Synthesis, structure, photophysical, electrochemical properties and antibacterial activity of brominated BODIPYs

Article abstract of DOI:10.1039/c6ra12258b

A series of mono- and di-brominated BODIPYs (1-5) was synthesized and characterized with a view to study the performance of dyes towards antibacterial activity. Regioselective bromination at the 2- and 2,6-positions of the BODIPY core was achieved with quantitative yield. The bromination of meso-(4-hydroxyphenyl) BODIPY (5) yielded an unexpected dibromo derivative, where the bromine groups were installed at the 3,5-positions of the phenyl ring rather than the 2,6-positions of the BODIPY core, which is confirmed and supported by UV-visible, fluorescence, and ¹H NMR spectroscopic analyses, electrochemical studies, and also by single crystal X-ray crystallography. We observed a red shift of ～16 nm in the absorption and 20-29 nm in the emission spectra in CH₂Cl₂ for the installation of each bromine group at the BODIPY core. The small difference between the first reduction potentials of the parent and dibromo derivative (5 and 5b) reveal that dibromination does not occur on the pyrrolic moiety. The intermolecular interactions involving C?H, F?H, H?H, and Br?H are the key factors in stabilizing the molecular crystal packing. The antibacterial properties of these dyes were investigated and the brominated derivatives showed better antibacterial effects than their corresponding parent BODIPYs, particularly the unusual dibromo derivative, 5b.

Full text of DOI:10.1039/c6ra12258b

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Page 1 of 35
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DOI: 10.1039/C6RA12258B
Synthesis, structure, photophysical, electrochemical properties and
antibacterial activity of brominated BODIPYs
Dijo Prasannana, Darpan Raghavb, Subramaniam Sujathaa, Haritha Hareendrakrishna
kumara, Krishnan Rathinasamyb*and Chellaiah Arunkumara*
aBioinorganic Materials Research Laboratory, Department of Chemistry, National Institute of Technology Calicut,
Kozhikode, Kerala, India ꢀ 673 601; Eꢀmail: arunkumarc@nitc.ac.in
bSchool of Biotechnology, National Institute of Technology Calicut, Kozhikode, Kerala, India ꢀ 673 601; Eꢀmail:
rathin@nitc.ac.in
Abstract:
A series of monoꢀ and diꢀbrominated BODIPYs (1ꢀ5) was synthesized, characterized with a view
to study the performance of dyes towards antibacterial activity. The regioselective bromination at
2ꢀ and 2,6ꢀpositions of the BODIPY core was achieved with quantitative yield. The bromination
of mesoꢀ(4ꢀhydroxyphenyl) BODIPY (5) yielded an unexpected diꢀbromo derivative where the
bromine groups installed at the 3,5ꢀpositions of the phenyl ring rather than 2,6ꢀpositions of
1
BODIPY core which is confirmed and supported by UVꢀVisible, fluorescence, H NMR
spectroscopic analyses, electrochemical studies and also by single crystal xꢀray crystallography.
We observed a red shift of ~ 16 nm in the absorption and 20ꢀ29 nm in the emission spectra in
CH2Cl2 for the installation of each bromine group at the BODIPY core. The small difference
between the first reduction potentials of parent and diꢀbromo derivative (5 and 5b) reveal that the
diꢀbromination does not occur on the pyrrolic moiety. The intermolecular interactions involving
CꢁꢁꢁH, FꢁꢁꢁH, HꢁꢁꢁH and BrꢁꢁꢁH are the key factors in stabilizing the molecular crystal packing. The
antibacterial properties of these dyes were investigated and the brominated derivatives showed
better antibacterial effect than their corresponding parent BODIPYs, especially the unusual diꢀ
bromo derivative, 5b.
Keywords: BODIPYs, Bromination, Crystal structure analysis, Electrochemistry, Antibacterial
activity.
________________________________________________________________________

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Introduction
Development of highly emissive dyes is of multifaceted interest for various applications
ranging from material chemistry to biology.1 Out of the numerous organic dyes like fluorescein,
cyanine, rhodamine etc., BODIPYs (4,4ꢀdifluoroꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene) are intriguing
because of their intense absorption profile, high fluorescent quantum yields, negligible triplet
state formation, redox properties, decent solvatochromism, good solubility and chemical
robustness.1,2 Architecting the BODIPYs with secondary units at the core / boron center / the
mesoꢀposition is a thriving subject of interest and makes the system so sensitive owing to their
unusual spectroscopic behaviors.2,3 Such renewed molecules could act as chemosensors, laser
dyes, energy transfer cassettes, organic photovoltaics, biomolecular labels, supramolecular
polymers, fluorescent pH probes, photosensitizers for solar cells and also in photodynamic
therapy.4
The synthetic difficulty in obtaining BODIPYs with unsubstituted pyrrole is that, none of
the pyrroleꢀbased carbons are blocked from electrophilic attack. However, effective synthetic
strategies are available in the literature for the synthesis of diverse BODIPY derivatives, for
unsubstituted pyrrole,2 modifications has to be allowed than substituted pyrroles in order to
prevent polymerization of pyrrole in the former. The effect of halogen substitution at the pyrrole
carbons of boron dipyrrin core on structural and spectral properties is frequently met.3,5 The
presence of heavy atoms such as bromine onto the BODIPY nucleus enhances intersystem
crossing and are potential sensitizers for PDT as an alternative to the porphyrin based
photosensitizers. As far as biological applications are concerned, fluorophores absorbing at
longer wavelength are of much importance because at higher wavelength absorption by cells and
water, light scattering and autoꢀfluorescence are reduced significantly, allowing for deeper light
penetration, resulting in efficient therapy and diagnosis.2 The regioselective electrophilic
bromination of the 1,2,3,5,6,7ꢀpositions of BODIPY has recently been reported using either
liquid bromine or Nꢀbromosuccinimide (NBS).5 It is also well documented in literature that 2,6ꢀ
halogenated BODIPYs could act as efficient singlet oxygen photosensitizers for photodynamic
therapy (PDT) due to heavy atom effect.6 Moreover, the introduction of a halogen atom onto the
BODIPY core may facilitates generation of longer wavelength BODIPY dyes with further
diverse derivatization through aromatic nucleophilic substitution or palladiumꢀcatalyzed
coupling reactions such as Suzuki, Stille, Heck and Sonogashira reactions.3,7

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The antibacterial8 and antimicrobial9 activities of BODIPY dyes are very limited in the
literature. Inspired by the report on singlet oxygen formation in brominated BODIPYs10, we
attempted the antibacterial studies of synthesized brominated BODIPYs towards Gram positive
and Gram negative bacteria under light and dark conditions. Furthermore, the halogenated
BODIPYs were not tested for any other biological activities so far. In order to delineate the
heavy atom (bromine) effect on the BODIPYs towards antibacterial activity, we report here for
the first time of monoꢀ / diꢀbromo derivatives of BODIPYs bearing 4ꢀmethyl, 4ꢀtꢀbutyl, 4ꢀN,N′ꢀ
dimethyl and 4ꢀhydroxyl groups at mesoꢀphenyl position (Scheme 1 and 2) and the present study
is also directed towards a comparison on spectral, electrochemical and photophysical properties.
Experimental
Materials and methods
All the chemicals used for the synthesis were reagent grade unless otherwise specified.
Pyrrole and borontrifluoride etherate (BF3⋅OEt2) purchased from Spectrochem (India) was
distilled over CaH2 before use. Benzaldehyde, 4ꢀhydroxybenzaldehyde, 4ꢀmethylbenzaldehyde,
4ꢀtertꢀbutylbenzaldehyde, 4ꢀN,Nꢀdimethylaminobenzaldehyde and 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀpꢀ
benzoquinone (DDQ) were obtained from Sigma Aldrich and were used as such. Nꢀ
bromosuccinimide (NBS) purchased from Merck (India) was recrystallized from hot water and
dried at 80 °C for 6 h. Trifluroacetic acid (TFA) purchased from Merck (India) and was distilled
over P2O5. Triethylamine (Et3N) purchased from Qualigens was used as such. Chloroform,
dichloromethane, hexane and methanol purchased from AVRA Synthesis Pvt. Ltd. were purified
by distilling over K2CO3. Toluene and acetonitrile purchased from Fischer Scientific and THF
1
obtained from SRL Chemicals Ltd. India. H NMR spectra were recorded with a Bruker 400
MHz FTꢀNMR spectrometer in CDCl3 / CD3COCD3 using tetramethylsilane as the internal
reference. IR data was collected on a JASCO FT / IRꢀ4700 with KBr pellets. Mass spectra of
BODIPY complexes were performed either using liquid chromatographyꢀmass
spectrometry (LCꢀMS) ShimadzuꢀLCMSꢀ2010 or with a Waters Xevo G2 QuadrapoleꢀTimeꢀofꢀFlight
(QꢀTOF) high resolution mass spectrometer (HRMS). UVꢀVisible spectra were recorded on a
Shimadzu double beam spectrometer 2450 instrument using 1 cm matched quartz cuvettes at
room temperature. Single crystal structure Xꢀray data collections were performed on a Bruker
AXS Kappa Apex II CCD diffractometer with graphite monochromated Mo Kα radiation (λ =

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0.71073 Å) at 298 K. Fluorescence spectra were recorded on a PerkinꢀElmer LSꢀ55
Luminescence spectrophotometer with a slit width of 10 at 470 nm excitation wavelength and
emission from 490 to 750 nm. The fluorescence quantum yield (Φf) was computed using the
below equation,
2
2
Φf = ΦfRFSARɳS / FRASɳR
where FS and FR are the integrated fluorescence intensities of the sample and reference, AS and
AR are the absorbance of the sample and reference at excitation wavelength, ɳS and ɳR are the
refractive indexes of the solvents used for the sample and reference. Fluorescein in 0.1 M NaOH
solution was used as the reference (Φf = 0.85, λexc= 470 nm). All Φf values were corrected for
changes in refractive index. Both the BODIPYs and reference solutions were prepared with the
same absorbance at the excitation wavelength (between 0.1 and 0.05 in a 1 cm quartz cell).
Electrochemical measurements were performed on a CHꢀinstruments Inc, USA (Model:
CHI660E) equipped with potentiostat / galvanostat with Fourier Transform AC voltammeter.
The electrochemical system utilized a threeꢀelectrode configuration consisting of a glassy carbon
(working), platinum wire (auxillary) and standard Calomel reference (SCE) electrodes. The
concentrations of the samples were maintained as 0.1 M containing tetrabutylammonium
hexafluorophosphate (NBu4PF6) as supporting electrolyte (0.1 M) in dichloromethane at 25 °C
under N2 atmosphere at a scan rate of 50 mV/s. The fluorescence imaging was performed using a
Nikon ECLIPSE Tiꢀmicroscope attached with a Cool SNAP digital camera at 40X
magnification. The images were processed using NIS Elements BR software.
Microorganism and growth conditions
Escherichia coli strain 2065 and Bacillus subtilis strain 168 (trpC2) were used for studying the
antibacterial activity of BODIPY compounds. Both the organisms were grown aerobically at 37
°C in the standard Luria–Bertani (LB) broth containing 10 g/L tryptone, 5 g/L yeast extract and
10 g/L NaCl. For preparing LB agar petriꢀplates, 12 g/L agar powder was added to LB broth
before autoclaving.
Agar well diffusion assay for determining the growth inhibition of Escherichia coli and
Bacillus subtilis under light and dark conditions

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Agar well diffusion assay was performed as described earlier.11a Briefly, Staphylococcus
aureus (SA) and Escherichia coli (EC) were grown overnight in 5 mL LB broth in a shaking
incubator maintained at 37 °C and 200 rotations per minute (RPM). LB agar containing petri
plates were prepared by pouring the sterile LB agar into sterile petri plates to a uniform depth of
4 mm which is equivalent to approximately 25 mL in a 90 mm plate. The medium was allowed
to solidify in the plates. The overnight culture of SA and EC was spread evenly on the solidified
agar with the help of a sterile cotton swab. Wells were made in the agar plates for addition of the
BODIPY solutions. All the BODIPYs were then added in equal volume and equal concentration
(25 ꢂg/well) into the wells. The petri plates were distributed into two sets, one set was kept in the
incubator with an in built light source while other was kept in dark incubator. Both incubators
were programmed to maintain 37 °C and organisms were allowed to grow overnight. In the
incubator with the light source, irradiation was done using a Quartz line lamp (300 W). Light
intensities were measured with a pyroelectric detector (RJPꢀ735 from Laser Probe) which was
connected to an energy ratio meter (RJꢀ7620 from Laser Probe). The study was conducted for an
irradiation time of 12 h and the average light fluence rate was 95 µJ/cm2. Tetracycline (10
ꢂg/well) was used as a positive control for this experiment and DMSO was kept as the solvent
control. After 12 h of incubation, the zone of inhibition was calculated using the formula given
below:11a
% of inhibition = (Diameter of the inhibition zone x 100) / 90 (diameter of petriꢀplate)
Determination of half maximal inhibitory concentration (IC50) and minimum inhibitory
concentration (MIC) values for the BODIPYs using LB broth assay
Overnight culture of EC and BS cells was inoculated in 5 mL LB medium containing
different concentrations of BODIPYs (5, 10, 20, 40, 60, and 80 µM). The growth inhibitory
effect of the compounds was studied by monitoring the absorbance at 600 nm (A600), at
different time intervals (0, 4, 8, and 12 hours). The A600 value of the control cells treated with
the solvent control (DMSO) was subtracted from the A600 value obtained immediately after
adding the compounds in all cases in order to eliminate the absorbance contributed by
compounds alone. The percentage of growth inhibition was determined for 12 h after the
addition of BODIPYs to the bacterial cultures. The percentage inhibition of the cell growth was
calculated using the equation:11

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% Inhibition = [1 – (XD600 / CD600)] × 100
where, XD600 represents the A600 value of X µM of the compound treated culture at the different
time points. CD600 represents the A600 value of the control culture of the same time. The IC50
values of the compounds were determined by plotting the percentage inhibition of cell growth
against various concentrations of BODIPYs. Furthermore, the concentration of the compound at
which there was no visible growth of organism was diluted and spread on to the LB agar plates
and incubated for an additional 12 h at 37 °C. The number of colonyꢀforming units was
calculated by counting the colonies on each plate. The concentrations were no visible growth
observed was considered as the MIC.11
Intracellular localization of BODIPYs in EC and BS cells by fluorescence microscopy
analysis
For studying the intracellular localization of the BODIPYs EC and BS cells were grown
in the liquid medium for 6 hours in the presence of compounds from the 5 series (5, 5a and 5b),
in a shaking incubator at 37 °C. The cells were then harvested and washed twice with 1X PBS in
order to remove the unꢀinternalized compounds. The cells were then incubated with Hoechst
33342 (1.5 µg/mL) for 30 minutes to visualize the DNA.11 This step was followed by washing
the cells twice in 1X PBS. The cells were then mounted on clean glass slides and sealed for
microscopic analysis. The compounds were excited using a green filter (490–510 nm) so as to
excite the BODIPYs. The fluorescence imaging was performed using a Nikon ECLIPSE Tiꢀ
microscope attached with a Cool SNAP digital camera at 40X magnification. The images were
processed using NIS Elements BR software.
Measurement of singlet oxygen species
1,3ꢀDiphenylisobenzofuran (DPBF) (4.9 x 10ꢀ5 M) solutions with and without BODIPYs
(2.25 x 10ꢀ6 M) in DMSO prepared under dark were irradiated at room temperature under gentle
magnetic stirring. The singlet oxygen (1O2) acceptor DPBF gradually decolorized upon reaction
with singlet oxygen species generated by BODIPYs. The breakdown of DPBF molecules were
monitored by measuring the decrease in absorbance at 410 nm at regular irradiation intervals.
The relative rate of singlet oxygen production in selected BODIPYs (1, 1a, 1b, 2a, 3a, 4b and
5b) were determined.

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Synthesis
Synthesis of dipyrromethanes
To a solution of corresponding aldehyde (1 equiv.) in freshly distilled pyrrole (25/50
equivalent) degassed with a stream of N2 was added catalytic amount of trifluoroacetic acid
(TFA).10 The mixture was stirred at room temperature for 10ꢀ25 min. Upon completion of the
reaction, dilute NaOH (0.1 M) was added to quench the reaction. The product extracted with
ethyl acetate was dried over anhydrous Na2SO4 and evaporated to dryness under vacuum. This
crude product was then purified by recrystallization in EtOH / water mixture or column
chromatographed on silica gel using CHCl3 / hexane.
5ꢀPhenyldipyrromethane: Prepared according to the general procedure.12 Yield: 62 %. 5ꢀ(4ꢀ
1
Methylphenyl)dipyrromethane: Yield: 30 %; H NMR (400 MHz, CDCl3, δ in ppm): 2.32 (s,
3H; methylꢀH), 5.43 (s, 1H; mesoꢀH), 5.91 (s, 2H; py), 6.15ꢀ6.13 (m, 2H; py), 6.68ꢀ6.66 (m, 2H;
py), 7.13ꢀ7.08 (m, 4H; ArꢀH), 7.89 (s br, 1H; pyꢀNH). 5ꢀ(4ꢀtertꢀButylphenyl)ꢀdipyrromethane:
Yield: 64 %; 1H NMR (400 MHz, CDCl3, δ in ppm): 1.30 (s, 9H; tꢀbutylꢀH), 5.45 (s, 1H; mesoꢀ
H), 5.94ꢀ5.92 (m, 2H; py), 6.16ꢀ6.14 (m, 2H; py), 6.69ꢀ6.67 (m, 2H; py), 7.15ꢀ7.13 (m, 2H; Arꢀ
H), 7.33ꢀ7.31 (m, 2H; ArꢀH), 7.91 (s br, 1H; pyꢀNH). 5ꢀ(4ꢀN,N'ꢀDimethylaminophenyl)ꢀ
1
dipyrromethane: Yield: 48 %; H NMR (400 MHz, CDCl3, δ in ppm): 2.92 (s, 6H;
dimethylaminoꢀH), 5.38 (s,1H; mesoꢀH), 5.94ꢀ5.92 (m, 2H; py), 6.15ꢀ6.13 (m, 2H; py), 7.08ꢀ7.06
(m, 2H; py), 6.70ꢀ6.67 (d, 2H; ArꢀH), 7.06ꢀ7.08 (d, 2H; ArꢀH), 7.89 (s br, 1H; pyꢀNH). 5ꢀ(4ꢀ
1
Hydroxyphenyl)ꢀdipyrromethane: Yield: 46 %; H NMR (400 MHz, acetone, δ in ppm): 5.33
(s, 1H; mesoꢀH), 5.71 (d, 2H; py), 5.96ꢀ5.94 (q, 2H; py), 6.65 (d, 2H; py), 6.73 (d, 2H; ArꢀH),
7.02 (d, 2H;ArꢀH), 9.57 (s br, 2H; pyꢀNH).
Synthesis of BODIPYs, 1ꢀ5
To the corresponding dipyrromethane dissolved in dry CH2Cl2, nitrogen was purged for
15 min. 3ꢀDichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (DDQ) was added and stirred vigorously for
0.5ꢀ1 h at room temperature till the complete consumption of dipyrromethane as monitored by
TLC. To this oxidized product dipyrromethene, triethylamine was added and stirred further at
room temperature for another 15 min, after which borontrifluoro etherate was added and
continued stirring. TLC analysis indicated the formation of expected compound as a fluorescent

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orangeꢀred spot. The reaction mixture was then washed with water, extracted with CH2Cl2,
organic layers were combined, dried over Na2SO4, filtered and the solvent was removed under
reduced pressure to give the crude product. This was further purified by silica gel column
chromatography using chloroform to afford pure compound as orange red / green fluorescent
solid.
4,4ꢀDifluoroꢀ8ꢀphenylꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (1): The compound 1 was obtained as
orange solid. Yield = 56 %; UV/vis (CH2Cl2): λmax (log ε) = 500 nm (4.66); Fluorescence
(CH2Cl2): λem = 527 nm (λex = 470 nm). IR (KBr, cmꢀ1): 3063, 2923, 2852, 2345, 1638, 1577,
1542, 1413, 1389, 1352, 1321, 1260, 1226, 1157, 1116, 1076, 910, 780, 775, 751, 722, 665.
4,4ꢀDifluoroꢀ8ꢀ(4ꢀmethylphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (2): Compound 2 was
1
afforded in pure form as reddish orange solid. Yield = 84 %. H NMR (400 MHz, CDCl3, δ in
ppm): 2.47 (s, 3H; ꢀCH3), 6.53ꢀ6.54 (d, 2H; py), 6.95ꢀ6.96 (d, 2H; py), 7.32ꢀ7.34 (d, 2H; ArꢀH),
7.46ꢀ7.48 (d, 2H; ArꢀH), 7.92 (s, 2H; py); UV/vis (CH2Cl2): λmax (log ε) = 499 nm (4.22);
Fluorescence (CH2Cl2): λem = 514 nm (λex = 470 nm). IR (KBr, cmꢀ1): 2923, 2958, 2853, 1730,
1574, 1545, 1476, 1413, 1387, 1262, 1223, 1186, 1156, 1121, 1098, 1081, 1050, 977, 911, 774,
757, 738.
4,4ꢀDifluoroꢀ8ꢀ(4ꢀtertꢀbutylphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (3): The crude solid was
purified chromatographically to yield the target compound 3 as a reddish orange solid in 57 %
1
yield; H NMR (400 MHz, CDCl3, δ in ppm): 1.39 (s, 9H), 6.53ꢀ6.54 (d, 2H; py), 6.98ꢀ6.99 (d,
2H; py), 7.50ꢀ7.55 (m, 4H; ArH), 7.93 (s, 2H; py).UV/vis (CH2Cl2): λmax (log ε) = 499 nm
(4.75); Fluorescence (CH2Cl2): λem = 515 nm (λex = 470 nm). IR (KBr, cmꢀ1): 3114, 2923, 2956,
2852, 1537, 1603, 1735, 1475, 1353, 1077, 1117, 1260, 1223, 1198, 1157, 1312, 1384, 1411,
1475, 719, 775, 764, 803, 836, 851, 916, 649.
4,4ꢀDifluoroꢀ8ꢀ(4ꢀN,N'ꢀdimethylaminophenyl)ꢀ4ꢀboraꢀ3a,4aꢀsꢀdiazaꢀsꢀindacene (4): Green
solid was obtained and the yield was noted as 16 %; 1H NMR (400 MHz, CDCl3, δ in ppm): 3.11
(s, 6H; dimethylaminoꢀH), 6.53ꢀ6.54 (d, 2H; py), 6.78ꢀ6.81 (d, 2H; py), 7.04ꢀ7.05 (d, 2H; ArꢀH),
7.55ꢀ7.57 (d, 2H; ArꢀH), 7.87 (s, 2H; py). λmax (log ε) = 489 nm (3.82); Fluorescence (CH2Cl2):
λem = 520 nm (λex = 470 nm); IR (KBr, cmꢀ1): 2853, 2714, 1734, 1662, 1603 1551, 1534, 1465,

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1411, 1389, 1374, 1263, 1232, 1200, 1167, 1120, 1079, 941, 910, 824, 812, 742, 728, 775, 742,
728, 699, 596, 2923, 1465, 1475.
4,4ꢀDifluoroꢀ8ꢀ(4ꢀhydroxyphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (5): An orange solid was
1
obtained and the yield was 56 %; H NMR (400 MHz, CDCl3, δ in ppm): 6.55 (d, 2H; py), 6.98
(d, 4H; ArꢀH), 7.49 (d, 2H; py), 7.92 (s, 2H; py); UV/vis (CH2Cl2): λmax (log ε) = 497 nm (4.77);
Fluorescence (CH2Cl2): λem = 512 nm (λex = 470 nm). IR (KBr, cmꢀ1): 3576, 3439, 2918, 2801,
2682, 2324, 1757, 1708, 1604, 1510, 1474, 1446, 1413, 1587, 1539, 1510, 1474, 1446, 1413,
1353, 1299, 1278, 1259, 1227, 1178, 1119, 1077, 1040, 1009, 978, 963, 909, 844, 780, 765, 744,
710, 647.
Synthesis of brominated BODIPYs, 1–5 (aꢀb)
To a solution of BODIPY dissolved in dry CH2Cl2 was added drop wise three equivalents
of Nꢀbromosuccinimide (NBS) in dry CH2Cl2 over a period of 20 min. This mixture was left
stirring at room temperature for an additional 15ꢀ17 h, washed with brine and extracted with
CH2Cl2. Organic layers were combined, dried over Na2SO4 and evaporated to dryness under
vacuum. The purification was performed by column chromatography on silica gel using CHCl3 /
hexane as eluent which afforded two products, monoꢀbrominated (1aꢀ5a) as well as diꢀ
brominated (1bꢀ5b) derivatives.
2ꢀBromoꢀ4,4ꢀdifluoroꢀ8ꢀphenylꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (1a) and 2,6ꢀDibromoꢀ4,4ꢀ
difluoroꢀ8ꢀphenylꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (1b).5 Purification was performed by column
chromatography on silica gel using CHCl3 / hexane (80 / 20) as eluent to provide compounds 1a
1
and 1b. Compound 1a: Yield = 20 %; H NMR (400 MHz, CDCl3, δ in ppm): 6.59 (s, 1H; py),
6.88 (d, 1H; py), 6.99 (s, 1H; pyꢀH), 7.53ꢀ7.59 (m, 5H; ArꢀH), 7.79 (d, 1H; py), 8.00 (d, 1H; py);
13C NMR (400 MHz, CDCl3): δ 147.1, 146.2, 142.1, 135.4, 134.3, 133.3 ꢀ 133.2, 131.16, 130.4,
130.3, 128.6, 119.61, 105.9. λmax (log ε) = 516 nm (4.70); Fluorescence (CH2Cl2): λem = 547 nm
(λex = 470 nm); IR (KBr, cmꢀ1): 3124, 2922, 2852, 2955, 1637, 1729, 1577, 1551, 1477, 1474,
1441, 1402, 1362, 1360, 1253, 1222, 1148, 1115, 1074, 1038, 985, 925, 903, 832, 772, 749, 723,
700, 665, 633, 614, 576. HRMS (ESIꢀTOF) m/z: 346.97 (calc.) 347.02 (found). Compound 1b:
Yield = 65 %; 1H NMR (400 MHz, CDCl3 , δ in ppm): 6.88 (s, 2H; py), 7.56 (m,1H; ArꢀH), 7.47
(m, 4H; Ar ꢀH), 7.78 (s, 2H; py); 13C NMR (400 MHz, CDCl3): δ 146.0, 143.2, 133.6, 131.8,

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130.7, 130.5, 129.3, 127.8, 106.2. λmax (log ε) = 538 nm (4.65); Fluorescence (CH2Cl2): λem
=
569 nm (λex = 470 nm); IR (KBr, cmꢀ1): 3114, 2923, 2852, 2959, 1733, 1638, 1576, 1553, 1479,
1464, 1435, 1375, 1346, 1253, 1099, 1073, 992, 916, 848, 802, 748, 715, 633, 614, 573. LCMS
m/z: 423.92 (calc.) 423 (found).
2ꢀBromoꢀ4,4ꢀdifluoroꢀ8ꢀ(4ꢀmethylphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (2a) and 2,6ꢀ
Dibromoꢀ4,4ꢀdifluoroꢀ8ꢀ(4ꢀmethylphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (2b): The reaction
mixture was stirred at 45 °C for 72 h. The purification was performed by column
chromatography on silica gel using CHCl3 / hexane (40 / 60) as eluent. Compound 2a: Yield = 24
%; 1H NMR (400 MHz, CDCl3, δ in ppm): 2.48 (s, 3H; methylꢀH), 6.59 (d, 1H; py), 6.90 (s,1H;
py), 7.02 (d, 1H; py), 7.35 (d, 2H; ArꢀH), 7.46 (d, 2H; ArꢀH), 7.77 (s, 1H; py), 7.98 (s, 1H; py);
13C NMR (400 MHz, CDCl3): δ 147.5, 145.8, 141.9, 141.8, 133.1, 135.3, 134.2, 130.5, 130.2,
129.4, 119.4, 105.7, 21.5. UV/vis (CH2Cl2): λmax (log ε) = 515 nm (4.66); Fluorescence
(CH2Cl2): λem = 538 nm (λex = 470 nm). IR (KBr, cmꢀ1): 3134, 2923 1731, 1604, 1566, 1536,
1474, 1402, 1474, 1365, 1360, 1256, 1227, 1365, 1360, 1075, 1032, 1146, 1121, 1104, 1018,
905, 925, 984, 869, 772, 756, 740, 703, 646. HRMS (ESIꢀTOF) m/z: 360.99 (calc.) 361 (found).
1
Compound 2b: Yield = 67 %; H NMR (400 MHz, CDCl3, δ in ppm): 2.41 (s, 3H; methylꢀH),
13
6.90 (s, 2H; py), 7.29 (d, 2H; ArꢀH), 7.38 (d, 2H; ArꢀH), 7.76 (s, 2H; py); C NMR (400 MHz,
CDCl3): δ 147.4, 143.8, 142.4, 134.6, 131.7, 130.5 – 130.5, 130.1, 129.6 – 129.5, 107.0, 21.5.
UV/vis (CH2Cl2): λmax (log ε) = 535 nm (4.84); Fluorescence (CH2Cl2): λem = 558 nm (λex = 470
nm). IR (KBr, cmꢀ1): 3123, 2922, 2852, 2345, 2360, 1735, 1684, 1606, 1569, 1541, 1569, 1541,
1569, 1479, 1352, 1362, 1379, 1263, 1183, 1126, 1103, 1001, 982, 915, 835, 824, 754, 743, 705,
620, 644, 575. HRMS (ESIꢀTOF) m/z: 439.89 (calc.) 440.91 (found).
2ꢀBromoꢀ4,4ꢀdifluoroꢀ8ꢀ(4ꢀtertꢀbutylphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (3a) and 2,6ꢀ
Dibromoꢀ4,4ꢀdifluoroꢀ8ꢀ(4ꢀtertꢀbutylphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene
(3b):
The
brominated derivatives were obtained by column chromatography on silica gel using CHCl3 /
hexane (80 / 20) as eluent. Compound 3a: Yield = 5 %; 1H NMR (400 MHz, CDCl3, δ in ppm):
1.35 (s, 9H), 6.53 (s, 1H; py), 6.87 (s, 1H; py), 6.99 (s, 1H; py), 7.44 (d, 2H; ArꢀH), 7.49 (d, 2H;
13
ArꢀH), 7.71 (s, 1H; py), 7.92 (s, 1H; py); C NMR (400 MHz, CDCl3): δ 154.0, 146.5, 144.7,
140.7, 134.3, 133.1, 132.2, 129.4 ꢀ 129.3, 124.6, 118.3, 104.7, 34.0, 30.1. λmax (log ε) = 515 nm
(4.57); Fluorescence (CH2Cl2): λem = 537 nm (λex = 470 nm); IR (KBr, cmꢀ1): 3119, 2923, 2959,

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2852, 1732, 1539, 1364, 1642, 1478, 1463, 1402, 1032, 1069, 1088, 1128, 1260, 1256, 1146,
649, 906, 830, 719, 775, 760, 744, 714. LCMS m/z: 402.07 (calc.) 402 (found). Compound 3b:
1
Yield = 89 %; H NMR (400 MHz, CDCl3, δ in ppm): 1.32 (s, 9H), 6.93 (s, 2H), 7.42 (m, 2H;
ArꢀH), 7.49 (d, 2H; ArꢀH), 7.75 (s, 2H; py); 13C NMR (400 MHz, CDCl3): δ 155.5, 144 ꢀ 143.7,
134.6, 131.8, 130.5 ꢀ 130.3, 129.5, 125.9 ꢀ 125.8, 114, 35.1, 31.1. λmax (log ε) = 544 nm (4.59);
Fluorescence (CH2Cl2): λem = 564 nm (λex = 470 nm); IR (KBr, cmꢀ1): 2923, 2959, 2852, 3116,
1556, 1640, 1746, 1352, 1464, 1075, 1101, 1227, 1243, 1352, 1397, 835, 755, 708, 1609, 1464,
1370, 1382, 741. HRMS (ESIꢀTOF) m/z: 481.97 (calc.) 481 (found).
2ꢀBromoꢀ4,4ꢀdifluoroꢀ8ꢀ(4ꢀN,N'ꢀdimethylaminophenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (4a)
and
2,6ꢀDibromoꢀ4,4ꢀdifluoroꢀ8ꢀ(4ꢀN,N'ꢀdimethylaminophenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀ
indacene (4b): Prepared according to the general procedure, 4,4ꢀdifluoroꢀ8ꢀ(4ꢀN,N'ꢀ
Dimethylaminophenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (4) was treated with NBS for 22 h at room
temperature. The crude reaction mixture after standard work up was purified on silica gel using
CHCl3 / hexane (30 / 70) as eluent. Compound 4a: Yield = 28 %; 1H NMR (400 MHz, CDCl3, δ
in ppm): 2.95 (s, 6H; dimethylaminoꢀH), 6.54 (s, 1H; py), 6.86 (s, 1H; py), 6.98 (d, 1H; py), 7.47
(m, 2H; ArꢀH), 7.64 (m, 2H; ArꢀH), 7.71 (s, 1H; py), 7.91 (s, 1H; py); 13C NMR (400 MHz,
CDCl3): δ 154.6, 145.7, 141.7, 136.2, 135.0, 133.9, 132.7, 130.7, 129.9, 127.6, 119.7 – 119.5,
117.3, 105.8, 43.6. UV/vis (CH2Cl2): λmax (log ε) = 518 nm (4.66); Fluorescence (CH2Cl2): λem=
542 nm (λex = 470 nm). IR (KBr, cmꢀ1): 3116, 2923, 2956, 2852, 1725, 1684, 1592, 1556, 1469,
1402, 1360, 1331, 1260, 1221, 1168, 1118, 1078, 1035, 1078, 988, 946, 933, 915, 825,773, 756,
742, 720, 665, 642. ESIꢀMS m/z: 390.03 (calc.) [M+Na]+ = 413 (found). Compound 4b: Yield =
42 %; 1H NMR (400 MHz, CDCl3, δ in ppm): 2.98 (s, 6H; dimethylaminoꢀH), 7.00 ꢀ7.02 (d, 2H;
Ar), 7.15ꢀ 7.13 (d, 2H; py), 7.50ꢀ7.45 (m, 2H; py), 7.78 – 7.60 (d, 2H; Ar); 13C NMR (400 MHz,
CDCl3): δ 154.9, 148.4, 143.6, 142.4, 136.2, 134.8, 134.7, 131.3, 130.8, 119.8, 107.1, 43.6.
UV/vis (CH2Cl2): λmax (log ε) =535 nm (4.60); Fluorescence (CH2Cl2): λem = 548 nm (λex = 470
nm). IR (KBr, cmꢀ1): 3107, 3125, 2851, 1688, 1590, 1553, 1499, 1474, 1375, 1349, 1263, 1189,
1166, 1120, 1090, 1035, 1375, 1349, 992, 946, 915, 886, 867, 821, 742, 720, 661, 643, 606, 566,
585. LCMS m/z: 468.93 (calc.) 468 (found).
2ꢀBromoꢀ4,4ꢀdifluoroꢀ8ꢀ(4ꢀhydroxyphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (5a) and 2,6ꢀ
Dibromoꢀ4,4ꢀdifluoroꢀ8ꢀ(3,5ꢀdibromoꢀ4ꢀhydroxyphenyl)ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene

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(5b): The reaction mixture was stirred for 15 h. Purification was performed by column
chromatography on silica gel using CHCl3 / hexane (80 / 20) as eluent. Compound 5a: Yield = 2
%. 1H NMR (400 MHz, CDCl3, δ in ppm): 6.45 (s br, 1H; ꢀOH), 6.59 (d, 1H; py), 6.95ꢀ6.68 (dd,
1H; py), 7.55ꢀ7.54 (m, 1H; py), 7.86ꢀ7.83 (m, 4H; ArꢀH), 7.98 (s, 1H; py), 8.04 (s, 1H; py).
UV/vis (CH2Cl2): λmax (log ε) = 514 nm (4.39); Fluorescence (CH2Cl2): λem = 541 nm (λex = 470
nm). IR (KBr, cmꢀ1): 3449, 2923, 2853, 2957, 1719, 1640, 1560, 1534, 1465, 1401, 1362, 1260,
1222, 1184, 1080, 1021, 908, 861, 808, 728, 645. ESIꢀMS m/z: 362.96 (calc.) 363.25 (found).
Compound 5b: Yield = 90 %; 1H NMR (400 MHz, CDCl3, δ in ppm): 6.23 (s, br, 1H; ꢀOH), 6.51
(d, 2H; py), 6.87 (d, 2H; pyꢀH), 7.88 (s, 2H; py), 7.62 (s, 2H; ArꢀH); 13C NMR (400 MHz,
CDCl3): δ 151.7, 144.8, 143.2, 134.6, 133.7, 131.1, 128.2, 119, 110. UV/vis (CH2Cl2): λmax (log
ε) = 510 nm (4.58); Fluorescence (CH2Cl2): λem = 529 nm (λex = 470 nm). IR (KBr, cmꢀ1): 3423,
2925, 2853, 2963, 1640, 1560, 1536, 1467, 1409, 1399, 1261, 1098, 863 801, 742, 727, 702, 663.
HRMS (ESIꢀTOF) m/z: 441.86 (calc.) 440.90 (found).
Results and discussion
Synthesis and characterization
The required appropriate boron dipyrromethane precursors were synthesized by adopting
the standard protocol.12 In the first step, the corresponding dipyrromethanes were prepared by the
condensation of substituted benzaldehyde with a large excess of pyrrole (25 / 50 equiv.), in
presence of catalytic amount of trifluoroacetic acid (TFA, 0.1 equiv.) for 10ꢀ20 min at room
temperature. The crude compounds were purified by column chromatography / recrystallization
technique, afforded mesoꢀ(4ꢀphenyl substituted) dipyrromethanes in 30ꢀ64 % yield. These
dipyrromethanes were then subjected to a twoꢀstep one pot reaction by oxidizing the
dipyrromethanes in the first step with DDQ and reacted in second step with Et3N followed by
BF3⋅OEt2 at room temperature. Column chromatography on silica gel yielded the mesoꢀ
substituted BODIPY dyes 1ꢀ3 and 5 in 56ꢀ84 % and 4 in 16 %. The yields of dipyrromethanes
and unbrominated BODIPYs (1ꢀ 3 and 5, except 4) are almost fair, reasonably comparable to the
literature.12 The synthetic route employed to prepare the mesoꢀsubstituted BODIPYs and its
monoꢀ and diꢀbromo derivatives are outlined in scheme 1 and 2.

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R
R
CHO
i) TFA, N2, RT
NBS, CH2Cl2, RT
Br
X
+
ii) DDQ, Et3N,
BF3⋅OEt2
N
F
N
F
N
F
N
F
N
H
B
B
R
R = H, PhBOBIPY; 1
R = CH3, 4ꢀMPhBOBIPY; 2
R = tBu, 4ꢀBPhBOBIPY; 3
R = H, X = H; PhBOBIPY(Br); 1a
R = H, X = Br; PhBOBIPY(Br)2; 1b
R = CH3, X = H; 4ꢀMPhBOBIPY(Br); 2a
R = CH3, X = Br; 4ꢀMPhBOBIPY(Br)2; 2b
R = tBu, X = H; 4ꢀBPhBOBIPY(Br); 3a
R = tBu, X = Br; 4ꢀBPhBOBIPY(Br)2; 3b
R = N(CH3)2, X = H; 4ꢀNDMPhBOBIPY(Br); 4a
R = N(CH3)2, 4ꢀNDMPhBOBIPY; 4
R = N(CH3)2, X = Br; 4ꢀNDMPhBOBIPY(Br)2; 4b
Scheme 1. Synthesis of various BODIPYs and its monoꢀ and diꢀbrominated derivatives.
It has been reported that the regioselective bromination of unsubstituted BODIPY with
1.2 and 2.4 equivalents of NBS in dichloromethane at room temperature could yield monoꢀ and
diꢀbrominated derivatives respectively in quantitative yield.7 In the present study, regioselective
bromination of BODIPYs were obtained at 2ꢀ, 2,6ꢀpositions (except in case of 4ꢀHPhBODIPY,
5 with three equivalents of NBS in dichloromethane at room temperature. TLC analysis of the
crude reaction mixture showed a major spot corresponding to the diꢀbrominated BODIPYs (1bꢀ
4b) along with a minor amount of monoꢀbrominated product (1aꢀ4a) (Scheme 1). The crude
products were then subjected to silica gel column chromatographic purification using CHCl3 /
hexane. Here, all the dibromo derivatives were obtained in good yield; higher yields of
monobromo derivative could be obtained with 1 ꢀ 1.2 equivalents of NBS.3,4 Hence, no further
optimization of the protocol was done. In addition, limiting the side product formation and low
separation yield has to be addressed to yield quantitative amount of products
chromatographically.
The synthesized compounds were freely soluble in common organic solvents and were
1
characterized by absorption, fluorescence, H NMR spectroscopic methods, electrochemical
studies and XRD techniques. In contrast to other derivatives, bromination of 4ꢀHPhBODIPY, 5,
occurred at 3,5ꢀphenyl positions rather than the desired 2,6ꢀpyrrole positions as supported by
spectroscopic and XRD analyses (Scheme 2).

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OH
OH
OH
Br
Br
Br
NBS, CH2Cl2, RT
+
N
F
N
N
F
N
F
N
N
F
B
B
B
F
F
4ꢀHPhBOBIPY; 5
4ꢀHPhBOBIPY(Br); 5a
4ꢀHPhBOBIPY(Br)2; 5b
Scheme 2. Bromination reaction of 4ꢀhydroxy BODIPY and its brominated derivatives.
1H NMR spectra for dipyrromethanes were taken and the integrated intensities of all the
proton signals are very well in concordance with the expected structure indicating their structural
confirmation. Unlike other BODIPYs (1ꢀ4), the resonance structures of compound 5, bear the
least positive charge at beta pyrrole positions and hence diꢀbromination occurred at the 3,5ꢀ
phenyl positions rather than the desired 2,6ꢀpyrrole positions. The absence of proton signals
corresponding to one and/or two
β
ꢀpyrrole hydrogen(s) at 2ꢀ and/or 2,6ꢀprotons respectively in
1
the spectra clearly indicated the successful bromination at beta pyrrole positions. In general, H
NMR spectra of BODIPYs, give six pyrrole protons whereas monoꢀbrominated and diꢀ
brominated products give five and four protons signals respectively. However, the number of
pyrrole proton signals and its spin multiplicity vary with the symmetric / asymmetric nature of
1
the compounds. A representative overlayed H NMR spectrum of compound 2 and its
brominated derivatives (2a and 2b) are shown in figure 1. The βꢀpyrrole proton signals at 6.95
ppm in the parent BODIPY is deshielded to 7.01 ppm in monoꢀbrominated product, 2a which in
turn disappeared from diꢀbrominated spectrum, 2b. The aromatic phenyl protons with four
proton intensity in 5 at 6.98 ppm was deshielded to 7.62 ppm with two proton intensity in the
corresponding brominated derivative (5b) clearly indicated the bromination on mesoꢀphenyl
1
group rather than pyrrole ring of the BODIPY core. The H NMR spectra of other
dipyrromethanes, BODIPYs and their brominated derivatives are presented in the supplementary
information.

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a
b
c
b
f
c
d
e
d
e
N
N
f
B
F
F
a
b
c
g
h
d
b
c
e
Br
N
N
e
f
B
g
F
F
h
f
d
a
c
d
b
b
c
e
d
Br
Br
N
N
e
B
F
F
Figure 1. Selected region of overlayed 1H NMR spectra of compounds, 2, 2a and 2b in CDCl3 at 25 oC.
The FTꢀIR spectra of the synthesized compounds were taken with KBr pellets and
overlayed spectra of selected BODIPYs is shown in figure S27. The IR spectra of all the
BODIPYs, showed three characteristic aromatic ꢀCꢀH vibration peaks near 2923, 2959 and 2852
cmꢀ1. The sp2 carbon (=CH) stretching frequencies were found between 3000 and 3019 cmꢀ1. The
vibrations corresponding to BꢀF and ꢀC=N of the BODIPY core were observed in the vicinity of
1537 and 1735 cmꢀ1 respectively.13 The βꢀH of pyrrole ring13 was observed near 775 cmꢀ1 in
unbrominated BODIPYs (1ꢀ5), but this peak was absent in the corresponding brominated
products, 1bꢀ4b (Figure S27). The BꢀN stretching frequency was observed at the proximity of
1411 cmꢀ1. The presence of paraꢀdisubstitution is shown by vibrations in the range of 800 to 850
cmꢀ1. The absorption in the range of 1032ꢀ1088 cmꢀ1 indicates the presence of aryl bromides. The
presence of alkyl CꢀH stretch at 2923 cmꢀ1 in compounds 2, 2a and 2b indicate the presence of ꢀ

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CH3. The ꢀCH3 deformations of tertꢀbutyl group in 3, 3a and 3b arise respectively at 1384, 1364
and 1352 cmꢀ1. The characteristic vibrations at 2852 cmꢀ1 point out the presence of ꢀN(CH3)2 in
BODIPYs 4, 4b and 4c and the CꢀN stretches around 1260 cmꢀ1. In the case of hydroxyl
BODIPYs 5, 5a and 5b, an OH stretching frequency of medium intensity around 3400 cmꢀ1
indicate the presence of Hꢀbonding in those molecules.
Photophysical properties
The absorption and emission properties of the BODIPYs in dichloromethane along with
their molar absorption coefficients and quantum yield data are summarized in table 1. The stock
solutions (10ꢀ5 M) were freshly prepared in doubly distilled dichloromethane. The absorption
spectra of BODIPYs show a narrow, intense transition between 490ꢀ545 nm (S0 → S1), a
shoulder at high energy centered around 480 ± 5 nm (0ꢀ1 vibrational transition) and a weak
broad absorption band around ~ 330ꢀ390 nm (S0 → S2) transition. Their molar absorption
coefficient ranges from 38000 to 46000 Mꢀ1 cmꢀ1.
1.0
0.8
0.6
0.4
0.2
0.0
2
2a
2b
2
2a
2b
300
400
500
600
Wavelength (nm)
Figure 2. Normalized optical and fluorescence spectra of BODIPYs, 2, 2a and 2b in CH2Cl2.
It has been documented that the introduction of substituent on the BODIPY affects the
spectroscopic characteristics of the dye particularly, with increasing number of bromine groups
at the pyrrole carbons of BODIPY core, a systematic increase in magnitude of bathochromic

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shift was observed and limited up to four bromine atoms.5 It is also reported that, in mesoꢀ(pꢀ
methoxyphenyl) BODIPY, each bromine substitution at the pyrrole carbon contributes an
additional ~ 10 nm red shift with respect to the absorption maxima.5 Consistent with the reported
literature, we also observed a red shift of 10 nm with the installation of each bromine substituent
at the pyrrole carbon of PhBODIPYs in toluene.14 But, in dichloromethane, a red shift of about
16 nm was observed in the absorption with each attached bromine group in compounds, 1ꢀ3 with
respect to their brominated derivatives (Table 1 and S1). Interestingly, this trend was not
observed in 4 and its brominated derivatives 4a, 4b which may be described as an antagonistic
inductive effect by bromines present at the pyrrole carbons of the BODIPY core.5 Whereas, a red
shift of ~ 13 nm was observed for the diꢀbrominated product 5b with respect to 5, in which
bromination occurs at the phenyl positions rather than at βꢀpyrrole positions.
Table 1. Photophysical properties of compounds, 1ꢀ5.
Max. brightness,
Stokes
shift,
max (cmꢀ1)
1024
1098
1012
584
Φfii
Code
λmax (nm)
Log ε
λem (nm)
Φ x ε / 103 Mꢀ1
cmꢀ1
1.37
4.02
3.63
∆
1i
1ai
1bi
2
2a
2b
3
3a
3b
4
4a
4b
5
500
516
538
499
515
535
499
515
544
489
518
535
497
514
510
4.66
4.70
4.65
4.22
4.66
4.84
4.75
4.57
4.59
3.82
4.66
4.60
4.77
4.39
4.58
527
547
569
514
538
558
515
537
564
520
542
548
512
541
529
0.03
0.08
0.08
0.05
0.83
0.06
0.02
0.04
0.05
0.04
0.06
0.00056
0.00014
0.06
2.77
1.39
2.25
1.87
1.57
0.40
0.03
0.006
3.53
830
770
623
796
652
1219
854
443
589
0.04
0.05
0.98
1.91
971
704
5a
5b
iSimilar data reported for 1, 1a and 1b in dichloromethane at room temperature by L. Jiao et al.
Taken from J. Org. Chem., 2011, 76, 9988−9996. iiFluorescence quantum yields (Φf) were calculated
using the standard, Rhodamine B in anhydrous ethanol (Φf = 0.49) for 1b and fluorescein in aqueous
NaOH (0.1) N (Φf = 0.90) for 1 and 1a.
When excited into either the S1 or S2 states, a narrow, slightly Stokesꢀshifted emission
band of mirror image shape with λmax positioned between 530ꢀ560 nm was also observed from
the S1 state. A remarkable red shift was also induced by bromine atoms in emission spectra;
which were observed to be higher in diꢀbrominated than that of monoꢀbrominated which was in
turn higher than that of parent BODIPYs. For the standard compounds 1, 1a and 1b in toluene,

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the extent of redꢀshift in emission maxima was same as reported.14 The low emission yields (<
0.1) in brominated derivatives is due to heavy atom quenching by bromine in which intersystem
crossing (S1 to T1) becomes prominent by spin orbit coupling and consequently, these derivatives
are likely to be sensitizers for PDT. Thus, the presence of bromine intensified the spinꢀforbidden
process either internal or external to the excited molecule.14 Yet another notable photophysical
property of a fluorescent dye is its brightness, the product of the fluorescence quantum yield (Φf)
and the molar extinction coefficient (ε) at given wavelength λ, is also compared in table 1. The
monoꢀbrominated derivatives show higher quantum yield than its corresponding diꢀbrominated
analogues and the brightness also follows the similar trend.14
More often than not, photophysical processes on BODIPYs are closely associated with
the intermolecular interactions between the dye and its immediate environment and this could be
easily interpreted by the solvatochromic studies which in turn are associated with both the
solvent polarity and viscosity. Both the absorption and emission spectra of all the compounds
were acquired in five different solvents with varying polarity / refractive indices
(dichloromethane, toluene, THF, acetonitrile and methanol) and are summarized in table S1. No
much change in absorption or emission was observed with changing polarity of the solvent. For
parent / unbrominated BODIPYs, the highest and lowest absorption and emission maxima were
observed respectively with toluene and methanol and hence the Stokes shift. The maximum red
shifts were observed for compound 4 in toluene. Both the absorption and emission maxima
shows bathochromic shift with decreasing solvent polarity, indicating the more polar character of
the ground state.14 In general, each bromine substitution at the pyrrole carbon contributes an
average of 18 nm bathochromic shift with respect to the parent / unbrominated BODIPY. The
highest red shift was observed in toluene and lowest in methanol. Further, the red shift could be
immediately noticed and identified just after the purification of the products as, diꢀbrominated
derivatives have a pinkish appearance much different from monoꢀbromo or parent derivatives, as
shown in the photoꢀimages of the compounds in visible and UV light (Figure S29).
Single crystal Xꢀray diffraction studies
The molecular structures of compounds (1, 2, 2a, 3, 5 and 5b) were explored with the
help of Xꢀray crystallography. Single crystals of compounds of suitable size were obtained by
evaporating a mixture of chloroform / hexane at ambient temperature over a period of one week.

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Compound, 1 crystallized with three rotamers in the asymmetric unit whereas 2, 2a, 3, 5 and 5b
crystallized with monomers. The crystal structures of compounds are presented in figure 3; the
crystallographic data and selected geometrical parameters are included in table 2, 3 respectively.
In all compounds, BODIPY core displays characteristic bond lengths and angles (Table 3) with
two pyrrole rings and boron atom in one plane featuring central sixꢀmembered ring and both
fluorine atoms in another plane.
Table 2. Crystal data and structure refinement parameters for compounds 1, 2, 2a, 3, 5, and 5b.
Parameters
Molecular
formula
Formula weight
CCDC number
Temperature / K
Crystal system
Space group
λ / Å
1
2
2a
3
5
C15H11BF2
N2O. H2O
302.08
1041760
293(2)
5b
C15H9BBr2F2
N2O
441.87
1434317
293(2)
C15H11BF2N2
C16H13BF2N2
C16H12BBrF2N2
C19H19BF2N2
307.86
1434321
293(2)
Triclinic
Pꢀ1
0.71073
8.1738(5)
10.1538(6)
27.9370(17)
80.058(3)
89.870(3)
72.418(3)
2174.1(2)
6
282.09
1434320
293(2)
Triclinic
Pꢀ1
0.71073
8.0364(3)
8.0696(4)
11.0822(5)
88.549(2)
84.664(2)
71.997(2)
680.53(5)
2
361.00
1434318
293(2)
Monoclinic
C2/c
0.71073
20.8544(14)
7.3456(5)
22.1366(17)
90
324.17
1434319
293(2)
Orthorhombic
P212121
0.71073
9.1279(6)
11.6936(9)
15.8335(12)
90
90
90
1690.0(2)
4
0.090
Orthorhombic
Pbca
Monoclinic
P21/c
0.71073
18.456(2)
7.9351(7)
19.4327(19)
90
90
90
2846.0(5)
8
0.111
0.71073
11.5754(8)
15.3509(11)
9.3750(5)
90
112.989(2)
90
1533.57(17)
4
a / Å
b / Å
c / Å
α (°)
β (°)
γ (°)
117.420(2)
90
3010.1(4)
8
Volume / Å3
Z
µ / mmꢀ1
0.279
0.100
2.748
5.310
Densitycalcd / mg
1.411
1.377
1.593
1.274
1.410
1.914
mꢀ3
F(000)
944
292
1440
680
1248
856
θ range (°)
e data / unique
Rint
2.373ꢀ24.997
57302 / 7632
0.0392
2.654ꢀ25.997
18363 / 2671
0.0227
2.200ꢀ25.994
24867 / 2950
0.0390
2.165ꢀ25.996
26059 / 3333
0.0393
2.207ꢀ24.369
34754 / 2330
0.0654
2.327ꢀ25.000
30194 / 2693
0.0459
Data / restraints /
parameters
GOF on F2
7632/183/615
2671/0/192
2950/0/200
3333/0/221
2330/3/208
2693/0/208
1.082
0.0548,
0.1220
0.0901,
0.1462
1.064
0.0392,
0.0851
0.0569,
0.1022
1.051
1.051
1.073
0.0390,
0.0907
0.0831,
0.1176
1.102
0.0439,
0.1011
0.0565,
0.1083
a
R1 , wR2b [I >
0.0444, 0.1117 0.0480, 0.1172
2σ(I)]
R1, wR2 (all data)
0.0712, 0.1273 0.0797, 0.1413
aR1 = Σ||Fo|–|Fc||/Σ|Fo|; Io > 2σ (Io). bwR2 = [Σ w(Fo –Fc2)2/Σw(Fo ) ] .
2
2 2 1/2
The BꢀF distances are nonꢀequivalent in all BODIPYs and present above and below the
plane of the pyrrole moieties, and the FꢀBꢀF plane is almost perpendicular (86.7° ꢀ 88.5°) to the
plane of the ring system. The boron atom in all BODIPYs exhibits a distorted tetrahedral
geometry with an average angle of NꢀBꢀN, 106.4° and FꢀBꢀF 108.9°. The average BꢀN distance
present in all BODIPYs is 1.53(5) Å which is in between a single bond (1.420 Å) and a

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coordination bond (1.630 Å) distance displaying that the electron density on the N atom is
delocalized through conjugation within the system.15
Table 3. Selected bond lengths and angles for compounds 1, 2, 2a, 3, 5, and 5b
Parameter PhBODIPYi
1ii
2
2a
4ꢀBPhBODIPYi
3
5
5b
CBodꢀCAryl
BꢀN1
BꢀN2
NꢀBꢀN
FꢀBꢀF
F1ꢀBꢀC5
F2ꢀBꢀC5
ΦAryl
1.481 (11)
1.547 (9)
1.547 (9)
106.4 (8)
110.3 (12)
124.9
124.9
ꢀ
ꢀ
1.476 (4) 1.473 (2)
1.529 (4) 1.534 (3)
1.535 (4) 1.541 (2)
106.4 (3) 106.2 (14) 106.0 (3) 105.9 (15)
109.4 (3) 108.5 (15) 109.9 (3) 109.9 (16)
1.482 (5) 1.474 (3)
1.536 (6) 1.546 (3)
1.541 (6) 1.546 (3)
1.466 (5) 1.459 (3)
1.530 (6) 1.517 (4)
1.539 (6) 1.523 (4)
106.2 (3) 106.9 (2)
109.4 (4) 107.7 (2)
1.486 (6)
1.529 (6)
1.537 (7)
106.9 (4)
108.2 (4)
120.9
131.0
50.2
175.5
123.2
127.4
51.4
119.5
132.1
54.6
117.9
132.2
53.6
115.8
134.3
ꢀ
ꢀ
116.3
134.3
52.9
120.8
131.5
47.7
ΦPyrrole
175.7
174.6
174.1
173.6
174.6
iTaken from J. Phys. Chem. B, 2005, 109, 20433ꢀ20443; iiAverage value obtained from three molecules in the asymmetric unit of 1.
In all compounds, the two pyrrole units along with boron atom forms a rigid six
membered plane and the mean plane deviations of the BODIPY core are ranging from ± 0.0405 ꢀ
± .0723 Å. The BODIPY core is nearly planar in all compounds, because the value of the
dihedral angle between the two pyrrole rings are nearly 180° (ΦPyrrole values varying from 174.1°
ꢀ 175.7°, Table 3).
1
2
2a
3
5
5b
Figure 3. ORTEP representation of molecular structures of 1, 2, 2a, 3, 5 and 5b shown at 40 % probability
ellipsoids. (Solvent molecules are omitted for clarity)
The dihedral angles between the mesoꢀaryl group and BODIPY core (ΦAryl values) are
51.4°, 54.6°, 53.6°, 52.9°, 47.7° and 50.2° for 1, 2, 2a, 3, 5 and 5b respectively indicate they are
midway to orthogonal and parallel arrangements comparable to the reported BODIPYs.15 Such

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arrangement of these two moieties restrict the possible resonance between the BODIPY core and
the aryl substituent and is confirmed by the presence of singleꢀbond distance, CBodꢀCAryl, 1.476,
1.473, 1.482, 1.466, 1.459, 1.486 Å in 1, 2, 2a, 3, 5 and 5b respectively.
(a)
(b)
(c)
(d)
(e)
(f)
Figure 4. Molecular crystal packing of (a) 1, viewed down ‘a’ axis; (b) 2, viewed along ‘bc’ plane; (c) 2a, viewed
along ‘bc’ plane; (d) 3, viewed along ‘ac’ plane; (e) 5, viewed down ac plane and (f) 5b, viewed along ‘bc’ plane.
The molecular crystal packing of 1 is shown in figure 4a which exhibits an antiꢀparallel
3D arrangement of molecules, and the solvent molecule (CHCl3) acting as a bridging unit
between the molecules through various intermolecular interactions as listed in table S2. Whereas,
the crystal packing of 2 forms a herringbone like structure (Figure 4b) in which the BODIPY
core is connected through BꢀFꢁꢁꢁH(Ph), (Pyr)HꢁꢁꢁC(Ph) and (Pyr)HꢁꢁꢁH(Pyr) interactions. Compound 2a
forms zigꢀzag 3D structural arrangements (Figure 4c) that are connected through the interactions

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such as C(Pyr)ꢁꢁꢁC(Pyr), NꢀBꢁꢁꢁH(Pyr), BꢀFꢁꢁꢁH(Ph), BꢀFꢁꢁꢁH(Pyr) and (Pyr)BrꢁꢁꢁC(Ph). Interestingly, the
packing of 3 forms an one dimensional array (Figure 4d) of BODIPY molecules connected
exclusively via BꢀFꢁꢁꢁH(Ph) interaction. The packing diagram of 5 is through the solvent water
molecules that connect the BODIPY molecules forming a trimerꢀlike structure through various
nonꢀcovalent interactions (Table S2) which further extended in all three directions to form an
intricate 3D structural motif. In the packing of 5b, a dimerꢀlike structure formed through various
interactions to form a complex 3D structural arrangement.
We have analyzed all the crystal structures quantitatively with the help of Crystal
Explorer 3.1 to know the presence and importance of various intermolecular interactions, the
data were presented in the form of Hirshfeld surfaces (HSs) and 2D fingerprint plots (FPs)16
(Figure 5). In most of the cases, there is a red spot observed for the intermolecular interactions
involving boron and fluorine atoms (BF2 moieties) with other atoms like carbon, hydrogen and
oxygen of different groups. Interestingly, in 5 and 5b there is an intense red spot on the hydroxyl
group indicates the presence of stronger Hꢀbonding interactions observed. The observed FPs for
each BODIPY is unique in nature with characteristic spike of various lengths and thickness
(Figure 5b).
(a)
(b)
Figure 5. (a) Hirshfeld surfaces with normalized contact distance ranging from ꢀ0.08 Å (red) to 1.67 Å (blue) and
(b) 2D fingerprint plots of all the intermolecular contacts with di and de ranging from 0.8 to 2.8 Å for 1, 2, 2a, 3, 5
and 5b.
Figure 6 displays the percentage distribution of individual intermolecular interactions
based on the results of HS analysis for all BODIPYs. It is observed that the relative contribution

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of CꢁꢁꢁH, FꢁꢁꢁH and HꢁꢁꢁH contacts are comparable in all BODIPYs, except for 2a and 5b where
BrꢁꢁꢁH contacts also contribute an important role in the molecular crystal packing.
Figure 6. Percentage contribution of nonꢀcovalent interactions in BODIPYs on the basis of HS analysis.
Electrochemical measurements
The electrochemical redox properties of BODIPYs were probed through cyclic
voltammetric and differential pulse voltammetric studies using tetrabutylammonium
hexafluorophosphate (NBu4PF6) as supporting electrolyte (0.1 M) in dichloromethane at room
temperature with a scan rate of 50 mV/s. It has been reported that the BODIPY species usually
exhibit one or two reductions, but oxidation is either irreversible or absent.17 In this study,
BODIPY derivatives exhibited a reversible first reduction and a quasiꢀreversible second
reduction in cyclic voltammetric studies; hence the differential pulse voltammetric studies were
carried out in order to get the second reduction potential and listed in the table 4.

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8 x 10ꢀ6
6 x 10ꢀ6
4 x 10ꢀ6
2 x 10ꢀ6
0
2
2a
2b
ꢀ2 x 10ꢀ6
ꢀ4 x 10ꢀ6
ꢀ6 x 10ꢀ6
0.0
ꢀ0.2
ꢀ0.4
ꢀ0.6
ꢀ0.8
ꢀ1.0
ꢀ1.2
Potential / V
Figure 7. Cyclic voltammograms of 2, 2a and 2b in CH2Cl2 under N2 at 25 oC.
Electrochemical redox data of all BODIPYs, 1ꢀ5 is presented in table 4; overlayed cyclic
voltammograms and differential pulse voltammograms of compounds 2, 2a and 2b are depicted
in figure 7 and figure S26 respectively. The first reduction potentials of 2, 2a and 2b are ꢀ0.85, ꢀ
0.73 and ꢀ0.55 V respectively; the more positive values of 2a and 2b compared to 2 indicate that
they can be more easily reducible which also confirms the electron deficient nature of the
compounds. Similar trend is observed in all other cases except in 5, 5a and 5b (Table 4). The less
positive reduction potential of 5b (ꢀ0.73 V) compared to 5 (ꢀ0.88 V) suggesting that the diꢀ
bromination does not occur on the pyrrolic moiety which is confirmed and further supported by
various spectroscopic data (UVꢀVisible, fluorescence and 1H NMR) and also by single crystal xꢀ
ray crystallographic studies.
The reduction potentials of unbrominated BODIPYs follow the trend, ꢀN(Me)2 > ꢀOH > ꢀ
tꢀBu > ꢀCH3 > ꢀH; and found to increase with increasing electron donating nature of the
substituent on the mesoꢀaromatic ring, may be due to the inductive stabilization effect of their
anionic form.17 The presence of alkyl or tertꢀbutyl (2 and 3 respectively) substituents in fact step
up the reduction potentials than that of compound 1. Moreover, in these unbrominated BODIPYs
1ꢀ5, the lack of substitutions at positions 2, 3, 5, or 6 tend to cause instability for radical cation
produced during oxidation and the presence of mesoꢀsubstitution usually results in less reactive
radical formation during reduction.

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The brominated BODIPYs 1aꢀ5a and 1bꢀ5b with their electron deficient nature displayed
one irreversible oxidation and reversible reduction peak.17 Bromination actually lowers the
oxidation potential14 and this small redox potential values hints to the fact that these dyes could
be easily reduced and oxidized. Their irreversible oxidation curve point out that the radical
cations produced on the anode could be quickly converted to other products.18 As reported
earlier, a successive anodic shift in reduction potential was observed with the introduction of
bromine than their respective parent BODIPY dyes, ie., the ease of reduction which follows the
order; 1b > 1a > 1, 2b > 2a > 2, 3b > 3a > 3, 4b > 4a > 4, 5a > 5b > 5.5 The absence of any
oxidation peaks between 0ꢀ2 V also throws light on the fact that the dyes are not electron rich.
Table 4. Electrochemical redox data of BODIPYs, 1ꢀ5.
Reduction
Reduction
potential (V)
potential (V)
Compound
Compound
I
II
I
II
ꢀ0.82
ꢀ0.71
ꢀ0.58
ꢀ0.85
ꢀ0.73
ꢀ1.72
ꢀ1.62
ꢀ1.52
ꢀ1.78
ꢀ1.67
ꢀ0.56
ꢀ0.93
ꢀ0.70
ꢀ0.59
ꢀ0.88
ꢀ1.46
ꢀ1.88
ꢀ1.57
ꢀ1.47
ꢀ1.11
1
3b
4
1a
1b
2
4a
4b
5
2a
ꢀ0.55
ꢀ1.50
ꢀ0.69
ꢀ0.73
ꢀ1.43
ꢀ1.55
2b
5a
5b
ꢀ0.86
ꢀ0.71
ꢀ1.79
ꢀ1.65
3
3a
Antimicrobial activities
Agar well diffusion assay for determining the antimicrobial effect of BODIPYs on Gram
positive and Gram negative bacteria
The diffusion method is a qualitative and quantitative screening technique to suggest the
antimicrobial activity of the molecules under study. The antimicrobial effect of BODIPYs was
assessed against EC and BS cultures by zone of inhibition assay using 25 µg of the compound
per well. It was found that the BODIPYs showed more potency towards BS (Gramꢀpositive
bacteria) as compared to EC (Gramꢀnegative bacteria). The experiment was divided into two
sets, one set being illuminated and the other left unilluminated. It was observed that the

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difference in the growth inhibitory effect of the compounds in the presence and absence of light
was very minimal in case of BS. For example, compound 5b showed 30 % and 32.22 % growth
inhibition under unilluminated and illuminated conditions respectively in BS. Similarly,
compounds 1a showed 18.88 % growth inhibition under dark and 18.33 % in light, whereas, the
compound 1b showed 15 % growth inhibition under dark and 15.55 % in light (Figure 8). Few
compounds like 4b, 5, and 2 exhibited moderate increase in the growth inhibitory effect under
illuminated conditions. For example, the inhibitory effect of the compounds 4b, 5, and 2 in the
unilluminated condition was found to be 15.55, 5.55 and 0 % respectively while their activities
increased to 21.11, 8.88 and 10 % respectively upon illumination. The antibacterial activity of
5b, under illuminated (32.22 %) and unilluminated conditions (30 %) are comparable to that of
the standard broad spectrum antiꢀbacterial drug tetracycline (10 ꢂg per well) which exhibited a
zone of inhibition of 22.22 %. Compound 3 showed no activity on BS under both illuminated and
unilluminated conditions.
(a)
B. subtilis
E. coli
30
20
10
0
1
1a
1b
2
2a
2b
3
3a
3b
4
4a
4b
5
5a
5b
R
Compound
(b)
B. subtilis
E. coli
30
20
10
0
1
1a
1b
2
2a
2b
3
3a
3b
4
4a
4b
5
5a
5b
R
Compound
Figure 8. Comparison of the percentage of inhibition by BODIPYs, (a) under dark and (b) light conditions. All the
BODIPYs were added in equal volume and equal concentration (25 ꢂg/well) into the wells. R refers to the standard
antiꢀbacterial drug tetracycline. Error bar represents standard deviation.

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In case of the Gramꢀnegative bacteria EC, 3, 3a and 3b showed no activities both in
illuminated and unilluminated conditions. The maximum growth inhibition activity was
exhibited by compound 4a, i.e., 25.55 % under unilluminated condition which was slightly
higher than the growth inhibitory activity of tetracycline. Compounds 5, 5a and 2a showed
higher growth inhibition activities of 16.66 %, 14.44 % and 15% respectively under illuminated
condition; while, in unilluminated condition their activities were found to be 13.33 %, 0 % and
11 % respectively.
The experiments were performed under dark conditions assuming little or no activity as
there won’t be any reactive singlet oxygen production with BODIPYs in the absence of light.
But, it was observed that, most of the compounds had a significant activity towards Gram
positive / negative bacteria under dark which indicates that the singlet oxygen production is not
the sole mechanistic pathway governing the antibacterial property.9 Hence, we advanced the
experiments under ambient light.
Determination of IC50 and MIC values for the BODIPYs using LB broth assay
Inhibition of growth of BS and EC cells in LB broth assay was studied only under
ambient light condition as we could not see any significant difference in the antiꢀbacterial
activity of compound under illuminated or unilluminated conditions. In case of BS, all the
compounds except compound 3 showed good antibacterial activity in a time and concentration
dependent manner (Figure 9). Compound 5b showed the best antiꢀbacterial activity with the IC50
values of 5 µM. Compound 2 showed the highest IC50 value of 36 µM, which makes it the least
potent compound amongst all the other studied test compounds against BS. In case of EC, all
compounds except 1, 2, 3, 3a and 3b showed good antiꢀbacterial activity. Compound 5 showed
the most potency with the lowest IC50 value of 17 µM. The compound 4a showed the least
potency with a high IC50 value of 41 µM. Overall, these results are in compliance with the results
obtained from the previous experiment where all the compounds exhibited more potency against
Gramꢀpositive bacteria than the Gramꢀnegative bacteria. The IC50 values of all the BODIPYs are
tabulated in table S3. Growth inhibitory effect of the most potent compound 5b against BS and
EC is shown in figure 9.

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0 ꢀM
5 ꢀM
(a)
0 ꢀ
5 ꢀM
M
(b)
1.8
1.2
0.6
0.0
1.8
1.2
0.6
0.0
10 ꢀM
20 ꢀM
40 ꢀM
60 ꢀM
80 ꢀM
10 ꢀM
20 ꢀM
40 ꢀM
60 ꢀM
80 ꢀM
0
2
4
6
8
10
12
0
2
4
6
8
10
12
Time (h)
Time (h)
Figure 9. Effect of BODIPY 5b at different time points on the growth of (a) BS and (b) EC cultured in LB broth.
The MIC values were also calculated for the active compounds against BS and EC. The
lowest concentration of the compound at which no visible growth observed was considered as
the MIC. In case of Gramꢀpositive bacteria BS, compounds 4b and 5b showed the lowest MIC
value of 60 µM whereas the other compounds showed the higher MIC values. A comparison of
MIC values of 5b against BS and EC is shown in figure 10. The dilution methods are considered
as the quantitative assays as they determine the MIC values; the observed discrepancy between
MIC and zone of inhibition values may be attributed to the extent of diffusion of molecules
within the solid and liquid media, which in turn is dependent on the structure and size of the
molecule.
100
75
50
25
0
100
75
50
25
0
(a)
(b)
5
5a
5b
5
5a
5b
0
20
40
60
80
100
0
20
40
60
80
100
Concentration (ꢀM)
Concentration (ꢀM)
Figure 10. Effect of different concentrations of BODIPY 5, 5a and 5b on the % growth inhibition of (a) BS and (b)
EC cultured in LB broth.

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The experiments with DPBF were done with the most bioactive compounds from each
series. The results proved that the generation of singlet oxygen could be one of the possible
mechanisms for the antibacterial properties of the dyes in presence of light and this was easily
visualized in presence of DPBF. It was observed that the gradual decolourisation of DPBF at 410
nm with increasing irradiation time was much quicker with 2 and 2,6ꢀ mono/diꢀbromo
derivatives than that with bromine substituted on the phenyl group (5b). The generation of
singlet oxygen in 2ꢀ and 2,6ꢀbrominated derivatives has been well reported by Elezcano et al.6
The plot of absorbance vs irradiation time reflects the singlet oxygen yield of the compounds and
1
the slope of the line with the relative rate of O2 generation was compared (Figure 11). The
1
higher the slope, the higher the O2 yield. From the plot, the order of the singlet oxygen
generation rate was found to be 3a > 2a > 1a > 4b > 5b.
1
The O2 production for series 1 was also performed and compared (Figure S28). The
gradual decolourisation of DPBF occurs due to the quenching of singlet oxygen generated by the
brominated derivatives. Figure S28 is a representative to show that the dibromoꢀ derivative
shows quicker decolourisation of DPBF than that of monoꢀ bromo as well as unbrominated
derivative which in turn reflects the fact that the singlet oxygen generation follows the order 1b >
1a > 1 if bromination occurred the 2ꢀ /2ꢀ,6ꢀpositions of the pyrrole moiety. These results are in
agreement with the antibacterial assays, except for 5b where bromination happened at the mesoꢀ
(3,5ꢀphenyl) group of the BODIPY. Therefore, we strongly suggest that, the formation of singlet
oxygen is probably not the sole antibacterial mechanism associated with BODIPYs. Still there
are several other factors which contribute towards the cell disruption.
1.0
0.9
1a
2a
0.8
3a
4b
5b
DPBF
0.7
0
1
2
3
4
5
Irradiation time (min.)
Figure 11. Plot of change in absorbance of DPBF vs. irradiation time in the presence of selected BODIPYs
.