Synthesis and biological properties of new glycosidic cationic lipids for DNA delivery

Article abstract of DOI:10.1016/S0960-894X(00)00251-1

Lipidic glycosides with amino alkyl pendent groups were synthesized and evaluated for in vitro DNA transfection activity. The first representative of this new class of cationic lipids showed good gene delivery and low toxicity to HeLa and 3T3 cells. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00251-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1393±1395
Synthesis and Biological Properties of New Glycosidic Cationic
Lipids for DNA Delivery
Michel Bessodes,* Catherine Dubertret, Gabrielle Jaslin and Daniel Scherman
Laboratoire de Vectorologie Moleculaire et Cellulaire, UMR7001 ENSCP/CNRS/Aventis Pharma Gencell,
13 quai Jules Guesde 94403 Vitry/Seine Cedex France
Received 23 February 2000; accepted 26 April 2000
AbstractÐLipidic glycosides with amino alkyl pendent groups were synthesized and evaluated for in vitro DNA transfection
activity. The ®rst representative of this new class of cationic lipids showed good gene delivery and low toxicity to HeLa and 3T3
cells. # 2000 Elsevier Science Ltd. All rights reserved.
Nucleic acid transfer into eukariotic cells represents a
considerable therapeutic challenge. The elaboration of
non-viral synthetic vectors have engendered wide inter-
est.1 Various synthetic molecules have been proposed,
among which cationic lipids seem to be particularly
attractive.2 Since the ®rst report of the use of a cationic
lipid as a synthetic vector for gene delivery into cells by
Felgner and co-workers,3 a number of articles have
described the synthesis of cationic lipids as gene carriers.4
It is generally accepted that cationic lipids form com-
plexes, called lipoplexes, with DNA by electrostatic
interaction with the negatively charged phosphates of
the nucleic acid. The structure of the DNA in lipoplexes
is highly compacted, which allows its cellular uptake
and its protection from degradation by endo-nucleases.
These supra-molecular structures are then supposed to
collapse in the cell, thus permitting the expression of the
transfected gene. However, the actual cationic lipids suf-
fer so far from severe drawbacks, such as cell toxicity and
poor eciency in vivo. In the course of our studies in
the ®eld of synthetic carriers for gene delivery, we
describe in this letter the synthesis and biological prop-
erties of a new class of cationic lipids bearing a glycosidic
cationic head. The rationale for this synthesis was an
organized spatial density of positive charges, by the gly-
cosidic core, which could give a tighter interaction with
DNA, and an eventual lower toxicity than the pre-
viously published cationic lipids.4
bromide in a Williamson type etheri®cation reaction,5
to give the corresponding 6-O-benzyloxy derivative 1.
Methanolysis of the isopropylidene protecting groups in
1 gave the methyl glycoside 2. This compound was then
either etheri®ed with bromoethylacetate to give 3, or
esteri®ed with N-Boc glycine to give 4. It is noteworthy
that in the case of compound 3, only the triacid was
isolated, the same reaction was actually performed with
bromoacetic acid, leading to the same product. From
that point the synthesis followed two divergent proto-
cols: boron hydride reduction of 3 led to the corre-
sponding triol 5, which was converted to the triazide 6 by
the Mitsunobu reaction.6 The azides were further
reduced with concomitant cleavage of the benzyloxy
protecting group (7). Compound 8 was then obtained
after selective protection of the amino groups with di-
tert-butyl dicarbonate. On the other hand, 4 was hydro-
genolyzed to the corresponding 6-unprotected glycoside
9. Compounds 8 and 9 were reacted with either succinic
or phtalic anhydride to give, respectively, products 10,
11 and 12, 13. Condensation of the latter compounds
with dioctadecylamine lead to 14, 15, 16 and 17, which
were subsequently deprotected to the ®nal products 18,
19, 20 and 21.
Compounds 18±21 were then evaluated for their ability
to deliver a plasmid bearing the sequence of the luciferase
reporter gene on two di€erent cell types. The pCMV-Luc
plasmid used included the luciferase reporter gene under
the dependence of the human cytomegalovirus promoter.
It was constructed from pGL2-basic Vector (Promega) by
insertion of the Mlu I-Hind III fragment containing the
CMV promoter, which was extracted from the pcDNA3
plasmid (Invitrogen). The DNA saline solutions (NaCl
This synthesis is outlined in Scheme 1. Substrate 1,2-3,4-
di-O-isopropylidene-d-galactose was reacted with benzyl
*Corresponding author. Tel.: +33-1-5571-3779; fax: +33-1-5571-
8132; e-mail: michel.bessodes@aventis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00251-1

1394
M. Bessodes et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1393±1395
Scheme 1. Reagents and conditions: (i) MeOH, HCl; (ii) BrCH2CO2Et, KOH, 18-Cr-6, THF; (iii) BH3/THF; (iv) Ph3P, DEAD, N3H, THF; (v) H2,
Pd/C, EtOH; (vi) Boc2O, Et3N, CH2Cl2; (vii) succinic anhydride (or phtalic anhydride), pyridine, DMAP; (viii) (C18H37)2NH, BOP, Et3N, CH2Cl2;
(ix) TFA, CH2Cl2; (x) Boc-glycine, BOP, Et3N, CH2Cl2.
0.15M) were adjusted to 40mg/mL. Compounds 18±21
were solubilized in water (80±800mM) and mixed (v/v) to
the nucleic acid solution to a ®nal NaCl concentration of
75mM. Cells were grown in 24 wells microplaques
(2cm2/well) and transfected in their exponential phase, to
50±70% con¯uence. Transfection was evaluated 48 h after
addition of DNA lipoplexes by measuring the luciferase
expression using a commercial kit. Toxicity was estimated
from a dosage of protein concentration in lysate cells.
and 19 was optimum for a ratio of 8 nmol vector/mg
DNA in the case of the HeLa cells (Figure 1, bars), and
for a ratio of 12 nmol vector/mg DNA in the case of the
NIH 3T3 cells (Figure 2, bars). The histograms relating
the dose e€ects of the vectors 18 and 19 were super-
imposable in either cell type. The two compounds were
non toxic to cells at optimal active ratio and only a 20%
loss in cellular proteins was observed for ratios >8 nmol
lipid/mg DNA (Figures 1 and 2, black dots).
In summary, we have described the synthesis and gene
transfection properties of new glycosidic cationic lipids.
Compounds 18 and 19 showed a transfection level
comparable to previously described cationic lipids.4j
Compounds 20 and 21 were inactive in these conditions,
Results and Discussion
Compounds 20 and 21 were found inactive in these
conditions. Transfection eciency with compounds 18

M. Bessodes et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1393±1395
1395
Acknowledgements
We thank the ``Ile de France'' district for a SESAME
®nancial support.
References and Notes
1. Scherman, D.; Bessodes, M.; Cameron, B.; Herscovici, J.;
Ho¯and, H.; Pitard, B.; Soubrier, F.; Wils, P.; Crouzet, J.
Curr. Opin. Biotechnol. 1998, 9, 480.
2. Miller, A. D. Angew. Chem., Int. Ed. Engl. 1998, 37, 1768.
3. Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan,
H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen,
M. Proc. Natl. Acad. Sci. USA 1987, 84, 7413.
Figure 1.
4. (a) Gao, X.; Huang, L. Biochem. Biophys. Res. Commun.
1991, 179, 280. (b) Felgner, J. H.; Kumar, R.; Sridhar, C. N.;
Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin,
M.; Felgner, P. L. J. Biol. Chem. 1994, 269, 2550. (c) Bennett,
M. J.; Malone, R. W.; Nantz, M. H. Tetrahedron Lett. 1995,
36, 2207. (d) Solodine, I.; Brown, C. S.; Bruno, M. S.; Chow,
C.; Jang, E.; Debs, R. J.; Heath, T. D. Biochemistry 1995, 34,
13537. (e) Wheeler, C. J.; Felgner, P. L.; Tsai, Y. J.; Marshall,
J.; Sukhu, L.; Doh, S. G.; Hartikka, J.; Nietupski, J.; Man-
thorpe, M.; Nichols, M.; Plewe, M.; Liang, X.; Norman, J.;
Smith, A.; Cheng, S. H. Proc. Natl. Acad. Sci. USA 1996, 93,
11454. (f) Lee, E. R.; Marshall, J.; Siegel, C. S.; Jiang, C.;
Yew, N. S.; Nichols, M. R.; Nietupski, J. B.; Ziegler, R. J.;
Lane, M. B.; Wang, K. X.; Wan, N. C.; Scheule, R. K.; Har-
ris, D. J.; Smith, A. E.; Cheng, S. H. Hum. Gen. Ther. 1996, 7,
1701. (g) Bennet, M. J.; Aberle, A. M.; Balasubramaniam, R.
P.; Malone, J. G.; Malone, R. W.; Nantz, M. H. J. Med. Chem.
1997, 40, 4069. (h) Wang, J. K.; Guo, X.; Xu, Y. H.; Barron, L.;
Szoka, F. C. Jr. J. Med. Chem. 1998, 41, 2207. (i) Cooper, R.
G.; Etheridge, C. J.; Stewart, L.; Marshall, J.; Rudginsky, S.;
Cheng, S. H.; Miller, A. D. Chem. Eur. J. 1998, 4, 137. (j) Byk,
G.; Dubertret, C.; Escriou, V.; Frederic, M.; Jaslin, G.; Ran-
gara, R.; Pitard, B.; Crouzet, J.; Wils, P.; Schwartz, B.; Scher-
man, D. J. Med. Chem. 1998, 41, 224. (k) Geall, A. J.;
Blagbrough, I. S. Tetrahedron Lett. 1998, 39, 443. (l) Blessing,
T.; Remy, J. S.; Behr, J. P. J. Am. Chem. Soc. 1998, 120, 8519.
5. Bessodes, M.; Shamsazar, J.; Antonakis, K. Synthesis 1988,
7, 560.
Figure 2.
suggesting the in¯uence of the pK value of the amino
groups on the interaction with the nucleic acids. All the
four compounds tested were found non toxic, which could
permit the use in vitro and eventually in vivo of higher
vector/DNA ratio. Such high ratios usually lead to lipo-
plexes displaying favorable physicochemical and biologi-
cal characteristics, such as size, stability and transfecting
eciency. Active work is in progress to develop this new
class of promising cationic DNA vectors.
6. Loibner, H.; Zbiral, E. Helv. Chim. Acta. 1976, 59, 2100.