Synthesis of new cationic lipids from an unsaturated glycoside scaffold.

Article abstract of DOI:10.1021/ol0159423

[see structure]. We report the synthesis of new cationic lipids. These amphiphiles present a hydrophobic domain connected to a guanidinium entity by an unsaturated glycoside scaffold. The synthetic strategy using amide or acetal linkage led to various mono- and bicatenar derivatives. Investigation of their physicochemical properties indicated that these new compounds compact DNA.

Full text of DOI:10.1021/ol0159423

ORGANIC
LETTERS
2001
Vol. 3, No. 12
1893-1896
Synthesis of New Cationic Lipids from
an Unsaturated Glycoside Scaffold
,†
Jean Herscovici,* Marie Jose´ Egron,† Alain Quenot,‡ Franc¸oise Leclercq,†
Nicolas Leforestier,‡ Nathalie Mignet,†,‡ Barbara Wetzer,‡ and Daniel Scherman†,‡
LCBBMC UMR 7001 CNRS-ENSCP-AVentis Gencell. ENSCP 11,
rue Pierre et Marie Curie 75231 Paris Cedex France, and AVentis Gencell CRVA13,
quai Jules Guesde BP 14 94403Vitry sur Seine France
herscoVi@ext.jussieu.fr
Received April 5, 2001
ABSTRACT
We report the synthesis of new cationic lipids. These amphiphiles present a hydrophobic domain connected to a guanidinium entity by an
unsaturated glycoside scaffold. The synthetic strategy using amide or acetal linkage led to various mono- and bicatenar derivatives. Investigation
of their physicochemical properties indicated that these new compounds compact DNA.
Positively charged polymers, peptides, and lipids have
emerged as promising alternatives to viral-based technology
for gene therapy.1 Since the first report about plasmid
transfection,2 many cationic lipids have been investigated3
for their ability to transfer foreign genes and oligonucleotides
into cells. However, there is a continuous need for the
development of new cationic lipids because of their low in
vivo transfection efficiency and some toxicity. To improve
the delivery of therapeutic DNA, recent efforts have focused
on structure activity relationship,4 linkages modification
between the polar and hydrophobic parts of the vector,5
biodegradable cationic lipids,6 template oligomerization,7 and
introduction of targeting ligands.8 As a part of our program
aimed toward the development of new vehicles for gene
delivery, we report a new approach for the synthesis of DNA
vectors, based on an unsaturated carbohydrate scaffold.
Unsaturated glycosides offer several advantages for the
design of gene delivery systems. First, each of the ring
substituents can be functionalized with a high selectivity
leading to a wide range of structures. Second, the unsaturated
acetal shows a moderate stability in acidic media, which
could facilitate the intracellular DNA release and lipid
biodegradability. Finally, the ring could allow the formation
of liposomes from single chain derivatives that usually led
to micelles.9
† ENSCP 11.
‡ Aventis Gencell CRVA13.
(6) (a) Wang, J.; Guo, X.; Xu, Y.; Barron, L.; Szoka, F. C.; Jr. J. Med.
Chem. 1998, 41, 2207-2215. (b) Tang, F.; Hughes, J. A. Biochem. Biophys.
Res. Commun. C 1998, 242, 141-145. (c) Rui, Y., Wang, S.; Low, P. S.;
Thompson, D. H. J. Am. Chem. Soc. 1998; 120, 11213-11218. (d) Tang,
F.; Hughes, J. A. Bioconjugate Chem. 1999, 10, 791-796. (e) Obika, S.;
Yu, W.; Shimoyama, A.. Uneda, T.; Miyashita, K.; Doi, T.; Imanishi, T.
Bioorg. Med. Chem. Lett. 1999, 7, 1817-1820.
(1) (a) Felgner, P. L. AdV. Drug DeliVery ReV. 1990, 5, 163-187. (b)
Behr, J. P. Bioconjugate Chem. 1994, 5, 382-389.
(2) Felgner, P. L.; Gadek, T. G.; Holm, M.; Roman, R.; Chan, H. W.;
Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad.
Sci. U.S.A. 1987, 84, 7413-7417.
(3) For a review, see: Miller, A. D. Angew. Chem., Int. Ed. Engl. 1998,
37, 7, 1768-1785.
(7) (a) Blessing, T.; Remy, J. S.; Behr, J. P. J. Am. Chem. Soc. 1998,
120, 8519-8520. (b) Blessing, T.; Remy, J. S.; Behr, J. P. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 1427-1431.
(4) Byk, G.; Dubertret, C.; Escriou, V.; Frederic, M.; Jaslin, G.; Rangara,
R.; Pitard, B.; Crouzet, J.; Wils, P.; Schwartz, B.; Scherman, D. J. Med.
Chem. 1998, 41, 224-235.
(8) (a) Jacopin, J.; Hofland, H.; Scherman, D.; Herscovici, J. Biomed.
Chem. Lett. 2001, 11, 419-422. (b) Reference 4. (c) Ren, T.; Liu, D..
Tetrahedron Lett. 1999, 40, 7621-7625.
(5) . (b) Ren, T.; Liu, D. Tetrahedron Lett. 1999, 40, 209-212 (c) Ren,
T.; Liu, D. Bioorg. Med. Chem. Lett. 1999, 9, 1247-1250.
10.1021/ol0159423 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/15/2001

Thus, we present herein our preliminary results regarding
the synthesis of cationic lipids, based on an unsaturated
glycoside scaffold. Our first approach involved the conden-
sation of an unsaturated glycosyl-amine with a BOC-
protected carboxyspermine. This first choice was made in
order to compare the properties of the new synthesized
compounds with those previously described by our group.4
However, preliminary observation demonstrated the high
sensitivity of the unsaturated acetal to the conditions needed
to remove the BOC groups. Thus, among the possible
cationic head, we selected guanidines10 that could be directly
prepared from amines without any protection. In this letter
we describe the preparation of a family of cationic lipids in
which the hydrophobic domain, made from various combina-
tions of alkyl chains, was connected by an unsaturated
glycosyl linker to a guanidinium salt.
without purification. Oleic acid was reacted with the crude
mixture in the presence of DCC to yield the amide 5 (50%,
two steps). Deprotection of 5 with a solution of potassium
carbonate in methanol/THF gave the amine 6 in 96% yield.
Finally, treatment of 6 with O-methylisourea hydrogen sulfate
or hydrochloride14 gave, respectively, the guanidines 7a and
7b (yields 70%).
Next, we investigated the preparation of bicatenar am-
phiphiles. To solve this issue, we selected an acetal strategy
to connect the secondary hydroxyl to a fatty alcohol. This
process offered several advantages. First, this group is
orthogonal to the trifluoroacetamide. Second, the use of an
acetal could increase the acid sensitivity of the cationic lipid.
Thus, we choose to prepare the bis C-14 guanidine 12
(Scheme 2). First, the myristamide 8 was prepared from azide
Our starting point was Ferrier condensation11 of triacetyl-
D-glucal 1 with 2,2,2-trifluoro-acetamidopropanol 2 (Scheme
1). The resulting glycoside was immediately deacetylated
Scheme 2a
Scheme 1a
a (i) (a) THF, 40 °C, PPh3/H2O; (b) CH2Cl2, rt, myristic acid,
DCC, two steps. 70%. (ii) THF, 60 °C, DIPEA, tBu4NI,
C14H29OCH2Cl (9) 3 equiv, 70%. (iii) (a) MeOH/THF, 40 °C,
K2CO3, 99%; (b) MeOH, 40 °C, O-methylisourea hydrochloride
50%.
4 by our reduction-condensation procedure. The methoxy-
tetradecane synthesis was carried out by adding 3 equiv of
the chloromethoxy-tetradecane15 9 to a THF solution of 8
in the presence of diisopropylethylamine and tetrabutylam-
monium iodide. After 2 h at 60 °C, the methoxy-acetal 10
was isolated in 80% yield. Removal of the trifluoroacetamide
and treatment with O-methylisourea hydrochloride yielded
the guanidine 12 (50%).
a (i) (a) CH2Cl2, 0 °C, 2,2,2-trifluoro-acetamidopropanol 2,
BF3‚Et2O, 1 h; (b) 2 N MeONa in MeOH, rt, 15 min (100%, two
steps). (ii) (a) CH2Cl2, 0 °C, TsCl 1 equiv, pyridine overnight, 70%;
(b) DMF, 60 °C, NaN3, NaI, 18-crown-6, overnight, 70%. (iii) (a)
THF, 40 °C, PPh3, H2O, 1 h; (b) CH2Cl2, rt, DCC oleic acid, 50%
(two steps). (iv) MeOH/THF 3:1, 40 °C, K2CO3, 96%. (v) MeOH,
40 °C, Et3N, O-methylisourea hydrogen sulfate or chloride,
overnight, 70%.
The methoxy alkyl chemistry offers a new approach for
the design of mono- and bicatenar amphiphiles. Examination
of the literature16 showed that at room temperature chlo-
to give quantitatively the diol 3. Selective tosylation of the
primary hydroxyl furnished the 6-p-toluenesulfonyloxy gly-
coside. Overnight treatment of this tosylate with NaN3 and
NaI in the presence of 18-crown-612 afforded the azide 4 in
70% yield. Reduction of 4 using the Staudinger procedure13
(PPh3/water) led to the amino glycoside, which was used
(9) Tang, F.; Hughes, J. A. J. Controlled Release 1999, 62, 345-358.
(10) For guanidine lipids, see: Vigneron, J. P.; Oudrhiri, N.; Fauquet,
M.; Vergely, L.; Bradley, J. C.; Bassville, M.; Lehn, P.; Lehn, J. M. Natl.
Acad. Sci. U.S.A. 1996, 93, 9682-9686.
(11) Ferrier, R. J.; Prasad, N. J. Chem. Soc. C. 1969, 570-575.
(12) For a recent example, see: Hansen, H. C.; Magnusson, G.
Carbohydr. Res. 1999, 322, 166-180.
(13) Staudinger, H. HelV. Chim. Acta 1919, 2, 635-646.
1894
Org. Lett., Vol. 3, No. 12, 2001

romethoxy-alkanes react selectively with a primary hydroxyl,
whereas reaction with a secondary alcohol requires heating.
Scheme 3 illustrates the preparation of guanidines from the
Scheme 3a
Figure 1. Unsaturated guanidine glycosides.
was then investigated. Lipoplex colloidal stability was first
studied. The UGG/DNA complexes were prepared at dif-
ferent UGG/DNA ratios by mixing the glycoside with
pXL3031 (3600 bp) plasmid17 in the presence (liposomes)
or in the absence (micelles) of dioleyl phosphatidylethano-
lamine (DOPE).
As a model for the physicochemical characterization of
the UGG, we have used 23, which exhibited a three-zone
model, termed A, B and C, of colloidal stability.18 In zone
A, lipoplexes have a mean diameter of 100 nm (Figure 2).
a (i) THF, rt, DIPEA, tBu4NI, C14H29OCH2Cl (9) or C18H35OCH2Cl
(13) 2 equiv. (ii) THF, 60 °C, DIPEA, tBu4NI, 9 5 equiv, 70%.
(iii) MeOH/THF, 40 °C, K2CO3, overnight. (iv) MeOH, 40 °C,
O-methylisourea hydrogen sulfate or hydrochloride overnight, 70%.
(v) Dowex 21K, THF/H2O 8:2, 43%.
diol 3. Addition of 2 equiv of 1-chloromethoxy-cis-undec-
9-ene 13 to a solution of 3 at room temperature gave the
6′-O-methoxy acetal 14 in 70% yield. In the same conditions,
reaction of 1-chloromethoxy-tetradecane 9 with 3 led to 15,
whereas treatment of the diol 3 with 5 equiv of 9 afforded
the bis acetal 16. Removal of the trifluoroacetyl group with
potassium carbonate afforded the amines 17-19. Reaction
of the amines with O-methylisourea hydrochloride or hy-
drogen sulfate gave, after purification by reverse phase
HPLC, the guanidines 20-22 in 70% yield. Finally hydro-
chloride 23 was prepared from sulfate 22 by ion-exchange
chromatography (Dowex 21K, 20% water in THF, 43%
yield).
The capacity of the unsaturated guanidine glycosides
(UGG) 7a, 7b, 12, 20, 21, 22, and 23 (Figure 1) to compact
DNA and to form stable lipid-DNA complexes (lipoplexes)
(14) Lee, Y. B.; Park, M. H.; Folk, J. E. J. Med. Chem. 1995, 38, 3053-
3061.
Figure 2. Colloidal stability and fluorescence experiments for 23-
DNA lipoplexes. 23-DNA lipoplexes of various charge ratios, at
10 µg DNA/mL in 150 mM NaCl, were obtained by mixing 23
micelles with the pXL 3031 plasmid. EtBr ) ethidium bromide.
(15) Shipov, A. G.; Savost’yanova, I. A.; Baukov, Y. I Zh. Obshch. Khim.
1984, 54, 2649-2650.
(16) For some examples, see: Mamedov, S.; Rzaev, A. S. J. Gen. Chem.
USSR 1963, 3, 3781-3784. Takeda, K.; Yano, S.; Sato, M.; Yoshii, E. J.
Org. Chem. 1987, 52, 4135-4137.
Org. Lett., Vol. 3, No. 12, 2001
1895

7b, 20, and 21 indicated that neither the lipid linkage (amide
or methoxy-acetal) nor the insaturation did significantly
modify the UGG behavior. The bismethoxy-acetal 23 gave
also small particles. However, the more hydrophobic amido-
acetal 12 displayed an increase in the size of lipoplexes, as
well as the broadness of the B zone. These results showed
that either a single chain or two acetal bridges favored the
formation of small and stable lipoplexes. This is consistent
with the idea that hydrophilicity is the main factor controlling
the formation of small size UGG lipoplexes.
Table 1. Size of the Cationic Lipid/DNA Complexes as a
Function of the Lipid/DNA Ratio
Finally, 7b and 12 have been used as models to evaluate
the lipid stability in acid media. Reaction with strong acids
such as trifluoroacetic acid or IR 120 led to the immediate
destruction of the unsaturated glycoside. On the other hand
7b and 12 were stable when dissolved in 0.01 N HCl.
a Lipoplexes made from micelles. b Lipoplexes made from liposomes.
c Size in nm.
In summary, we have prepared a series of new cationic
lipids that compact DNA and form colloidally stable li-
poplexes. Transfection experiments are underway and will
be published in due course.
In the B zone, the lipoplexes are unstable and precipitate.
This precipitation is due to a charge ratio near neutrality,
inducing lipoplex aggregation by reducing electrostatic
repulsion. Finally, zone C lipoplexes were colloidally stable
with a mean diameter around 60 nm. Ethidium bromide
fluorescence experiments were performed to evaluate DNA
entrapment in the lipoplexes. Ethidium bromide displays high
fluorescence when intercalated between DNA bases. Fluo-
rescence sharply decreased in zone A from 100% to about
25% in zone B and C, indicating ethidium bromide exclusion
due to compaction in the lipoplexes. Gel retardation experi-
ments confirmed these results, showing that all DNA was
retained for a charge ratio above 1 (see Supporting Informa-
tion Figure 1). Examination of Table 1 deserves further
comments. All UGG that can be formulated behave like 23,
exhibiting the three-zone scheme. At the ratio of 3 nmoles
lipid/µg of DNA, particles around 200 nm were obtained
for the sulfate 7a. In addition, attempts to formulate 22 and
the sulfate analogue of 12 led to highly insoluble complexes.
These results prompted us to synthesize hydrochloride UGG.
For all the chloride except 12, small particles of less than
100 nm sizes were observed. Comparison of the data for
Acknowledgment. This work was financially supported
by the CNRS, Aventis-Gencell, the ARC, the MENRT, and
the Re´gion Ile de France (SESAME).
Supporting Information Available: Experimental pro-
cedures and NMR data for compounds 5-23 and DNA
complexation experiments (7a, 7b, 12, 20, 21, 23). This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0159423
(17) Soubrier, F.; Cameron, B.; Manse, B.; Somarriba, S.; Dubertret,
C.; Jaslin, G.; Jung, G.; Caer, C. L.; Dang, D.; Mouvault, J. M.; Scherman,
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Org. Lett., Vol. 3, No. 12, 2001