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

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.