Thymidine\nPhosphodiester Chemiluminescent Probe for\nSensitive and Selective Detection of Ectonucleotide Pyrophosphatase\n1

ENPP-1 is a transmembrane enzyme involved in nucleotide\nmetabolism,\nand its overexpression is associated with various cancers, making\nit a potential therapeutic target and biomarker for early tumor diagnosis.\nCurrent detection methods for ENPP-1 utilize a colorimetric probe, <b>TMP-</b><i><b>p</b></i><b>NP</b>, which\nhas significant limitations in sensitivity. Here, we present probe <b>CL-ENPP-1</b>, the first nucleic acid-based chemiluminescent probe\ndesigned for rapid and highly sensitive detection of ENPP-1 activity.\nThe design of probe <b>CL-ENPP-1</b> features a phenoxy-adamantyl-1,2-dioxetane\nluminophore linked to thymidine via a phosphodiesteric bond. Upon\ncleavage of the enzymatic substrate by ENPP-1, the probe undergoes\nan efficient chemiexcitation process to emit a green photon. Probe <b>CL-ENPP-1</b> demonstrates an exceptional signal-to-noise ratio\nof 15000 and a limit of detection value approximately 4500-fold lower\nthan 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\nversus alkaline phosphatase (ALP) reveals a complete lack of selectivity.\nRemoval 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.\nThe ability of probe <b>CL-ENPP-2</b> to detect ENPP-1 activity\nin mammalian cells was assessed using the human breast cancer cell\nline MDA-MB-231. This probe demonstrated a 19.5-fold improvement in\nthe signal-to-noise ratio, highlighting its superior ability to detect\nENPP-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\nprobes for the detection of ENPP-1 catalytic activity. We anticipate\nthat our new chemiluminescent probes will be valuable for various\napplications requiring ENPP-1 detection, including enzyme inhibitor-based\ndrug discovery assays. The insights gained from our probe design principles\ncould advance the development of more selective probes for ENPP-1\nand contribute to future innovations in chemiluminescence research.