Diphenylpolyene-cholesterol conjugates as fluorescent probes for microheterogeneous media

Article abstract of DOI:10.1016/j.jphotochem.2014.02.013

Extrinsically conjugated fluorescent diphenylpolyene cholesterol derivatives are synthesized and spectroscopic investigations in homogeneous and aqueous micellar solutions are described. The emission of these cholesterol conjugates reveals characteristic

Full text of DOI:10.1016/j.jphotochem.2014.02.013

Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Diphenylpolyene-cholesterol conjugates as fluorescent probes for
microheterogeneous media
Veerabhadraiah Palakollu, Sriram Kanvah∗
Department of Chemistry, Indian Institute of Technology Gandhinagar, Ahmedabad 382424, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Extrinsically conjugated fluorescent diphenylpolyene cholesterol derivatives are synthesized and spec-
troscopic investigations in homogeneous and aqueous micellar solutions are described. The emission
of these cholesterol conjugates reveals characteristic intra-molecular charge transfer (ICT) behaviour
in homogeneous solvents with a mono-exponential decay. Spectroscopic evidence in micellar aqueous
solutions reveals a bi-exponential decay. This indicates the presence of two preferred locations of the
cholesterol conjugated diphenylpolyenes sites of lower polarity and interfacial sites. The sensitivity of
these fluorophores was utilized to determine the critical micelle concentrations.
Received 30 November 2013
Received in revised form 20 February 2014
Accepted 26 February 2014
Available online 12 March 2014
Keywords:
Fluorescent cholesterol
Intramolecular charge transfer
Determination of CMC
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Amphipathic cholesterol is an essential component of these biolog-
ical membranes and has been used as a building block for creating
␲-Conjugated materials based on dyes that fluoresce at
various wavelengths and exhibit charge transfer are of great
interest towards various optoelectronic [1–4] as well as diagnos-
tic/analytical applications [5–9]. The unique modulation of the
ground and excited state dynamics of diphenylpolyenes using
variously substituted donor and acceptor groups have rendered
them with potential applications in organic optoelectronic devices
[3,10] and as photoresponsive materials [10,11]. Molecules having
donor and acceptor groups on the aromatic ring can lead to strong
intramolecular charge transfer (ICT) character responsible for envi-
ronment sensitive emission behaviour [12]. For such systems,
fluorescence quantum yield decreases with increase in solvent
polarity accompanied by large bathochromic emission spectral
shifts [13]. This unique behaviour can be utilized for fluorometric
characterization of biological membranes [14]. Diphenylpolyenes
substituted with suitable donor or acceptor groups have been
examined as membrane probes in homogeneous and microhetero-
geneous environments [7,15,16]. A well-known example of an
extensively studied non-covalent probe was diphenylhexatriene
(DPH) which preferentially resides in the nonpolar regions of the
cell membrane [17].
photo responsive materials [18–20] and in bionanotechnology [21].
The rigid planar subunit of the rings and a flexible iso-octyl side
chain ‘tail’ allows cholesterol to modulate various functions in the
biological membrane organization. Many fluorescent cholesterol
conjugates [22–27] containing fluorophores such as NBD [28,29],
BODIPY [30,31], fluorescein [23] have been used to examine vari-
ous functionalities of cholesterol in membranes. Functions such as
cholesterol organization, trafficking, lipid interactions and modula-
tion of activity of membrane proteins can be monitored by utilizing
changes in fluorescence response with respect to their polar or
non-polar environments.
In this paper, we report the synthesis and photophysical
properties of two novel fluorescent diphenylpolyene-cholesterol
analogues linked at the 3␤-OH position. Due to the lack of 3-
OH group these cholesterol probes lose the amphipathic property
[14,32], but may show preference for partitioning into ordered
micro domains and therefore can be a valuable tool for explo-
ration of such dynamics. This structural design yields a fluorophore
that can be monitored using non-invasive fluorescence meth-
ods. Presence of cholesterol also enables the fluorophores to be
embedded in the biological hydrophobic environment rendering
biosensing applications [33,34]. The fluorophores (7, 8) and their
cholesterol derivatives (10, 11) (Scheme 1) that we utilized exhibit
strong solvent dependent emission characteristics attributed to ICT
behaviour [12]. We intended to tap into their emission proper-
ties and understand the feasibility of using these covalently linked
cholesterol fluorophores for probing microenvironments.
Biological membranes are composed of complex assemblies of
lipids and proteins that allow many important cellular functions.
∗
Corresponding author. Tel.: +011 91 7932419500.
E-mail address: kanvah@gmail.com (S. Kanvah).
http://dx.doi.org/10.1016/j.jphotochem.2014.02.013
1010-6030/© 2014 Elsevier B.V. All rights reserved.

V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
19
Scheme 1. Synthesis of cholesterol conjugated stilbene (10) and diphenylbutadiene (11). Reagents & conditions used: (a) B(OMe)3, BF3·Et2O, BH(Py), EDC, RT to 0 ◦C, 3 h; (b)
CBr4, DCM, PPh3, 0 ◦C to RT, 3 h; (c) P(OEt)3, DMF, 140 ◦C, 2 h; (d) NaH, THF, 0 ◦C to RT, 12 h and (e) pyridine, benzene, 80 ◦C, 24 h.
2. Experimental
quantitative yields [36]. The hydroxyl group was replaced with bro-
mide using tetrabromomethane (CBr4) yielding a yellow crystalline
solid (3) with 83% yield [37]. Phosphonate (4) was obtained upon
treatment of (3) with triethylphosphite in dimethylformamide
(DMF). The phosphonate ester (4) was then subjected to a reaction
with suitable aldehyde, 4-dimethylaminobenzaldehyde yielding
(7) and 4-dimethylaminocinnamaldehyde yielding (8) with sodium
hydride (NaH) as a base and THF as a solvent [37]. The final step of
conjugation with cholesterol was done utilizing cholesteryl chloro-
formate to yield the carbonate linked fluorescent sterol derivatives
(10) and (11).
2.1. Materials and methods
The chemicals, surfactants (CTAB, Triton X-100 and SDS) and
other reagents used for this study were obtained from Aldrich,
Alfa Aesar, Acros or S.D. fine chemicals Ltd. Solvents were dried
using reported procedures before their use in synthesis and optical
spectroscopic studies. Double distilled Millipore water was used to
prepare solutions of the desired concentration. A 20 ␮L tetrahydro-
furan (THF) solution of the fluorophore (1 × 10−4 M) was added to
the surfactant solution maintaining a uniform dye-concentration.
1H and 13C NMR characterization was done using Bruker Avance
500 (500 MHz) spectrometer and accurate mass analysis was per-
formed using Waters-synapt G2S (ESI-QToF) mass spectrometer.
UV–vis absorption spectra were recorded using Analytik Jena
Specord 210 plus and steady state fluorescence emission studies
were performed using Horiba Jobin Yvon fluolorog-3 spectrofluo-
rimeter using a slit-width of 1 nm. The fluorescence quantum yields
were determined using a reference solution with a known quantum
yield of fluorescence [35] with similar optical density. Picosecond
pulsed diode laser-based time-correlated single photon counting
(TCSPC) instrument from Horiba Jobin Yyon IBH (UK) set at a
magic angle at 54.7◦ was used to determine the fluorescence life-
times. The excitation sources used were 406 nm and 440 nm with
the corresponding fwhm of 249, 248 ps having resolutions of 7
and 14 ps/channel, respectively. 406 nm excitation was used for
molecule (10) and 440 nm excitation was used for molecules (7), (8)
and (11). The excitation wavelength corresponds to their observed
absorption maxima. The number of channels per decay was 5000
for both resolutions. The decays were fitted by using IBH DAS v6.2
software in mono and biexponential models by deconvolution by
iterative reconvolution. The error associated with the determina-
tion of lifetime studies is 0.1–1.5%.
2.3. General procedure for the synthesis of diphenylpolyene
derivatives (7) and (8)
To a solution of phosphonate (4) (1 mmol) in dry THF (5 mL) at
0 ◦C, NaH (2.5 mmol) was added under N2 atmosphere. After stir-
ring for five minutes, aldehyde (1 mmol) in dry THF (5 mL) was
added drop wise. Stirring was continued for further 30 min while
maintaining the temperature at 0 ◦C. The reaction was later allowed
to stir at room temperature for 12 h and the mixture was poured
into ice-cold water. Extraction with dichloromethane (DCM) and
concentrating under reduced pressure yields a dark red residue,
which on elution by column chromatography (silica gel, 15% ethyl
acetate/petroleum ether) afforded the desired product as dark red
solid.
2.3.1. (E)-5-(4-(dimethylamino)Styryl)-2-nitrophenol (7)
Yield: 30% (85 mg); dark red solid; 1H NMR (500 MHz, CDCl3)
3.03 (s, 6H), 6.71 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 16.5 Hz, 1H), 7.09
(d, J = 9.0 Hz, 1H), 7.13 (s, 1H), 7.19 (d, J = 16.5 Hz, 1H), 7.44 (d,
J = 8.5 Hz, 2H), 8.04 (d, J = 8.5 Hz, 1H), 10.79 (s, 1H). 13C 125 MHz,
CDCl3) 40.3, 111.9, 115.9, 127.2, 128.6, 131.1, 141.1. HRMS [ESI]
[M−1]− 283.1449.
2.2. Synthesis of fluorescent cholesterol conjugates
2.3.2. 5-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,
3-dienyl)-2-nitrophenol (8)
The synthesis of cholesterol analogues of diphenylpolyenes (10)
and (11) was achieved in five steps (Scheme 1). Carboxylic group of
3-hydroxy, 4-nitrobenzoic acid (1) was reduced to the correspond-
ing alcohol using trimethylborate and borane-pyridine complex in
Yield: 27% (83 mg); dark red solid; 1H NMR (500 MHz, CDCl3)
3.03 (s, 6H), 6.51 (d, J = 15 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 6.83 (d,
J = 16.5 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 7.13(s, 1H), 7.19 (d, J = 16.5 Hz,
2H), 7.44 (d, J = 8.5 Hz, 2H), 8.04 (d, J = 8.5 Hz, 1H), 10.79 (s, 1H).

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V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
13C (125 MHz, CDCl3) 40.4, 112.3, 115.9, 116.0, 124.0, 125.6, 127.2,
128.4, 135.6, 137.9.HRMS [ESI] [M + 1]+ 311.1792.
responsive properties than a free dye [15,16]. The diphenylbuta-
diene and the cholesterol groups were covalently linked through a
short “carbonate” linker. This structural design enables monitoring
of solvent sensitivity through the solvatochromic fluorescence of
these derivatives and helps to understand the properties of orga-
nized assemblies. Furthermore the short covalent spacer, linking
fluorophore to the cholesterol appendage, could enable transfer
of the probe to its natural resident sites leading to environment
specific optical properties.
2.4. Synthesis of cholesterol conjugated polyenes (10) and (11)
To a solution of stilbene or diene [(7), (8)] (0.3 mmol, 85 mg) in
benzene (4 mL), pyridine was added (1.2 mmol, 145 ␮L) and stirred
for 5 min. To this, cholesteryl chloroformate (0.3 mmol, 134 mg) in
benzene (4 mL) was added drop wise. After complete addition of
the cholesteryl chloroformate, the reaction was allowed to reflux
for 24 h. Pyridine and benzene were removed under reduced pres-
sure and was followed by extraction of the compound using DCM.
Conjugates were recrystallized using DCM and methanol.
3.1. Absorption behaviour in homogeneous solvents
There is no discernible difference in absorption maxima (ꢀa) of
(8) when compared to (7) although the conjugation is extended by
a double bond (Table 1). However, solvent polarity changes yields
moderate ꢀa shifts in (7) and (8). In the case of (7), the observed
shift is about 13 nm and in the case of diene (8) the shift is lesser
(∼8 nm) (Fig. 1C and D). The maximum absorption wavelength is
observed in acetonitrile and the lowest in heptane. Planarity of
the molecules in the ground state and solute–solvent H-bonding
interaction accounts for the observed absorption spectral shifts.
Conjugation of cholesterol with stilbene or diene moiety leads to
intense absorption in the long wavelength region similar to the
un-substituted derivatives. It is expected that bulky cholesterol
substitution and subsequent loss of conjugation due to modifi-
cation of the phenolic hydroxyl group may affect the absorption
maxima. Likewise, butadiene conjugated (11) exhibits blue shifted
absorption as compared to the free diene (8) (Fig. 1A) with a shift
of 10–15 nm in non-polar solvents. On the other hand, cholesterol
conjugated stilbene (10) has a large hypsochromic shift of up to
57 nm in the solvents studied (Fig. 1B) in comparison to the free stil-
bene (7). A combination of factors such as loss of H-bonding because
of hydroxyl modification, bulkiness of the cholesterol moiety and
aggregation or association because of the tendency due to the pres-
ence of cholesterol [22] contribute to the observed blue shift.
Thus among the molecules studied, it is found that (8) exhibits
maximum ꢀa and (10) minimum ꢀa. Cholesterol linked fluoropho-
res (10) and (11) also show moderate shifts in absorption maxima
as the solvent polarity is varied from heptane to methanol (Fig. 1C
and D). Unlike (7) and (8), where additional double bond has no
influence on ꢀa, cholesterol conjugated diene (11) exhibit a strong
bathochromic shift (∼up to 47 nm) as compared to (10).
2.4.1. Stilbene-(10)
Yield: 55%; dark red solid; 1H NMR (500 MHz, CDCl3) 0.71 (s, 3H),
1.87 (m, 38H), 2.55 (s, 2H), 3.07 (s, 6H), 5.45 (m, 1H), 5.46 (s, 1H),
6.90 (m, 3H), 7.22 (d, J = 16.5 Hz, 1H), 7.38 (s, 1H), 7.47 (m, 3H), 8.15
(d, J = 8.5 Hz, 1H). 13C (125 MHz, CDCl3) 18.9, 19.4, 21.2, 22.7, 23.0,
24.0, 24.4, 27.8, 28.2, 28.4, 32.0, 32.1, 36.0, 36.3, 36.7, 37.0, 38.1,
39.7, 39.9, 40.4, 42.5, 50.2, 56.3, 56.9, 112.1, 121.7, 123.4, 126.7,
131.5, 133.5, 139.3, 142.8, 152.9. HRMS [ESI] [M + 1]+ 697.4642.
2.4.2. Diene-(11)
Yield: 55%; dark red solid; 1H NMR (500 MHz, CDCl3) 0.71 (s,
3H), 1.87 (m, 38H), 2.49 (s, 2H), 3.04 (s, 6H), 5.45 (d, J = 4.5 Hz, 1H),
5.46 (s, 1H), 6.55 (d, J = 15.5 Hz, 1H), 6.75 (m, 2H), 6.79 (m, 2H), 7.11
(dd, J = 6.0/9.5 Hz, 1H), 7.26 (s, 1H), 7.39 (dd, J = 5.0/7.0 Hz, 3H), 8.13
(d, J = 8.5 Hz, 1H). 13C (125 MHz, CDCl3) 12.0, 14.3, 18.9, 19.4, 21.2,
22.7, 22.8, 23.0, 24.0, 24.4, 27.7, 28.2, 28.4, 29.5, 29.8, 29.9, 32.0,
32.1, 35.9, 35.3, 35.7, 37.0, 38.0, 39.0, 39.7, 39.9, 42.5, 50.2, 56.3,
56.8, 121.4, 123.5, 123.8, 126.8, 128.4, 139.2, 145.3, 153.0. HRMS
[ESI] [M + 1]+ 723.4721.
3. Results and discussion
Utilizing intensely solvatochromic probes to understand the
micelle properties should lead to a molecule that can either reside
in the interior or span the interface region of the media [12]. (7) and
(8) are examples of such environmentally sensitive dyes with pro-
nounced solvatochromic shifts in homogeneous solutions [12]. In
this reported work, we have targeted synthesis of cholesterol ana-
logues of diphenylpolyenes that preserves the ability to undergo
charge transfer from donor to acceptor. To achieve this we have
incorporated a phenolic hydroxyl group as a synthetic handle to
conjugate receptors of choice which could lead to better photo
3.2. Emission in homogeneous solvents
Both the cholesterol free and cholesterol linked molecules
exhibit solvent dependent emission behaviour. As the solvent
Table 1
Absorption, emission data for the molecules (7), (8), (10) & (11) in homogeneous solvents.
a
a
Solvent
ꢀa (nm)
ꢀf (nm)
˚
f
Stokes shift
(nm)
Solvent
ꢀa (nm)
ꢀf (nm)
˚
f
Stokes shift
(nm)
Heptane
Cyclohexane
Toluene
Dioxane
THF
442
447
462
448
455
454
455
523
529
605
633
0.09
0.11
0.09
0.02
81
82
143
185
Heptane
Cyclohexane
Toluene
Dioxane
THF
443
449
463
448
458
451
446
523, 547
530
609
634
686
–
0.10
0.13
0.11
0.03
80
81
146
186
7
8
CH3CN
CH3OH
–
–
CH3CN
CH3OH
–
Heptane
Cyclohexane
Toluene
Dioxane
THF
392
396
405
400
412
400
400
503
503
554
573
–
0.29
0.29
0.18
0.09
111
Heptane
Cyclohexane
Toluene
Dioxane
THF
433
434
453
447
454
454
448
502, 531
505, 535
595
620
677
0.28
0.29
0.17
0.08
69
71
142
173
107
149
173
10
11
CH3CN
CH3OH
–
–
CH3CN
CH3OH
–
–
a
Quinine sulphate (0.545 in 1 N H2SO4)/rhodamine B (0.92 in ethanol)/fluorescein (0.79 in ethanol) were used as fluorescence standards [35] in determining the fluorescence
quantum yield.

V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
21
0.25
10 nm
A
(8)
B
0.20
0.15
0.10
0.05
0.00
(7)
(10)
57 nm
(11)
0.20
0.15
0.10
0.05
0.00
300
350
400
450
500
550
600
300
400
500
600
Wavelength (nm)
Wavelength (nm)
Heptane
Cyclohexane
Toluene
Dioxane
THF
C
0.20
Heptane
D
0.20
cyclohexane
Toluene
Dioxane
THF
Acetonitrile
Methanol
0.15
0.10
0.05
0.00
0.15
0.10
0.05
0.00
Acetonitrile
Methanol
300
400
500
600
300
400
500
600
Wavelength (nm)
Wavelength (nm)
Fig. 1. Absorption spectra of the molecules investigated. (A) Absorption of (8) & (11) in toluene; (B) absorption of (7) & (10) in toluene; (C and D) absorption of (8) & (11) in
solvents of varying polarity.
polarity is increased, a strong bathochromic shift in emission max-
ima (ꢀf) is observed. Reorganization of polar solvent molecules
around the fluorophore and emission arising from a non-planar,
highly polar twisted intramolecular charge transfer (TICT) state
contribute to these observed strong emission shifts [13]. Further
large Stokes shift observed indicates the formation of the high
dipole moment excited charge separated states. Decreased life-
times with an increase in solvent polarity present other evidence of
the presence of such charge transfer states. In contrast to the little
or no absorption shifts, the additional double bond in (8) yields a
stronger bathochromic shift (up to 110 nm) than the stilbene (7)
(up to 70 nm) (Table 1). The spectral data are shown in Fig. 2A and
B. This behaviour is akin to molecules, as reported in the litera-
ture [15], that do not contain the phenolic hydroxyl group. The
quenched emission in polar solvents is strongly influenced by inter-
molecular H-bonding interactions as well as efficient nonradiative
Heptane
Cyclohexane
Toluene
Dioxane
THF
B
A
Heptane
Cyclohexane
Toluene
Dioxane
THF
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
500
600
700
800
500
C
600
700
800
Wavelength (nm)
Wavelength (nm)
Heptane
Cyclohexane
Toluene
Dioxane
THF
Heptane
Cyclohexane
Toluene
Dioxane
THF
D
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
450
500
550
600
650
700
750
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Fig. 2. Normalized emission spectra of the molecules investigated in solvents of varying polarity. A (7); B (8); C (10); D (11).

22
V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
decay processes [15]. This observation is also confirmed by greater
quantum yields of these compounds in non-polar solvents than in
polar solvents.
Conjugating a cholesterol moiety that is largely unresponsive
to light is expected to maintain the fluorescence properties of the
cholesterol linked stilbenes or dienes as the core donor–acceptor
structure is conserved. But in the case of cholesterol conjugated
stilbene (10), hypsochromic shifts of emission are observed with
a minimum shift of 20 nm in heptane and a maximum shift of
60 nm in dioxane as compared to free-cholesterol derivative (7).
Along with the hypsochromic shift, an overall reduction in solva-
tochromic effect [70 nm (10) and ∼110 nm (7)] is also seen (Fig. 2C).
Bulky cholesterol closer to nitro moiety may slightly offset the
donor–acceptor conjugation causing the reduction in the observed
solvatochromism. Similar hypsochromic shifts are observed for
(11) with a shifts up to 14–21 nm in nonpolar heptane or dioxane
as compared to free dye (8). The solvatochromic shift of 163 nm
from heptane to THF (Fig. 2D) is moderately lower than observed
Fig. 3. Effect of change in surfactant concentration on fluorescence emission Spectra of (10) and (11). A, C and E are for (10) and B, D and F are for (11) in CTAB, SDS and
Triton X-100 respectively.

V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
23
5.0x105
4.5x105
4.0x105
3.5x105
3.0x105
A
1.0
0.8
0.6
0.4
0.2
0.0
B
4.0x10-3 8.0x10-3 1.2x10-2 1.6x10-2 2.0x10-2
Concentration (M)
1.0x10-4 2.0x10-4 3.0x10-4 4.0x10-4 5.0x10-4 6.0x10-4
Concentration (M)
0.0
0.0
7.0x105
C
6.3x105
5.6x105
4.9x105
4.2x105
0.0
4.0x10-4 8.0x10-4 1.2x10-3 1.6x10-3 2.0x10-3
Concentration (M)
Fig. 4. Plots of emission intensity versus surfactant concentration for molecules (10-Red) and (11-Blue). A, B and C are for SDS, Triton X-100 and CTAB respectively. (For
interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
in free diene (175 nm). Apart from a moderate reduction in the sol-
vatochromic emission properties, conjugating a rigid cholesterol
moiety contributes to 2–3 fold enhancement in the quantum yield
of the molecules. The emission of both compounds, similar to the
cholesterol-free dyes, was completely quenched in polar solvents.
Table 1 summarizes the absorption and emission behaviour in the
solvents studied.
3.3. Absorption and emission of (10) and (11) in surfactant media
The ability of surfactants to form micelles and their microen-
vironmental similarity with biological macromolecules has signifi-
cant applications in materials science as well as in biology [38,39].
The critical micelle concentration (CMC) is an important parameter
to characterize micelle formation and various optical techniques
B
Prompt
A
Prompt
C
Prompt
SDS
Triton X-100
CTAB
Dioxane
Heptane
Toluene
Heptane
Toluene
Dioxane
103
102
101
103
102
101
103
102
101
4
8
12
16
6
9
12
15
18
6
9
12
Time (ns)
15
18
Time (ns)
Time (ns)
Fig. 5. Excited state decay profile of (8) and (11) in homogeneous and surfactant media. A (8) in homogeneous solvents, B (11) in homogeneous solvents and C (11) in
surfactant media. Decay profile for (8) in surfactant media could not be observed (the samples were excited at 440 nm).

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V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
are used to measure the CMC of micelles [40]. Solubilization of
probes at a number of different sites in these surfactant media leads
to interesting medium and probe dependent solvatochromic prop-
erties. Such fluorophores have therefore been utilized as probes to
detect the CMC of various surfactants or related systems [15,41–43]
and as solvent polarity indicators [44]. The cholesterol conjugated
fluorophores (10) and (11) were utilized to determine the CMC of
surfactants in aqueous solutions. The structural changes induced
by cholesterol addition, the type of microenvironment or tem-
perature could affect the emission of these probes. It is therefore
hypothesized that hydrophobic cholesterol appendage allows the
fluorophores to interact with the hydrophobic chains of the surfac-
tant. Subsequently we observed smooth changes to fluorescence
intensity for these probes in the surfactant media.
The absorption of (10) in surfactant media (CTAB, SDS, Triton
X-100) exhibits a moderate red-shift in comparison to non-polar
solvents but is comparable to other solvents. Diene (11) has a blue-
shifted absorption as compared to the organic solvents. Table 2
lists the absorption and emission maxima of these molecules in
the micelle media utilized.
Similar to the absorption behaviour of these fluorophores,
cholesterol probes have emission maxima comparable to those
obtained in homogeneous solvents such as dioxane. The emission
maxima show a slight red-shift and moderate blue shift in (10) and
(11) respectively. The observations suggest that diene (11) has a
greater propensity to occupy and interact with less polar domains
in micellar media than (10). The emission of cholesterol free fluo-
rophores (7) and (8) was completely quenched in the surfactant
media owing to polar interactions. The quantum yields of the fluo-
rophores in the micellar media are expected to increase because
of their partitioning into the hydrophobic core as well due to the
confinement provided by the micellar core. The observed emission
quantum yields for (10) and (11) are comparable to that obtained
in dioxane. This similarity indicates the possible localization of the
fluorophores towards hydrophobic regions of the micelle.
As the concentration of surfactant is increased, the fluorescence
intensity of (10) and (11) increases in all three media (SDS, Tri-
ton X-100, CTAB). The corresponding fluorescence intensity values
are given in Table 1 in supplementary information. This concen-
tration increase has minor effect on ꢀf of the molecules studied.
The minor variations in emission maxima are not consistent across
all the three micelles and local perturbations may be a contribut-
ing factor for such behaviour. Fig. 3A–F summarizes the emission
intensity changes as a function of wavelength and surfactant con-
centrations for different micelle media. As can be seen clearly, in
the case of neutral surfactant Triton X-100 (Fig. 3E and F), a distinct
jump in fluorescence intensity was observed whereas in other sur-
factants the change is not demarcated. Nevertheless, this intensity
change due to increase in surfactant concentration indicates par-
titioning of the probe to preferential domains of the micelle. This
also explains the observed blue shifted emission of diene (11) in
comparison to dioxane. As soon as the surfactants reach a CMC,
measurable emission intensity changes were observed because of
probes preferential localization.
3.4. Determination of critical micelle concentration
As part of our investigation to determine the CMC and to prove
the efficacy of our compounds as fluorescence probes, we chose
non-ionic (Triton X-100), anionic (SDS) and cationic (CTAB) sur-
factants as a representative selection. The CMC data for these
surfactants were well documented in the literature [42,45,46] and
offer an easy comparison to probe the fluorophore’s suitability for
such measurements. The plot of emission intensity changes versus
the change in concentration of surfactant has been utilized as a
tool to determine the CMC. As described earlier, a sharp increase

V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
25
in emission intensity for both the cholesterol linked fluorophores
was observed as soon as the surfactant adopts the micelle con-
formation. Fig. 4A–C depicts plots of emission intensity changes
of fluorophore with the surfactant concentration. The inflection
point of the curve has been taken as the CMC for all the surfac-
tants. Table 2 summarizes the obtained values of CMC and these
results are in harmony with results obtained with similar probes
[12,13,15]. Substitution of cholesterol moiety preferentially pushes
the fluorophore to more non-polar locations and this attribute can
be useful in probing complex biological membranes. As a control
experiment, when cholesterol free dye (7) was used to determine
the CMC, the fluorescence was heavily quenched and no changes
either to the intensity or wavelength were observed indicating that
presence of cholesterol moiety is advantageous in probing these
hydrophobic environments.
3.5. Fluorescence lifetimes (7)–(11) in homogeneous and
microheterogeneous media
The fluorescence decay profile of substituted diphenylbutadi-
ene derivatives in homogeneous solvents (heptane, toluene and
dioxane) and micellar media when excited at 406 or 440 nm are
shown in Fig. 5 (Fig. S1 for (7) and (10)). The obtained lifetime
values are listed in Table 3. In organic solvents, all the molecules
(7), (8), (10) & (11) decays single-exponentially with a lifetime
of ∼0.41–2.00 ns depending on the polarity of solvent used. The
observed lifetimes show a decreasing trend with an increase in
solvent polarity. Reliable fluorescence lifetimes in highly polar
solvents (acetonitrile/methanol/H2O) could not be obtained due
to very low fluorescence quantum yields and rapid non-radiative
decay. On cholesterol substitution (10, 11) the observed lifetime
increases as compared to cholesterol free dyes (7, 8) in the given
solvents.
Interestingly, in the case of micellar media, a bi-exponential
decay was observed for cholesterol linked fluorophores (Fig. 5C)
indicating the ability of the molecules to reside in two different
microenvironments leading to more than one emitting species.
Alternately one part of a solute could have an inclination towards
polar properties associated with the interface while another part
has a disposition towards non-polar interior. The shorter lifetimes,
0.33 ns (SDS), 0.20 ns (Triton X 100), 0.20 ns (CTAB) for (10) and
0.25 ns, 0.17 ns and 0.21 ns for (11) observed could be correlated
to the solute occupying the polar interface (Table 3). The relatively
low quantum yields obtained for these fluorophores in polar sol-
vents as well as in surfactant media also support the interaction
of the fluorophore with polar surroundings. The decay profile of
non-cholesterol conjugated molecules (7, 8) in surfactant media
data could not be resolved to generate a best fit as a result of heavy
quenching of the free fluorophore in these media. Based on the
structure of our molecule, it is likely that the cholesterol end favours
the non-polar domain and dimethyl amino moiety favours polar
domain.
4. Conclusions
The investigation describes the synthesis and fluorescence
spectroscopic investigations of cholesterol free [(7), (8)] and choles-
terol conjugated donor–acceptor diphenylpolyenes [(10), (11)].
The diphenylpolyene moiety containing dimethylamino group as
a donor and a nitro group as an acceptor is extrinsically linked
to the 3-hydroxyl position via a carbonate linker. The spectro-
scopic investigations in homogeneous solvents reveal preservation
of charge-transfer emission for these molecules despite choles-
terol modification. The fluorescence emission behaviour of these
molecules in surfactant media indicates assistance of cholesterol

26
V. Palakollu, S. Kanvah / Journal of Photochemistry and Photobiology A: Chemistry 281 (2014) 18–26
in preferentially placing the fluorophore to less polar locales of the
micelle yielding a bi-exponential decay profile. Thus cholesterol
conjugated fluorophores can be excellent biological microenvi-
ronment distinguishing reporters [22,47]. This study, although
limited to cholesterol conjugation, can pave the way for similar
donor–acceptor based fluorophore conjugated biological receptors
to probe complex biological media.
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The authors appreciate financial grant from the Department
of Science and Technology, India and Council of Scientific and
Industrial Research (CSIR), India (01(2487)/11/EMR-II). The time-
resolved experiments were performed at Department of Chemistry,
Indian Institute of Technology Bombay, India. The authors grate-
fully acknowledge Prof. Anindya Datta’s and Mr. Avinash’s kind
co-operation and assistance for the time-resolved experiments.
Supplementary material related to this article can be
found, in the online version, at http://dx.doi.org/10.1016/
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