Synthesis of novel cholesterol-based cationic lipids for gene delivery

Article abstract of DOI:10.1016/j.bmcl.2009.04.036

The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with carbamoyl linkage were also synthesized in order to com

Full text of DOI:10.1016/j.bmcl.2009.04.036

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2986–2989
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel cholesterol-based cationic lipids for gene delivery
Bieong-Kil Kim a, , Kyung-Oh Doh b, , Joo Hyeung Nam , Hyungu Kang , Jong-Gu Park , Ik-Jae Moon ,
Young-Bae Seu
a
a
c
d
d
a,*
School of Life Sciences and Biotechnology, Kyungpook National University, Sankyukdong, Bookgu, Daegu 702-701, Republic of Korea
Department of Physiology, Yeungnam University College of Medicine, 317-1 Daemyung-dong, Daegu 705-717, Republic of Korea
b
c
POSTECH Biotechcenter, San31, Hyoja-Dong, Nam-gu, Pohang 790-784, Republic of Korea
d
WelGENE Inc., 71B-4L, Hightech Sector 2, Sungseo Industrial Park 3, Dalseogu, Daegu 704-230, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 February 2009
Revised 9 April 2009
Accepted 10 April 2009
Available online 18 April 2009
The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by
using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with car-
bamoyl linkage were also synthesized in order to compare the difference in transfection efficiency of
the two linkage types. To this end, GFP expression of these cationic lipids was confirmed respectively.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Cationic lipids
Gene delivery
Cholesterol derivatives
Cyanoethylation
Gene therapy represents a medicine of the future owing to its
potential to treat various diseases including cancer. In gene ther-
apy, it is exceedingly important not only to manufacture the ther-
apeutic materials such as plasmid DNA and antisense
oligonucleotides but also to delivery these therapeutics to target
cholesterol-based cationic lipid has been used as the major lipid
1
1
12,13
of liposomes for the delivery of genes and chemical drugs
1
4
due to it being less toxic than other cationic lipids. The capacity
of the gene delivery using these cationic lipids depends on the
combination of cationic head, degree of hydrophobicity of the tails,
and the bond that links the lipophilic tails. Neutral lipid, for exam-
1
cells. Generally, the vectors for an successful delivery of genetic
materials can be classified into two main types: viral or nonviral.
Although viral vectors are highly efficient for delivery of DNA into
cells, they have many disadvantages such as antigenicity, toxicity,
limited size of cargo, and the difficulty of large-scale virus produc-
ple, 1,2-dioleoyl-
L-a-glycero-3-phosphatidylethanolamine (DOPE),
is also play a key role when forming cationic liposome for high
1
5,16
activity.
Especially, the presence of serum in gene delivery by
almost commercial liposomes decreases the degree of gene
2
,3
17
tion. As an alternative to the limitations of viral vectors, non-vir-
al vectors offer the new hope for DNA delivery systems. Among the
non-viral vectors, cationic lipids have been intensively investigated
because of the advantages of handy synthesis, low immune re-
expression.
This study focused on the development of optimum cationic lip-
ids for cationic liposome delivery systems that can improve serum
stability and transfection efficiency. The synthesis of novel choles-
terol-based cationic lipids B, C, and D (Scheme 1) having aminopro-
pyl head groups, ether linker, and cholesterol tail is reported.18 The
current synthetic strategy is based on the cyanoethylation of cho-
lesterol and direct Boc protection via reduction of nitrile group.
This study first introduced the cationic lipids that aminopropyl
cationic head groups attached via carbamoyl and ether linkers to
cholesterol as shown in Scheme 1. According to the current re-
4
–8
sponse, and safety.
The cationic lipids are also a class of vectors
easily applying rational design and studying structure–activity
9
relationships.
Cationic lipids are commonly composed of three parts (positive-
1
0
charged polar head group, linker, and lipophilic domain). The
head group often consists of amines or those extended formation
such as ethylamine, propylamine, lysine, spermidine, and sperm-
ine. The linker mostly composes of ether, ester and carbamoyl (ure-
thane) structure, and the lipophilic tails compose long chain fatty
acids or cholesterol derivatives. Among these components, the
1
9
port , ether linker is more efficient than other linkers on the same
structure used for gene delivery system. To our knowledge, lipid B
having an aminopropyl cationic head group has not been compared
with other carbamoyl linked cationic lipids having an aminopropyl
cationic head group. Therefore, to demonstrate the predominance
of the ether linkage, lipid A having the carbamoyl linkage was syn-
*
Corresponding author. Tel.: +82 53 950 5380; fax: +82 53 955 5522.
E-mail address: ybseu@knu.ac.kr (Y.-B. Seu).
2
0
Both authors contributed equally to this work.
thesized according to the method of Tsutomu et al. as shown in
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.036

B.-K. Kim et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2986–2989
2987
Chol
with 2 equiv of NiCl
added. After the mixture stirred 5 min, 10 equiv of NaBH were
4
2
Á6H
2
O and 3 equiv of crystalline Boc-ON were
O
slowly added under nitrogen atmosphere at room temperature
and gave the carboxyamine 3 by the usual workup. Compounds
Chol
H2N
N
O
H
2, 5, 8 were reduced only by NiCl
2 2 4
Á6H O and NaBH to directly ob-
Lipid A
tain final compounds. However, the yield was low (up to 50% in the
case of compound 3) because of by-products and treatment pro-
cess that is fastidious due to the gluey reaction mixture. Therefore,
Boc-protection method was used in this study in order to obtain
high yields and easy treatment. Trifluoroacetic acid (TFA) treat-
ment is necessary procedure to retain cationic form of amine head
groups and Boc-deprotection is easily reacted by TFA. Ether spacer
linked amine 4 attached to cholesterol was obtained by Boc-depro-
tection using TFA. The intermediate 5 and 8 were obtained by
reacting 1 equiv of the lipid 4 with 5 equiv of acrylonitrile in meth-
anol under reflux condition in one step. Finally, the one or two
elongated 3-aminopropyl chain cationic lipids 7, 10 were synthe-
sized by reductative Boc protection and deprotection as described
above. Structures of all of the synthetic intermediates and the tar-
H
H
H
Lipid B: R = -NH2
C: R = -NH(CH2)3NH2
D: R = -N((CH2)3NH2)2
R
O
head
linker
lipophilic part
Scheme 1. Structure of cholesterol-based cationic lipids.
a
H
H
O
O
H
H
H
H
Cl
O
H2N
N
H
O
1
get lipids as shown in Scheme 3 were confirmed by H NMR. Final
1
1
12, A
compounds 4, 7, and 10 as shown in Scheme 3 were also character-
ized by mass spectrometry including GC or FAB (fast atomic bom-
bardment) to confirm the identity of the molecular ions.
Scheme 2. Synthesis of carbamoyl linked cationic lipid A. Reagent and condition:
a) 1,3-diaminopropane, toluene/r.t.
(
This study next examined the GFP expression of liposomes con-
taining lipids A–D. The liposomes were prepared from cationic lip-
3
ids with appropriate volume of DOPE in CHCl and the solvent was
Scheme 2. This study also designed with a view to probe the role of
an additional elongated cationic amine center in modulating the
gene transfer properties of novel cholesterol-based cationic lipids
evaporated in a rotary evaporator under reduced pressure. The thin
lipid films were resuspended in sterile water and vigorously vor-
texed. The mixture was subjected to extrusion 10 times through
a polycarbonate membrane of 200 nm pore size using an extruder
device. The optimized medium for transfection (TOM) and plasmid
coding for Green Fluorescent Protein (pCMVTnT-GFP) were ob-
tained from Welgene (Korea) to test the ability of the lipoplexes
to transfer DNA in AGS cell (Human Stomach Epithelial—Gastric
7
, 10.
A typical procedure for the synthesis of lipids B–D from choles-
terol is as follows: Acrylonitrile was added to a mixture of aqueous
KOH, 18-crown-6 and cholesterol 1, 5-cholesten-3b-ol in CH Cl at
2
2
room temperature. Compound 2, 2-cyanoethyl-O-b-cholesterol
ether synthesized by cyanoethylation was dissolved in dry MeOH
H
H
H
HO
Chol
Chol
N
N
H
O
H2N
N
H
O
1, Cholesterol
5
7
, C
a
c
b
Boc
Chol
d
N
N
O
Chol
Chol
Chol
4
H
N
O
Boc
2
N
H2N
6
b
Chol
Boc
Chol
N
O
N
O
Boc
H
N
N
H
O
8
c
N
H2N
10, D
3
b
Chol
N
O
c
9
Boc
N
H
H2N
O
4, B
Scheme 3. Synthesis of ether linked cationic lipids B–D. Reagents and conditions: (a) acrylonitrile, 18-crown-6, aqueous KOH/CH
2
Cl
2
(yield: 100%); (b) NiCl
2
Á6H
2 2
O, Boc O,
NaBH /MeOH (yield: 3, 93%; 6, 80%; 9, 68%); (c) TFA/CH Cl (yield: 4, 85%; 7, 65%; 10, 67%); (d) acrylonitrile/MeOH, reflux (yield: 5, 42%; 8, 51%; overall, 93%).
4
2
2

2
988
B.-K. Kim et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2986–2989
Figure 1. Expression of GFP using lipids A–D. Plasmids pCMVTnTGFP (0.3 lg) complexed with lipids A–D were added to AGS cell observed under fluorescence microscope.
4
17
Adenocarcinoma). The day before transfection, AGS cells (4 Â 10
per well) were seeded into a 48-well plates. Each well was briefly
gene expression. However, these cholesterol-based cationic lip-
ids successfully delivered plasmid DNA in the presence of serum.
Transfection biology and characterization of lipids A–D are now
under investigation.
washed with 200
l
L of sterile PBS (Phosphate Buffered Saline), and
L of TOM before transfection. Each liposome
L of TOM, and mixed with
g) in 50 L of TOM. The lipo-
plexes, consists of liposomes and DNA, were incubated for
added with 100
l
(
0.6
lg) were diluted to 100 l
pCMVTnT-GFP plasmid DNA (0.3
l
l
Acknowledgment
1
2
5 min in room temperature, and then added to each well. After
4 or 48 h of incubation, the media was removed, and cells were
This research was supported by the Yeungnam University re-
search Grant in 2008 (208-A-054-034).
washed twice with PBS. Fluorescence expression was observed
with a Leica DM-IRE2 fluorescence microscope (Germany). The
ether linked cationic lipid B containing aminopropyl head group
was shown to be more effective in vitro transfection when com-
pared to carbamoyl linked cationic lipid A. Also, this study con-
firmed higher transfection efficiency in the presence of serum
than in the absence of serum and elongating one or two 3-amino-
propyl chain was benefited to plasmids DNA uptake as shown in
Figure 1.
In conclusion, this study describes the synthesis of the novel
cholesterol-based cationic lipids B–D and confirmed that the ether
linker is more effective than carbamoyl linker in the cholesterol-
based cationic lipids in vitro transfection. Generally, the presence
of serum decreases the transfection efficiency and intracellular
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