Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine derivatives: Potential fluorinated probes for the study of membrane topology

Article abstract of DOI:10.1039/c2ob06570c

The synthesis of three monofluorinated dimyristoylphosphatidylcholine derivatives (F-DMPC), with the fluorine atom located on the acyl chain in position 2 of the glycerol (sn-2) is described. The synthetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) and three different fluorinated fatty acids. The latter were obtained from two different and complementary synthetic routes. Preliminary FTIR studies suggest that the presence of the fluorine atom does not significantly perturb the lipid conformational order and phase transition temperature and that these monofluorinated PC derivatives could be used as probes for the study of membrane topology, i.e. the location of drugs, peptides or proteins in membranes. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob06570c

Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Volume 10 | Number 6 | 14 February 2012 | Pages 1125–1312
ISSN 1477-0520
COMMUNICATION
Paquin et al.
Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine
derivatives: Potential fluorinated probes for the study of membrane topology

Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2012, 10, 1145
www.rsc.org/obc
COMMUNICATION
Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine
derivatives: Potential fluorinated probes for the study of membrane topology†
Jonathan Guimond-Tremblay,a,b Marie-Claude Gagnon,a,b Jozy-Ann Pineault-Maltais,a,b Vanessa Turcotte,a,b
Miche`le Auger*a and Jean-Franc¸ois Paquin*b
Received 13th September 2011, Accepted 21st November 2011
DOI: 10.1039/c2ob06570c
The synthesis of three monofluorinated dimyristoylphos-
phatidylcholine derivatives (F-DMPC), with the fluorine
atom located on the acyl chain in position 2 of the glycerol (sn-
2) is described. The synthetic strategy relies on the coupling
of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-
PC) and three different fluorinated fatty acids. The latter were
obtained from two different and complementary synthetic
routes. Preliminary FTIR studies suggest that the presence
of the fluorine atom does not significantly perturb the lipid
conformational order and phase transition temperature and
that these monofluorinated PC derivatives could be used as
probes for the study of membrane topology, i.e. the location
of drugs, peptides or proteins in membranes.
the surface of negatively charged membranes through electro-
static interactions, thus conferring selectivity towards bacterial
membranes.2
Several techniques have been developed to investigate the
interactions between drugs or peptides and lipid membranes.1a
Among these, solid-state nuclear magnetic resonance (NMR) is
a method of choice to study the effects of drugs and peptides on
model membranes.3 In addition, the measurement of internuclear
distances between specifically labelled nuclei such as 13C and
15N can help to probe local structure at the molecular level.4
However, these nuclei suffer from their low natural abundance
and sensitivity. The use of 19F offers several advantages for
NMR studies, including its high sensitivity and 100% natural
abundance.3c In addition, the absence of native 19F in membrane
components allows the selective detection of 19F labelled sites.3c It
is therefore of great interest to develop efficient methods for the
synthesis of fluorinated lipids labelled both on the lipid acyl chains
and the polar head groups.
We report herein the synthesis of three monofluorinated
dimyristoylphosphatidylcholine derivatives (F-DMPC) with the
fluorine atom located on one of the two acyl chains (Fig. 1). The
synthetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-
sn-glycero-3-phosphocholine (lyso-PC) and three different fluo-
rinated fatty acids. The latter were obtained from two different
and complementary synthetic routes. Characterization of the three
monofluorinated DMPC derivatives by FTIR is also reported.
Introduction
Understanding the interactions between lipid membranes and
drugs, peptides or proteins is of primary importance to determine
their mechanism of action.1 For example, several drugs with
diverse structures and applications (anticancer, local anaesthetic,
non-steroidal anti-inflammatory, antibiotic, etc.) can interact with
cell membranes and modulate their physical properties. The
membranes can also affect the pharmacological properties of the
drugs being administered.
Biological membranes are made of a variety of lipids differing
in terms of their headgroup, chain length and degree of saturation.
For example, eukaryotic membranes are composed mainly of
zwitterionic (neutral) lipids such as phosphatidylcholine (PC) and
phosphatidylethanolamine (PE) while prokaryotic cell membranes
also contain negatively charged lipids such as cardiolipin (CL)
and phosphatidylglycerol (PG). A key factor in determining drug
and peptide selectivity is the composition of the membranes. For
examples, many antimicrobial peptides are cationic and bind to
aDepartment of Chemistry, PROTEO, CERMA, 1045 avenue de la
Me´decine, Universite´ Laval, Que´bec, Canada. E-mail: michele.auger@
chm.ulaval.ca; Fax: +1-418-656-7916; Tel: +1-418-656-3393
bCanada Research Chair in Organic and Medicinal Chemistry, Department
of Chemistry, PROTEO, 1045 avenue de la Me´decine, Universite´ Laval,
Que´bec, Canada. E-mail: jean-francois.paquin@chm.ulaval.ca; Fax: +1-
418-656-7916; Tel: +1-418-656-2131 ext. 11430
† Electronic supplementary information (ESI) available: Experimental
procedures, isolation and spectroscopic information for the new com-
pounds prepared. See DOI: 10.1039/c2ob06570c
Fig. 1 Targeted monofluorinated 1,2-dimyristoyl-sn-glycero-3-phospho-
choline (F-DMPC) and their retrosynthetic analysis.
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1145–1148 | 1145
©

Results and discussion
We initially decided to focus on fluorinated derivatives of 1,2-
dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as this lipid is
commonly used as model membrane for eukaryotic cells.5 We
focused on the introduction of only one fluorine atom at different
specific locations on the acyl chain located in position 2 of the
glycerol (sn-2) as it has been shown that having two fluorinated acyl
chains led to very different physical properties of non-fluorinated
lipids preventing their use as model membranes.6 Also, the side
chain will bear only one fluorine atom because it has been shown
that two fluorine atoms (vs CH2) led to an underestimation of the
order parameters,7 again limiting their use in model membranes.
Finally, two fluorinated derivatives of 1,2-dipalmitoyl-sn-glycero-
3-phosphocholine (DPPC) has been described previously in the
literature8 but to the best of our knowledge, monofluorinated
derivatives of DMPC have not yet been reported.
The synthesis of 4F-DMPC (1a) starts with commercially
available or readily prepared 4-benzyloxy-1-butanol (3)9 which
is first oxidized to the aldehyde 410 under Albright–Onodera
conditions using Taber’s modification (Scheme 1).11 Addition of
a freshly prepared solution of decanemagnesium bromide gave
the secondary alcohol in good yield. Deoxofluorination using
diethylaminosulfur trifluoride (DAST)12 furnished compound 6 in
80% isolated yield. The latter is however contaminated by ca. 10%
of an unexpected isomer, 4-benzyloxy-1-fluorotetradecane (9) and
a possible mechanism for its formation is shown in Fig. 2. Upon
activation of the alcohol (5) with DAST, two pathways would
be possible. First, as expected for a deoxofluorination reaction,
fluoride could displace the activated alcohol on 7 through a SN2
reaction leading to compound 6 (pathway not shown). In addition,
the oxygen of the protected alcohol could displace the leaving
group via an intramolecular SN2 reaction, thus leading to the
intermediate 8. Fluoride ion could then attack either carbon (C1
or C4) where attack at C4 would lead to the expected product (6
via path b) and attack at C1 would give the isomeric compound (9
Fig. 2 Possible mechanism for the formation of 9.
via path a). Nevertheless, the impure fluoro compound 6 is carried
on by removing the benzyl protecting group. The primary alcohol
is then directly oxidized to the carboxylic acid using TEMPO13
to give compound 2a in 86% yield. Finally, the fluorinated
fatty acid 2a is coupled with 1-myristoyl-2-hydroxy-sn-glycero-
3-phosphocholine (lyso-PC) to give pure 4F-DMPC (1a) in 72%
yield after purification by flash chromatography on silica gel using
a 1 : 1 MeOH/CH2Cl2 mixture as the eluent.14 An overall yield of
33% was obtained for the 6 synthetic steps.
As shown in Scheme 2, the synthesis of 7F-DMPC (1b) starts
with readily available 5-hexynyl tetrahydro-2H-pyran-2-yl ether
(10).15 Deprotonation of the alkyne and addition of the resulting
alkynyl anion onto octanal produced alcohol 1116 in 70% yield.
Deoxyfluorination of the alcohol with DAST12 gave the fluoro
compound 12 in good yield. Hydrogenation of the alkyne under
palladium catalysis and simultaneous deprotection of the THP
ether17 gave the fluorinated alcohol 13 in 91% yield. Oxidation
of the primary alcohol to the carboxylic acid was achieved using
TEMPO13 and gave the desired acid in 80% yield. Finally, the latter
Scheme 1 Synthesis of 4F-DMPC: Reagents and conditions: a) DMSO,
P2O5, Et3N, CH2Cl2, 0 ◦C to rt, 98%; b) 1-bromodecane, Mg, THF, 0 ◦
C
Scheme 2 Synthesis of 7F-DMPC: Reagents and conditions: a) i) n-BuLi,
THF, -78 ◦C, ii) Octanal, THF, 0 ◦C to rt, 70%; b) DAST, CH2Cl2, -78 ◦C
to rt, 64%; c) H2 (1 atm), 10% Pd/C, PdCl2 (5 mol%), MeOH/hexane
(1/1), 91%; d) TEMPO (7 mol%), NaClO2, NaOCl (2 mol%), phosphate
buffer (0.67 M, pH = 6.7), CH3CN, 80%; e) Lyso-PC, 2,6-dichlorobenzoyl
chloride, 1-methylimidazole, CHCl3, 85%.
to rt, 83%; c) DAST, CH2Cl2, -78 ◦C to rt, 80% (contaminated with
ca. 10% of 9); d) H2 (2 atm), 10% Pd/C, AcOH/THF (1/1), 82%; e)
TEMPO (7 mol%), NaClO2, NaOCl (2 mol%), phosphate buffer (0.67
M, pH = 6.7), CH3CN, 86%; f) Lyso-PC, 2,6-dichlorobenzoyl chloride,
1-methylimidazole, CHCl3, 72%.
1146 | Org. Biomol. Chem., 2012, 10, 1145–1148
This journal is
The Royal Society of Chemistry 2012
©

was coupled with lyso-PC to give 7F-DMPC (1b) after purification
by flash chromatography on silica gel using a 1 : 1 MeOH/CH2Cl2
mixture as the eluent. An overall yield of 28% was obtained for
the 5 synthetic steps.
Finally, the synthesis of 10F-DMPC, conducted similarly to 4F-
DMPC, starts with 1,10-decanediol (14) which is first monoben-
zylated followed by oxidation of the remaining alcohol to the
aldehyde 15 (Scheme 3).18 Addition of butylmagnesium bromide
gave the secondary alcohol.19 Deoxofluorination using DAST
furnished compound 17 in good yield. Removal of the benzyl
group20 followed by oxidation of the newly revealed alcohol using
TEMPO gave compound 2c in 77% yield.20 Finally, the fluorinated
fatty acid 2c is coupled with 1-myristoyl-2-hydroxy-sn-glycero-
3-phosphocholine (lyso-PC) to give pure 10F-DMPC (1c) after
purification by flash chromatography on silica gel using a 1 : 1
MeOH/CH2Cl2 mixture as the eluent. An overall yield of 19%
was obtained for the 7 synthetic steps.
Fig. 3 Temperature dependence of the wavenumber of the CH2 symmetric
stretching vibration for DMPC, 4F-DMPC, 7F-DMPC and 10F-DMPC.
The error is estimated to be 0.1 cm-1.
10F-DMPC derivative and the 4F-derivative. However, a single
phase transition temperature is observed for all three derivatives,
indicating that the lipids form a homogeneous mixture.
Our results are in agreement with previous studies on lipids
monofluorinated on both acyl chains which have shown that the
introduction of the fluorine atom does not significantly perturb
the order of the lipid bilayer.8a However, the introduction of more
than one fluorine atom on one or two acyl chains has been shown
to induce a significant decrease of the lipid gel-to-fluid phase
transition temperature.7b,22 More specifically, the phase transition
temperature is decreased by about 5–11 ◦C with the introduction
of two fluorine atoms on the sn-2 acyl chains7b and by ~21 ◦C with
the introduction of six fluorine atoms on both acyl chains.22 In
addition, the introduction of six fluorine atoms on both acyl chains
results in an increase of the CH2 stretching vibration wavenumber
of ~2.5 c◦m-1 in the gel phase and a significant broadening (by
about 12 C) of the phase transition.22 It is also interesting to note
that the introduction of two fluorine atoms at position 4 of the
sn-2 acyl chain has less effect than the introduction of two fluorine
atoms at positions 8 and 12, which is in agreement with the results
obtained in the present study with monofluorinated lipids.7b
The wavenumbers of the CH2 stretching vibration as a function
of temperature obtained for pure commercial DMPC and for 4F-
DMPC incorporated in DMPC at different percentages ranging
from 2.5 to 100% are shown in Fig. 4. These results indicate that
the presence of the fluorine atom does not significantly perturb
the lipid phase transition temperature and conformational order
at percentages between 2.5 and 25%. A small decrease of the lipid
phase transition temperature and lipid order is observed at higher
ratios of fluorinated lipids (percentages of 50 and 100%). Similar
results have also been obtained as a function of the fluorinated
lipid molar ratio with the 7F-DMPC and 10F-DMPC derivatives
(results not shown).
Scheme 3 Synthesis of 10F-DMPC: Reagents and conditions: a) BnBr,
Ag2O, CH2Cl2/CHCl3 (1/1), 64%; b) DMSO, P2O5, Et3N, CH2Cl2, 0 ◦
C
to rt, 64%; c) 1-bromobutane, Mg, THF, 0 ◦C to rt, 94%; d) DAST, CH2Cl2,
-78 ◦C to rt, 81%; e) H2 (2 atm), 10% Pd/C, AcOH/THF (1/1), 98%; f)
TEMPO (7 mol%), NaClO2, NaOCl (2 mol%), phosphate buffer (0.67
M, pH = 6.7), CH3CN, 77%; g) Lyso-PC, 2,6-dichlorobenzoyl chloride,
1-methylimidazole, CHCl3, 80%.
We have used FTIR spectroscopy to determine the effect of the
introduction of a fluorine atom on the acyl chain conformational
order. More specifically, the study of the CH2 symmetric (2850
cm-1) stretching vibration, which is sensitive to trans/gauche
isomerization in the lipid acyl chains, can provide valuable
information about the order of the acyl chains and the lipid gel-
to-fluid phase transition temperature.21
Fig. 3 shows the wavenumbers of the CH2 stretching vibration as
a function of temperature for pure commercial DMPC and pure
4F-DMPC, 7F-DMPC and 10F-DMPC. These results indicate
that the presence of a fluorine atom in the three derivatives
does not significantly perturb the properties of the lipid bilayer.
However, it induces a small decrease in the lipid phase transition
temperature (by about 1–2 ◦C), a small broadening (by about 1–
2
◦C) of the phase transition and a small increase in the CH2
Conclusions
stretching vibration wavenumber (~0.5–1 cm-1) associated with a
small increase in the number of gauche conformers. This effect
is more important for the 7F-DMPC derivative then for the
We have described a practical synthesis of three monofluorinated
dimyristoylphosphatidylcholine derivatives in 5–7 synthetic steps
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1145–1148 | 1147
©

Notes and references
1 (a) A. M. Seddon, D. Casey, R. V. Law, A. Gee, R. H. Templer and O.
Ces, Chem. Soc. Rev., 2009, 38, 2509; (b) J. M. Sanderson, Org. Biomol.
Chem., 2005, 3, 201.
2 D. I. Chan, E. J. Prenner and H. J. Vogel, Biochim. Biophys. Acta,
Biomembr., 2006, 1758, 1184.
3 (a) E. Strandberg and A. S. Ulrich, Concepts Magn. Reson., Part
A, 2004, 23A, 89; (b) M. Ouellet and M. Auger, Annu. Rep. NMR
Spectrosc., 2008, 63, 1; (c) A. S. Ulrich, Prog. Nucl. Magn. Reson.
Spectrosc., 2005, 46, 1; (d) F. Cronier, A. Patenaude, R. C. Gaudreault
and M. Auger, Chem. Phys. Lipids, 2007, 146, 125; (e) M. Ouellet, J.-D.
Doucet, N. Voyer and M. Auger, Biochemistry, 2007, 46, 6597.
4 (a) D. P. Raleigh, M. H. Levitt and R. G. Griffin, Chem. Phys. Lett.,
1988, 146, 71; (b) T. Gullion, Concepts Magn. Reson., 1998, 10, 277.
5 M. Ouellet, G. Bernard, N. Voyer and M. Auger, Biophys. J., 2006, 90,
4071.
6 J. M. Sturtevant, C. Ho and A. Reinman, Proc. Natl. Acad. Sci. U. S.
A., 1979, 76, 2239.
7 (a) E. Oldfield, R. W. K. Lee, M. Meadows, S. R. Dowd and C. Ho,
J. Biol. Chem., 1980, 255, 11652; (b) S. R. Dowd, J. M. Sturvenant, V.
Simplaceanu and C. Ho, J. Phys. Chem., 1993, 97, 2946.
8 (a) B. McDonough, P. M. Macdonald, B. D. Sykes and R. N.
McElhaney, Biochemistry, 1983, 22, 5097; (b) D. J. Hirsh, N. Lazaro,
L. R. Wright, J. M. Boggs, T. J. McIntosh, J. Schaefer and J. Blazyk,
Biophys. J., 1998, 75, 1858.
Fig. 4 Temperature dependence of the wavenumber of the CH2 symmetric
stretching vibration for various mixtures of 4F-DMPC and DMPC. The
error is estimated to be 0.1 cm-1.
9 L. Nielsen, K. B. Lindsay, J. Faber, N. C. Nielsen and T. Skrydstrup, J.
Org. Chem., 2007, 72, 10035.
10 J. S. Yadav, M. Sreenivas, A. Srinivas Reddy and B. V. Subba Reddy, J.
Org. Chem., 2010, 75, 8307.
11 D. F. Taber, J. C. Amedio Jr. and K.-Y. Jung, J. Org. Chem., 1987, 52,
5621.
12 R. P. Singh and J. M. Shreeve, Synthesis, 2002, 2561.
13 M. Zhao, J. Li, E. Mano, Z. Song, D. M. Tschaen, E. J. J. Grabowski
and P. J. Reider, J. Org. Chem., 1999, 64, 2564.
14 H. P. Acharya and Y. Kobayashi, Synlett, 2005, 2015.
15 L. Zhao, X. Lu and W. Xu, J. Org. Chem., 2005, 70, 4059.
16 M. J. Cryle and J. J. De Voss, Chem. Commun., 2004, 86.
17 (a) L. H. Kaisalo and T. A. Hase, Tetrahedron Lett., 2001, 42,
7699; (b) T. Ikawa, H. Sajiki and K. Hirota, Tetrahedron, 2004, 60,
6189.
and 19–33% overall yields from commercially or readily available
starting materials. Preliminary data obtained using FTIR indicate
that the presence of the fluorine atom does not significantly perturb
the lipid conformational order and phase transition temperature.
This suggests that these novel monofluorinated lipids could
potentially be used as NMR probes for the study of membrane
topology and for the investigation at the molecular level of the
interaction between drugs or peptides and lipid membranes.
Acknowledgements
This work was supported by PROTEO (fellowships to JGT, VT,
MCG), the Canada Research Chair Program (JFP), the Natural
Sciences and Engineering Research Council of Canada (MA and
JFP), the Canada Foundation for Innovation (JFP), the Fonds
de Recherche sur la Nature et les Technologies (MA and JFP),
and Universite´ Laval (MA and JFP). We thank Mr. Jean-Franc¸ois
Rioux-Dube´ for his assistance with the FTIR experiments and
Prof. Michel Pe´zolet for helpful discussions.
18 T. Shioiri, Y. Terao, N. Irako and T. Aoyama, Tetrahedron, 1998, 54,
15701.
19 B. Jakob and H. Gerlach, Liebigs Ann., 1996, 2123.
20 J.-L. Abad, G. Villorbina, G. Fabria`s and F. Camps, Lipids, 2003, 38,
865.
21 H. H. Mantsch and R. N. McElhaney, Chem. Phys. Lipids, 1991, 57,
213.
22 S. Schuy, S. Flaiss, N. C. Yoder, V. Kalsani, K. Kumar, A. Janshoff and
R. Vogel, J. Phys. Chem. B, 2008, 112, 8250.
1148 | Org. Biomol. Chem., 2012, 10, 1145–1148
This journal is
The Royal Society of Chemistry 2012
©