Real‐Time Tracking of ASCT2‐Mediated Glutamine Uptake in Living Tumors With a Bioorthogonal Bioluminescent Probe

Abstract Glutamine addiction, as a hallmark of tumor metabolism, drives malignant progression via proliferation, survival, and metastasis. Alanine‐serine‐cysteine transporter 2 (ASCT2), the primary glutamine transporter, is overexpressed in tumors to meet metabolic demands, making it a promising therapeutic target. Accurately monitoring ASCT2‐mediated glutamine uptake is essential for investigating tumor metabolism and developing ASCT2‐targeted therapeutics. However, current methods lack specificity, require laborious sample processing, and do not support real‐time measurements in living systems. To overcome these issues, BLGLN is designed, an innovative bioluminescent reporter system exploiting Staudinger ligation. BLGLN comprises two components: 1) BL568, a caged D‐luciferin derivative protected with 2‐diphenylphosphinobenzoic acid, and 2) AA201, an azide‐modified glutamine mimetic taken up by ASCT2. Once inside, AA201 undergoes Staudinger ligation with membrane‐permeable BL568, releasing D‐luciferin that is converted by luciferase into a bioluminescent signal, allowing real‐time tracking of ASCT2‐dependent glutamine uptake in tumors. BLGLN provides simplified synthesis, eliminates complex sample preparation, and enables real‐time tracking and evaluation of glutamine uptake rate in living tumors.