br The synthesis of TAB GSH TAB Cys
The synthesis of TAB-3-GSH, TAB-3-Cys-Gly, TAB-3-Cys, and TAB-3-GSH-Et are shown in supporting information (Scheme S1).
2.3. Cell culture and imaging
Human umbilical vein endothelial cells (HUVEC-1) were cultured in Bronchial Epithelial Cell Growth Medium supplemented with 10% fetal bovine serum (FBS). Human ovarian cancer cells （SKOV-3） and Mouse fibroblast cells (NIH/3T3) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with glucose (4.5 g/L), L-glutamine, sodium pyruvate, and 10% fetal bovine serum (FBS). The cells were plated on glass bottomed dishes at 37 °C under 5% CO2 Coelenterazine before ima-ging. Cell imaging were conducted using a confocal microscope FV1000-IX81 and were analyzed with FV10-ASW software.
Cells, pre-washed twice, were incubated with 10 μM of probes in cultured medium without FBS at 37 °C under 5% CO2 for 15min. Then the cells were washed with PBS to remove unbounded probes for six times before in situ imaging by Olympus FV1000-IX81 confocal laser scanning microscopy using oil objective, with excitation by 405 nm.
2.4. Tumor model and in vivo imaging
Nude mice 6–7 weeks old were provided by the Laboratory Animal Center of North Sichuan Medical College, Nanchong, China. All pro-cedures involving animals were performed according to a protocol approved by the Institutional Animal Care and Treatment Committee of North Sichuan Medical College. These nude mice were subcutaneously injected with 1 × 106 SKOV-3 cells in the right rear thigh under aseptic conditions. Then they were individually housed under specific pa-thogen-free conditions with free access to food and water until the formed tumor grow to approximately 0.5 cm in diameter by measuring caliper; tumor growth to this size took about a month. These tumor-bearing mice were fasting for 24 h and then were anesthetized by in-traperitoneal injection of 0.05 mL 3% aqueous solution of pento-barbital. The mice were then placed into the small animal imager and injected intraperitoneally with a certain amount of probe solution for imaging.
3. Results and discussion
3.1. Fluoresent response for GGT in vitro
We firstly measured the fluorescence spectra of TAB-3-GSH with the addition of diﬀerent amounts GGT to test whether it can be used as a
fluorescent probe for GGT. Fig. 1a shows its fluorescence spectra, which demonstrated a slight change when GGT concentration was less than 100 U/L, which corresponds to the concentration range in normal human body. With increasing concentrations of GGT, the fluorescence intensity achieved by the probe at 480 nm increased and displayed a ∼50-fold enhancement when the concentration of GGT reached 1000 U/L. In addition, maximum emission wavelength showed an obvious blue shift, providing a ratiometric readout, accompanied by visible fluorescence color change from weak yellow to intense green. The other three reference compounds, TAB-3-Cys-Gly, TAB-3-Cys, and TAB-3-GSH-Et, demonstrated similar fluorescence response tendency with TAB-3-GSH but a smaller magnitude of response in presence of GGT. Among them, TAB-3-Cys-Gly showed a 10-fold fluorescence enhance-ment, which was larger than that achieved with TAB-3-Cys and TAB-3-GSH-Et. This may imply that GGT can induce some reaction in the structure of TAB-3-Cys-Gly. Therefore, we further explored the re-cognition mechanism involved by utilizing high-resolution mass spec-trometry to detect the reaction products.
The mixture of TAB-3-GSH and low concentration of GGT (100 U/L) demonstrated an obvious MALDI mass peak at 1233.55, indicating the producing of TAB-3-Cys-Gly (Fig. S3). The mixture of TAB-3-GSH and high concentration of GGT (1000 U/L) manifested an ESI mass peak at 237.09, exactly corresponding the product after the further hydrolysis of amide linkage in TAB-3-Cys-Gly structure (Fig. S4). The hydrolysis can also be confirmed by the presence of a series of MALDI mass peaks of related products after mixing TAB-3-Cys-Gly with high concentration of GGT (1000 U/L) (Fig. S5). Accordingly, we propose a mechanism that GGT catalyzes a two-step hydrolysis reaction of TAB-3-GSH as follows: (1) The γ-glutamyl bond of GSH group can be firstly hydro-lyzed under low concentration of GGT to produce TAB-3-Cys-Gly; (2) The amide linkage of TAB-3-Cys-Gly may then be further hydrolyzed under higher concentration of GGT (Scheme 2). The proposed me-chanism also can help us understand their diﬀerent fluorescence re-sponse for GGT. TAB-3-GSH demonstrates greater fluorescence change than TAB-3-Cys-Gly after reacting with GGT because it undergoes a complete two-step hydrolysis process and TAB-3-Cys-Gly only happen the second hydrolysis reaction. The possible explanation for the less fluorescence response of TAB-3-GSH-Et and TAB-3-Cys for GGT can be