Towards parahydrogen-induced polarization of acetoacetate

Abstract Hyperpolarized acetoacetate is a promising probe for the detection of redox status inside mitochondria using 13 C MRI. In this pilot study we used parahydrogen-induced polarization by means of a side arm hydrogenation (PHIP-SAH) approach for the hyperpolarization of [1,3- 13 C 2 ]acetoacetate. The propargyl [1,3- 13 C 2 ]acetoacetate precursor was synthesized and subjected to homogeneous hydrogenation with parahydrogen. After optimization, the observed 1 H polarization was 25.5% while achieving 100% chemical conversion of the propargyl moiety, and 80% selectivity to allyl [1,3- 13 C 2 ]acetoacetate product. After hydrogenation, a linear magnetic field sweep was performed for the 1 H-to- 13 C polarization transfer in allyl [1,3- 13 C 2 ]acetoacetate, with maximum observed 13 C polarizations: P( 13 C-1) = 4.1% and P( 13 C-3) = 2.7%. Next, we analyzed the products of allyl [1,3- 13 C 2 ]acetoacetate hydrolysis under different conditions. Alkaline hydrolysis results in the formation of stereoisomeric enolates of allyl [1,3- 13 C 2 ]acetoacetate, which can then be converted into hyperpolarized [1,3- 13 C 2 ]acetoacetate under harsh alkaline conditions with a subsequent decomposition to hyperpolarized [2- 13 C]acetone and [ 13 C]carbonate. These results expand PHIP-SAH studies beyond pyruvate to the metabolically relevant acetoacetate probe and identify the key chemical factors that currently limit the direct probe production. Beyond the achieved polarization levels, this work provides a useful mechanistic insight into allyl acetoacetate hydrolysis and offers guidance for future PHIP-SAH development of β-ketoacid-derived probes.