Synthesis, characterization and photophysics of new photoactive esipt lipophilic dyes. partition experiments with different composed liposomes

Article abstract of DOI:10.1016/j.dyepig.2014.04.024

New lipophilic dyes were synthesized from a fluorescent precursor and used to produce photoactive liposomes and ethosomes made of different phospholipids. The lipophilic dyes absorbed in the ultraviolet region due to π-π* transitions and had a dual fluorescence emission in the violet-yellow regions, depending on solvent polarity. The long alkyl chain lipophilic dyes had a narrow emission band with a very regular shape, and their photophysical behaviour indicates that these dyes were inserted into the lipid bilayer. The dye with short alkyl chain appeared to be in a polar environment, which indicated this fluorophore experienced a more hydrophobic environment. Fluorescence titration experiments were also performed with this compound and the partition coefficient was determined, which showed higher values than those found in the literature for similar intramolecular proton transfer dyes, likely due to better interaction between this dye and the phospholipids, due to the alkyl chain present in the dyes.

Full text of DOI:10.1016/j.dyepig.2014.04.024

Dyes and Pigments xxx (2014) 1e9
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis, characterization and photophysics of new photoactive
ESIPT lipophilic dyes. Partition experiments with different composed
liposomes
*
*
Felipe Lange Coelho, Fabiano Severo Rodembusch , Leandra Franciscato Campo
Grupo de Pesquisa em Novos Materiais Orgânicos e Fotoquímica, Instituto de Química, Departamento de Química Orgânica, Universidade Federal do Rio
Grande do Sul, UFRGS, CEP 91501-970 Porto Alegre, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
New lipophilic dyes were synthesized from a fluorescent precursor and used to produce photoactive
liposomes and ethosomes made of different phospholipids. The lipophilic dyes absorbed in the ultra-
violet region due to pep* transitions and had a dual fluorescence emission in the violeteyellow regions,
Received 2 March 2014
Received in revised form
8 April 2014
Accepted 16 April 2014
Available online xxx
depending on solvent polarity. The long alkyl chain lipophilic dyes had a narrow emission band with a
very regular shape, and their photophysical behaviour indicates that these dyes were inserted into the
lipid bilayer. The dye with short alkyl chain appeared to be in a polar environment, which indicated this
fluorophore experienced a more hydrophobic environment. Fluorescence titration experiments were also
performed with this compound and the partition coefficient was determined, which showed higher
values than those found in the literature for similar intramolecular proton transfer dyes, likely due to
better interaction between this dye and the phospholipids, due to the alkyl chain present in the dyes.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probes
Liposome
Ethosome
Membrane probes
Lipophilic dyes
ESIPT
1. Introduction
very similar composition, shape, chemical and physical properties
[6,7], which allow for the extension of results obtained in liposomes
Since the discovery by Bangham et al. which showed that
phospholipids could self-assemble in aqueous solutions to form
highly-organized structures (liposomes) [1], the interest in these
artificially-prepared vesicles has increased considerably due to
their properties. Liposomes are defined as microscopic spherical-
shaped vesicles formed by one or more multiple concentric phos-
pholipid bilayers composed with an aqueous interior, which allow
interaction with hydrophilic or hydrophobic molecules by encap-
sulation in the aqueous interior or entrapment within the lipid
bilayer, respectively [2]. Liposomes can have differential perme-
ability, where low-polarity molecules tend to partition into interior
more readily, and thus permeate faster. On the other hand, polar
compounds permeate the membrane more slowly, and very polar
solutes do not permeate at all (over several minutes) [3]. Addi-
tionally, these structures can also present high chemical and
physical stability [4]. Based on these properties, liposomes can
mimic cell membranes [5], as liposomes and cell membranes have
to cell membranes [8].
Fluorescence spectroscopy has been extensively applied since
the late 1960s to study different aspects of membrane-related
phenomena [9e11]. This technique provides information about
the organization, motion and surface of the membrane, which
helps understand the motion of fatty acyl chains, the dynamics of
phospholipids in the bilayer(s), as well as the distribution and
orientation of lipids and other components [12e18]. Furthermore,
some fluorescent features, such as a large Stokes’ shift, stable
fluorescence emission as well as thermal and photochemical sta-
bility [9,19] are needed to design new fluorescent membrane
probes; excited state intramolecular proton transfer (ESIPT) com-
pounds well fit these requirements [20,21]. The ESIPT mechanism is
due to the absorption of radiation from an enol-cis conformer (1E),
which is usually the most stable conformer in the ground state
(solution or solid state). In the excited state, this conformer un-
dergoes ESIPT to form a keto tautomer (1K*) giving rise to an
emission with a large Stokes’ shift. Additional conformers such as
the enol open structure (Fig. 1, right) do not undergo ESIPT [22].
These non ESIPT species are responsible for short wavelength
emission bands which can compete with the keto tautomer, and
evidence of this conformational equilibrium in the ground state can
* Corresponding authors. Tel.: þ55 51 3308 6299; fax: þ55 51 3308 7304.
E-mail addresses: rodembusch@gmail.com, rodembusch@iq.ufrgs.br, fabiano.
rodembusch@ufrgs.br (F.S. Rodembusch), leandra.campo@ufrgs.br (L.F. Campo).
http://dx.doi.org/10.1016/j.dyepig.2014.04.024
0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Coelho FL, et al., Synthesis, characterization and photophysics of new photoactive ESIPT lipophilic dyes.
Partition experiments with different composed liposomes, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.04.024

2
F.L. Coelho et al. / Dyes and Pigments xxx (2014) 1e9
responses such as wavelength characteristics of the mono-
chromator or detector using Rhodamine B as an external standard
(quantum counter). All experiments were performed at room
temperature (25 ꢀC) at
a M concentration (Electronic
10ꢁ5
Supporting Information, Table ESI1). The quantum yield of fluo-
rescence (Ffl) was measured at 25 ꢀC using spectroscopic grade
solvents in the optical dilute methodology. Quinine sulfate (Riedel)
was used as the quantum yield standard [24].
2.2. Synthesis
2.2.1. 2-(50-Amino-20-hydroxyphenyl)benzoxazole (3)
The aminobenzoxazole 3 was obtained in a condensation reac-
tion of equimolar amounts of 5-aminosalicylic acid (1) and 2-
aminophenol (2) in polyphosphoric acid (PPA) at 180 ꢀC for 5 h,
followed by TLC using dichloromethane as the eluent. The reaction
mixture was poured onto ice, the precipitate filtered, neutralized
with NaHCO3 (20%) and dried at 60 ꢀC. The material was purified by
column chromatography (eluted with dichloromethane) to yield
the corresponding aminobenzoxazole 3. Yield: 74%. Yellow powder,
m.p.: 174e175 ꢀC (Lit. 173e175 ꢀC [25]). FTIR (KBr, cmꢁ1): 3410 (nas
Fig. 1. Simplified photophysics of the ESIPT mechanism illustrated by the enol-cis
conformer from the 2-(50-amino-20-hydroxyphenyl)benzoxazole (left).
A normal
emission from an enol open conformer was also presented for comparison (right).
NH2); 3330 (ns NH2), 1630 and 1545 (
300 MHz, TMS):
n
C]C). 1H NMR (CDCl3,
d
(ppm) ¼ 10.94 (broad s, 1H, OH), 7.83e6.74 (m,
be related to the dual fluorescence emission of these compounds
[22].
7H, AreH), 3.54 (broad s, 2H, NH2). Anal. Calcd. for C13H10N2O2: C,
69.02; H, 4.46; N, 12.38%; Anal. Found: C, 69.06; H, 4.56; N, 12.06%.
This work described the synthesis of new lipophilic ESIPT dyes,
their photophysical behaviour in solution and their efficiency as
membrane probes after incorporation into liposomes and etho-
somes. Ethosomes are vesicular systems embodying ethanol in
relatively high concentrations, and are very efficient at enhancing
the skin permeation of a number of drugs as already reported in the
literature [23]. Additionally, the partition coefficient (Kp) of these
probes was calculated and discussed.
2.2.2. Synthesis of 2-(50-N-octylamino-20-hydroxyphenyl)
benzoxazole (4)
Compound 3 (0.61 g, 2.65 mmol), iodooctane (1.27 g, 5.3 mmol)
and potassium carbonate (0.36 g, 2.65 mmol) in 2-butanone
(15 mL) were mixed and stirred for 18 h at reflux temperature.
The solvent was removed under reduced pressure and a dark-
brown oil was obtained which was purified by column chroma-
tography using chloroform: hexane (1:1) as the eluent. Yield: 45%.
2. Experimental
Dark-yellow crystals, m.p.: 92e94 ꢀC. FTIR (KBr, cmꢁ1): 3440 (
n
OH),
3400 (
n
NH), 2930 (n CeH), 2840 (n
CeH). 1H NMR (CDCl3, 300 MHz,
2.1. Materials and methods
TMS):
d
(ppm) ¼ 10.89 (broad s, 1H, OH), 7.75e7.67 (m, 1H, AreH),
7.63e7.55 (m, 1H, AreH), 7.40e7.32 (m, 2H, AreH), 7.24e7.19 (m,
1H, AreH), 7.01e6.95 (m, 1H, AreH), 6.83e6.76 (m, 1H, AreH), 3.14
(t, 2H, NeCH), 2.84 (broad s, 1H, NH), 1.64 (m, 3H, CH), 1.34 (m, 8H,
CH), 0.89 (t, 3H, CH). 13C NMR (CDCl3, 75.4 MHz, TMS):
All reagents were obtained from Aldrich and used without
further purification. Chicken egg L-a-phosphatidylcholine (ECPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-
dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased
from Avanti Polar Lipids, Inc. and used without purification. Chro-
matographic purification of products was accomplished using 230e
400 mesh silica gel and thin-layer chromatography (TLC) was per-
formed on 0.20 mm silica gel plates (Whatman Al Sil G/UV). All
solvents were used as received or purified using standard pro-
cedures, while spectroscopic grade solvents (Merck or Aldrich)
were used for fluorescence and UVeVis measurements. Melting
points were measured with a Thermolyne apparatus and were
uncorrected. 1H and 13C NMR spectra were recorded on a Varian
Inova 300 MHz spectrometer or Varian VNMRs 300 MHz spec-
d
(ppm) ¼ 162.9, 152.0, 149.1, 140.7, 140.2, 125.38, 124.9, 121.2, 119.2,
118.1, 110.6, 110.3, 109.8, 44.9, 31.7, 29.5, 29.3, 29.0, 27.13, 22.6, 14.0.
Anal. Calcd. for C21H26N2O2: C, 74.25; H 7.96; N 7.90%; Anal. Found:
C, 74.55; H, 7.69; N, 8.28%.
2.2.3. Synthesis of 2-(50-N-cis-9-octadecenamide-20-
hydroxyphenyl)benzoxazole (5)
Oleic acid (0.497 g, 1.76 mmol), N,N0-dicyclohexylcarbodiimide
(DCC) (0.439 g, 2.13 mmol) and a catalytic amount of de 4-N,N-
dimethylaminopyridine (DMAP) in dry dichloromethane (30 mL)
were mixed and the reaction stirred under argon for 3 h at room
temperature (25 ꢀC). To this reaction mixture, the benzoxazole
precursor 3 (0.400 g,1.76 mmol) was added and stirred overnight at
room temperature. The precipitate, mostly composed by DCU, was
removed by filtration and washed with dichloromethane
(3 ꢂ 20 mL). The organic layers containing the product were
combined, washed with water (4 ꢂ 50 mL), dried (MgSO4),
concentrated under reduced pressure and the residue purified by
column chromatography, eluting with dichloromethane. Yield:
trometer. The chemical shifts were expressed as d (ppm) relative to
tetramethylsilane (TMS) as the internal standard and using CDCl3 or
CF3COOD (as the solvents) at room temperature. Data for 1H NMR
were reported as follows: multiplicity (s ¼ singlet, d ¼ doublet,
t ¼ triplet, m ¼ multiplet), integration, coupling constant (Hz) and
assignment. Data for 13C APT were reported in terms of chemical
shift (ppm) and infrared spectra were recorded on a Varian 640-IR
using KBr pellets. Elemental analysis was performed in a Perkine
Elmer CHN 2400, UVeVis absorption spectra were determined
using a Shimadzu UV-2450 spectrophotometer and steady state
fluorescence spectra were measured using a Shimadzu spectroflu-
orometer model RF-5301PC. Spectrum correction was performed to
allow measurements of true spectra by eliminating instrumental
50%. White powder, m.p.: >300 ꢀC. FTIR (KBr, cmꢁ1): 3281 (
2921 ( CeH), 2853 ( CeH), 1709 ( C]C), 1648 ( C]O), 1531 (
C]C), 1451 ( C]C), 1250 (
CeN). 1H NMR (CDCl3, 300 MHz, TMS):
(ppm) ¼ 11.31 (broad s,1H, OH), 8.35 (s,1H, NH), 7.73e7.66 (m,1H,
AreH), 7.60e7.53 (m, 1H, AreH), 7.47e7.32 (m, 4H, AreH), 7.08e
n NH),
n
n
n
n
n
n
n
d
Please cite this article in press as: Coelho FL, et al., Synthesis, characterization and photophysics of new photoactive ESIPT lipophilic dyes.
Partition experiments with different composed liposomes, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.04.024

F.L. Coelho et al. / Dyes and Pigments xxx (2014) 1e9
3
7.02 (m, 1H, AreH), 5.34 (t, 2H, C]CeH), 2.39 (t, 2H, COCeH), 2.00
(t, 2H, CeH), 1.76 (m, 2H, CeH), 1.32 (m, 25H, CeH), 0.88 (t, 3H, Ce
Partition experiment were performed using a titration method
where the dye solution (1 mL) was added to different concentra-
tions of liposomes (1 mL) in water (pH 7.0) to yield solutions with
phospholipid: dye ratios of 1e50. After the addition of dye, the
solutions was kept at 20 ꢀC for 1 h before experiments. Blank so-
lutions were prepared by adding pure methanol instead of dye to
the liposome solutions.
H). 13C NMR (CDCl3, 75.4 MHz, TMS):
d
(ppm) ¼ 171.6, 162.5, 155.5,
149.1, 139.9, 130.1, 130.0, 129.7, 126.2, 125.5, 125.0, 119.2, 118.8, 117.7,
110.8, 110.3. Anal. Calcd. for C31H42N2O3: C, 75.88; H, 8.63; N, 5.71%;
Anal. Found: C, 76.34; H, 9.33; N, 5.93%.
2.2.4. Synthesis of 2-(50-N-octadecanamide-20-hydroxyphenyl)
benzoxazole (6)
3. Results and discussion
Lipophilic derivative 6 was prepared using two different meth-
odologies. Method 1: Stearic acid (0.629 g, 2.21 mmol), N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDCI)
(0.429 g, 2.21 mmol) and a catalytic amount of 4-N,N-dimethyla-
minopyridine (DMAP) in dry dichloromethane (30 mL) were stirred
under argon for 3 h at room temperature (25 ꢀC), 3 (0.500 g,
2.21 mmol) added and stirred overnight at room temperature. The
solid was separated by filtration and TLC analysis showed no
product in filtrate. The residue, composed mainly of urea and
product, was suspended in 1 L of water, stirred for 12 h and pure
product obtained by filtration. Method 2: In a round-bottom flask
equipped with a condenser, stearic acid (0.500 g, 1.76 mmol) and
thionyl chloride (0.500 mL, 6.85 mmol) were stirred under reflux
for 6 h. The excess thionyl chloride was removed by evaporation
followed by the addition of 3 (0.400 g, 1.76 mmol) and triethyl-
3.1. Synthesis
The aminobenzazole
3 was prepared using a previously
described methodology [25], which involved the condensation of
an equimolar amount of 5-aminosalicylic acid (1) with the o-
substituted aniline 2 in polyphosphoric acid (PPA) (Scheme 1).
The lipophilic derivatives 4e6 were prepared according to
Scheme 2. The procedure for the synthesis of dye 4 consisted of a
bimolecular nucleophilic reaction using iodooctane as the alkylat-
ing agent in 2-butanone [32,33]. Derivatives 5 and 6 were prepared
by the Steglich esterification method using dicyclohex-
ylcarbodiimide and 4-N,N-dimethylaminopyridine as reactants
[34]. Dye 5 was prepared by the reaction of 3 with oleic acid using
DCC/DMAP as coupling reagents [35,36]. Despite the use of deac-
tivated carboxylic acid, this methodology provided good yields,
though the urea (by-product) was difficult to remove. As the urea
could not be removed by washing, column chromatography was
performed twice to obtain product free from urea. A similar strat-
egy was applied to 6, modifying the coupling agent, as the by-
product formed by EDCI was water-soluble [37], where urea was
removed by stirring the crude product with water followed by
filtration. An alternative methodology was also tested, where the
carboxylic acid was converted in acyl chloride [38] using SOCl2
(Method 2), followed by reaction with 3 to form the desired amide
lipophilic dye 6 at a low yield.
amine (200 mL) in dry dichloromethane (20 mL). The reaction was
stirred overnight at room temperature (25 ꢀC), the precipitate
filtered and washed with water to yield compound 6. Yield: 50%.
White powder, m.p. >300 ꢀC. FTIR (KBr, cmꢁ1): 3295 (
n
NH), 2921 (
C]C), 1470 ( C]C),
CeN). 1H NMR (CF3COOD, 300 MHz, TMS):
(ppm) ¼ 11.66
n
CeH), 2853 (
n
CeH), 1648 (
n
C]O), 1540 (
n
n
1256 (
n
d
(broad s, 1H, OH), 8.87 (s, 1H, NH), 8.21e8.10 (m, 2H, AreH), 8.07e
7.92 (m, 4H, AreH), 7.62e7.54 (m, 1H, AreH), 2.89 (t, 2H, COCeH),
2.09 (m, 2H, CeH), 1.78e1.41 (m, 28H, CeH), 1.09 (t, 2H, CeH). 13
C
NMR (CF3COOD, 75.4 MHz, TMS):
d
(ppm) ¼ 178.8, 161.3, 156.8,
148.0, 133.9, 130.2, 129.6, 128.9, 127.6, 123.7, 118.3, 115.2, 112.3,
105.9, 36.4, 31.6, 29.3, 29.3, 29.3, 29.3, 29.2, 29.0, 29.0, 28.8, 28.8,
25.8, 22.2, 12.5. Anal. Calcd. for C31H44N2O3: C, 75.57; H, 9.00; N,
5.59%; Anal. Found: C, 75.54; H, 8.67; N, 5.67%.
3.2. Photophysical characterization of the lipophilic dyes
The UVeVis absorption spectra of lipophilic dyes 4e6 are shown
in Fig. 2 and relevant data from UVeVis absorption spectroscopy
are presented in Table 1.
Dyes 4e6 had absorption maxima at 385, 344 and 343 nm,
respectively. The molar absorptivity coefficient ε values, as well as
the calculated radiative rate constant (k0e) for all dyes indicated spin
2.3. Labelling of liposome for photophysical study
The liposomes were prepared based on a previously published
methodology [26,27], where stock solutions of the lipophilic dyes
4e6 were prepared in chloroform (w10ꢁ5 M) in which the lipid
(ECPD, DPPC or DOPC) (50e60 mg) was solubilized (round-bottom
flask). After total solubilization of the lipid, the solvent was
removed under reduced pressure, yielding a thin film deposited on
the inner walls of the round-bottomed flask. The films were hy-
drated with 4 mL water, dispersed by stirring and sonicated for
30 min.
allowed electronic transitions, which could be related to
pep*
transitions. No significant solvatochromism in the ground state was
observed. Dye 4 had an absorption maximum redshifted compared
to the precursor 3 [22], indicating that alkylation of the amino
group enhanced the electron-donating effect. It is well-known that
a qualitative understanding of the effects of substituents on the
position of the UVeVis spectrum can be considered by classifying
the substituents into electron-donating and electron-withdrawing
groups. Since dyes 5 and 6 possess electron-withdrawing amide
2.4. Labelling of ethosome for photophysical study
Ethosomes were prepared based on published methodologies
[23,28]. Ethosomes were composed of 1% lipid, 1% dye, 40% ethanol
and 58% water (w/w) where 1 mg of dye (4, 5 or 6) and lipid (ECPD,
DPPC or DOPC) were solubilized in ethanol (40 mL) and water
(60 mL) was added with stirring. The mixture was stirred for an
additional 10 min.
moieties, a decrease in the
p-system length takes place through
resonance, and shifts the absorption maxima to shorter wave-
lengths (when compared to 3). Additionally, the intense absorption
2.5. Labelling of liposomes for partition experiments
The partition experiments were performed as previously
described [29e31]. Stock solutions of each dye in methanol
(5.0 ꢂ 10ꢁ5 M) and ECPC, DPPC and DOPC liposomes were prepared.
Scheme 1. Synthesis of the 2-(50-amino-20-hydroxyphenyl)benzoxazol (3).
Please cite this article in press as: Coelho FL, et al., Synthesis, characterization and photophysics of new photoactive ESIPT lipophilic dyes.
Partition experiments with different composed liposomes, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.04.024

4
F.L. Coelho et al. / Dyes and Pigments xxx (2014) 1e9
Scheme 2. Synthesis of the lipophilic benzoxazoles 4e6, where (i) iodooctane, K2CO3, 2-butanone, reflux, 18 h; (ii) oleic acid, dicyclohexylcarbodiimide (DCC), 4-N,N-dimethy-
laminopyridine (DMAP), dichloromethane, overnight; Method 1: stearic acid, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDCI), 4-N,N-dimethylaminopyr-
idine (DMAP), dichloromethane, overnight; Method 2: stearic acid, SOCl2, reflux; NEt3, dichloromethane, overnight.
bands observed at 280e310 nm was observed in similar com-
pounds [22] and can be associated to the absorption of the ben-
zoxazole moiety [39].
The normalized fluorescence emission spectra of lipophilic dyes
4e6 are presented in Fig. 3, where emission curves were obtained
using the absorption maxima as the excitation wavelength, while
relevant data from fluorescence emissions are summarized in
Table 2.
At least for one solvent, the fluorescence spectra showed a dual
emission (as expected), since it is known that the ESIPT mechanism
in amino derivatives is dependent on solvent polarity [22]. The
emission at short wavelengths could be ascribed to normal
emission from an enol conformer (1E*), while the redshifted
emission located in the greeneyellow region was related to the keto
tautomer (1K*), which arises from the ESIPT in the excited state.
These results indicated the photophysics of these compounds were
complex, and the results from one benzoxazole fluorophore cannot
be extended to parent compounds, as usually observed for classical
aromatic photoactive dyes with different substituents [40]. In
ethanol, for instance, the precursor 3 had a single emission band
due to stabilization in the ground state of an enol conformer which
does not exhibit phototautomerism in the excited state. The
derivatization of this compound seemed to affect the photophysics
of the fluorophore benzoxazole, since 5 and 6 in the same solvent
showed ESIPT bands at longer wavelengths, indicating ethanol
could stabilize the enol cis conformer which would transfer a
proton in the excited state to produce the keto tautomer.
3.3. Photophysical characterization of the liposomes and ethosomes
Fig. 4 shows the electronic absorption spectra of ESIPT dyes 4e6
in ECPC, DPPC and DOPC liposomes and ethosomes. Upon incor-
poration, the absorption maxima of lipophilic dyes 5 and 6 did not
significantly change shape and spectral position. Additionally, the
phenylbenzoxazole derivatives can possess two distinct absorption
regions, the first at 280e310 nm (ascribed to the benzoxazole
chromophore) and the other above 330 nm
(p-extended
Table 1
Relevant photophysical data from the UVeVis spectra of the lipophilic dyes 4e6,
where labs is the absorption maxima, ε is the molar absorptivity coefficient, fe is the
calculated oscillator strength, k0e is the calculated radioactive rate constant and n0-0 is
the frequency of purely electronic transition.
Dye Solvent
labs (nm) ε (Mꢁ1 cmꢁ1
)
fe
ke0
n0-0 (cmꢁ1
)
4
5
6
Chloroform 380
Ethanol 380
1,4-Dioxane 391
Acetonitrile 388
Chloroform 341
6220
0.12 8.58 ꢂ 107 23,294
0.07 4.65 ꢂ 107 22,743
0.22 1.47 ꢂ 108 22,541
0.20 1.30 ꢂ 108 22,497
0.17 1.46 ꢂ 108 26,606
0.04 3.34 ꢂ 107 26,618
0.26 2.18 ꢂ 108 26,128
0.13 1.09 ꢂ 108 26,377
0.45 3.91 ꢂ 108 26,658
0.32 2.76 ꢂ 108 26,521
0.30 2.49 ꢂ 108 26,111
0.16 1.32 ꢂ 108 26,323
3189
11,288
9661
8246
2890
14,706
21,961
24,198
18,899
17,488
22,759
Ethanol
342
1,4-Dioxane 348
Acetonitrile 343
Chloroform 340
Ethanol
342
Fig. 2. Normalized UVeVis absorption spectra of the lipophilic ESIPT dyes 4e6. The
UVeVis spectra of the precursor 3 in MeCN (dash line) and EtOH (dot line) was also
presented for comparison.
1,4-Dioxane 348
Acetonitrile 343
Please cite this article in press as: Coelho FL, et al., Synthesis, characterization and photophysics of new photoactive ESIPT lipophilic dyes.
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F.L. Coelho et al. / Dyes and Pigments xxx (2014) 1e9
5
liposomes and ethosomes (Fig. 4), dyes 5 and 6 had lower FP (w1)
values (similar to those observed in solution), while dye 4 exhibited
a very particular photophysical behaviour. In liposomes, the
average value for FP was higher (w2) than those observed for the
parent compounds, while in ethosomes, despite having the same
absorption region, the spectra changed drastically and the planarity
factor increased to an average value of w6. In ECPC ethosomes, FP
reached the maximum value of 11.6, which indicated the phenyl
ring was not conjugated with the benzoxazole backbone in etho-
somes. Thus, the absorption spectrum may be described by two
independent absorption bands for each subunit, as observed for
similar structures [41]. These data suggested the intensity of the
higher absorption maxima was reinforced conjugation enabled by
the intramolecular hydrogen bond.
The fluorescence emission curves of lipophilic ESIPT dyes 4e6 in
liposomes and ethosomes are presented in Fig. 5. The curves were
obtained using the absorption maxima for each dye as the excita-
tion wavelength and relevant data from the liposome and etho-
some photophysical studies are summarized in Table 3.
Dyes 5 and 6 had one narrow emission band with a very regular
shape and location in liposomes and ethosomes (w500 nm, with a
Stokes’ shift w9500 cmꢁ1) ascribed to the ESIPT mechanism.
Although it is well-known the ESIPT mechanism in benzazoles is
dependent on solvent [22] and polarity microenvironments [43],
the surroundings provided by the different phospholipids in lipo-
somes or ethosomes, as well as the alkyl chain length of dyes 5 and
6, appeared to play a fundamental role in the photophysics of the
Fig. 3. Normalized fluorescence emission spectra of the lipophilic ESIPT dyes 4e6
(10ꢁ5 M). The fluorescence emission spectra of the precursor 3 in MeCN (dash line) and
EtOH (dot line) was also presented for comparison.
chromophore phenylbenzoxazole) [22,41], and the difference in
absorption band intensities can be associated with a difference in
planarity in some dyes [22,42]. The observed changes in absorption
band intensities for dyes 4e6 incorporated in liposomes and
ethosomes, can yield interesting results (as follows).
An empirical ratio could be calculated, so-called planarity factor
Fp by the equation FP ¼ Ibz/Imax, where Ibz is the intensity of the
absorption band located between 280 and 310 nm and Imax is the
intensity of the absorption maxima, where the lower the FP the
higher the planarity of the
p-extended chromophore. Considering
the non-normalized absorption spectra (Electronic Supporting
Information, Figs. ESI16e18), dyes 5 and 6 had a value of FP w1.6,
and dye 4 had a calculated value of w3.2 (Electronic Supporting
Information, Tables ESI3e5). This difference indicated that in so-
lution, dye 4 tended to be less planar than dyes 5 and 6. In
Table 2
Relevant photophysical data from the fluorescence emission spectra of the lipophilic
dyes 4e6, where lem is the emission maxima, DlST is the Stokes’ shift and Ffl is the
quantum yield of fluorescence.
Dye Solvent
Enol conformer
lem (nm) DlST (nm/cmꢁ1
91/5084.8
Keto tautomer
Ffl (ꢂ10ꢁ2
)
)
lem (nm) DlST (nm/cmꢁ1
)
4
5
6
Chloroform 471
Ethanol 481
1,4-Dioxane 464
Acetonitrile 486
Chloroform
Ethanol
1,4-Dioxane 391
Acetonitrile 391
568
e
188/8710.2
e
2.31
1.12
0.41
0.24
1.51
0.75
0.83
1.11
1.90
0.67
0.71
0.43
101/5525.8
73/4023.7
98/5197.1
e
587
587
509
510
518
516
505
511
516
516
196/8539.6
199/8737.4
168/9679.1
168/9631.9
170/9430.6
173/9774.7
165/10,031.9
169/9859.9
168/9355.8
173/9774.7
e
403
61/4425.9
43/3160.2
48/3579.1
e
Chloroform
Ethanol
e
Fig. 4. Normalized UVeVis absorption spectra of the lipophilic ESIPT dyes 4e6 in li-
posomes (left) and ethosomes (right), where ECPC, DPPC and DOPC are the phos-
406
64/4609.3
42/3094.6
45/3381.3
1,4-Dioxane 390
Acetonitrile 388
pholipids egg chicken L-
a-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine, respectively.
Please cite this article in press as: Coelho FL, et al., Synthesis, characterization and photophysics of new photoactive ESIPT lipophilic dyes.
Partition experiments with different composed liposomes, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.04.024

6
F.L. Coelho et al. / Dyes and Pigments xxx (2014) 1e9
Scheme 3. Pictorial representation of the lipophilic dyes 4e6 into the lipid bilayer of
liposomes (H2O in the interior) and ethosomes (ethanol in the interior), based on the
fluorescence emission profile of the lipophilic dyes.
benzoxazole fluorophore, which allowed these dyes to experience
an apolar environment. These results indicated the dyes were
inserted into the lipid bilayer without contact with the water or
ethanolic surroundings (Scheme 3). On the other hand, dye 4 had a
blueshifted band (compared to dyes 5 and 6) located around
477 nm, ascribed to the normal emission (DlST w5211 cmꢁ1),
despite the emission band at long wavelengths (w566 nm) being
related to the ESIPT mechanism. The photophysical behaviour
indicated that unlike the parent compounds, dye 4 experienced a
polar environment, which showed this fluorophore was located in a
more hydrophobic environment (Scheme 3) and may be associated
with the size of the alkyl chains of the dyes. Dyes 5 and 6 have
longer alkyl chains and were more similar to the lipid backbone
than dye 4, and the intermolecular van der Waals interactions were
favoured, keeping the dyes within the tangle of alkyl chains.
Additionally, stronger interactions between 5 and 6 and the phos-
pholipids (absent in dye 4) existed, since ECPC, DPPC and DOPC
(Electronic Supporting Information, Figs. ESI13e15) contained ester
groups in their structures which could be responsible for the
photophysical behaviour, and could present as dipoleedipole and
hydrogen bond interactions with the amides 5 and 6.
Fig. 5. Normalized fluorescence emission spectra of the lipophilic ESIPT dyes 4e6 in
liposomes (left) and ethosomes (right), where ECPC, DPPC and DOPC are the phos-
pholipids egg chicken L-a-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine, respectively.
Table 3
Relevant photophysical data from the UVeVis absorption and fluorescence emission
spectra of the liposomes and ethosomes, where labs and lem are the absorption and
emission maxima, respectively and DlST is the Stokes’ shift.
3.4. Fluorescence titration study
Fluorescence titration experiments were performed to study the
influence of phospholipid concentration [PL] (mol/L) on the fluo-
rescence intensity maxima of dyes. In the presence of phospholipid,
the fluorescence intensity of dye 6 remained constant (Fig. 6), not
seen in the absence of the phospholipid (also observed for dye 5,
Electronic Supporting Information, Figs. ESI19e21). These results
indicated that for the phospholipid concentrations (mM) studied,
these dyes interacted strongly with phospholipids, as at the lowest
concentration, the fluorescence intensity reached the maximum
value (maintained up to 2.5 mM), which was different from other
ESIPT dyes [31].
Dye Phospholipid labs (nm) Fluorescence emission
Enol conformer
Keto tautomer
lem (nm) DlST
lem (nm) DlST
(nm/cmꢁ1
)
(nm/cmꢁ1
)
Liposomes
4
5
6
ECPC
DPPC
DOPC
ECPC
DPPC
DOPC
ECPC
DPPC
DOPC
396
391
387
336
343
338
337
338
338
482
482
487
e
86/4505.6 581
91/4828.6 563
100/5305.9 559
e
e
e
e
e
e
185/8040.8
172/7813.5
172/7950.7
166/9841.6
159/9234.2
164/9665.5
165/9753.3
164/9665.5
164/9665.5
502
502
502
502
502
502
e
e
e
However, dye
4 had a fluorescence intensity which was
e
dependent on phospholipid concentration [PL] (Fig. 7), also seen for
DPPC and DOPC (but at lower concentrations, 1.0e2.5 mM)
(Electronic Supporting Information, Figs. ESI22 and 23). Addition-
ally, the enhanced fluorescence intensity of dye 4 exhibited satu-
ration at w1 mM PL (Fig. 8, top).
The partition coefficients (Kp) of dye 4 in ECPC, DPPC and DOPC
phospholipids were calculated from the slope of the double
reciprocal linear plot of 1/F vs. 1/[PC] according to 1/F ¼ 55.6/
(KpFmax[PC]) þ 1/Fmax, where Fmax was the maximum fluorescence
resulting from total probe incorporation into the membrane (Fig. 8,
e
Ethosomes
4
5
6
ECPC
DPPC
DOPC
ECPC
DPPC
DOPC
ECPC
DPPC
DOPC
379
366
373
339
340
341
342
338
348
459
477
473
e
80/4598.7
111/6358.0 563
100/5668.0 565
e
e
e
e
e
e
e
e
197/9560.4
192/9110.5
164/9617.8
163/9531.0
160/9365.4
161/9359.1
165/9705.1
153/8775.6
503
503
501
503
503
501
e
e
e
e
e
Please cite this article in press as: Coelho FL, et al., Synthesis, characterization and photophysics of new photoactive ESIPT lipophilic dyes.
Partition experiments with different composed liposomes, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.04.024

F.L. Coelho et al. / Dyes and Pigments xxx (2014) 1e9
7
Fig. 7. Fluorescence emission spectra of dye 4 in ECPC for a concentration range of
0.05e2.5 mM at 25 ꢀC. Dye 4 in absence of phospholipids was also presented for
comparison (light grey line).
bottom) [30,31]. From the slope and 1/F intercepts of this linear
plot, the Kp was found to be 6.67 ꢂ 105, 7.30 ꢂ 105 and 7.04 ꢂ 105 for
ECPC, DPPC and DOPC, respectively, at 25 ꢀC with an R2 w1
(Electronic Supporting Information, Table ESI6). The partition co-
efficient values found in this work were higher than those found in
the literature for ESIPT dyes [31,44], likely due to better interactions
between the dye and PC allowed by the alkyl chains.
4. Conclusions
In conclusion, three new fluorescent lipophilic dyes (4e6) were
synthesized from an ESIPT fluorescent precursor, and used to create
photoactive liposomes and ethosomes prepared from chicken egg
L-a
-phosphatidylcholine (ECPC), 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-
phosphocholine (DOPC). The dyes absorbed in the UV region
(w340e390 nm) which was related to * transitions, where no
pep
significant solvatochromism in the ground state was observed and
dual fluorescence emissions in the violeteyellow regions were
seen. The emission band at short wavelengths could be related to
normal emission from an enol conformer (1E*), and the long
wavelength, located in the greeneyellow region related to the keto
tautomer (1K*), which arose from the ESIPT in the excited state. The
lipophilic dyes 5 and 6 exhibited one narrow emission band with a
very regular shape and location in liposomes and ethosomes
ascribed to the ESIPT mechanism, which indicated the surround-
ings provided by the different phospholipids seemed to play a
fundamental role in the photophysics of these dyes, creating an
apolar environment. On the other hand, dye 4 seemed to be located
in a polar environment which suggested the fluorophore was
located in a more hydrophobic medium. Fluorescence titration
experiments performed to study the influence of the phospholipid
concentration on the fluorescence intensity indicated the partition
coefficient values obtained for dye 4 was higher than those found in
the literature for ESIPT dyes, and may be due to better interaction
between dye and phospholipids (ascribed to the alkyl chain pre-
sent). These data suggested a model for the interaction between
lipophilic ESIPT dyes and some liposomes/ethosomes which
Fig. 6. Fluorescence emission spectra of dye 6 in ECPC (top), DPPC (middle) and DOPC
(bottom) phospholipids for a concentration range of 0.05e2.5 mM at 25 ꢀC. Dye 6 in
absence of phospholipids was also presented for comparison (light grey line).
Please cite this article in press as: Coelho FL, et al., Synthesis, characterization and photophysics of new photoactive ESIPT lipophilic dyes.
Partition experiments with different composed liposomes, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.04.024

8
F.L. Coelho et al. / Dyes and Pigments xxx (2014) 1e9
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Partition experiments with different composed liposomes, Dyes and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.04.024