Natural pH regulatory mechanisms can be overruled during several pathologies such as cancer, inflammation and ischaemia, leading to local pH changes in the human body. Here we demonstrate that ¹³C-labelled zymonic acid (ZA) can be used as hyperpolarized magnetic resonance pH imaging sensor. ZA is synthesized from [1-¹³C]pyruvic acid and its ¹³C resonance frequencies shift up to 3.0 p.p.m. per pH unit in the physiological pH range. The long lifetime of the hyperpolarized signal enhancement enables monitoring of pH, independent of concentration, temperature, ionic strength and protein concentration. We show in vivo pH maps within rat kidneys and subcutaneously inoculated tumours derived from a mammary adenocarcinoma cell line and characterize ZA as non-toxic compound predominantly present in the extracellular space. We suggest that ZA represents a reliable and non-invasive extracellular imaging sensor to localize and quantify pH, with the potential to improve understanding, diagnosis and therapy of diseases characterized by aberrant acid-base balance.

PH is a tightly regulated physiological parameter that is often altered in diseased states like cancer. The development of biosensors that can be used to non-invasively image pH with hyperpolarized (HP) magnetic resonance spectroscopic imaging has therefore recently gained tremendous interest. However, most of the known HP-sensors have only individually and not comprehensively been analyzed for their biocompatibility, their pH sensitivity under physiological conditions, and the effects of chemical derivatization on their logarithmic acid dissociation constant (pK_a). Proteinogenic amino acids are biocompatible, can be hyperpolarized and have at least two pH sensitive moieties. However, they do not exhibit a pH sensitivity in the physiologically relevant pH range. Here, we developed a systematic approach to tailor the pK_a of molecules using modifications of carbon chain length and derivatization rendering these molecules interesting for pH biosensing. Notably, we identified several derivatives such as [1-¹³C]serine amide and [1-¹³C]-2,3-diaminopropionic acid as novel pH sensors. They bear several spin-1/2 nuclei (¹³C, ¹⁵N, ³¹P) with high sensitivity up to 4.8 ppm/pH and we show that ¹³C spins can be hyperpolarized with dissolution dynamic polarization (DNP). Our findings elucidate the molecular mechanisms of chemical shift pH sensors that might help to design tailored probes for specific pH in vivo imaging applications.