Detection Sensitivity Enhancement of Naphthalimide PET Fluorescent Probes by 4-Methoxy-Substitution

Naphthalimide photoinduced electron transfer (PET) fluorescent probes are widely used in fluorescence imaging. Thereinto, detection sensitivity is the vital parameter of PET probes. However, the modulation of detection sensitivity is yet to be reported for naphthalimide PET probes. Herein, the detection sensitivity enhancement of naphthalimide PET fluorescent probes through 4-methoxy-substitution is proposed in this work. Taking Zn2+ detection an example, 4-methoxy-naphthalimide PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPNM) and control PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPN) are separately synthesized. The addition of 4-methoxy group with ability of strong electron donating to naphthalimide facilitates the construction of electronic push-pull system in the fluorophore resulting in the bathochromic shift of absorption and fluorescence emission spectra of BPNM and is further conducive to the enhancement of molar extinction coefficient ε and fluorescence quantum yield Φf of BPNM. Compared with BPN, BPNM shows lower Zn2+ detection limit in titration assays. Meanwhile, the fluorescence signal change (off-on) before and after Zn2+ addition of intracellular BPNM is more obvious and easier to control in confocal laser scanning imaging. Therefore, 4-methoxy-substitution improves the detection sensitivity of naphthalimide PET probe, which is favorable for the precise sensing of analyte, and further lays a good foundation for the synthesis of PET probe with high sensitivity.


Synthesis routes of probe BPN and BPNM
Scheme S1 Synthesis routes of probe BPN and BPNM

Synthesis of compound 1
Under the condition of vigorous stirring at 0°C, Boc2O (5.8 mL, 25.0 mM) dissolved (100 mL) in 100 mL of dichloromethane was slowly dropped into the dichloromethane solution (100 mL) of ethylenediamine (10.0 mL, 150 mM) within 3 h and the above mixture was stirred for 16 h. After the reaction, the solvent was evaporated, the oily residue was dissolved in 60 mL of sodium carbonate aqueous solution, and extracted with dichloromethane (2×60 mL).
The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure to yield compound 1 (a colorless viscous liquid, 1.44 g, 100%).

Synthesis of compound 2
Compound 1 (0.8 g, 5.0 mmol), sodium carbonate (2.4 g, 22.6 mmol) and 2-pyridylmethyl chloride hydrochloride (1.8 g, 10.9 mmol) were dissolved in 3 15 mL of absolute ethanol. The above mixture was refluxed for 12 h under nitrogen protection. After the reaction, the solvent was distilled under reduced pressure. The residue was dissolved in 50 mL of sodium hydroxide aqueous solution and extracted with dichloromethane (3×30 mL). Then the organic layer was separated, dried with potassium carbonate, and evaporated.

Synthesis of compound 3
At 0°C, compound 2 (130 mg, 0.39 mmol) dissolved in 8 mL of dichloromethane was added dropwise to 0.5 mL of trifluoroacetic acid solution. The mixture reacted at room temperature for 25 h. After the reaction, the solvent was evaporated. The residue was dissolved in sodium hydroxide aqueous solution and extracted with dichloromethane. The solvent was evaporated to obtain a yellow oil, compound 3 (85 mg, yield 90%).

Synthesis of compound 4
Compound 3 (52.5 mg, 0.217 mM) was dissolved by 5 mL of absolute ethanol in 10 mL round bottom flask,. After adding 4-bromo-1,8-naphthalic anhydride (56.76 mg, 0.205 mM) and a small amount of anhydrous Na2SO4, the reaction solution was refluxed for 6 h under the protection of nitrogen.
After the reaction, the solution was evaporated and the residue was separated through silica gel flash chromatography. Thereinto, compound 4 (yellow oil) was collected using the eluent composed of methane dichloride: methanol (V1: V2 = 40: 1). Compound 4 was obtained as yellow solid after vacuum drying (43 mg, yield 42%).

Synthesis of probe BPN
In a 10 mL round-bottom flask, compound 3 was dissolved in 5 mL of absolute ethanol. After adding 1,8-naphthalic anhydride (100 mg, 0.51 mM) and a small amount of anhydrous Na2SO4, the mixture solution refluxed for 6 h under nitrogen protection. After the reaction, the solvent was evaporated and the residue was further separated through silica gel flash chromatography.

1 H-NMR, 13 C-NMR, and TOF-MS spectra of BPN, BPNM, and intermediates.
Figure S1 1 H NMR spectrum of compound 1 recorded in CDCl3      / 6-31g (d, p) calculation. (The geometries of the molecules at the ground state (S0) and the excited state (S1) were optimized by density functional theory (DFT) and time-dependent DFT (TD-DFT) methods with B3-LYP functional and 6-31G (d, p) basis set. No constraints to bonds/angles/dihedral angles were applied in the calculations and all atoms were free to optimize. The frontier molecular orbital energy of DPA, DPA-Zn 2+ , and fluorophore moieties were studied by B3LYP/6-31 g (d, p) calculation. All theoretical calculations were carried out using the Gaussian 09 program package.)