Lastly, the development of logic-gated ABS probes, where activation depends on the presence of two biomarkers (e.g., GLUT1 and Cu), hold tremendous promise for accurate and reliable assessment of a disease state. For instance, we have shown that mvpCu is able to effectively target cell populations overexpressing the GLUT1 protein, whereas PACu-1 cannot, by monitoring the PA signal of the tumor site upon irradiation with 670 nm light (Figure ). Moreover, because interaction with Cu(I) is required to red shift the λPA via TPA trigger cleavage, observing the PA signal change at 765 nm allows us to confirm probe activation (Figure ). We would not expect to see significant change if the second biomarker was absent from the tumor site or if an imaging agent was not equipped with the Cu-responsive trigger (i.e., mvGlu-AB3). ). To a round-bottom flask, 1 (693 mg, 1.5 mmol, 1 equiv.) and 2 (1.1 g, 3.1 mmol, 2 equiv.) were suspended in n-butanol (9.3 mL). The solution was heated to 130 °C for 45 minutes in an oil bath containing rice bran oil. Then, ammonium acetate (1.8 g, 23 mmol, 15 equiv.) was added, and the reaction was allowed to heat at 130 °C overnight. The reaction was cooled to room temperature, diluted with water, and extracted with DCM (3×). The organic layers were combined, dried with sodium sulfate, and concentrated via rotary evaporation. The excess nbutanol was removed azeotropically with toluene. The resulting shiny blue solid was used without further purification. The solid was dissolved in DCM (150 mL) and cooled to 0 °C. DIPEA (2.7 mL, 15 mmol, 10 equiv.) and BF3•OEt2 (2.7 mL, 22 mmol, 14 equiv.) were added sequentially. The reaction was warmed to room temperature and was allowed to stir overnight. The reaction was quenched with aqueous sodium bicarbonate and extracted with DCM (3×).