**3. Materials and methods**

### **3.1. Synthesis of fluorescent nitrones**

*3.1.1. p-Nitrostilbene-tert-butyl-nitrone was synthesized as previously described (Hauck et al., 2009).* 

### *3.1.2. Coumaryl-styryl-tert-butyl-nitrone*

2-(4-Trifluoromethyl-2-oxo-2H-chromen-7-yl)-*E*-vinyl-1-(N-(1,1-dimethyl))-phenyl-4-nitrone was synthesized according to the scheme in Fig. 4.

### *(Trifluoromethyl)-2-oxo-2H-chromene-7-diazoniumtetrafluoroborate*

To a solution of 2.62 mmol of commercially available 7-amino-4-(trifluoromethyl)-coumarin (coumarin 151, Sigma-Aldrich, Taufkirchen, Germany) in 18 ml of hydrochloric acid (18%), a solution of sodium nitrite (2.72 mmol) was added dropwise and stirred for 1 h at 0 °C. Then, 41 mmol tetrafluoroboric acid was added dropwise over 1 h, with maintaining the reaction temperature lower than 0 °C. (Trifluoromethyl)-2-oxo-2H-chromene-7-diazoniumtetrafluoroborate (1.52 mmol, yield: 58 %) was separated and rinsed with cold water, methanol and ethyl acetate. 1*H*-NMR: 6.45 (s, 1H), 6.51 (d, 1H, 2.4 Hz), 6.66 (dd, 1 H, 8.8 Hz and 2.0 Hz), 7.37 (dd, 1H, 8.8 Hz and 2.0 Hz).

### *4-Bromobenzaldehyde-dimethyl-acetal*

4-Bromobenzaldehyde (94.9 mmol) was dissolved in 120 ml of dry methanol under an argon atmosphere, 96 µl titanium chloride was added and the solution stirred for 20 minutes. After addition of 240 µl trietylamine and 15 minutes of stirring, water was added and the product was extracted with diethyl ether. The organic phase was dried and the acetal isolated by evaporation of the solvent.

### *[2-(4-Dimethoxymethylphenyl)-vinyl]-trimethyl-silane*

4-Bromobenzaldehyde-dimethyl-acetal (20.6 mmol) was dissolved in 20 ml of dry acetonitrile under an argon atmosphere. The solution was boiled under reflux for 24 h after addition of 51.6 mmol tetrabutylammonium acetate, 4 % (by mol) palladium(II) acetate and 41.3 mmol trimetyl-vinyl-silane. Then, 50 ml PBS buffer (pH = 7.4) was added and the product was extracted with diethyl ether. The organic phase was dried and the solvent removed by evaporation. The product was formed in 86 % yield (17.8 mmol). 1*H*-NMR: 0.18 (s, 9H), 3.34 (s, 1H), 5.42 (s, 1H), 6.53 (d, 1 H, 19.07 Hz), 6.91 (d, 1H, 19.41 Hz), 7.45 (m, 2H).

### *(4-Trifluoromethyl-2-oxo-2H-chromen-7-yl)-E-2-vinyl]-benzaldehyde*

2 mmol (Trifluoromethyl)-2-oxo-2H-chromene-7-diazoniumtetrafluoroborate and 2 mmol [2- (4-dimethoxymethylphenyl)-vinyl]-trimethyl-silane were dissolved in 40 ml of dry methanol and stirred for 5 h with 2 % (by mol) palladium(II) acetate (Hiyama reaction). Water was added and the product was extracted with diethyl ether. The solvent was removed under vacuum and the product was washed with ethyl acetate. 1*H*-NMR: 7.28 (s, 2H), 7.33 (s, 1H), 7.72 (t, 2 H, 8.86 Hz), 7.94 (d, 2H, 8.51), 8.28 (d, 1H, 8.86), 8.45 (d, 1H, 8.51), 10.05 (s, 1H).

### *2-(4-Trifluoromethyl-2-oxo-2H-chromen-7-yl)-E-vinyl-1-(N-(1,1-dimethylethyl))-phenyl-4-nitrone (2, CSN)*

1.25 mmol 4-[(4-Trifluoromethyl-2-oxo-2H-chromen-7-yl)-E-2-vinyl]-benzaldehyde and 1.85 mmol *N*-tert-butylhydroxylamine hydrochloride were dissolved in benzene and ptoluenesulfonic acid was added as catalyst. The solution was boiled in a Dean-Stark apparatus (reflux and water removal) for 12 h. Water was added and the product was extracted with ethyl acetate. 1*H*-NMR: 1.66 (s, 1H), 6.78 (s, 1H), 7.22 (d,1H, 16.04 Hz), 7.28 (s, 1H), 7.30 (d, 1H, 16.04 Hz), 7.63 (d, 3H, 8.61 Hz), 7.73 (t, 2 H, 8.61 Hz), 8.20 (d, 1H, 8.61 Hz), 8.35 (d, 2H, 8.60 Hz).

### *3.1.3. 4-Pyrrolidine-1,8-naphthylimido-methylphenyl-tert-butyl-nitrone, 3*

4-Pyrrolidine-1,8-naphthylimido-methylphenyl-*tert*-butyl-nitrone was synthesized according to the scheme in Fig. 17.

#### *6-(Pyrrolidin-1-yl)-benzo[de]isochromene-1,3-dione*

362 Nitroxides – Theory, Experiment and Applications

**3.1. Synthesis of fluorescent nitrones** 

*3.1.2. Coumaryl-styryl-tert-butyl-nitrone* 

was synthesized according to the scheme in Fig. 4.

and 2.0 Hz), 7.37 (dd, 1H, 8.8 Hz and 2.0 Hz).

*[2-(4-Dimethoxymethylphenyl)-vinyl]-trimethyl-silane* 

*(4-Trifluoromethyl-2-oxo-2H-chromen-7-yl)-E-2-vinyl]-benzaldehyde* 

*4-Bromobenzaldehyde-dimethyl-acetal* 

evaporation of the solvent.

*(Trifluoromethyl)-2-oxo-2H-chromene-7-diazoniumtetrafluoroborate* 

*3.1.1. p-Nitrostilbene-tert-butyl-nitrone was synthesized as previously described (Hauck et* 

2-(4-Trifluoromethyl-2-oxo-2H-chromen-7-yl)-*E*-vinyl-1-(N-(1,1-dimethyl))-phenyl-4-nitrone

To a solution of 2.62 mmol of commercially available 7-amino-4-(trifluoromethyl)-coumarin (coumarin 151, Sigma-Aldrich, Taufkirchen, Germany) in 18 ml of hydrochloric acid (18%), a solution of sodium nitrite (2.72 mmol) was added dropwise and stirred for 1 h at 0 °C. Then, 41 mmol tetrafluoroboric acid was added dropwise over 1 h, with maintaining the reaction temperature lower than 0 °C. (Trifluoromethyl)-2-oxo-2H-chromene-7-diazoniumtetrafluoroborate (1.52 mmol, yield: 58 %) was separated and rinsed with cold water, methanol and ethyl acetate. 1*H*-NMR: 6.45 (s, 1H), 6.51 (d, 1H, 2.4 Hz), 6.66 (dd, 1 H, 8.8 Hz

4-Bromobenzaldehyde (94.9 mmol) was dissolved in 120 ml of dry methanol under an argon atmosphere, 96 µl titanium chloride was added and the solution stirred for 20 minutes. After addition of 240 µl trietylamine and 15 minutes of stirring, water was added and the product was extracted with diethyl ether. The organic phase was dried and the acetal isolated by

4-Bromobenzaldehyde-dimethyl-acetal (20.6 mmol) was dissolved in 20 ml of dry acetonitrile under an argon atmosphere. The solution was boiled under reflux for 24 h after addition of 51.6 mmol tetrabutylammonium acetate, 4 % (by mol) palladium(II) acetate and 41.3 mmol trimetyl-vinyl-silane. Then, 50 ml PBS buffer (pH = 7.4) was added and the product was extracted with diethyl ether. The organic phase was dried and the solvent removed by evaporation. The product was formed in 86 % yield (17.8 mmol). 1*H*-NMR: 0.18 (s, 9H), 3.34 (s, 1H), 5.42 (s, 1H), 6.53 (d, 1 H, 19.07 Hz), 6.91 (d, 1H, 19.41 Hz), 7.45 (m, 2H).

2 mmol (Trifluoromethyl)-2-oxo-2H-chromene-7-diazoniumtetrafluoroborate and 2 mmol [2- (4-dimethoxymethylphenyl)-vinyl]-trimethyl-silane were dissolved in 40 ml of dry methanol and stirred for 5 h with 2 % (by mol) palladium(II) acetate (Hiyama reaction). Water was added and the product was extracted with diethyl ether. The solvent was removed under vacuum and the product was washed with ethyl acetate. 1*H*-NMR: 7.28 (s, 2H), 7.33 (s, 1H), 7.72 (t, 2 H, 8.86 Hz), 7.94 (d, 2H, 8.51), 8.28 (d, 1H, 8.86), 8.45 (d, 1H, 8.51), 10.05 (s, 1H).

**3. Materials and methods** 

*al., 2009).* 

To a solution of 37.4 mmol of commercially available 6-bromo-benzo[de]isochromene-1,3 dione (Sigma-Aldrich, Taufkirchen, Germany) in 50 ml of methyl glycol was added 56.7 mmol pyrrolidine and a catalytic amount of *p*-toluenesulfonic acid and was boiled under reflux for 6 h. After cooling to 0 °C the precipitation was filtered off and washed with cold ethanol. The crude product was recrystallized from n-hexane and dried. 6-(Pyrrolidin-1-yl) benzo[de]isochromene-1,3-dione was formed in 90 % yield (33.7 mmol). C16H13NO3 (267.3 g/mol) calc.: C, 71.90 %; H, 4.90 %; N, 5.24 %. found: C, 71.76 %; H, 4.88 %; N, 5.23 %. Mp**.:** 284 °C. *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 2.07 – 2.19 (m, 4 H), 3.75 – 3.89 (m, 4 H), 6.77 (d, 1 H, 8.81 Hz), 7.52 (t, 1 H, 7.30 Hz), 8.31 (d, 1 H, 8.81 Hz), 8.50 (d, 1 H, 7.30 Hz), 8.64 (d, 1 H, 8.81 Hz). *13C*{*1H*}-NMR (chloroform-d, 100 MHz): δ [ppm] = 160.9, 153.2, 135.4, 134.0, 133.4, 133.0, 123.2, 122.3, 118.6, 108.8, 53.3, 26.1.

### *4-(1,3-Dioxacyclopent-2-yl)-benzonitrile*

To a solution of 79.5 mmol of commercially available 4-cyanobenzaldehyde (Merck, Darmstadt, Germany) in 150 ml toluene, was added 0.32 mol of ethylene glycol and *p*toluenesulfonic acid was added as catalyst. The solution was boiled in a Dean-Stark apparatus for 20 h. After complete removal of water, the solution was stirred for 20 h at room temperature. Then, 60 ml of a 5 % sodium bicarbonate aqueous solution was added. The organic layer was washed three times with 25 ml bidest. water each and dried over magnesium sulfate. The solvent was removed under vacuum and the remaining colorless liquid was crystallized from n-hexane and dried under reduced pressure. The hygroscopic product was formed in 95 % yield (75.9 mmol). C10H9NO2 (175.2 g/mol) calc.: C, 68.56 %; H, 5.18 %; N, 8.00 %. found: C, 68.58 %; H, 5.12 %; N, 8.09 %. Mp.: -. *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 4.02 – 4.11 (m, 4 H), 5.83 (s, 1 H), 7.58 (d, 2 H, 8.56 Hz), 7.66 (d, 2 H, 8.06 Hz). *13C*{*1H*}-NMR (chloroform-d, 100 MHz): δ [ppm] = 143.1, 132.2, 127.2, 118.6, 112.9, 102.4, 65.4.

#### *4-(1,3-Dioxolan-2-yl)-phenylmethanamine*

4-(1,3-Dioxolan-2-yl)-benzonitrile (42.2 mmol) was dissolved in 50 ml of dry diethyl ether under an argon atmosphere and was added dropwise at 0 °C to a well-stirred suspension of LiAlH4 (84.5 mmol) in 100 ml of dry diethyl ether. The suspension was stirred at this temperature for 4 h, then the mixture was allowed to warm up to room temperature and was stirred for a further 18 h. Afterwards, the mixture was hydrolyzed by 35 ml of 95 % ethanol and then by 35 ml of 50 % ethanol. The ether layer was separated and the aqueous phase lyophilized. The resulting solid was extracted with 80 ml dichloromethane. The combined organic layers were dried over MgSO4 and the yellowish-white solid product was obtained by evaporation of the solvent and crystallized in the fridge in 94 % yield (39.5 mmol). *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 1.49 (s, 2 H), 3.87 (s, 2 H), 3.99 – 4.15 (m, 4 H), 5.80 (s, 1 H), 7.32 (d, 2 H, 8.19 Hz), 7.44 (d, 2 H, 8.19 Hz). 13C{*1H*}-NMR (chloroformd, 100 MHz): δ [ppm] = 144.4, 136.4, 127.1, 126.7, 103.6, 65.3, 46.3.

#### *2-(4-(1,3-Dioxolan-2-yl)-benzyl)-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione*

7.11 mmol of 6-pyrrolidin-1-yl)-benzo[de]isochromene-1,3-dione and 10.7 mmol of 4-(1,3 dioxolan-2-yl)-phenylmethanamine were dissolved in 130 ml of ethanol. The mixture was boiled under reflux for 23 h and then cooled to 0 °C. The resulting orange solid was filtered off and washed four times with 20 ml of cold ethanol each. The crude product was recrystallized from toluene and dried over MgSO4. The product was obtained as an orange solid in 88 % yield (6.26 mmol). C26H24N2O4 (428.5 g/mol) calc.: C, 72.88 %; H, 5.65 %; N, 6.54 %. found: C, 72.68 %; H, 5.70 %; N, 6.36 %. Mp.: 189 °C. *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 2.05 – 2.12 (m, 4 H), 3.74 – 3.80 (m, 4 H), 3.95 – 4.09 (m, 4 H), 5.37 (s, 2 H), 5.78 (s, 1 H), 6.80 (d, 1 H, 8.61 Hz), 7.38 (d, 2 H, 7.83 Hz, 2 H), 7.51 (t, 1 H, 8.61 Hz), 7.56 (d, 2 H, 8.22 Hz), 8.41 (d, 1 H, 8.86 Hz), 8.56 (t, 2 H, 5.09 Hz). *13C*{*1H*}-NMR (chloroform-d, 100 MHz): δ [ppm] = 164.8, 164.0, 152.7, 138.9, 136.9, 133.6, 132.0, 131.2, 128.9, 126.4, 123.0, 122.6, 122.5, 110.5, 108.5, 103.6, 65.2, 53.2, 43.0, 26.1.

#### *4-((1,3-Dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)-methyl)-benzaldehyde*

To a solution of 2-(4-(1,3-dioxolan-2-yl)-benzyl)-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (8.40 mmol) in 100 ml THF, was added 25 ml of 3 % hydrochloric acid. The solution was boiled under reflux for 4 h and then cooled to 0 °C. Afterwards, the solution was brought to pH 8 with triethylamine upon which an orange precipitate was formed. The crude product was filtered off, washed three times with 20 ml of water each. The orange aldehyde was obtained in 98 % yield (8.22 mmol) and was stored under argon unless used immediately. C24H20N2O3 (384.4 g/mol) calc.: C, 74.98 %; H, 5.24 %; N, 7.29 %. found: C, 74.60 %; H, 5.27 %; N, 6.91 %. Mp.: 160 °C. *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 2.05 – 2.14 (m, 4 H), 3.74 – 3.84 (m, 4 H), 5.43 (s, 2 H), 6.81 (d, 1 H, 8.61 Hz), 7.53 (t, 1 H, 7.43 Hz), 7.65 (d, 2 H, 8.22 Hz), 7.80 (d, 2 H, 8.22 Hz), 8.43 (d, 1 H, 8.61 Hz), 8.60 (t, 2 H, 7.43 Hz), 9.95 (s, 1 H). *13C*{*1H*}-NMR (chloroform-d, 100 MHz): δ [ppm] = 192.0, 163.9, 152.9, 144.9, 135.4, 133.8, 132.3, 131.4, 129.9, 129.1, 126.4, 123.1, 122.6, 122.2, 108.6, 53.2, 43.2, 26.1.

*(Z)-N-(4-((1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl-)methyl)-benzylidene)-2 methylpropan-2-amine oxide* 

A suspension of 4-((1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl) methyl)-benzaldehyde (3 mmol), N-(*tert*-butyl)hydroxylamine hydrochloride (6 mmol), triethylamine (6.6 mmol) and sodium sulfate (17.6 mmol) as desiccant in 50 ml CH2Cl2 was stirred for 9 days at room temperature under argon. Afterwards, the mixture was filtered off and the solid was washed three times with 20 ml CH2Cl2 each. The organic layer was dried and the crude product isolated by evaporation of the solvent. Then, the orange solid was recrystallized from ethanol and the nitrone was purified by column chromatography (200– 400 mesh chromagel; solvent: methylene chloride/acetone/triethylamine, 12:1:0.1) in 38 % yield (1.14 mmol). *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 1.57(s, 9 H), 2.07 – 2.10 (m, 4 H), 3.75 – 3.79 (m, 4 H), 5.37 (s, 2 H), 6.80 (d, 1 H, 8.80 Hz), 7.47 (s, 1 H), 7.51 (dd, 1 H, 8.56 Hz, 7.43 Hz), 7.56 (d, 2 H, 8.56 Hz, 2 H), 8.19 (d, 2 H, 8.56 Hz), 8.41 (d, 1 H, 8.80 Hz), 8.57 (m, 2 H). *13C*{*1H*}-NMR (chloroform-d, 100 MHz): δ [ppm] = 164.8, 164.0, 152.8, 140.0, 133.7, 132.1, 131.3, 130.0, 129.6, 128.8, 128.7, 123.0, 122.6, 122.5, 110.5, 108.5, 70.6, 53.1, 43.2, 28.3, 26.1.

### **3.2. UV/VIS and fluorescence spectroscopy**

500 µM stock solutions of the spin traps in dimethyl sulfoxide containing 1 % C12E9 were employed. For fluorescence spectra the solutions were added to 25 mM phosphate buffer, pH = 7.2, with 1 % Triton-X 100 to a final concentration of 1 µM. To simulate photobleaching in the fluorescence microscope, the solutions were irradiated for three minutes with a blue LED light source (λ = 366 nm) and compared to samples without prior irradiation.

### **3.3. Cytotoxicity**

364 Nitroxides – Theory, Experiment and Applications

*4-(1,3-Dioxolan-2-yl)-phenylmethanamine* 

110.5, 108.5, 103.6, 65.2, 53.2, 43.0, 26.1.

d, 100 MHz): δ [ppm] = 144.4, 136.4, 127.1, 126.7, 103.6, 65.3, 46.3.

*2-(4-(1,3-Dioxolan-2-yl)-benzyl)-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione* 

*4-((1,3-Dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)-methyl)-benzaldehyde* 

133.8, 132.3, 131.4, 129.9, 129.1, 126.4, 123.1, 122.6, 122.2, 108.6, 53.2, 43.2, 26.1.

To a solution of 2-(4-(1,3-dioxolan-2-yl)-benzyl)-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (8.40 mmol) in 100 ml THF, was added 25 ml of 3 % hydrochloric acid. The solution was boiled under reflux for 4 h and then cooled to 0 °C. Afterwards, the solution was brought to pH 8 with triethylamine upon which an orange precipitate was formed. The crude product was filtered off, washed three times with 20 ml of water each. The orange aldehyde was obtained in 98 % yield (8.22 mmol) and was stored under argon unless used immediately. C24H20N2O3 (384.4 g/mol) calc.: C, 74.98 %; H, 5.24 %; N, 7.29 %. found: C, 74.60 %; H, 5.27 %; N, 6.91 %. Mp.: 160 °C. *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 2.05 – 2.14 (m, 4 H), 3.74 – 3.84 (m, 4 H), 5.43 (s, 2 H), 6.81 (d, 1 H, 8.61 Hz), 7.53 (t, 1 H, 7.43 Hz), 7.65 (d, 2 H, 8.22 Hz), 7.80 (d, 2 H, 8.22 Hz), 8.43 (d, 1 H, 8.61 Hz), 8.60 (t, 2 H, 7.43 Hz), 9.95 (s, 1 H). *13C*{*1H*}-NMR (chloroform-d, 100 MHz): δ [ppm] = 192.0, 163.9, 152.9, 144.9, 135.4,

7.11 mmol of 6-pyrrolidin-1-yl)-benzo[de]isochromene-1,3-dione and 10.7 mmol of 4-(1,3 dioxolan-2-yl)-phenylmethanamine were dissolved in 130 ml of ethanol. The mixture was boiled under reflux for 23 h and then cooled to 0 °C. The resulting orange solid was filtered off and washed four times with 20 ml of cold ethanol each. The crude product was recrystallized from toluene and dried over MgSO4. The product was obtained as an orange solid in 88 % yield (6.26 mmol). C26H24N2O4 (428.5 g/mol) calc.: C, 72.88 %; H, 5.65 %; N, 6.54 %. found: C, 72.68 %; H, 5.70 %; N, 6.36 %. Mp.: 189 °C. *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 2.05 – 2.12 (m, 4 H), 3.74 – 3.80 (m, 4 H), 3.95 – 4.09 (m, 4 H), 5.37 (s, 2 H), 5.78 (s, 1 H), 6.80 (d, 1 H, 8.61 Hz), 7.38 (d, 2 H, 7.83 Hz, 2 H), 7.51 (t, 1 H, 8.61 Hz), 7.56 (d, 2 H, 8.22 Hz), 8.41 (d, 1 H, 8.86 Hz), 8.56 (t, 2 H, 5.09 Hz). *13C*{*1H*}-NMR (chloroform-d, 100 MHz): δ [ppm] = 164.8, 164.0, 152.7, 138.9, 136.9, 133.6, 132.0, 131.2, 128.9, 126.4, 123.0, 122.6, 122.5,

4-(1,3-Dioxolan-2-yl)-benzonitrile (42.2 mmol) was dissolved in 50 ml of dry diethyl ether under an argon atmosphere and was added dropwise at 0 °C to a well-stirred suspension of LiAlH4 (84.5 mmol) in 100 ml of dry diethyl ether. The suspension was stirred at this temperature for 4 h, then the mixture was allowed to warm up to room temperature and was stirred for a further 18 h. Afterwards, the mixture was hydrolyzed by 35 ml of 95 % ethanol and then by 35 ml of 50 % ethanol. The ether layer was separated and the aqueous phase lyophilized. The resulting solid was extracted with 80 ml dichloromethane. The combined organic layers were dried over MgSO4 and the yellowish-white solid product was obtained by evaporation of the solvent and crystallized in the fridge in 94 % yield (39.5 mmol). *1H*-NMR (chloroform-d, 400 MHz): δ [ppm] = 1.49 (s, 2 H), 3.87 (s, 2 H), 3.99 – 4.15 (m, 4 H), 5.80 (s, 1 H), 7.32 (d, 2 H, 8.19 Hz), 7.44 (d, 2 H, 8.19 Hz). 13C{*1H*}-NMR (chloroform-

> Cytotoxicity of the spin traps was determined by the sulforhodamine assay. 170 µl of a cell suspension of 8000 cells/ml of Hela or MCF-7 cells were added to 20 ml of RPMI medium and 1 ml each was pipetted into a 24 wells plate and incubated for 48 h at 37 °C under 5 % CO2. After this time, the cells were incubated for another 72 h with RPMI medium containing 0.5 % DMSO and the spin trap at six concentrations varying from 0 to 200 µM. The reaction was stopped by addition of 100 µl of 50 % trichloroacetic acid for 1 h at 4 °C and subsequently washed four times with cold water and then dried for 24 h at room temperature followed by addition of 250 µl of sulforhodamine B solution (0.4 % in 1 % acetic acid; Sigma, Taufkirchen). After 30 min wells were washed three times with cold water and another three times with 1 % acetic acid, dried for 24 h at room temperature and extinction measured at 570 nm after addition of 1 ml of 10 mM Tris base solution, pH 10.0.

> In case of **3** concentrations were varied from 0 to 5 µM in RPMI medium containing 0.5 % DMSO and 0.5 % ethanol in addition.

#### **3.4. Fluorescence microscopy measurements**

These were carried out using a quasi-confocal microscope (Axiovert 440 equipped with an ApoTome, Carl Zeiss, Jena) as previously described (Hauck et al., 2009). For corresponding measurements with **3** a Nikon Eclipse E 600 confocal microscope equipped with a Hamamatsu ORCA-ER camera was employed. After 20 minutes of incubation with 1 µM spin trap and 0.5 % DMSO the cells were washed three times with RPMI medium and the coverslip was mounted on a chamber and put under the microscope. Imaging was achieved by laser excitation at 488 nm.

To determine the half-life of fluorescence, a representative cell was defined as region of interest (ROI) and the evolution of average intensity of the ROI was investigated in the presence and absence of various inhibitors of components of mitochondrial proteins.
