The goal of this research is to evaluate [¹³C]-tert-butanol-2-β-D-galactose (tert-butanol-bGal) as a hyperpolarizeable agent for in vivo imaging of senescent cells.Senescent cells, cells that have lost the ability to divide, are now understood to be central to arresting the proliferation of potential cancers and driving the degenerative changes underlying aging and age-related disease^1,2 and there has been ongoing search for methods to remove or reduce the accumulation of senescent cells as a potential therapeutic intervention^3,4. Multiple agents have been suggested for the detection and imaging of senescent cells, including colorimetric assays, fluorescence, SPECT, PET, and MRI probes. Most agents are specific to β-galactosidase (β-gal or b-gal), an enzyme that accumulates to high levels in senescent cells^5,6, however none of the identified probes have sufficiently low toxicity, high sensitivity and specificity to be currently applicable to human studies. The high accumulation of β-gal in senescent cells, combined with its rapid enzymatic turnover rate, makes β-gal an ideal enzyme for an in vivo metabolic imaging target using hyperpolarized ¹³C MRSI. Galactose-linked versions of the simple alcohols, such as tert-butanol-2-β-D-galactose (tert-butanol-bGal) should be readily polarized with favorable T₁ relaxation times for in vivo studies.Figure 1 outlines the salient steps involved in synthesizing tert-butanol-bGal(Figure 1d) based on the method by Kartha et. al.⁷ starting with the dissolution of 1,2,3,4,6-Penta-O-acetyl-β-D-galactopyranose(Figure 1a) and the intermediate compounds. The carbon-13 resonance of the quaternary carbon in tert-butanol and in the synthesized tert-butanol-bGal was determined to be 69.6ppm and 77ppm respectively using a 400 MHz Varian Inova spectrometer, with a 7.4ppm chemical shift extremely favorable for in vivo studies. 436mg of tert-butanol-bGal was dissolved in 60% Glycerol to a total volume of 770uL, resulting in a 2.4M solution. A mixture of 190uL of this solution and 86uL of 5.2M tert-butanol (in order to measure the T₁ of the quaternary carbon in both the compounds) was polarized for over 12hours in GE SPINlab polarizer. Using 5ml of substrate dispensed from the polarizer with 16g of 0.1g/L Na₂EDTA in water as dissolution medium, a simple FID sequence with a 512us, 11.25⁰ flip angle hard RF pulse centered at 73ppm and 5kHz bandwidth was used to acquire the spectrum every 3 seconds for T₁ measurement at 3T (GE Scanner).Over expression of the enzyme β-galactosidase in senescence cells cleaves the sugar from the tert-butanol-bGal compound resulting in free tert-butanol (Figure 2). The chemical shift of the quaternary carbon between tert-butanol and tert-butanol-bGal allows in vivo imaging of the enzyme using hyperpolarized substrate. We were successfully able to synthesize tert-butanol-bGal, measure a 7.4 ppm chemical shift difference for the quaternary carbon between tert-butanol and tert-butanol-bGal, hyperpolarized both tert-butanol and tert-butanol-bGal (Figure 3), and measure their respective T₁ decay times at 3T of 34s and 22s respectively(Figure 4). While in this study 1% naturally occurring ¹³C nuclei contributed to the NMR signal, ¹³C isotopic enrichment of the targeted compound will provide almost 100 fold increase in sensitivity and better polarizability, thus enabling use of millimolar concentration for in vivo studies. As suggested by published hyperpolarized ¹³C studies of tert-butanol, these T₁ relaxation times can likely be lengthened to close to 1 min with deuteration. Both the detected chemical shift and observed T₁ relaxation times are quite favorable for further in vivo studies of animal models exhibiting senescence, in order to determine the uptake dynamics and in vivo turnover rates of tert-butanol-bGal.Hyperpolarized [¹³C]-tert-butanol-bGal has the potential to be a viable in vivo imaging agent, and the ultimate success of this project would provide an important new preclinical and clinical imaging tool for the evaluating the buildup of senescent cells and assessing the efficacy of senescent cell targeting agents in the treatment of cancer and age-related pathologies.