Synthesis and evaluation of new phosphonolipid compounds for gene delivery

Article abstract of DOI:10.1016/j.ejmech.2007.11.002

The preparation of a series of novel water soluble cationic lipid derivatives possessing phosphonate ester groups linked to the para-position of N-methyl pyridinium moieties and bearing either identical or different alkyl chains is reported. The obtained phospholipids were tested for transfection efficiency into three different mammalian cell lines alone and in conjunction with diphytanoylphosphatidylethanolamine (DiPPE) or dioleylphosphatidylethanolamine (DOPE), using an assay adapted for 96-well microplates based on the detection of a colorimetric change caused by the production of a chromogen induced by expressed secreted human placental alkaline phosphatase. In our conditions, the highest transfection activities of cells HEK293 and hard-to-transfect cell lines B16 and CHO were achieved with a 4-phosphonobutylpyridinium compound used at 1:5, 1:10 or 3:6 DNA/lipid ratio bearing two myristyl chains in the presence of the fusogenic helper lipid DiPPE.

Full text of DOI:10.1016/j.ejmech.2007.11.002

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1758e1766
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and evaluation of new phosphonolipid
compounds for gene delivery
a,b
Angelique Durand Dal-Maso , Jerome Dellacasagrande , Frederic Legendre ,
b
b
´
´ ˆ
Gerard Tiraby , Casimir Blonski , Pascal Hoffmann
´ ´
b
a
a,
*
´
a Laboratoire de Synthe`se et Physico-Chimie de Mole´cules d’Inte´reˆt Biologique, Groupe de Chimie Organique Biologique,
UMR/CNRS 5068, Universite´ Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9, France
b Laboratoire de Recherche CAYLA-InvivoGen, 5 rue Jean Rodier, 31405 Toulouse cedex 4, France
Received 23 March 2007; received in revised form 29 October 2007; accepted 12 November 2007
Available online 19 November 2007
Abstract
The preparation of a series of novel water soluble cationic lipid derivatives possessing phosphonate ester groups linked to the para-position of
N-methyl pyridinium moieties and bearing either identical or different alkyl chains is reported. The obtained phospholipids were tested for trans-
fection efficiency into three different mammalian cell lines alone and in conjunction with diphytanoylphosphatidylethanolamine (DiPPE) or
dioleylphosphatidylethanolamine (DOPE), using an assay adapted for 96-well microplates based on the detection of a colorimetric change
caused by the production of a chromogen induced by expressed secreted human placental alkaline phosphatase. In our conditions, the highest
transfection activities of cells HEK293 and hard-to-transfect cell lines B16 and CHO were achieved with a 4-phosphonobutylpyridinium
compound used at 1:5, 1:10 or 3:6 DNA/lipid ratio bearing two myristyl chains in the presence of the fusogenic helper lipid DiPPE.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Transfection; Cationic lipid; Non-viral gene transfer
1. Introduction
been completed or approved in different countries, using
over 100 genes [2]. One of the major problems of gene therapy
is the efficiency of introduction and stabilization of nucleic
acids in the target cells, which are currently ensured by gene
transfer systems based on viral [3] or synthetic non-viral vec-
tors [4]. Delivery systems based on non-viral vectors, such as
cationic lipids or cationic polymers, are presently less efficient
than viral systems, which have been used in the two-thirds of
the therapy clinical trials performed to date [2]. Nevertheless,
non-viral systems have proved to be generally less toxic or
immunogenic, more easily produced, and to exhibit a greater
stability. Cationic lipids, like other non-viral gene transfer vec-
tors, interact with the negatively charged phosphate backbone
of nucleic acids to form a compact structure able to enter
a cell. A number of cationic lipids have been synthesized,
including monovalent and polyvalent cationic lipids, choles-
terol derivatives or guanidine-containing compounds, some
of them being now commercially available, such as DOTMA
Gene therapy constitutes a mode of treatment of genetic
disorders in which genes are inserted into eukaryotic cells
[1], and to date over 1340 gene therapy clinical trials have
Abbreviations: DMEM, Dulbecco’s modified eagle’s medium; FCS, Foetal
calf serum; DOTAP, 1,2-Dioleoyl-3-trimethylammonium propane; DOTMA,
N-(2,3-Dioleoyloxy)propyl-N,N,N-trimethylammoinum chloride; DC-Chol,
3b[N-(N’,N’-Dimethylaminoethane)carbamoyl]cholesterol; DOGS, 5-Carbox-
yspermylglycine dioctadecylamide; DOSPA, 2,3-Dioleoyloxy-N-{2-[1,4-bis(3-
aminopropymino)-2-butylcarboxamido]ethyl}-N,N-dimethyl-1-propylammonium
bromide; CHO, Chinese hamster ovarian adenocarcinoma; B16, Mouse mela-
noma; C2C12, Mouse muscle myoblasts; HepG2, Human hepatoblastoma;
GFP, Green fluorescent protein; DOPE, 1,2-Dimyristoyl-sn-glycero-3-phosphoe-
thanolamine; DiPPE, 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine; SEAP,
Secreted embryonic alkaline phosphatase.
* Corresponding author. Tel.: þ33 5 61 55 61 44; fax: þ33 5 61 55 60 11.
E-mail address: hoffmann@cict.fr (P. Hoffmann).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.002

A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
1759
(LipofectinÒ) [5], DOSPA (LipofectAMINEÒ) [6], DOTAP
[7], DC-Chol [8] or DOGS (TransfectamÒ) [9]. All these cat-
ionic lipids carry a DNA-interacting head group with a net
positive charge in physiological conditions or at the lower
pHs found in the endosome environment, a hydrophobic lipid
anchor group such as cholesterol or fatty acid chains of various
lengths and unsaturation states, and a linker group that binds
the polar group to the lipidic moiety. Spacer groups are com-
monly ester [7], amide [9] or urethane [10] groups, but other
groups such as phosphate [9] or phosphonate [11] have also
been employed. The positively charged polar groups com-
monly used are also of various natures, including quaternary
ammonium ions, polycations such as spermine, or cationic
polymeric compounds such as polyethylenimine, poly-lysine
or their derivatives. In 1997, a series of double-chained pyridi-
nium compounds were described by van der Woude et al. that
displayed interesting transfection properties, and were found
to be essentially non-toxic toward cells [12]. In this work,
we report the synthesis and the evaluation of new pyridi-
nium-based phosphonolipids, bearing either two identical or
two different alkyl chains, as transfection agents. The lipid
chains C14-saturated (myristyl), C18-unsaturated (oleyl) and
phytanyl moieties were examined (Fig. 1).
by anion exchange using Dowex (Clꢀ) gave the N-methyl
pyridinium chloride compounds 1e5, respectively. All spec-
trophotometric and spectroscopic data were consistent with
the assigned structures.
2.1.2.2. Synthesis of 4-(dialkylphosphono)-1-methyl pyridinium
compounds (1, n ¼ 0, Fig. 1). Several methods for the synthe-
sis of diethyl-4-pyridylphosphonate compounds have been
reported such as the reaction of 4-bromopyridine hydrochlo-
ride with diethylphosphite in the presence of triethylamine
and tetrakis(triphenylphosphine)palladium as catalyst [14].
Here, compounds 1 were obtained in two steps, as depicted
in Scheme 1, with overall yields ranging from 19e33%. 4-
Pyridylphosphonate intermediates (10) were first prepared
with a one-pot procedure following a modified method de-
scribed by Kers and Stawinski [15]. This method involves
the transient in situ formation of stable N-protected dihydro-
pyridine intermediates by means of trityl chloride, which
allow a regiospecific nucleophilic attack on the 4-position of
the pyridine ring by the H-phosphonate anion of the corre-
sponding dialkylphosphite, followed by a spontaneous rearo-
matization of the pyridine ring with loss of the trityl moiety.
In our experimental conditions, DBU was used as base to
form the phosphanion. In a second step, quaternarization by
methyl iodide afforded the N-methyl pyridinium chloride com-
pounds 1.
2. Results
2.1. Chemistry
2.1.2.3. Synthesis of 4-(1-(dialkylphosphono)methyl)-1-methyl
pyridinium compounds (2, n ¼ 1, Fig. 1). Compounds 2
were synthesized in two steps from commercially available
4-(chloromethyl)pyridine hydrochloride (Scheme 2). Pyridine
intermediates 20 were first prepared using a protocol previ-
ously described for the synthesis of the diethylphosphono de-
rivative: 4-(chloromethyl)pyridine hydrochloride was reacted
with 1.5 eq. of the sodium salt of dialkylphosphite in THF
to afford the corresponding (dialkylphosphono)methyl pyri-
dine intermediates 20, with yields ranging from 22 to 38%.
These intermediates 20 were then N-alkylated by methyl iodide
to yield the N-methyl pyridinium chloride compounds 2.
2.1.1. Synthesis of dialkylphosphite compounds
Phosphite derivatives bearing two identical chains (homo-
dialkylphosphites) were obtained by direct esterification of
phosphorus trichloride by the corresponding alcohol, as previ-
ously described [11]. On the other hand, the unsymmetrical
introduction of two different alkyl chains leading to hetero-
dialkylphosphites has not been reported. Consequently, a pro-
cedure for their preparation was developed, based on two
successive transesterification reactions of commercially avail-
able diphenylphosphite (Fig. 2) [13].
2.1.2. Synthesis of pyridine (10e50) and N-methyl-pyridinium
compounds (1e5)
2.1.2.4. Synthesis of 4-(2-(dialkylphosphono)ethyl)-1-methyl
pyridinium compounds (3, n ¼ 2, Fig. 1). Attempts to synthe-
size the esters of 2-(pyridyl)ethylphosphonic acids 30 either
from 4-picoline in the presence of halogenomethyl dialkyl-
phosphonate or from the reaction of 4-(halogenomethyl)pyri-
dine with a methyl dialkylphosphonate derivative failed,
leading to a complex mixture in the former case, and affording
the autocondensation product of the methylphosphonate deriv-
ative as major product in the latter case. Nevertheless, one
member of the series was obtained with a satisfactory yield
(compound 30a, R1 ¼ R2 ¼ C14H29) using a method described
by Maruszewska-Wieczorkowska and Michalski [16] based on
nucleophilic addition of the corresponding dialkylphosphite on
4-vinylpyridine, known to behave as a typical a,b-unsaturated
compound, in the presence of sodium ethanoate (Scheme 3).
However, this compound was found to readily undergo retro-
addition, and thus to be rather instable in our experimental
2.1.2.1. General strategy. The appropriate pyridine deriva-
tives 10, 20 and 40 (Schemes 1, 2 and 4, respectively) were
obtained using a MichaeliseBecker type coupling reaction
between the H-phosphonate anion of a homo- or hetero-
dialkylphosphite, and pyridine or a halogenated pyridine.
Due to the instability of 4-(chloropropyl)pyridine, compounds
40 were obtained in poor yields, and only compound 4a was
assessed as a vector for transfection. For 30, a similar strategy
was used with 4-vinylpyridine as electrophile instead of a halo-
genated pyridine (Scheme 3). However, as for compounds 40,
they were obtained in poor yields and only compound 3a was
assessed. The butylated pyridine compounds 50 were synthe-
sized by addition of the carbanion of 4-methyl pyridine to
dialkyl-3-bromopropylphosphonates (Scheme 5). Treatment
of all pyridylphosphonates 10e50 by methyl iodide, followed

1760
A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
O
OR1
OR2
O
P
OR1
OR2
P
n
n
N
N
Cl
CH3
n
0
1
2
3
4
n
0
1
2
3
4
Cpds
1'
2' 3'
4' 5'
Cpds
1
2
3
4
5
(C14H29, myristyl)
R1,R2
=
(C18H35, oleyl)
(C20H41, phytanyl)
Compounds
n
0
R
R
2
1
1’a, 1a
1’b, 1b
1’c, 1c
1’d, 1d
1’e, 1e
1’f, 1f
C H
C H
14 29
14 29
C H
C H
18 35
18 35
C H
C H
20 41
20 41
C H
C H
14 29
18 35
C H
C H
14 29
20 41
C H
C H
18 29
20 41
2’a, 2a
2’b, 2b
2’c, 2c
2’d, 2d
2’e, 2e
2’f, 2f
C H
C H
14 29
14 29
C H
C H
18 35
18 35
C H
C H
20 41
20 41
1
C H
C H
14 29
18 35
C H
C H
14 29
20 41
C H
C H
18 35
20 41
3’a, 3a
4’a, 4a
2
3
C H
C H
14 29
14 29
C H
14 29
C H
14 29
5’a, 5a
5’b, 5b
5’c, 5c
5’d, 5d
5’e, 5e
5’f, 5f
C H
C H
14 29
14 29
C H
C H
18 35
18 35
4
C H
C H
20 41
20 41
C H
C H
14 29
18 35
C H
C H
14 29
20 41
C H
C H
18 35
20 41
Fig. 1. Structures of pyridine and pyridinium chloride phosphonolipid compounds.
conditions. Alkylation of 30a with methyl iodide gave the
corresponding methyl pyridinium.
synthesis for phosphonate compounds from dialkylphosphites
and alkyl halides has been reported using cesium carbonate
[17]. Using these reported conditions, we successfully synthe-
sized, but in poor yields, the ester of 2-(pyridyl)propylphos-
phonic acid 40a starting from 4-(chloropropyl)pyridine,
2.1.2.5. Synthesis of 4-(3-(dialkylphosphono)propyl)-1-methyl
pyridiniumcompounds(4,n ¼ 3,Fig.1). Recently, a convenient

A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
1761
OR1
H
depicts SEAP activity of cells CHO treated with selected phos-
phonolipids at different DNA/lipid ratios in the presence or the
absence of DiPPE or DOPE. All other compounds (1aed, 2b,
2def, 3a, 4a and 5cef) were found to be inactive or poorly active
as vectors of transfection. As shown in Fig. 3, except for com-
pounds 1e and 2c, the absence of lipid helpers dramatically
reduced the transfection activity. Overall, phosphonolipids that
comprise two identical lipid chains were found to induce trans-
fection of CHO cells more efficiently than those bearing two dif-
ferentchains(notshown). Unliketheothermembersoftheseries,
compound 2f, bearing two different alkyl chains (phytanyl and
oleyl), was found to display suitable transfection activities in
the presence of DOPE, comparable to that of FuGENEÒ 6, but
only at a DNA/lipid ratio of 1/10. The most effective compound
on CHO cells was 4-(4-(ditetradecanylphosphono)butyl)-
1-methyl pyridinium chloride (5a) in combination with lipid
helper DiPPE, and to a lesser extent with DOPE, the best results
being obtained for a DNA/lipid ratio of 3/6 (Fig. 3), and no
toxicity for this compound was evidenced in our experimental
conditions. The mixture 5a/DIPPE has also been tested and
proved to be effective on human kidney HEK 293 cells and other
hard-to-transfect cell lines, such as mouse melanoma B16 cells
and mouse myoblast C2C12 cells.3 Surprinsingly, compound
5b with two oleyl chains exhibited satisfactory activities in the
presence of lipid helpers at DNA/lipid ratios of 1/5 and 3/6, but
totally lost its transfection capacity at a ratio of 1/10.
OPh
H
OR2
OPh
H
OPh
i
ii
O
O
P
P
O
P
OR2
Fig. 2. Synthesis of dialkylphosphite compounds: (i) pyridine (0.8 eq.), R1OH
(0.8 eq.), 2 h; (ii) pyridine (0.8 eq.), R2OH (0.8 eq.), 3 h.
prepared from 4-pyridinepropanol, and the corresponding di-
myristyl phosphite (Scheme 4). Alkylation using methyl io-
dide of intermediate 40a gave the desired pyridinium
compound 4a.
2.1.2.6. Synthesis of 4-(4-(dialkylphosphono)butyl)-1-methyl
pyridinium compounds (5, n ¼ 4, Fig. 1). Esters of 2-(pyridyl)-
butylphosphonic acids (50) were synthesized as outlined in
Scheme 5 according to an established procedure based on the
nucleophilic attack of the carbanion of 4-methyl pyridine,
generated with lithium diisopropylamide, to the appropriately
dialkyl-3-bromopropylphosphonate derivatives, readily ob-
tained in three steps from commercially available 1,3-dibromo-
propane. The pyridyl intermediates were then alkylated by
methyl iodide to yield the corresponding methyl pyridinium
compounds 5.
2.2. Transfection properties of phosphonolipids
To evaluate transfection activities of the phosphonolipids,
a rapid colorimetric assay adapted for a 96-well microplate was
developed that greatly decreases the analysis time over tradi-
tional transfection assays. This spectrophotometric assay, based
on the quantification of SEAP activity in supernatants of cell
cultures by means of a colorimetric enzyme assay (QUANTI-
BlueÔ, InvivoGen), was validated by comparison to a classical
method based on SEAP-catalyzed p-nitrophenyl phosphate
hydrolysis.1 At the same time, the efficacy of transfection was
also assessed visually by looking at GFP-expressing cells with
a fluorescence microscope (data not shown), and the toxicity of
each lipid/DNA complex was roughly estimated by examination
of monolayer confluency. Transfection was examined in hard-
to-transfect Chinese hamster ovary (CHO) cells, and transfected
DNA consisted of a plasmid (pVITRO14, InvivoGen) encoding
green fluorescent protein (GFP) and secreted human placental
alkaline phosphatase (SEAP). Phosphonolipids were assayed at
various DNA/lipid ratios (1:5, 1:10 and 3:6, w:w) either alone
or in the presence of neutral lipid helpers dioleylphosphatidyle-
thanolamine (DOPE) or diphytanoylphosphatidylethanolamine
(DiPPE). As control, FuGENEÒ 6 (Roche) was used.2 Fig. 3
3. Discussion
Cationic lipids have been widely used as a safe non-viral
method of delivery of nucleic acids both in vitro and in
vivo, but the mechanism by which DNA is transfected is com-
plex and not well understood. For this purpose, a number of
cationic lipids have been synthesized, some of them have al-
ready been used in the clinical setting, having advantage of
safety and non-immunogenesity over viral vectors, but the
problems of low efficiency and cytoxicity have not been
solved yet. The aim of this study was to extend previous
work on cationic lipid compounds as non-viral vectors for
gene delivery by investigating a series of new pyridinium-
based phosphonolipids, including for the first time compounds
bearing two different alkyl chains that would enhance the
understanding of the structure-function relationship in design-
ing such compounds. Multiple factors influence the delivery
process including the physical structure and stability of the
lipid/DNA complex and its interaction with the cell mem-
brane. Moreover, transfection activities of the complexes are
cell type dependent since the cellular membrane varies with
1
For the detection of SEAP, QUANTI-BlueÔ (InvivoGen) solution has
been compared to a classical method based on pNPP hydrolysis: purified hu-
man alkaline phosphatase (Calbiochem) has been serially diluted in DNEM
and measured spectrophotometrically with SEAP Reporter Assay Kit (Invivo-
Gen) at 405 nm or QUANTI-BlueÔ (InvivoGen) at 655 nm (see Supplemen-
tary data associated with this article).
3
On HEK293 cells, compound 5a was found to display transfection activi-
ties in the absence or presence of lipid helpers DiPPE or DOPPE comparable
to that of our reference FuGENEÒ 6 at both 1/6 and 1/12 DNA/lipid ratios.
Comparable transfection activities were also observed on B16 cells and
C2C12 cells in the presence of lipid helpers DiPPE or DOPPE, but only for
a 1/12 DNA/lipid ratio, and strongly decreased for a 1/6 DNA/lipid ratio. In
the absence of lipid helper, a significantly (three-fold) higher activity than
that of FuGENEÒ 6 for B16 cells transfection was observed.
2
FuGENEÒ 6 transfection reagent is a proprietary secret mixture of lipids
and other components supplied in 80% ethanol by Roche that has been shown
to form a complex with DNA and to transport it into animal cells, and to trans-
fect many common cell types, including HeLa, NIH 3T3, COS-1, COS-7, and
CHO-K1.

1762
A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
O
OR1
P
OR2
O
ii
i
OR1
OR2
1
+
H
P
N
N
1'
Scheme 1. Synthesis of 4-(dialkylphosphono)-1-methyl pyridinium chloride: (i) dialkylphosphite (1 eq.), trityl chloride (1.2 eq.), DBU (2.4 eq.), pyridine, 6 h (RT);
(ii) methyl iodide (4 eq.), acetone, 1 h (RT); DowexÒ ion exchange (Clꢀ) resin.
cell type and cycle. It is therefore important to understand the
factors governing the relationship between the lipid structure
and the activity of transfection. The alkyl chains (length,
degree of saturation) and the linker are, for instance, important
elements influencing the transfection reagent structure, as well
as the nature of the head group that can also dramatically alter
the charge interactions with DNA and consequently the trans-
fection capability of the final DNA/lipid complex. Indepen-
dently of the chemical structure, other parameters like the
DNA/lipid ratio influence the transfection activity of the lipid
compounds, as observed with compound 5b. Likewise, the
presence of lipid helper plays also a crucial role for transfec-
tion. In this work, we prepared a series of new pyridinium-
based phosphonolipids, bearing two identical or different alkyl
chains, and some of them were found to exhibit good transfec-
tion activities. However, the results of the current study show
that no significant correlation can be observed between struc-
ture and transfection properties, and thus support the notion
that transfection activity strongly depends on complex multi-
factorial components. Consequently further work will be
needed to understand the structureefunction relationships of
these compounds. One of the compounds (5a) was found to
display a higher transfection efficiency of mammalian cells
in the presence of lipid helpers than the commercially avail-
able gene delivery lipid FuGENEÒ 6, and thus have a great
potency as vector for gene delivery. Moreover, considering
the importance of cationic lipids for gene delivery, compounds
bearing two lipid chains of different nature have received
surprisingly little attention. Here, we showed that such com-
pounds also display significant transfection activities, and
would seem to merit further investigation.
gel plates 60F-254 nm (0.2-mm thickness, Merck) were used
for thin layer chromatography. Nuclear magnetic resonance
spectra were recorded on a Brucker AC 300. Mass spectrom-
etry was performed on a quadripolar Nermag R10-10C
spectrometer.
4.2. General procedure for the preparation and
characterization of selected compounds (1e, 1f, 2a, 2c,
2f, 5a, 5b)
4.2.1. Synthesis of compounds 1
To a solution of phosphite (1 mmol) in pyridine (10 mL)
were added trityl chloride (1.2 mmol) and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) (2.4 mmol), successively. After
6 h under magnetic stirring at room temperature, the solvent
was evaporated under reduced pressure. The residue was dis-
solved in dichloromethane, and washed (3ꢁ) with a 5% aque-
ous solution of sodium hydrogenocarbonate. The organic layer
was dried on magnesium sulfate, and evaporated to dryness
under reduced pressure. The residue was then column-purified
on silica gel using a mixture of heptane and diethyl ether (1:1)
then ethyl ether as eluent to obtain pyridine compounds 10.
4.2.1.1. Compound 10e. Yield, 50% (yellow oil). 1H NMR
(300 MHz, CDCl3) d (ppm) 0.80e0.87 (m, 24 H), 0.90 (d,
3
6H, JHeH ¼ 6.0 Hz), 0.98e1.78 (m, 48H), 3.96e4.10 (m,
3
3
4H), 7.58 (dd, 2H, JHeH ¼ 6.0 Hz, JHeP ¼ 15.0 Hz), 8.70
3
(t, 2H, JHeH ¼ 6.0 Hz). 13C NMR (75 MHz, CDCl3)
d (ppm) 14.0, 19.5, 19.7, 22.6, 22.7, 22.8, 24.4, 24.5, 24.8,
3
26.1, 28.0, 29.1e29.6, 30.6 (d, JCeP ¼ 6.4 Hz), 31.9, 32.8,
2
2
37.0e37.8, 39.4, 65.2 (d, JCeP ¼ 6.3 Hz), 66.8 (d, JCeP
¼
¼
2
1
6.2 Hz), 125.5 (d, JCeP ¼ 7.5 Hz) 137.5 (d, JCeP
3
4. Experimental protocols
186.4 Hz), 150.1 (d, JCeP ¼ 12.8 Hz). 31P NMR (81 MHz,
CDCl3) d (ppm) 14.8. ES-MS (m/z): 658 (MNaþ).
4.1. Chemistry
4.2.1.2. Compound 10f. Yield, 40% (yellow oil). 1H NMR
(300 MHz, CDCl3) d (ppm) 0.80e0.87 (m, 15H), 0.90 (d,
All chemicals were obtained from SigmaeAldrich and used
without further purification. Silica gel 60 (200e400 mesh,
Merck) was used for column flash chromatography, and silica
3
3H, JHeH ¼ 6.1 Hz), 1.00e1.77 (m, 48H), 1.89e2.08 (m,
2
4H), 3.11 (d, 2H, JHeP ¼ 22.3 Hz), 3.89e4.09 (m, 4H),
O
Cl
OR1
P
OR2
O
i
ii
OR1
+
2
H
P
OR2
N
N
2'
Scheme 2. Synthesis of 4-(1-(dialkylphosphono)methyl)-1-methyl pyridinium chloride: (i) a e NaH (2.5 eq.), dialkylphosphite (1.5 eq.), THF, 30 min (RT); b e 4-
(chloromethyl)pyridine hydochloride (1 eq.), 18 h (reflux); (ii) methyl iodide (4 eq.), acetone, 1 h (RT); DowexÒ ion exchange (Clꢀ) resin.

A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
1763
O
OR1
P
OR2
O
OR1
i
ii
+
H
P
3
OR2
N
N
3'
Scheme 3. Synthesis of 4-(2-(dimyristylphosphono)ethyl)-1-methyl pyridinium chloride (3a, R1 ¼ R2 ¼ C14H29): (i) Na (0.15 eq.), ethanol, 18 h (50 ꢂC); (ii)
methyl iodide (4 eq.), acetone, 1 h (RT); DowexÒ ion exchange (Clꢀ) resin.
3
3
5.29e5.43 (m, 4H), 7.58 (dd, 2H, JHeH ¼ 6.8 Hz, JHeP
¼
24.4, 24.5, 24.8, 25.6, 27.2, 28.0, 29.1e29.6, 29.4, 30.7 (d,
1
15.7 Hz), 8.72 (t, 2H, JHeH ¼ 6.8 Hz). 13C NMR (75 MHz,
3JCeP ¼ 5.9 Hz), 31.9, 32.8, 33.2 (d, JCeP ¼ 146.0 Hz),
3
2
CDCl3) d (ppm) 14.0, 19.5, 19.7, 22.6, 22.7, 22.8, 24.4,
¼
37.0e37.8, 39.5, 49.6, 65.2 (d, JCeP ¼ 6.3 Hz), 66.8 (d,
24.5, 24.8, 25.6, 27.2, 28.0, 29.1e29.6, 29.4, 30.7 (d, 3JCeP
2JCeP ¼ 6.8 Hz), 129.9 (d, JCeP ¼ 5.8 Hz), 146.2 (d, JCeP
¼
3
1
1
3
5.9 Hz), 31.9, 32.8, 33.2 (d, JCeP ¼ 146.0 Hz), 37.0e37.8,
186.3 Hz), 146.1 (d, JCeP ¼ 10.8 Hz). 31P NMR (81 MHz,
CDCl3) d (ppm) 8.3. ES-MS (m/z): 704 (MþeClꢀ).
39.5, 65.2 (d, 2JCeP ¼ 6.1 Hz), 66.8 (d, 2JCeP
¼
2
6.7 Hz), 125.3 (d, JCeP ¼ 7.5 Hz), 129.9, 129.8, 137.6 (d,
1JCeP ¼ 186.4 Hz), 159.9 (d, JCeP ¼ 12.8 Hz). 31P NMR
3
4.2.2. Synthesis of compounds 2
(81 MHz, CDCl3) d (ppm) 14.6. ES-MS (m/z): 712 (MNaþ).
Methyl iodide (1.2 mmol) was added to a solution of 10
(0.4 mmol) in dry acetone (5 mL) under magnetic stirring at
room temperature. After 1 h, the solvent was removed under
reduced pressure and the residue was purified by chromatogra-
phy on alumine (act IV) (dichloromethane/methanol, 98:2),
and on ion exchange resin (Dowex 1X8-200 Clꢀ) with meth-
anol as eluent. Recrystallization from a mixture of acetonitrile
and acetone gave pure pyridinium chloride compounds 1.
The adequate phosphite (0.8 mmol) compound was slowly
added to a suspension of sodium hydride (1.33 mmol) in dry
THF (8 mL), and the solution was stirred at room temperature
for 1 h. 4-(Chloromethyl)pyridine (0.53 mmol) was then
added to this solution, and the mixture was heated at reflux
for 18 h. After cooling, water was added, and the solution
was extracted with ethyl acetate (3ꢁ). The organic layer was
then dried over magnesium sulfate, and evaporated to dryness.
The crude product was purified by chromatography on silica
gel (ethyl ether, then ethyl acetate as eluent) to afford pyridine
compounds 20.
4.2.1.3. Compound 1e. Yield, 60% (pale yellow solid). 1H
NMR (300 MHz, CDCl3) d (ppm) 0.80e0.87 (m, 24H), 0.90
3
1
4.2.2.1. Compound 20a. Yield, 38% (pale yellow solid). H
(d, 6H, JHeH ¼ 6.1 Hz), 0.98e1.78 (m, 48H), 4.09e4.30
3
(m, 4H), 4.90 (s, 3H), 8.21e8.32 (m, 2H), 9.70e9.87 (m,
2H). 13C NMR (75 MHz, CDCl3) d (ppm) 14.0, 19.5, 19.7,
22.6, 22.7, 22.8, 24.4, 24.5, 24.8, 26.1, 28.0, 29.1e29.6,
NMR (300 MHz, CDCl3) d (ppm) 0.87 (t, 6H, JHeH
6.6 Hz), 1.20e1.45 (m, 44H), 1.60e1.70 (m, 4H), 3.07 (d,
¼
2H, JHeP ¼ 21.0 Hz), 3.93 (q(td), 4H, JHeH ¼ 3JHeP
¼
2
3
3
6.0 Hz), 7.18e7.20 (m, 2H), 8.46e8.52 (m, 2H). 13C NMR
(75 MHz, CDCl3) d (ppm) 14.0, 22.6, 25.5, 29.1e29.6, 30.6
30.6 (d, JCeP ¼ 6.4 Hz), 31.9, 32.8, 37.0e37.8, 39.4, 49.7,
2
2
65.2 (d, JCeP ¼ 6.3 Hz), 66.8 (d, JCeP ¼ 6.7 Hz), 129.9 (d,
2JCeP ¼ 5.7 Hz), 146.2 (d, 1JCeP ¼ 186.0 Hz), 146.1 (d, 3JCeP
¼
(d,
3JCeP ¼ 6.4 Hz),
31.8,
33.4
(d,
1JCeP
¼
10.3 Hz). 31P NMR (81 MHz, CDCl3) d (ppm) 8.1. ES-MS
(m/z): 650 (MþeClꢀ).
2
3
146.3 Hz), 66.4 (d, JCeP ¼ 6.8 Hz), 125.0 (d, JCeP
¼
2
4
6.0 Hz), 141.4 (d, JCeP ¼ 8.3 Hz), 149.7 (d, JCeP ¼ 3.0
Hz). 31P NMR (81 MHz, CDCl3) d (ppm) 24.2. ES-MS (m/z):
566 (MHþ).
4.2.1.4. Compound 1f. Yield, 55% (pale yellow solid). 1H
NMR (300 MHz, CDCl3) d (ppm) 0.80e0.87 (m, 15H), 0.90
3
4.2.2.2. Compound 20c. Yield, 27% (yellow oil). 1H NMR
(300 MHz, CDCl3) d (ppm) 0.81e0.89 (m, 24H), 0.90 (d,
(d, 3H, JHeH ¼ 6.1 Hz), 1.00e1.77 (m, 48H), 1.89e2.08
(m, 4H), 4.07e4.29 (m, 4H), 4.91 (s, 3H), 5.29e5.43 (m,
4H), 8.26e8.35 (m, 2H), 9.71e9.86 (m, 2H). 13C NMR
(75 MHz, CDCl3) d (ppm) 14.0, 19.5, 19.7, 22.6, 22.7, 22.8,
6H, 3JHeH ¼ 6.1 Hz), 0.98e1.78 (m, 48H), 3.06 (d, 2H, 2JHeP
¼
22.0 Hz), 3.82e4.03 (m, 4H), 7.15e7.28 (m, 2H), 8.42e8.61
O
Cl
OR1
P
OR2
O
OR1
i
ii
+
H
P
4
OR2
N
4'
N
Scheme 4. Synthesis of 4-(3-(dimyristylphosphono)propyl)-1-methyl pyridinium chloride (4a, R1 ¼ R2 ¼ C14H29): (i) a e Cs2CO3 (3 eq.), dialkylphosphite (1 eq.),
TBAI (3 eq.), DMF, 1 h (RT); b e 4-(chloropropyl)pyridine (3 eq.), 48 h (RT); (ii) methyl iodide (4 eq.), acetone, 1 h (RT); DowexÒ ion exchange (Clꢀ) resin.

1764
A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
O
OR1
P
OR2
CH3
O
OR1
i
ii
Br
+
P
5
OR2
N
N
5'
Scheme 5. Synthesis of 4-(4-(dialkylphosphono)butyl)-1-methyl pyridinium compounds: (i) a e BuLi (1 eq.), diisopropylamine (1 eq.), THF, 30 min
(ꢀ15 ꢂC); b e picoline (1 eq.), 15 min (ꢀ15 ꢂC). c e ReBr (1 eq.), 18 h (RT); (ii) methyl iodide (4 eq.), acetone, 1 h (RT); DowexÒ ion exchange (Clꢀ)
resin.
3
2
(m, 2H). 13C NMR (75 MHz, CDCl3) d (ppm) 19.5, 19.7, 22.6,
¼ 6.2 Hz), 125.4 (d, JCeP ¼ 5.7 Hz), 145.3, 152.4 (d, JCe
(m/z): 748 (MþeClꢀ).
P
P
1
22.7, 24.4, 24.5, 24.8, 28.0, 29.4, 32.8, 33.4 (d, JCeP
¼
¼ 8.1 Hz). 31P NMR (81 MHz, CDCl3) d (ppm) 21.2. ES-MS
2
137.3 Hz), 37.0e37.8, 39.4, 64.9 (d, JCeP ¼ 6.0 Hz), 125.3 (d,
2
4
3JCeP ¼ 6.7 Hz), 141.2 (d, JCeP ¼ 9.0 Hz), 149.7 (d, JCeP
¼
2.9 Hz). 31P NMR (81 MHz, CDCl3) d (ppm) 24.2. ES-MS
(m/z): 756 (MNaþ).
4.2.2.6. Compound 2f. Yield, 55% (yellow solid). H NMR
(300 MHz, CDCl3) d (ppm) 0.80e0.87 (m, 15H), 0.90 (d,
1
3
3H, JHeH ¼ 6.1 Hz), 1.00e1.77 (m, 48H), 1.89e2.08 (m,
4.2.2.3. Compound 20f. Yield, 28% (yellow oil). 1H NMR
(300 MHz, CDCl3) d (ppm) 0.80e0.87 (m, 15H), 0.90 (d,
4H), 3.11 (d, 2H, JHeP ¼ 22.3 Hz), 3.49 (d, 2H, JHeP ¼
2
2
23.1 Hz), 3.96e4.18 (m, 4H), 4.68 (s, 3H), 5.29e5.43 (m,
4H), 7.88e8.07 (m, 2H), 9.19e9.30 (m, 2H). 13C NMR
(75 MHz, CDCl3) d (ppm) 14.0, 19.5, 19.7, 22.6, 22.7, 22.8,
24.4, 24.5, 24.8, 25.6, 27.2, 28.0, 29.1e29.6, 29.4, 30.7 (d,
3
3H, JHeH ¼ 6.1 Hz), 1.00e1.77 (m, 48H), 1.89e2.08 (m,
2
4H), 3.11 (d, 2H, JHeP ¼ 22.3 Hz), 3.94e4.05 (m, 4H),
5.29e5.43 (m, 4H), 7.15e7.30 (m, 2H), 8.46e8.63 (m, 2H).
13C NMR (75 MHz, CDCl3) d (ppm) 14.0, 19.5, 19.7, 22.6,
22.7, 22.8, 24.4, 24.5, 24.8, 25.6, 27.2, 28.0, 29.1e29.6,
3JCeP ¼ 5.9 Hz), 31.9, 32.8, 34.4 (d, JCeP ¼ 133.1 Hz),
1
37.0e37.8, 39.5, 48.7, 65.8 (d, 2JCeP ¼ 6.2 Hz), 67.3 (d, 2JCeP
¼
3
1
3
29.4, 30.7 (d, JCeP ¼ 5.9 Hz), 31.9, 32.8, 33.2 (d, JCeP
¼
6.7 Hz), 129.2 (d, JCeP ¼ 7.5 Hz), 129.9, 129.8, 145.3, 152.4
(d, 2JCeP ¼ 8.1 Hz). 31P NMR (81 MHz, CDCl3) d (ppm) 21.2.
ES-MS (m/z): 718 (MþeClꢀ).
2
146.0 Hz), 37.0e37.8, 39.5, 64.9 (d, JCeP ¼ 6.3 Hz), 66.6
2
3
(d, JCeP ¼ 6.8 Hz), 125.1 (d, JCeP ¼ 8.9 Hz), 129.9, 129.8,
141.5 (d, JCeP ¼ 8.3 Hz), 149.8 (d, JCeP ¼ 2.7 Hz). 31P
NMR (81 MHz, CDCl3) d (ppm) 24.2. ES-MS (m/z): 727
(MNaþ).
2
4
4.2.3. Synthesis of compounds 5
A 1.6 M butyllithium solution in hexane (0.43 mL) was
added at ꢀ15 ꢂC to a 0.3 M solution of diisopropylamine in
dry THF (2.4 mL) and magnetically stirred for 30 min. To
the LDA solution were added dropwise at ꢀ15 ꢂC freshly dis-
tilled 4-picoline (70 mL), and after 15 min, a solution of the
corresponding dialkyl-3-bromopropylphosphonate (0.7 mmol)
in dry THF (5 mL). The solution was stirred overnight at
room temperature, and the solvent was removed under reduced
pressure. The residue was dissolved in ethyl acetate, and
washed with a saturated aqueous ammonium chloride solution.
The organic layer was then dried over magnesium sulfate and
evaporated to dryness. Compounds 50 were then purified by
chromatography on silica gel (eluent: petroleum ether/ethyl
acetate 3:2).
The pyridinium compounds 2 were obtained from com-
pounds 20 by the same procedure used for the preparation of
compounds 1.
1
4.2.2.4. Compound 2a. Yield, 60% (white solid). H NMR
3
(300 MHz, CDCl3) d (ppm) 0.87 (t, 6H, JHeH ¼ 6.6 Hz),
1.20e1.45 (m, 44H), 1.60e1.70 (m, 4H), 3.07 (d, 2H, 2JHeP
21.0 Hz), 3.62 (d, 2H, JHeP ¼ 24.0 Hz), 3.94e4.10 (m, 4H),
4.58 (s, 3H), 7.93e8.00 (m, 2H), 9.10e9.18 (m, 2H). 13C
NMR (75 MHz, CDCl3) d (ppm) 14.0, 22.6, 25.5, 29.1e29.6,
¼
2
3
1
30.6 (d, JCeP ¼ 6.4 Hz), 31.8, 33.8 (d, JCeP ¼ 133.9
2
3
Hz), 48.6, 67.4 (d, JCeP ¼ 6.8 Hz), 129.1 (d, JCeP ¼ 5.8
Hz), 145.2, 152.6 (d, JCeP ¼ 8.2 Hz). 31P NMR (81 MHz,
2
CDCl3) d (ppm) 21.4. ES-MS (m/z): 580 (MþeClꢀ).
1
4.2.3.1. Compound 50a. Yield, 58% (pale yellow solid). H
1
3
4.2.2.5. Compound 2c. Yield, 61% (yellow solid). H NMR
(300 MHz, CDCl3) d (ppm) 0.81e0.89 (m, 24H), 0.90 (d,
NMR (300 MHz, CDCl3) d (ppm) 0.87 (t, 6H, JHeH
6.6 Hz), 1.20e1.45 (m, 44H), 1.57e1.79 (m, 14H), 2.60 (t,
¼
6H, 3JHeH ¼ 6.1 Hz), 0.98e1.78 (m, 48H), 3.49 (d, 2H, 2JHeP
¼
2H, 3JHeH ¼ 6.7 Hz), 3.90e4.00 (m, 4H), 7.06 (d, 2H, 3JHeH
¼
3
23.1 Hz), 3.96e4.18 (m, 4H), 4.68 (s, 3H), 7.88e8.09 (m, 2H),
9.19e9.30 (m, 2H). 13C NMR (75 MHz, CDCl3) d (ppm) 19.5,
19.7, 22.6, 22.7, 24.4, 24.5, 24.8, 28.0, 29.4, 32.8, 34.3 (d,
5.8 Hz), 8.44 (d, 2H, JHeH ¼ 5.8 Hz). 13C NMR (75 MHz,
2
CDCl3) d (ppm) 14.0, 22.1 (d, JCeP ¼ 5.3 Hz), 22.6, 25.3
(d, 1JCeP ¼ 141.9 Hz), 29.1e29.6, 30.6 (d, 3JCeP
¼
2
3
4
1JCeP ¼ 133.5 Hz), 37.0e37.8, 39.4, 48.5, 64.9 (d, JCe
6.4 Hz), 31.0 (d, JCeP ¼ 16.6 Hz), 31.8, 34.7 (d, JCeP ¼ 0.8

A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
1765
3
3
30.4 (d, JCeP ¼ 5.8 Hz), 31.0 (d, JCeP ¼ 16.6 Hz), 31.8, 34.7
A
3
2
1
4
2
(d, JCeP ¼ 0.8 Hz), 65.7 (d, JCeP ¼ 6.7 Hz), 123.8, 129.9,
129.8, 149.5, 150.8. 31P NMR (81 MHz, CDCl3) d (ppm)
31.9. ES-MS (m/z): 716 (MHþ).
The pyridinium compounds 5 were obtained from com-
pounds 50 by the same procedure used for the preparation of
compounds 1.
1
4.2.3.3. Compound 5a. Yield, 96% (yellow solid). H NMR
3
(300 MHz, CDCl3) d (ppm) 0.87 (t, 6H, JHeH ¼ 6.6 Hz),
1.20e1.45 (m, 44H), 1.50e1.82 (m, 14H), 2.89 (t, 2H, 3JHeH
¼
7.2 Hz), 3.98 (q(td), 4H, 3JHeH ¼ 3JHeP ¼ 7.0 Hz), 4.65 (s, 3H),
0
3
3
3
1e
1f
1f
1f
2a
2c
2f
2f
2f
5a
5a
5a
5b
5b
5b
Ref
Ref
Ref
7.09 (d, 2H, JHeH ¼ 6.1 Hz), 9.10 (d, 2H, JHeH
¼
6.1 Hz). 13C NMR (75 MHz, CDCl3) d (ppm) 14.0, 22.6 (d,
B
2JCeP ¼ 4.6 Hz), 24.7 (d, JCeP ¼ 140.1 Hz), 29.1e29.6, 30.3
1
3
2
(d, JCeP ¼ 18.0 Hz), 31.8, 35.7, 48.9, 66.4 (d, JCeP
¼
+ DIPPE
6.8 Hz), 130.7, 145.0, 161.4. 31P NMR (81 MHz, CDCl3)
2
1
d (ppm) 31.2. ES-MS (m/z): 662 (MþeClꢀ).
1
4.2.3.4. Compound 5b. Yield, 30% (colorless oil). H NMR
3
(300 MHz, CDCl3) d (ppm) 0.87 (t, 6H, JHeH ¼ 6.6 Hz),
1.30e1.45 (m, 44H), 1.50e1.82 (m, 14H), 2.62 (t, 2H, 3JHeH
6.8 Hz), 3.91e4.07 (m, 4H), 4.65 (s, 3H), 5.29e5.43 (m, 4H),
¼
3
3
7.09 (d, 2H, JHeH ¼ 6.1 Hz), 8.48 (d, 2H, JHeH
¼
6.1 Hz). 13C NMR (75 MHz, CDCl3) d (ppm) 14.1, 22.6 (d,
0
3
2JCeP ¼ 4.6 Hz), 22.7, 25.1 (d, JCeP ¼ 139.7 Hz), 25.5, 27.2,
1
1e
2a
2c
3
3
29.0e29.6, 30.4 (d, JCeP ¼ 5.8 Hz), 30.6 (d, JCeP
¼
C
3
6.4 Hz), 30.7 (d, JCeP ¼ 16.5 Hz), 31.8, 35.7, 48.9, 66.4 (d,
2JCeP ¼ 6.8 Hz), 129.9, 129.8, 130.7, 145.0, 161.4. 31P NMR
(81 MHz, CDCl3) d (ppm) 31.2. ES-MS (m/z): 730 (MþeClꢀ).
+ DOPE
2
1
0
4.3. Transfection evaluation
Transfection assay were performed as follows: phosphono-
lipids (5 mg/mL in ethanol) were tested alone or mixed (vol/
vol) with DOPE or DIPPE (5 mg/mL in ethanol). Lipid/
DNA complexes were prepared as following: lipid was first di-
luted in DMEM and incubated for 5 min at room temperature.
Diluted lipid was added to plasmid DNA (pVITRO14 LGFP
hSEAP; 0.5 mg/ml in water), at various DNA/lipid ratio (w/
w, 1:5, 1:10 and 3:6) and incubated for 15 min at room temper-
ature. FuGENEÒ 6 (Roche) was used as control according to
manufacturer’s instructions. Cells (HEK 293, B16 and CHO)
were obtained from ATCC and MEF cells were purified
from mouse embryos; all were grown in the recommended
medium. For the transfection assay, cells were washed and re-
suspended at 1.25 ꢁ 105 cells/ml in DMEM containing 10%
heat-inactivated FCS, and 200 mL (25 000 cells) of cell sus-
pension was added to each well of a flat-bottomed 96-well
plate. Lipid/DNA complexes (10 mL/well) were then dropped
onto the cells and the plates were incubated at 37 ꢂC in
a CO2 incubator for 48 h (for a more detailed protocol on
transfection, see Supplementary data associated with this arti-
cle). Toxicity was evaluated by examination of the monolayer
confluency. The efficacy of transfection was assessed visually
by looking at GFP-expressing cells with a fluorescence micro-
scope or spectrophotometrically by quantifying the amount of
1e
2a
2c
Fig. 3. CHO cells transfection activities of selected phosphonolipids without
lipid helpers (A), and in the presence of DiPPE (B) or DOPE (C) at different
lipid/DNA ratios ( 1/5, /10, ■ 3/6). As control (Ref), FuGENEÒ 6 transfec-
■
tion reagent (Roche) was used. In vitro transfection experiments were per-
formed as described in the Section 4. Each bar represents the mean of three
independent measurements. For clarity, error bars are not included, but are typ-
ically about ꢃ 0.2 Abs in size for values absorbance-values superior to 0.5.
2
Hz), 65.5 (d, JCeP ¼ 6.8 Hz), 123.8, 149.7, 150.8. 31P NMR
(81 MHz, CDCl3) d (ppm) 31.9. ES-MS (m/z): 608 (MHþ).
1
4.2.3.2. Compound 50b. Yield, 50% (colorless oil). H NMR
3
(300 MHz, CDCl3) d (ppm) 0.87 (t, 6H, JHeH ¼ 6.6 Hz),
1.13e1.45 (m, 44H), 1.56e1.81 (m, 14H), 2.61 (t, 2H, 3JHeH
6.7 Hz), 3.90e4.00 (m, 4H), 5.29e5.43 (m, 4H), 7.08 (d, 2H,
¼
3
3JHeH ¼ 5.8 Hz), 8.45 (d, 2H, JHeH ¼ 5.8 Hz). 13C NMR
2
(ppm) 14.1, 22.3 (d, JCeP
5.3 Hz), 22.7, 25.5 (d, JCeP ¼ 140.3 Hz), 27.2, 29.1e29.6,
(75 MHz, CDCl3)
d
¼
1

1766
A.D. Dal-Maso et al. / European Journal of Medicinal Chemistry 43 (2008) 1758e1766
[5] P.L. Felgner, T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M. Wenz,
J.P. Northrop, G.M. Ringold, M. Danielsen, Proc. Natl. Acad. Sci.
U.S.A. 84 (1987) 7413e7417.
SEAP released by transfected cells using QUANTI-BlueÔ
(InvivoGen): 20 ml of supernatant were mixed with 180 ml of
QUANTI-BlueÔ solution followed by 3 h incubation at
37 ꢂC and the absorbance was measured at 655 nm.
[6] P. Hawley-Nelson, V. Ciccarone, G. Gebeyehu, J. Jessee, Focus 15 (1993)
73e79.
[7] R. Leventis, J.R. Silvius, Biochim. Biophys. Acta 1023 (1990) 124e132.
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Acknowledgements
[9] J.P. Behr, B. Demeneix, J.P. Loeffler, J. Perez-Mutul, Proc. Natl. Acad.
Sci. U.S.A. 86 (1989) 6982e6986.
We thank Elise Faure for her excellent technical help.
´
Angelique Durand Dal-Maso was supported by the National
Association for Technical Research (ANRT).
[10] E.R. Lee, J. Marshall, C.S. Siegel, C. Jiang, N.S. Yew, M.R. Nichols,
J.B. Nietupski, R.J. Ziegler, M.B. Lane, K.X. Wang, N.C. Wan,
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Appendix. Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.ejmech.2007.11.002.
[12] I. van der Woude, A. Wagenaar, A.A. Meekel, M.B. ter Beest,
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diphenyl-H-phosphonate, Synth. Commun., submitted for publication.
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