Thymidine Phosphodiester Chemiluminescent Probe for Sensitive and Selective Detection of Ectonucleotide Pyrophosphatase 1

ENPP-1 is a transmembrane enzyme involved in nucleotide metabolism, and its overexpression is associated with various cancers, making it a potential therapeutic target and biomarker for early tumor diagnosis. Current detection methods for ENPP-1 utilize a colorimetric probe, <b>TMP-</b><i><b>p</b></i><b>NP</b>, which has significant limitations in sensitivity. Here, we present probe <b>CL-ENPP-1</b>, the first nucleic acid-based chemiluminescent probe designed for rapid and highly sensitive detection of ENPP-1 activity. The design of probe <b>CL-ENPP-1</b> features a phenoxy-adamantyl-1,2-dioxetane luminophore linked to thymidine via a phosphodiesteric bond. Upon cleavage of the enzymatic substrate by ENPP-1, the probe undergoes an efficient chemiexcitation process to emit a green photon. Probe <b>CL-ENPP-1</b> demonstrates an exceptional signal-to-noise ratio of 15000 and a limit of detection value approximately 4500-fold lower than the widely used colorimetric probe <b>TMP-</b><i><b>p</b></i><b>NP</b>. A comparison of <b>TMP-</b><i><b>p</b></i><b>NP</b> activation by ENPP-1 versus alkaline phosphatase (ALP) reveals a complete lack of selectivity. Removal of the self-immolative spacer from probe <b>CL-ENPP-1</b> resulted in a new chemiluminescent probe, <b>CL-ENPP-2</b>, with an 18.4-fold increase in selectivity for ENPP-1 over ALP. The ability of probe <b>CL-ENPP-2</b> to detect ENPP-1 activity in mammalian cells was assessed using the human breast cancer cell line MDA-MB-231. This probe demonstrated a 19.5-fold improvement in the signal-to-noise ratio, highlighting its superior ability to detect ENPP-1 activity in a biological sample. As far as we know, to date, <b>CL-ENPP-1</b> and <b>CL-ENPP-2</b> are the most sensitive probes for the detection of ENPP-1 catalytic activity. We anticipate that our new chemiluminescent probes will be valuable for various applications requiring ENPP-1 detection, including enzyme inhibitor-based drug discovery assays. The insights gained from our probe design principles could advance the development of more selective probes for ENPP-1 and contribute to future innovations in chemiluminescence research.