Synthesis, photophysical properties and solvatochromism of meso-substituted tetramethyl BODIPY dyes

Article abstract of DOI:10.1007/s10895-013-1293-8

The 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene fluorescent dyes (BODIPYs) were first synthesized almost 50 years ago; however, the exploration of their technological application has only begun in the last 20 years. These dyes possess interesting photophys

Full text of DOI:10.1007/s10895-013-1293-8

J Fluoresc
DOI 10.1007/s10895-013-1293-8
ORIGINAL PAPER
Synthesis, Photophysical Properties and Solvatochromism
of Meso-Substituted Tetramethyl BODIPY Dyes
Lucas Cunha Dias de Rezende & Miguel Menezes
Vaidergorn & Juliana Cristina Biazzotto Moraes &
Flavio da Silva Emery
Received: 24 April 2013 /Accepted: 21 August 2013
#
Springer Science+Business Media New York 2013
.
.
.
Abstract The 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
fluorescent dyes (BODIPYs) were first synthesized almost
50 years ago; however, the exploration of their technological
application has only begun in the last 20 years. These dyes
possess interesting photophysical properties, increasing inter-
est in their application as fluorescent markers and/or dyes.
Herein, we report the synthesis of tetramethyl BODIPY and
four meso-substituted dyes (2-thienyl, 4-pyridinyl, 4-
fluorophenyl and 4-nitrophenyl derivatives). Their photo-
physical characterization (absorption spectra, emission spec-
tra, fluorescence quantum yields and time-resolved fluores-
cence) and solvatochromic behavior were studied. Absorption
and emission were barely affected by substituents, with a
slightly higher stokes shift observed in the substituted dyes.
Substitutions could be associated with a shorter fluorescence
lifetime and lower quantum yields. Good correlations were
observed between the Catalán solvent descriptors and the
photophysical parameters. Also, better correlation was ob-
served between the solvent polarizability descriptor (SP)
and photophysical parameters. Overall, only slight
solvatochromism was observed. The 4-pyridinyl derivative
was the subject of a relatively significant solvatochromism
regarding the wavelengths of the emission spectra, with the
observation of a bathochromically shifted emission in metha-
nol. The fluorescence quantum yield of the 4-nitrophenyl
substituted BODIPY was approximately 30 times higher in
hexane, which may be of interest for practical applications.
Keywords BODIPY Solvatochromism Photophysical
.
.
Fluorescence Quantum yield Lifetime
Introduction
An increased interest for fluorescent small molecules has
inspired the development of a large variety of fluorescence-
based spectroscopic and imaging techniques. These fluores-
cent compounds, also known as fluorochromes or
fluorophores, are used as dyes and/or markers and are funda-
mental for several scientific experimental procedures; these
procedures can encompass biological, chemical or physical
interests. Though a diverse set of fluorophores are commer-
cially available, several research groups are engaged in the
synthesis of novel, more selective and efficient compounds.
The 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)
group of fluorescent dyes meet the criteria for a good fluores-
cent probe (high photostability and quantum yields) and have
significant chemical and application versatility.
BODIPY dyes were first synthesized in 1968 by Treibs and
Kreuzer [1] and the application and chemical modification of
these dyes has dramatically risen since the late 1980’s [2].
BODIPYs are the product of complexation between a dipyrrin
dye and a difluoroboryl unity, which compose a tricyclic flat
structure with six resonating pairs of π electrons. These dyes
are also usually associated with good photophysical parame-
ters, easy chemical manipulation, high stability and tunable
emission, making them highly interesting to the scientific
community [3].
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-013-1293-8) contains supplementary material,
which is available to authorized users.
Photophysical processes are closely associated with the
intermolecular interactions between a dye and its immediate
environment. Fluorescence intensity, absorption and emission
spectra, as well as the lifetime of the excited state are usually
influenced by the interaction of the dye with the solvent and
other solutes; this process is known as solvatochromism. This
:
:
L. Cunha Dias de Rezende M. Menezes Vaidergorn
:
J. C. Biazzotto Moraes F. da Silva Emery (*)
Faculty of Pharmaceutical Sciences at Ribeirao Preto, University of
São Paulo, Ribeirão Preto 14040-903, Brazil
e-mail: flavioemery@fcfrp.usp.br

J Fluoresc
environmental effect is the result of changes in the
fluorophore’s dipole due to the interaction between the dye
and solvent molecules. This effect is usually associated with
solvent polarity, solvent viscosity and solvent relaxation,
among other solvent parameters [4]. The influence of the
molecular environment around fluorochromes is important
due to the potential analytical applications of solvent-
dependent fluorescent dyes for obtaining information regard-
ing a microenvironment in a cell or macromolecule [5–7].
Some research groups have published articles pertaining to
the solvatochromic effects on BODIPY dyes, and in the
present study we aimed to gather new data on this subject by
comparing the behavior of four meso-substituted tetramethyl
BODIPY dyes and an unsubstituted tetramethyl BODIPY dye
in different solvents varying in polarity. For this analysis, the
steady-state absorption and fluorescence emission spectra,
fluorescence quantum yields and the time-resolved fluores-
cence profiles were collected in six solvents: hexane,
dichloromethane, acetonitrile, ethanol, methanol and dimethyl
sulfoxide.
HRMS-ESI: [M + H]+ calculated for C13H16BF2N2
249.1369; found, 249.1343.
Synthesis of 8 To a stirring solution of 3 (570 mg, 6 mmol)
and 4 (300 mg, ≈ 2.7 mmol) in CH2Cl2 at room temperature
under inert atmosphere, three drops of TFAwere added. After
1 h of stirring under these conditions, DDQ (613 mg,
2.7 mmol) was added, and the mixture was stirred for another
6 h. The mixture was washed 3 times with 0.1 M NaOH(aq)
,
dried with Na2SO4, filtered, and combined with TEA (3.2 mL,
20 mmol) and BF3.OEt2 (2.6 mL, 20 mmol) at room temper-
ature. The solution was washed with water (3 times) and dried
under Na2SO4. The solvent was removed by distillation under
reduced pressure and the oily residue was purified by flash
column chromatography (230–400 mesh, hexane/ethyl ace-
1
tate 3:1) to yield 135.7 mg (0.411 mmol) of 8 (15.2 %). H
NMR (500 MHz, CDCl3) δH: 7.50 (d, J=5.0 Hz, 1H), 7.13
(dd, J=5.0, 3.5 Hz, 1H), 6.99 (d, J=3.5 Hz, 1H), 6.00 (s, 2H),
2.55 (s, 6H), 1.58 (s, 6H). 13C NMR (125 MHz, CDCl3) δC:
156.50, 143.89, 135.09, 132.82, 128.24, 127.98, 127.79,
125.91, 121.90, 15.01, 13.91 HRMS-ESI: [M + H]+ calcu-
lated for C17H18BF2N2S 331.1246; found, 331.1254. IR (ν -
cm−1): 2923; 1544; 1303; 1243; 1172; 1078; 971; 806; 753;
474.
Experimental
Synthesis
Synthesis of 9 To a stirring solution of 3 (864 mg, 8.4 mmol)
and 5 (428 mg, ≈ 4 mmol) in CH2Cl2 at room temperature
under inert atmosphere, three drops of TFAwere added. After
3 h of stirring under these conditions, a solution of DDQ
(907 mg, 4 mmol) in CH2Cl2 was added to the reaction, and
the mixture was stirred for another 12 h. The mixture was
washed 3 times with 0.1 M NaOH(aq), dried under Na2SO4,
filtered, and combined TEA (4.8 mL, 30 mmol) and BF3.OEt2
(3.9 mL, 30 mmol) at room temperature. The solution was
washed with water (3 times) and dried under Na2SO4. The
solvent was removed by distillation under reduced pressure
and the oily residue was purified by flash column chromatog-
raphy (230– 400 mesh, hexane/ethyl acetate/TEA 75:23:2) to
yield 14480 mg (0.446 mmol) of 9(11.1 %). 1H NMR
(500 MHz, CDCl3) δH: 8.78 (d, J=5.3 Hz, 1H), 7.30 (d, J=
5.3 Hz, 1H), 6.00 (s, 1H), 2.55 (s, 3H), 1.40 (s, 3H). 13C NMR
(125 MHz, CDCl3) δC: 156.59, 150.70, 150.04, 143.78,
142.77, 137.71, 130.44, 123.45, 121.93, 113.83, 77.16,
14.77, 14.75. HRMS-ESI: [M + H]+ calculated for
C18H19BF2N3 326.1635; found, 326.1643. IR (ν - cm−1):
2918; 1654; 1508; 1466; 1410; 1306; 1156; 1120; 1076; 980;
812; 720.
Reagents were obtained from Sigma-Aldrich Brasil Ltda. (São
Paulo, SP—Brazil) and were readily used in the synthetic
procedures. Solvents were obtained from local suppliers and
treated according to established purification protocols. The
structures of the BODIPYs synthesized herein were deter-
mined by 125 MHz 13C-NMR and 500 MHz 1H-NMR using
a Bruker DRX 500-MHz NMR system from Bruker
Daltonics® (Billerica, MA, USA), infrared spectroscopy (IR)
using a Shimadzu IR-Prestige 21 system from Shimadzu
(Kyoto, Japan), and a high-resolution electrospray mass spec-
trometer (HRMS-ESI) using the ultrOTOFQ—ESI-TOF sys-
tem from Bruker Daltonics ® (Billerica, MA, USA).
Synthesis of 2 To a stirring solution of 1 (123 mg, 1 mmol) in
CH2Cl2 at 0 °C under inert atmosphere, POCl3 (92 μL,
1 mmol) was slowly added. After 3 h at room temperature,
the starting material was completely consumed. Subsequently,
DIPEA (800 μL, ≈5 mmol) and BF3.OEt2 (650 μL, ≈5 mmol)
were added to the reaction, and the mixture was stirred for 1 h.
The fluorescent solution was washed with water (3 times) and
dried with Na2SO4. The solvent was removed by distillation
under reduced pressure and the oily residue was purified by
flash column chromatography (230–400 mesh, hexane/ethyl
acetate 9:1) to yield 53.9 mg (0.217 mmol) of 2 (43.5 %). 1H
NMR (400 MHz, CDCl3) δH: 7.04 (s, 1H), 6.04 (s, 2H), 2.53
(s, 6H), 2.24 (s, 6H). 13C NMR (101 MHz, CDCl3) δC:
156.83, 141.34, 133.52, 120.21, 119.14, 77.16, 14.80, 11.41.
Synthesis of 10 To a stirring solution of 3 (570 mg, 6 mmol)
and 6 (181 mg, ≈ 3 mmol) in CH2Cl2 at room temperature
under inert atmosphere, three drops of TFAwere added. After
3 h of stirring under these conditions, a solution of DDQ
(680 mg, 3 mmol) in CH2Cl2 was added to the reaction, and

J Fluoresc
the mixture was stirred for another 6 h. The mixture was
washed 3 times with NaOH(aq) 0.1 M, dried under Na2SO4,
filtered, and reacted at room temperature with TEA (3.2 mL,
20 mmol) and BF3.OEt2 (2.6 mL, 20 mmol). The solution was
washed with water (3 times) and dried under Na2SO4. The
solvent was distilled off under reduced pressure and the oily
residue was purified by flash column chromatography (230–
400 mesh, hexane/ethyl acetate 3:1) to yield 183.0 mg
(0.535 mmol) of 10 (17.8 %).: 1H NMR (500 MHz, CDCl3)
δH: 7.27 (t, J=7.6 Hz, 1H), 7.20 (t, J=8.4 Hz, 1H), 5.99 (s,
1H), 2.55 (s, 3H), 1.40 (s, 3H). 13C NMR (125 MHz,
CDCl3) δC: 155.94, 143.11, 143.08, 140.60, 131.70,
131.69, 131.66, 131.09, 131.06, 130.15, 130.08, 121.55,
121.53, 116.61, 116.43, 77.16, 14.72, 14.65. HRMS-ESI:
[M + H]+ calculated for C19H19BF3N2 343.1588; found,
343.1599. IR (ν - cm−1): 2909; 1654; 1508; 1466; 1410;
1306; 1156; 1120; 1076; 980; 812; 720.
The EasyLife™ V (Optical Building Blocks) fluorescence
lifetime system was used to obtain the time-resolved fluores-
cence spectroscopy. A dilute solution of colloidal silica was
used to obtain the instrument response function. Fluorescence
lifetimes were calculated using the EasyLife™ V software
package by fitting an exponential decay curve to the obtained
data. Chi-square statistics (χ2), Durbin-Watson statistics
(DW) and Z statistics (Z) were calculated, and every fitted
curve showed results within reasonable statistical limits: 0.9
<χ2<1.2; DW>1.7; Z>−1.96.
Quantum yields were obtained by a comparative method
[8] using fluorescein in 0.1 M NaOH(aq) as the standard (φ=
0.91, λexc=470 nm) [9]. The emission spectra from five sam-
ples of each fluorophore (absorbance between 0.1 and 0.01 at
the excitation wavelength −470 nm) were obtained. The re-
sults were plotted with the integrated fluorescence intensity
vs. absorbance to obtain the slope of the curve. A curve was
obtained for each tested compound and the standard. The
quantum yield of the tested compound (ϕx) was calculated
using formula 1, where ϕst is the quantum yield of the stan-
dard, mx and mst are the slopes for the test compound and
standard compound, and nx and nst are the refractive indexes
of the solvents.
Synthesis of 11 To a stirring solution of 3 (200 mg, 2.1 mmol)
and 7 (151 mg, 1 mmol) in CH2Cl2 at room temperature under
inert atmosphere, three drops of TFAwere added. After 1 h of
stirring under these conditions, a solution of DDQ (227 mg,
1 mmol) in CH2Cl2 was added to the reaction, the mixture was
stirred for another 2 h. The mixture was washed 3 times with
0.1 M NaOH(aq), dried under Na2SO4, filtered, and reacted
with TEA (1.6 mL, 10 mmol) and BF3.OEt2 (1.3 mL,
10 mmol) at room temperature. The solution was washed with
water (3 times) and dried under Na2SO4. The solvent was
distilled off under reduced pressure and the oily residue was
purified by flash column chromatography (230–400 mesh,
hexane/ethyl acetate 3:1) to yield 159.3 mg (0.431 mmol) of
10 (43.1 %). 1H NMR (500 MHz, CDCl3) δH: 8.39 (d, J=
8.7 Hz, 1H), 7.54 (d, J=8.7 Hz, 1H), 6.02 (s, 1H), 2.56 (s, 3H),
1.36 (s, 3H). 13C NMR (125 MHz, CDCl3) δC: 156.82,
148.45, 142.67, 142.11, 138.45, 130.75, 129.78, 124.52,
122.01, 77.16, 14.85, 14.82. HRMS-ESI: [M + H]+ calculat-
ed for C19H19BF2N3O2 370.1533; found, 370.1517. IR (ν -
cm−1): 2924; 1734; 1543; 1512; 1462; 1261; 1076; 1028;
804.
ꢀ
ꢁꢀ
ꢁ
2
mx
nx
ϕx ¼ ϕst
mst
nst
The influence of solvent parameters and descriptors over
BODIPYs’ characteristics was analyzed via a simple linear
regression and multilinear regression analyses. Statistical cal-
culations were made using Bioestat 5.0 free software [10], and
spectra were constructed and manipulated by plotting raw data
with Spekwin32 free software [11].
Results and Discussion
Synthesis
The synthetic routes used to obtain the five BODIPY deriva-
tives for this work are shown in Fig. 1. Non-meso-substituted
tetramethyl-BODIPY (2) was obtained in a 43.5 % yield from
the self-condensation of 3,5-dimethyl-2-carbaldehyde (1) in
the presence of POCl3, followed by complexation with
BF3.OEt2 using diisopropylethylamine (DIPEA) [12]. The
meso-substituted BODIPY dyes 8, 9, 10 and 11 were
obtained as red solids with variable green fluorescence when
in solution (Fig. 2), from the reaction of 2,4-dimethyl pyrrole
(3 ) with thiophene-2-carbaldehyde (4 ), pyridine-4-
carbaldehyde (5 ), 4-fluorobenzaldehyde (6 ) or 4-
nitrobenzaldehyde (7), respectively. For these compounds,
we applied the trifluoroacetic acid method, as described by
Photophysical Parameters
Absorption spectra were obtained on an Agilent 8453 UV-
visible spectrophotometer at room temperature in the solvents
described above. Steady state fluorescence spectra were
obtained on a Shimadzu RF5301PC spectrofluorimeter with
a xenon arc lamp as the light source while using an excitation
wavelength (λexc) of 470 nm. Molar extinction coefficients (ε)
were obtained in ethanol. The absorbance of five solutions of
known concentration of 2, 8, 9, 10 or 11 were obtained and
plotted against the concentration, and ε was calculated from
the slope of the regression analyses of the plotted data.

J Fluoresc
Fig. 1 Synthetic procedure used to obtain compounds 2, 8, 9, 10, 11
Lindsey, for meso-substituted dipyrromethanes [13] followed
by oxidation with DDQ and complexation with BF3.OEt2 in
triethylamine (TEA).
likely the result of some specific effects of this solvent on
11 , it was excluded from the averaging and was shown as a
separate curve. While a relatively narrow emission peak
near 510 nm was observed for compound 2, peak broaden-
ing was observed for 8, 9, 10, and 11 . Compounds 8, 9 and
11 correlated with a weak bathochromic shift in the emis-
sion, which can be observed by the slight different green
fluorescence of compound 9 in Fig. 2.
Photophysical Properties
Photophysical properties of the synthesized BODIPYs
were tested in six solvents, which were varied in polarity
and were either protic or aprotic: ethanol (EtOH), hexane
(HEXN), dichloromethane (DCM), acetonitrile (MeCN),
dimethyl sulfoxide (DMSO) and methanol (MeOH). In
Fig. 3a, normalized absorption spectra representing the
average of the six spectra for each of the five compounds
are shown (for complete absorption and emission data for
each compound, please see the supplementary material).
The shapes of the absorption spectra are similar to those
previously collected from other BODIPY derivatives in the
literature [14 –16]. Commonly, a strong relatively narrow
peak near 500 nm (attributed to the S0-S1 transition), a
shoulder near 475 nm (resulting from the 0 –1 vibrational
transition), and a weak broad band near 350 nm (attributed
to the S0-S2 transition) are observed [16 –18]. The averaged
normalized emission spectra of 2, 8, 9, 10, and 11 are
shown in Fig. 3b. Due to the unusual shape of the emission
spectrum collected from 11 in dichloromethane, which is
Table 1 shows detailed results regarding absorption and
emission of the synthesized compounds. The maximum wave-
length for the main absorption band (λabs) of the unsubstituted
BODIPY 2 goes from 500 nm in MeCN to 506 nm in hexane.
Compound 8 showed a bathochromic shift (6 – 9 nm) in the
absorption maxima while compound 10 showed a weak
hypsochromic shift (2 –5 nm) with respect to 2. No significant
difference was observed among the λabs of meso-substituted
dyes 9 and 11 . The effects of the aromatic substituents over the
emission spectra were more evident. No significant shift of the
wavelength at emission maxima (λem) was observed for com-
pound 10; however, a red-shifted emission was observed in
compounds 8 (15 – 20 nm), 9 (6 – 25 nm) and 11 (9– 16 nm).
The Stokes shift (Δv ), which was calculated from the
wavenumber of maximum absorption and emission intensities,
was invariably increased with the addition of the aromatic
substituents to the BODIPY core. Compounds 8, 9 and 11
showed a more remarkable increase in Δv than compound 10.
The subtle effects of meso-substitution over photophysical
parameters of BODIPYs can be attributed to the lack of resonance
interaction between the aromatic system of BODIPY core and the
aromatic substituents. The presence of two methyl substituents
near the central position is associated with a nearly perpendicular
configuration of meso-substituted tetramethyl BODIPYs; this
arrangement precludes the resonance interaction [19].
Bathochromic effects observed in the emission or absorption of
some meso-substituted dyes can be explained by the increase in
LUMO stabilization due to the aromatic substituents [16, 19].
Electron withdrawing substituents at central position of the
Fig. 2 From left to right: ethanolic Solutions of 2, 8, 9, 10, and 11 under
black light

J Fluoresc
Fig. 3 a Averaged and normalized absorption spectra of 2 (black), 8 (red), 9 (green), 10 (blue), and 11 (cyan). b Averaged and normalized emission
spectra of 2 (black), 8 (red), 9 (green), 10 (blue), 11 (cyan) and the spectra of 11 in dichloromethane (purple)
BODIPY core have already been associated with bathochromism
[16], and the four substituents varied in this work seem to having
the same effect.
The broadening of FWHMem upon solvent polarity increase was
usually less evident but was also observed, except in the case of
11 . In this excepted case, the opposite effect was observed. The
slightly positive slopes in the linear regressions of 2, 8, 9 and 10
and the negative slope for 11 corroborate this observation. The
inverse trend of 11 results from the unusual shape of its emission
peak in dichloromethane, which increases the FWHMem in this
low polarity solvent and influenced the regression analysis. In
the linear regression analysis of absorption and emission peak
broadening of compounds 2, 8, 10, and 11 , higher correlation
coefficients and slopes were obtained for the dipole moment
parameter, while for compound 9 this occurred for the Dimroth-
Reichardt solvent polarity parameter. Polar solvents are com-
monly related to broader peaks due to the enhanced interaction
of the solvent molecules with the transition dipole moment of the
fluorophores.
The absorption spectrum maxima (λabs) were barely affected
by solvent polarity; only slight solvatochromic effects were
observed. This weak trend can be rationalized by the absence
of significant differences between the dipole moment of the S0
and S1 states of the synthesized molecules [21]. It was observed
that there was a negative relationship between solvent polarity
and λabs. Moreover, methanol and acetonitrile were observed to
be analogous, with shorter absorption wavelengths, while
dichloromethane and hexane were similarly related, with longer
absorption wavelengths. Additionally, λem was not highly
influenced by the choice of solvent and the relationship of λem
with solvent polarity was not as clear. The different shape of the
absorption spectra of 11 in DCM and, additionally, the relatively
significant increase of λem and Δv observed for compound 9 in
methanol are worth noting in terms of solvatochromism.
To better understand the solvatochromism of these systems,
the relationship between solvent parameters, emission maxima
(vem ), stokes shift (Δv ) and wavenumber (cm−1) of absorption
maxima (vabs ) were analyzed via a multilinear regression. This
type of analysis allows the simultaneous study of the effect of
several solvent parameters through the following equation:
In Table 2 the results of time-resolved fluorescence spec-
troscopy and the quantum yields are shown. The fluorescence
lifetime (τ) of 2 was in the range of 5 to 6 ns, which was in
agreement with previous publications [20]. In the literature the
fluorescence quantum yield of 2 ranges from 0.85 to ~1, with
our results also being near unity. This discrepancy among the
results found in the literature is due to inherent error within the
comparative method of fluorescence quantum yield calcula-
tion. The substituted BODIPYs invariably showed shorter
lifetimes and lower quantum yields than 2. The rate of non-
radiative decay (Knr) was also significantly increased among
the substituted compounds, especially for compounds 8 and
11 . The rate of radiative decay (Kf) was relatively less
influenced, except in the case of 11 , where a significant
lowering was observed. The results suggest that the insertion
of aromatic moieties at the central position of 2 result in
higher energy dissipation via non-radiative pathways.
Solvatochromic Effects
Tables 1 and 2 show the results of photophysical profile of the
BODIPY derivatives in six solvents, which enabled the study of
solvatochromism among the synthesized compounds. Table 3
shows the values of the dipole moment, the dielectric constant
(εr) and the Dimroth-Reichardt solvent ionizing power index
(E N), which were collected to rationalize the obtained results.
T
Solvent descriptors proposed by Kamlet-Taft and by Catalán,
which were used for multilinear regression analysis, are also
shown in Table 3 and are going to be discussed ahead.
The effect of solvent polarity on the broadening of the ab-
sorption peak is easily observed; peak narrowing occurred for
every compound when measured in hexane. The linear regres-
sion of the plot of FWHMabs against the polarity parameters
(Fig. 4) highlights this relationship, which was especially intense
for 2. In the case of 2, the slope was approximately 3 times
higher than the observed for the other compounds (a list of linear
regression parameters can be found at supplementary material).
y ¼ y0 þ Caa þ Cbb þ Ccc…

J Fluoresc
Table 1 Parameter from absorption and emission spectra of 2, 8, 9, 10,
and 11 in six solvents
Table 2 Fluorescence lifetime, quantum yield, coefficient of radiative
and non-radiative decay of 2, 8, 9, 10, and 11 in six solvents
Subst. effecta
τ (ns)
φ
Kf
Knr
Δv
(cm−1
Compound λabs logε λem
(nm) (nm)
FWHM
(cm−1
)
)
2
Δv
abs em λabs λem
EtOH
5.6
5.8
5.3
5.9
5.5
5.7
~1
0.178
0.164
0.179
0.163
0.180
0.172
~ 0
HEXN
DCM
0.95
0.95
0.96
0.99
0.98
0.009
0.009
0.007
0.002
0.003
2
EtOH
HEXN 506
DCM 506
503 5.96 510 272
906 603
694 516
824 506
933 651
939 723
904 627
MeCN
DMSO
MeOH
512 232
513 270
508 315
514 425
509 274
MeCN 500
DMSO 503
MeOH 502
8
EtOH
0.4
0.5
0.5
0.4
0.6
0.5
0.06
0.06
0.07
0.05
0.11
0.06
0.150
0.120
0.140
0.125
0.183
0.120
2.35
1.88
1.86
2.37
1.48
1.88
HEXN
DCM
8
EtOH
HEXN 513
DCM 512
510 5.69 530 740
764 798 +7 +20 + 468
712 773 +7 +15 + 286
762 775 +6 +17 + 393
798 826 +8 +18 +359
789 808 +9 +16 + 238
762 805 +7 +17 + 361
MeCN
DMSO
MeOH
527 518
530 663
526 674
530 663
526 635
MeCN 508
DMSO 512
MeOH 509
9
EtOH
1.4
1.1
1.6
1.5
2.5
1.1
0.23
0.18
0.28
0.26
0.45
0.15
0.164
0.163
0.175
0.173
0.180
0.136
0.550
0.746
0.450
0.493
0.220
0.773
HEXN
DCM
9
EtOH
HEXN 503
DCM 504
503 5.77 525 833
899 985
0
+15 + 561
MeCN
DMSO
MeOH
523 760
523 721
519 772
520 610
796 908 −3 +11 + 528
848 938 −2 +10 + 451
854 921 −1 +11 + 457
857 986 +1 +6 + 185
MeCN 499
DMSO 504
MeOH 501
10
EtOH
3.6
2.8
3.7
3.5
4.6
3.0
0.62
0.55
0.71
0.69
0.82
0.60
0.17
0.20
0.19
0.20
0.18
0.20
0.11
0.16
0.08
0.09
0.04
0.13
534 1233 859 1127 −1 +25 + 959
HEXN
DCM
10
EtOH
500 5.83 511 431 821 840 −3 +1 + 159
MeCN
DMSO
MeOH
HEXN 501
513 467
514 465
510 472
515 503
510 432
770 843 −5 +1 + 235
822 835 −4 +1 + 195
856 856 −2 +2 + 157
861 873 −2 +1 + 78
821 846 −3 +1 + 158
DCM
502
MeCN 498
DMSO 502
MeOH 499
11
EtOH
0.6
0.9
1.6
0.4
1.0
0.6
0.007
0.123
0.004
0.002
0.004
0.005
0.012
0.136
0.002
0.006
0.004
0.008
1.65
0.97
0.62
2.49
0.99
1.66
HEXN
DCM
11
EtOH
HEXN 504
DCM 505
502 5.66 524 836
875 1278 −1 +14 + 564
838 1312 −2 +9 + 415
882 2006 −1 +15 + 593
890 1368 +1 +14 + 488
895 1296 +1 +12 + 405
883 1291 −1 +16 + 638
MeCN
DMSO
MeOH
521 647
528 863
522 803
526 830
525 912
MeCN 501
DMSO 504
MeOH 501
Catalán solvent parameters [25], which are solvent acidity
(SA), basicity (SB), dipolarity (SdP), and polarizability (SP).
Each approach was realized by applying the following equa-
tions.
where a, b and c are solvent descriptors; Ca, Cb and Cc are the
coefficient for each descriptor; y is the predicted value of the
photophysical property under study; and y0 is the interception,
which would be the value for y in gas phase, where Ca, Cb and
Cc are zero.
y ¼ y0 þ Cαα þ Cββ þ Cπ* π* ðKamlet ꢀ TaftÞ
y ¼ y0 þ CSASA þ CSBSB þ CSdPSdP þ CSPSP ðCatal"nÞ
Two solvent sets of descriptors were chosen to perform the
multilinear regression: the Kamlet-Taft solvent parameters
[22–24] (Table 3), which are solvent acidity (α), solvent
basicity (β), and solvent polarity/polarizability (π *), and the
The analysis using the Catalán solvent parameters resulted
in a better correlation coefficient (R) thus reflecting the most
reliable results obtained by this method (Table 4). It is

J Fluoresc
Table 3 Values of solvent polar-
ity parameters used for
solvatochromism analyses
Kamlet-Taft
Catalán
SP
α
β
π*
0.54
SdP
SA
SB
E Na
DMb
εr
c
T
EtOH
0.86
0.00
0.13
0.19
0.00
0.98
0.75
0.00
0.10
0.40
0.76
0.66
0.633
0.616
0.761
0.645
0.83
0.783
0
0.4
0.658
0.056
0.178
0.286
0.647
0.545
51.9
31.0
40.7
45.6
45.0
55.5
1.66
0.08
1.14
3.44
4.10
2.87
24.6
1.9
HEXN
DCM
− 0.04
0.82
0.75
1.00
0.60
0
a Dimroth-Reichardt ionizing
power parameter
b Dipole moment (debye)
c Relative permittivity (dielectric
constant)
0.769
0.974
1
0.04
0.044
0.072
0.605
8.9
MeCN
DMSO
MeOH
37.5
46.7
32.7
0.608
0.904
interesting to note that the Catalán parameters were reviewed
in 2009 with the substitution of the old polarity/polarizability
parameter (SPP) for two new parameters (SP and SdP).
Multilinear regression analyses using the old parameters were
also performed in this work, and the correlation coefficients,
which are shown in Table 4, were not as reliable as those
obtained with the new parameters. To our knowledge, only a
few studies addressed the novel Catalán descriptors to analyze
solvatochromism in BODIPY derivatives, and excellent fits
were unambiguously obtained when using this set of descrip-
tors [26–29]. Due to these better fits, the results will be
discussed based on the coefficients CSA, CSB, CSdP, and CSP
from the analysis using the Catalán solvent descriptors, while
results obtained using other descriptors can be found in the
supplemental material.
In the analysis of vabs and vem , high negative values of Csp
were observed, which was similar to a recent study applying
the same descriptors to analyze solvatochromism in BODIPY
dyes [26]. This result indicates that the polarizability of the
environment surrounding the fluorophore is the main factor
influencing its absorption and emission maxima. Because
both the absorption and emission maxima are highly
influenced by solvent polarizability, the effects over Δv are
not as prominent. Another interesting set of observations are
the high negative values of CSA and the high positive values
of CSB obtained in the analysis of vem and Δv of compound
9. This can be rationalized by the presence of a pyridinyl
nitrogen within the π-conjugated system, whose protonation/
deprotonation process directly influences electron distribution
throughout the molecule.
The high quantum yield values collected for compound 2
were not greatly influenced by solvents nor was a significant
relationship between polarity and quantum yield observed
(Fig. 5). Compound 11 yielded very low quantum yield values,
a situation usually associated with high analytical error, and we
found it prudent not to include this compound in the
solvatochromism study of this property and related coefficients
(Kf and Knr). The most striking observation was the quantum
yield of 11 in hexane, which was at least 30 times higher than
the quantum yield in the other solvents, and the fluorescence
could be easily observed under black light (Fig. 6). In the linear
regression analysis, the solvent dipole moment was related to
slightly higher slopes and regular correlation coefficients, es-
pecially for 9 and 10; these data indicate that the solvent dipole
moment is most likely related to fluorescence quantum yields.
The relatively good correlation coefficients observed using the
Fig. 4 Linear regression of emission and absorption peak lengths against solvent parameters. 2 (black), 8 (red), 9 (green), 10 (blue), and 11 (cyan)

J Fluoresc
Table 4 Correlation coefficients
and regression coefficients in the
analysis of vabs , vem and Δv
Catalán Parameters
R1a
R2b
CSP
CSdP
CSA
CSB
R
2
vabs
vem
0.59
0.59
0.92
0.83
0.54
0.92
−1097.24
−1257.57
160.33
246.58
202.28
44.30
−304.79
−129.75
−175.04
197.17
−2.00
0.95
0.99
0.93
199.17
Δv
8
vabs
vem
0.67
0.59
0.82
0.75
0.19
0.89
− 867.82
− 659.27
− 208.55
227.38
95.59
−104.52
−19.37
− 85.15
3.70
0.99
0.79
0.84
−128.42
132.12
131.80
Δv
9
vabs
vem
0.43
0.96
0.97
0.41
0.79
0.65
−1065.53
− 926.92
−138.61
218.40
111.35
107.06
−193.53
−1214.67
1021.14
− 42.39
490.75
−533.14
0.94
0.99
0.97
Δv
10
vabs
vem
0.5
0.49
0.62
0.72
− 944.84
−1036.32
91.48
154.12
152.31
1.81
−138.46
− 48.30
− 90.16
23.19
−5.49
28.67
0.98
0.99
0.92
0.68
0.89
a Correlation coefficients using
old Catalán solvent descriptors
(SPP, SA and SB)
b Correlation coefficients using
Kamlet-Taft solvent descriptors
c Correlation coefficients using re-
cent Catalán solvent descriptors
(SdP, SP, SA and SB)
Δv
11
vabs
vem
0.64
0.68
0.98
0.41
0.61
0.88
− 874.19
−1374.27
500.07
131.33
− 45.01
176.34
−101.13
− 458.82
357.69
96.89
0.98
0.96
0.99
301.52
−204.63
Δv
dielectric constant also corroborate the existence of
solvatochromic effects; however, the low slopes indicate that
only subtle variations of quantum yield with polarity can be
expected. As for the Dimroth-Reichardt solvent polarity pa-
rameter, good correlation was obtained only for compound 2,
while values near zero were observed for this coefficient with
the other compounds. Interestingly, the highest fluorescence
quantum yields were observed for 8, 9 and 10 in DMSO, and
this effect was specially observed in 9, where the quantum
yield in DMSO was 3 times higher than methanol. Higher
quantum yields in polar solvents are related to the better inter-
action of the polar solvent with the π system of the dyes, which
makes the molecule less flexible and better able to avoid non-
radiative decay [28].
Linear regressions of fluorescence lifetime are also shown
in Fig. 5. Fluorescence lifetimes of 2 and 8 were hardly
Fig. 5 Linear regression of fluorescence quantum yield and lifetime against solvent parameters. 2 (black), 8 (red), 9 (green), and 10 (blue)

J Fluoresc
Similar results were recently obtained with other BODIPYs
[28]. It is interesting to note that for compound 9, values of
CSA, CSB were near CSP, suggesting an important influence of
solvent acidity and basicity on photophysical parameters of
this compound. The reasoning behind this observation was
discussed earlier in the text and involves the proton-accepting
pyridinic nitrogen. Regarding Kf, none of the coefficients
obtained in each solvent were significantly higher than the
others, ruling out any significant influence of a particular
solvent parameter over Kf. This result corroborates the very
small variation of Kf observed at Table 2.
Fig. 6 Six solutions of 11 observed under black light, showing the
intense solvatochromism. From left to right: EtOH, HEXN, DCM,
MeCN, DMSO, and MeOH
influenced by solvent parameters, as can be observed graph-
ically by noting the low correlation coefficients and slopes.
The relatively higher correlation coefficients obtained for 9
and 10 demonstrated a stronger relation between this param-
eter and solvent properties, mainly when the dielectric con-
stant and dipole moment were used in the calculations. It is
worth noting that the effect of DCM in the emission of 11
(which was observed as a change in the shape of emission
spectra) was also observed in the time-resolved fluorescence
analysis; the fluorescence lifetime was approximately two
times longer in this solvent. Similarly to what was observed
for fluorescence quantum yields, the use of DMSO was also
related to higher fluorescence lifetimes in 8, 9 and 10.
We reasoned that the effect of DMSO in the enhancement
of the fluorescence quantum yield and fluorescence lifetime
might result from the effect of solvent viscosity. Relatively
good correlation coefficients and high slopes were obtained
from the linear regression analysis of solvent viscosity [30]
versus quantum yield and fluorescence lifetimes of 8, 9, and
10 (please see the supplementary material for details). This
result suggests an important effect of viscosity over the
photophysical parameters. Viscosity slows molecular rotation
and/or twisting, possibly serving as a barrier to processes of
non-radiative decay and thus influencing both quantum yield
and lifetime [31]. This hypothesis is supported by the smaller
values of Knr obtained in DMSO. In compound 2, no depen-
dence on solvent viscosity was observed, which can be
explained by its lower molecular volume and the absence of
non-radiative processes so that it is less subject to viscosity
effects.
Conclusions
Overall, we observed only slight effects of meso-substitutions
on the absorption and emission spectra of 2; however, the
addition of aromatic substituents seems to invariably lead to a
new path of non-radiative decay, which then influences the
fluorescence quantum yield and fluorescence lifetime. Re-
garding solvatochromism, the emission and fluorescence
spectra were not highly influenced by solvent, except for the
unusual shape of the absorption spectra observed in
dichloromethane for compound 11 and higher λem observed
in methanol for compound 9. Additionally, compound 11
generally exhibited higher quantum yields in hexane and
may be applied as a polarity fluorescent marker. With regard
to the compounds synthesized in this work, Catalán solvent
parameters served as excellent descriptors in the multilinear
regression analysis of solvatochromism, and solvent polariz-
ability seemed to be the most important parameter influencing
the photophysical characteristics of the BODIPYs.
Acknowledgments This work was financed by São Paulo Research
Foundation (FAPESP– grant #2011/23342-9), NAP-FTO—USP, INCT-
IF. We are grateful to prof. Roberto Santana da Silva and the analytical
centre of the institution.
References
1. Treibs A, Kreuzer FH (1968) Difluoboryl-komplexe von di- and
tripyrrylmethen. Liebigs Ann Chem 718:208–223
The multilinear regression approach was also applied for
the analysis of Kf and Knr. The recent set of descriptors
proposed by Catalán (SA, SB, SP, and SdP) resulted in supe-
rior fits for almost every analysis when compared to the results
found when using Kamlet-Taft descriptors or the old Catalán
descriptors (SA, SB, SPP). In the analysis of Knr, CSP was the
highest (negative) coefficient obtained for the substituted
BODIPYs. The negative CSP obtained for substituted dyes
indicates that solvent polarizability leads to a decrease of the
non-radiative deactivation, supporting the concept of polar
solvent effects in non-radiative decay discussed earlier.
2. Ulrich G, Ziessel R, Harriman A (2008) The chemistry of fluorescent
bodipy dyes: versatility unsurpassed. Angew Chem Int Ed 7(7):
1184–1201
3. Loudet A, Burgess K (2007) BODIPY dyes and their derivatives:
syntheses and spectroscopic properties. Chem Rev 107(11):4891–4932
4. Zakerhamidi MS, Moghadam M, Ghanadzadeh A, Hosseini S (2012)
Anisotropic and isotropic solvent effects on the dipole moment and
photophysical properties of rhodamine dyes. J Lumin 132(4):931–937
5. Toutchkine A, Kraynov V, Hahn K (2003) Solvent-sensitive dyes to
report protein conformational changes in living cells. J Am Chem
Soc 125(14):4132–4145
6. Bergstrom F, Hagglof P, Karolin J, Ny T, Johansson LBA (1999) The
use of site-directed fluorophore labeling and donor-donor energy

J Fluoresc
migration to investigate solution structure and dynamics in proteins.
Proc Natl Acad Sci U S A 96(22):12477–12481
7. Karolin J, Johansson LBA, Strandberg L, Ny T (1994) Fluorescence
and absorption spectroscopic properties of dipyrrometheneboron
difluoride (bodipy) derivatives in liquids, lipid-membranes, and pro-
teins. J Am Chem Soc 116(17):7801–7806
8. Williams ATR, Winfield SA, Miller JN (1983) Relative fluorescence
quantum yields using a computer-controlled luminescence spectrom-
eter. Analyst 108(1290):1067–1071
9. Resch-Genger U, Derose PC (2010) Fluorescence standards: classi-
fication, terminology, and recommendations on their selection, use,
and production (IUPAC Technical Report). Pure Appl Chem 82(12):
2315–2335
10. Ayres M, Ayres-Júnior M, Ayres DL, Santos AS (2007) BioEstat 5.0-
Aplicações Estatísticas nas Áreas das Ciências Biológicas e Médicas.
MCT; IDSM; CNPq, Belém
11. Spekwin32—optical spectroscopy software. Version 1.71.6.1, 2012,
http://www.effemm2.de/spekwin/. Accessed 23 March 2013
12. Wu L, Burgess K (2008) A new synthesis of symmetric boraindacene
(BODIPY) dyes. Chem Commun 2008(40):4933–4935
13. Littler BJ, Miller MA, Hung CH, Wagner RW, O’Shea DF, Boyle PD
et al (1999) Refined synthesis of 5-substituted dipyrromethanes. J
Org Chem 64(4):1391–1396
analogue of the laser dye PM567 in different solvents: internal
conversion mechanisms. Chem Phys Lett 385(1–2):29–35
20. Johnson ID, Kang HC, Haugland RP (1991) Fluorescent membrane
probes incorporating dipyrrometheneboron difluoride fluorophores.
Anal Biochem 198:228–237
21. Arbeloa FL, Prieto JB, Martinez VM, Lopez TA, Arbeloa IL (2004)
Intramolecular charge transfer in pyrromethene laser dyes:
photophysical behaviour of PM650. Chemphyschem 5(11):1762–1771
22. Kamlet MJ, Taft RW (1976) The solvatochromic comparison meth-
od. I. The β-scale of solvent hydrogen-bond acceptor (HBA) basic-
ities. J Am Chem Soc 98(2):377–383
23. Kamlet MJ, Abboud JL, Taft RW (1977) The solvatochromic com-
parison method. 6. The π* scale of solvent polarities. J Am Chem Soc
99(18):6027–6038
24. Taft RW, Kamlet MJ (1976) The Solvatochromic Comparison Meth-
od. 2. The α-scale of Solvent Hydrogen-Bond Donor (HBD) Acid-
ities. J Am Chem Soc 98(10):2886–2894
25. Catalan J (2009) Toward a generalized treatment of the solvent effect
based on four empirical scales: dipolarity (SdP, a new scale), polar-
izability (SP), acidity (SA), and basicity (SB) of the medium. J Phys
Chem B 113(17):5951–5960
26. Leen V, Qin W, Yang W, Cui J, Xu C, Tang X et al (2010) Synthesis,
spectroscopy, crystal structure determination, and quantum chemical
calculations of BODIPY dyes with increasing conformational restric-
tion and concomitant Red-shifted visible absorption and fluorescence
spectra. Chem-Asian J 5(9):2016–2026
14. Bura T, Retailleau P, Ulrich G, Ziessel R (2011) Highly substituted
bodipy dyes with spectroscopic features sensitive to the environment.
J Org Chem 76(4):1109–1117
15. Leen V, Miscoria D, Yin S, Filarowski A, Ngongo JM, Van der
Auweraer M et al (2011) 1,7-Disubstituted boron dipyrromethene
(BODIPY) dyes: synthesis and spectroscopic properties. J Org Chem
76(20):8168–8176
16. Qin WW, Baruah M, Van der Auweraer M, De Schryver FC, Boens N
(2005) Photophysical properties of borondipyrromethene analogues
in solution. J Phys Chem A 109(33):7371–7384
17. Baruah M, Qin WW, Flors C, Hofkens J, Vallee RAL, Beljonne D
et al (2006) Solvent and pH dependent fluorescent properties of a
dimethylaminostyryl borondipyrromethene dye in solution. J Phys
Chem A 110(18):5998–6009
27. Galangau O, Dumas-Verdes C, Meallet-Renault R, Clavier G (2010)
Rational design of visible and NIR distyryl-BODIPY dyes from a
novel fluorinated platform. Org Biomol Chem 8(20):4546–4553
28. Banuelos-Prieto J, Agarrabeitia AR, Garcia-Moreno I, Lopez-Arbeloa
I, Costela A, Infantes L et al (2010) Controlling optical properties and
function of BODIPY by using asymmetric substitution effects. Chem
Eur J 16(47):14094–14105. doi:10.1002/chem.201002095
29. Boens N, Leen V, Dehaen W, Wang L, Robeyns K, Qin W et al
(2012) Visible absorption and fluorescence spectroscopy of
conformationally constrained, annulated BODIPY dyes. J Phys
Chem A 116(39):9621–9631
18. Pardoen JA, Lugtenburg J, Canters GW (1985) Optical properties of
pyrromethene derivatives. Possible excited-state deactivation through
proton tunneling. J Phys Chem 89(20):4272–4277
19. Prieto JB, Arbeloa FL, Martinez VM, Lopez TA, Amat-Guerri F,
Liras M et al (2004) Photophysical properties of a new 8-phenyl
30. Lide DR (2004) CRC Handbook of Chemistry and Physics. CRC
Press, Boca Raton
31. Kuimova MK, Yahioglu G, Levitt JA, Suhling K (2008) Molecular
rotor measures viscosity of live cells via fluorescence lifetime imag-
ing. J Am Chem Soc 130(21):6672–6673. doi:10.1021/ja800570d