Evaluation of the effect of fluorination on the property of monofluorinated dimyristoylphosphatidylcholines

Article abstract of DOI:10.1039/c4ob00934g

The synthesis of three monofluorinated dimyristoylphosphatidylcholines (F-DMPC's), with the fluorine atom located at the extremities of 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 (14:0 lyso-PC) and three different fluorinated fatty acids. FTIR results suggest that the presence of the fluorine atom does not significantly perturb the lipid phase transition temperature and conformational order even though a small increase in the phase transition temperature is observed for the 14F derivative. Overall, comparison with previously reported F-DMPC's where the fluorine atom is located in the middle or close from either side supports the fact that monofluorination of the acyl chain in sn-2 brings minimal perturbation to the lipid bilayer. F-DMPC's could therefore potentially be used as NMR probes for the investigation at the molecular level of the interaction between drugs or peptides and lipid membranes and for the study of membrane topology.

Full text of DOI:10.1039/c4ob00934g

Organic &
Biomolecular Chemistry
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Evaluation of the effect of fluorination on the
property of monofluorinated
dimyristoylphosphatidylcholines†
Cite this: Org. Biomol. Chem., 2014,
12, 5126
Marie-Claude Gagnon,a,b Bianka Turgeon,a,b Jean-Daniel Savoie,a,b
Jean-François Parent,a,b Michèle Auger*a and Jean-François Paquin*b
The synthesis of three monofluorinated dimyristoylphosphatidylcholines (F-DMPC’s), with the fluorine
atom located at the extremities of the acyl chain in position 2 of the glycerol (sn-2), is described. The syn-
thetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 lyso-
PC) and three different fluorinated fatty acids. FTIR results suggest that the presence of the fluorine atom
does not significantly perturb the lipid phase transition temperature and conformational order even
though a small increase in the phase transition temperature is observed for the 14F derivative. Overall,
comparison with previously reported F-DMPC’s where the fluorine atom is located in the middle or close
from either side supports the fact that monofluorination of the acyl chain in sn-2 brings minimal pertur-
bation to the lipid bilayer. F-DMPC’s could therefore potentially be used as NMR probes for the investi-
gation at the molecular level of the interaction between drugs or peptides and lipid membranes and for
the study of membrane topology.
Received 6th May 2014,
Accepted 25th May 2014
DOI: 10.1039/c4ob00934g
www.rsc.org/obc
nuclei provides a more facile access to the labelled compounds
especially given the recent development in synthetic organo-
Introduction
Fluorine is increasingly used for nuclear magnetic resonance fluorine chemistry.2
spectroscopy studies of biological systems since this element
Overall, these characteristics make 19F NMR spectroscopy
offers several advantages over other nuclei. 19F is a 100% natu- very attractive to study biological systems with fluorine labeled
rally abundant spin-1/2 nucleus with a high gyromagnetic molecules such as drugs, peptides, proteins and nucleic
ratio. This allows for fast accumulation of highly resolved acids.3,4 While 19F solution NMR spectroscopy has been widely
spectra which are simplified compared to nuclei with a quad- used for structural and functional studies1b,5 or for drug disco-
rupolar moment.1 Notably, 19F relative sensitivity is about 83% very,1b,6 19F solid-state NMR spectroscopy is ideal for the study
that of 1H, which is much greater than the relative sensitivities of complex of biomolecules with lipid bilayers1a as exemplified
of 13C (1.6%) and 15N (0.1%). In addition, its large spectral with recent reports.7 In these systems, the fluorine atom is
width and the absence of fluorine in native biological com- located directly on the biomolecules under investigation.8 An
ponents allow respectively the detection of even slight vari- interesting complementary alternative is to incorporate the 19F
ations of the fluorine chemical environment and the selective label into the lipid bilayer.9
detection of the labeled sites.1 Finally, compared to several
Natural cell membranes contain several components,
other nuclei such as 13C and 15N, for which expensive isotope including various lipids, proteins and cholesterol, which
labeling is necessary,1b the use of the 100% abundant 19F render spectroscopic analyses very difficult.10 Since the major
component of cell membranes is lipid, model membranes
composed of phospholipids are widely used in the literature to
simplify the study of cell membranes. In particular, dimyristoyl-
aDepartment of Chemistry, PROTEO, CERMA, 1045 avenue de la Médecine,
Université Laval, Québec, Canada. E-mail: Michele.auger@chm.ulaval.ca;
phosphatidylcholine (DMPC) is often used to mimic eukaryotic
Fax: +1-418-656-7916; Tel: +1-418-656-3393
membranes.11
bCanada Research Chair in Organic and Medicinal Chemistry, Department of
Chemistry, PROTEO, CGCC, 1045 avenue de la Médecine, Université Laval, Québec,
Canada. E-mail: jean-francois.paquin@chm.ulaval.ca; Fax: +1-418-656-7916;
The incorporation of fluorine atoms on phospholipids has
been explored before and those studies have shown that the
number of fluorine atoms and their specific positions influ-
ence significantly the perturbation of the lipid bilayers
observed. While the introduction of one or two perfluorinated
Tel: +1-418-656-2131 ext. 11430
†Electronic supplementary information (ESI) available: Copy of the NMR spectra
for the new compounds prepared and experimental details for the characteriz-
ation by FTIR. See DOI: 10.1039/c4ob00934g
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Fig. 1 Polyfluorinated and monofluorinated phosphatidylcholines previously reported.
side chains (number of fluorine per side chain >2) on phos- interest to use lipids with the fluorine atom located at the
phatidylcholines systematically disturbs the properties of the extremities of the lipid acyl chain. Herein, we therefore report
lipids,12 less fluorinated lipids behave differently (Fig. 1). For the synthesis and characterization by FTIR of three novels
instance, the presence of fluorine atoms on DMPC analogs 113 F-DMPC’s with the fluorine located at both ends of the acyl
and 2,14 containing gem-difluorinated groups, and DPPC chain (2F, 12F and 14F-DMPC) (Fig. 2).
(dipalmitoylphosphatidylcholine) analogs 3,15 which contain
one fluorine atom on each acyl chain, induces a significant
disorder of the lipid bilayers characterized by a decrease in the
phase transition temperature of up to 12, 11 and 5 °C respecti-
vely compared to the non-fluorinated analog. In contrast,
DPPC monofluorinated analog 4 was shown to stabilise the
Results and discussion
Synthesis of monofluorinated
dimyristoylphosphatidylcholines
lipid bilayers, increasing its phase transition temperature by Our synthetic strategy for the synthesis of the targeted
about 10 °C as a result of the formation of interdigitated F-DMPC (9–11) relied on the coupling of commercially avail-
bilayers.16 Nevertheless, it was shown that mixture of fluori- able 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0
nated lipids (4 and 5) with non-fluorinated analogs may mini- lyso-PC, 15) and fluorinated fatty acids (12–14) (Fig. 3). The
mise the perturbation.17
latter were obtained from different and complementary syn-
With the goal of using fluorinated lipids as molecular thetic routes.
probes in 19F solid-state NMR for the study of biomolecules,
The synthesis of 2F-DMPC was inspired by the work per-
we have reported the synthesis of three monofluorinated formed by Haufe and coworker (Scheme 1).19 Thus, bromo-
dimyristoylphosphatidylcholines (F-DMPC’s) with the fluorine fluorination20 of commercially available 1-tetradecene (16)
atom located in the middle (7F-DMPC) or close from either using NBS and Et3N·3HF produced 1-bromo-2-fluorotetra-
side (4F and 10F-DMPC) on the acyl chain at position two of decane (16) in moderate yield after flash chromatography con-
the glycerol (Fig. 2).18 FTIR studies indicated that the presence taminated with small amount (ca. 2%) of the inseparable
of the fluorine atom in pure lipid or in mixtures with DMPC regioisomer, 2-bromo-1-fluorotetradecane.21 Formation of
brought minimal perturbation. However, for extensive and acetate 18 using potassium acetate followed by hydrolysis
complete studies of membrane topology, it would also be of under basic conditions produced alcohol 19. Both steps
Fig. 2 Previously reported and targeted monofluorinated 1,2-dimyris-
toyl-sn-glycero-3-phosphocholines (F-DMPC’s). The colored circle rep-
resents the carbon bearing the fluorine atom.
Fig. 3 Retrosynthetic analysis.
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Scheme 1 Synthesis of 2F-DMPC.
proceeded efficiently. Oxidation of 19 to the carboxylic acid 12 the racemic fluorinated acid 13 that was coupled with 14:0
was problematic. Indeed, of the numerous methods investi- lyso-PC (15) in moderate yield22 after purification by flash
gated, only Jones oxidation provided the desired acid in a poor chromatography on silica gel. An overall yield of 19% was
22% yield. Finally, the racemic fluorinated fatty acid 12 was obtained for the 6 synthetic steps starting from 20.
coupled with 14:0 lyso-PC (15) in good yield22 to give pure
Finally, 14F-DMPC (11) was prepared starting from readily
2F-DMPC (9) in 86% yield after purification by flash chromato- available monobenzylether 24 (Scheme 3).28 Deoxofluorination
graphy on silica gel. An overall yield of 8% was obtained for with XtalFluor-E ([Et2NSF2]BF4)29 and Et3N·3HF as an external
the 5 synthetic steps.
source of fluoride provided fluoride 25 in good yield. Deprotec-
The synthesis of 12F-DMPC was conducted similarly to tion of the benzyl ether with DDQ26 followed by TEMPO oxi-
4F-DMPC (Scheme 2).18 Readily prepared 1,12-dodecanediol, dation gave the acid 14. Finally, coupling with 14:0 lyso-PC (15)
monobenzyl ether (20),23 was oxidized to the aldehyde24 fol- provided 14F-DMPC (11) in 46% yield22 after purification by
lowed by addition of ethylmagnesium bromide to give the sec- flash chromatography on silica gel. An overall yield of 26% was
ondary alcohol (21). Deoxofluorination using Me-DAST25 gave obtained for the 4 synthetic steps starting from 24.
the fluoro compound 22 in moderate yield. Surprisingly, the
Characterization of monofluorinated
dimyristoylphosphatidylcholines
removal of the benzyl under hydrogenolysis conditions proved
difficult and DDQ was employed for this step.26 Finally, oxi-
dation of the primary alcohol in one step to the carboxylic acid FTIR spectroscopy was used in the present study to investigate
using TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl)27 provided how the introduction of a fluorine atom affects the order of
Scheme 2 Synthesis of 12F-DMPC.
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Scheme 3 Synthesis of 14F-DMPC.
the lipid acyl chains. More specifically, monitoring the CH2 case. However, the introduction of a fluorine atom results in a
symmetric (2850 cm−1) stretching vibration, which has been small increase in the CH2 stretching vibration wavenumber
shown to be sensitive to anti/gauche isomerization in the lipid associated with a small increase in the number of gauche con-
acyl chains, yields valuable information about the lipid gel-to- formers for the 12F and 14F derivatives, while a small decrease
fluid phase transition temperature and the conformational in the lipid phase transition temperature and a slight broaden-
order of the acyl chains.30
ing of the phase transition are observed for the 2F and 12F
The wavenumbers of the CH2 stretching vibration as a derivatives. On the other hand, the lipid phase transition
function of temperature for pure commercial DMPC, for the temperature is slightly increased for the 14F derivative. Com-
three novel pure F-DMPC derivatives investigated in the parison with results obtained for the previously reported 4F,
present study (2F, 12F and 14F-DMPC) and the three pre- 7F and 10F derivatives indicates that the increase in the CH2
viously reported F-DMPC derivatives (4F, 7F and 10F-DMPC) stretching vibration wavenumber is more important for the 7F
are shown in Fig. 4 and the lipid phase transition temp- and the 10F-DMPC derivatives than for the other derivatives,
eratures for these six pure F-DMPC derivatives are reported suggesting that the perturbation of lipid conformational order
in Table 1. The results first indicate that the properties induced by the fluorine atom is greater towards the center of
of the lipid bilayer are not significantly affected by the the lipid hydrophobic core. However, the results indicate that
presence of a fluorine atom at positions 2, 12 and 14 of the the lipids form a homogeneous mixture as a single phase tran-
lipid acyl chains. The result with 2F-DMPC is somewhat sur- sition temperature is observed for all six derivatives.
prising given that the fluorine atom is located closely to the
Previous studies on lipids monofluorinated on both acyl
carbonyl group which might influence the conformational pos- chains have shown that the order of the lipid bilayers is not
sibilities. Nonetheless, the IR results suggest that it is not the significantly perturbed by the introduction of the fluorine
atom.15 These results are therefore in agreement with the
results obtained in the present study with the three novel
monofluorinated lipid derivatives and results previously
reported with the 4F, 7F and 10F derivatives. However, other
studies have shown a significant decrease of the lipid gel-to-
fluid phase transition temperature upon the introduction
of more than one fluorine atom on one or two acyl
chains.13,14,31,32 In particular, the introduction of two fluorine
atoms on the sn-2 acyl chain results in a decrease of the phase
transition temperature by about 5–11 °C31 and the introduc-
tion of 13 fluorine atoms on both acyl chains results in a
decrease of the phase transition temperature by ∼21 °C.32 On
the other hand, an increase of the CH2 stretching vibration
wavenumber of ∼2.5 cm−1 in the gel phase and a significant
broadening (by about 12 °C) of the phase transition are
observed with the introduction of 13 fluorine atoms on both
acyl chains.32 It has also been shown that the introduction of
two fluorine atoms at position 4 of the sn-2 acyl chain has less
Fig. 4 Temperature dependence of the wavenumber of the CH2 sym-
effect than the introduction of two fluorine atoms at positions
metric stretching vibration for DMPC and F-DMPC’s. The error is esti-
mated to be 0.1 cm−1
8 and 12,31 which is in agreement with our results with mono-
.
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Table 1 Phase transition temperature for pure F-DMPC’s, DMPC and for mixtures with DMPC. The error is estimated to be 0.5 °C
Phase transition temperature (°C)
F-DMPC : DMPC
2F-DMPC
4F-DMPC
7F-DMPC
10F-DMPC
12F-DMPC
14F-DMPC
100 : 0
25 : 75
10 : 90
5 : 95
22.1
22.5
22.5
23.2
23.5
22.5
22.5
23.5
23.6
23.7
20.4
23.5
23.6
24.4
24.5
22.0
22.5
22.9
22.6
23.7
19.5
21.7
22.2
22.5
23.5
25.6
24.5
24.3
23.7
23.5
0 : 100a
a Different phase transition temperatures are reported for pure DMPC as these temperatures have been measured for every series with the
different F-DMPC derivatives.
fluorinated lipids indicating that the most significant pertur- previously reported 4F, 7F and 10F-DMPC derivatives indicates
bation of the lipid conformational order is observed with the that the effect on the phase transition temperature is more
7F and 10F-DMPC derivatives. Similar results have also been important for the 7F and 12F-DMPC derivatives. However, the
obtained upon the introduction of two fluorine atoms at posi- results presented in Fig. 5 and Table 1 clearly indicate that the
tions 4, 8 and 12 of the two acyl chains.13 2H solid-state NMR presence of the fluorine atom does not significantly perturb
spectroscopy results also indicate that the order of the methyl- the lipid phase transition temperature and conformational
ene groups close to two fluorine atoms at position 8 of the sn-2 order at smaller ratios for every fluorinated lipids, namely at
acyl chain is decreased by about 30% due to the increased size F-DMPC percentages between 2.5 and 25%. These results
of the 19F label.14 It is also interesting to note that the intro- therefore indicate that all F-DMPC derivatives could be suc-
duction of a fluorine atom at position 16 in the sn-2 chain cessfully used as probes for membrane topology at percentages
of dipalmitoylphosphatidylcholine (DPPC) results in inter- up to 25% in non-fluorinated lipids without significantly
digitated lipid bilayers with increased order.16 However, only a affecting the properties of the lipid bilayers.
small increase in the phase transition temperature is observed
for the novel 14F derivative investigated in the present study,
indicating that, interestingly, the introduction of a fluorine
atom at position 14 of the sn-2 acyl chain of DMPC does not
Conclusions
lead to the formation of fully interdigitated bilayers. The small Three monofluorinated dimyristoylphosphatidylcholines with
increase in the phase transition temperature might however be the fluorine atom located at the extremities of the acyl chain in
associated with partial interdigitation. Nevertheless, this effect position 2 of the glycerol were synthesized in 4–6 synthetic
is small and the 14F derivative could therefore be used for steps and 8–26% overall yields from commercially or readily
membrane topology studies, as opposed to the 16F derivative. available starting materials. FTIR results indicate that the pres-
Finally, as all the fluorinated fatty acids were racemic (with the ence of the fluorine atom on the three novel derivatives does
exception of 14F-DMPC as the fluorine is not located on a not significantly perturb the lipid phase transition tempera-
stereocenter), the corresponding F-DMPC’s were isolated and ture and conformational order at ratios of fluorinated lipids
characterized as a 1 : 1 mixture of diastereoisomers. The up to 25%, in agreement with results previously reported for
results obtained so far suggest that this has no impact on the the 4F, 7F and 10F derivatives. Overall, monofluorination of
behavior of the lipids. Indeed, 2F-DMPC, for which the C–F the acyl chain in sn-2 brings minimal perturbation to the lipid
stereocenter is the closest to the chiral backbone of the lipid bilayer. These monofluorinated lipids could therefore poten-
(i.e. the glycerol), does not show a different behavior compared tially be used as NMR probes for the investigation at the mole-
to the other ones, for which the C–F stereocenter is further.
cular level of the interaction between drugs or peptides and
Fig. 5 shows the wavenumbers of the CH2 stretching lipid membranes and for the study of membrane topology.
vibration as a function of temperature obtained for mixture of
pure commercial DMPC, for the three novel F-DMPC deriva-
tives and the three previously reported F-DMPC derivatives at
different percentages ranging from 2.5 to 100% (F-DMPC–
DMPC). The lipid phase transition temperatures for the pure
Experimental
Materials and methods
DMPC and F-DMPC lipids, as well as for various mixtures, are
also reported in Table 1. The lipid phase transition tempera-
ture and lipid order are slightly decreased at high ratios of
fluorinated lipids (percentages of 50 and 100%) for the 2F and
12F derivatives, while the lipid phase transition temperature is
slightly increased at high fluorinated lipid ratios for the 14F
derivative. Comparison with the results obtained for the three
All reactions were carried out under a nitrogen or argon atmos-
phere with dry solvents under anhydrous conditions. Unless
otherwise noted, all commercial reagents were used without
further purification. Dichloromethane, toluene, ether, tetra-
hydrofuran and acetonitrile were purified by using a Vacuum
Atmospheres Inc. Solvent Purification System. Thin-layer
chromatography (TLC) analysis of reaction mixtures was per-
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Fig. 5 Temperature dependence of the wavenumber of the CH2 symmetric stretching vibration for various mixtures of F-DMPC and DMPC.
The error is estimated to be 0.1 cm−1
.
formed using Silicycle silica gel 60 Å F254 TLC plates, and at ambient temperature using tetramethylsilane (1H NMR) or
visualized under UV or by staining with ceric ammonium residual CHCl3 (1H and 13C NMR) as the internal standard,
molybdate, phosphomolybdic acid or molybdenum blue. Flash CFCl3 (19F NMR) and H3PO4 (31P NMR) as external standards.
column chromatography was carried out on Silicycle Silica Gel Coupling constants (J) are measured in hertz (Hz). Multiplici-
1
60 Å, 230 × 400 mesh. H, 13C, 19F and 31P NMR spectra were ties are reported using the following abbreviations: s = singlet,
recorded on a Agilent DD2 500 or a Varian Inova 400 in CDCl3 d = doublet, t = triplet, q = quartet, m = multiplet, br = broad
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resonance. High-resolution mass spectra were obtained on a JF–H = 47.5, 21.8 Hz, minor regioisomer). HRMS-ESI calcd for
LC/MS-TOF Agilent 6210 using electrospray ionization (ESI). A C16H32O2F [M + H]+ 275.2381 found 275.2396.
low-resolution mass spectrum was obtained using EI ioniza-
2-Fluorotetradecan-1-ol (19). A mixture of 18 (212 mg,
tion on a GC-MS. Infrared spectra were recorded on a Thermo 0.77 mmol) in 1.5 mL of methanol is added to a mixture of
Scientific Nicolet 380 FT-IR spectrometer (for characterization) potassium hydroxide (130 mg, 2.31 mmol) in 2.5 mL of metha-
and on a Nicolet Magna-IR 560 or 760 spectrometer (for the nol. After stirring 4 h, the solution is poured into 15 mL of ice/
lipid phase temperature study). Melting points were recorded water and extracted five times with CH2Cl2. The combined
on a Stanford ResearchSystem OptiMelt capillary melting organic layers are washed three times with water, dried over
point apparatus and are uncorrected. Lyso-PC was purchased MgSO4 and the solvent was removed under reduced pressure.
from Avanti Polar Lipids (Alabaster, AL). The synthesis of The product 19 is obtained as a white solid (322 mg, 94%) con-
4F-DMPC, 7F-DMPC and 10F-DMPC has been reported taminated with 2% of 1-fluorotetradecan-2-ol. mp 56–58 °C; IR
previously.18
(ATR, ZnSe) 2952, 2924, 2847, 1470, 1099, 1061, 1000,
728 cm−1 1H NMR (400 MHz, CDCl3) δ 4.57 (dm, 1H, JH–F
.
=
50 Hz), 3.76–3.61 (m, 2H), 2.29 (bs, 1H), 1.73–1.40 (m, 4H),
1.36–1.21 (m, 18H), 0.88 (t, 3H, J = 6.9 Hz). 13C NMR
Synthesis of 2F-DMPC
1-Bromo-2-fluorotetradecane (17). To a solution of 1-tetra- (100 MHz, CDCl3) δ 95.0 (d, JC–F = 168 Hz), 65.2 (d, JC–F = 21.7
decene (16) (333 mg, 1.69 mmol) and Et3N·3HF (0.43 mL, Hz), 32.1, 31.1 (d, JC–F = 20.3 Hz), 29.79, 29.77, 29.7, 29.59,
2.63 mmol) in CH2Cl2 (5 mL) at 0 °C was added NBS (332 mg, 29.56, 29.5, 25.1, 25.0, 22.8, 14.2. 19F NMR (376 MHz, CDCl3)
1.86 mmol). The reaction was stirred at 0 °C for 15 min and δ −190.0 (m, 1F, major regioisomer), −228.6 (td, 1F, JF–H
=
then warmed to room temperature over 6 h. The reaction 47.8, 18.0 Hz, minor regioisomer). HRMS-ESI calcd for
mixture was then poured into 20 mL of ice/water, neutralized C14H33NOF [M + NH4]+ 250.2541 found 250.2540.
with 25% aq. NH4OH and extracted three times with CH2Cl2.
2-Fluorotetradecanoic acid (12). To a mixture of 19 (201 mg,
The organic layer was then washed with twice with 0.1 N HCl, 0.87 mmol) in acetone (0.5 mL) at 0 °C was added Jones
three times with 5% aq. NaHCO3, dried over MgSO4 and the reagent (prepared from CrO3 (173 mg, 1.73 mmol), 1.7 mL of
solvent was removed under reduced pressure. The crude was concentrated H2SO4 and 6 mL of water) dropwise. The reaction
purified by silica gel chromatography (100% hexane) affording was stirred for 70 h at room temperature. Afterward, the reac-
17 as a colorless solid (314 mg, 63%) contaminated with 2% of tion mixture was extracted three times with ether. The com-
2-bromo-1-fluorotetradecane. mp 30–31 °C; IR (ATR, ZnSe) bined organic layers were washed once with water, dried over
2915, 2848, 1464, 1131, 1097, 817, 719, 666 cm−1 1H NMR MgSO4 and the solvent was removed under reduced pressure.
.
(400 MHz, CDCl3) δ 4.62 (dm, 1H, JH–F = 48 Hz), 3.47 (ddd, 2H, The crude was purified by silica gel chromatography (10%
J = 19.7, 5.2, 1.9 Hz), 1.79–1.61 (m, 2H), 1.53–1.19 (m, 20H), EtOAc–hexane) affording 12 as a white solid (47 mg, 22%). mp
0.88 (t, 3H, J = 6.9 Hz). 13C NMR (100 MHz, CDCl3) δ 92.2 (d, 72–74 °C; IR (ATR, ZnSe) 2916, 2848, 1709, 1468, 1097, 1075,
JC–F = 175 Hz), 33.9 (d, JC–F = 25.4 Hz), 33.5 (d, JC–F = 20.5 Hz), 720, 679 cm−1 1H NMR (500 MHz, CDCl3) δ 4.97 (dm, 1H,
.
32.1, 29.81, 29.79, 29.77, 29.7, 29.6, 29.5, 29.4, 24.9, 24.8, 22.8, JH–F = 39.6 Hz), 2.04–1.86 (m, 2H), 1.54–1.46 (m, 2H), 1.40–1.21
14.3. 19F NMR (376 MHz, CDCl3) δ −178.1 (m, 1F); regioisomer: (m, 18H), 0.89 (t, 3H, J = 7.0 Hz). 13C NMR (125 MHz, CDCl3)
δ −210.0 (td, 1F, JF–H = 47.2, 13.7 Hz). GC-MS calcd for δ 175.3 (d, JC–F = 21.7 Hz), 88.7 (d, JC–F = 185 Hz), 32.4 (d, JC–F
=
C14H28Br [M − F]+ 275 found 275.33
20.8 Hz), 32.1, 29.79, 29.78, 29.7, 29.6, 29.50, 29.46, 29.2, 24.5
1-Acetoxy-2-fluorotetradecane
(18). Potassium
acetate (d, JC–F = 2.7 Hz), 22.8, 14.3. 19F NMR (470 MHz, CDCl3)
(417 mg, 4.3 mmol) was added to a solution of 17 (314 mg, δ −192.2 (m, 1F). HRMS-ESI calcd for C14H31NO2F [M + NH4]+
1.1 mmol) in DMF (6 mL) and heated at reflux under an argon 264.2333 found 264.2331.
atmosphere for 26 h. The reaction mixture was then cooled
1-Myristoyl-2-(2-fluoromyristoyl)-sn-glycero-3-phosphocholine
down to room temperature and 10 mL of a 1 : 1 mixture of (9, 2F-DMPC). To a solution of 12 (30 mg, 0.12 mmol), 14:0
hexane and ethyl acetate was added and the solid was filtered lyso-PC (15) (58 mg, 0.12 mmol) and 1-methylimidazole
off and washed carefully with the same solvent. The organic (29 µL, 0.37 mmol) in CHCl3 (1.3 mL) was added 2,6-dichloro-
layer was then washed six times with water, dried over MgSO4 benzoyl chloride (54 µL, 0.38 mmol) and the resulting mixture
and the solvent was removed under reduced pressure, was stirred for 16 h at room temperature. The reaction mixture
affording 18 as a yellow oil (212 mg, 73%) contaminated with was then concentrated under reduced pressure. The crude was
2% of 2-acetoxy-1-fluorotetradecane. IR (ATR, ZnSe) 2923, purified by silica gel chromatography (1 : 1 MeOH–CH2Cl2)
2854, 1746, 1466, 1368, 1230, 1048, 722 cm−1 1H NMR affording 9 as a light yellow wax (74 mg, 86%) in a 1 : 1 diastereo-
.
(500 MHz, CDCl3) δ 4.66 (dm, 1H, JH–F = 49.5 Hz), 4.26–4.09 isomeric mixture. IR (ATR, ZnSe) 2916, 1737, 1090, 1051, 969,
(m, 2H), 2.11 (s, 2H), 1.75–1.44 (m, 4H), 1.40–1.21 (m, 19H), 823, 763, 720 cm−1. 1H NMR (400 MHz, CDCl3) δ 5.33 (m, 1H),
0.88 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 171.0, 5.14–4.78 (m, 1H), 4.40 (td, J = 12.5, 2.9 Hz, 1H), 4.30 (bs, 2H),
91.6 (d, JC–F = 172 Hz), 66.1 (d, JC–F = 21.9 Hz), 32.1, 31.5 (d, 4.23–4.09 (m, 1H), 4.00 (t, J = 6.0 Hz, 2H), 3.78 (bs, 2H), 3.43
JC–F = 20.4 Hz), 29.81, 29.79, 29.77, 29.67, 29.57, 29.50, 29.49, (s, 2H), 3.34 (bs, 7H), 2.37–2.21 (m, 2H), 1.92–1.78 (m, 2H),
24.9 (d, JC–F = 4.5 Hz), 22.9, 21.0, 14.3. 19F NMR (470 MHz, 1.63–1.52 (m, 2H), 1.49–1.37 (m, 2H), 1.34–1.21 (m, 38H), 0.88
CDCl3) δ −187.4 (m, 1F, major regioisomer), −230.6 (td, 1F, (t, J = 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 173.5, 173.4,
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169.8 (d, JC–F = 25.0 Hz), 169.7 (d, JC–F = 23.2 Hz), 89.3 (d, J = at reflux for 48 h. The reaction mixture was then cooled down
186 Hz), 89.0 (d, JC–F = 181 Hz), 72.2, 72.1, 66.4, 63.6 (d, J = to room temperature and 10 mL of distilled water was added.
15.6 Hz), 63.5 (d, JC–F = 15.7 Hz), 62.7, 59.5, 54.5 (3C), 34.2, The aqueous layer was extracted three times with CH2Cl2 and
32.6, 32.5 (d, JC–F = 20.8 Hz), 32.0 (2C), 30.0–29.2 (m, 15C), the combined organic layers were washed twice with sat. aq.
25.0, 24.6 (d, JC–F = 12.3 Hz), 24.5 (d, JC–F = 12.2 Hz), 22.8 (2C), NaHCO3. The organic layer was then dried over MgSO4 and the
14.2 (2C). 19F NMR (470 MHz, CDCl3) δ −192.3 (m, 1F). solvent was removed under reduced pressure. The crude was
31P NMR, (152 MHz, CDCl3) δ −0.96 (s, 0.5P), −1.05 (s, 0.5P). purified by silica gel chromatography (10% EtOAc–hexane)
HRMS-ESI calcd for C36H72NO8FP [M + H]+ 696.4974 found affording 23 as a white solid (59 mg, 91%). mp 43–44 °C; IR
696.4993.
(ATR, ZnSe) 3266, 2914, 2847, 1462, 1061, 935, 856, 729 cm−1
.
1H NMR (500 MHz, CDCl3) δ 4.48–4.42 (m, 0.5H), 4.38–4.32
(m, 0.5H), 3.65 (t, 2H, J = 6.6 Hz), 1.70–1.40 (m, 8H), 1.37–1.24
Synthesis of 12F-DMPC
12-(Benzyloxy)tetradecan-1-ol (21). To a solution of 12-(benzyl- (m, 14H), 0.97 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3)
oxy)dodecanal (93 mg, 0.32 mmol, prepared from the Swern δ 95.9 (d, JC–F = 167 Hz), 63.2, 34.8 (d, JC–F = 20.8 Hz), 33.0,
oxidation of 10-(benzyloxy)decan-1-ol (20)23 as previously 29.73, 29.69, 29.68, 29.67, 29.66, 29.6, 28.2 (d, JC–F = 21.5 Hz),
described)24 in THF (3 mL) at 0 °C was added a solution of 25.9, 25.3 (d, JC–F = 4.5 Hz), 9.54 (d, JC–F = 5.8 Hz). 19F NMR
ethyl magnesium bromide (214 µL, 0.64 mmol, 3 M in Et2O). (470 MHz, CDCl3) δ −181.2 (m, 1F). HRMS-ESI calcd for
The resulting mixture was stirred 1 hour at 0 °C and then C14H33NOF [M + NH4]+ 250.2541 found 250.2541.
warmed to room temperature over 1 hour. The reaction
12-Fluorotetradecanoic acid (13). To a mixture of TEMPO
mixture was then cooled to 0 °C and quenched with 5% aq. (3 mg, 0.02 mmol) in CH3CN (1.1 mL) and a phosphate buffer
H2SO4. The aqueous layer was extracted twice with ethyl (pH 6.7, 0.67 M, 360 µL) at 35 °C was added 23 (56.3 mg,
acetate. The combined organic layers were washed twice with a 0.24 mmol). Solutions of sodium chlorite (44 mg, 0.49 mmol)
sat. aq. Na2SO3, once with water, dried over MgSO4 and con- in distilled water (228 µL) and sodium hypochlorite (37 µL of a
centrated under reduced pressure. The product 21 is obtained 10% aqueous solution, 5 µmol) diluted in distilled water
as a colorless oil (605 mg, 89%). IR (ATR, ZnSe) 2924, 2853, (117 µL) were added dropwise over 2 h in two separate syringe.
1455, 1239, 1101, 1046, 733, 697 cm−1 1H NMR (500 MHz, The reaction was stirred for 4 h at 35 °C and then cooled to
.
CDCl3) δ 7.38–7.25 (m, 5H), 4.52 (s, 2H), 3.53 (bs, 1H), 3.48 (t, room temperature. Water (1 mL) was added and the pH was
2H, J = 6.7 Hz), 1.68–1.1.59 (m, 2H), 1.57–1.33 (m, 8H), adjusted to 8 with a 2 M NaOH solution. The reaction mixture
1.33–1.24 (m, 12H), 0.95 (t, 3H, J = 7.5 Hz). 13C NMR was poured in sat. aq. Na2SO3 and maintained under 20 °C
(125 MHz, CDCl3) δ 138.9, 128.5 (2C), 127.8 (2C), 127.6, 73.5, (pH was around 8.5–9.0) After stirring for 10 minutes, the solu-
73.0, 70.7, 37.1, 30.3, 29.91, 29.89, 29.8, 29.7 (4C), 29.6, 26.3, tion was extracted with Et2O (1 mL) and the organic phase was
25.8, 10.0. HRMS-ESI calcd for C21H37O2 [M + H]+ 321.2788 discarded. The aqueous phase was then acidified to pH 3–4
found 321.2784.
using a 2 M HCl solution. Sat. aq. NaCl was added to the
((12-Fluorotetradecyloxy)methyl)benzene (22). To a solution mixture before extracting with EtOAc (3×). Organic phases were
of 21 (605 mg, 1.89 mmol) in CH2Cl2 (19 mL) at −78 °C was combined, dried over MgSO4 and concentrated under reduced
added Me-DAST (210 µL, 1.89 mmol) dropwise. The reaction pressure. The crude was purified by silica gel chromatography
mixture was stirred for 3 h at −78 °C and allowed to warm up (20% EtOAc–hexane) affording 13 as a white solid (53 mg,
to room temperature over 3 h. The solution was carefully 90%). mp 65–66 °C; IR (ATR, ZnSe) 2912, 2847, 1699, 1471,
poured to a sat. aq. CaCO3 cooled at 0 °C. The organic layer 1265, 1216, 929, 718 cm−1 1H NMR (500 MHz, CDCl3)
.
was separated and the aqueous layer was extracted with δ 4.48–4.43 (m, 0.5H), 4.39–4.33 (m, 0.5H), 2.36 (t, 2H, J =
CH2Cl2. The combined organic layers were dried over MgSO4 7.5 Hz), 1.68–1.42 (m, 8H), 1.38–1.25 (m, 12H), 0.97 (t, 3H, J =
and concentrated under reduced pressure. The crude was puri- 7.5 Hz). 13C NMR (125 MHz, CDCl3) δ 180.6, JC–F = 167 Hz),
fied by silica gel chromatography (1% Et2O–hexane) affording 34.8 (d, JC–F = 20.9 Hz), 34.2, 29.64 (2C), 29.62, 29.5, 29.4, 29.2,
22 as a colorless oil (418 mg, 69%). IR (ATR, ZnSe) 2925, 2853, 28.2 (d, JC–F = 21.5 Hz), 25.3 (d, JC–F = 4.5 Hz), 24.8, 9.5 (d,
1455, 1239, 1101, 943, 733, 696 cm−1 1H NMR (500 MHz, JC–F = 5.8 Hz). 19F NMR (470 MHz, CDCl3) δ −181.3 (m, 1F).
.
CDCl3) δ 7.37–7.25 (m, 5H), 4.51 (s, 1H), 4.50–4.42 (m, 0.5H), HRMS-ESI calcd for C14H31NO2F [M + NH4]+ 264.2333 found
4.39–4.30 (m, 0.5H), 3.47 (t, 2H, J = 6.7 Hz), 1.68–1.46 (m, 6H), 264.2332.
1.45–1.25 (m, 16H), 0.97 (t, 3H, J = 7.5 Hz). 13C NMR
1-Myristoyl-2-(12-fluoromyristoyl)-sn-glycero-3-phospho-
(100 MHz, CDCl3) δ 138.9, 128.5 (2C), 127.7 (2C), 127.6, 95.8 choline (10, 12F-DMPC). To solution of 13 (28 mg,
a
(d, JC–F = 167 Hz), 73.0, 70.7, 34.8 (d, JC–F = 20.9 Hz), 29.9, 0.11 mmol), 14:0 lyso-PC (15) (54 mg, 0.11 mmol) and
29.71, 29.71, 29.69, 29.68, 29.66, 29.62, 28.2 (d, JC–F = 21.5 Hz), 1-methylimidazole (27 µL, 0.34 mmol) in CHCl3 (1.1 mL) was
26.3, 25.3 (d, JC–F = 4.5 Hz), 9.54 (d, J = 5.7 Hz). 19F NMR added 2,6-dichlorobenzoyl chloride (50 µL, 0.35 mmol) and
(470 MHz, CDCl3) δ −181.2 (m, 1F). HRMS-ESI calcd for the resulting mixture was stirred for 16 h at room temperature.
C21H39NOF [M + NH4]+ 340.3010 found 340.3013.
The reaction mixture was then concentrated under reduced
12-Fluorotetradecan-1-ol (23). DDQ (126 mg, 0.55 mmol) pressure. The crude was purified by silica gel chromatography
was added to a solution of 22 (90 mg, 0.28 mmol) in a (1 : 1 MeOH–CH2Cl2) affording 10 as a light yellow wax (35 mg,
18 : 1 mixture of chloroform–distilled water (3 mL) and heated 44%). IR (ATR, ZnSe) 2918, 1736, 1238, 1089, 1064, 969, 760,
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691 cm−1. 1H NMR (500 MHz, CDCl3) δ 5.21 (m, 1H), 4.48–4.43
14-Fluorotetradecanoic acid (14). To a mixture of TEMPO
(m, 4H), 4.18–4.10 (m, 1H), 4.01–3.91 (m, 2H), 3.89–3.78 (bs, (8.3 mg, 0.05 mmol) in CH3CN (0.22 mL) and a phosphate
2H), 3.48 (s, 1H), 3.39 (s, 9H), 2.34–2.48 (m, 2H), 1.69–1.51 (m, buffer (pH 6.7, 0.67 M, 0.67 mL) at 35 °C was added 26
8H), 1.36–1.21 (m, 34H), 0.97 (t, 3H, J = 7.5 Hz), 0.89 (t, 3H, J = (155 mg, 0.67 mmol). Solutions of sodium chlorite (121 mg,
6.9 Hz). 13C NMR (125 MHz, CDCl3) δ 173.6, 173.2, 95.7 (d, JC–F 1.33 mmol) in distilled water (0.63 mL) and sodium hypochlor-
= 168 Hz), 71.1 (d, JC–F = 7.2 Hz), 67.0, 63.7 (d, JC–F = 5.0 Hz), ite (96 µL of a 10% aqueous solution, 13 µmol) diluted in dis-
63.3, 59.5 (d, JC–F = 4.7 Hz), 54.9, 34.9 (d, JC–F = 21.0 Hz), 34.5 tilled water (0.32 mL) were added dropwise over 2 h in two
(d, JC–F = 25.0 Hz), 32.1 (2C), 29.9–29.3 (m, 14C), 28.3 (d, JC–F
=
separate syringe. The reaction was stirred for 4 h at 35 °C and
21.7 Hz), 25.3 (d, JC–F = 4.6 Hz), 25.15 (d, JC–F = 7.4 Hz), 22.8 then cooled to room temperature. Water (1 mL) was added and
(2C), 14.1, 9.4 (d, JC–F = 5.9 Hz). 19F NMR (470 MHz, CDCl3) the pH was adjusted to 8 with a 2 M NaOH solution. The reac-
δ −181.2 (m, 1F). 31P NMR, (152 MHz, CDCl3) δ 0.76 (s, 1P). tion mixture was poured in sat. aq. Na2SO3 and maintained
HRMS-ESI calcd for C36H72NO8FP [M + H]+ 696.4974 found under 20 °C (pH was around 8.5–9.0). After stirring for
696.4985.
10 minutes, the solution was extracted with Et2O (1 mL) and
the organic phase was discarded. The aqueous phase was then
acidified to pH 3–4 using a 2 M HCl solution. Some sat. aq.
Synthesis of 14F-DMPC
((14-Fluorotetradecyloxy)methyl)benzene (25). To a solution NaCl was added to the mixture before extracting with EtOAc
of XtalFluor-E, [Et2NSF2]BF4, (288 mg, 1.3 mmol) in CH2Cl2 (3×). Organic phases were combined, dried over MgSO4 and
(4.5 mL) at −78 °C was added Et3N·3HF (170 mg, 1.68 mmol). concentrated under reduced pressure. The crude was purified
A mixture of 2428 (268 mg, 0.84 mmol) in CH2Cl2 (4.5 mL) was by silica gel chromatography (20% EtOAc–hexane) affording 14
slowly added and the reaction mixture was stirred and allowed as a white solid (135 mg, 83%). mp 65–67 °C; IR (ATR, ZnSe)
to warm up to room temperature for 22 h. The reaction 2912, 2847, 1704, 1470, 1250, 1210, 948, 718 cm−1 1H NMR
.
mixture was then quenched with sat. aq. NaHCO3. The (400 MHz, CDCl3) δ 4.49 (t, 1H, J = 6.2 Hz), 4.37 (t, 1H, J =
aqueous layer was extracted twice with CH2Cl2 and the com- 6.2 Hz), 2.34 (t, 2H, J = 7.5 Hz), 1.77–1.56 (m, 4H), 1.46–1.18
bined organic layers were dried over MgSO4 and concentrated (m, 20H). 13C NMR (125 MHz, CDCl3) δ 180.7, 84.4 (d, JC–F
=
under reduced pressure. The crude was purified by silica gel 164 Hz), 34.2, 30.5 (d, JC–F = 19.3 Hz), 29.71, 29.69, 29.66, 29.6,
chromatography (1% EtOAc–hexane) affording 25 as a yellow 29.5, 29.4 (2C), 29.2, 25.3 (d, JC–F = 5.6 Hz), 24.8. 19F NMR
oil (217 mg, 80%). IR (ATR, ZnSe) 2925, 2854, 1455, 1362, (376 MHz, CDCl3) δ −217.9 (m, 1F). HRMS-ESI calcd for
1
1101, 1028, 909 cm−1. H NMR (500 MHz, CDCl3) δ 7.41–7.22 C14H31NO2F [M + NH4]+ 264.2333 found 264.2329.
(m, 5H), 4.52 (s, 1H), 4.50 (t, J = 6.3 Hz), 4.40 (t, J = 6.2 Hz),
1-Myristoyl-2-(14-fluoromyristoyl)-sn-glycero-3-phospho-
3.48 (t, J = 6.7 Hz), 1.75–1.59 (m, 4H), 1.44–1.25 (m, 20H). choline (11, 14F-DMPC). To a solution of 14 (40 mg,
13C NMR (125 MHz, CDCl3) δ 138.9, 128.5 (2C), 127.8 (2C), 0.16 mmol), 14:0 lyso-PC (15) (73 mg, 0.15 mmol) and
127.6, 84.4 (d, JC–F = 164 Hz), 73.0, 70.7, 30.6 (d, JC–F = 19.4 1-methylimidazole (37 µL, 0.46 mmol) in CHCl3 (1.6 mL) was
Hz), 29.9, 29.78, 29.77, 29.74 (2C), 29.69, 29.66, 29.6, 29.4, added 2,6-dichlorobenzoyl chloride (69 µL, 0.48 mmol) and
26.3, 25.3 (d, JC–F = 5.6 Hz). 19F NMR (475 MHz, CDCl3) the resulting mixture was stirred for 16 h at room temperature
δ −218.0 (m, 1F). HRMS-ESI calcd for C21H39NOF [M + NH4]+ and for 6 h at 45 °C. The reaction mixture was then concen-
340.3010 found 340.3006.
trated under reduced pressure. The crude was purified by
14-Fluorotetradecan-1-ol (26). DDQ (187 mg, 0.82 mmol) silica gel chromatography (1 : 1 MeOH–CH2Cl2) affording 11 as
was added to a solution of 25 (132 mg, 0.41 mmol) in a a light yellow wax (49 mg, 46%). IR (ATR, ZnSe) 3356, 2918,
18 : 1 mixture of chloroform–distilled water (4.2 mL) and 2850, 1651, 1469, 1094, 765, 694 cm−1 1H NMR (400 MHz,
.
heated at reflux for 48 h. The reaction mixture was then cooled CDCl3) δ 5.19 (bs, 1H), 4.47 (t, 1H, J = 6.1 Hz), 4.38 (m, 2H),
down to room temperature and 10 mL of distilled water was 4.30 (bs, 2H), 4.12 (m, 1H), 3.93 (bs, 2H), 3.76 (bs, 2H), 3.33 (s,
added. The aqueous layer was extracted three times with 9H), 2.34–2.22 (m, 4H), 1.74–1.62 (m, 2H), 1.57 (bs, 4H),
CH2Cl2 and the combined organic layers were washed twice 1.43–1.13 (m, 38H), 0.87 (t, 3H, J = 6.5 Hz). 13C NMR
with sat. aq. NaHCO3. The organic layer was then dried over (125 MHz, CDCl3) δ 173.7, 173.3, 84.3 (d, J = 164 Hz), 70.5 (d,
Na2SO4 and the solvent was removed under reduced pressure. J = 6.4 Hz), 63.4, 63.6, 63.0, 59.5, 54.4 (3C), 34.4, 34.2, 32.1,
The crude was purified by silica gel chromatography (10% 30.5 (d, J = 19.3 Hz), 29.9–29.3 (m, 16C), 25.3 (d, J = 5.6 Hz),
EtOAc–hexane) affording 26 as a white solid (82 mg, 86%). mp 25.1, 25.0, 22.8, 14.3. 19F NMR (475 MHz, CDCl3) δ −218.0 (m,
41–43 °C; IR (ATR, ZnSe) 3322, 2915, 2848, 1463, 1059, 1049, 1F). 31P NMR, (152 MHz, CDCl3) δ −1.06 (s, 1P). HRMS-ESI
1
1030, 730 cm−1. H NMR (500 MHz, CDCl3) δ 4.48 (t, 1H, J = calcd for C36H72NO8FP [M + H]+ 696.4974 found 696.4985.
6.2 Hz), 4.38 (t, 1H, J = 6.2 Hz), 3.62 (t, 2H, J = 6.7 Hz),
1.75–1.61 (m, 3H), 1.60–1.50 (m, 2H), 1.42–1.23 (m, 19H).
13C NMR (125 MHz, CDCl3) δ 84.4 (d, JC–F = 164 Hz), 63.1, 32.9,
Acknowledgements
30.5 (d, JC–F = 19.3 Hz), 29.74, 29.73 (2C), 29.70, 29.66, 29.63,
29.56, 29.4, 25.9, 25.3 (d, JC–F = 5.5 Hz). 19F NMR (475 MHz, This work was supported by PROTEO (fellowships to MCG, BT,
CDCl3) δ −218.0 (m, 1F). HRMS-ESI calcd for C14H33NOF and JDS), the Canada Research Chair Program (JFP), the
[M + NH4]+ 250.2541 found 250.2535.
Natural Sciences and Engineering Research Council of Canada
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(MA and JFP), the Canada Foundation for Innovation (JFP), the
Fonds de recherche sur la nature et les technologies (MA and
JFP), and Université Laval (MA and JFP). We thank Mr Jean-
François Rioux-Dubé for its assistance with the FTIR experi-
ments and Mrs Jozy-Ann Pineault-Maltais for preliminary
experiments. OmegaChem is thanked for a gift of XtalFluor-E.
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