Figure 1. ¹⁵N CSI of HP [¹⁵N]TPA-d₆ in HEPES buffered phantom solutions containing different ratios of Zn²⁺/[¹⁵N]TPA-d₆. A diagram of the phantom arrangement (middle), ¹⁵N images of free, uncomplexed [¹⁵N]TPA-d₆(left), images of the Zn²⁺-[¹⁵N]TPA-d₆ complex (right).

     Although MR imaging is typically used to study biochemical systems by detecting the NMR signal of protons present in water molecules, the majority of biochemical reactions involve carbon or nitrogen containing molecules. Thus, making carbon-13 or nitrogen-15 based molecular probes are potentially very useful in biochemical studies in DNP. For example, MRI studies have demonstrated that a gadolinium-based Zn²⁺-responsive contrast agent can be used to monitor Zn²⁺secretion from pancreatic islets and prostate in mice.  Given that the Zn²⁺ content of prostate tissue is known to be much lower in prostate cancer, this technology has the potential to differentiate between healthy versus malignant tissue in a simple MRI exam in combination with a Zn²⁺-sensitive contrast agent.²  However, it should be pointed out that this test does not detect total Zn²⁺ content of prostate tissue but rather only the Zn²⁺ ions released in response to glucose.  Hence, an imaging method that provided a direct measure of total Zn²⁺ content could perhaps be even more informative. In the present study, we shows the use of  ¹⁵N labeled hyperpolarized probe to detect molecular interactions with biologically important metal ions such as Zn²⁺.  This Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn²⁺ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. We prepared the cell-permeable, ¹⁵N-enriched, d₆-deuterated version of the well-known Zn²⁺chelator, tris(2-pyridylmethyl)amine (TPA), and demonstrate that the polarized ligand had favorable T₁ and linewidth characteristics for ¹⁵N MRI. ¹⁵N resonances of the free and Zn-bound [¹⁵N]TPA-d₆ were observed at different chemical shifts in N-15 NMR spectra (Figure 1). This probe was successfully used to quantify free Zn-levels in human prostate tissue homogenate and human prostate epithelial cells (Figure 2).   Zn²⁺ in the prostate is known to drop dramatically in malignant tissue, the current results demonstrate that the use of HP-[¹⁵N]TPA-d₆ to measure freely available Zn²⁺ in prostate tissues in vivo during progression of prostate cancer would likely be diagnostically informative. Furthermore, hyperpolarized probes have various applications such as real time metabolic profiling, probing drug binding, monitoring drug mechanism, and covalent chemical labeling of amino acid.

Figure 2. Zinc detection in intact human prostate epithelial cells by hyperpolarized [¹⁵N]TPA-d_6. Time-dependent ¹⁵N spectra collected on a sample of PNT1A cells (left) after adding HP [¹⁵N]TPA-d_6, and its first ¹⁵N spectrum (right).

We thank The National Center for In Vivo Metabolism all the supports for this research. http://www.invivometabolism.org

Learn more about our discovery published at communication Chemistry (2020).

1. Ardenkjær-Larsen, J. H. et al. Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR. PNAS, 100, 10158-10163 (2003).
2. Clavijo Jordan, M. V. et al. Zinc-sensitive MRI contrast agent detects differential release of Zn(II) ions from the healthy vs. malignant mouse prostate. PNAS, 113, E5464-E5471, (2016).