Synthesis of a photoactivatable phospholipidic probe containing tetrafluorophenylazide

Article abstract of DOI:10.1016/j.tetlet.2005.06.125

In order to study lipid-lipid and lipid-protein interactions using a photolabeling approach, we synthesized and characterized a phospholipidic probe in which the photoactivatable tetrafluorophenylazido group is incorporated into the fatty acid chain. This

Full text of DOI:10.1016/j.tetlet.2005.06.125

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 5893–5897
Synthesis of a photoactivatable phospholipidic probe containing
tetrafluorophenylazide
Qing Peng,a Yi Xia,a Fanqi Qu,a Xiaojun Wu,a Daniel Campeseb and Ling Penga,b,
*
aCollege of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China
b
´
Departement de chimie, CNRS UMR 6114, 163, avenue de Luminy, 13288 Marseille, France
Received 16 May 2005; revised 15 June 2005; accepted 22 June 2005
Available online 7 July 2005
Abstract—In order to study lipid–lipid and lipid–protein interactions using a photolabeling approach, we synthesized and charac-
terized a phospholipidic probe in which the photoactivatable tetrafluorophenylazido group is incorporated into the fatty acid chain.
This probe is stable in the dark and becomes highly reactive upon being exposed to photoirradiation. It shows fast, clear-cut photo-
chemical reactions and holds promise for further use to study lipid–lipid and protein–lipid interactions.
Ó 2005 Elsevier Ltd. All rights reserved.
Biological membranes containing lipids and proteins
serve as a barrier protecting the cells they surround, as
well as being involved in some essential biological pro-
cesses.1 The lipids present in biomembranes are mainly
phospholipids, which play an important role in various
cellular processes.2 Proteins associated with biomem-
brane lipid bilayers also mediate many biological func-
tions, such as signal transduction, energy conversion,
and the transport of ions and molecules across the mem-
brane. In addition, membrane proteins are important
targets for drug development, since more than 50% of
the drugs on the market are targeted to membrane pro-
teins.3 Studies on lipid–lipid and lipid–protein interac-
tions throw useful light on both the functional and
structural properties of biological membranes as well
as identifying novel therapeutic targets and contributing
to drug development programs. However, structural
information about membrane proteins and biomem-
branes is scarce because both X-ray crystallography
and NMR spectroscopy are of limited applicability in
this context.
of photoactivatable lipidic probes. When exposed to
light, these probes generate highly reactive species such
as nitrene or carbene, and this leads to a process of cova-
lent cross-linkage with the protein or lipid present at the
interaction site. This process can be used to identify the
proteins and lipids that interact with the lipid probes
and to map the lipid-binding sites on proteins and bio-
membranes. This information can therefore be used to
investigate specific lipid–protein interactions and the
topology and patterns of distribution of membrane pro-
teins in biomembranes.
The preparation of photoactivatable phospholipids was
pioneered by Chakrabarti and Khorana,7 and a number
of additional examples have subsequently been synthe-
sized, usually containing azide, or diazirine,8 or benzo-
phenone,9 or a diazo or diazonium group,10 for
example. Among these photophores, aryl azides are
those most frequently used as photoaffinity probes
because they are small and can easily be synthesized,
as well as being chemically stable in the dark and highly
reactive upon being exposed to photoirradiation.
Among the aryl azides, fluorinated aryl azides are par-
ticularly promising candidates because upon being
photoactivated, they give much more efficient photo-
labeling than non-fluorinated aryl azides.11 In addition,
the presence of fluorine atoms in phospholipids can be
used to probe specific lipid–lipid and lipid–protein inter-
actions in biomembranes using 19F NMR methods,
which are highly suitable for investigating structural
and dynamic features because of the wide range of
Photoaffinity labeling4–6 provides a useful chemical
approach for obtaining the structural and functional
data on biomembranes. This approach involves the use
Keywords: Photoactivatable phospholipidic probe; Phospholipids;
Photolabeling; Photolabeling probe; Biomembranes.
*
Corresponding author. Tel.: +33 4 91 82 91 54; fax: +33 4 91 82 93
01; e-mail: ling.peng@univmed.fr
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.06.125

5894
Q. Peng et al. / Tetrahedron Letters 46 (2005) 5893–5897
O
P
O
F
N+
F
N3
F
O
O
O
-
O
P
OH
O
O
H
F
F
O
F
N
F
O
O
O
O
O
n
F
O
O
O
N3
n
O
2
1 (n = 11, 13 or 15)
Scheme 1. Photoactivatable phospholipidic probes 1 and 2.
chemical shifts and the extreme sensitivity of these meth-
ods to environmental changes.12 We therefore chose the
tetrafluorophenylazido group as the starting point for
preparing the photoactivatable phospholipidic probes.
have previously reported on the synthesis and character-
ization of probe 1.13 Here we report on probe 2.
To synthesize probe 2, it was necessary to first pre-
pare the tetrafluorophenylazido containing fatty acid
surrogate carboxylic acid 3 (Scheme 2). Coupling
12-hydroxydodecanoic acid with N-succinimidyl-4-
azidotetrafluorobenzoate14 yielded only tiny amounts
of 3 in our hands (Scheme 2a),15 while condensing
12-hydroxydodecanoic acid with 4-azidotetrafluoroben-
zoic acid resulted in a mixture mainly containing the
self-condensation product of 12-hydroxydodecanoic
acid (Scheme 2b), which was difficult to remove. We
therefore attempted to protect the acid group of
12-hydroxydodecanoic acid before introducing the tetra-
fluorophenylazido group.
In order to probe various areas of proteins and other lip-
ids with which phospholipids interact, it was proposed
to prepare two complementary types of photoactivat-
able phospholipidic probes 1 and 2 (Scheme 1), contain-
ing the tetrafluorophenylazido group at the polar head
and in the fatty acid chain, respectively. Probe 1 has
the photoactivatable moiety at the polar head and is
designed for probing the lipid/water interface of bio-
membranes, while 2, where the photoactivatable group
is located in one fatty acid chain of the phospholipid,
serves to probe the hydrophobic membrane core. We
O
F
O
F
a)
F
O N
F
F
O
F
O
N3
O
F
OH
O
HO
F
OH
O
N3
Et3N, CH2Cl2, r.t.
3 (9%)
F
F
O
F
O
b)
F
OH
O
HO
OH
N3
O
O
(major)
+
HO
F
O
F
OH
DCC, DMAP, CH2Cl2, r.t.
F
OH
O
O
3 (minor)
N3
F
O
c)
HO
O
O
O
F
F
O
F
4
5
F
OH
F
N3
O
F
O
O
O
HO
O
DCC, DMAP, CH2Cl2, r.t.
N3
F
4
F
F
O
F
OH
3
O
N3
F
F
Scheme 2. Attempted synthesis of 3.

Q. Peng et al. / Tetrahedron Letters 46 (2005) 5893–5897
5895
F
F
O
F
F
OH
N3
O
O
SH
HO
HO
OH
S
DCC, DMAP, CH2Cl2
80.9 %
DCC, MeCN
6
80.0 %
F
F
O
F
F
F
O
AgNO3
F
S
F
OH
O
O
dioxane, H2O
92.5 %
O
O
7
3
N3
N3
F
O
P
O
O
N+
O
lyso-PC
O
O-
F
F
O
F
DCC, DMAP, CHCl3
55.0 %
F
O
O
O
2
N3
Scheme 3. Synthesis of 2.
First we attempted to protect 12-hydroxydodecanoic
acid by transforming it into a methyl ester (4 in Scheme
2c), before coupling it with 4-azidotetrafluorobenzoic
acid. However, the ester of 4-azidotetrafluorobenzoic
acid is more reactive and more readily hydrolyzed than
the methyl ester in 5. Hydrolysis of 5 led to the hydro-
lysis of the ester of 4-azidotetrafluorobenzoic acid,
giving 4 instead of 3.
We then protected 12-hydroxydodecanoic acid by trans-
forming it into thiolester 6 (Scheme 3),16 which was fur-
ther transformed into 7 by subsequently coupling the
latter with 4-azidotetrafluorobenzoic acid.17 The thiol-
Figure 1. UV spectral recording of the increased solubility of 2 in
buffer solution (100 mM phosphate, pH 7.4 at 20 °C) when adding the
detergent, sodium dodecylsulfate (SDS).
18
ester in 7 was selectively deprotected by AgNO3 and
gave 3 with a yield of more than 90%. Finally, coupling
3 with lyso-phosphocholine (lyso-PC) gave 2 with a yield
of 55%.19 It is worth noting that the coupling reactions
with lyso-phosphocholine have to be performed under
strictly anhydrous conditions in order to obtain satisfac-
tory yields.20
went a fast, clear-cut process of photodecomposition in
response to irradiation at P300 nm (Fig. 2). Irradiation
of the probe 2 quickly led to the disappearance of the
absorption band. The isobestic points observed indi-
cated that the photochemical reaction was a unique photo-
decomposition process. One important point is the fact
that 2 can be activated at wavelength P300 nm, which
ensures that the biological macromolecules will not be
damaged by UV irradiation.
The synthesized probe 2 is stable in the dark. It is
soluble in MeOH–CH2Cl2, but not readily soluble in
phosphate buffer. We attempted to solubilize it in phos-
phate buffer in the presence of the detergent sodium
dodecylsulfate (SDS). The fact that the solubility of 2
in the buffer can be significantly increased by adding
SDS (Fig. 1) suggests that 2 conserves the amphiphilic
characteristics of phospholipids.
In conclusion, a photoactivatable phospholipidic probe
with fluorinated aryl azide introduced into the fatty acid
chain was synthesized and characterized here as a
potential tool for studying lipid–lipid and lipid–protein
interactions using a photolabeling approach. The photo-
chemical reactions occurring with this probe were inves-
tigated in both organic solvent and buffer solution, and
it consistently showed a fast, clear-cut photochemical
reaction, which suggests that it will provide promising
tool for use in photolabeling studies. Furthermore, the
We further characterized the photochemical properties
of probe 2. Its maximum UV absorption is around
265 nm, and the molar absorption coefficient is around
15,000 (mol/L)À1 cmÀ1: this value is characteristic of
fluorinated aryl azides.11 A photochemical study was
carried out with 2 in both organic solvent (CH2Cl2–
MeOH, 1:1) and phosphate buffer. Compound 2 under-

5896
Q. Peng et al. / Tetrahedron Letters 46 (2005) 5893–5897
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Figure 2. UV spectral recording of the photochemical reaction
occurring with 2 in (a) MeOH–CH2Cl2 and (b) 100 mM phosphate
buffer (pH 7.4) in response to irradiation at 300 nm at 20 °C.
15. Compound 3: white solid. Mp: 71 °C; 1H NMR
(300 MHz, CDCl3): d 4.36 (t, 2H, J = 6.6 Hz), 2.35 (t,
2H, J = 7.5 Hz), 1.72–1.76 (m, 2H), 1.61–1.65 (m, 2H),
1.28 (m, 14H); 13C NMR (150 MHz, CDCl3): d 176.2,
1
presence of fluorine atoms in the probe makes it a use-
ful means of studying specific lipid–lipid and lipid–
protein interactions using 19F NMR methods. Studies
on the use of this probe for investigating biomembranes
and protein–lipid interactions via a photolabeling
approach and 19F NMR are now underway at our
laboratories.
155.5, 140.4–142.1 (ds, 2C, JCF = 255.9 Hz), 135.5–137.2
(dd, 2C, JCF = 249.6 Hz, JCF = 15.9 Hz), 119.1, 104.0–
1
2
2
104.1 (d, 1C, JCF = 15.3 Hz), 62.8, 30.0, 25.4, 25.3, 25.2,
25.1, 25.0, 24.4, 21.7, 20.6; 19F NMR (564.6 MHz,
CDCl3): d À139.2 (m, 2F), À151.4 (m, 2F); IR (KBr):
2138.1 cmÀ1; MS (ESI): 431.8 (MÀH)À, 864.6 (2MÀH)À;
UV (CH2Cl2): kmax = 265 nm, e = 20,665 (mol/L)À1 cmÀ1
.
16. Compound 6: white solid. Mp: 35 °C; 1H NMR
(300 MHz, CDCl3): 3.64 (m, 2H), 2.84 (t, 2H,
d
J = 6.9 Hz), 2.53 (t, 2H, J = 7.5 Hz), 1.54–1.67 (m, 6H),
1.27 (m, 14H), 0.96 (t, 3H, J = 7.2 Hz); 13C NMR
(150 MHz, CDCl3): d 200.2, 63.2, 44.4, 33.0, 30.9, 29.8,
29.7, 29.6, 29.6, 29.4, 29.1, 25.9, 25.9, 23.2, 13.5; MS (ESI):
274.5 (M)+.
Acknowledgments
We thank Dr. Jessica Blanc for revising the English
manuscript. Financial support from the Ministry of
Science and Technology in China (Nos. 2003CB114400
and 2003AA2Z3506), National Science foundation
of China (20372055), Cheung Kong Scholar Founda-
tion, Wuhan University and CNRS is gratefully
acknowledged.
17. Compound 7: white solid. Mp: 34 °C; 1H NMR
(300 MHz, CDCl3): d 4.35 (t, 2H, J = 6.6 Hz), 2.84 (t,
2 H, J = 7.2 Hz), 2.53 (t, 2H, J = 7.5 Hz), 1.57–1.73 (m,
6H), 1.27–1.39 (m, 14H), 0.95 (t, 3H, J = 7.2 Hz); 13C
NMR (150 MHz, CDCl3): d 200.0, 159.6, 144.6–146.3
1
1
(ds, 2C, JCF = 256.3 Hz), 139.7–141.5 (dd, 2C, JCF
=
2
258.5 Hz, JCF = 15.5 Hz), 123.3, 108.3, 67.0, 44.3, 30.9,
29.6, 29.6, 29.4, 29.3, 29.1, 28.6, 26.0, 25.9, 23.2, 13.6; 19F
NMR (564.6 MHz, CDCl3): d À139.3 (dd, 2F, J = 20.6,
11.0 Hz), À151.4 (dd, 2F, J = 21.5, 10.2 Hz); IR (CH2Cl2):
2127.5 cmÀ1; MS (ESI): 515.0 (M+Na)+; UV (CH2Cl2):
References and notes
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max = 265 nm, e = 20,954 (mol/L)À1 cmÀ1
.
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19. Compound 2: waxy solid. 1H NMR (300 MHz, CDCl3/
CD3OD): d 5.21 (m, 1H), 4.34–4.42 (m, 3H), 4.10–4.17 (m,
1H), 3.99 (t, 2H, J = 6.0 Hz), 3.38 (m, 2H), 3.22 (s, 9H),
2.27–2.34 (m, 4H), 1.72–1.77 (m, 2H), 1.59 (m, 6H), 1.25–
1.28 (m, 38H), 0.88 (t, 3H, J = 6.6 Hz); 13C NMR
(150 MHz, CDCl3/CD3OD): d 174.0, 173.6, 159.7, 144.5–
4. Peng, L.; Alcaraz, M.-L.; Klotz, P.; Kotzyba-Hibert, F.;
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1
146.3 (ds, 2C, JCF = 261.6 Hz), 139.7–141.5 (dd, 2C,
2
1JCF = 250.5 Hz, JCF = 14.9 Hz), 123.4, 108.1, 70.5, 67.0,
66.6, 63.7, 62.9, 59.3, 54.5, 34.5, 34.3, 32.1, 31.1, 30.0, 29.8,

Q. Peng et al. / Tetrahedron Letters 46 (2005) 5893–5897
5897
29.7, 29.6, 29.5, 29.4, 29.3, 28.6, 26.0, 25.0, 22.9, 14.3; 19F
NMR (564.6 MHz, CDCl3/CD3OD): d À139.4 (m, 2F),
À151.5 (m, 2F); 31P NMR (242.9 MHz, CDCl3/CD3OD):
d 0.26; IR (CH2Cl2): 2127.4 cmÀ1; MS (ESI): 933.9
(MÀH+Na)+, 1845.3 (2MÀH+Na)+; UV (CH2Cl2):
k
max = 265 nm, e = 13,933 (mol/L)À1 cmÀ1
.
20. Sun, M. J.; Deng, Y. J.; Batyreva, E.; Sha, W.; Salomon,
R. G. J. Org. Chem. 2002, 67, 3575.