Cationic dialkylarylphosphates: A new family of bio-inspired cationic lipids for gene delivery

Article abstract of DOI:10.1039/c4ob01770f

In this work that aims to synthesize and evaluate new cationic lipids as vectors for gene delivery, we report the synthesis of a series of cationic lipids in which a phosphate functional group acts as a linker to assemble on a molecular scale, two lipid chains and one cationic polar head. The mono or dicationic moiety is connected to the phosphate group by an aryl spacer. In this work, two synthesis strategies were evaluated. The first used the Atherton-Todd coupling reaction to introduce a phenolic derivative to dioleylphosphite. The second strategy used a sequential addition of lipid alcohol and a phenolic derivative on POCl₃. The two methods are efficient, but the latter allows larger yields. Different polar head groups were introduced, thus producing amphiphilic compounds possessing either one permanent (N-methyl-imidazolium, pyridinium, trimethylammonium) or two permanent cationic charges. All these cationic lipids were formulated as liposomal solutions and characterized (size and zeta potential). They formed stable liposomal solutions both in water (at pH 7.0) and in a weakly acidic medium (at pH 5.5). Finally, this new generation of cationic lipids was used to deliver DNA into various human-derived epithelial cells cultured in vitro. Compared with Lipofectamine used as a reference commercial lipofection reagent, some cationic dialkylarylphosphates were able to demonstrate potent gene transfer abilities, and noteworthily, monocationic derivatives were much more efficient than dicationic analogues. This journal is

Full text of DOI:10.1039/c4ob01770f

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Cationic dialkylarylphosphates: new family of bio-
inspired cationic lipids for gene delivery.
Cite this: DOI: 10.1039/x0xx00000x
Stéphanie S. Le Corre,a Nawal Belmadi,c Mathieu Berchel,a,b Tony Le Gall,b,c Jean-Pierre
Haelters,a Pierre Lehn,c Tristan Montier,b,c,d Paul-Alain Jaffrèsa,b*
Received 00th January 2012,
Accepted 00th January 2012
In this work, that aims to synthesize and evaluate new cationic lipids as vectors for gene
delivery, we report the synthesis of a series of cationic lipids in which a phosphate functional
group acts as a linker to assemble at a molecular scale two lipid chains and one cationic polar
head. The cationic moiety, mono or di-cationic is connected to the phosphate group by an aryl
spacer. In this work, two synthetic strategies were evaluated. The first one used the Atherton-
Todd coupling reaction to introduce a phenolic derivative to dioleylphosphite. The second
strategy used a sequential addition of lipid alcohol and a phenolic derivative on POCl3. The
two methods are efficient but the later allows larger yields. Different polar head groups were
DOI: 10.1039/x0xx00000x
www.rsc.org/
introduced thus producing amphiphilic compounds possessing either one permanent (N-methyl-
imidazolium, pyridinium, trimethylammonium) or two permanent cationic charges. All these
cationic lipids were formulated as liposomal solutions and characterized (size and zeta
potential). They form stable liposomal solutions both in water (at pH 7.0) and in a weakly
acidic medium (at pH 5.5). Finally, this new generation of cationic lipids was used to deliver
DNA into various human-derived epithelial cells cultured in vitro
Lipofectamine used as reference commercial lipofection reagent, some cationic
. Compared with
a
dialkylarylphosphates were able to demonstrate potent gene transfer abilities and noteworthy
monocationic derivatives were much more efficient than dicationic analogues.
Introduction
production from readily available lipid building blocks (e.g.
dioleylphosphite). Moreover, structural modifications can be
readily implemented to modify the lipid domain,6 the polar
head group (guanidinium,7 trimethylphosphonium or
Cationic lipids constitute a broad family of synthetic vectors
that were first used as gene carriers by Felgner,1 Behr2 and
Wu3. The design of new cationic lipids for nucleic acid delivery
aims to identify carriers with high transfection efficacies and a
reduced toxicity. Interestingly this type of vectors can be
readily synthesised according to simple and efficient
procedures which is an important feature with the aim to
anticipate the need of large productions required for in vivo
experiments. One important parameter, which must be carefully
addressed when cationic lipids are used for transfection, is the
stability of the supramolecular aggregates (lipoplexes) formed
when cationic liposomes are mixed with nucleic acid
constructs. Indeed, if robust lipoplexes are required outside the
cell, weak supramolecular aggregates are needed after cell
internalisation in order to facilitate nucleic acid release from the
endosomes. Cationic lipophosphoramidates (Figure 1A) is an
efficient family of synthetic vectors for gene delivery (Figure
1A) and, noteworthy, cationic polymers possessing this
functionality (phosphoramidates) were also designed.4 One
interesting feature of lipophosphoramidates, in addition to their
in vitro and in vivo gene delivery efficacies,5 is their facility of
trimethylarsonium8)
or
the
phosphorus-based
linker
(phosphoramidate or thiophosphoramidate9). All these
possibilities also permitted the introduction of additional
functionalities to produce fluorescent carriers (e.g. fluorescent
probe),10 or red/ox sensitive cationic lipids.11 In addition, we
observed that some lipophosphoramidates have shown
interesting bactericidal action simultaneously with their
capacity to transfect eukaryotic cells.12 In the present work, we
have synthesised a new series of cationic lipids possessing a
phosphate functional group (dialkyl aryl phosphate; Figure 1B)
that acts as a linker between the hydrophobic domain and the
cationic polar region. With cationic lipids possessing an
arylphosphate linker, it can be expected to produce less stable
lipoplexes (a characteristic that should favour the endosomal
escape), when compared to lipophosphoramidates, due to i) the
replacement of the NH group by an oxygen atom that removes
a group acting as a hydrogen bond donor; ii) arylphosphate
function is known to be a good leaving group after nucleophilic
addition on the phosphorus atom. iii) natural enzymatic system
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(e.g. phospholipases) could be able to cleave phosphate groups,
thus facilitating the catabolism and subsequently the
elimination of these cationic lipids after transfection. A recent
View Article Online
DOI: 10.1039/C4OB01770F
study
reporting
the
use
of
phenyl
ester
of
hydroxymethylenediphosphonate as prodrug indicates that the
P-O-Ar bond can be indeed cleaved by phosphodiesterase.13
We report herein the synthesis of a series of cationic
arylphosphate-based amphiphiles possessing different type of
cationic
polar
heads
(e.g.
trimethylammonium,
methylimidazolium, and pyridinium groups). Additionally, the
synthesis of dicationic derivatives is also reported. Then, the
capacity of these cationic lipids to produce liposomal solutions
is studied jointly with the ability of these liposomal solutions to
produce lipoplexes after mixing with a plasmid DNA (pDNA).
Finally, this series of new vectors were evaluated as gene
carriers in in vitro assays and their cytotoxicity was determined.
Fig. 2 Synthesis of cationic diethylphosphoramidates via Atherton-
O
CH3
O
Todd reaction.
A
P
N
H
(CH2)n
Z
CH3
O
expected compound was purified by chromatography on silica
gel. In all our attempts, the product that would have resulted
from the reaction of the alcohol functional group was never
CH3
I
Z = N, P, As
O
P
Lipid chain
Lipid chain
O
O
B
detected. Then, compound
produce the expected bromo derivative
1
was engaged in Appel reaction to
in high yield. The
Z
CH2
Z
O
O
2
exchange of the halogen (Finkelstein reaction) to introduce
iodide was readily achieved (not shown in Figure 2) but its
replacement was not necessary to produce cationic lipid by
O
P
Lipid chain
Lipid chain
O
O
reaction with
that the aryl group present in compound
N
-methylimidazole or pyridine. It must be noted
or may act as a
Z
3
4
good leaving group especially due to the presence of the
cationic charge at the benzylic position. It can be expected that
the stability of this kind of cationic arylphosphate towards
nucleophilic species could be influenced by the presence of a
cationic group in benzylic position. In other studies, it was
indeed shown that the substitution on triarylphosphate (e.g.
Fig. 1 Chemical structure of cationic lipophosphoramidates (A) and
cationic or dicationic dialkylarylphosphates reported in this study (B).
Results and discussion
The synthesis of cationic lipophosphates reported herein was
directly inspired from our previous works dedicated to the
synthesis and use of cationic lipophophoramidates. We aimed
to keep the synthetic scheme unchanged because the lipid
building blocks (e.g. dioleyl phosphite) were readily
synthesised. Accordingly, the first step is based on an Atherton-
Todd coupling14 between a selected phosphite and a functional
phenol. It is noteworthy that Atherton-Todd reaction is efficient
with phenol whereas it is almost inefficient with alcohol due to
the lower nucleophilicity. Consequently, we used 4-
hydroxymethylphenol as substrate because the alcohol
functional group can subsequently be transformed in a suitable
functional group that allows the incorporation of a cationic
group. To evaluate the stability of the targeting compounds
(Figure 1-B) the synthesis was first achieved with
diethylphosphite as starting compound (Figure 2). The
Atherton-Todd reaction was achieved in 62 % yield. This
modest yield was attributed to the low solubility of 4-
hydroxymethylphenol in dichloromethane. After different
attempts, the best method consisted to incorporate this
compound as its powder form directly in the reaction media.
Despite the formation of phosphate as side products, the
tris(p-nitrophenyl)phosphate) deeply influenced the rate of
hydrolysis at pH 6 or 9.15 However other aryldialkylphosphates
like some derived from homocamptothecin exhibited good
stability in water at pH 3 or 7.4.16 In view of these data from the
literature, the stability of compound
3 in water was evaluated.
This compound was placed in D2O and was left at 20°C for 20
days. NMR spectroscopy assessed its stability in these
conditions meaning that the synthesis of amphiphilic
compounds with this motif (dialkylarylphosphate) should have
sufficient water stability to prepare liposomal solutions. With
more drastic conditions (HEPES buffer at pH 5.5) that aim to
mimic the acidity of endosomes, we actually found that the
phosphate functional group was kept unaffected either at 20°C
or at 37°C after 24h. This result indicated that in these
experimental conditions the cationic aryldialkyl-lipophosphate
exhibited a high stability.
The synthesis of the lipophosphate
7, which is a bis-oleyl
analogue of compound , was first attempted by using the same
1
synthetic pathway as those reported in Figure 2. The yield was
modest (38%) due to the formation of side products and also,
due to the purification step which is more laborious than
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Fig. 3 Synthesis of dioleylarylphosphate
7
via Atherton-Todd reaction or the POCl3 procedure.
with 75% yield. However, the workup of this reaction and the
. However, this reaction can be reproduced purification of compound with this method were tedious due
with compound
1
7
and sufficient quantities were isolated (200 mg per batch) for to the amount of salt present in the reaction media. Altogether
the subsequent synthetic steps (Figure 4). An alternative the methods A and B were chosen for the production of
method was achieved (method B, Figure 3), which was based compound
on a recent protocol applied to produce cationic lipids from Compound
7
in sufficient quantities to achieve the next steps.
was transformed into the bromo derivative
7
8
POCl317. Herein, two equivalents of oleyl alcohol were first (Appel reaction) in 54% yield (Figure 4). Before the synthesis
added to POCl3 in diethylether. The completeness of this step of the cationic lipid, a halogen exchange reaction (Finkelstein
was checked by 31P NMR before the addition of reaction) can be applied to compound
8 to replace bromide
dichloromethane and 4-hydroxymethylphenol (powder). After atom by iodine atom. This exchange is not necessary for the
purification, the lipophosphate was isolated in 45% yield. introduction of the cationic group. Accordingly, the bromo
Interestingly, these conditions of reaction can be readily applied derivative
to produce 1.0 to 2.7 g of the pure phosphate . Finally, a third yield). Then, three different cationic polar heads were
method was attempted (method C, Figure 3). Herein, 4- introduced. It can be indeed anticipated that the nature of the
hydroxybenzaldehyde, which is fully soluble in cationic polar head can influence the transfection efficacies as
dichloromethane, was engaged in an Atherton-Todd reaction previously observed.8,19 Herein, the cationic group was either
with dioleylphosphite . The aldehyde was isolated with an -methylimidazolium 10), pyridinium 11 or
excellent yield (97%). The higher solubility of this reagent in trimethylammonium (12).
8 was transformed into the iodo compound 9 (89%
7
5
6
N
(
(
)
dichloromethane but also the higher acidity of the phenol After the synthesis of monocationic lipophosphates, the
functional group (pKa = 7 for 4-hydroxybenzaldehyde whereas procedure was further used to produce dicationic derivatives. It
the pKa of 4-hydroxymethylphenol is 15) likely explained the is first important to underline the interest of dicationic
excellent yields. Interestingly, the nucleophilic addition of the amphiphilic compounds for gene transfer experiments. Cationic
deprotonated phosphite on the aldehyde functional group lipid/DNA complexes are characterized by their charge ratio
(Pudovik reaction18) was never observed. The reduction of the (CR) which corresponds to the number of positive charges
aldehyde functional group with NaBH4 produced the alcohol
7
(provided by the cationic lipid) to the number of negative
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charges (from the nucleic acid). The reduction of the quantities conduct gene transfection experiments. The monocationic
of cationic lipids in a selected formulation may be beneficial, in phosphates 10-12 and 17-22 were formulated as liposomal
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O
Et2O, 0°C to RT
CBr4/Ph3P
CH2Cl2
O
O
DOI: 10.1039/C4OB01770F
Br
O
P
OH
1) R-OH (2 éq)
O
P
RO
RO
O
NEt3
P
O
0°C, 2h
Cl
Cl
(1eq)
Cl
2) Br
Br
54%
7
Br
13
OH
14
15
R = oleyl
NaI,
Acetone
27 %
57 %
O
P
(1eq)
O
O
R = Phytanyl
Br
20°C
16h
O
N
N
8
or
89%
N
I
I
NaI (2.6 eq)
Acetone
O
N
O
or
(2 eq)
O
RO
RO
P
O
I
N
N
P
RT, 16h
86 %
or
or
40°C, 16h, neat
O
O
9
N
100 %
N
16 R = oleyl
40°C
16h
100%
N(CH3)3
17 (BSV 125) Z =
N
X
R = oleyl
X = I
Z
Z
N
O
P
18 (BSV 126)
19 (BSV 127)
Z =
Z =
O
O
Z
RO
N
P
O
O
O
I
RO
N
N
N
Z =
20 (BSV 128)
21 (BSV 129)
22 (BSV 130)
R = Phytanyl
X = Br
12 (BSV 122)
N(CH3)3
Z =
Z =
10 (BSV 120)
N
N
Z =
Z =
X
Z = N
11 (BSV 121)
N
(C18H35, oleyl)
Fig. 4 Synthetic scheme used to produce monocationic aryl-lipophosphates.
R =
(C20H41, phytanyl)
particular in a view to reduce potential side effects for the host
cells or animals. However, the reduction of the quantities of
cationic lipid directly impacts the CR that can, consequently,
produce unstable lipoplexes solution (colloidal instability).
Accordingly, the use of dicationic20 or polycationic amphiphiles
permits to reduce the quantity of cationic lipid while keeping
the CR unchanged. This strategy is also valuable for the
delivery of gene by aerosolisation or instillation since high
quantities of pDNA must be placed in a reduced volume.21 The
synthesis of dicationic aryldialkylphosphates was performed
from POCl3 (Figure 5) and was inspired from a methodology
that we have developed to produce cationic lipids incorporating
two different lipid domains.17 This methodology started by the
addition of two equivalents of oleyl alcohol or phythanol
followed by the addition of 3,5-dibromomethylphenol. The
expected dibromophosphates were isolated respectively with
27% (14) and 57% (15) at a 2.5 to 5.7 grams scale. Oleyl6b and
phytanyl6a chains were selected as lipid domains on the basis of
previous reports that have shown the high transfection
efficacies obtained with cationic lipids possessing this type of
hydrophobic domain. The halogen exchange was applied only
to the oleyl derivatives to produce the bis-iodide derivative 16
(86% yield). Then compound 15 or 16 was mixed with two
Fig. 5 Synthetic scheme used to produce dicationic aryl-lipophosphates.
solutions by hydration of a lipid film (1.5 mmol.L-1) in water
for 18 h. After mixing (vortex) and sonication, all these mono
or dicationic lipids produced liposomal solutions that were
thereafter characterized by DLS and zeta measurements (Table
SI-1). The sizes varied from 70 to 250 nm, dicationic
derivatives producing smaller particles when compared to
monocationic ones. In every instance, the zeta potential was
clearly positive, varying from +40 to +50 mV for monocationic
derivatives and from +50 to +84 mV for dicationic derivatives.
Next, the ability of monocationic and dicationic lipophosphates
to interact with a pDNA was evaluated. First, DNA retardation
assays by agarose gel electrophoresis were achieved at different
CR, from CR1 to CR8. The results obtained (Figures SI-2)
showed that the dicationic compounds were able to fully
compact DNA from CR2 or CR4 whereas monocationic
derivatives were much less efficient, traces of free pDNA being
still observed at CR8. With the aim to evaluate the stability of
the lipoplexes formed, a dismantling experiment was then
carried out, 11 and 19 being used as representative
monocationic and dicationic lipophosphates, respectively. For
this purpose, lipoplexes formed at CR2, CR4, and CR8 were
incubated with increasing quantities of dextran sulfate (DS). In
this experiment, DS was used as a counter-poly-anion capable
to compete with DNA for electrostatic interaction with the
cationic lipids. The dismantling of preformed lipoplexes led to
equivalents of
to produce the dicationic derivatives possessing two oleyl
chains (17 19) or two phytanyl chains (20 22). The compounds
17 22 were isolated in sufficient quantities (between 0.12 to
N-methylimidazole, pyridine or trimethylamine
-
-
-
0.23 g) to study their capacity to compact a pDNA and to
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the release of free DNA. By quantifying the recovery of
fluorescence on agarose gel, it was thus possible to compare the
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Panel A:
Panel B:
Panel C:
Fig. 7 Transfection efficacies of mono (Panel A) and dicationic dialkylarylphosphates (Panel B and C) as determined using a luciferase-encoding pDNA and three lung
cell lines (A549, 16HBE and CFBE). The transfection efficacy is expressed in the unit of RLU/mg of proteins. LFM, lipofectamine (Invitrogen).
resistance to dissociation – i.e. the stability – of the various naked uncomplexed DNA (i.e. 100% fluorescence) can be
lipoplexes considered. This showed that the fluorescence of recovered from 11-based lipoplexes incubated with DS at 2 or 4
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µg/µg DNA whereas only residual fluorescence was obtained With respect to the toxicity of this series of compounds (see SI-
from 19-based lipoplexes incubated with DS at the same doses 5) which was evaluated by the extracellular quantification of a
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(Figure 6). Altogether, these results demonstrated that the cytosolic enzyme (adenylate kinase), no significant differences
DOI: 10.1039/C4OB01770F
dicationic lipophosphates are much more efficient than their were observed between the different cationic lipids neither with
monocationic analogues both to condense and to retain DNA.
the references.
DS (µg/µg DNA)
0.25 0.5
DS (µg/µg DNA)
0.25 0.5
A
Conclusion
Ctrl
0
1
2
4
Ctrl
0
1
2
4
a
b
c
a
b
c
We report herein the synthesis of cationic lipids characterized
by a dialkylarylphosphate group that acts as a linker between
the hydrophobic domain and the cationic polar head. The access
of this type of cationic amphiphiles was achieved according to
two methods that made use of the Atherton-Todd coupling of
lipid-based phosphite with a phenol derivative and a third
method that started from POCl3. Different cationic polar heads
B
120
100
80
60
40
20
0
120
100
80
60
40
20
0
a
b
c
(N-methyl imidazolium, pyridinium, and trimethylammonium)
and various lipid chains (oleyl or phytanyl) were incorporated
into the structure of these new cationic lipids. They were
formulated as liposomal solutions. It was first noticed that they
exhibited good stability during storage, no degradation being
evidenced after a few days in water or in an acidic media (pH
5.5). Next, the liposomes prepared from these various cationic
lipids were tested as vectors for gene delivery into four human-
derived cell lines. They demonstrated positive gene transfer
abilities, despite great differences of efficiency were observed
between all these derivatives. Especially, monocationic
derivatives were much more efficient than dicationic lipids. The
particular geometry of the polar head of dicationic derivatives
likely contribute to their great affinity for DNA that in turn may
explain their poor gene delivery potency – as compared with
monocationic analogues – due to an inefficient release of DNA
inside the cells. The use of the most promising cationic
a
b
c
0
1
2
3
4
0
1
2
3
4
DS (µg/µg DNA)
DS (µg/µg DNA)
Fig. 6 Ability of a monocationic (11, left panels) and a dicationic (19, right panels)
aryl-lipophosphates to maintain condensed the super-coiled form of a pDNA in
presence of dextran sulfate (DS). Agarose gel electrophoresis (A) and
quantification of fluorescence intensities (B) for lipoplexes prepared at CR2 (a),
CR4 (b), and CR8 (c) incubated at RT with either OptiMEM or OptiMEM
containing increasing quantities of DS. Ctrl, naked uncomplexed DNA; its
fluorescence intensity is given the value of 100%.
Next, we evaluated the transfection efficacies of monocationic
and dicationic derivatives. For this purpose, four human-
derived cell lines were used (A549, 16HBE, CFBE, and
SKMel28). Lipoplexes were formed at different CR by mixing
the various cationic lipids with a luciferase-encoding pDNA.
The best results were obtained at CR2 and CR4 (see Figure 7
and SI-S3). Each figure summarises the experiments that were
realized in triplicate the same day on the same batch of cultured
cells. It is first observed that the monocationic amphiphilic
compounds (Figure 7, panel A) are more efficient than the
dicationic derivatives (Figure 7 panel B and C). Among the
monocationic compounds, their efficacy is clearly optimum at
lipophosphates (the mono cationic compounds 10, 11 and 12)
will be tested in vivo either by i.v. injection6 or by
aerosolization with the aim to transfect lung epithelial cells.
Experimental section
Solvents were dried with a solvent purification system MBraun-
SPS (THF, CH2Cl2). All compounds were fully characterized
1
by H (500.13 or 400.133 or 300.135 MHz), 13C (125.773 or
75.480 MHz) and 31P (161.970 or 121.498 MHz) NMR
spectroscopy (Bruker AC 300, Avance DRX 400 and Avance
DRX 500 spectrometers). The lipids were purified by flash
chromatography (GRACE REVELERIS Flash Chromatography
System) equipped with UV and DLS detectors allowing to
attest the high purity (> 95%) of the purified compounds.
Coupling constants J are given in Hertz. The following
abbreviations were used: s for singlet, d doublet, t triplet, q
quadruplet, qt quintuplet, m for multiplet and dt for doublet of
triplets. When needed, 13C and 31P heteronuclear HMQC and
HMBC were used to unambiguously establish molecular
structures. Mass spectroscopy analyses were performed at Brest
(service commun de spectrometrie de masse) on a Bruker
Autoflex MALDI TOF-TOF III LRF200 CID. Commercial
compounds [Oleyl alcohol 85 %, 2-bromoethylamine
hydrobromide] were used as received except DIPEA which was
CR4. The transfection efficacies of compounds 10, 11 and 12
are actually very close with a slight advantage to the pyridinium
derivative 11. The dicationic derivatives were 10 to 100 fold
less efficient than the monocationic derivatives. In this series of
dicationic compounds the lipid chain is either an oleyl (17
-19)
or a phytanyl chain (20 22). However, we cannot point out any
-
major difference of transfection efficacies between these two
series of compounds. It is hypothesized that the higher
compaction property of dicationic derivatives which is likely
explained by the cooperative effect of the two cationic charges
might limit the dissociation of pDNA from the cationic lipid
after endocytosis. The lipoplexes dismantling experiment
(Figure 6) and a complementary transfection assay (Figure SI-
4) support this hypothesis. The geometry of the cationic polar
head may also explain this result since we found that linear
shaped dicationic lipids produced high transfection efficacies.20
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distilled over KOH. O,O-dioleylphosphite 5 22
,
and 3,5- 4H, 2 x O-CH2-CH3) ; 5.57 (s, 2H, CH2-N) ; 7.24 (m, 4H, C2-H
3
Bis(bromomethyl)phenol23 13, have been synthesized according and C6-H and N-CH=CH) ; 7.53 (d, JHH = 8 Hz, 2H, C3-H and
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to a reported procedure. The mean particle diameter and zeta C5-H) ; 10.57 (s, 1H, CH=N). 13C-NMR D(1O0I:01.05.910M39H/Cz4,OCB0D17C7l03F)
potential (ξ) of the liposomes and lipoplexes were measured : 16.10 (d, JCP = 6 Hz, 2 x O-CH2-CH3) ; 36.72 (s, CH3-N) ;
3
2
using a Zetasizer Nano ZS (Malvern Instruments) at 25 °C (see 52.58 (s, CH2-N) ; 64.90 (d, JCP = 6 Hz, 2 x O-CH2-CH3) ;
supplementary material).
120.94 (s, C2 and C6) ; 121.71 (s, NCH=CHN) ; 123.10 (s,
NCH=CHN) ; 129.84 (s, C4) ; 130.84 (s, C3 and C5) ; 151.60 (s,
C1). 31P-NMR (161.97 MHz, CDCl3) : -5.4.
General protocol for the synthesis of dialkyl-arylphosphate from
dialkylphosphite.
{4-[(O,O-diethylphosphoryl)oxy]phenyl}methylpyridinium
O
,
O
-diethyl 4-(hydroxymethyl)phenyl phosphate 1
bromide
4
To a solution of 4-hydroxybenzyl alcohol (1.98 g ; 15.9 mmol)
in CH2Cl2 (30 mL) with molecular sieves was added DIPEA To a solution of diethyl 4-(bromomethyl)phenyl phosphate
(5.61 g ; 43.4 mmol). The reaction mixture is stirred at room (0.10 g ; 0.31 mmol) was added pyridine (0.25 mL ; 3.09
temperature for 5 min and then cooled down with an ice water mmol). The reaction mixture is stirred at 40°C for 15h. Then
1
bath. Diethylphosphite (2.00 g ; 14.5 mmol) and bromotri- evaporation (124 mg ; 90%). H-NMR (400.08 MHz, CDCl3) :
chloromethane (5.74 g ; 28.9 mmol) were then added 1.35 (t, 3JHH = 8 Hz, 6H, 2 x O-CH2-CH3) ; 4.20 (m, 4H, 2 x O-
successively. The reaction mixture was stirred at 0-5°C for 1h, CH2-CH3) ; 6.31 (s, 2H, CH2-N) ; 7.20 (d, 3JHH = 8 Hz, 2H, C2-
3
then at room temperature for 5h. The evaporation of the solvent H and C6-H) ; 7.77 (d, JHH = 8 Hz, 2H, C3-H and C5-H) ; 7.98
produced a residue that was dissolved in diethyl ether. After (t, 3JHH = 8 Hz, 2H, Hpyr) ; 8.42 (t, 3JHH = 8 Hz, 1H, Hpyr) ; 9.61
3
filtration and evaporation of the solvent, the crude product was (t, JHH = 8 Hz, 2H, Hpyr). 13C-NMR (100.59 MHz, CDCl3) :
3
purified by flash chromatography using hexane/ethyl acetate as 16.12 (d, JCP = 6 Hz, 2 x O-CH2-CH3) ; 63.18 (s, CH2-N) ;
eluent to produce a yellow oil (2.32 g ; 62%).1H-NMR (400.08 64.91 (d, 2JCP = 6 Hz, 2 x O-CH2-CH3) ; 121.26 (s, C2 and C6) ;
MHz, CDCl3) : 1.34 (t, 3JHH = 8 Hz, 6H, 2 x O-CH2-CH3) ; 4.21 128.20 (s, Cpyr) ; 129.69 (s, C4) ; 131.62 (s, C3 and C5) ; 145.12
(m, 4H, 2 x O-CH2-CH3) ; 4.66 (s, 2H, CH2-OH) ; 7.19 (d, , (s, Cpyr) ; 151.98 (s, C1). 31P-NMR (161.97 MHz, CDCl3) : -5.4.
3JHH = 8 Hz, 2H, C2-H and C6-H) ; 7.32 (d, 3JHH = 8 Hz, 2H, C3-
O,O-dioleyl 4-formylphenyl phosphate 6
H and C5-H). 13C-NMR (125.80 MHz, CDCl3) : 15.55 (d, 3JCP
=
6 Hz, 2 x O-CH2-CH3) ; 63.15 (s, CH2-OH) ; 64.31 (d, 2JCP = 6 To a solution of 4-hydroxybenzaldehyde (0.46 g ; 3.77 mmol)
Hz, 2 x O-CH2-CH3) ; 119.20 (s, C2 and C6) ; 127.72 (s, C3 and in CH2Cl2 (20 mL) with molecular sieves was added DIPEA
C5) ; 138.18 (s, C4) ; 149.07 (s, C1). 31P-NMR (161.97 MHz, (1.33 g ; 10.29 mmol). The reaction mixture is stirred at room
CDCl3): -5.1. MS (MALDI-TOF): m/z calcd for C11H18O5P temperature for 5 min and then cooled down with an ice water
(M+H) : 261.089 ; found 261.049.
bath. Dioleylphosphite (2.00
g
;
3.43 mmol) and
bromotrichloromethane (1.36 g ; 6.86 mmol) were then added
successively. The reaction mixture was stirred at 0-5°C for 1h,
O,O
-diethyl 4-(bromomethyl)phenyl phosphate 2
To the solution of carbon tetrabromide (0.77 g ; 2.31 mmol) then at room temperature for 5h. Evaporation of the solvent
and diethyl 4-(hydroxymethyl) phenyl phosphate (0.50 g ; 1.92 produced a residue that was dissolved in diethyl ether. Filtration
mmol) in 8 mL of CH2Cl2 was added triphenylphosphine (0.61 and evaporation of the solvent. The compound was purified by
g ; 2.31 mmol) at 0°C. The resultant mixture was stirred for 2h. flash chromatography using CH2Cl2/methanol as eluent to
1
The compound was purified by flash chromatography using produce a colourless oil (2.29g ; 95% yield). H-NMR (400.08
3
hexane/ethyl acetate as eluent in a yield 97 % (0.60 g ; 97%). MHz, CDCl3) : 0.88 (t, JHH = 8 Hz, 6H, CH3) ; 1.26 (s, 44H,
3
1H-NMR (400.08 MHz, CDCl3) : 1.35 (t, JHH = 8 Hz, 6H, 2 x CH2) ; 1.69 (m, 4H, CH2 β) ; 2.00 (m, 8H, CH2-CH=CH) ; 4.15
3
O-CH2-CH3) ; 4.21 (m, 4H, 2 x O-CH2-CH3) ; 4.47 (s, 2H, (m, 4H, CH2 α) ; 5.34 (m, 4H, CH=CH) ; 7.38 (d, JHH = 8 Hz,
3
CH2-Br) ; 7.19 (d, JHH = 8 Hz, 2H, C2-H and C6-H) ; 7.36 (d, 2H, C3-H and C5-H) ; 7.89 (d, 3JHH = 8 Hz, 2H, C2-H and C6-H)
3JHH = 8 Hz, 2H, C3-H and C5-H). 13C-NMR (100.59 MHz, ; 9.97 (s, 1H, CHO). 13C-NMR (125.75 MHz, CDCl3) : 14.50
3
CDCl3) : 15.88 (d, JCP = 6 Hz, 2 x O-CH2-CH3) ; 32.46 (s, (s, 2 x CH3) ; 22.81 (s, CH2) ; 25.60 (s, CH2) ; 27.13 (s,
2
CH2-Br) ; 64.51 (d, JCP = 6 Hz, 2 x O-CH2-CH3) ; 120.05 (s, CH=CH-CH2) ; 29-30 (s, CH2) ; 68.94 (d, 2JCP = 6 Hz, CH2 α) ;
C2 and C6) ; 130.33 (s, C3 and C5) ; 134.36 (s, C4) ; 150.41 (s, 120.46 (s, C2 and C6) ; 129.66 (s, CH=CH) ; 129.95 (s,
C1). 31P-NMR (161.97 MHz, CDCl3) : -5.3. MS (MALDI-TOF) CH=CH) ; 131.60 (s, C3 and C5) ; 133.21 (s, C4) ; 155.50 (s, C1)
: m/z calcd for C11H17BrO4P (M+H) : 323.005 ; found 322.979.
; 190.63 (s, CHO). 31P-NMR (161.97 MHz, CDCl3) : -5.6. MS
(MALDI-TOF) : m/z calcd for C43H75NaO5P (M+Na) : 725.525
; found 725.484.
{4-[(O,O-diethylphosphoryl)oxy]phenyl}methylmethyl
imidazolium bromide 3
O,O-dioleyl 4-(hydroxymethyl)phenyl phosphate 7
To a solution of diethyl 4-(bromomethyl)phenyl phosphate
(0.10 g ; 0.31 mmol) was added 1-methylimidazole (25 µL; To a solution of 4-hydroxybenzyl alcohol (0.23 g ; 1.9 mmol)
0.31 mmol). The reaction mixture is stirred at 40°C for 15h in CH2Cl2 (9 mL) with molecular sieves was added DIPEA
1
3
(125 mg ; 90%). H-NMR (400.08 MHz, CDCl3) : 1.36 (t, JHH (0.66 g ; 5.1 mmol). The reaction mixture is stirred at room
= 8 Hz, 6H, 2 x O-CH2-CH3) ; 4.02 (s, 3H, CH3-N) ; 4.20 (m, temperature for 5 min and then cooled down with an ice water
8 | J. Name., 2014, 00, 1-3
This journal is © The Royal Society of Chemistry 2014

Page 9 of 13
Journal Name
Organic & Biomolecular Chemistry
ARTICLE
bath. Dioleyle phosphite (1.00
g
;
1.7 mmol) and CH2-I) ; 14.16 (s, 2 x CH3) ; 22.73 (s, CH2) ; 25.43 (s, CH2) ;
bromotrichloromethane (0.66 g ; 3.4 mmol) were then added 27.27 (s, CH=CH-CH2) ; 29-30 (s, CH2) ; 30.27 (d, 3JCP = 7 Hz,
successively. The reaction mixture was stirred at 0-5°C for 1h, CH2 β) ; 31.96 (s, CH2) ; 32.66 (s, CH2) ;D6O8I.:6190.1(0d3V,9ie2/wJCCA4PrOti=cBle061O7nH7li0nzFe,
then at room temperature for 5h. The evaporation of the solvent CH2 α) ; 120.33 (s, C2 and C6) ; 129.79 (s, C3 and C5) ; 130.04
produced a residue that was dissolved in diethyl ether. After (s, CH=CH) ; 130.16 (s, CH=CH) ; 135.99 (s, C4) ; 150.23 (s,
filtration and evaporation of the solvent, the crude product was C1). 31P-NMR (161.97 MHz, CDCl3) : -5.0. MS (MALDI-TOF)
purified by flash chromatography using CH2Cl2 /methanol as : m/z calcd for C43H76INaO4P (M+Na) : 837.442 ; found
eluent to produce a yellow oil (0.45 g ; 38%). 1H-NMR (400.04 837.541.
3
MHz, CDCl3) : 0.87 (t, JHH = 6 Hz, 6H, CH3) ; 1.26 (s, 44H,
CH2) ; 1.66 (m, 4H, CH2 β) ; 1.99 (m, 8H, CH2-CH=CH) ; 4.12
{4-[(O,O-dioleylphosphoryl)oxy]phenyl}methylmethyl
imidazolium) iodide 10
(m, 4H, CH2 α) ; 4.64 (s, 2H, CH2-OH) ; 5.34 (m, 4H, CH=CH)
3
3
; 7.18 (d, JHH = 8 Hz, 2H, C2-H and C6-H) ; 7.31 (d, JHH = 8 To a solution of dioleyl 4-(bromomethyl)phenyl phosphate
Hz, 2H, C3-H and C5-H). 13C-NMR (125.75 MHz, CDCl3) : (0.10 g ; 0.13 mmol) was added methylimidazole (0.01 mL ;
14.12 (s, 2 x CH3) ; 22.70 (s, CH2) ; 25.41 (s, CH2) ; 27.24 (s, 0.13 mmol). The reaction mixture is stirred at 40°C for 15h
CH=CH-CH2) ; 29-30 (s, CH2) ; 30.22 (d, 3JCP = 6 Hz, CH2 β) ; (110 mg ; 100% yield). H-NMR (400.04 MHz, CDCl3) : 0.87
1
3
31.93 (s, CH2) ; 32.63 (s, CH2) ; 64.57 (s, CH2-OH) ; 68.65 (d, (t, JHH = 6 Hz, 6H, CH3) ; 1.26 (s, 44H, CH2) ; 1.68 (m, 4H,
2JCP = 6 Hz, CH2 α) ; 115.38 (s, C2 and C6) ; 127.72 (s, C3 and CH2 β) ; 2.00 (m, 8H, CH2-CH=CH) ; 4.04 (s, 3H, CH3-N) ;
C5) ; 129.56 (s, CH=CH) ; 129.76 (s, CH=CH) ; 137.72 (s, C4) ; 4.12 (m, 4H, CH2 α) ; 5.34 (m, 4H, CH=CH) ; 5.58 (s, 2H,
150.13 (s, C1). 31P-NMR (161.97 MHz, CDCl3) : -4.9. MS CH2-N) ; 7.20 (d, 4H, C2-H, C6-H and Himid) ; 7.51 (d, 3JHH = 8
(MALDI-TOF) : m/z calcd for C43H77NaO5P (M+Na) : 727.541 Hz, 2H, C3-H and C5-H) ; 10.72 (s, 1H, CH=N). 13C-NMR
; found 727.514.
(125.75 MHz, CDCl3) : 14.05 (s, 2 x CH3) ; 22.70 (s, CH2) ;
25.40 (s, CH2) ; 27.17 (s, CH=CH-CH2) ; 29-30 (s, CH2) ; 36.77
(s, CH3-N) ; 52.56 (s, CH2-N) ; 68.84 (d, JCP = 6 Hz, CH2 α) ;
O,O-dioleyl 4-(bromomethyl)phenyl phosphate 8
2
To the solution of carbon tetrabromide (0.42 g ; 1.28 mmol) 120.92 (s, C2 and C6) ; 121.75 (NCH=CHN) ; 123.19
and dioleyl 4-(hydroxymethyl)phenyl phosphate (0.43 g ; 0.61 (NCH=CHN) ; 129.74 (s, CH=CH) ; 129.97 (s, CH=CH) ;
mmol) in 7 mL of CH2Cl2 was added triphenylphosphine (0.33 129.77 (s, C4) 130.48 (s, C3 and C5) ; 138.01 (s, NCHN) ;
;
g ; 1.28 mmol) at 0°C. The resultant mixture was stirred for 2h. 152.00(s, C1). 31P-NMR (161.97 MHz, CDCl3) : -5.1. MS
The compound was purified by flash chromatography using (MALDI-TOF) : m/z calcd for C47H82N2O4P (M+) : 769.601 ;
1
CH2Cl2/methanol as eluent in a yield 54 % (0.25 g ; 54%). H- found 769.530.
3
NMR (400.04 MHz, CDCl3) : 0.87 (t, JHH = 6 Hz, 6H, CH3) ;
1.26 (s, 44H, CH2) ; 1.67 (m, 4H, CH2 β) ; 1.99 (m, 8H, CH2-
{4 -[(O,O-dioleylphosphoryl)oxy]phenyl}methylpyridinium)
iodide 11
CH=CH) ; 4.12 (m, 4H, CH2 α) ; 4.45 (s, 2H, CH2-Br) ; 5.33
3
(m, 4H, CH=CH) ; 7.17 (d, JHH = 8 Hz, 2H, C2-H and C6-H) ; To a solution of dioleyl 4-(bromomethyl)phenyl phosphate
3
7.34 (d, JHH = 8 Hz, 2H, C3-H and C5-H). 13C-NMR (125.75 (0.10 g ; 0.13 mmol) was added pyridine (0.10 mL ; 1.30
MHz, CDCl3) : 14.14 (s, 2 x CH3) ; 22.69 (s, CH2) ; 25.38 (s, mmol). The reaction mixture is stirred at 40°C for 15h. Then
3
1
CH2) ; 27.22 (s, CH=CH-CH2) ; 29-30 (s, CH2) ; 30.24 (d, JCP evaporation (110 mg ; 100% yield). H-NMR (400.04 MHz,
3
= 6 Hz, CH2 β) ; 31.86 (s, CH2-Br) ; 32.61 (s, CH2) ; 68.67 (d, CDCl3) : 0.86 (t, JHH = 6 Hz, 6H, CH3) ; 1.25 (s, 44H, CH2) ;
2JCP = 6 Hz, CH2 α) ; 120.31 (s, C2 and C6) ; 129.75 (s, C3 and 1.67 (m, 4H, CH2 β) ; 1.98 (m, 8H, CH2-CH=CH) ; 4.11 (m,
C5) ; 130.21 (s, CH=CH) ; 130.47 (s, CH=CH) ; 134.46 (s, C4) ; 4H, CH2 α) ; 5.32 (m, 4H, CH=CH) ; 6.37 (s, 2H, CH2-N) ;
150.20 (s, C1). 31P-NMR (161.97 MHz, CDCl3) : -5.1. MS 7.19 (d, 3JHH = 8 Hz, 2H, C2-H and C6-H) ; 7.78 (d, 3JHH = 8 Hz,
3
(MALDI-TOF) : m/z calcd for C43H76BrNaO4P (M+Na) : 2H, C3-H and C5-H) ; 7.97 (t, JHH = 8 Hz, 2H, Hpyr) ; 8.40 (t,
3
789.456 ; found 789.598.
3JHH = 8 Hz, 1H, Hpyr) ; 9.63 (d, JHH = 8 Hz, 2H, Hpyr). 13C-
NMR (125.75 MHz, CDCl3) : 14.49 (s, 2 x CH3) ; 22.75 (s,
O,O-dioleyl 4-(iodomethyl)phenyl phosphate 9
CH2) ; 25.50 (s, CH2) ; 27.17 (s, CH=CH-CH2) ; 29-30 (s, CH2)
2
Sodium iodide (76 mg ; 0.51 mmol) was added to a solution of ; 62.95 (s, CH2-N) ; 68.85 (d, JCP = 6 Hz, CH2 α) ; 121.11 (s,
dioleyl 4-(bromomethyl)phenyl phosphate (300 mg ; 0.39 C2 and C6) ; 128.22 (s, C8 and C10) ; 129.74 (s, CH=CH) ;
mmol) in 5 mL of acetone. The mixture was stirred at R.T. for 129.98 (s, CH=CH) ; 131.60 (s, C3 and C5) ; 144.93 (s, C7, C9
16h. Acetone was removed. The residue was dissolved in and C11) ; 151.93(s, C1). 31P-NMR (161.97 MHz, CDCl3) : -5.0.
petroleum ether and filtered on celite. The filtrate was MS (MALDI-TOF) : m/z calcd for C48H81NO4P (M+) : 766.590
concentrated and the expected compound was afforded as a ; found 766.508.
yellow oil (285 mg ; 89 % yield). 1H-NMR (400.04 MHz,
CDCl3) : 0.88 (t, JHH = 8 Hz, 6H, CH3) ; 1.26 (s, 44H, CH2) ;
3
{4-[(O,O-dioleylphosphoryl)oxy]phenyl}methyltrimethyl
ammonium iodide 12
1.66 (m, 4H, CH2 β) ; 2.00 (m, 8H, CH2-CH=CH) ; 4.12 (m,
4H, CH2 α) ; 4.43 (s, 2H, CH2-I) ; 5.35 (m, 4H, CH=CH) ; 7.13 To a solution of dioleyl 4-(iodomethyl)phenyl phosphate (0.066
(d, 3JHH = 8 Hz, 2H, C2-H and C6-H) ; 7.34 (d, 3JHH = 8 Hz, 2H, g ; 0.08 mmol) was added trimethylamine (3.0 mL). The
C3-H and C5-H). 13C-NMR (125.75 MHz, CDCl3) : 4.55 (s, reaction mixture is stirred at RT for 15h. After evaporation of
This journal is © The Royal Society of Chemistry 2014
J. Name., 2014, 00, 1-3 | 9

Organic & Biomolecular Chemistry
Page 10 of 13
Journal Name
ARTICLE
the volatiles the expected compound 12 was isolated (71 mg ; phytanyl) ; 24.30 to 24.90 (s, CH2 phytanyl) ; 28.02 (s, CH
100% yield). 1H-NMR (400.04 MHz, CDCl3) : 0.87 (t, 3JHH = 8 phytanyl) ; 29.39 (s, CH phytanyl) ; 31.85 (s, CH2-Br) ; 32.95
View Article Online
Hz, 6H, CH3) ; 1.26 (s, 44H, CH2) ; 1.69 (m, 4H, CH2 β) ; 2.00 (s, CH phytanyl) ; 37.20 to 37.60 (s, CH2 phytanyl) ; 39.41 (s,
DOI: 10.1039/C4OB01770F
2
(m, 8H, CH2-CH=CH) ; 3.36 (s, 9H, N-(CH3)3) ; 4.15 (m, 4H, CH2 phytanyl) ; 67.39 (d, JCP = 6 Hz, CH2 α) ; 120.11 (s, C2
CH2 α) ; 5.03 (s, 2H, CH2-N) ; 5.34 (m, 4H, CH=CH) ; 7.31 (d, and C6) ; 126.05 (s, C4) ; 140.11 (s, C3 and C5) ; 150.99 (s, C1).
3JHH = 8 Hz, 2H, C2-H and C6-H) ; 7.70 (d, 3JHH = 8 Hz, 2H, C3- 31P-NMR (161.97 MHz, CDCl3) : -5.8. MS (MALDI-TOF) :
H and C5-H). 13C-NMR (125.75 MHz, CDCl3) : 7.29 (s, CH2-I) m/z calcd for C48H89Br2O4P (M+Na+) : 641.476 ; found :
; 14.13 (s, 2 x CH3) ; 22.69 (s, CH2) ; 25.43 (s, CH2) ; 27.23 (s, 941.474.
CH=CH-CH2) ; 29-30 (s, CH2) ; 30.20 (s, CH2) ; 31.92 (s, CH2)
; 32.62 (s, CH2) ; 52.57 (s, N(CH3)3) ; 68.69 (d, JCP = 6 Hz,
2
O,O-dioleyl 3,5-bis(iodomethyl)phenyl phosphate 16
CH2 α) ; 120.33 (s, C2 and C6) ; 129.79 (s, C3 and C5) ; 130.04 Sodium iodide (330 mg ; 2.2 mmol) was added to a solution of
(s, CH=CH) ; 130.16 (s, CH=CH) ; 135.99 (s, C4) ; 150.23 (s, dioleyl 3,5-bis(bromomethyl)phenyl phosphate (725 mg ; 0.8
C1). 31P-NMR (161.97 MHz, CDCl3) : -5.3. MS (MALDI-TOF) mmol) in 10 mL of acetone. The mixture was stirred at R.T. for
: m/z calcd for C46H85NO4P (M+) : 746.621 ; found 746.586.
16h. Acetone was removed. The residue was dissolved in ether
petroleum and filtered on celite. The filtrate was concentrated
and the expected compound was afforded as a yellow oil (86 %
O,O-dioleyl- -[3,5-bis(bromomethyl)phenyl]phosphate 14
O
Triethylamine (3.13 mL ; 22.5 mmol) was added to a cooled yield ; 0.69 g). 1H-NMR (400.04 MHz, CDCl3) : 0.87 (t, 3JHH
=
(0°C) solution of POCl3 (1.64 g ; 10.7 mmol) in diethylether 8 Hz, 6H, CH3) ; 1.26 (s, 44H, CH2) ; 1.68 (m, 4H, CH2 β) ;
(15 mL) and placed under argon atmosphere. Oleyl alcohol 1.99 (m, 8H, CH2-CH=CH) ; 4.12 (q, 3JHH = 8 Hz, 4H, CH2 α) ;
(5.75 g ; 21.4 mmol) in diethylether (5 mL) was then added 4.35 (s, 4H, CH2-I) ; 5.34 (m, 4H, CH=CH) ; 7.10 (s, 2H, C2-H
dropwise. This solution was further stirred for 5h while the and C6-H) ; 7.17 (s, 1H, C4-H). 13C-NMR (125.75 MHz,
temperature was slowly increased up to 20°C. Then, a solution CDCl3) : 3.44 (s, CH2-I) ; 14.14 (s, 2 x CH3) ; 22.68 (s, CH2) ;
of 3,5-bis(bromomethyl)phenol (3.00 g ; 10.7 mmol) and 25.41 (s, CH2) ; 27.21 (s, CH=CH-CH2) ; 29-30 (s, CH2) ; 31.94
triethylamine (1.49 mL ; 10.7 mmol) in diethylether (8 mL) was (s, CH2) ; 32.67 (s, CH2) ; 68.87 (s, CH2 α) ; 119.91 (s, C2 and
added and the solution was stirred for 3h. Filtration and C6) ; 125.54 (s, C4) ; 129.76 (s, CH=CH) ; 129.98 (s, CH=CH)
evaporation of the solvent. The compound was purified by ; 141.64 (s, C3 and C5) ; 150.85 (s, C1). 31P-NMR (161.97 MHz,
chromatography on silica gel using CH2Cl2 as eluent to produce CDCl3) : -5.9.
1
a colorless oil (27% yield ; 2.47 g). H-NMR (400.04 MHz,
CDCl3) : 0.87 (t, JHH = 8 Hz, 6H, CH3) ; 1.26 (s, 44H, CH2) ;
1.68 (m, 4H, CH2 β) ; 1.99 (m, 8H, CH2-CH=CH) ; 4.13 (q,
3
{3,5-bis-[(O,O-dioleylphosphoryl)oxy]phenyl}methylmethyl
imidazolium iodide 17
3JHH = 8 Hz, 4H, CH2 α) ; 4.42 (s, 4H, CH2-Br) ; 5.34 (m, 4H, To a solution of dioleyl 3,5-bis(iodomethyl)phenyl phosphate
CH=CH) ; 7.20 (s, 2H, C2-H and C6-H) ; 7.22 (s, 1H, C4-H). (0.10 g ; 0.105 mmol) was added methylimidazole (17 µL ;
13C-NMR (125.75 MHz, CDCl3) : 14.11 (s, 2 x CH3) ; 22.68 (s, 0.209 mmol). The reaction mixture is stirred at 40°C for 15h
1
CH2) ; 25.40 (s, CH2) ; 27.22 (s, CH=CH-CH2) ; 29-30 (s, CH2) (0.12 g ; 100 % yield). H-NMR (400.04 MHz, CDCl3) : 0.87
3
; 30.24 (s, CH2-Br) ; 31.90 (s, CH2) ; 32.61 (s, CH2) ; 68.87 (s, (t, JHH = 7 Hz, 6H, CH3) ; 1.24 (s, 44H, CH2) ; 1.68 (m, 4H,
CH2 α) ; 120.61 (s, C2 and C6) ; 126.03 (s, C4) ; 129.75 (s, CH2 β) ; 2.00 (m, 8H, CH2-CH=CH) ; 4.00 (s, 6H, CH3-N) ;
CH=CH) ; 130.00 (s, CH=CH) ; 140.09 (s, C3 and C5) ; 150.97 4.16 (m, 4H, CH2 α) ; 5.34 (m, 4H, CH=CH) ; 5.58 (s, 4H,
(s, C1). 31P-NMR (161.97 MHz, CDCl3) : -5.9.
CH2-N) ; 7.32 (s, 2H, NCH=CHN) ; 7.37 (s, 2H, C2-H and C6-
H) ; 7.66 (s, 2H, NCH=CHN) ; 7.78 (s, 1H, C4-H) ; 10.02 (s,
2H, CH=N). 13C-NMR (125.75 MHz, CDCl3) : 14.12 (s, 2 x
O,O-diphytanyl 3,5-bis(bromomethyl)phenyl phosphate 15
Triethylamine (3.13 mL ; 22.5 mmol) was added to a cooled CH3) ; 22.68 (s, CH2) ; 25.45 (s, CH2) ; 27.22 (s, CH=CH-CH2)
(0°C) solution of POCl3 (1.64 g ; 10.7 mmol) in diethylether ; 29-30 (s, CH2) ; 31.90 (s, CH2) ; 32.61 (s, CH2) ; 37.42 (s,
2
(15 mL) and placed under argon atmosphere. Phytanol (6.40 g ; CH3-N) ; 52.32 (s, CH2-N) ; 69.31 (d, JCP = 6 Hz, CH2 α) ;
21.4 mmol) in diethylether (10 mL) was then added dropwise. 121.24 (s, C2 and C6) ; 122.80 (NCH=CHN) ; 123.71
This solution was further stirred for 5h while the temperature (NCH=CHN) ; 126.38 (s, C4) ; 129.72 (s, CH=CH) ; 130.01 (s,
was slowly increased up to 20°C. Then, a solution of 3,5- CH=CH) ; (s, NCHN) ; 136.13 (s, C3 and C5) ; 151.54 (s, C1).
bis(bromomethyl)phenol (3.00
g
;
10.7 mmol) and 31P-NMR (161.97 MHz, CDCl3) : -6.1. MS (MALDI-TOF) :
triethylamine (1.49 mL ; 10.7 mmol) in diethylether (5 mL) was m/z calcd for C52H89IN4O4P (M-I-) : 991.567 ; found : 991.472.
added and the solution was stirred for 3h. Filtration and
{3,5-bis-[(O,O-dioleylphosphoryl)oxy]phenyl}methyltrimethyl
ammonium iodide 18
evaporation of the solvent. The compound was purified by
chromatography on silica gel using CH2Cl2 as eluent to produce
a colorless oil (57% yield ; 5.66 g). H-NMR (400.04 MHz,
CDCl3) : 0.87 (m, 30H) ; 1.00-1.70 (m, 48H, CH2) ; 4.17 (m,
4H, CH2 α) ; 4.42 (s, 4H, CH2-Br) ; 7.19 (s, 2H, C2-H and C6-
H) ; 7.22 (s, 1H, C4-H). 13C-NMR (125.80 MHz, CDCl3) :
19.30 to 19.80 (s, CH3 phytanyl) ; 22.70 to 22.80 (s, CH3
1
To a solution of dioleyl 3,5-bis(iodomethyl)phenyl phosphate
(0.17 g ; 0.18 mmol) was added trimethylamine (2.0 mL). The
reaction mixture is stirred at RT for 15h. Then evaporation
1
(0.19 g ; 100 % yield). H-NMR (400.04 MHz, CDCl3) : 0.88
3
(t, JHH = 8 Hz, 6H, CH3) ; 1.26 (s, 44H, CH2) ; 1.71 (m, 4H,
10 | J. Name., 2014, 00, 1-3
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ARTICLE
CH2 β) ; 2.00 (m, 8H, CH2-CH=CH) ; 3.41 (s, 18H, N(CH3)3) ; {3,5-bis-[(O,O-diphytanylphosphoryl)oxy]phenyl}methyl
4.20 (m, 4H, CH2 α) ; 4.99 (s, 4H, CH2-N) ; 5.34 (m, 4H, trimethylammonium bromide 21
View Article Online
CH=CH) ; 7.65 (s, 2H, C2-H and C6-H) ; 8.13 (s, 1H, C4-H).
13C-NMR (125.75 MHz, CDCl3) : 14.16 (s, 2 x CH3) ; 22.72 (s,
CH2) ; 25.50 (s, CH2) ; 27.27 (s, CH=CH-CH2) ; 29-30 (s, CH2)
; 31.94 (s, CH2) ; 32.67 (s, CH2) ; 53.69 (s, N(CH3)3) ; 67.51 (s,
CH2-N) ; 69.64 (d, 2JCP = 6 Hz, CH2 α) ; 126.97 (s, C2 and C6) ;
129.76 (s, CH=CH) ; 130.03 (s, CH=CH) ; 130.49 (s, C3 and
C5) ; 134.52 (s, C4) ; 151.52 (s, C1). 31P-NMR (161.97 MHz,
CDCl3): -6.0. MS (MALDI-TOF) : m/z calcd for C50H95IN2O4P
(M-I-) : 945.607 ; found : 947.590.
DOI: 10.1039/C4OB01770F
solution of diphytanyl 3,5-bis(bromomethyl)phenyl
To
a
phosphate (0.17 g ; 0.18 mmol) was added trimethylamine (2.0
mL). The reaction mixture is stirred at RT for 15h. Then
evaporation (190 mg ; 100% yield). H-NMR (400.04 MHz,
1
CDCl3) : 0.87 (m, 30H) ; 1.00-1.70 (m, 48H, CH2) ; 3.46 (s,
18H, N(CH3)3) ; 4.22 (m, 4H, CH2 α) ; 5.07 (s, 4H, CH2-N) ;
7.65 (s, 2H, C2-H and C6-H) ; 8.19 (s, 1H, C4-H). 13C-NMR
(125.75 MHz, CDCl3) : 19.30 to 19.80 (s, CH3 phytanyl) ; 22.60
to 22.80 (s, CH3 phytanyl) ; 24.30 to 24.90 (s, CH2 phytanyl) ;
28.01 (s, CH phytanyl) ; 29.45 (s, CH phytanyl) ; 32.84 (s, CH
phytanyl) ; 37.30 to 37.50 (s, CH2 phytanyl) ; 39.41 (s, CH2
phytanyl) ; 53.41 (s, N-(CH3)3) ; 67.53 (s, CH2-N) ; 67.95 (s,
CH2 α) ; 126.68 (s, C2 and C6) ; 130.52 (s, C3 and C5) ; 134.65
(s, C4) ; 151.50 (s, C1). 31P-NMR (161.97 MHz, CDCl3) : -6.0.
{3,5-bis-[(O,O-dioleylphosphoryl)oxy]phenyl}methylpyridinium
iodide 19
To a solution of dioleyl 3,5-bis(iodomethyl)phenyl phosphate
(0.15 g ; mmol) was added pyridine (0.6 mL ; 7.38 mmol). The
reaction mixture is stirred at RT for 15h. After evaporation of {3,5-bis-[(O,O-diphytanylphosphoryl)oxy]phenyl}methyl
the volatiles the expected compound 18 was isolated (0.21 g ; pyridinium bromide 22
100 % yield). 1H-NMR (400.04 MHz, CDCl3) : 0.86 (t, 3JHH = 6
To
a solution of diphytanyl 3,5-bis(bromomethyl)phenyl
Hz, 6H, CH3) ; 1.25 (s, 44H, CH2) ; 1.68 (m, 4H, CH2 β) ; 1.99
(m, 8H, CH2-CH=CH) ; 4.17 (m, 4H, CH2 α) ; 5.33 (m, 4H,
CH=CH) ; 6.20 (s, 4H, CH2-N) ; 7.53 (s, 2H, C2-H and C6-H) ;
phosphate (0.20 g ; 0.22 mmol) was added pyridine (0.60 mL ;
7.38 mmol). The reaction mixture is stirred at RT for 15h (230
mg ; 100% yield). H-NMR (400.04 MHz, CDCl3) : 0.87 (m,
1
3
3
8.12 (t, JHH = 8 Hz, 5H, C4-H and Hpyr) ; 8.51 (t, JHH = 8 Hz,
30H) ; 1.00-1.70 (m, 48H, CH2 phytanyl) ; 4.18 (m, 4H, CH2 α)
; 6.28 (s, 4H, CH2-N) ; 7.59 (s, 2H, C2-H and C6-H) ; 8.12 (t,
3JHH = 7 Hz, 4H, Cb-H and Cd-H) ; 8.41 (s, 1H, C4-H) ; 8.42 (t,
3JHH = 8 Hz, 2H, Cc-H) ; 9.96 (d, 3JHH = 8 Hz, 4H, Ca-H and Ce-
H). 13C-NMR (125.75 MHz, CDCl3) : 19.30 to 19.80 (s, CH3
phytanyl) ; 22.70 to 22.80 (s, CH3 phytanyl) ; 24.40 to 24.90 (s,
CH2 phytanyl) ; 27.99 (s, CH phytanyl) ; 29.39 (s, CH phytanyl)
; 32.82 (s, CH phytanyl) ; 37.30 to 37.50 (s, CH2 phytanyl) ;
39.38 (s, CH2 phytanyl) ; 62.65 (s, CH2 α) ; 67.85 (s, CH2-N) ;
122.14 (s, C2 and C6) ; 128.21 (s, C4) ; 128.69 (s, Cb and Cd) ;
135.65 (s, C5 and C3) ; 145.33 (s, Ca and Ce) ; 145.76 (s, Cc) ;
151.70 (s, C1). 31P-NMR (161.97 MHz, CDCl3): -6.1.
3
2H, Hpyr) ; 9.62 (d, JHH = 8 Hz, 4H, Hpyr). 13C-NMR (125.75
MHz, CDCl3) : 13.89 (s, 2 x CH3) ; 22.40 (s, CH2) ; 24.05 (s,
CH2) ; 26.95 (s, CH=CH-CH2) ; 29-30 (s, CH2) ; 31.62 (s, CH2)
; 32.64 (s, CH2) ; 62.34 (s, CH2-N) ; 69.05 (d, 2JCP = 6 Hz, CH2
α) ; 121.94 (s, C2 and C6) ; 128.48 (s, Cpyr) ; 129.71 (s, CH=CH)
; 129.90 (s, CH=CH) ; 135.96 (s, C3 and C5) ; 145.38 (s, Cpyr) ;
151.40 (s, C1). 31P-NMR (161.97 MHz, CDCl3) : -6.1. MS
(MALDI-TOF) : m/z calcd for C49H82INO4P+ (M-C6H7NI-) :
906.502 ; found : 906.435.
{3,5-bis-[(O,O-diphytanylphosphoryl)oxy]phenyl}methylmethyl
imidazolium bromide 20
Liposomal formulations
To
a solution of diphytanyl 3,5-bis(bromomethyl)phenyl
The cationic lipids were formulated as liposomal solution by
using the hydratation of a lipid film. First, an aliquot of a
concentrated solution of a desired lipid in chloroform (e.g. 2, 22
mg, 1.0 mL) was placed in a glass tube and the solvent was
evaporated to produce a thin lipid film. Then 1 mL of water
was added on this film. After a hydratation period (12 h except
for compound 20: 2 days) at 4°C the solution was vortexed (1
min) and sonicated (6 x 10 min) at 45 kHz using a VWR
ultrasonic bath. The size and the zeta potential were determined
for each liposomal solution.
phosphate (0.20 g ; 0.22 mmol) was added methylimidazole (35
µL ; 0.43 mmol). The reaction mixture is stirred at 40°C for
15h (230 mg ; 100% yield). H-NMR (400.04 MHz, CDCl3) :
1
0.87 (m, 30H) ; 1.00-1.70 (m, 48H, CH2) ; 4.04 (s, 6H, CH3-N)
; 4.18 (m, 4H, CH2 α) ; 5.59 (s, 4H, CH2-N) ; 7.31 (s, 2H,
NCH=CHN) ; 7.37 (s, 2H, C2-H and C6-H) ; 7.76 (s, 2H,
NCH=CHN) ; 7.79 (s, 1H, C4-H) ; 10.03 (s, 2H, CH=N). 13C-
NMR (125.80 MHz, CDCl3) : 19.20 to 19.70 (s, CH3 phytanyl)
; 22.50 to 22.70 (s, CH3 phytanyl) ; 24.20 to 24.80 (s, CH2
phytanyl) ; 27.89 (s, CH phytanyl) ; 29.32 (s, CH phytanyl) ;
32.71 (s, CH phytanyl) ; 36.90 (s, CH3-N) ; 37.20 to 37.40 (s,
CH2 phytanyl) ; 39.29 (s, CH2 phytanyl) ; 51.93 (s, CH2-N) ;
DNA condensation and relaxation. The condensation of a
pDNA by the varous cationic lipids studied was investigated
using ethidium bromide intercalation into DNA. Upon
condensation, ethidium bromide is expelled from DNA and
thus the fluorescence signal decreases. Conversely, DNA
relaxation from the complexes results in recovery of
fluorescence. Assays were performed as previously reported.21
2
67.57 (d, JCP = 6 Hz, CH2 α) ; 120.53 (s, C2 and C6) ; 122.72
(NCH=CHN) ; 123.57 (NCH=CHN) ; 126.25 (s, C4) ; 136.48 (s,
C3 and C5) ; 139.99 (s, NCHN) ; 151.39 (s, C1). 31P-NMR
(161.97 MHz, CDCl3) : -6.1.
This journal is © The Royal Society of Chemistry 2014
J. Name., 2014, 00, 1-3 | 11

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determined using the BC assay kit (Uptima). Data are expressed
Cells and culture conditions. Four cell lines were used. A549, as relative light units (RLU) per milligram of total proteins
View Article Online
DOI: 10.1039/C4OB01770F
bronchial alveolar type II epithelial cells derived from a human (mean ± SD with n = 3).
pulmonary carcinoma (ATCC No. CCL-185), and SKMel28,
human melanoma cells (ATCC No. HTB-72), were obtained
Evaluation of early toxicity
from the American Type Culture Collection (USA). 16HBE The toxicity of the different lipid/DNA complexes was
14o-, human bronchial epithelial cells, and CFBE 41o-, cystic determined by using a chemiluminescent assay (Toxilight -
fibrosis bronchial epithelial cells, were kindly provided by D. Cambrex, Liège, Belgium). Briefly, this test consists of the
Gruenert, University of Vermont, Burlington, VT, USA.24. measurements of the release of a normally cytoplasmic enzyme
They were grown in Dulbecco’s Modified Eagle Medium (adenylate kinase, AK) from damaged cells into the
(DMEM, for A549 and SKMel28), or Eagle’s Minimum surrounding medium. The reaction involves two steps. The first
Essential Medium (EMEM, for 16HBE and CFBE), one requires the addition of ADP as a substrate for AK. In the
supplemented with 100 units/mL penicillin, 100
µg/mL presence of AK, the ADP is converted to ATP for assay by
streptomycin, 10% heat-inactivated FBS, and incubated at 37 bioluminescence. Then, the bioluminescence method utilizes an
°C, in a humidified atmosphere containing 5% CO2. DMEM, enzyme Luciferase, which catalyzes the emission of photons
EMEM, FBS, penicillin, and streptomycin were purchased from from ATP and luciferin. By combining both reactions, the
Invitrogen (UK).
emitted light intensity is directly related to the AK
concentration. The relative light units (RLU) are conversely
proportional to the damages induced to the cell’s membranes,
In vitro reporter gene measurements: luciferase assays.
The protocol of the experiments was similar to those previously as a consequence of their exposure to the complexes studied.
reported6a-b. Shortly, cells were seeded 24 h before transfection Untransfected cells were used to evaluate the AK background
onto a 24-well plate at a density of 100,000 cells per well and activity.
incubated overnight in a humidified 5% CO2 atmosphere at 37
◦C. About 200 µL of a solution of lipoplex was added to each
well. Lipofectamine (LFM) was used as a positive control.
After 2h30 of incubation at 37 ◦C, 20 µL of the surrounding
was picked up to evaluate the toxicity of each formulation (see
below). The culture medium was removed to be replaced with
fresh. Following a further 48 h incubation at 37 ◦C, the cells
were lysed in order to be assayed for luciferase expression
using a chemiluminescent assay (Luciferase Assay System,
Promega). The total protein content of the cell lysate was
Acknowledgements
This work was supported by grants from “Association
Française contre les Myopathies” (Evry, France), “Vaincre La
Mucoviscidose” (Paris, France), “Association de Biogenétique
et de Transfusion Sanguine Gaetan Saleun” (Brest, France),
“Conseil Régional de Bretagne”. We thank the “Services
Communs” of the University of Brest (RMN-RPE and mass
spectrometry) and FEDER (Fond Européen de Développement
Régional)
Notes and references
a
UMR CNRS 6521; SFR ScInBioS, Université de Bretagne Occidentale,
Université Européenne de Bretagne, Faculté des Sciences, 6 avenue
Victor Le Gorgeu, 29238 Brest (France). E-mail : pjaffres@univ-brest.fr
b
IBiSA SynNanoVect platform, SFR ScInBIoS, Université de Bretagne
Occidentale, 29238 Brest Cedex 3, France.
c
Unité INSERM 1078; SFR ScInBioS, Université de Bretagne
Occidentale, Université Européenne de Bretagne, Faculté de Médecine et
des Sciences de la Santé, 22 avenue Camille Desmoulins, 29238 Brest
(France)
d
CHRU de Brest, Service de Génétique moléculaire et de
d’Histocompatibilité, 5 avenue du maréchal Foch, 29609 Brest cedex ;
DUMG, Faculté de Médecine et des Sciences de la Santé, 22 avenue
Camille Desmoulins, 29238 Brest (France)
†
Supplemental data include zeta and size measurements of liposomal
solutions, additional in vitro transfection assays and 1H, 13C, 31P NMR
spectra of mono and dicationic derivatives.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
12 | J. Name., 2014, 00, 1-3
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ARTICLE
View Article Online
DOI: 10.1039/C4OB01770F
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