Development of alkyne-BODIPYs as viscosity sensitive fluorescent probes for enumeration of bacterial cells

Article abstract of DOI:10.1142/S1088424620500194

We report a series of alkyne-functionalized meso-aryl boron dipyrrin (BODIPY) molecular rotors sensitive to viscosity. The planar and twisted conformation within the molecular structure decides the viscosity-dependent behavior. The variations in fluorescence lifetime and intensity were appreciable to the local viscosity. Hence, the dye has been successfully employed in the enumeration of microbes by considering the proportionate fluorescence intensity of the BODIPYs as an index of the number of cells per mL. With increasing cells per mL, the viscosity of the bacterial solution is increased. Consequently, the fluorescence intensity of the sample containing BODIPY tends to increase due to the restricted rotation in the viscous medium. The BODIPY probe offers high sensitivity and is easier than other conventional techniques of colony-forming unit (CFU) determination. The theoretical studies indicate that intramolecular charge transfer is responsible for the enhanced fluorescence intensity in a highly viscous solvent.

Full text of DOI:10.1142/S1088424620500194

This is a JAM - Just Accepted Manuscript
Journal of Porphyrins and Phthalocyanines
Article Title:
Development of alkyne-BODIPYs as viscosity sensitive fluorescent probes
for enumeration of bacterial cells
Author(s):
DOI:
Dijo Prasannan, Suchithra Tharamel Vasu, Chellaiah Arunkumar, Pattiyil
Parameswaran
10.1142/S1088424620500194
To be cited as:
Dijo Prasannan, Suchithra Tharamel Vasu, Chellaiah Arunkumar, Pattiyil Parameswaran,
Development of alkyne-BODIPYs as viscosity sensitive fluorescent probes for enumeration of
bacterial cells, Journal of Porphyrins and Phthalocyanines, doi: 10.1142/S1088424620500194
Received: 17 January 2020
Accepted: 16 April 2020
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Accepted manuscript to appear in JPP
Development of alkyne-BODIPYs as viscosity sensitive fluorescent probes for enumeration
of bacterial cells
a
b
a
c
Dijo Prasannan, Suchithra Tharamel Vasu, Chellaiah Arunkumar and Pattiyil Parameswaran *
a
Bioinorganic Materials Research Laboratory, Department of Chemistry, National Institute of
Technology Calicut, NIT Campus P.O., Calicut, India-673 601.
b
School of Biotechnology, National Institute of Technology Calicut, NIT Campus P.O., Calicut, India-673 601.
c
Theoretical and Computational Chemistry Laboratory, Department of Chemistry, National Institute of Technology
Calicut, NIT Campus P.O., Calicut, India-673 601.
Abstract
We report a series of alkyne-functionalized meso-aryl boron dipyrrin (BODIPY) molecular
rotors sensitive to viscosity. The planar and twisted conformation within the molecular structure
decides the viscosity dependent behavior. The variations in fluorescence lifetime and intensity
were appreciable to the local viscosity. Hence, the dye has been successfully employed in the
enumeration of microbes by considering the proportionate fluorescence intensity of the
BODIPYs as an index of the number of cells per ml. With increasing cells per mL, the viscosity
of the bacterial solution is increased. Consequently, the fluorescent intensity of the sample
containing BODIPY tends to increase due to the restricted rotation in the viscous medium. The
BODIPY probe offers high sensitivity and is easier than other conventional techniques of
colony-forming unit (CFU) determination.
The theoretical studies indicate that the
intramolecular charge transfer is responsible for the enhanced fluorescence intensity in a high
viscous solvent.
Keywords: BODIPY, viscosity, fluorescence, bacterial enumeration, restricted rotation
Introduction
Intracellular viscosity plays an essential role in cellular biophysics (eg., signaling,
diffusions of nutrients or metabolites, changes in proteins configurations, etc.), and any subtle
alterations are linked to diseases.[1] Accumulation of misfolded proteins in the endoplasmic
reticulum during diabetes, increase in mitochondrial viscosity in Alzheimer’s disease, reduced
viscosity of nucleoplasm with Hutchinson–Gilford progeria syndrome, etc., are some of such
biological processes.[2,3] Hence, monitoring real-time viscosity variations is of great importance
in biological research as well as clinical diagnoses. Meso-substituted BODIPYs involving
twisted intramolecular charge transfer (TICT) or Forster resonance energy transfer (FRET) have
1

Accepted manuscript to appear in JPP
emerged as fluorescent molecular rotors (FMRs) to probe micro-viscosity of cell domains.[4,5]
The rotation of the meso-aryl ring and accompanying distortion of the dipyrrin framework
hinders free rotation in a viscous medium. This hindered rotation prohibits the non-radiative
processes of the electronically excited dyes resulting in increased emission intensity, quantum
yield, and lifetime. BODIPY based fluorescent rotors help in monitoring viscosity variations
during apoptosis,[6] lipid oxidation, cell lysis in microbes via protein or DNA leakage, bacterial
sporulation and deactivation, bacterial membrane viscosity changes in response to variations in
temperature, identifying tumor on the basis viscosity of normal cells, etc.[5,7–10]
Bacterial enumeration is an important parameter to understand the ecological role of
microbes in any environment to determine its rate of growth/ death, particularly in the food and
dairy industry. Conventionally, bacterial enumeration has been accomplished via serial dilution-
viable plate count or turbidometric methods which are time-consuming, and suffer from high
variability as well as a limited dynamic range due to self-chemical or microbial action on the
substrate.10 Both clinical analyses and industry demand fast and reliable quantification of
bacterial suspensions. Here, we exploit the viscosity sensitive property of a BODIPY compound
as a novel method for the enumeration of bacteria. It is based on the fact that the viscosity due to
cell density is proportional to the fluorescence characteristics of BODIPY. Hence the proposed
method, which is based on the emission properties of BODIPYs, offers a more accurate, quick,
and repeatable process in the bacterial enumeration.
Here, para- and meta-derivatives of three structurally similar fluorescent probes (1b-6b)
are developed. The restricted rotation is certainly more pronounced in ortho derivatives as
compared to meta and para derivatives. However, the effect of ortho-substituents is well studied
earlier [11] Also, symmetrically substituted FMRs are reported to show a declined viscosity
sensing property.[12] Here we have designed structurally asymmetric meta-derivatives, and the
effect of structural alterations in the deactivation of the excited state through the non-radiative
pathway is investigated theoretically. Since the propargyl groups are susceptible for facile
functionalization and precursors group for synthesizing click products, we have selected one of
the substituents as the propargyl group (1b – 4b). The practical utility of viscosity sensitive
molecules as a novel method for the enumeration of bacteria is also demonstrated using the
model organism Escherichia coli.
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Accepted manuscript to appear in JPP
Results and discussion
The stepwise synthesis of dipyrromethane precursors and their BODIPYs were afforded by
following general procedure.[13,14] The synthetic route employed is outlined in scheme 1. The
crude compounds purified by column chromatography afforded meso-aryl substituted
dipyrromethanes in 30-64 % and meso-aryl substituted BODIPY dyes in 56-84 % yield.
OH
CH2 C CH
O
CHO
OH
CHO
R
CH2 C CH
O
R'
(
i) Pyrrole, TFA
RT, N2
CH C CH Br
2
CHO
CHO
(
ii) DDQ, Et N
COOH
O
C
3
DMF, K CO
2
3
BF OEt
3
2
0
O CH2 C CH
N
N
F
8
0 C, N2
B
F
CHO
O CH2 C CH,
R' = H : 1b
R =
COOH
CHO
R = H, R' =
O
O CH2 C CH : 2b
O
C O CH2 C CH
R =
C O CH2 C CH, R' = H : 3b
CHO
O
R = H, R' =
C O CH2 C CH : 4b
CHO
COOH
R
R'
(
i) Pyrrole, TFA
RT, N2
CHO
R = COOH, R' = H : 5b
R = H, R' = COOH : 6b
COOH
(
ii) DDQ, Et N
3
BF OEt
3
2
N
N
F
B
CHO
F
Scheme 1. Synthetic scheme of BODIPYs, 1b-6b.
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Accepted manuscript to appear in JPP
The integrated intensities of all the proton and carbon signals in H and 13C NMR
respectively are very well in concordance with the expected structure. The NMR and mass
spectra are given in the supplementary information (Figures S1 to S22). The IR spectra of all the
1
-1
BODIPYs showed characteristic -C≡C and terminal alkyne -CH near 2120 and 3400 cm ,
respectively. The aromatic =C-H, -C-H, and Ar-O and stretching frequencies were found at
3
100, 2900, and 1263cm-1
respectively. The vibrations corresponding to B-N, B-F, and β-H of
,
pyrrole ring[15] of the BODIPY core appeared in the vicinity of 1534-1414 and 762-781 cm-1
respectively.
Single crystal X-ray diffraction studies
The structural characterization of all the compounds was obtained by single-crystal X-ray
diffraction analyses. ORTEP diagrams are shown in Figure 1. The packing diagrams, crystal
structure parameters, and interactions present in the compounds were shown respectively in
Tables S2, S3, and Figure S23.
1
b
2b
3b
4
b
5b
6b
Fig. 1. ORTEP diagrams of compunds 1b - 6b shown at 40 % probability ellipsoids. (Solvent
molecules are omitted for clarity). Atom colors: Gray-Carbon; Blue-Nitrogen; Green-Fluorine;
Pink-Boron; White-Hydrogen; Red-Oxygen).
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Accepted manuscript to appear in JPP
Single crystals of suitable diffraction quality of compounds were obtained by a slow
evaporation method using a variety of solvents, including chloroform, hexane, methanol, etc. at
room temperature for 6-7 days. Compounds, 1b and 3b, are crystallized in the monoclinic
system, whereas 2b, 4b, 5b.DMF and 6b are crystallized in triclinic.
The bond lengths and bond angles of BODIPY derivatives were similar to those reported
previously.[14] The nitrogen atom of two pyrrole units and two fluorine atoms are tetrahedrally
coordinated to the boron atom. The B−N and B−F bond distances are in the range of 1.527-1.545
Å and 1.336-1.393 Å, respectively. The F–B–F plane is twisted 86.8-89.8 with respect to the
BODIPY core structure. The mean plane displacement of the 12-atoms of BODIPY core deviates
from ~0.0262 to ~0.0988 Å, indicating its planar nature. Further, the aryl ring present in the
meso-position is oriented perpendicular to the plane of the BODIPY core with the dihedral angle
ranging from 52.7-64.4º, which is comparable to the literature values.[14] This indicates that the
aromatic ring is non-conjugated to the core structure.
Compounds, 1b, and 2b exhibit a similar kind of anti-parallel and herringbone-like
structure, as reported previously through various intermolecular interactions (Figure S23a, b;
Table S2). The crystal packing of para-carboxyl (5b) and para-alkyne (3b) derivatives form a
2
D network. BODIPY core is connected through the solvent lattice (DMF) and B∙∙∙H, F∙∙∙H,
F∙∙∙C, C∙∙∙C, H∙∙∙O interactions in 5b and 3b, respectively (Figure S23c, e). The meta-carboxyl
6b) and meta-alkyne (4b) derivatives form zig-zag 3D structural arrangements that are
(
connected through exclusively as F∙∙∙H and C∙∙∙H interactions (Figure S23d, f).
Photophysical properties
The absorption spectra of BODIPYs show a narrow, strong S
nm with a 0-1 vibrational transition as a shoulder at around 480 nm as well as a broad S
band around 380 nm. These values are comparable to the structurally similar BODIPY
molecules.[14] The mirror shaped emission peak stems from S →S transition. The peaks appear
0
→S
1
transition around 502
0
→S
2
1
0
sharp due to the virtually identical geometries of the ground and excited states in the Franck-
Condon region.[2,16] The optical transition from the ground state to the second excited singlet
state, centered at 480 nm, is more pronounced due to the reduced splitting between the two
states. The steady-state absorption and fluorescence features examined at the different polarities
and are summarized in Table S4. The blue shift in absorption with solvents of increasing polarity
5

Accepted manuscript to appear in JPP
is explained as the lower electric dipole moment of LUMO as compared to HOMO.[16] The
geometry relaxation of BODIPY molecules upon photoexcitation affects the energy levels of
molecular orbitals.[15] The geometries of excited states of S
1
and S of the BODIPY framework
0
being similar, the difference between absorption and the emission energy will be small. The
small Stokes' shift between the two bands is thus consistent with the rigid and neutral dipyrrin
that hampers major geometrical relaxations at excited state associated with emission
broadening.[2]
Fluorescence response to solvent viscosity
Here, we have designed meso-substituted BODIPYs, 1b-6b with unsubstituted pyrrole
rings. For a BODIPY molecular rotor, the alkyl groups usually found on the pyrrole units are
excluded because excited-state dynamics are mainly governed by meso-phenyl rotation.[12,16]
The viscosity and polarity sensitivities are two exclusive properties of FMRs. Hence, those rotors
which show solvent-polarity-dependent emission, do not show the FMR property and vice
versa.[17] A maximum of 5 nm shift in emission wavelength was observed when the solvent was
changed from less viscous ethanol to high viscous glycerol. Glycerol and ethanol are completely
miscible and are important in the pharmaceutical industry. Hence we used glycerol-ethanol
mixtures as viscosity model. The glycerol-ethanol mixtures as viscosity model has been reported
earlier by Muggeridge and co-workers [22]. BODIPYs 1b-6b are insensitive to the polarity of
the solvent and hence be considered as perfect FMRs. The fluorescence intensity of all
compounds increases linearly with increasing viscosity at room temperature. While increasing
the viscosity from ethanol (1.20 cP) to glycerol (1457 cP), the compounds 1b-6b exhibited 8-40-
fold increase in emission intensity respectively. (Figures 2 and S25-29). The fluorescence
quantum yield of BODIPYs also exhibited a linear increase with viscosity.[2] The fluorescence
quantum yield decreases with increasing electron-withdrawing nature of the substituent at the
para position of the phenyl group [18]. However, this effect is very negligible for the compounds
1
b-6b. The fluorescence quantum yield of compounds 1b-6b in ethanol has an average value of
0
.03.
The viscosity dependence of the fluorescence intensity of FMRs is expressed using the
Forster–Hoffmann theory as,
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Accepted manuscript to appear in JPP
where I is the emission intensity, ƞ is the viscosity of the solvent, C is an experimental
constant depending on the temperature, and x is the viscosity sensitivity of the rotors.[19] The
higher the x value, the more sensitive is the emission intensity of an FMR toward the viscosity
variation. The slope of the log(fluorescence intensity) versus log (viscosity) plot gives the
sensitivity for a rotor. According to Foster and Hoffman, a perfect rotor exhibits a sensitivity of
0
.6[19,20] Among the dyes, the best sensitivity was observed for 3b (0.52) and 4b (0.50)
respectively. As predicted by Forster and Hoffmann, the log-log relationship between viscosity
and quantum yield also holds well in all cases (Figure S31).
The internal motions, like the rotation of the aryl substituent and accompanying distortion
of the dipyrrin core due to the frictional forces with the surrounding medium, provide an active
non-radiative decay channel for the rotors.[7] These changes are reflected in the emission
properties with varying viscosity of the solvent. The fluorescence intensity of rotors was found to
be less in non-viscous solvents, which are attributed to the free rotation of a meso-aryl group of
BODIPY.[4] The free rotation about the C≡C bonds in the excited state may cause twisting of
molecular configuration. Consequently, the emission appears weak in low viscous solutions. The
free rotation of the single bond connecting the meso-aryl carbon and oxygen of –O-CH -C≡CH
2
in molecular rotors (1b-4b) causes the non-radiative decay. As the energy of the excited state is
released through non-radiative transitions, the molecules are weakly fluorescent with a quantum
yield (Φ ) ranging from 0.01 to 0.05 in ethanol (Table S8). The rotation through the meso-phenyl
f
C-C bond is restricted in the presence of a highly viscous solvent, such as glycerol[4], and the
excited energy is reserved for emission without non-radiative energy dissipation. With increasing
viscosity, the non-radiative processes caused by configurational relaxation from the planar
locally-excited state (S
1
LE) to the twisted intramolecular charge transfer excited state (S TICT) are
1
suppressed. There arises a restriction in the rotation (or twisting) of phenyl groups with respect to
LE
the -O-C bridge of the propargyl group. Subsequently, the radiative emission from S
1
increases, which in turn reduces the fluorescence efficiencies. Thus, molecular rotors may
perceive the local viscosity via intramolecular twisting motion that occurs in an excited state.[21]
These twisting motions may slow down with increasing viscosity due to which compounds 1b-
6
b displays higher emission intensity in 100% glycerol than other mixtures of
ethanol/glycerol[22] (FigureS 2 and S25-S29).[2,23] Thus, kinetically-restricted rotation of the
phenyl group with respect to the dipyrrin core hampers the non-radiative decay channel.[2]
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Accepted manuscript to appear in JPP
600
500
400
300
200
100
0
-0.4
-
0.6
0.8
0
1
2
3
4
5
6
7
8
9
1
%
-
0 %
0 %
0 %
0 %
0 %
0 %
0 %
0 %
0 %
00 %
-1.0
-1.2
-1.4
-1.6
-1.8
-2.0
-2.2
-2.4
0
.5
1.0
1.5
2.0
2.5
3.0
500
550
600
650
log(viscosity)
Wavelength (nm)
Fig 2. (a) Emission spectra of 3b in ethanol-glycerol mixtures of different viscosity and (b) log
(quantum yield) vs. log (viscosity) of 3b.
The viscosity sensitivity of molecular rotors 3b and 4b are comparable to the other classes
of benchmark molecular rotors such as homodimeric BODIPYs.[19] The good linear relationship
between log (emission intensity) and log (viscosity) with a correlation coefficient of 0.98
indicates that the probes can be used for the quantitative determination of viscosity (Figure S30).
Although all the fluorescent probes 1b-6b described here were found to be sensitive to local
viscosity, fluorescence quantum yield of 3b was greatly enhanced by about 63 fold with when
viscosity was increased from ethanol to glycerol. Hence, we advanced further application studies
with 3b.
Fluorescence lifetime analyses
The fluorescence lifetime (τ) of a selected molecular rotor, 3b, has been determined to get
more insight into the variations in local viscosity.[1] The lifetime evaluated in 100% ethanol,
5
0/50 ethanol/glycerol, and 100% glycerol was best fitted with biexponential function and was
observed to be 0.25, 1.96, and 2.49 ns respectively (Figure 3). The fluorescence lifetime was
found to increase with increasing viscosity. As Forster–Hoffmann equation is also applicable to
the lifetime and viscosity as,[23]
)
, where C is constant.
0
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Accepted manuscript to appear in JPP
This confirms that 3b as an ideal rotor to monitor intracellular viscosity. A calibration
curve of viscosity versus lifetime can be constructed using solvents of different viscosities and
subsequently applied to map the viscosity inside the cells. Further, radiative and non-radiative
rate constants, K
r
and Knr, were calculated from the experimentally measured quantum yields φ
f
and the fluorescence lifetime, τ using the following equation,[20]
-1
The values of K (0.04 to 0.35 ns ) do not vary with change in viscosity. On the other
r
-1
hand, Knr shows a sharp decrease with increasing viscosity from 4.02 to 0.06 ns when solvent
changes from ethanol to glycerol. This strongly suggests that the increase in quantum yield with
increasing viscosity is principally through the suppression of non-radiative processes.[20]
Figure 4 shows the fluorescence lifetime imaging of BODIPY 3b, in 100% ethanol, 50/50
ethanol/glycerol, and 100% glycerol, which further reflects the possibility of biological
application of the molecule in probing viscosity.
1
0000
EtOH
:1 EtOH: glycerol
1
glycerol
1
000
1
00
1
0
2
4
6
8
10
12
14
16
Time ns
Fig. 3. Fluorescence lifetime of 3b in (a) ethanol, black; (b) 1:1 ethanol:glycerol, red; and (c)
glycerol, blue.
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Accepted manuscript to appear in JPP
(
a)
(b)
(c)
Fig. 4. Fluorescence lifetime imaging of 3b in (a) ethanol; (b) 1:1 ethanol: glycerol and (c)
glycerol.
Theoretical studies
To explore the charge transfer in fluorescent molecular rotors, 1b-6b, TDDFT calculations
were performed. Tables S5 and S6 list the lowest-energy transitions and respective oscillator
strength at ground state and transition state for the rotation of phenyl ring. The ground-state
geometry optimizations were performed by DFT functional BP86[24,25] with Def2-SVP[26]
basis set. The single point calculations were performed at the M06[27] level with Def2-
3
0
TZVPP[28] basis set using Gaussian 09 software package. The rotational energy barrier was
studied computationally at the M06/Def2-TZVPP level for BODIPYs 1b-6b. The main
electronic transitions in 1b-6b are given in the Tables S5 and S6. Figure 5 shows the
representative molecular orbital diagram indicating intermolecular charge transfer in the ground
state and rotational transition state of 3b.
The HOMO and LUMO of 1b are mainly localized on the BODIPY core, whereas HOMO-
1
and LUMO+1 are localized mainly on the phenyl ring. The TDDFT calculations reveal that the
HOMO-1 to LUMO transition has considerable oscillator strength (f = 0.2320), and the
corresponding transition can be considered as intramolecular charge transfer (ICT) from phenyl
ring to BODIPY core. However, the rotational barrier for rotation of the phenyl group with
respect to the boradiacene unit computed for 1b is only 1.63 kcal/mol. Hence, the phenyl group
undergoes free rotation in a non-viscous solvent. The oscillator strength corresponding to a
similar transition in the transition state where the boradiacene unit is perpendicular to the phenyl
ring is only 0.0026. All other transitions in the ground and transition state have similar oscillator
strength. Hence, the HOMO-1 to LUMO transition is responsible for the variation in
fluorescence intensity when the viscosity of solvent changes. In viscous solvents, due to the
hindered rotation, the probability of HOMO-1 to LUMO transition will be high (Table S5).
1
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Accepted manuscript to appear in JPP
0
1
2
3
4
5
6
7
8
-
-
-
-
-
-
-
-
-
1.83
3.19
LUMO+1
LUMO
-1.88
-
-3.23
f = 0.3369
f = 0.001
HOMO
-6.58
-6.52
HOMO-1
HOMO-2
-7.70
-7.85
-7.77
-7.94
Transition state
for rotation
Ground state
Fig. 5. Molecular orbital diagram of 3b in the ground state and rotational transition state.
The rotational barriers calculated for 2b-6b are also marginal (0.99 (2b), 0.94 (3b), 0.94
4b), 0.92 (5b) and 1.28 (6b)). Hence, similar to 1b, the compounds 2b-6b also undergoes
(
rotation of phenyl ring with respect to boradiacene unit. For rotors 3b-6b, the HOMO-
→LUMO transition accounts for the ICT. The oscillator strength for this transition has
2
significant value, which reduces significantly in the transition state (0.3369 to 0.001 (3b), 0.2121
to 0.0007 (4b), 0.3038 to 0.0005 (5b) and 0.2350 to 0.0006 (6b)). On the other hand, ICT from
phenyl ring to boradiacene unit arises from HOMO-3→LUMO, in 2b (f = 0.2032). The
theoretical studies indicate that the electronic transition responsible for the enhanced
fluorescence intensity of 1b-6b in the high viscous solvent is attributed to intramolecular charge
transfer.
1
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Accepted manuscript to appear in JPP
Enumeration of bacterial cells using molecular rotors
The practical applicability of the FMRs (1b-6b) in microbiology has been illustrated using
b and model organism E. coli, and we anticipate that this can be extended to clinical
3
analyses.[29] The bacterial strain, E. coli was grown in nutrient broth at 37 ºC for 18-24 hours.
The cell densities of five serially diluted cultures were estimated using absorbance and
McFarland standard table and recorded in CFU/mL. Each sample was treated with 3b for 30 min,
and the unbound probe compound was removed from the biomass by repeated washing and
centrifugation with PBS 1X. Fluorescence spectra of the cell suspensions were recorded, and the
experiments were performed in triplicate. The fluorescent intensity was compared with cell
density and found that an increase in the fluorescent intensity is proportional to the cell density
2
(
Figure 6; R = 0.99).
One of the common features of BODIPYs is that it will be internalized easily into the
bacterial cell.[14] The viscosity of bacterial culture is directly dependent on increasing cell
density. From Figure 6, it is obvious that the fluorescence intensity of this compound can be
taken as an index of the number of cells per ml. However, the cell density varies with the type of
organism in McFarland standards. Therefore, a different calibration curve has to be plotted for
determining the cell number for various microbes, and these investigations are underway in our
laboratory. The rate of internalization of the probe, as well as the viscosity of the bacterial
suspension, can ensure the accuracy of the experiment. Thus, fluorescence measurement allows a
quick assessment of microbial growth, enabling bacterial enumeration independent of growth
characteristics.
2
2
2
2
1
1
1
1
1
60
40
20
00
80
60
40
20
00
0
1
2
3
4
5
6
8
Cell density (X10 cfu/mL)
-5
Fig. 6. Fluorescence intensity of 3b (2.4 x 10 M) at different cell density, λex = 480 nm.
1
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Accepted manuscript to appear in JPP
Conclusions
Herein, we propose meso- 3/4-phenyl substituted BODIPYs (1b-6b) as fluorescent
molecular rotors to identify viscosity variations. The rotors are insensitive to solvent polarity;
hence, they are perfect rotors showing sensitivity comparable to benchmark FMRs. We have also
demonstrated the application of rotor 3b in the bacterial enumeration with E. coli as a model
organism. The present rotors can also be used to determine local viscosity variations via
fluorescence lifetime imaging microscopy (FLIM). The FLIM using rotors 1b-6b can be further
employed in determining the colony-forming unit (CFU) of various microbes. The DFT
calculations indicate that the hindered rotation of the phenyl ring with respect to boradiacene unit
enhances the intramolecular charge transfer, which in turn leads to high fluorescence intensity in
the viscous solvent.
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
Department of Science and Technology (DST), New Delhi through the EMR/2016/002396
project, is gratefully acknowledged for funding to CA. We thank Dr. Babu Varghese, SAIF, IIT
Madras for the single crystal data collection, structure solution, and refinement.
References
1
2
3
4
5
6
. Dziuba D, Jurkiewicz P, Cebecauer M, Hof M and Hocek M. Angew. Chemie Int. Ed. 2016;
5: 174-178.
. Ferri G, Nucara L, Biver T, Battisti A, Signore G and Bizzarri R. Biophys. Chem. 2016; 208:
7-25.
5
1
. Yang Z, He Y, Lee JH, Chae W-S, Ren W X, Lee J H, Kang C and Kim J S. Chem.
Commun. 2014; 50: 11672-11675.
. Yang Z, He Y, Lee J H, Park N, Suh M, Chae W S, Cao J, Peng X, Jung H, Kang C and Kim
JS. J. Am. Chem. Soc. 2013; 135: 9181-9185.
. Gupta N, Reja S I, Bhalla V, Gupta M, Kaur G and Kumar M. J. Mater. Chem. B 2016; 4:
1
968-1977.
. Kuimova MK, Botchway S W, Parker AW, Balaz M, Collins H A, Anderson H L, Suhling K
1
3

Accepted manuscript to appear in JPP
and Ogilby PR. Nat. Chem. 2009; 1: 69-73.
7
8
. Zhu H, Fan J, Li M, Cao J, Wang J and Peng X. Chem. A Eur. J. 2014; 20: 4691-4696.
. Mika J T, Thompson A J, Dent M R, Brooks N J, Michiels J, Hofkens J and Kuimova M K.
Biophys. J. 2016; 111: 1528-1540.
9
1
. Suhling K. Biophys. J. 2016; 111: 1337-1338.
0. S. Clais, G. Boulet, M. Van kerckhoven, E. Lanckacker, P. Delputte, L. Maes and P. Cos,
Lett. Appl. Microbiol.2015; 60, 79-84.
1
1
1. Lee S C, Heo J, Woo HC, Lee J A, Seo YH, Lee C L, Kim S and Kwon O P. Chem. A Eur.
J. 2018; 24: 13706-13718.
2. Bahaidarah E, Harriman A, Stachelek P, Rihn S, Heyer E and Ziessel R. Photochem.
Photobiol. Sci. 2014; 13: 1397-1401.
1
1
3. Lee C H and S. Lindsey J. Tetrahedron 1994; 50: 11427-11440.
4. Prasannan D, Raghav D, Sujatha S, Hareendrakrishna kumar H, Rathinasamy K and
Arunkumar C. RSC Adv. 2016; 6: 80808-80824.
1
1
5. Chen Y, Zhao J, Guo H and Xie L. J. Org. Chem. 2012; 77: 2192-2206.
6. Kee H L, Kirmaier C, Yu L, Thamyongkit P, Youngblood W J, Calder ME, Ramos L, Noll
B C, Bocian D F, Scheidt WR, Birge R R, Lindsey J S and Holten D. J. Phys. Chem. B
2
005; 109: 20433-20443.
7. Zhou F, Shao J, Yang Y, Zhao J, Guo H, Li X, Ji S and Zhang Z. European J. Org. Chem.
011: 4773-4787.
8. Qin W, Baruah M, Van Der Auweraer M, De Schryver F C and Boens N. J. Phys. Chem. A
005; 109: 7371-7384.
1
1
1
2
2
2
9. Lee S C, Heo J, Ryu J W, Lee C L, Kim S, Tae J-S, Rhee B O, Kim S W and Kwon O P.
Chem. Commun. 2016; 52: 13695-13698.
0. Raut S, Kimball J, Fudala R, Doan H, Maliwal B, Sabnis N, Lacko A, Gryczynski I, Dzyuba
S V. and Gryczynski Z. Phys. Chem. Chem. Phys. 2014; 16: 27037-27042.
1. Haidekker M A and Theodorakis E A. Org. Biomol. Chem. 2007; 5: 1669-1678.
2. Alkindi A S, Al-Wahaibi Y M and Muggeridge A H. J. Chem. Eng. Data 2008; 53: 2793-
2
2
2
796.
3. Stöckl M T, Bizzarri R and Subramaniam V. In Springer Series on Fluorescence. , 2012;
15-240.
2
2
2
4. Becke A D. Phys. Rev. A 1988; 38: 3098-3100.
1
4

Accepted manuscript to appear in JPP
2
2
2
2
2
3
5. Perdew J P. Phys. Rev. B 1986; 33: 8822-8824.
6. Schäfer A, Horn H and Ahlrichs R. J. Chem. Phys. 1992; 97: 2571-2577.
7. Zhao Y and Truhlar D G. Theor. Chem. Acc. 2008; 120: 215-241.
8. Weigend F, Häser M, Patzelt H and Ahlrichs R. Chem. Phys. Lett. 1998; 294: 143-152.
9. Bodio E, Denat F and Goze C. J. Porphyr. Phthalocyanines 2019; 23: 1159-1183.
0. Frisch M J, Trucks G W, Schlegel H B, Scuseria G E, Robb M A, Cheeseman J R, Scalmani
G, Barone V, Mennucci B, Petersson G A, Nakatsuji H, Caricato M, Li X, Hratchian H P,
Izmaylov A F, Bloino J, Zheng G, Sonnenberg J L, Hada M, Ehara M, Toyota K, Fukuda R,
Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery J
A, Jr., Peralta J E, Ogliaro F, Bearpark M, Heyd J J, Brothers E, Kudin K N, Staroverov V
N, Keith T, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant J C, Iyengar S S,
Tomasi J, Cossi M, Rega N, Millam J M, Klene M, Knox J E, Cross J B, Bakken V, Adamo
C, Jaramillo, J, Gomperts R, Stratmann R E, Yazyev O, Austin A J, Cammi R, Pomelli C,
Ochterski J W,. Martin R L, Morokuma K, Zakrzewski V G, Voth G A, Salvador P,
Dannenberg J J, Dapprich S, Daniels A D, Farkas O, Foresman J B, Ortiz J V, Cioslowski J
and Fox, D J Gaussian 09 (Revision B.01), Gaussian, Inc., Wallingford CT, 2010.
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Electronic Supplementary Information
For
Development of alkyne-BODIPYs as viscosity sensitive fluorescent probes for
enumeration of bacterial cells
a
b
a
c
Dijo Prasannan, Suchithra Tharamel Vasu, Chellaiah Arunkumar and Pattiyil Parameswaran *
a
Bioinorganic Materials Research Laboratory, Department of Chemistry, National Institute of Technology
Calicut, NIT Campus P.O., Calicut, India-673 601.
b
School of Biotechnology, National Institute of Technology Calicut, NIT Campus P.O., Calicut, India-673 601.
c
Theoretical and Computational Chemistry Laboratory, Department of Chemistry, National Institute of Technology
Calicut, NIT Campus P.O., Calicut, India-673 601.
Contents
EXPERIMENTAL
Materials and Methods
Pyrrole purchased from Spectrochem (India) was distilled over CaH
Hydroxybenzaldehyde, 3-Hydroxybenzaldehyde, Propargyl bromide and 3-Dichloro-5,6-dicyano-
,4-benzoquinone (DDQ) were obtained from Sigma Aldrich and were used as such. 3-
Carboxaldehyde procured from Alfa Aesar was used without further purification. Trifluroacetic
acid (TFA) was purchased from AVRA Synthesis Pvt. Ltd. and was distilled over P
Triethylamine (Et N) purchased from Qualigens was used without further purification. 4-Formyl
benzoicacid and Boron trifluoride etherate (BF .OEt ) were purchased from Spectrochem (India).
BF .OEt and dimethylformamide (DMF) (SRL, India) were purified by vacuum distillation over
CaH whereas 4-Formyl benzoicacid was used as such. Chloroform, dichloromethane, hexane, and
2
before use. 4-
1
2
5
O .
3
3
2
3
2
2
ethanol were purchased from Merck (India) and purified by distilling over K
THF was obtained from and was used as such.
2
CO (except ethanol).
3
1H NMR spectra were recorded with a Bruker 400 MHz FT-NMR spectrometer in CDCl
3
/
CD
3
COCD using tetramethylsilane as the internal reference. IR data was collected on a JASCO
3
FT/IR-4700 with KBr pellets. UV-Visible spectra were recorded on a Shimadzu double beam
spectrometer 2450 instrument using 1 cm matched quartz cuvettes at room temperature.
Fluorescence spectra were recorded on a Perkin-Elmer LS-55 Luminescence spectrophotometer

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with a slit width of 10 at 470 nm excitation wavelength and emission from 490 to 750 nm. The
fluorescence quantum yield (Φ ) was computed using the below equation,
f
Φ
f
= Φ
are the integrated fluorescence intensities of the sample and reference, A
are the absorbance of the sample and reference at excitation wavelength, ɳ and ɳ 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 (Φ = 0.85, λexc= 470 nm). All Φ values were corrected for changes in
f
RF
A
S R
ɳ
S
2
/ F
R
S
A ɳ
R
2
where F
S
and F
R
S
and A
R
S
R
f
f
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). The
fluorescence lifetime was done on a MicroTime 200 timeresolved confocal fluorescence setup
from PicoQuant equipped with an inverted microscope from Olympus IX 71 containing a water
immersion objective of Olympus UplansApo NA 1.2 60X. A 485 nm pulsed diode laser
(
PicoQuant) was used as the excitation source with a stable 10 MHz repetition rate and 60 nW
excitation power.
Electrochemical measurements were performed on a CH-instruments Inc, USA (Model:
CHI660E) equipped with potentiostat / galvanostat with Fourier Transform AC Voltammeter. The
electrochemical system utilized a three-electrode 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 (NBu PF ) as supporting electrolyte (0.1 M) in dichloromethane at 25 C
under N atmosphere at a scan rate of 50 mV/s. The crystal structure data was collected on a Bruker
AXS Kappa Apex II CCD diffractometer with Mo K radiation ( = 0.7107 Å). The structures
4
6
2
α
were solved using direct methods and refined by full matrix least-squares using the program
SHELXL-97/SIR 92 (WINGX). The fluorescence imaging was performed using an
Olympus- IX73 microscope attached with a digital camera at 100x magnification. The images
were processed using cellSens software.
General procedure for the synthesis of substituted benzaldehydes
1
The substituted aldehydes were synthesized using the standard procedure described
below. To the corresponding benzaldehyde (1 equiv.) in DMF was added potassium carbonate (0.5
equivalents). To this stirring solution under nitrogen, propargyl bromide (1.1 equivalents) was

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added and refluxed at 80ºC for 3 – 6 h. The reaction mixture after cooling to room temperature
was diluted with water, extracted with CH
2
Cl
2
and the combined organic layers were washed
SO , solvent
several times with water to remove DMF. The organic phase was then dried over Na
2
4
was evaporated and the residue was purified by silica gel column chromatography using
chloroform/hexane as eluent to obtain the pure compound.
Compound 1, 2: The crude residue was purified by silica gel column chromatography using
chloroform/hexane, 60/40 as eluent to obtain the pure compounds 1 and 2 in 77 % yield.
Compound 3, 4: The reaction mixture was refluxed for 6 h and the crude compounds after
standard work up were purified on silica gel chromatography using 66/34 chloroform/hexane as
eluent. Yield = 83 % for compound 3 and 70 % for compound 4.
General procedure for the synthesis of dipyrromethanes, 1a - 6a
To a solution of corresponding benzaldehyde (1 equivalent) in pyrrole (25/50 equivalent)
under N atmosphere, was added catalytic amount of trifluoroacetic acid (TFA, 0.1 equivalents).
2
The mixture was stirred at room temperature for 10-15 minutes. Upon completion of the reaction,
dilute NaOH (0.1 M) was added to quench the reaction. The product was extracted with ethyl
acetate washed with water, dried over Na
crude product 2-3 drops of triethylamine was added to arrest the degradation of dipyrromethane.
The compound was purified by column chromatography on silica gel using CHCl /hexane. For
2
SO
4
, and evaporated to dryness under vacuum. To this
3
dipyrromethanes, 5a and 6a, the concentrated pale brown oil was dissolved in a 1:20 mixture of
water and ethanol and was allowed to crystallize at 0 °C overnight, affording solid that was filtered
and dried in vacuo.
General procedure for the synthesis of BODIPYs, 1b - 6b
2
The BODIPYs were prepared by following the standard procedure. Briefly, to the
corresponding dipyrromethane dissolved in DCM, nitrogen was purged for 15 min. 3-Dichloro-
5
3
,6-dicyano-1,4-benzoquinone (DDQ), (1.1 equivalents) was then added and stirred vigorously for
0 min at room temperature. To this oxidized product, triethylamine was added. After 15 min,
boron trifluroetherate was added and stirring was continued. The observation of orange red
fluorescent spot confirms the formation of desired BODIPY. The reaction mixture was extracted
with dichloromethane, the combined organic fractions were then washed with water, dried over

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Na
2
SO
4
, 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 (unless specified)
to afford pure compound as orange red fluorescent solid.
meso-(4-Propargyloxyphenyl)dipyrromethane, 1a: The compound was purified by with 70/30
1
chloroform/hexane to yield pale yellow coloured compound 1a. Yield = 34 %. H-NMR (400
MHz, CDCl
α to pyrrole NH), 6.15 (br m, 2H; CH β to pyrrole NH), 5.88 (s, 2H; py-H), 5.39 (s, 1H; meso-H),
.65 (d, J = 2.4 Hz, 2H), 2.5 (t, J = 2.0 Hz 2.4 Hz, 1H, long range coupling).
3
, δ in ppm) 7.87 (br s, 2H; NH protons), 7.13-6.66 (d, 4H; Ar-H), 6.66 (br d, 2H; CH
4
meso-(3-Propargyloxyphenyl)dipyrromethane, 2a: The crude compound was purified by silica
1
gel column chromatography using chloroform. Yield = 68.56 %. H-NMR (400 MHz, CDCl
3
, δ in
ppm) 7.92 (br s, 2H; NH protons), 7.26-6.84 (br m, 4H; Ar-H), 6.68 (br d, 2H; CH α to pyrrole
NH), 6.15 (br m, 2H; CH β to pyrrole NH), 5.93 (s, 2H; py-H), 5.44 (s, 1H; meso-H), 4.63 (d, J =
2
.4 Hz, 2H), 2.48 (t, J = 2.4 Hz, 1H).
meso-(Prop-2-yn-1-yl-4-formylbenzoate)dipyrromethane, 3a: The crude residue was
chromatographed using 58:48 chloroform: hexane as eluent to obtain the pure compound 3a. Yield
1
=
51 % . H-NMR (400 MHz, CDCl
3
, δ in ppm) 7.25 (br, d, 2H; NH protons), 7.9 (d, 2H; Ar-H,
NH protons), 6.68 (br d, 2H; CH α to pyrrole NH), 6.13 (br m, 2H; CH β to pyrrole NH), 5.86 (s,
H; py-H), 5.49 (s, 1H; meso-H), 4.87 (d, 2H), 2.48 (t, 1H).
2
meso-(Prop-2-yn-1-yl-3-formylbenzoate)dipyrromethane, 4a: The pale yellow oily
dipyrromethane was eluted with 60/40 chloroform/hexane to yield the pure compound 4a. Yield =
1
7
2 % (0.5133 g). H-NMR (400 MHz, CDCl
3
, δ in ppm) 8.10 (br, s, 2H; NH protons), 7.99 (s, 1H;
Ar-H), 7.91(d, 1H; Ar-H), 7.32 (m, 2H; Ar-H) 6.63 (d, 2H; CH α to pyrrole NH), 6.11 (m, 2H; CH
β to pyrrole NH), 5.83 (s, 2H; py-H), 5.43 (s, 1H; meso-H), 4.82 (d, 2H), 2.46 (t, 1H).
1
5
-(4-Carboxyphenyl)dipyrromethane, 5a: Yield = 50 %. H NMR (acetone-d
6
) δ (ppm): 5.549
(s, 1H), 5.751(s, 2H), 5.981 (t, 2H), 6.697 (s, 2H), 7.321 (d, 2H), 7.938 (d, 2H), 9.77 (s, br, 2H).
1
meso-(4-Propargyloxyphenyl) BODIPY, 1b:. Yield = 34.49 % H-NMR (400 MHz, CDCl
3
, δ in
ppm) 2.596 (1H, t), 4.803 (2H, d), 6.559 (2H, d; py-H), 6.981 (2H, d; py-H), 7.930 (1H, s; py-H),
.149 (2H, d; Ar-H), 7.576 (2H, d; Ar-H); 55.91, 76.30, 76.94, 114.80, 118.28, 127.12, 131.19,
7

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1
32.19, 134.85, 143.62, 147.12, 160.03. TOF-MS (ES): m/z : Calcd= 322.12; found: 322.00. IR
(KBr): 3448, 3276, 3130, 2120, 1601, 1577, 1544, 1475, 1449, 1413, 1390, 1353, 1298, 1261,
1
225, 1178, 1079, 976, 840, 762, 744, 709, 678, 593, 535, 429.
meso-(3-Propargyloxyphenyl) BODIPY, 2b: The crude product after standard work up was
purified by silica gel column chromatography (chloroform: hexane = 7:3) to yield the pure orange
1
red fluorescent dye. Yield = 15 % (42.83 mg). H-NMR (400 MHz, CDCl
3
, δ in ppm) 2.572 (1H,
t), 4.761(2H, d), 6.545 (2H, d; py-H), 6.997 (2H, d; py-H), 7.947 (2H, s; py-H) 7.198 - 7.438 (4H,
1
3
m; Ar-H). C-NMR (400 MHz, CDCl
23.82, 129.56, 131.66, 134.86, 144.29, 146.80, 157.29. TOF-MS (ES): m/z : Calcd= 322.12;
found: 322.00. IR (KBr): 3850, 3272, 3105, 2973, 2119, 1601, 1584, 1557, 1475, 1450, 1408,
3
, δ in ppm) 55.94, 76.29, 78.14, 116.83, 117.54, 118.57,
1
1
387, 1354, 1290, 1263, 1229, 1169, 1081, 990, 849, 773, 751, 670, 612, 593, 533, 416.
1
meso-(Prop-2-yn-1-yl-4-formylbenzoate)BODIPY, 3b: Yield = 14.5 %. H-NMR (400 MHz,
CDCl
3
, δ in ppm) 2.55 (1H, t), 4.99 (2H, d), 6.56 (2H, d; py-H), 6.87 (2H, d; CH β to pyrrole NH),
1
3
7
.65 (2H, s; CH α to pyrrole NH) 7.97 - 8.22 (2H, m; Ar-H). C-NMR (400 MHz, CDCl , δ in
3
ppm) 52.92, 75.45, 77.50, 76.74, 119.00, 129.83, 130.47, 131.38, 134.59, 138.40, 144.92, 164.95.
TOF-MS (ES): m/z : Calcd= 350.10; found: 350.21. IR (KBr): 3432, 3293, 3118, 2947, 2129,
1
7
725, 1573, 1543, 1478, 1439, 1414, 1389, 1353, 1368, 1262, 1227, 1161, 1095, 977, 871, 771,
54, 1725, 642, 591, 420.
meso-(Prop-2-yn-1-yl-3-formylbenzoate)BODIPY, 4b: After completion of the reaction, the
crude product was purified by silica gel column chromatography using chloroform/hexane (90/10)
1
to afford orange red solid. Yield = 14 %. H-NMR (400 MHz, CDCl
3
, δ in ppm) 2.53 (1H, t), 4.96
(2H, d), 6.56 (2H, d; py-H), 6.87 (2H, d; CH β to pyrrole NH), 7.97 (2H, s; CH α to pyrrole NH),
1
3
7
5
1
2
7
.63 (1H, t; Ar-H), 7.77 (d, 1H; Ar-H), 8.26 (t, 2H; Ar-H). C-NMR (400 MHz, CDCl , δ in ppm)
3
2.94, 75.45, 76.72, 77.35, 118.95, 128.81, 131.37, 131.45, 134.20, 134.82, 144.81, 145.69,
64.86. TOF-MS (ES): m/z : Calcd= 350.10; found: 350.21. IR (KBr): 3423, 3250, 3105, 2932,
133, 1720, 1601, 1584, 1556, 1534, 1476, 1409, 1385, 1353, 1161, 1294, 1263, 1221, 986, 853,
81, 750, 599, 429. The crude product was purified by silica gel column chromatography
(chloroform : acetone = 90/10) to yield the pure orange fluorescent dye.

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1
8
-(4-Carboxyphenyl) BODIPY, 5b: Yield = 72 %. H NMR in CDCl
3
(300 MHz): δ ppm: 8.27
, δ in
ppm) 29.36, 30.94, 119.05, 130.19, 131.24, 134.72, 138.80, 144.98, 145.50, 169.96. IR (KBr, cm-
1)
: 3435, 3116, 2928, 2665, 2538, 2371, 2345, 1688, 1573, 1546, 1478, 1412, 1388, 1356, 1284,
262, 1224, 1158, 1115, 1081, 1046, 1071, 978, 912, 752, 731, 417.
1
3
(
d, 2H), 7.98 (s, 2H), 7.69 (d, 2H), 6.90 (s, 2H), 6.58 (s, 2H). ). C-NMR (400 MHz, CDCl
3
1
8
-(3-Carboxyphenyl) BODIPY, 6b: Yield = 60 %. UV/vis (CHCl
3
): λmax (log ε) = 505 nm (4.81);
(300 MHz): δ ppm: 8.34
1
Fluorescence (CHCl
3
): λem= 522 nm (λex = 505 nm) H NMR in CDCl
3
13
(
d, 2H), 7.98 (s, 2H), 7.83 (m 1H), 7.68 (m 1H), 6.89 (d, 2H), 6.58 (d, 2H). C-NMR (400 MHz,
CDCl , δ in ppm) 76.70, 118.99, 128.91, 129.78, 131.82, 132.21, 134.23, 135.32, 144.87, 145.68,
3
-
1
1
1
70.37. IR (KBr, cm ): 3433, 3118, 2931, 2738, 2676, 2490, 2370, 2345, 2233, 1700, 1604, 1550,
451, 1413, 1387, 1357, 1258, 1224, 1111, 1078, 992, 940, 893, 776, 730, 689, 633, 411.
1
Figure S1. H NMR spectrum of 1a.
1
Figure S2. H NMR spectrum of 2a.
1
Figure S3. H NMR spectrum of 3a.
1
Figure S4. H NMR spectrum of 4a.
1
Figure S5. H NMR spectrum of 5a.
1
Figure S6. H NMR spectrum of 1b.
1
Figure S7. H NMR spectrum of 2b.
1
Figure S8. H NMR spectrum of 3b.
1
Figure S9. H NMR spectrum of 4b.
1
Figure S10. H NMR spectrum of 5b.
1
Figure S11. H NMR spectrum of 6b.
Figure S12. C-13 NMR spectrum of 1b.
Figure S13. C-13 NMR spectrum of 2b.
Figure S13. C-13 NMR spectrum of 3b.
Figure S14. C-13 NMR spectrum of 4b.
Figure S15. C-13 NMR spectrum of 5b.
Figure S16. C-13 NMR spectrum of 6b.
Figure S17. Mass spectrum of 1b.
Figure S18. Mass spectrum of 2b.

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Figure S19. Mass spectrum of 3b.
Figure S20. Mass spectrum of 4b.
Figure S21. Mass spectrum of 5b.
Figure S22. Mass spectrum of 6b.
Figure S23. Figure S23. Packing diagrams of (a) 1b, viewed down ‘c’ axis; (b) 2b, viewed along
‘a’ axis; (c) 3b, viewed along ‘b’ axis; (d) 4b, viewed along ‘b’ axis; (e) 5b, viewed down ‘a’ axis
and (f) 6b, viewed along ‘a’ axis.
Figure S24. Normalized UV-Visible (solid) and fluorescence (dash) spectra of compounds, 1b-
4
b.
Figure S25. (a) Fluorescence intensity versus wavelength (b) log (quantum yield) versus log
viscosity) for 1b.
Figure S26. (a) Fluorescence intensity versus wavelength (b) log (quantum yield) versus log
viscosity) for 2b.
Figure S27. (a) Fluorescence intensity versus wavelength (b) log (quantum yield) versus log
viscosity) for 4b.
Figure S28. (a) Fluorescence intensity versus wavelength (b) log (quantum yield) versus log
viscosity) for 5b.
Figure S29. (a) Fluorescence intensity versus wavelength (b) log (quantum yield) versus log
viscosity) for 6b.
(
(
(
(
(
Figure S30. Log FI versus log viscosity of (a) 1b (b) 2b (c) 3b (d) 4b (e) 5b (f) 6b.
Figure S31.
Table S1. Electrochemical redox data of BODIPYs, 1b-6b.
Table S2. Crystallographic data of compounds, 1b-6b.
Table S3. Distances (in Å) for different types of interactions in 1b, 2b, 3b, 4b, 5b and 6b.
Table S4. Photophysical properties of 1b-6b in different solvents.
Table S5. Calculated oscillator strength (f), excitation energy, wavelength, coefficient and its
percentage contribution of transitions in transition state.

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*
1
Figure S1 H NMR spectrum of compound 1a

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*
**
1
Figure S2 H NMR spectrum of compound 2a

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1
Figure S3 H NMR spectrum of compound 3a

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*
1
Figure S4 H NMR spectrum of compound 4a

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1
Figure S5 H NMR spectrum of compound 5a

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*
*
*
1
Figure S6 H NMR spectrum of compound 1b

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*
*
*
1
Figure S7 H NMR spectrum of compound 2b