Amphiphilic ESIPT benzoxazole derivatives as prospective fluorescent membrane probes

Article abstract of DOI:10.1016/j.tetlet.2014.03.103

Fluorescent amphiphilic benzoxazole derivatives were synthesized and used to produce photoactive phosphatidylcholine (PC) liposomes by reserve-phase evaporation. The dyes absorbed in the UV region and were fluorescent in the blue-green region (determined

Full text of DOI:10.1016/j.tetlet.2014.03.103

Tetrahedron Letters 55 (2014) 3024–3029
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Amphiphilic ESIPT benzoxazole derivatives as prospective
fluorescent membrane probes
⇑
⇑
Priscila Franken Dick, 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:
Fluorescent amphiphilic benzoxazole derivatives were synthesized and used to produce photoactive
phosphatidylcholine (PC) liposomes by reserve-phase evaporation. The dyes absorbed in the UV region
and were fluorescent in the blue-green region (determined by solvent polarity). The alkyl chain length
seemed to play a fundamental role in the photophysics of the benzoxazole fluorophore in reverse
liposomes, and despite the same ESIPT core and phospholipid building block, each amphiphilic dye
had a particular emission profile related to the dye location in the liposome. The fluorescence emission
spectra from dye 5 showed that its fluorophore experienced a polar environment, due to the single nor-
mal emission, while dyes 6–7 had (in part) a normal emission, and the main fluorescent band ascribed to
the ESIPT emission indicated a more hydrophobic environment. Despite the complex fluorescent profiles,
the benzoxazole derivatives could be successfully introduced into the reverse liposome structure due to
the interaction between the alkyl chain and PC bilayer.
Received 3 March 2014
Revised 16 March 2014
Accepted 20 March 2014
Available online 31 March 2014
Keywords:
Fluorescent probes
Reverse liposome
Membrane probes
Reserve-phase evaporation method
Ó 2014 Elsevier Ltd. All rights reserved.
Fluorescence spectroscopy is widely used as a tool to study the
properties of lipid membranes1 and this technique can provide
information about organization, motion and surface aspects of
the membrane. Additionally, the permeability of membranes, as
well as lipid packing and diffusion, visualization of membrane
domains, elasticity,2 perturbation of a substrate insertion in mem-
branes3 and interfacial polarity4 can be investigated. In order to
obtain these parameters, fluorescent probes can be used, interact-
ing with the membrane lipids or even part of its structure.5 Several
different dyes have been reported in the literature,6 including
probes containing pyrene derivates,7 anthrylvinyl and perylenoyl
moieties,8,9 diphenylhexatriene groups (DPH),9 6-lauryl-2-dimeth-
ylaminonaphthalene (Laurdan) and most popular 7-nitrobenz-2-
oxa-1,3-diazol-4-yl (NBD),10 which contained alkyl chains of
different lengths which behaved as an aggregation agent with
the membrane.11
hydrophobic–hydrophilic environments in systems using polymers
for drug delivery.14 As shown in Figure 1, this proton transfer
mechanism starts with the absorption of radiation from an enol-
cis conformer (1E) (usually the most stable conformer in the ground
state) and in the excited state, this conformer undergoes ESIPT to
form a keto tautomer (1K⁄) giving rise to an emission with a large
Stokes shift. Additional conformers are described which do not
undergo ESIPT15 and these species are responsible for short wave-
length emission bands, which can compete with the ESIPT-exhibit-
ing dyes. Evidence of these conformational equilibria in solution in
the ground state were indicated by dual fluorescence emissions.15
Aggregation studies of photoactive probes within membranes
can be performed using synthetic liposomes, consisting of
phospholipid layers which can simulate the comportment of cellu-
lar membranes.16 The addition of a fluorescent probe to the
phospholipid solution during vesicle formation led to a doped lipo-
some, where the probe was incorporated into the structure, useful
in evaluating probe-membrane interactions by fluorescence spec-
troscopy.17 This work reported the synthesis of three new amphi-
philic ESIPT probes, their photophysical behaviour in solution
and their incorporation into reverse liposomes.
Amphiphilic fluorescent dyes with a large Stokes’ shift and
stable fluorescence emission as well as thermal and photochemical
stability, can be used as potential membrane probes. Excited state
intramolecular proton transfer (ESIPT) compounds fit these
requirements,12,13 and are used (for instance) to investigate
The benzazole precursor 3 was prepared using a previously
described methodology involving a condensation of an equimolar
amount of 2-aminophenol (1) with 5-aminosalicylic acid (2) in
polyphosphoric acid (PPA) at 180 °C for 5 h.18 The reaction was
accompanied by TLC using dichloromethane as eluent. The reaction
⇑
Corresponding authors. Tel.: +55 51 3308 6299; fax: +55 51 3308 7304 (F.S.R.).
Tel.: +55 51 3308 7199; fax: +55 51 3308 7304 (L.F.C.).
E-mail addresses: fabiano.rodembusch@ufrgs.br (F.S. Rodembusch), leandra.
campo@ufrgs.br (L.F. Campo).
http://dx.doi.org/10.1016/j.tetlet.2014.03.103
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

P. Franken Dick et al. / Tetrahedron Letters 55 (2014) 3024–3029
3025
produce 5. The general methodology consisted in stirring a mixture
of 4 and N,N-dimethyloctadecylamine in toluene in ice bath for
30 min, followed by an increase in the temperature until reflux
for 48 h. The solvent was removed under reduced pressure and
the residue was washed with hexane to remove small portions of
the unreacted aliphatic amine to yield 5.21
The same strategy was applied to derivatives 6 and 7, using
1-iodododecane and 1-iodooctane in 2-butanone, respectively, as
alkylating agents through a bimolecular nucleophilic substitution
reaction.22,23 The reactions were stirred under reflux for 18 h.
The solvent for each reaction was removed under reduced pressure
and yellow oils were obtained, which were purified by column
chromatography using silica gel and a mixture of chloroform/
hexane as eluent mixture (1:1) to yield 6 and 7.
The UV–vis absorption emission spectra were determined in
different dielectric constant organic solvents (Fig. 2) and all the
Figure 1. Simplified photophysics of the ESIPT mechanism (right). Illustrated by
2-(20-hydroxyphenyl)benzoxazole (HBO). A normal emission was also presented for
comparison (left).
mixture was poured into ice and the obtained precipitate was fil-
tered, neutralized with sodium carbonate and dried at room tem-
perature. The product 3 was purified on a silica gel column using
dichloromethane as eluent. The amphiphilic derivatives 5–7 were
prepared according to Scheme 1.19
The procedure for the synthesis of the amphiphilic derivative 5
consisted of a ring-opening reaction between epichlorohydrin and
the amino derivative 3, where an ethanolic solution of the benzox-
azole precursor 3 was added dropwise in epichlorohydrin at 45 °C.
After addition, the mixture was refluxed for 24 h. The solvent was
removed under reduced pressure and the residue was purified by
column chromatography using silica gel and a mixture of chloro-
form/acetone (1:1) as eluent to yield N-3-chloro-2-hydroxypropyl
derivative 4.20 The second step was an N-alkylation of the
N,N-dimethyloctadecylamine using 4 as an alkylating agent to
Figure 2. Normalized UV–vis absorption spectra of the amphiphilic ESIPT deriva-
tives 5–7. The UV–vis spectra of the precursor in MeCN were also presented for
comparison (dash line).
Table 1
Relevant photophysical data from the UV–vis spectra of the amphiphilic derivatives
5–7, where kabs is the absorption maxima (nm) and
e is the molar absorptivity
coefficient (103 L molꢀ1 cmꢀ1
)
Dye
Solvent
kabs (e)
5
1,4-Dioxane
Chloroform
AcOEt
Ethanol
Acetonitrile
382 (1.82); 307sh; 296 (5.90)
373 (2.60); 306sh; 295 (7.00)
384 (2.12); 307sh; 295 (6.60)
375 (1.64); 307sh; 296 (5.74)
379 (1.12); 306sh; 294 (4.40)
6
7
1,4-Dioxane
Chloroform
AcOEt
Ethanol
Acetonitrile
389 (3.38); 309sh; 297 (10.6)
335 (8.12); 324 (8.74); 294 (8.80)
390 (3.98); 309sh; 295 (13.3)
380 (1.76); 309sh; 296 (6.12)
387 (4.66); 307sh; 295 (15.2)
1,4-Dioxane
Chloroform
AcOEt
Ethanol
Acetonitrile
389 (3.96); 309sh; 297 (12.6)
337 (9.76); 324 (10.4); 295 (10.6)
388 (2.76); 308sh; 296 (9.48)
378 (2.60); 339 (2.54); 296 (9.00)
385 (2.66); 309sh; 296 (9.20)
Scheme 1. Synthesis of the amphiphilic benzoxazole derivatives 5–7, where (i)
polyphosphoric acid, 5 h; (ii) 1-iodooctane, K2CO3, 2-butanone, reflux, 18 h; (iii) 1-
iodododecane, K2CO3, 2-butanone, reflux, 18 h; (iv) epichlorohydrin, ethanol, reflux,
24 h; (v) N,N-dimethyloctadecylamine, toluene.

3026
P. Franken Dick et al. / Tetrahedron Letters 55 (2014) 3024–3029
Table 2
experiments were performed at room temperature (25 °C) at a con-
centration of 10ꢀ5 M. The relevant data from UV–vis analyses are
summarized in Table 1. The dyes had absorption maximum at
Relevant photophysical data from fluorescence emission spectra of the amphiphilic
derivatives 5–7 (10ꢀ5 M), where kem is the emission maxima (nm) and
kST is the
Stokes’ shift (nm/cmꢀ1
D
)
ꢁ377 nm with molar absorptivity coefficient
e values in agreement
with
p–
p⁄ transitions (Fig. 2). No significant solvatochromism in
Dye
Solvent
Fluorescence emission
Enol
Keto
the ground state was observed for these dyes. Moreover, the
absorption maxima for derivatives 6 and 7 were red-shifted
20 nm (compared to precursor 3),15 which indicated that alkylation
of the amino groups enhanced the electron donating effect. How-
ever, a similar photophysical behaviour could not be observed for
dye 5, likely due to the presence of the hydroxyl group in the alkyl
chain, where the donating effect was reduced. In chloroform, dyes
6 and 7 had blue-shifted ꢁ50 nm absorption maxima (not seen for
other solvents), and the amino group did not appear to contribute
significantly to the electronic content of these dyes, as the absorp-
tion maxima region and shape were similar to 2-(20-hydroxy-
phenyl)benzoxazole (HBO).24
Figure 3 shows normalized fluorescence emission spectra of
derivatives 5–7, which were obtained using the absorption max-
ima as the excitation wavelengths. The relevant data from fluores-
cence emission are summarized in Table 2. As expected, dual
fluorescence emissions could be observed for all dyes.15 Despite
the presence of the same fluorophore in all dyes, it can be seen
these compounds behaved independently, which indicated the
photophysical behaviour could not be extended to parent struc-
tures, or even solvents with similar polarities. The precursor in ace-
tonitrile (dashed line) showed a dual fluorescence emission, a band
at ca. 581 nm attributed to an excited keto tautomer (keto emis-
sion) due to the enol-cis conformer in an the excited state, and a
blue-shifted band at ca. 458 nm due to additional enol conformers
stabilized in solution with normal relaxation (enol emission).25
With respect to the amphiphilic characteristic of these deriva-
tives, additional fluorescence emission spectra (Fig. 4) were
obtained (in the same solvents) for more dilute solutions
(10ꢀ6 M) to observe the influence of the dye concentration on the
kem
D
kST
kem
DkST
5
1,4-Dioxane
Chloroform
AcOEt
499
454
455
479
481
117:6138
79:4640
71:4064
106:5933
102:5595
558
565
567
567
561
176:8257
190:8968
183:8405
194:9173
182:8560
Ethanol
Acetonitrile
6
7
1,4-Dioxane
Chloroform
AcOEt
466
—
474
488
484
77:4248
—
84:4544
108:5824
97:5179
568
490
565
—
179:8101
155:9443
175:7942
—
Ethanol
Acetonitrile
566
179:8172
1,4-Dioxane
Chloroform
AcOEt
457
—
481
469
470
68:3825
—
93:4983
91:5133
85:4697
553
488
559
—
164:7624
151:9182
171:7884
—
Ethanol
Acetonitrile
561
176:8149
ESIPT mechanism, which could relate the ESIPT bands (Fig. 3) to
solvent or hydrophobic environment effects. The fluorescence
emission was different for all dyes in ethanolic media, where a
blue-shifted main emission (normal emission) was seen. The dilute
solution allowed a better interaction with the solvent due to inter-
molecular hydrogen-bonding which stabilized the non-ESIPT con-
formers. Although the photophysical behaviour in ethanol
seemed to be particular, it was expected, as ESIPT compounds
can exhibit specific and strong interactions with polar protic
solvents. Additionally, only dye 5 appeared to be affected by solu-
tion concentration due to its larger alkyl chain, which in more
Figure 3. Normalized fluorescence emission spectra of the amphiphilic ESIPT
derivatives 5–7 (10ꢀ5 M). The fluorescence emission spectra of the precursor in
MeCN were also presented for comparison (dash line).
Figure 4. Normalized fluorescence emission spectra of the amphiphilic ESIPT
derivatives 5–7 in dilute solution (10ꢀ6 M). The fluorescence emission spectra of the
precursor in MeCN were also presented for comparison (dash line).

P. Franken Dick et al. / Tetrahedron Letters 55 (2014) 3024–3029
3027
Figure 5. Normalized UV–vis absorption spectra of the amphiphilic ESIPT deriva-
tives 5–7 in liposome suspension in a dye concentration of 10ꢀ5 M.
Scheme 2. Representation of the amphiphilic dyes 5 (left) and 6 (right) into the
lipid bilayer in reverse liposomes based on the fluorescence emission profile.
concentrated solutions can provide a more hydrophobic environ-
ment allowing the observed redshifts (Fig. 3) in spite of the fluores-
cence maxima at 10ꢀ5 M.
the excited state was solvent- and polarity-dependent,30–32
achieved in this study by the location of the dye in the reverse lipo-
some tailored by the alkyl chain length. All amphiphilic compounds
had at least one emission band located around 475 nm, ascribed to
The reverse liposomes containing probes 5–7 were prepared
from a chloroform solution of purified soy lecithin phosphatidylcho-
line (PC)26,27 by reserve-phase evaporation.28,29 Figure 5 shows the
UV–vis spectra of the dyes in reverse liposome media, where the
absorption maxima from the dyes could not be clearly observed.
The UV–vis spectra exhibit a monotonic featureless decrease in
absorbance with increasing wavelength, despite differences in
intensity among the dyes and an absorption-like band for dye 7
located at 425 nm (likely related to the benzoxazole chromophore).
The photophysical behaviour of dyes 5–7 are presented in
Figure 6. The spectra were obtained using an average of the absorp-
tion maxima for each dye as the excitation wavelength (ca. 377 nm).
Changes in the alkyl chain-length seemed to play a fundamental
role in the photophysics of the benzoxazole fluorophore for this
phospholipid, and despite having the same ESIPT benzoxazole core
and phospholipid building block, each amphiphilic dye had a partic-
ular emission profile. This complex photophysical behaviour could
be related to the ESIPT mechanism, since this phototautomerism in
normal emission (
additional red-shifted band could be observed for dyes 6 and 7
kST = 173 nm/8343 cmꢀ1 and 223 nm/9859 cmꢀ1, respectively),
D
kST lower than 100 nm/5473 cmꢀ1), while an
(D
ascribed to the ESIPT mechanism. This photophysical behaviour
indicated all dyes experienced a polar environment due to the
blue-shifted emission and that the fluorophore of compounds 6
and 7 was located in a more hydrophobic environment (Scheme 2).
Figure 7. Fluorescence emission spectra of 2.5 ꢂ 10ꢀ5 M of 5 (top), 6 (middle) and 7
(bottom) in PC liposome for a concentration of lipid in the range 5–200 lM at 25 °C,
Figure 6. Normalized fluorescence emission spectra of the amphiphilic ESIPT
derivatives 5–7 in reverse liposomes in a dye concentration of 10ꢀ5 M.
kex = 379 nm (5), 376 nm (6) and 375 nm (7). The inset plots of fluorescence
maxima intensity with varying PC concentrations.

3028
P. Franken Dick et al. / Tetrahedron Letters 55 (2014) 3024–3029
5. Maier, O.; Oberle, V.; Hoekstra, D. Chem. Phys. Lipids 2002, 116, 3–18.
6. Kwiatek, J. M.; Owen, D. M.; Abu-Siniyeh, A.; Yan, P.; Loew, L. M.; Gaus, K. PLoS
ONE 2013, 8, e52960.
7. Subuddhi, U.; Haldar, S.; Sankararaman, S.; Mishra, A. K. J. Photochem.
Photobiol., A Chem. 2008, 200, 381–387.
8. Bergelson, L. D.; Molotkovsky, J. G.; Manevich, Y. M. Chem. Phys. Lipids 1985, 37,
165–195.
9. Trevors, J. T. J. Biochem. Biophys. Methods 2003, 57, 87–103.
10. Loura, L. M. S.; Ramalho, J. P. P. Biochim. Biophys. Acta 2007, 1768, 467–478.
11. Cardoso, R. M. S.; Filipe, H. A. L.; Gomes, F.; Moreira, N. D.; Vaz, W. L. C.;
Moreno, M. J. J. Phys. Chem. B 2010, 114, 16337–16346.
12. Kwon, J. E.; Park, S. Y. Adv. Mater. 2011, 23, 3615–3642.
13. Zhao, J. Z.; Ji, S. M.; Chen, Y. H.; Guo, H. M.; Yang, P. Phys. Chem. Chem. Phys.
2012, 14, 8803–8817.
14. Jäger, A.; Stefani, V.; Guterres, S. S.; Pohlmann, A. R. Int. J. Pharm. 2007, 338,
297–305.
15. Rodembusch, F. S.; Leusin, F. P.; Campo, L. F.; Stefani, V. J. Lumin. 2007, 126,
728–734.
16. Alessenko, A. V.; Burlakova, E. B. Bioelectrochemistry 2002, 58, 13–21.
17. Dennison, S. M; Guharay, J.; Sengupta, P. K. Spectrochim. Acta, Part A 1999, 55,
1127–1132.
18. dos Santos, R. C.; Faleiro, N. V. S.; Campo, L. F.; Scroferneker, M. L.; Corbellini, V.
A.; Rodembusch, F. S.; Stefani, V. Tetrahedron Lett. 2011, 52, 3048–3053.
19. General procedure for the synthesis of the compounds 4–7: 2-(50-(N-3-Chloro-2-
hydroxypropylamino)-20-hydroxyphenil)benzoxazole (4). An ethanol solution
Figure 8. Double reciprocal plot of 1/F versus 1/[PL] of dyes 5–7 in different PC
concentrations at 25 °C (bottom).
of
3 (0.29 g, 1.29 mmol) was added dropwise in epichlorohydrin 0.477 g
(5.16 mmol) at 45 °C. After addition, the mixture had been stirred in the
temperature of reflux for 24 h. The solvent was removed under reduced pressure
and the residue was purified by column chromatography using silica gel and a
mixture of chloroform/acetone (1:1) as eluent. Yield: 20%; orange solid; mp:
The fluorescence intensity was enhanced with increasing phos-
phatidylcholine concentration [PC] (Fig. 7, inset) for all studied
dyes, and the partition coefficients (Kp) were calculated from the
slope of the double reciprocal linear plot of 1/F versus 1/[PC]
according to the equation 1=F ¼ 55:6=ðKpFmax½PCꢃÞ þ 1=Fmax, where
Fmax was the fluorescence maximum resulting from total probe
incorporation into the membrane.33,34
117–120 °C. FTIR (KBr, cmꢀ1): 3322 ( arom C@C). 1
vNH); 2904; 1622 and 1587 (v H
NMR (300 MHz, CDCl3): d (ppm) = 11 (s, 1H, OH); 7.25–6.25 (m, 7H), 4.21–4.09
(m, 1H), 3.75 (dd, J = 4.5 Hz and 4.8 Hz, 1H), 3.70 (dd, J = 6.0 and 5.7 Hz, 1H), 3.42
(dd, J = 3.9, 1H), 3.27 (dd, J = 7.8 Hz, 1H). 13C APT (75.4 MHz, CDCl3):
d
(ppm) = 47.8, 48.1, 69.9, 109.8, 110.3, 110.6, 118.9, 119.2, 121.2, 124.9, 125.3,
140.2, 140.7, 149.1, 152.0, 162.9. N-(3-((3-(Benzoxazol-2-yl)-4-hydroxy-
phenylamino)-2-hydroxypropyl)-N,N-dimethyl-1-octadecanoamonium (5).
A
Despite the good results from ESIPT flavonols,34 where a linear-
ity was obtained in the corresponding double reciprocal plot of 1/F
versus 1/[PC], saturation could be observed only for dye 5 at the
studied concentrations. From the slope and 1/F intercept of this lin-
ear plot, the partition coefficient Kp was 3.61 ꢂ 106–1.15 ꢂ 108 at
25 °C with an R2 ꢁ1 (Fig. 8), which were higher than those found
in ESIPT dye literature,34,35 due to better interaction between dye
and PC due to the alkyl chains present in the dyes.
In conclusion, three new fluorescent amphiphilic benzoxazole
derivatives were synthesized and used as prospective fluorescent
membrane probes. The new dyes were fluorescent in the blue-
green region, dependent upon solvent polarity. The dyes were suc-
cessfully used to obtain photoactive phosphatidylcholine lipo-
somes by reverse-phase evaporation. The values obtained for the
partition coefficient indicated the alkyl chain allowed a higher
interaction between these dyes and the phospholipids (in compar-
ison to other ESIPT dyes).
mixture of 4 (0.29 g, 0.94 mmol) and N,N-dimethyloctadecylamine (0.16 g,
0.54 mmol) in toluene was slowly stirred in ice bath for 30 min, followed by an
increase in the temperature until reflux for 48 h. The solvent was removed under
reduced pressure and the residue was washed with hexane to remove small
portions of N,N-dimethyloctadecylamine until TLC analysis showed only the
desired product 5. Yield: 68%; dark-brown solid; mp: 200 °C (decomp.). Anal.
calcd for C36H58N3O3 (580.86 g molꢀ1): C 70.39, H 9.19, N 6.84. Found: C 70.12, H
9.83, N 6.12. FTIR (KBr, cmꢀ1): 2916 (
vas C–H), 2849 (vs C–H), >3000 (vO–H), 1636
(v
C@C). 1H NMR (300 MHz, CDCl3): d (ppm) = 10.74 (s, 1H, OH), 7.62–6.95
(m, 7H), 4.64 (m, 1H) 4.02–3.53 (m, 4H), 3.52–3.05 (m, 8H), 1.70–2.50 (m, 4H),
1.10 (m, 28H), 0.78 (t, 3H). 13C NMR (75.4 MHz, CDCl3): d (ppm) = 13.9, 22.5,
26.9, 27.2, 28.8–29.5, 109.8, 110.3, 110.6, 118.1, 119.2, 121.2, 124.9, 125.4,
140.2, 140.7, 149.1, 152.0, 162.9. 2-(50-N-dodecylamino-20-hydroxyphenyl)
benzoxazole (6). In a 100 mL round-bottom flask was added a solution of 3
(0.51 g, 2.21 mmol) in 2-butanone, 1-iodododecane (0.98 g, 3.31 mmol) and
potassium carbonate (0.30 g, 2.21 mmol). The reaction was stirred under reflux
for 18 h. The solvent was removed under reduced pressure and a yellow oil was
obtained, which was purified by column chromatography using silica gel and a
mixture of chloroform/hexane as eluent mixture (1:1). Yield: 45%; yellow solid;
mp: 96–98 °C. Anal. calcd for C25H34N2O2 (394.55 g molꢀ1): C 85.11, H 7.97, N
7.37. Found: C 86.44, H 7.63, N 7.11. FTIR (KBr, cmꢀ1): 3390 (
v NH), 2915 (vas
C–H), 2850 (vs C–H). 1H NMR (300 MHz, CDCl3): d (ppm) = 11 (s, 1H, OH);
7.23–6.25 (m, 7H), 3.23 (t, 2H), 1.67 (m, 2H), 1.22 (m, 18H), 0.8 (t, 3H). 13C NMR
(75.4 MHz, CDCl3): d (ppm) = 14.4, 22.9, 26.2, 29.4-30.0, 32.2, 33.8, 52.2, 109.8,
110.4, 110.6, 118.1, 119.2, 121.2, 124.9, 125.3, 140.2, 140.7, 149.1, 152.0, 162.9.
Acknowledgements
2-(50-N-octylamino-20-hydroxyphenyl)benzoxazole (7). In
a 100 mL round-
bottom flask was added a solution of 3 (0.61 g, 2.65 mmol) in 2-butanone,
iodooctane (1.27 g, 5.3 mmol) and potassium carbonate (0.36 g, 2.65 mmol). The
reaction was stirred under reflux for 18 h. The solvent was removed under
reduced pressure and a dark-brown oil was obtained. The oil was purified by
column chromatography using silica gel and a mixture of chloroform/hexane
(1:1) as eluent. Yield: 45%; dark-yellow crystals; mp: 92–94 °C. Anal. calcd for
We are grateful for financial support and scholarships from the
Brazilian agencies CNPq and CAPES and Instituto Nacional de Ino-
vação em Diagnósticos para a Saúde Pública (INDI-Saúde).
Supplementary data
C
21H26N2O2 (338.44 g molꢀ1): C 74.25, H 7.96, N 7.90. Found: C 74.55, H 7.69, N
7.28. FTIR (KBr, cmꢀ1): 3390 ( NH), 2930 (vas C–H), 2840 (vs C–H). 1H NMR
v
(300 MHz, CDCl3): d (ppm) = 11 (s, 1H, OH), 7.22–6.25 (m, 7H), 3.05 (t, 2H), 1.68
(m, 2H), 1.23 (m, 10H), 0.84 (t, 3H). 13C NMR (75.4 MHz, CDCl3): d (ppm) = 14.0,
22.6, 27.1, 29.0, 29.3, 29.5, 31.7, 44.9, 109.8, 110.3, 110.6, 118.1, 119.2, 121.2,
124.9, 125.4, 140.2, 140.7, 149.1, 152.0, 162.9.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.03.
103.
20. Liu, Y. N.; Shu, W. G.; Zeng, D. M.; Huang, K. L. Trans. Nonferrous Met. Soc. China
2000, 10, 691–694.
21. Singh, C. B.; Kavala, V.; Samal, A. K.; Patel, B. K. Eur. J. Org. Chem. 2007, 8, 1369–
1377.
22. Raimundo, J. M.; Blanchard, P.; Gallego-Planas, N.; Mercier, N.; Ledoux-Rak, I.;
Hierle, R.; Roncali, J. J. Org. Chem. 2002, 67, 205–218.
23. Fang, J. Y.; Whitaker, C.; Weslowski, B.; Chen, M. S.; Naciri, J.; Shashidhar, R. J.
Mater. Chem. 2001, 11, 2992–2995.
References and notes
1. Topics in Fluorescence Spectroscopy; Lakowicz, J. R., Ed.Biochemical Applications;
Kluwer Academic Publishers, 2002; Vol. 3,.
2. Bouvrais, H.; Pott, T.; Bagatolli, L. A.; Ipsen, J. H.; Méléard, P. Biochim. Biophys.
Acta 2010, 1798, 1333–1337.
3. Teague, H.; Ross, R.; Harris, M.; Mitchell, D. C.; Shaikh, S. R. J. Nutr. Biochem.
2013, 24, 188–195.
4. Epand, R. M.; Kraayenhof, R. Chem. Phys. Lipids 1999, 101, 57–64.
24. Rodembusch, F. S.; Brand, F. R.; Correa, D. S.; Pocos, J. C.; Martinelli, M.; Stefani,
V. Mater. Chem. Phys. 2005, 92, 389–393.
25. Santra, S.; Dogra, S. K. Chem. Phys. 1998, 226, 285–296.

P. Franken Dick et al. / Tetrahedron Letters 55 (2014) 3024–3029
3029
26. Mertins, O.; Sebben, M.; Schneider, P. H.; Pohlmann, A. R.; da Silveira, N. P.
Quim. Nova 2008, 31, 1856–1859.
concentration was 10ꢀ4 M, which was diluted to obtain the working
concentrations of 10ꢀ5 and 10ꢀ6 M.
27. Ponpipom, M. M.; Bugianesi, R. L. J. Lipid Res. 1980, 21, 136–139.
28. Szoka, F., Jr.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 4194–
4198.
30. Paul, B. K.; Guchhait, N. J. Lumin. 2012, 132, 2194–2208.
31. Jayabharathi, J.; Thanikachalam, V.; Jayamoorthy, K.; Srinivasan, N.
Spectrochim. Acta, Part A 2013, 105, 223–228.
29. Reverse liposome preparation: To a solution of purified phosphatidylcholine
(60 mg) in chloroform (9 ml) was added the amphiphilic derivatives 5–7 in
32. Wang, R.; Liu, D.; Xu, K.; Li, J. J. Photochem. Photobiol., A Chem. 2009, 205,
61–69.
chloroform (1 mg/ml). To this solution was added 200
l
L of water in order to
33. Huang, Z.; Haugland, R. P. Biochem. Biophys. Res. Commun. 1991, 181, 166–171.
34. Chaudhuri, S.; Basu, K.; Sengupta, B.; Banerjee, A.; Sengupta, P. K. Luminescence
2008, 23, 397–403.
obtain the liposomes in reverse phase. The mixture was then sonicated for
4 min and the solvent was removed under reduced pressure resulting in an
organogel, which was hydrated with 10 mL of water. The final dye
35. Shyamala, T.; Mishra, A. K. Photochem. Photobiol. 2004, 80, 309–315.