General remarks. All chemicals were obtained from commercial sources and were used without further purification. Solvents were dried according to published procedures.28 The course of the reactions was monitored with thin-layer chromatography (TLC), using aluminum sheets (0.2 mm) coated with silica gel 60 with fluorescence indicator (silica gel 60 F254). Purification of the products was carried out by flash column chromatography, using silica gel 60 (230−400 mesh). Nuclear Magnetic Resonance (NMR) spectra were obtained with a Bruker Avance 400MHz or a Varian Mercury 200MHz spectrometer. Chemical shifts are reported in ppm. High-resolution mass spectral (HRMS) spectra were recorded in a QTOF maXis impact (Bruker) spectrometer under electron spray ionization conditions (the 1H and 13C NMR data and spectra, as well as HRMS data are reported in Supplementary Information, Fig. 8).
Steady-state and time-resolved fluorescence measurements were performed with a FLS1000 spectrofluorometer (Edinburgh instruments), equipped with a N-DMM double-emission monochromator, a N-G11 PMT-980 detector and equipped with a pulsed LED (λexc 300 nm) as excitation source. The kinetic traces were fitted by one monoexponential decay function, using a deconvolution procedure to separate them from the lamp pulse profile. All experiments were performed in a quartz cuvette of 1 cm of optical path. These experiments were performed in a PBS solution at pH 8. The phthalate solutions were prepared with a concentration of 10−4 M while a stock solution of ETPP (0.4 M) was prepared, so it was only necessary to add microliter volumes (250 µL) to the sample cell to obtain appropriate concentration of the quencher. The rate constants (kq and k´q) for the reaction were obtained from the Stern-Volmer plots following equations 1 and 2, respectively:
I0/I = 1 + kq x τ0 x [ETPP], (4)
τ0/τ = 1 + k´q x τ0 x [ETPP], (5)
where I0 and I are the intensities at the emission maxima in the absence and after addition of a quencher concentration [ETPP], τ0 is the fluorescence lifetime of the phthalate in the absence of ETPP and τ is the lifetime after addition of a quencher concentration [ETPP].
Monitoring of the CL at pH 10 of luminol in the absence and presence of ETPP was performed using the same spectrofluorometer with its own lamp switched off. The set was ran in the timebased mode with the detection dialed at 425 nm. Each experiment was performed at least 10 times. For triggering the chemiluminescence, luminols were dissolved in aqueous basic solutions giving a final concentration of 7.5 µM. Then, 2 mL of luminol or luminol plus ETPP were introduced in a quartz cuvette and the CL was triggered by addition of 2.5 µL of H2O2 (50% w/w) and 8 µL of K3[Fe(CN)6] 75 mM while vigorously stirring.
For the detection of transient species, time-resolved kinetic analyses were performed using a laser flash photolysis (LFP) system equipped with a Nd:YAG SL404G-10 Spectron Laser at the excitation wavelength of 355 nm. The single pulses were of ca. 10 ns duration, and the energy was lower than 30 mJ per pulse. The detecting light source was a pulsed Lo255 Oriel Xenon lamp. In addition to the pulsed laser, the LFP included the pulsed Lo255 Oriel Xe lamp, a 77200 Oriel monochromator, a photomultiplier (Oriel, model 70705PMT) system and a TDS-640A Tektronix oscilloscope. A customized Luzchem Research LFP-111 system was employed to collect and transfer the output signal from the oscilloscope to a personal computer to process the data. A quartz cell of 1 cm optical path length was employed for all kinetic measurements. These experiments were performed in a PBS solution at pH 8. The phthalate solutions were prepared with a concentration of 0.0016 M (absorbance at λexc = 355 nm was 0.25) while a stock solution of ETPP (0.04 M) was prepared.
Cyclic voltammetry measurements were performed with a VersaSTAT 3 potentiostat (Princeton Applied Research) and using a three electrode standard configuration with a carbon sheet as working electrode, a platinum wire as counter electrode and Ag/AgCl in saturated KCl as reference electrode. Measurements were carried out on DMF or PBS at pH 8 solutions with 0.1 M Bu4NI or LiClO4 as electrolyte of ETPP or 3AP (1 mM) respectively at a scan rate of 0.05 V·s−1. All the solutions were previously purged with N2 for at least 15 min before the measurements.
Synthesis procedures. 4-Amino-2-(sec-butyl)isoindoline-1,3-dione 2 was synthesized as previously published.23
4-Amino-5,7-dibromo-2-( sec -butyl)isoindoline-1,3-dione (3). A round-bottom flask was charged with aminophthalimide 2 (1 g, 4.6 mmol) and sodium acetate (762 mg, 9.3 mmol) in acetic acid (11 mL) and the resulting solution was stirred at r.t. for 30 mins. Then, a solution of bromine (0.5 mL, 9.4 mmol) in acetic acid (4 mL) was added dropwise, and the reaction mixture was stirred at r.t. for 18 hours. After that, the mixture was poured into ice-cold water (100 mL) forming a yellow precipitate. This was collected by filtration, washed with water and dried under reduced pressure, yielding compound 3 as a yellow powder (1.6 g, 92%).
4-Amino-5,7-dimethyl-2-( sec -butyl)isoindoline-1,3-dione (4). A round-bottom flask under inert atmosphere was charged with brominated phthalimide 3 (1.5 g, 4 mmol), trimethylboroxine (2.5 M in THF, 2.5 mL, 8.8 mmol) and potassium carbonate (3.3 g, 24.2 mmol) in a mixture of water (22 mL) and 1,4-dioxane (22 mL). The solution was purged with argon for approximately 30 mins. Tetrakis(triphenylphosphine)palladium(0) (92 mg, 0.08 mmol) was then added and the mixture was purged once again for 15 mins. Subsequently, the reaction mixture was stirred under an inert atmosphere at 105oC for 18 hours. After cooling to ambient temperature, the solvent was partially evaporated and water (80 mL) was added to the flask. The aqueous phase was extracted with EtOAc (150 mL). The organic phase was then washed with 1N HCl (50 mL) and brine (50 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was subjected to column chromatography (P.E./EtOAc 9:1 to 7:3) and, after evaporation of the solvent, 4 was acquired as a yellow solid (552 mg, 56%).
General procedure for the N-alkylation of 4-aminophthalimides (5a-5b)
Aminophthalimide 2 or 4 (2.72 mmol) was dissolved in N-methyl-pyrrolidone (0.6 mL). 1-iodohexane (0.5 mL, 3.3 mmol) was added and the reaction mixture was stirred at 110oC for two days. After cooling down to ambient temperature, the mixture was quenched with water and was extracted with EtOAc. The organic phase was separated, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was subjected to column chromatography (P.E./EtOAc 9:1) and the product (eluted first) was acquired after evaporation of the solvent.
General procedure for the synthesis of 3-aminophthalic anhydrides (6a-6c)
To a solution of aminophthalimide (0.3 mmol) in ethanol (3mL) was added an aqueous solution of potassium hydroxide (15 N, 3 mL) and the resulting solution was heated at reflux for 3 days. After cooling to ambient temperature, ethanol was evaporated, water (10 ml) was added to the flask and the solution was washed with DCM (3 x 15 mL). The aqueous phase was collected and acidified with an aqueous solution of 1N HCl until pH = 2. Upon acidification, the aqueous solution slowly turned from colourless to fluorescent green, indicative of the condensation of the phthalic acid to the corresponding anhydride. The solution was then extracted with EtOAc (3 x 20 mL). The combined organic phase was dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was subjected to column chromatography (P.E./EtOAc 9.5:0.5), the solvent was evaporated and the residue was washed with hexane to yield the desired anhydride.