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M.L. Agazzi et al. / European Journal of Medicinal Chemistry 126 (2017) 110e121
stirred for 12 h. The organic layer containing the crude product was
washed with water and the organic solution was dried over Na2SO4.
The solvent was evaporated under reduced pressure. Flash column
chromatography (silica gel) using CH2Cl2 as eluent afforded 275 mg
(25%) of BODIPY 1 as orange needles. TLC (CH2Cl2) Rf ¼ 0.60. 1HNMR
Volmer's Equation (1):
I0
I
¼ 1 þ kqt0½Qꢁ ¼ 1 þ KSV½Qꢁ
(1)
where I0 and I are the fluorescence intensity of BODIPY in the
absence and in the presence of quencher, kq represents the
biomolecule quenching rate constant, t0 the excited state lifetime
of BODIPY in the absence of KI, [Q] is the KI concentration and KSV is
the Stern-Volmer quenching constants.
(CDCl3, TMS)
d [ppm] 1.48 (s, 6H), 2.55 (s, 6H), 3.04 (s, 6H,
-N(CH3)2), 5.97 (s, 2H, pyrrole), 6.82 (d, 2H, J ¼ 8.7 Hz, Ar), 7.08 (d,
2H, J ¼ 8.7 Hz, Ar). APPI-MS [m/z] 368.2110 (M þ H)þ (367.2031
calculated for C21H24BF2N3).
8-[4-(3-(N,N-Dimethylamino)propoxy)phenyl]-4,4-difluoro-4-
bora-3a,4a-diaza-s-indacene 2. A solution of 4-[3-(N,N-dimethyla-
mino)propoxy]benzaldehyde (2 mL, 9.95 mmol) and pyrrole
(15.0 mL, 216.2 mmol) was treated as previously described to
obtain 1.77 g (55%) of meso-[4-(3-N,N-dimethylaminopropoxy)
phenyl]dipyrromethane [16]. A mixture of this dipyrromethane
(829 mg, 2.57 mmol) and DDQ (1.06 g, 4.67 mmol) in 130 mL of
CH2Cl2 was stirred for 3 h at room temperature. Then, TEA (12 mL,
86.1 mmol) and BF3$OEt2 (12 mL, 97.2 mmol) were added. The
mixture was stirred for an additional 12 h at room temperature and
then was washed with water for two times (10 mL each). The sol-
vent was evaporated under reduced pressure. Flash column chro-
matography (silica gel, CH2Cl2/Et3N 1%) yielded 114 mg (12%) of
pure BODIPY 2. TLC (CH2Cl2/Et3N 1%) Rf ¼ 0.74. 1HNMR (CDCl3,
2.4. Photooxidation of DPBF
Solutions of DPBF (20 mM) and BODIPY in DMF or n-heptane/
AOT (0.1 M)/water (W0 ¼ 10) media were irradiated in 1 cm path
length quartz cells (2 mL) with monochromatic light at
lirr ¼ 500 nm (BODIPY absorbance 0.1). The kinetics of DPBF
photooxidation were studied following the decrease of the absor-
bance (A) at lmax ¼ 413 nm. The observed rate constants (kobs) were
obtained by a linear least-squares fit of the semilogarithmic plot of
Ln A0/A vs. time. Values of quantum yields of O2(1Dg) production
(FD) in DMF were calculated comparing the kobs for the corre-
sponding BODIPY with that for C60, which was used as a reference
(
FD ¼ 1) [19]. Measurements of the sample and reference under the
TMS)
d [ppm] 1.96 (m, 2H), 2.30 (s, 6H, -N(CH3)3), 2.67 (t, 2H,
same conditions afforded FD for photosensitizers by direct com-
parison of the slopes in the linear region of the plots. Also, photo-
oxidation of DPBF by BODIPYs was evaluated in the presence of
different concentrations of KI (10 and 50 mM) in DMF/5% water.
J ¼ 6.0 Hz), 4.11 (t, 2H, J ¼ 6.1 Hz), 6.55 (dd, 2H, J ¼ 1.7, 4.2 Hz,
pyrrole), 6.97 (d, 2H, J ¼ 4.2 Hz, pyrrole), 7.04 (d, 2H, J ¼ 8.3 Hz, Ar),
7.53 (d, 2H, J ¼ 8.3 Hz, Ar), 7.92 (br, 2H, pyrrole). APPI-MS [m/z]
370.1902 (M þ H)þ (369.1824 calculated for C20H22BF2N3O).
1,3,5,7-Tetramethyl-8-[4-(N,N,N-trimethylamino)phenyl]-4,4-
difluoro-4-bora-3a,4a-diaza-s-indacene 3. A mixture of BODIPY 1
2.5. Photooxidation of Trp
(20 mg, 0.052 mmol) and methyl iodide (200 mL, 3.21 mmol) in
Solutions of Trp (20 mM) and BODIPY in DMF were treated as
described above for photodecomposition of DPBF. Photooxidation
2 mL of N,N-dimethylformamide (DMF) was stirred for 72 h at
40 ꢀC. The solvents were removed under vacuum to obtain 25 mg
(96%) of BODIPY 3. 1HNMR (DMSO-d6, TMS)
d [ppm] 1.53 (s, 6H),
of Trp was studied by exciting the samples at lexc ¼ 290 nm and
following the decrease of the fluorescence intensity at
l
¼ 341 nm.
2.46 (s, 6H), 3.66 (s, 9H, -N(CH3)3), 6.21 (s, 2H, pyrrole), 7.71 (d, 2H,
J ¼ 8.8 Hz, Ar), 8.14 (d, 2H, J ¼ 8.8 Hz, Ar). APPI-MS [m/z] 383.2344
(M þ H)þ (382.2261 calculated for C22H27BF2N3).
Control experiments showed that under these conditions the
fluorescence intensity correlates linearly with Trp concentration.
The observed rate constants (kobs) were obtained by a linear least-
squares fit of semi-logarithmic plots of ln (I0/I) vs. time. Similarly,
photooxidation of Trp was performed in presence of IK (50 mM) in
DMF/10% water. Also, decomposition of Trp by BODIPYs in DMF/10%
water was investigated by adding diazabicyclo[2.2.2]octane
(DABCO, 50 mM) and D-mannitol (50 mM).
8-[4-(3-(N,N,N-Trimethylamino)propoxy)phenyl]-4,4-difluoro-
4-bora-3a,4a-diaza-s-indacene 4. A mixture of BODIPY 2 (10 mg,
0.027 mmol) and methyl iodide (200 mL, 3.21 mmL) in 2 mL of DMF
was stirred for 72 h at 40 ꢀC. The solvents were removed under
vacuum to yield 15 mg (95%) of BODIPY 4. 1HNMR (DMSO-d6, TMS)
d
[ppm] 2.18 (m, 2H), 3.04 (s, 9H, -N(CH3)3), 3.37 (t, 2H, J ¼ 6.2 Hz),
4.17 (t, 2H, J ¼ 6.0 Hz), 6.69 (dd, 2H, J ¼ 1.8, 4.2 Hz, pyrrole), 7.02 (d,
2H, J ¼ 4.2 Hz, pyrrole), 7.17 (d, 2H, J ¼ 8.3 Hz, Ar), 7.66 (d, 2H,
J ¼ 8.3 Hz, Ar), 8.09 (br, 2H, pyrrole). APPI-MS [m/z] 441.2763
(M þ H)þ (440.2679 calculated for C25H33BF2N3O).
2.6. Generation of iodine species
UV-visible spectra of the solution containing BODIPY (1 mM) and
10 mM of KI in DMF/5% water was performed before and after
irradiation with visible light at different times (5, 10, 15 and 30 min)
under aerobic condition. Also, the same experiment was carried out
2.3. Spectroscopic studies
in an argon atmosphere. Diluted Lugol's solution (20 mM) was used
UV-visible absorption and fluorescence spectra of BODIPYs
to obtain reference spectra in DMF/5% water. Also, a control was
performed irradiating KI in absence of the BODIPYs.
(3.4
25.0
m
M) were recorded in a quartz cell of 1 cm path length at
0.5 ꢀC. Reverse micelles of n-heptane/AOT (0.1 M)/water
(W0 ¼ 10) were prepared as previously described [17]. The steady-
state fluorescence emission spectra were performed exciting the
samples at lexc ¼ 470 nm. Absorbances (<0.05) were matched at
the excitation wavelength and the areas of the emission spectra
were integrated in the range of 480e700 nm. The energy of the
singlet-state (Es) was calculated from the intersection of the
normalized absorption and fluorescence curves. The fluorescence
quantum yield (FF) of BODIPYs were calculated by comparison of
the area below the corrected emission spectrum with that of
fluorescein as a reference (FF ¼ 0.92 in 0.1 M NaOH) and taking into
account the refractive index of the solvents [18]. Singlet excited
state deactivation of BODIPY by KI was investigated using Stern-
2.7. Photosensitized inactivation of microorganisms
The microorganisms used in this study were the strains of
S. aureus ATCC 25923, E. coli (EC7) and C. albicans (PC31), which
were previously characterized and identified [20]. Microbial cell
suspensions (2 mL, ~108 colony forming units (CFU)/mL bacteria
and ~106 CFU/mL yeast) in 10 mM phosphate-buffered saline (PBS,
pH ¼ 7.4) solution were incubated with 50 mM KI for 10 min in dark
at 37 ꢀC. After that, cells were treated with BODIPY 3 or 4 (1
mM for
S. aureus and 5 mM for E. coli and C. albicans) for 30 min in dark at
37 ꢀC in Pyrex culture tubes (13 ꢂ 100 mm). KI were added from a