optionally, intramolecular NOE to aliphatic protons. On the basis of these two- and three-site models, average exchange (kex) and cross-relaxation rates (σ) obtained were kex = 12 s–1, σ = −0.33 s–1 for pH 7.5 and kex = 1.8 s–1, σ = −0.72 s–1 for pH 6.0 at a temperature of 304 K. These values were validated using conventional EXSY and NOESY measurements. In general, a rapid measurement of exchange and cross-relaxation rates may be of interest for the study of structural changes of the protein occurring on the same time scale. Besides protein–water interactions, interactions with cosolvent or solutes can further be investigated using the same methods.

Toward Quantitative Measurements of Enzyme Kinetics by Dissolution Dynamic Nuclear Polarization

Miclet E, Abergel D, Bornet A, Milani J, Jannin S, Bodenhausen G. Toward Quantitative Measurements of Enzyme Kinetics by Dissolution Dynamic Nuclear Polarization. The Journal of Physical Chemistry Letters. 2014;5(19):3290-5.

http://dx.doi.org/10.1021/jz501411d

Dissolution dynamic nuclear polarization (D-DNP) experiments enabled us to study the kinetics of the enzymatic phosphorylation reaction of glucose to form glucose-6-phosphate (G6P) by hexokinase (HK), with or without the presence of an excess of G6P, which is known to be an inhibitor of the enzyme. Against all expectations, our observations demonstrate that the phosphorylation of both α and ? glucose anomers occurs with comparable kinetics. The catalytic constant of the reaction was estimated based on a simple kinetic model tailored for hyperpolarized systems.

Detection of Living Anionic Species in Polymerization Reactions Using Hyperpolarized NMR

Lee, Y., et al., Detection of Living Anionic Species in Polymerization Reactions Using Hyperpolarized NMR. J. Am. Chem. Soc., 2013. 135(12): p. 4636-4639.

http://dx.doi.org/10.1021/ja4001008

Intermediates during the anionic polymerization of styrene were observed using hyperpolarized NMR. Dissolution dynamic nuclear polarization (DNP) of monomers provides a sufficient signal-to-noise ratio for detection of 13C NMR signals in real time as the reaction progresses. Because of its large chemical shift dispersion, 13C is well-suited to distinguish and characterize the chemical species that arise during the reaction. At the same time, incorporation of hyperpolarized small-molecule monomers is a unique way to generate polymers that exhibit a transient signal enhancement at the active site. This strategy is applicable despite the decay of the hyperpolarization of the polymer due to rapid spin-lattice relaxation. Real-time measurements on polymerization reactions provide both mechanistic and kinetic information without the need for stable isotope labeling of the molecules of interest. These capabilities are orthogonal to currently established methods that separate synthesis and analysis into two steps, making dissolution DNP an attractive method to study polymerization reactions.

Detection of Living Anionic Species in Polymerization Reactions Using Hyperpolarized NMR

Lee, Y., et al., Detection of Living Anionic Species in Polymerization Reactions Using Hyperpolarized NMR. J. Am. Chem. Soc., 2013. 135(12): p. 4636-4639.

http://dx.doi.org/10.1021/ja4001008

Intermediates during the anionic polymerization of styrene were observed using hyperpolarized NMR. Dissolution dynamic nuclear polarization (DNP) of monomers provides a sufficient signal-to-noise ratio for detection of 13C NMR signals in real time as the reaction progresses. Because of its large chemical shift dispersion, 13C is well-suited to distinguish and characterize the chemical species that arise during the reaction. At the same time, incorporation of hyperpolarized small-molecule monomers is a unique way to generate polymers that exhibit a transient signal enhancement at the active site. This strategy is applicable despite the decay of the hyperpolarization of the polymer due to rapid spin-lattice relaxation. Real-time measurements on polymerization reactions provide both mechanistic and kinetic information without the need for stable isotope labeling of the molecules of interest. These capabilities are orthogonal to currently established methods that separate synthesis and analysis into two steps, making dissolution DNP an attractive method to study polymerization reactions.

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