Design Of Near-Infrared Unsymmetrical Squaraine And Its Application In Detection Of Leucine Aminopeptidase

Unsymmetrical squaraine dyes have excellent optical properties in the near-infrared region(650-900nm).However,they exhibit a strong aggregation trend in the water medium,which often leads to fluorescence quenching due to their poor water solubility.As a result,their ability of detection and imaging of enzymes in the physiological environment are restricted.Leucine aminopeptidase(LAP)has abnormaliy high level of expression in various pathological processes.Thus,it is of great significance to accurately monitor the activity of LAP and conduct real-time imaging on LAP.In this study,we designed an ethylene glycol oligomer-modified asymmetric squaraine fluorescent probe to detect and image the activity of endogenous leucine aminopeptidase.The structure of USSQ-Leu has been confirmed by ~1H NMR and HR-MS.The relationship between probe structure and solubility was investigated by solubility test,and it was proved that the structure designed by us can effectively improved the solubility of the probe.By studying the spectral properties of the probe and the response behavior of the probe to the enzyme,we found that USSQ-Leu has good near infrared absorption properties and has maximum absorption peak at 650nm.The probe can react with leucine aminopeptidase to release fluorescence under physiological conditions and the largest emission peak is at 710nm.Besides,the response of the probe to the enzyme has time dependence,concentration dependence,good sensitivity and specificity(0.61ng/mL).HPLC and HR-MS were used to demonstrate the detection mechanism with L-leucine group being cleaved from USSQ-Leu to restore fluorescence under the reaction with LAP.MTT and flow cytometry showed that the probe had low toxicity and good cell uptake ability.Fluorescence imaging showed that the probe had good ability to detect endogenous LAP.In xenograft tumor mouse model,the probe was used for sensitive detection and imaging of LAP in mice.