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A Potentially New Class of Redox-Sensitive CEST Agents: Preliminary Thiol-CEST Studies with Glutathione and Cysteine1School of Science and Health, Western Sydney University, Sydney, Australia, 2Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, United States, 3F. M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, Baltimore, MD, United States
Synopsis
In this study, we show that thiol-water proton exchange can generate MRI contrast by performing proton chemical exchange saturation transfer (1H-CEST) experiments on glutathione (GSH) and cysteine (Cys). The thiol proton exchange was quantified at various pHs, with Cys thiol exhibiting faster and more base-catalyzed exchange than GSH. However, both GSH and Cys thiol exchange were too fast to generate contrast at physiological pH. Potential applications of thiol compounds as redox-sensitive CEST agents are suggested.
Introduction
Unlike amides, amines, and hydroxyls, thiol-water proton exchange have not been exploited for MRI contrast using proton chemical exchange saturation transfer (1H-CEST).1 In 1H-CEST, labile protons are continuously saturated using RF irradiation thereby destroying the associated NMR signal. Proton exchange with the water then attenuates the water signal by an amount dependent on the exchange rate (kex), RF pulse intensity (B1) and duration (tsat). This CEST effect on the water signal can be several orders of magnitude larger than the observable solute labile proton resonances, resulting in enhanced sensitivity to labile protons. Since proton exchange is sensitive to pH, 1H-CEST can be used to monitor pH changes.2
Glutathione (GSH) and its precursor cysteine (Cys) play important roles in maintaining tissue redox environment. These molecules contain thiols which can be oxidized to form disulfides, allowing them to act as a redox buffer.3, 4 The oxidizability of thiol compounds depends on the fractional concentration of the thiolate (i.e., deprotonated) form;3 or simply put, on the lability of the thiol protons that can exchange with water protons. In this study, we use 1H-CEST to quantify the thiol-water proton kex of GSH and Cys to determine the potential of thiol-containing compounds as CEST agents.
Methods
1. GSH and Cys samples (pH 2.6 – 7.1) were prepared using PBS under N2 atmosphere.
2. 1H-CEST NMR data was obtained on a Bruker Avance III HD 600WB spectrometer at 310 K using saturation (tsat = 5 s; B1 = 1, 3, 5 µT) followed by a slice-selective spin-echo sequence.
3. Thiol-water proton kex values were obtained by fitting the Bloch-McConnell equations to the CEST data numerically.5, 6
Results
The 1H-CEST spectra in Figure 1 show the water signal attenuation by GSH and Cys thiol and amine proton exchange, with the thiol resonances appearing upfield from water as shown in 1H-NMR spectra. The thiol CEST signals coalesced as the pH approached 7, with Cys thiol protons coalescing at a lower pH than GSH thiol protons (Figure 2). The pH dependence of the thiol and amine kex is shown in Figure 3. The fitted exchange contributions, from Figure 3, for thiol exchange were determined to be: k0 = 985 s-1, ka = 0 s-1, and kb = 1.92 × 1011 s-1 (GSH); k0 = 1180 s-1, ka = 7.92 × 104 s-1, and kb = 2.95 × 1012 s-1 (Cys).
Discussion
Thiol-based CEST has been demonstrated with GSH and Cys. Unfortunately, the thiol kex of both compounds were too large to show CEST contrast near neutral pH. Interestingly, the Cys thiol kex was larger and more base-catalyzed than GSH, suggesting it has a higher propensity to be oxidized due to increased proton lability. The upfield thiol signal provides greater specificity compared to downfield amide, amines, and hydroxyls provided confounding upfield magnetisation transfer effects (e.g., NOE) are properly accounted for.7, 8Conclusion
The results presented here demonstrate that thiol-water proton exchange can generate MRI contrast by attenuating the water signal in the same way amide, amine, and hydroxyls can. Although GSH and Cys do not generate thiol-based CEST at physiological pH, there are still many other thiol-containing compounds4, 9 which should be investigated. Thiol-containing compounds can be oxidized to form disulfides, resulting in a reduced free thiol/thiolate concentration; this can lead to reduced thiol-based CEST contrast. Therefore, the thiol-based CEST contrast is redox-sensitive and can potentially be used to monitor the uptake of thiol-containing compounds into regions with high concentrations of oxidizing species (e.g, to map oxidative stress). Future work will include identifying thiol compounds suitable for CEST at neutral pH, and modulation of the thiol-based CEST contrast by redox reactions.Acknowledgements
This work was supported by Western Sydney University (WSU) Research Training Program (RTP) Funds, Australian National Health and Medical Research Council Program Grant APP1132471, Sigma Xi Grants-in-Aid of Research (GIAR) Grant G2017100194478959, and National Institute of Health’s (NIH) National Institute of Biomedical Imaging and Bioengineering (NIBIB) Grant 1R21EB025295-01A1. Dr Scott A. Willis is thanked for providing experimental assistanceReferences
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