Synthesis and spectral properties of fluorescent linear alkylphosphocholines labeled with all-(E)-1,6-diphenyl-1,3,5-hexatriene

Article abstract of DOI:10.1016/j.jphotochem.2010.09.010

A synthetic method has been designed to produce alkylphosphatidylcholine lipid molecules with the fluorescent group all-(E)-1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated to the end of a polymethylene chain of variable length. The resulting compounds ma

Full text of DOI:10.1016/j.jphotochem.2010.09.010

Journal of Photochemistry and Photobiology A: Chemistry 216 (2010) 79–84
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Synthesis and spectral properties of fluorescent linear alkylphosphocholines
labeled with all-(E)-1,6-diphenyl-1,3,5-hexatriene
Valentín Hornillosa,b, Laura Tormoa, Francisco Amat-Guerrib, A. Ulises Acun˜aa,∗
a Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
b Instituto de Química Orgánica General, CSIC, Juan de la Cierva 3, 20006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2010
Received in revised form 6 September 2010
Accepted 8 September 2010
Available online 17 September 2010
A synthetic method has been designed to produce alkylphosphatidylcholine lipid molecules with the
fluorescent group all-(E)-1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated to the end of a polymethy-
lene chain of variable length. The resulting compounds may be viewed as single-chain fluorescent lipid
probes terminated with a phosphocholine (PHC) polar head-group. The method was applied to the syn-
thesis of two lipids with different alkyl chain length, PHC-C4-DPH (1) and PHC-C6-DPH (2), as well as the
corresponding alcohols OH-C4-DPH (8) and OH-C6-DPH (9). The absorption and fluorescence properties
of these compounds in fluid solution were very similar to those of the well-known lipid probe propionic
acid-DPH (PA-DPH). In addition, it was observed that the alkylphosphocholines were easily incorporated
into unilamellar lipid vesicles, in which the DPH group would be positioned most likely near the centre
of the bilayer. In these conditions, 1 shows a fluorescence quantum yield of 0.4 0.1 and a relatively
simple decay time, with ꢀav close to 5.0 ns. Fluorescence polarization experiments also indicate that the
reorientational motions of the DPH group of phosphocholine 1 are sensitive to the fluidity changes that
accompany the main thermal phase transition of the bilayer. As a result, both compounds may be of
utility as novel fluorescent lipid probes, characterized by a biomimetic zwitterionic polar head-group.
© 2010 Elsevier B.V. All rights reserved.
Keywords:
Diphenylhexatriene
Lipid probes
Fluorescence
Synthesis
Alkylphosphocholines
1. Introduction
in the saturated alkyl chain as emitting labels, hopefully mini-
mally perturbing the original physico-chemical properties of the
The study of lipid function in natural or artificial systems could
be greatly simplified by means of the current large array of exper-
imental methods based on fluorescence spectroscopy. However,
since lipids usually lack intrinsic fluorescence, the application of
these methods requires first attaching a suitable emitting group
to the lipid molecule, either at its hydrophilic head-group or at
the hydrophobic alkyl chain [1]. As a result of that, the delicate
amphipatic balance of the original molecule is altered, frequently
perturbing one or several properties of the parent compound.
We were interested in the application of fluorescence microscopy
techniques to understand the antiparasite mechanism of single-
chain alkyl-lipids, as the phosphocholine (PHC) ester of the fatty
alcohol n-hexadecanol (miltefosine, Fig. 1), a drug used to treat
human leishmaniasis infections and other diseases [2]. The PHC
head-group of the molecule is important for its bioactivity and,
therefore, fluorescence labeling of the alkyl-lipid can only be car-
ried out at the polymethylene chain. Simple conjugated polyenes,
like tetraene [3,4] or pentaene [5,6] groups, may be inserted
drug [1]. Unfortunately, the UV excitation (ca. 350 nm) required
by these weakly emitting groups is inconvenient for living-cell
fluorescence imaging methods, although in some instances two-
photon excitation techniques may overcome this limitation [7].
Conjugated phenyl-polyenes show red-shifted absorption tran-
sitions and, in fact, a fluorescent analogue of miltefosine with
potent antiparasite activity was produced by attaching a phenyl-
tetraene chromophore to the parent drug [8]. Both, the excitation
wavelength (ca. 380 nm) and the photochemical stability of this
compound were more favorable than those of the simpler linear
conjugated polyenes mentioned above. Further improvement in
the photochemical and photophysical properties of the analogues
may be achieved using all-(E)-1,6-diphenyl-1,3,5-hexatriene (DPH)
as emitting tag.
The lipophilic DPH fluorophore became popular in lipid mem-
brane research because of its large absorption coefficient and
fluorescence yield, extensive dynamic characterization in fluid
solution and lipid bilayers and, importantly, high photochemical
stability [9–11]. A series of lipid probes have been produced by
attaching simple polar head-groups to the DPH fluorophore, such as
the trimethylammonium cation [12–15] or the negatively charged
propionic acid group [16–18]. In this way, the transverse location of
the fluorescent tag of the probe in a lipid membrane can be better
∗
Corresponding author at: Instituto de Química Física Rocasolano, CSIC, Serrano
119, 28006 Madrid, Spain. Tel.: +34 91 7459501; fax: +34 91 5642431.
E-mail address: roculises@iqfr.csic.es (A.U. Acun˜a).
1010-6030/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2010.09.010

80
V. Hornillos et al. / Journal of Photochemistry and Photobiology A: Chemistry 216 (2010) 79–84
O
P
N
N
O
O
O
O
n-hexadecylphosphocholine (miltefosine)
O
P
O
O
PHC-C4-DPH (1)
PHC-C6-DPH (2)
O
P
O
O
O
N
OH
OH
Scheme 1. Reagents and conditions: (a) MeONa, THF, 0 ◦C to r.t., 1 h, 98% (6), 80% (7);
(b) tetrabutylammonium fluoride, THF, r.t., 50 min, 96% (8), 97% (9); (c) 2-chloro-
1,3,2-dioxaphospholane-2-oxide, NMe3, MeCN, r.t., 3 h, then 70 ◦C, 4 h, 38% (1), 17%
(2).
8
OH-C4-DPH ( )
were used to incorporate the fluorescent alkylphosphocholines to
the LUV bilayer. In the first case, a few microliters of a concen-
trated ethanol solution of the compound were added to the aqueous
LUV suspension. In a second method, the phosphocholine was dis-
solved at the start with DMPC, before LUV preparation. Scattering
and background emission contributions were subtracted from the
absorption and fluorescence anisotropy spectra, respectively, using
blank LUV suspensions free of the emitting compound.
OH-C6-DPH (9)
Fig. 1. Structure of the fluorescent alkylphosphocholines 1 and 2, the corresponding
alcohols 8 and 9 and of miltefosine.
defined [19]. In addition, the DPH fluorophore has been incorpo-
rated to one of the acyl chains of different glycerophospholipids
with the same purpose [20].
2.2. Synthesis
Herein we report the synthesis and spectral properties of fluo-
rescent alkylphosphocholines with a structure that is intermediate
between these two types of compounds. In these novel single-chain
DPH-tagged lipids, the emitting group is bound to a saturated C4 or
C6 alkyl chain tethered to a phosphocholine head-group (Fig. 1). The
all-(E) stereochemistry of the three conjugated double bonds was
selected to minimize the perturbation of the lipid arrangement in
the membrane. The data presented here indicate that these com-
pounds may be of general utility as probes of lipid function and
structure in natural and artificial membranes.
Compounds PHC-C4-DPH (1) and PHC-C6-DPH (2) were
designed as to preserve the linear dimension of the parent
lipid n-hexadecylphosphocholine. They were obtained through a
stereoselective Horner–Wadsworth–Emmons (HWE) coupling, a
procedure used previously for the synthesis of a conjugated phenyl-
trienyne alcohol analogue of n-hexadecylphosphocholine [6]. In
brief, diethyl [(2E,4E)-5-phenylpenta-2,4-dienyl]phosphonate (3)
– prepared in four steps from (2E)-cinnamaldehyde, as described
previously [18] – was made to react with the silylated aldehydes 4
and 5, yielding with good yields the corresponding silylated all-(E)
compounds 6 and 7 (Scheme 1). 1H NMR spectra of 6 confirmed
the (E) stereochemistry of the generated double bond, with less
than 1% of the corresponding (Z) isomer. Irradiation of the pro-
ton signals of the central double bond of the DPH system allowed
the simplification of the two AB systems assigned to the protons
of the new double bond and of the Ph–CH CH– double bond,
with coupling constants close to 15 Hz and, hence, with (E) con-
figuration. This agrees with previous results on the simulated 1H
NMR spectrum from another DPH compound [18]. The silyl group
was separated with tetrabutylammonium fluoride in THF, yielding
the corresponding alcohols 8 and 9 with good yields. Eventually,
the introduction of the phosphocholine moiety by reaction with
2-chloro-1,3,2-dioxaphospholane-2-oxide and trimethylamine [8]
provided the target compounds PHC-C4-DPH (1) and PHC-C6-DPH
(2), respectively, both with all-(E) stereochemistry, as confirmed
by 1H NMR spectra. This synthetic method is easily scalable and
allows the preparation of other analogues with different alkyl chain
length and different terminal polar groups, as well as with different
substituents in the terminal phenyl group.
2. Experimental
2.1. Methods
UV/vis absorption spectra were recorded on a Varian CARY 3E
spectrophotometer and corrected for scattering when necessary
(lipid vesicles suspensions). Steady-state fluorescence excitation
and emission spectra and anisotropy were recorded in an ISS
PC1 photon counting fluorimeter and corrected for instrumental
factors. Relative fluorescence quantum yields of optically diluted
samples were determined by the comparative method [21], by
reference to that of quinine sulfate in 0.05 M H2SO4 (˚F = 0.51)
[22], with an uncertainty of 10–20%. Fluorescence lifetimes were
recorded in a picosecond laser spectrometer described elsewhere
[23], using the time-correlated single-photon counting technique.
Data analysis was carried out by a non-linear least-squares for-
malism using a commercial package (Globals, Laboratory for
Fluorescence Dynamics, Univ. California, Irvine).
Large unilamellar vesicles (LUV) of 1,2-dimyristoyl-sn-glycero-
3-phosphatidylcholine (DMPC, 99.0% purity, from Avanti Polar
Lipids, USA) in 10 mM pH 7 aqueous phosphate buffer were
prepared by extrusion of a suspension of multilamellar vesicles
through 0.1 ␮m filters, as described elsewhere [23]. The average
LUV diameter was ca. 80 nm, as determined by standard light-
scattering techniques. Total lipid concentration was determined
by the phosphomolybdate method [24]. Two different procedures
Both phosphochlines 1 and 2, as well as the corresponding alco-
hols 8 and 9, are photostable as DMF, DMSO or MeOH solutions. In
solid form and kept at −20 ◦C in the dark, they are stable for years.
While analogues 1 and 2 are soluble only in polar solvents (MeOH,
EtOH, DMSO, DMF), the corresponding alcohols 8 and 9 are rather
soluble (>10−3 M) in chloroform, dichloromethane, diethyl ether,
THF, ethyl acetate and acetonitrile.

V. Hornillos et al. / Journal of Photochemistry and Photobiology A: Chemistry 216 (2010) 79–84
81
1.0
OH
n-2
Br
(a)
a1 = 0.41;
a2 = 0.53;
a3 = 0.06;
τ
1 = 0.21 ns
2 = 1.90 ns
3 = 5.10 ns
0.8
0.6
0.4
0.2
0.0
O
O
τ
10
11
: n = 4
: n = 6
τ
χ
2 = 1.05
n OH
(b)
(c)
4
5
: n = 4
: n = 6
O
12
: n = 4
13: n = 6
Scheme 2. Reagents and conditions: (a) 3-butyn-1-ol (n = 4) or 5-hexyn-1-ol (n = 6),
Pd(PPh3)2Cl2, CuI, THF, Et3N, r.t., 48 h, 91% (10), 85% (11); (b) Pd/C (10%), 40 psi,
AcOEt, r.t., 3 h, 95% (12), 70% (13); (c) TBDMSCl, imidazole, THF, r.t., 3 h, 95% (4),
100% (5).
0
2
4
6
8
10
12
Aldehydes
4
and
5
were prepared by
a
three-step
t / ns
sequence (Scheme 2) starting with the reaction between
4-bromobenzaldehyde and 3-butyn-1-ol or 5-hexyn-1-ol, respec-
tively, under Sonogashira–Hagihara cross-coupling conditions
[25], yielding the corresponding compounds 10 and 11. After
triple-bond hydrogenation in ethyl acetate, in order to reduce the
hydrogen reactivity and, hence, to preserve the aldehyde group,
p-hydroxyalkylbenzaldehydes 12 and 13 were obtained. Their
reaction with tert-butyldimethylsilyl chloride (TBDMSCl) yielded
4 and 5, respectively. See Supplementary Data for additional
information on the synthesis of all the former compounds.
3
2
1
0
-1
-2
-3
0
2
4
6
8
10
12
t / ns
Fig. 3. Fluorescence lifetime of PHC-C4-DPH (1) in dimethylformamide solution
ca. 10−6 M. Up: experimental decay and three-exponential best-fitting function
(overlaid). Down: weighted residuals distribution; ꢁexc = 372 nm, ꢁF = 535 8 nm,
T = 23 ◦C.
3. Results
The absorption and fluorescence spectra of PHC-C4-DPH (1) in
dimethylformamide (DMF) solution are shown in Fig. 2. The absorp-
tion and emission spectra of the other DPH compounds obtained
in this work (Fig. 1) are very similar to those in Fig. 2, and only
the spectral maxima are reported in Table 1, together with the
values of the fluorescence quantum yield (˚F) and lifetime (ꢀF).
The decay of the fluorescence intensity in DMF solution is com-
plex and requires a multi-exponential best-fitting function. This is
illustrated for the case of PHC-C4-DPH in Fig. 3, that shows over-
laid the experimental data and a three-exponential fitting function.
All these spectral characteristics remind those described for the
free DPH chromophore, and do not differ substantially from those
observed before in other DPH-labeled compounds. Thus, the shape
of the absorption band centered at ca. 361 nm is nearly identical to
that of the unsubstituted DPH chromophore, but is red-shifted rel-
ative to that of the spectrum recorded in non-polar solvents. This
shift is due to the sensitivity of the DPH main absorption transition
to solvent polarity and polarizability [11].
The emission yield and lifetime of alkylphosphocholines 1 and
2, and those of the corresponding alcohols 8 and 9, are very similar,
indicating very small perturbation of the spectroscopic properties
of the pending chromophore by the polar head-group in DMF solu-
tion. These fluorescence properties are also very similar to those of
the parent DPH fluorophore in polar solvents, as e.g. ethanol [11].
The absorption and emission spectra of PHC-C4-DPH (1) incor-
porated into unilamellar lipid vesicles (LUV) of DMPC are shown
in Fig. 4, and the corresponding spectral parameters are listed in
1.0
0.8
0.6
1.0
0.8
0.6
A
F
A
F
0.4
0.2
0.0
0.4
0.2
0.0
300
350
400
450
500
550
600
650
280
320
360
400
440
λ / nm
480
520
560
600
λ / nm
Fig. 4. Absorption (A) and corrected fluorescence (F) spectrum of PHC-C4-DPH (1)
incorporated into DMPC large unilamellar lipid vesicles. pH 7.0 phosphate buffer,
probe:lipid molar ratio 1:300, [DMPC] = 6 × 10−4 M, ꢁexc = 380 1 nm, T = 35 ◦C.
Fig. 2. Absorption (A) and corrected fluorescence (F) spectrum of PHC-C4-DPH (1)
in dimethylformamide solution ca. 10−6 M; ꢁex = 360 2 nm, T = 23 ◦C.

82
V. Hornillos et al. / Journal of Photochemistry and Photobiology A: Chemistry 216 (2010) 79–84
Table 1
Absorption (ꢁabs) and fluorescence (ꢁF,
˚
ꢀF) properties of lipid analogues containing the all-(E)-1,6-diphenyl-1,3,5-hexatriene (DPH) emitting group. T = 23 ◦C.
F,
Compd.
Solventa
ꢁabs/nmb
ꢁF/nm
˚
F
a1
ꢀ1/ns
a2
ꢀ2/ns
a3
ꢀ3/ns
ꢀav/nsc
PHC-C4-DPH
DMF
344, 361, 382
347, 362, 381
347, 363, 383
344, 361, 382
345, 361, 381
345, 361, 381
434
433
433
433
458
460
0.16
0.36d
0.38d
0.21
0.17
0.16
0.40
–
–
0.40
0.30
0.30
0.22
–
–
0.24
0.23
0.22
0.54
0.14
0.15
0.56
0.55
0.53
1.9
1.3
2.0
1.9
1.8
1.8
0.06
0.86
0.85
0.04
0.15
0.17
5.1
5.0
5.5
5.0
5.2
5.1
2.5
4.9
5.3
2.2
3.2
3.3
DMPC, 35 ◦
DMPC, 15 ◦
DMF
C
C
OH-C4-DPH
PHC-C6-DPH
OH-C6-DPH
DMF
DMF
a
DMF: dimethylformamide; DMPC: large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine.
Spectrum maximum underlined.
b
c
ꢀ
ꢀ
Average lifetime, ꢀav
=
aiꢀi2
/
aiꢀ1.
d
Uncertainty 30%.
pounds [26]. By analogy, this absorption band can be assigned to
the symmetry-allowed transition to the S2 electronic state, as was
established [27] for the parent DPH (i.e. 11Bu ← 11Ag). This is con-
sistent with the red-shift of the alkylphosphocholine absorption in
DMF solution relative to that of unsubstituted DPH in hydrocar-
bon solvents (e.g. ꢁmax = 355 nm in n-hexane [9d]), due to lowering
of the energy of the 11Bu state in high-polarizability solvents.
The absorption coefficient of PHC-C4-DPH (1) in ethanol solution
(60100 2000 M−1 cm−1, ꢁabs = 356 nm) is ca. 25% lower than that
of the parent DPH, but very similar to that of the propionic acid-DPH
probe (PA-DPH) [16]. The fluorescence emission of the alkylphos-
phocholines 1 and 2 shows a large Stokes shift and lack of mirror
symmetry, indicating that the emission should take place largely
from the lowest S1 excited state (the symmetry-forbidden 21Ag
state in DPH). The complex fluorescence decay of these compounds
in DMF (Table 1) is likely due to interactions between the fluo-
rophore excited-states promoted by solvent-induced decrease of
the S1–S2 energy gap. These aspects of the fluorescence of the DPH
group have been studied in great detail by Saltiel and coworkers
[28]. It is interesting that the average fluorescence lifetime of PHC-
C4-DPH (1) in DMF solution reproduces that of PA-DPH in the same
solvent [16].
The solubility of compounds 1 and 2 in water and in aqueous
phosphate buffers is very low. In the 10−5 to 10−6 M concentra-
tion range, these phosphocholines form non-emitting aggregates,
which are detected by increased scattered light (data not shown). In
these conditions, it is very likely that these compounds are present
as micellar aggregates, since they are essentially zwitterionic sur-
factant molecules. In that case, the DPH chromophores would be
oriented towards the micelle core, favoring electronic interactions
that would quench the fluorescence. The critical micelle concentra-
tion (c.m.c.) of the parent compound n-hexadecylphosphocholine
(Fig. 1) has been determined several times using a variety of
techniques [29]. Although the reported values span a wide range
(2–160 ␮M), most of them cluster around the lower end of this
range. The c.m.c. of 1 and 2 should be even lower, considering the
strong lipophility of the pending DPH group.
0.28
0.26
0.24
0.22
0.20
0.18
0.16
10
15
20
25
T / ºC
30
35
40
45
Fig. 5. Steady-state fluorescence anisotropy (r) as a function of temperature of PHC-
C4-DPH (1) incorporated into DMPC unilamellar lipid vesicles. [DMPC] = 5 × 10−4 M,
probe:lipid = 1:200, ꢁexc = 360 1 nm, ꢁF = 450 10 nm, pH 7.0 phosphate buffer.
Table 1. The contribution due to LUV scattering in the absorption
spectrum has been subtracted. The spectra in the lipid bilayer are
similar to that reported above in DMF solution, although the fluo-
rescence yield and average lifetime increased by ca. 100%. These
larger values are due to the suppression of the subnanosecond
decay channel, accompanied by a large increase of the fractional
contribution of the 5 ns lifetime component (Table 1). At tempera-
tures below the DMPC gel/liquid-crystalline transition (Tm = 23 ◦C),
the alkylphosphocholine fluorescence is strongly polarized (Fig. 5).
By increasing the temperature of the LUV suspension, the value of
the emission anisotropy of the phosphocholine decreases substan-
tially, revealing the bilayer fluidification process.
4. Discussion
When added to
a DMPC vesicle suspension, the DPH-
The emitting lipids studied here have in common with natural
fatty acids and acylphospholipids the main physical and chem-
ical properties. Thus, for example, the estimated length of the
phosphocholines bind quickly to the lipid vesicles, as detected by
the increased fluorescence emission of the bound-form (data not
shown), while the fraction remaining in the aqueous buffer is virtu-
ally non-fluorescent. Preliminary measurements of the absorption
and fluorescence spectra of PHC-C4-DPH embedded in LUV (Fig. 4
and Table 1) show values similar to those of the PA-DPH probe in
lipid vesicles. The quantum yield of PHC-C4-DPH embedded in the
LUV lipid bilayer went up to ca. 0.4 (the PA-DPH value in similar
conditions is 0.73 [16]). There is also a large change in the fluo-
rescence lifetime of phosphocholine 1 from DMF solution to the
lipid bilayers. In the LUV, the decay becomes simpler, and can be
described by a biexponential function dominated by a large fraction
(85%) of the 5 ns component (Table 1). As indicated above, phos-
phocholine 1 was incorporated into LUV in two different ways. In
˚
C4-DPH chain in a fully extended conformation is ca. 20 A, which
is identical to that of the palmitoyl residue, and similar to the
average half-width of a fluid lipid bilayer. As a result of that, DPH-
phosphocholine esters 1 and 2 may show enough compatibility
with natural lipids to probe a large variety of lipid physical phe-
nomena. For that purpose, some understanding of the fundamental
photophysical properties of these compounds would be of utility.
As noted above, the main absorption band of these compounds
is due to the DPH group, and it is not affected by the type of
polar head-group. The small red-shift (2–3 nm) of this band rel-
ative to that of all-(E)-diphenylhexatriene in the same solvent [13]
is similar to that observed in other alkyl-substituted DPH com-

V. Hornillos et al. / Journal of Photochemistry and Photobiology A: Chemistry 216 (2010) 79–84
83
the first one, compound 1 was added to the LUV buffer suspen-
sion, while in the second compound 1 was dissolved together with
DMPC before preparing the vesicles. In the first case compound 1
would be associated only with the external lipid monolayer, due
to the slow flip-flop rate, while in the second procedure the probe
would be randomly distributed between the external and internal
bilayer leaflets. Nevertheless, the spectral properties of PHC-C4-
DPH recorded here were virtually the same in both cases. In the
case of the hydroxy-terminated compounds 8 and 9 the rate of
interchange between the two bilayer surfaces is expected to be
faster.
The polarized fluorescence of PHC-C4-DPH (1) is very sensitive
to changes in lipid order and fluidity that take place at the bilayer
main thermal transition (Fig. 5), which indicates that the emitting
molecules are incorporated to the membrane. The relatively low
anisotropy value in the gel phase and the broadened temperature
range of the recorded change have been also observed for other
probes in small unilamellar vesicles [30]. These experiments indi-
cate that the chromophore is very likely buried in the most fluid part
of the lipid bilayer, as would also be expected from the phospho-
choline chain length. To determine more precisely the transverse
location of the DPH group in the bilayer additional experiments
would be necessary [19].
[4] (a) B.S. Hudson, S. Cavalier, in: L.M. Loew (Ed.), Spectroscopic Membrane Probes,
Vol. 1, CRC Press, Boca Raton, FL, 1988, pp. 43–62;
(b) C.R. Mateo, A.U. Acun˜a, J.C. Brochon, Liquid-crystalline phases of choles-
terol/lipid bilayers as revealed by the fluorescence of trans-parinaric acid,
Biophys. J. 68 (1995) 978–987.
[5] (a) A.A. Souto, A.U. Acun˜a, F. Amat-Guerri, A general and practical synthesis of
linear conjugated pentaenoic acids, Tetrahedron Lett. 35 (1994) 5907–5910;
(b) C.R. Mateo, A.A. Souto, F. Amat-Guerri, A.U. Acun˜a, New fluorescent octade-
capentaenoic acids as probes of lipid membranes and protein-lipid interactions,
Biophys. J. 71 (1996) 2177–2191;
(c) E. Quesada, A.U. Acun˜a, F. Amat-Guerri, New transmembrane polyene
bolaamphiphiles as fluorescent probes in lipid bilayers, Angew. Chem. Int. Ed.
40 (2001) 2095–2097.
[6] E. Quesada, J. Delgado, V. Hornillos, A.U. Acun˜a, F. Amat-Guerri, Synthesis and
spectral properties of amphiphilic lipids with linear conjugated polyene and
phenylpolyene fluorescent groups, Eur. J. Org. Chem. (2007) 2285–2295.
[7] L. Kuerschner, C.S. Ejsing, K. Ekroos, A. Schevchenko, K.I. Anderson, C. Thiele,
Polyene-lipids: a new tool to image lipids, Nat. Methods 2 (2005) 3945.
[8] J.M. Saugar, J. Delgado, V. Hornillos, J.R. Luque-Ortega, F. Amat-Guerri, A.U.
Acun˜a, L. Rivas, Synthesis and biological evaluation of fluorescent leish-
manicidal analogues of hexadecylphosphocholine (Miltefosine) as probes of
antiparasite mechanisms, J. Med. Chem. 50 (2007) 5994–6003.
[9] (a) M. Shinitzky, Y. Barenholz, Dynamics of the hydrocarbon layer in liposomes
of lecithin and sphingomyelin containing dicetylphosphate, J. Biol. Chem. 249
(1974) 2652–2657;
(b) J.R. Lackowicz, F.G. Prendergast, D. Hogen, Differential polarized phase flu-
orometric investigations of diphenylhexatriene in lipid bilayers. Quantitation
of hindered depolarizing rotations, Biochemistry 18 (1979) 508–519;
(c) L.W. Engel, F.G. Prendergast, Values for and significance of order parame-
ters and “cone angles” of fluorophore rotation in lipid bilayers, Biochemistry
20 (1981) 7338–7345;
(d) B.R. Lentz, in: L.M. Loew (Ed.), Spectroscopic Membrane Probes, vol.1, CRC
Press, Boca Raton, FL, 1988, pp. 13–41.
5. Conclusions
[10] (a) B.R. Lentz, Use of fluorescent probes to monitor molecular order and motions
within liposome bilayers, Chem. Phys. Lipids 64 (1993) 99–116;
(b) C.R. Mateo, M.P. Lillo, J.C. Brochon, M. Martínez-Ripoll, J. Sanz-Aparicio, A.U.
Acun˜a, Rotational dynamics of 1,6-diphenyl-1,3,5-hexatriene and derivatives
from fluorescence depolarization, J. Phys. Chem. 97 (1993) 3486–3491.
[11] E.D. Cehelnik, R.B. Cundall, J.R. Lockwood, T.F. Palmer, Solvent and temperature
effects on the fluorescence of all-trans-1, 6-diphenyl-1,3,5-hexatriene, J. Phys.
Chem. 79 (1975) 1369–1376.
[12] M. Cranney, R.B. Cundall, G.R. Jones, J.T. Richards, E.W. Thomas, Fluorescence
lifetime and quenching studies of some interesting diphenylhexatriene mem-
brane probes, Biochim. Biophys. Acta Biomembr. 735 (1983) 418–425.
[13] F.G. Prendergast, R.P. Haugland, P.J. Callahand, 1-[4-(Trimethylamino)phenyl]-
6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties and use as a
fluorescence probe of lipid bilayers, Biochemistry 20 (1981) 7333–7338.
[14] (a) J.-G. Kuhry, G. Duportail, C. Bronner, G. Laustriat, Plasma membrane fluidity
measurements on whole living cell by fluorescence anisotropy of trimethy-
lammoniumdiphenylhexatriene, Biochim. Biophys. Acta Biomembr. 845 (1985)
60–67;
A general method for the synthesis of fluorescent alkylphos-
phocholines is detailed here. It can be used to prepare single-chain
lipids with different length and polar head-group, incorporating
DPH or its derivatives as fluorescent tag of the alkyl chain. This
procedure was used to obtain two single-chain emitting alkylphos-
phocholines incorporating the DPH chromophore, PHC-C4-DPH (1)
and PHC-C6-DPH (2), as well as the corresponding alcohols. The
spectroscopic and photochemical properties of 1 and 2, both in
solution and in unilamellar vesicles of DMPC, are similar to those
of the lipid probe propionic acid-DPH and well adapted for a vari-
etyoflipid-relatedstudies. Moreover, these compounds present the
added advantage that contain a biocompatible phosphatidylcholine
head-group, which would be in zwitterionic form at physiological
pH. Overall, the data presented here show the potential utility of
these alkylphosphocholines and warrant a more detailed assess-
ment of PHC-C4-DPH and PHC-C6-DPH as fluorescent molecular
probes of lipid structure and dynamics in bilayers and cell mem-
branes.
(b) A. Beck, D. Heissler, G. Duportail, Influence of the length of the spacer on the
partitioning properties of amphiphilic fluorescent membrane probes, Chem.
Phys. Lipids 66 (1993) 135–142.
[15] S. Kitagawa, E. Tachikawa, T. Kashimoto, Transbilayer transport of a propy-
ltrimethylammonium derivative of diphenylhexatriene (TMAP-DPH) in bovine
blood platelets and adrenal chromaffin cells, J. Pharm. Pharmacol. 54 (2002)
1631–1636.
[16] P.J. Trotter, J. Storch, 3-[p-(6-Phenyl)-1,3, 5-hexatrienyl]phenylpropionic acid
(PA-DPH): characterization as a fluorescent membrane probe and binding to
fatty acid proteins, Biochim. Biophys. Acta, Biomembr. 982 (1989) 131–139.
[17] J.C. Sanders, M.F. Ottaviani, A. van Hock, A.J.W. Visser, M.A. Hemminga, A small
protein in model membranes: a time-resolved fluorescence and ESR study on
the interaction of M13 coat protein with lipid bilayers, Eur. Biophys. J. 21 (1992)
305–311.
[18] A. Beck, D. Heissler, G. Duportail, Synthesis of fluorescent probes for localized
membrane fluidity measurements, Tetrahedron 47 (1991) 1459–1472.
[19] M. Castanho, M.E. Prieto, A.U. Acun˜a, The transverse location of the fluorescent
probe trans-parinaric acid in lipid bilayers, Biochim. Biophys. Acta Biomembr.
1279 (1996) 164–168.
Acknowledgments
We thank Dr B. Reija for helpful discussions. Work supported
by Projects BQU2003/4413 and CTQ2009-9452 from the Spanish
Ministry of Science and Technology (M.C. y T.) and PI06115 from
the Spanish Ministry of Health. V.H. and L.T. acknowledge the award
of a Scholarship from M.C. y T.
[20] R.A. Parente, B.R. Lentz, Advantages and limitations of 1-palmitoyl-2-
Appendix A. Supplementary data
[{2-[4-(6-phenyl-trans-1,
phosphatidylcholine as
(1985) 6178–6185.
3,5-hexatrienyl)phenyl]ethyl}carbonyl]-3-sn-
a
fluorescent membrane probe, Biochemistry 24
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jphotochem.2010.09.010.
[21] C.A. Parker, Photoluminescence of Solutions, Elsevier Pub. Co., Amsterdam,
1968, pp. 261.
[22] R.A. Velapoldi, K.D. Mielenz, A Fluorescence Standard Reference Material: Qui-
nine Sulfate, N.B.S. Special Pub. 260-64, Washington DC, 1980, p. 50.
[23] A.U. Acun˜a, F. Amat-Guerri, E. Quesada, M. Vélez, Dynamics of bolaamphiphilic
fluorescent polyenes in lipid bilayers from polarization emission spectroscopy,
Biophys. Chem. 122 (2006) 27–35.
[24] R.J. Mrsny, J.J. Volwerk, O.H. Griffith, A simplified procedure for lipid phospho-
rus analysis shows that digestion rates vary with phospholipid structure, Chem.
Phys. Lipids 39 (1986) 185–191.
References
[1] R.W. Sinkeldam, N.J. Greco, Y. Tor, Fluorescent analogs of biomolecular building
blocks: design, properties, and applications, Chem. Rev. 110 (2010) 2579–2619.
[2] S.L. Croft, M.P. Barrett, J.A. Urbina, Chemotherapy of trypanosomiasis and leish-
maniasis, Trends Parasitol. 21 (2005) 508–512.
[3] L.A. Sklar, B.S. Hudson, R.D. Simoni, Conjugated polyene fatty acids as fluo-
rescent probes: synthetic phospholipid membrane studies, Biochemistry 16
(1977) 819–828.
[25] R. Chinchilla, C. Najera, The Sonogashira reaction: a booming methodology in
synthetic organic chemistry, Chem. Rev. 107 (2007) 874–922.

84
V. Hornillos et al. / Journal of Photochemistry and Photobiology A: Chemistry 216 (2010) 79–84
[26] P.C. Alford, T.F. Palmer, Photophysics of derivatives of all-trans-1,6-diphenyl-
1,3,5-hexatriene (DPH). Part 1, J. Chem. Soc., Faraday Trans. 2 (79) (1983)
433–447.
(b) P.M. Macdonald, J.R. Rydall, S.C. Kuebler, F.M. Winnik, Synthesis and
characterization of a homologous series of zwitterionic surfactants based on
phosphocholine, Langmuir 7 (1991) 2602–2606;
[27] B.S. Hudson, B.E. Kohler, K. Schulten, in: E.C. Lim (Ed.), Excited States, Vol. 6,
Academic Press, New York, 1982, p. 1.
(c) E.C. Kang, S. Kataoka, K. Kato, Synthesis and properties of alkyl phospho-
rylcholine amphiphiles with linear and asymmetrically branched alkyl chain,
Bull. Chem. Soc. Jpn. 78 (2005) 1558–1564;
(d) M. Yaseen, T.J.S. Wang, J.R. Lu, Surface adsorption of zwitterionic surfactants:
n-Alkyl phosphocholines characterised by surface tensiometry and neutron
reflection, J. Colloid Interface Sci. 288 (2005) 361–370.
[28] (a) E.F. Hilinski, McGowan W.M. Sears Jr., J. Saltiel, Evolutions of singlet excited-
state absorption and fluorescence of all-trans-1,6-diphenyl-1,3,5-hexatriene in
the picosecond time domain, J. Phys. Chem. 100 (1996) 3308–33011;
(b) A.M. Turek, G. Krishnamoorthy, D.F. Sears, I. Garcia, O. Dmitrenko, J. Saltiel,
Resolution of three fluorescence components in the spectra of all-trans-1,6-
diphenyl-1,3,5-hexatriene under isopolarizability conditions, J. Phys. Chem. A
109 (2005) 293–303.
[29] (a) P. Soares de Araujo, M.Y. Rosseneau, J.M.H. Kremer, E.J.J. van Zoelen, G.H.
Haas, Structure and thermodynamic properties of complexes between phos-
pholipasa A2 and lipid micelles, Biochemistry 18 (1979) 580– 586;
[30] J. Suurkuush, B.R. Lentz, Y. Barenholz, R.L. Biltonen, T.E. Thompson, A calorimet-
ric and fluorescent probe study of the gel-liquid crystalline phase transition
in small, single-lamellar DMPC vesicles, Biochemistry 15 (1976) 1393–
1401.