Aromatic secondary amine-functionalized fluorescent NO probes: improved detection sensitivity for NO and potential applications in cancer immunotherapy studies

Fluorescent NO probes reported herein display high sensitivity for NO by responding to both N2O3 and ONOO– and robust abilities for evaluating the repolarization of tumor-associated macrophages (TAMs).

The organic layer was washed with brine and dried over Na 2 SO 4 . Filtration, evaporation and purification of the residue by silica gel chromatography (EtOAc/PE = 1:4) gave 1 as an orange solid (48 mg, 52 % yield). 1  Compound 6: To a solution of 4-hydroxybenzaldehyde (2 g, 16.4 mmol) in 50 mL of DMF were added 1, 3-dibromopropane (3.3 mL, 32.8 mmol) and K 2 CO 3 (4.5 g, 32.8 mmol), and the solution was stirred at room temperature for 12 hours. Then the mixture was poured into 150 mL of ice water and extracted with EtOAc for 3 times.
The combined organic extracts were dried over Na 2 SO 4 and evaporated under reduced pressure to obtain the crude compound, which was purified by column chromatography (EtOAc/PE = 1:3) to afford 6 as a white solid (10.7 g, 92% yield).
Compound 5: Compound 6 (2.6 g, 10 mmol) and triphenylphosphine (7.3 g, 30 mmol) were dissolved in 50 mL of anhydrous acetonitrile, and the solution was heated under reflux for 12 hours. The solvent was evaporated under reduced pressure. The residue was added 5 ml of anhydrous dichloromethane and then poured into 50 ml EtOAc. Compound Mito1: A solution of 2 (73.8 mg, 0.2 mmol), aldehyde 5 (300 mg, 0.6 mmol), and AcOH (0.3 mL) in solvent (5 mL CH 2 Cl 2 and 3 mL MeOH) was stirred at room temperature for 10 min then NaBH 3 CN (188.4 mg, 3 mmol) was added, and the obtained solution was stirred for 30 min. The reaction was quenched with water and extracted with CHCl 3 . The organic layer was washed with brine and dried over

Preparation of the test solution
Stock solution of 1 or Mito1 in CH 3 CN (2 mM) was used to prepare the working solutions in PBS (50 mM, pH 7.4, containing 20% CH 3 CN) with a final concentration of 4.0 μM. For assays in chemical system, the NO stock solution in deionized water was used, which was prepared by bubbling NO gas into a NaOH solution to eliminate NO 2 generated from the reaction of NO and O 2 , and then into deoxygenated deionized S6 water for 30 min. The concentration of the resulting NO stock solution was determined to be 1.8 mM by Griess method. For assays in cells, a commercially available NO donor NOC-9 (dissolved in 0.1 M NaOH solution) was used. For assays in chemical system, a ONOO  solution, which was synthesized according to a reported procedure, 1 was used, and its concentration was determined using an extinction coefficient of 1670 M -1 cm -1 at 302 nm. For cell imaging assays, ONOO  was generated from a commercially available ONOO  donor SIN-1 (dissolved in 0.1 M NaOH solution). O 2  was prepared by adding KO 2 (7.1 mg) and 18-Crown-6 (1 equiv) to dry dimethyl sulfoxide (5 mL) and stirring vigorously for 10 min. HO  was generated in situ by the Fenton reaction, and its concentration was equal to the Fe(II) concentration. 1

Cell costaining studies.
To evaluate the subcellular localization of 1 or Mito1, HeLa cells were incubated with

MTT assays
HeLa Cells were seeded in 96-well microplates in DMEM medium supplemented with 10 % FBS (Fetal Bovine Serum) at 37 ºC in humidified environment of 5% CO 2 .
After 24 h of cell attachment, the plates were washed with PBS, followed by addition of increasing concentrations of 1 (250 M) or Mito1 (114 M) in DMEM. The cells were then incubated at 37 ºC in an atmosphere of 5% CO 2 and 95% air for 24 h, followed by standard MTT assays (n= 6). Untreated assays (n = 6) were also conducted under the same conditions.