Synthesis and delivery activity of new cationic cholesteryl glucosides

Article abstract of DOI:10.1016/j.carres.2010.09.012

Cholesterol amphiphiles containing positively charged groups (pyridinium, N-methylimidazolium, N-methylmorpholinium, and N-methylpiperidinium) linked via β-glucosyl spacer were prepared by alkylation of the corresponding bases with 6 - mesyl-β-d-cholesteryl glucopyranoside. IC₅₀ values were in the range 20-35 μM for the series of compounds and liposomal formulations with DOPE (1:1) were significantly less toxic. The liposomal formulations provided the accumulation of FITC-labeled oligonucleotide in cells, and the efficiency of this process was comparable to that of Lipofectamine 2000. Cationic liposomes were able to deliver siRNA into the cells, and the liposomal formulation 7d/DOPE provided the most pronounced down-regulation of EGFP expression both in the presence and in the absence of serum (up to 30%).

Full text of DOI:10.1016/j.carres.2010.09.012

Carbohydrate Research 345 (2010) 2438–2449
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and delivery activity of new cationic cholesteryl glucosides
Mikhail A. Maslov a, , Nina G. Morozova a, Evgeniya I. Chizhik a, Dmitriy A. Rapoport b, Elena I. Ryabchikova b,
⇑
Marina A. Zenkova b, Galina A. Serebrennikova a
a Lomonosov State Academy of Fine Chemical Technology, Vernadsky Ave. 86, Moscow 119571, Russian Federation
b Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Ave. 8, Novosibirsk 630090, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2007
Received in revised form 4 September 2010
Accepted 10 September 2010
Available online 17 September 2010
Cholesterol amphiphiles containing positively charged groups (pyridinium, N-methylimidazolium, N-
methylmorpholinium, and N-methylpiperidinium) linked via b-glucosyl spacer were prepared by alkyl-
ation of the corresponding bases with 6-O-mesyl-b-
D-cholesteryl glucopyranoside. IC50 values were in
the range 20–35 M for the series of compounds and liposomal formulations with DOPE (1:1) were sig-
l
nificantly less toxic. The liposomal formulations provided the accumulation of FITC-labeled oligonucleo-
tide in cells, and the efficiency of this process was comparable to that of LipofectamineÒ 2000. Cationic
liposomes were able to deliver siRNA into the cells, and the liposomal formulation 7d/DOPE provided the
most pronounced down-regulation of EGFP expression both in the presence and in the absence of serum
(up to 30%).
Keywords:
Cationic lipids
Heterocyclic bases
Nonviral vectors
Gene delivery
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
carbamoyl, ester, or ether bond have been synthesized.5,7–9
Targeted modification of each domains gives rise to a series of
Gene therapy, as an alternative to the conventional medicine,
implies eliminating the cause of a disease by introducing DNA or
oligonucleotides (antisense oligonucleotide or siRNA). Before ther-
apeutic nucleic acids reach the nucleus, they have to overcome a
number of biological barriers, in particular, cell, endosomal, and
nuclear membranes. This is attained by using appropriate delivery
systems, protecting the genetic material from the destructive ac-
tion of enzymes and promoting their penetration into the intracel-
lular space, transfer through the nuclear membrane, and further
expression in the nucleus.1–3 In addition, these delivery systems
should be nontoxic, nonimmunogenic and biocompatible.
Lipofection, a method based on the use of cationic liposomes for
gene transfer, occupies an important place among the methods of
gene delivery into eukaryotic cells.4 To perform lipofection, it is
necessary first to form cationic liposomes or micelles and then
their complexes with plasmid DNA or oligonucleotides, called lipo-
plexes. Cationic liposomes are formed using cationic amphiphiles,
comprising a wide range of chemical compounds with common
structural features, namely, the presence of positively charged
and hydrophobic domains separated by spacers of various
lengths.5–9 Biodegradable cationic amphiphiles of natural origin
are of most interest now. Recently, glycerol- and cholesterol-
containing cationic amphiphiles with various cationic ‘heads’
attached to the hydrophobic moiety via a spacer by means of a
amphiphiles, which can be used to study the structure–activity
relationship. As a rule, it is possible to compare results only in
experiments with the same cell line.
It is known that the efficiency of liposome-mediated gene deliv-
ery is determined not only by the structure of cationic and helper
lipids, properties of the plasmid, but also by the size and f-poten-
tial of the lipoplex.10 The structure of supramolecular DNA–lipid
complexes depends on both external (pH, degree of hydration,
temperature, and the presence of doubly charged cations, that is,
Ca2+, Mg2+ 6,11
and internal factors. The internal factors mean the
)
phase state of the lipid molecule (lamellar or hexagonal), which
is governed by the ratio of space areas of the polar and hydropho-
bic domains.6,7,12,13 The incorporation of a helper lipid, such as
dioleoyl phosphatidylethanolamine (DOPE), in liposomal formula-
tions can significantly enhance the gene delivery activity of the cat-
ionic lipid. The zwitterionic phospholipid DOPE induces a strong
destabilizing effect on lipid bilayers due to its spatial organiza-
tion.14,15 This results in a facilitated disruption of the endosomal
membrane in transfected cells and subsequent cytosolic release
of a delivered cargo. To increase the transfection level at the endo-
somal release stage, especially ‘sensitive’ elements irreversibly
transformed by the action of cell medium have been introduced
into the cationic lipid structure.7,16–18 Therefore, the efficiency of
lipid vehicles for the nucleic acid delivery finally depends on the
structure of cationic amphiphile whose chemical design requires
a tailored approach taking into account the lipid composition of
membranes, the nature of cell receptors, and the chemical
processes in the intracellular environment.
⇑
Corresponding author. Tel.: +7 495 936 8903; fax: +7 495 936 8901.
E-mail address: mamaslov@mail.ru (M.A. Maslov).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.09.012

M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
2439
Cationic amphiphiles containing cholesterol as a hydrophobic
2. Results and discussion
residue possess a high transfection activity and a low toxicity and
find use in studies of both the structure–activity relationships and
the membrane fusion mechanisms. Among synthesized cholesterol
derivatives, there is a commercially available lipid, 3-b-[N-(N0,N0-
dimethylaminoethyl)carbamoyl]-cholesterol (DC-Chol). Liposomes
prepared with DC-Chol and DOPE lipids are widely used to deliver
the plasmid DNA for tumor immunotherapy19,20 or artificial
immunization21 and are under clinical trials for the therapy of
mucoviscidosis.22
As mentioned above, lipids with different positively charged
groups based on aliphatic or heterocyclic bases were synthesized
to reveal the structure–activity relationships between the structure
of cationic lipids and the transfection activities. It was found
that heterocyclic cationic lipids- containing imidazolium23 or
pyridinium polar heads24–27 displayed higher transfection effi-
ciency and reduced cytotoxicity when compared to classical trans-
fection systems. Recently, we performed systematic study on
the synthesis of lipofection mediators with the hydrophobic
component represented by diglycerides, cholesterol, and fatty acid
residues. Heterocyclic bases pyridine, N-methylimidazole, and
N-methylmorpholine were used for the creation of a cationic ‘head’
in diglycerides28,29 and cholesterol.30–32 Previously, we also synthe-
sized a number of cationic carbohydrate-containing glycerolipids
differing in the structure and the position of the positively charged
group and the type of linker between the carbohydrate and the
glycerol fragment.33,34 To get further development in this direction,
herein we describe the synthesis of novel cationic cholesteryl gluco-
sides with different heterocyclic headgroups, which may serve as
mediators of gene transfer into eukaryotic cells.
2.1. Synthesis of cationic glucolipids
It is known that the presence of a carbohydrate residue in-
creases the colloidal stability of DNA–cationic liposome complexes,
enhances DNA delivery, and decreases the cytotoxicity of cationic
amphiphiles.6,35–37 Our synthetic strategy considers the glycosyl
unit as a sort of biodegradable linker. According to the preliminary
biological assays, the efficiency of transfection of eukaryotic cells
using cationic glycerolipids increases when a carbohydrate spacer
group is introduced into the structure.38
The goal of this work was to prepare cholesterol-containing
amphiphiles 7a–d, containing a b-glucosyl unit as a spacer, in which
cationic groups are attached directly to C-6 of the carbohydrate
residue (see Scheme 1). We used pyridinium, N-methylimidazolium,
N-methylmorpholinium, and N-methylpiperidinium groups as cat-
ionic heads and employed these cationic heads in our previous study
to obtain cationic glycerolipids.28–32 Cholesterol was chosen as a
hydrophobic component due to its biological compatibility and the
ability to stabilize membranes and to form rigid liposomes.
The glucosides 2a,b were obtained by condensation of acetob-
romoglucose 1 and cholesterol at a 1:2 molar ratio under Helferich
reaction conditions39 in toluene in the presence of Hg(CN)2 and
dried molecular sieves (4 Å) at 100 °C. This approach is widely used
in steroid glycoside chemistry owing to a high promoting activity
of mercury salts and the easy accessibility of the glycosyl donor.40
The total yield and the stereoselectivity of this reaction largely
depend on the nature and the structure of the steroid alcohol.
The condensation gave two anomers with a total yield of 63%.
OAc
OAc
OH
O
a
b
c
AcO
AcO
O
O
AcO
AcO
HO
HO
OR1
OR1
AcO
AcO
HO
Br
1
2a, α-isomer
3
2b, β-isomer
R2 X-
R2 X-
OMs
j
d
O
O
O
AcO
AcO
AcO
AcO
HO
HO
OR1
OR1
OR1
AcO
HO
7a-d
AcO
5a-d
4
e
f
R2
R2
O
Compound
X
AcO
AcO
OR1
AcO
5a, 7a
MsO
N
2
6a
, R =
N
N
O
N
N Me
O
5b, 7b
5c, 7c
MsO
I
2
6b
, R
=
N
Me
I
N
Me
5d, 7d
R1 =
Scheme 1. Reagents and conditions: (a) cholesterol, Hg(CN)2, 63% or CdCO3, MePh, 52%; (b) MeONa/MeOH, 90%; (c) MsCl, then Ac2O, Py, 70%; (d) Py or MeIm, 78% (80%); (e)
morpholine or piperidine, then Ac2O, 52% (58%); (f) MeI, 82% (72%); and (g) 0.04 N MeONa/MeOH.

2440
M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
Chromatographic separation and analysis of 1H and 13C NMR data
suggested that - and b-glucosides were formed in a ratio of 1:2.3.
A sharp increase in the glycosylation stereoselectivity ( /b, 1:51)
glucosides. Target cationic cholesteryl glucosides 7a–d were
isolated in 45–89% yields by reverse phase column chromatogra-
phy on LiChroprepÒ RP-18.
a
a
with a slight decrease in the total yield (52%) was attained by
replacing Hg(CN)2 with CdCO3 and conducting the process in a
Soxhlet apparatus for 3 h according to the known procedure.41 b-
Anomer 2b was subsequently deacetylated by Zemplen reaction
with 0.2 N sodium methoxide in methanol yielding cholesteryl b-
2.2. Biological testing of cationic glycolipids
To assess the potential of the synthesized cationic glycolipids as
new gene delivery agents, we estimated biological effects of these
compounds on cell viability and cytosolic delivery of a 25-mer
fluorescein-labeled oligodeoxyribonucleotide (FITC-ODN) and a
small interfering RNA (siRNA). These biological tests were per-
formed for both individual glycolipids and liposomes. The liposo-
mal formulations were composed of one of the cationic
glycolipids 7a–d and the zwitterionic phospholipid DOPE at a mo-
lar ratio of 1:1. At this molar ratio, maximum levels of transfection
were observed for a number of cationic lipids.42,43 For the lipo-
somes preparation, a thin lipid film was hydrated and sonicated
to give the desired formulation.
D-glucoside (3) after recrystallization in 90% yield.
The mesyl group was used as a leaving group when introducing
the cationic ‘head’ into the glucoside molecule. A regioselective
mesylation at the carbohydrate C-6 atom of cholesteryl b- -gluco-
D
side (3), which was accomplished by the treatment with an equi-
molar amount of mesyl chloride at ꢀ15 °C,19 resulted in
a
complete disappearance of the starting glucoside 3 from the reac-
tion mixture to give a series of new products containing different
numbers of mesyl groups with predominance of the 6-O-mesyl
derivative. For more convenient purification of the product and
prevention of by-product formation during subsequent elimination
of the mesyl group under basic conditions, acetylation with acetic
anhydride and chromatographic separation were performed to
give the major reaction product 4 in 70% yield.
2.2.1. Estimation of the cytotoxicity of cationic glycolipids 7a–d
and liposomal formulations
The cytotoxicity of the cationic glycolipids 7a–d was evaluated
by the MTT assay.44 In these experiments, BHK cells were incu-
bated with various concentrations of cationic glycolipids 7a–d or
cationic liposomes composed of glycolipids and DOPE (molar ratio
of 1:1) for 24 h and then treated with MTT. The toxic effect of
cationic lipids (Fig. 1A) followed a typical S-shaped curve both in
the absence and in the presence of serum in the cell medium. In
the absence of serum, the IC50 values for lipids 7a–d were equal
Two approaches were used to introduce the cationic group into
cholesteryl glucoside. In the first approach, direct quaternarization
of heterocyclic bases with mesyl ester 4 was used. Heterocyclic
bases were quaternarized either in the medium of the base itself
(in the case of pyridine) or in the presence of an organic solvent,
methyl ethyl ketone (in the case of other bases). The reactions of
mesyl ester 4 with pyridine and N-methylimidazole proceeded
for 9 and 19 h at 115 and 100 °C, respectively. After recrystalliza-
tion, yields of compounds 5a and 5b were 78% and 80%, respec-
tively. Quaternarization of N-methylmorpholine and N-
methylpiperidine was unsuccessful and gave cationic lipids 5c,d
in low yields (<5–10%); therefore, another approach was proposed
in order to obtain lipids 5c,d. A two-stage route comprising alkyl-
ation of morpholine and piperidine with mesylate 4 followed by
quaternarization with methyl iodide provided lipids 5c and 5d un-
der milder conditions in good yields. Alkylation of cyclic amines
was accompanied by partial deacetylation of the carbohydrate
component due to a high basicity of used bases, which was con-
firmed by 1H NMR data for fractions obtained upon chromato-
graphic separation of the reaction products. Therefore, after
alkylation of heterocyclic amines, the mixture of compounds with
different numbers of acetyl groups was acetylated to give com-
pounds 6a and 6b in 52% and 58% yields after chromatographic
purification. At the next stage, tertiary amines 6a and 6b were
quaternized with methyl iodide to give target cationic lipids 5c
and 5d in 82% and 72% yields, respectively.
or exceeded 25 lM, while a small enhancement in glycolipid
cytotoxicity was observed in the presence of serum (Fig. 1A). The
structure of the positively charged group in cationic lipids 7a–d
had no significant influence on the cell growth; however, lipid 7c
with the N-methylmorpholinium head was slightly less toxic as
compared with the other cationic lipids.
An addition of DOPE to cationic glycolipids significantly reduced
their cytotoxicity: IC50 values exceed 100
served that all liposomes stimulated cell proliferation at concentra-
tions from 20 to 80 M.
lM (Fig. 1B). It was ob-
l
2.2.2. Cellular delivery of FITC-labeled oligonucleotides in the
presence of cationic glycolipids
To estimate the ability of cationic glycolipids to mediate nucleic
acid transfer across the plasmatic membrane, the delivery of FITC-
ODN into BHK cells was studied by FACS analysis using lipid con-
centrations of 10 and 20
lM. The FITC-ODN concentrations were
0.8 M and 5 M corresponding to the range of concentrations at
l
l
2D Homonuclear (COSY) and 1H/13C heteronuclear (HSQC) NMR
spectroscopy techniques were used to assign signals from the glu-
cosyl residue and to confirm structures of compounds 5a–d. We
found that in the case of compounds 5c and 5d, the signals for
the anomeric proton (d1H 5.40–5.48 ppm) and the proton at C-5
(d5H 4.70–5.00 ppm) shifted downfield as compared with the sig-
nals of these protons for compounds 5a and 5b, which occur at
high field (d1H 4.60–4.70 ppm, d5H 4.12–4.25 ppm). Presumably,
the presence of a positively charged nitrogen atom near the pyra-
nose ring in compounds 5c and 5d induces a decrease in the elec-
tron density on the ring oxygen atom. In turn, this results in the
electron density redistribution on the neighboring C-1 and C-5
atoms, which is evident from the NMR spectrum. In the case of
compounds 5a and 5b, the positive charge is delocalized through
which a down-regulation of a gene expression can be observed
and concentrations of glycolipids did not exceed the IC50 values
of individual compounds. In the first screening study, complexes
of glycolipids 7a–d with FITC-ODN were formed at four different
ODN/cationic lipid molar ratios (5:10, 5:20, 0.8:10, and 0.8:20) that
correspond to N/P (nitrogen to phosphate) ratio of 0.08, 0.16, 0.5,
and 1, respectively. Lipoplexes were preformed in OptiMem med-
ium for 30 min at room temperature and added to BHK cells. After
4 h incubation with FITC-ODN/glycolipids complexes, the cells
were fixed and analyzed. The percentage of the FITC-positive cells
and the cell-associated fluorescence intensity was measured. The
results showed that FITC-ODN delivery into BHK cells mediated
by glycolipids 7a–d was poor: no more than 8% of the BHK cell
population was transfected with FITC-ODN. Interestingly, the cel-
lular accumulation of FITC-ODN/glycolipid (20 lM) complexes
the aromatic ring
p-orbitals; therefore, the nitrogen atom is less
was two to threefolds higher in the presence of serum as compared
with serum-free conditions (5% and less than 1.5% of FITC positive
cells, respectively).
electrophilic and cannot interact with the pyranose oxygen atom.
Zemplen deacetylation of quaternarization products 5a–d was
the final stage in the synthesis of the cationic cholesteryl b-D-

M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
2441
120
100
80
60
40
20
0
A
160
B
140
120
100
80
60
40
20
0
0
5
10
15
20
25
30
35
40
0
20
40
60
80
100
120
Concentration, μM
Concentration, μM
Figure 1. Viability of BHK IR-780 cells treated with cationic glycolipids: (A) cationic liposomes, (B) for 24 h. Cells were incubated with cationic glycolipids 7a–d or with
cationic liposome, consisting of glycolipids 7a–d and DOPE (1:1) in the presence of 10% fetal bovine serum in the medium. Square for lipid 7a, circle for lipid 7b, up triangle for
lipid 7c, down triangle for lipid 7d. Cell viability was calculated as the percentage of survival cells, the amount of living cells in controls was set as 100%, error bars represent
standard deviations.
To study how the presence of DOPE can modify the cellular
accumulation of FITC-ODN mediated by glycolipids 7a–d, four
types of lipoplexes were prepared using different ratios of FITC-
ODN to cationic liposomes. Under serum-free conditions (Fig. 2A
and B), the efficiency of FITC-ODN liposomal delivery was 3–30-
fold higher than that of cationic glycolipids. The liposomes com-
posed of lipid 7d with N-methylpiperidinium ‘head’ and DOPE
were the most efficient formulation over the entire used concen-
tration range; the levels of FITC-ODN cellular delivery observed
in the case of 7d/DOPE liposomes (40%) were somewhat lower than
those of LipofectamineÒ 2000 (50%). Note that the cellular delivery
of FITC-ODN mediated by cationic liposomes at a concentration of
20
l
M only slightly depends on the concentration of ODN, thus,
M of FITC-ODN to 20 M of lipo-
indicating that the ratio of 0.8
somes (the N/P ratio of 1) provides an efficient cellular accumula-
tion of this oligonucleotide. (Fig. 2A).
l
l
In the presence of serum, FITC-ODN cellular accumulation med-
iated by cationic liposomes (10
whereas the cellular accumulation of FITC-ODN at a liposome con-
centration of 20 M was almost unaffected. Under these conditions
(10% FBS and 20 M liposome), the liposomes with glycolipid 7d
were the most active formulation providing the delivery of the oli-
gonucleotide into 40% of the cells (Fig. 2C and D). Similarly to Lipo-
fectamine, the cellular delivery of FITC-ODN by 7d/DOPE liposomes
lM) was reduced two to threefolds,
l
l
A
B
60
50
40
30
20
10
0
60
50
40
30
20
10
0
N/P
0.5
1
N/P
0.08
0.16
7a/DOPE
7b/DOPE
7c/DOPE
7d/DOPE
Lipofect
7a/DOPE
7b/DOPE
7c/DOPE
7d/DOPE
Lipofect
Cationic liposomes
Cationic liposomes
C60
D
60
50
40
30
20
10
0
N/P
N/P
50
40
30
20
10
0
0.5
1
0.08
0.16
7a/DOPE
7b/DOPE
7c/DOPE
7d/DOPE
Lipofect
7a/DOPE
7b/DOPE
7c/DOPE
7d/DOPE
Lipofect
Cationic liposomes
Cationic liposomes
Figure 2. Cellular delivery of FITC-ODN into BHK cells in the absence (A and B) or presence (C and D) of 10% fetal bovine serum in the medium. Lipoplexes were composed of
cationic liposomes (10 M, white bar and 20 M, gray bar) and FITC-ODN (A and C) ODN concentration of 0.8 M; (B and D) liposome concentration of 5 M) at various ratios.
The percentage of FITC-positive cells was measured by FACS analysis after 4 h incubation with the corresponding FITC-ODN/liposome complex. Transfection with
LipofectamineÒ 2000 was carried out according to the manufacturer’s protocol using 5
M FITC-ODN. Standard deviations were within 5%.
l
l
l
l
l

2442
M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
(20
lM) remained constant both in the absence and in the pres-
the siRNA penetration across the cell membrane. Therefore, to en-
ence of 10% FBS in the culture medium.
hance the biological effect of siRNA, it is necessary to perform dee-
per additional studies, including the optimization of liposomal
compositions, siRNA-to-carrier ratio, and delivery conditions.
2.2.3. Delivery of siRNA into eukaryotic cells within siRNA/
cationic liposome complexes
The ability of new cationic glycolipids and liposomes to mediate
a cytosolic delivery of short nucleic acids was confirmed in the
anti-EGFP-siRNA delivery experiments into the transgenic BHK
IR780 cells expressing green fluorescent protein (EGFP). The cho-
sen siRNA-EGFP (50 nM) is able to inhibit the EGFP expression in
transgenic cells; thereby, a reduction in green fluorescence of
BHK IR780 cells indicates the delivery activity of lipid or liposomal
formulations. The percentage of EGFP-positive cells was estimated
by flow cytometry at 72 h post-transfection. Untreated cells were
used as a negative control (fluorescence intensity, 100%). The siR-
NA delivery with LipofectamineÒ 2000 was used as a positive con-
trol. Cationic lipids insufficiently promoted the siRNA transfer into
the cells. No gene-silencing effect was detected under serum-free
2.3. Physicochemical characteristics of liposomes and
lipoplexes
Physicochemical properties of liposomes and their complexes
with nucleic acids (lipoplexes) are important characteristics that
can influence the transfection efficiency. We evaluated sizes and
surface potentials of liposomal formulations alone and complexes
formed with 25-mer FITC-ODN using the dynamic light scattering
instrument equipped with f-sizing capacity.
The average size of the liposomes with cationic glycolipids 7b–
d was found to be identical ꢁ100 nm. All liposomal formulations
were positively charged, and f potentials were found to be within
the range +13.07 to +34.38 mV (Table 2). The oligonucleotide
added to the liposomes induced the formation of significantly big-
ger particles: for the liposomal formulations tested (7a/DOPE and
7d/DOPE), formation of the particles of about 300 nm and
700 nm were observed. It is worse, noting that the initially opales-
cent solution of liposome/ODN complexes became turbid as the N/
P ratio was increased from 0.08 to 1. The surface potential of the
complexes prepared from liposomal formulation at N/P 1 was
found to be negative. Biological experiments showed that the
FITC-ODN cellular accumulation mediated by liposomal formula-
tions was not affected by the size or the f potential of the lipoplex-
es. It seems that these two characteristic parameters of lipoplexes
have midrange values and do not clarify the structure–activity
relationships, therefore, to answer what supramolecular structures
are formed by ODN and liposomal formulations, we have used the
transmission electron microscopy (TEM).
Electron microscopic images (Fig. 3) showed the large heteroge-
neity in sizes and structures of liposomal formulations. The biggest
amount was represented by homogeneous punctiforme particles
with sizes from 20 to 100 nm (Fig. 3A). Liposomal formulations
also contained closed lamellar structures within the range 50–
120 nm (Fig. 3B) which can be sort to liposomes.45 Interestingly,
in the case of 7d/DOPE, TEM picture showed the presence of a fin-
gerprint pattern (Fig. 3C).46
conditions for complexes at lipids concentration 2.5 or 5
EGFP expression was reduced by 10–17% as compared with the
control (100%) when the 10 M of glycolipids was used. When
lM, and
l
the medium was supplemented with 10% FBS, the cellular delivery
of siRNA by glycolipids 7a–d was completely inhibited (primary
data not shown).
In the case of the siRNA delivery with liposomal formulations,
the reliable down-regulation of EGFP expression was observed
(Table 1). Thus, in the absence of serum, cationic liposomes 7a/
DOPE, 7b/DOPE, and 7c/DOPE mediated the cellular delivery of siR-
NA, which resulted in the inhibition of EGFP synthesis by 27–30%.
In the presence of serum, the efficiency was approximately halved
for all tested liposomal formulations except for 7c/DOPE. For lipo-
somes 7c/DOPE (40 lM), similar inhibition levels of EGFP expres-
sion were observed in the presence and in the absence of serum
in the culture medium (29% and 31%, respectively). Under similar
conditions, when Lipofectamine was used as a delivery agent,
77% inhibition of EGFP expression was observed.
In our study, we characterized the delivery of siRNA mediated
by glycolipids/liposomal formulations by a down-regulation of
EGFP expression appearing when siRNA reached the cytosol. Thus,
the level of a biological effect depends on (1) cellular delivery of
siRNA and (2) endosomal and/or liposomal release of siRNA. Our
results showed that the cationic liposomes-containing lipids 7a–
d do deliver siRNA into the cells both in the presence and in the ab-
sence of serum. However, the down-regulation of EGFP expression
in the case of LipofectamineÒ 2000-mediated siRNA delivery was
2.5-fold higher. The results of our screening tests suggest that
low levels of siRNA-mediated EGFP silencing can be associated
with the endosomal or liposomal siRNA release rather than with
Samples of liposomal formulations entrapping oligonucleotide
at the N/P ratio of 1 (ODN (0.8 lM)–liposomes (20 lM)) contained
globular aggregates with rod-like elements, and the average size
was approximately 200–500 nm (Fig. 4A and B). The small lipid
particles appeared to aggregate in the presence of the ODN to form
multimodular complex particulates. Also we observed striped fine
structures, which were in a close contact with a punctiforme
Table 1
siRNA mediated inhibition of EGFP expression in BHK IR-780 cellsa
Delivery agent
Inhibition of EGFP expressionb (%)
Lipids concentration (
lM)
5
10
20
40
5
10
20
40
10% FBS
7a/DOPE
7b/DOPE
7c/DOPE
ꢀ
ꢀ
9
7
3
13
ꢀ
ꢀ
+
ni
13
ni
11
85.6
+
6
ni
ni
12
+
11
9
ni
15
+
5
9
31
ni
12
25
21
13
76.9
12
27
16
20
nic
13
29
22
7d/DOPE
LipofectamineÒ 2000
Flow cytometry data.
a
BHK IR-780 cells were treated with lipoplex composed of siRNA (50 nM) and liposomal formulation (from 5 to 40
l
M). Fluorescence of the cells was determined by FACS
analysis after 72 h of incubation with the corresponding lipoplexes. Transfection with LipofectamineÒ 2000 (10
lM) was carried out according to the manufacturer’s protocol.
Standard deviations were within 5%.
b
Level of EGFP expression in the control cells (without treatment with siRNA/liposome) was set at 100%. Inhibition of EGFP expression was calculated as follows [(EGFP
expressioncontrol ꢀ EGFP expressionexperiment)/EGFP expressioncontrol] ꢂ 100%.
c
ni, no inhibition.

M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
2443
Table 2
Hydrodynamic diameters and f potentials of liposomal formulations and lipoplexes
Lipids
Liposomal formulations cationic lipids/DOPE (1:1 mol)
Liposomal formulation/ODN complexesc
Average size and polydispersity (nm) f-potential (mV)
Average size and polydispersity (nm)
f-Potential (mV)
7a
72.9 11.3a
261 39.6b
+13.07
337.3 27.9
742.7 107.5
nd
ꢀ9.82
7b
7c
7d
100.5 18.7a
105.2 20.8a
134.4 24.1a
+23.12
+30.73
+34.38
ꢀ13.27
ꢀ18.91
ꢀ18.93
nd
361.0 31.7
708.3 136.9
The surface potential of ODN/liposomal formulation at N/P ratio of 1 was found to be negative.
nd, not determined.
a
Major population.
Minor population.
b
c
ODN (0.8 lM) was incubated with liposomal formulation (20 lM) (theoretical N/P ratio of 1) at 24 °C for 15 min.
an increase in the sizes of the lipoplexes (Fig. 4D). At the same
time, fingerprint-like fine structures were also clearly distinct
(Fig. 4E). Lipoplexes 7d/DOPE-ODN possessing higher transfection
activity contained more fingerprint structures than lipoplexes
formed by other liposomal formulations. This fact, that is, the pres-
ence of ‘transfection promoting’ fingerprint-like structures in 7d/
DOPE-ODN complexes, may explain the most pronounced transfec-
tion activity of liposomal formulation 7d/DOPE as compared with
other liposomal formulations tested.
Some distinct morphological differences were revealed for the
lipoplexes prepared at low N/P ratios. TEM images of 7a/DOPE-
ODN lipoplexes at N/P of 0.16 showed the presence of clustered
complexes formed by closely fine particles and the absence of
striped structures (Fig. 4F). The complexes prepared from cationic
liposomes and ODN at the minimal N/P ratio of 0.08 had shapeless
structures bound with occasional spherical particles (Fig. 4G).
Thus, we synthesized new cholesteryl glucosides containing
positively charged heterocyclic groups at the glucose C-6 atom.
The goal of this study was to determine the most promising candi-
date for further in vitro and in vivo laboratory applications, among
the tested compounds. Better understanding of the morphology of
lipoplexes is important for further rational development of liposo-
mal gene delivery systems. Our results give a starting point for the
subsequent enhancement of the gene delivery activity of systems
involving cationic cholesteryl glucosides.
Figure 3. Morphology of the liposomal formulations. Figure shows TEM images
(negative staining with phosphotungstic acid) of 1:1 7a/DOPE (A and C) and 7d/
DOPE (B) liposomal formulation. Lipid concentration is 20 lM. Notice, in C ordered
regions, similar structures are also found in 7d/DOPE liposome samples.
central region of lipoplexes (Fig. 4C). These stripes may be due to a
fingerprint-like structure, similar to those of complexes formed by
ODNs and cationic liposomes (cryo-EM data).47,48 The fingerprint-
striped structure is related to the formation of the lamellar lipid
phase47,49 but inverted hexagonal phases are also observed with
lipoplexes resulting from the association of ODN48 or DNA46 with
cationic liposomes. It is worse, noting that the fingerprint structure
was predominantly found for 7d/DOPE-ODN complexes. Decreas-
ing the N/P ratio to 0.5 (ODN 0.8 lM–liposomes 10 lM), led to
Figure 4. Morphology of the lipoplexes prepared from the ODN and liposomal formulations. (A and B) Lipoplexes composed of FITC-ODN and cationic liposomes 7a/DOPE at
the N/P ratios of 1 were of various sizes and shapes. (E) Striped fine structures, which were in close contact with a punctate central region of 7a/DOPE-ODN complexes were
observed. (C and F) Complexes composed of FITC-ODN and 7d/DOPE at the N/P ratio of 0.5. Fingerprint-like fine structures were also clearly distinct. (D and F) Lipoplexes
prepared at a low N/P (0.16 and 0.08) ratios showed clustered complexes and the absence of striped structures. TEM, negative staining with phosphotungstic acid.

2444
M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
d 170.56, 170.25, 169.40, and 169.23, (4 C(O)CH3), 140.56 (C-5),
3. Experimental
3.1. General methods
122.15 (C-6), 94.47 (C-1 Glc), 80.14 (C-3), 71.31 (C-3 Glc), 70.45
(C-2 Glc), 69.15 (C-4 Glc), 67.53 (C-5 Glc), 62.38 (C-6 Glc), 56.96
(C-14), 56.43 (C-17), 50.44 (C-9), 42.50 (C-13), 39.97 (C-4), 39.67
(C-12), 39.08 (C-24), 37.17 (C-1), 36.88 (C-10), 36.37 (C-22),
35.85 (C-20), 32.06 (C-7,8), 29.64 (C-2), 28.28 (C-16), 28.10 (C-
25), 24.38 (C-15), 23.98 (C-23), 22.84 and 22.66 (C-26,27), 21.30
(C-11), 20.64, 20.62, 20.56 (4 C(O)CH3), 19.42 (C-19), 18.87 (C-
21), 11.98 (C-18); C41H64O10 (716.95, 716.4499), MALDI-TOFMS:
m/z 739.9 [M+Na]+; Anal. Calcd for C41H64O10: C, 68.69; H, 9.00.
Found: C, 68.71; H, 8.58.
Toluene, ethyl methyl ketone, diethyl ether, and all amines
were purified and dried by distillation from CaH2 immediately be-
fore use. All solvents for column chromatography were distilled be-
fore use. Molecular sieves were activated at 180 °C under
diminished pressure for 2 h. Thin layer chromatography was per-
formed using pre-coated aluminum plates (Kieselgel 60 F254
,
Merck), which were visualized with the phosphomolybdic acid–
b-Anomer (2b): mp 152–154 °C, lit.51 157–159 °C; ½a D25
ꢀ20.15
ꢃ
ceric sulfate reagent.50 Flash column chromatography (FC) was
(c 1.0, CHCl3), lit.51
½
a 2D0
ꢃ
ꢀ26; 1H NMR (200 MHz): d 5.26–5.31 (m,
performed on Kieselgel 60 (40–63
l
m, Merck), silica gel (L
m, Chemapol, Czech Republic), and LichroprepÒ RP-18
m, Merck). Melting points were determined with
1H, H-6), 5.14 (t, 1H, J3,2, J3,4 9.4 Hz, H-3 Glc), 4.98 (t, 1H, J4,3, J4,5
9.4 Hz, H-4 Glc), 4.89 (dd, 1H, J2,1 7.7 Hz, J2,3 9.4 Hz, H-2 Glc),
4.52 (d, 1H, J1,2 7.7 Hz, H-1 Glc), 4.19 (dd, 1H, J6b,5 2.5 Hz, J6b,6a
12.0 Hz, Hb-6 Glc), 4.03 (dd, 1H, J6a,5 5.0 Hz, J6a,6b 12.0 Hz, Ha-6
Glc), 3.61 (ddd, 1H, J5,6b 2.5 Hz, J5,6a 5.0 Hz, J5,4 9.4 Hz, H-5 Glc),
3.32–3.49 (m, 1H, H-3), 2.04–2.19 (m, 2H, Ha,b-4), 2.01 (s, 3H),
1.98 (s, 3H), 1.95 (s, 3H), and 1.93 (s, 3H, 4 COCH3), 1.02–1.92
(m, 26H, H cholesterol), 0.92 (s, 3H, CH3-19), 0.84 (d, 3H, J21,20
6.4 Hz, CH3-21), 0.80 (d, 6H, J26,25, J27,25 6.4 Hz, CH3-26,27), 0.61
(s, 3H, CH3-18); 13C NMR (50 MHz): d 170.55, 170.26, 169.41, and
169.23 (4 C(O)CH3), 140.67 (C-5), 122.22 (C-6), 99.88 (C-1 Glc),
80.18 (C-3), 73.30 (C-3 Glc), 71.98 (C-2,4 Glc), 69.11 (C-5 Glc),
62.46 (C-6 Glc), 57.06 (C-14), 56.54 (C-17), 50.55 (C-9), 42.61 (C-
13), 40.04 (C-12), 39.75 (C-24), 39.16 (C-4), 37.45 (C-1), 36.95 (C-
10), 36.44 (C-22), 35.93 (C-20), 32.17 (C-7,8), 29.71 (C-2), 28.35
(C-16), 28.13 (C-25), 24.46 (C-15), 24.05 (C-23), 22.88 and 22.66
(C-26,27), 21.30 (C-11), 20.72, 20.70, 20.64 (4 C(O)CH3), 19.50 (C-
19), 18.94 (C-21), 12.03 (C-18); C41H64O10 (716.95, 716.4499),
MALDI-TOFMS: m/z 739.6 [M+Na]+; Anal. Calcd for C41H64O10: C,
68.69; H, 9.00. Found: C, 68.86; H, 8.67.
40–100
(40–63
l
l
a
Boetius apparatus (Germany). Optical rotations were recorded on
a Digytor Yasco DIP 360 (Japan) photoelectric spectropolarimeter
as [a]
D values. 1H and 13C NMR spectra were recorded at 24 °C with
Bruker MLS-200, Bruker AMX-400, and Bruker MSL-500 spectrom-
eters in CDCl3 as solvent unless otherwise stated. The signals of
SiMe4 (d = 0.00 ppm) and CDCl3 (d = 77.16 ppm) were used as
internal references. J values are given in Hertz. Signals were as-
signed by 2D proton–proton (COSY) and proton–carbon (HSQC)
shift correlation spectra. Mass spectra were recorded with a VG
ZAB-HSQ (VG Analytics) using 3-nitrobenzyl alcohol as a matrix
(FAB MS), a Vision 2000 (Thermo BioAnalysis) time-of-flight mass
spectrometer using 2,5-dihydroxybenzoic acid as a matrix (MALDI-
TOF MS), or a MS 7 T APEX II (FT ICR, Bruker-Daltonics) high-reso-
lution mass spectrometer in the positive mode (HRESIMS).
3.2. Cholest-5-en-3b-yl 2,3,4,6-tetra-O-acetyl-D-glucopyranoside
(2)
3.2.1. Procedure using mercury cyanide
3.3. Cholest-5-en-3-yl b-D-glucopyranoside (3)
HgCN2 (1.5 g, 6.09 mmol) and 4 Å molecular sieves (2.0 g) were
added to a soln of cholesterol (4.7 g, 12.19 mmol) in dry toluene
(26 mL) at 20 °C under stirring. After 30 min, the soln of 2,3,4,6-tet-
NaOMe (0.2 N, 8 mL) solution was added to the soln of 2b
(1.92 g, 2.68 mmol) in MeOH (20 mL) and CHCl3 (8 mL). After 3 h
at 20 °C. the reaction mixture was treated with Dowex 50w ꢂ 8
(H+), filtered, and concentrated under diminished pressure. The
crude product was recrystallized from MeOH to give the desired
product 3 as a white solid (1.47 g, 90%); mp 265–267 °C (from
ra-O-acetyl-a-D-glucopyranosyl bromide (1) (2.5 g, 6.09 mmol) in
dry toluene (8 mL) was added dropwise within 30 min. After 7 h
at 100 °C, the reaction mixture was cooled to ambient temperature,
filtered, and washed with 20% aq KI (2 ꢂ 5 mL) and water
(2 ꢂ 5 mL). The organic layer was dried (Na2SO4), filtered, and con-
centrated under diminished pressure. The residue was purified by
MeOH); ½a 2D5
ꢃ
ꢀ41.42 (c 0.8, CHCl3), lit.53
½
a 2D0
ꢃ
ꢀ48.5 (c 2.45, pyri-
dine); 1H NMR (400 MHz, Py-d5): d 5.32–5.37 (m, 1H, H-6), 5.05
(d, 1H, J1,2 7.6 Hz, H-1 Glc), 4.55 (dd, 1H, J6b,5 2.5 Hz, J6b,6a 11.9 Hz,
Hb-6 Glc), 4.40 (dd, 1H, J6a,5 5.3 Hz, J6a,6b 11.9 Hz, Ha-6 Glc), 4.22–
4.32 (m, 2H, H-3 Glc, H-5 Glc), 4.04 (dd, 1H, J2,1 7.6, J2,3 8.6 Hz,
H-2 Glc), 3.88–4.00 (m, 2H, H-3, H-4 Glc), 2.64–2.80 (m, 1H, Hb-
4), 2.38–2.56 (m, 1H, Ha-4), 0.99–2.20 (m, 24H, H cholesterol),
0.96 (d, 3H, J21,20 6.4 Hz, CH3-21), 0.93 (s, 3H, CH3-19), 0.89 (d,
6H, J26,25, J27,25 6.6 Hz, CH3-26,27), 0.65 (s, 3H, CH3-18); 13C NMR
(100 MHz): d 140.99, 121.98, 102.65, 78.63, 78.52, 78.19, 75.37,
71.75, 62.89, 56.90, 56.44, 50.44, 42.55, 40.04, 39.79, 39.41, 37.56,
36.99, 36.10, 36.55, 32.25, 32.13, 30.33, 28.57, 28.30, 24.56, 24.21,
FC (40/100 lm, 4:1 petroleum ether–diethyl ether,) to give a-(2a
0.84 g, 19%) and b-anomers (2b 1.92 g, 44%).
3.2.2. Procedure using cadmium carbonate
A
mixture of cholesterol (7.37 g, 19.06 mmol) and CdCO3
(1.64 g, 9.53 mmol) in dry toluene (80 mL) was refluxed in a Soxh-
let extractor filled with activated granular silica gel for 3 h. Bro-
mide 1 (3.51 g, 8.54 mmol) was then added portionwise within
1 h, and the mixture was additionally refluxed for 3 h and cooled.
Cadmium salts were filtered off, the solvent was evaporated, and
the residue was purified on silica gel to give
and b-anomers (2b 3.15 g, 51%) as white solids.
a-(2a 0.06 g, 1%)
23.00, 22.75, 21.36, 19.49, 19.01, 12.06;
548.4077), MALDI-TOFMS: m/z 571.7 [M+Na]+; Anal. Calcd for
33H56O6: C, 72.22; H, 10.29. Found: C, 72.11; H, 10.19.
C33H56O6 (548.00,
a
-Anomer (2a): mp 188–190 °C, lit.51 193–195 °C; ½a D25
ꢃ
+88.05
C
(c 1.0, CHCl3), lit.51
½
a 2D0
ꢃ
+92, lit.52
½
a 2D0 +88; 1H NMR (200 MHz):
ꢃ
d 5.42 (dd, 1H, J3,2 9.4 Hz, J3,4 10.2 Hz, H-3 Glc), 5.24–5.32 (m, 1H,
H-6), 5.16 (d, 1H, J1,2 3.8 Hz, H-1 Glc), 4.96 (t, 1H, J4,3, J4,5 9.4 Hz,
H-4 Glc), 4.74 (dd, 1H, J2,1 3.8 Hz, J2,310.2 Hz, H-2 Glc), 3.95–4.21
(m, 3H, H-5 Glc, Ha,b-6 Glc), 3.28–3.41 (m, 1H, H-3), 2.17–2.30
(m, 2H, Ha,b-4), 2.02 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H) and 1.95 (s,
3H, 4 COCH3), 0.97–1.92 (m, 26H, H cholesterol), 0.95 (s, 3H,
3.4. Cholest-5-en-3b-yl 2,3,4-tri-O-acetyl-6-O-methanesulfonyl-
b-D
-glucopyranoside (4)
Methanesulfonyl chloride (0.06 g, 0.55 mmol) in anhyd pyridine
(1.8 mL) cooled to ꢀ15 °C was added rapidly to the cooled soln of 3
(0.3 g, 0.55 mmol) in anhyd pyridine (25 mL). After 20 h at ꢀ15 °C,
the reaction mixture was warmed to 20 °C, Ac2O (0.31 mL,
3.3 mmol) was added, and the reaction mixture was kept for 3 h.
CH3-19), 0.84 (d, 3H, J21,20 6.4 Hz, CH3-21), 0.79 (d, 6H, J26,25
,
J27,25 6.4 Hz, CH3-26,27), 0.62 (s, 3H, CH3-18); 13C NMR (50 MHz):

M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
2445
The organic solvents were removed under diminished pressure,
and the resulting residue was purified by FC (40/100 , 7:3 petro-
4.06–4.20 (m, 1H, H-5 Glc), 4.03 (s, 3H, CH3N+), 3.40–3.47 (m,
1H, H-3), 2.73 (s, 3H, SCH3), 2.26 (s, 3H, COCH3), 2.17–2.21 (m,
1H, Hb-4), 2.03 (s, 3H, COCH3), 1.99 (m, 1H, Ha-4), 1.97 (s, 3H,
COCH3), 0.95–1.95 (m overlapped with s at 0.98, 26H, H choles-
terol), 0.98 (s, 3H, CH3-19), 0.91 (d, 3H, J21,20 6.5 Hz, CH3-21),
0.86 (d, 6H, J26,25, J27,25 6.6 Hz, CH3-26,27), 0.67 (s, 3H, CH3-18);
13C NMR (75 MHz, APT): d 170.97 (+), 169.95 (+) and 169.56 (+,
3C(O)CH3), 139.82 (+, C-5), 122.78 (ꢀ, C-6), 123.67 (ꢀ) and
122.31 (ꢀ, CH@CHN@CH), 99.83 (ꢀ, C-1 Glc), 80.63 (ꢀ, C-3),
72.29 (ꢀ, C-3 Glc), 71.66 (ꢀ, C-2 Glc), 71.29 (ꢀ, C-4 Glc), 68.31
(ꢀ, C-5 Glc), 56.84 (ꢀ, C-14), 56.28 (ꢀ, C-17), 50.20 (ꢀ, C-9 and
CH3N+),49.61 (+, C-6 Glc), 42.41 (+, C-13), 39.82 (+, C-12), 39.63
(+, C-24), 39.00 (+, C-4), 37.24 (+, C-1), 36.82 (ꢀ, SCH3), 36.75 (+,
C-10), 36.30 (+, C-22), 35.90 (ꢀ, C-20), 32.02 (+, C-7), 31.95 (ꢀ, C-
8), 29.80 (+, C-2), 28.35 (+, C-16), 28.14 (ꢀ, C-25), 24.40 (+, C-15),
23.95 (+, C-23), 23.00 and 22.70 (ꢀ, C-26, 27), 21.47 (ꢀ,
C(O)CH3), 21.19 (+, C-11), 20.82 (ꢀ) and 20.66 (ꢀ, 2 C(O)CH3),
l
leum ether–Et2O) to give 4 as a white crystalline solid (0.29 g,
70%); mp 170–172 °C; ½a D25
ꢃ
ꢀ15.35 (c 1.0, CHCl3); 1H NMR
(200 MHz): d 5.31–5.36 (m, 1H, H-6), 5.19 (t, 1 H, J3,2, J3,4 9.4 Hz,
H-3 Glc), 5.01 (dd, 1H, J4,3 9.4 Hz, J4,5 9.8 Hz, H-4 Glc), 4.92 (dd,
1H, J2,1 8.1 Hz, J2,3 9.4 Hz, H-2 Glc), 4.59 (d, 1H, J1,2 8.1 Hz, H-1
Glc), 4.27 (d, 2H, J6a,5, J6b,5 3.8 Hz, Ha,b-6 Glc), 3.75 (dt, 1 H, J5,6a
,
J5,6a 3.8 Hz, J5,4 9.8 Hz, H-5 Glc), 3.36–3.52 (m, 1H, H-3), 3.03 (s,
3H, SCH3), 2.07–2.21 (m, 2H, Ha,b-4), 2.03 (s, 6H) and 1.98 (s, 3H,
3 COCH3), 0.99–1.97 (m, 26H, H cholesterol), 0.97 (s, 3H, CH3-19),
0.88 (d, 3H, J20,21 6.4 Hz, CH3-21), 0.84 (d, 6H, J26,25, J27,25 6.4 Hz,
CH3-26,27), 0.66 (s, 3H, CH3-18); 13C NMR (50 MHz): d 170.12,
169.49 and 169.12 (3C(O)CH3), 140.34 (C-5), 122.33 (C-6), 99.80
(C-1 Glc), 80.25 (C-3), 72.90 (C-3 Glc), 71.80 (C-2 Glc), 71.69 (C-4
Glc), 68.78 (C-5 Glc), 67.31 (C-6 Glc), 56.91 (C-14), 56.43 (C-17),
50.41 (C-9), 42.50 (C-13), 39.93 (C-12), 39.67 (C-24), 39.05 (C-4),
37.80 (SCH3), 37.36 (C-1), 36.84 (C-10), 36.37 (C-22), 35.85 (C-
20), 32.07 (C-7, 8), 29.75 (C-2), 28.28 (C-16), 28.06 (C-25), 24.38
(C-15), 23.98 (C-23), 22.84 and 22.62 (C-26, 27), 21.22 (C-11),
20.56 (3 C(O)CH3), 19.42 (C-19), 18.87 (C-21), 11.96 (C-18);
19.48 (ꢀ, C-19), 18.85 (ꢀ, C-21), 12.00 (ꢀ, C-18); C44H70N2O11
S
(835.10, 834.4700), MALDI-TOFMS: m/z 739.9 [MꢀMsO+H]+; HRE-
SIMS: found 739.4876, calcd for [MꢀMsO]+ 739.4892.
C40H64O11
S
(753.00, 752.4169), MALDI-TOFMS: m/z 776.0
3.7. Cholest-5-en-3b-yl 2,3,4-tri-O-acetyl-6-deoxy-6-(N-
methylmorpholinio)-b-D-glucopyranoside iodide (5c)
[M+Na]+, 792.3 [M+K]+; Anal. Calcd for C40H64O11S: C, 63.80; H,
8.57; S, 4.26. Found: C, 63.63; H, 8.37; S, 4.16.
3.5. Cholest-5-en-3b-yl 2,3,4-tri-O-acetyl-6-deoxy-6-pyridinio-b-
3.7.1. Cholest-5-en-3 b-yl 2,3,4-tri-O-acetyl-6-deoxy-6-
morpholino-b- -glucopyranoside (6a)
D
-glucopyranoside methanesulfonate (5a)
D
A mixture of 4 (0.08 g, 0.102 mmol) and anhyd morpholine
(2 mL) was heated for 9 h at 70 °C. The reaction mixture was cooled
to 20 °C to add Ac2O (0.05 mL, 0.53 mmol). After 3 h, the mixture
was concentrated under diminished pressure and purified by FC
The soln of 4 (0.08 g, 0.11 mmol) in anhyd pyridine (2 mL) was
heated for 9 h at 115 °C. The residue obtained after removing the sol-
vent and traces of pyridine (0.1 Torr, 40 °C) was recrystallized from
dry Et2O to give 5a as a slightly beige solid (0.07 g, 78%); mp 247–
(40/63
l, CHCl3) to yield 6a as a white solid (0.048 g, 52%); mp
249 °C, ½a 2D5
ꢃ
ꢀ12.69 (c 1.0, CHCl3); 1H NMR (500 MHz): d 9.20 (m,
211–213 °C; ½a 2D5
ꢃ
ꢀ17.70 (c 1.0, CHCl3); 1H NMR (200 MHz): d
2H), 8.48 (m, 1H) and 8.03 (m, 2H, C5H5N+), 5.30–5.44 (m, H, H-6),
5.26–5.30 (m, 2H, H-2 Glc, H-3 Glc), 4.76–4.87 (m, 3H, H-4 Glc,
5.28–5.34 (m, 1H, H-6), 5.19 (t, 1H, J3,2, J3,4 9.4 Hz, H-3 Glc), 5.01
(dd, 1H, J4,3 9.4 Hz, J4,5 9.8 Hz, H-4 Glc), 4.92 (dd, 1H, J2,1 8.1 Hz,
J2,3 9.4 Hz, H-2 Glc), 4.51 (d, 1H, J1,2 8.1 Hz, H-1 Glc), 3.54–3.69
(m, 5H, H-5 Glc, CH2OCH2), 3.32–3.49 (m, 1H, H-3), 2.43–2.52
(m, 6H, Ha,b-6 Glc, CH2NCH2), 2.05–2.25 (m, 2H, Ha,b-4), 2.00 (s,
3H), and 1.99 (s, 3H) and 1.96 (s, 3H, 3 COCH3), 0.92–1.94 (m over-
lapped with s at 0.92 and 0.98, 26H, H cholesterol), 0.98 (s, 3H,
H
a,b-6 Glc), 4.60 (d, 1H, J1,2 8.1 Hz, H-1 Glc), 4.23–4.26 (m, 1H, H-5
Glc), 3.17–3.25 (m, 1H, H-3), 2.69 (s, 3H, SCH3), 2.21 (s, 3H, COCH3),
2.17–2.20 (m, 1H, H-4b), 2.07–2.12 (m, 1H, H-4a), 2.02 (s, 3H) and
1.99 (s, 3H, 2 COCH3), 1.92–1.95 (m, 3H, Hb-7, Ha,b-12), 1.72–1.86
(m, 3H, Hb-1, Hb-2, Hb-16), 0.95–1.59 (m, 20H, H cholesterol), 0.94
(s, 3H, H-19), 0.91 (d, 3H, J21,20 6.5 Hz, CH3-21), 0.86 (d, 6H, J26,25
,
CH3-19), 0.95 (d, 3H, J21,20 6.4 Hz, CH3-21), 0.83 (d, 6H, J26,25,
J27,25 6.6 Hz, CH3-26,27), 0.67 (s, 3H, CH3-18); 13C NMR (50 MHz): d
170.85, 169.82 and 169.38 (C(O)CH3), 139.83 (C-5), 146.95, 145.70
and 127.85 (C5H5N+),122.66 (C-6), 99.88 (C-1 Glc), 80.58 (C-3),
72.50 (C-3 Glc), 71.72 (C-2 Glc), 71.47 (C-4 Glc), 69.67 (C-5 Glc),
61.54 (C-6 Glc), 56.88 (C-14), 56.40 (C-17), 50.26 (C-9), 42.51
(C-13), 39.90 (C-12), 39.79 (SCH3), 39.67 (C-24), 38.94 (C-4), 37.21
(C-1), 36.77 (C-10), 36.37 (C-22), 35.89 (C-20), 32.03 (C-7, 8), 29.71
(C-2), 28.32 (C-16), 28.13 (C.25), 24.42 (C-15), 24.02 (C-23), 22.88
and 22.66 (C-26, 27), 21.22 (C-11), 21.04, 20.75 and 20.67 (3
J27,25 6.4 Hz, CH3-26,27), 0.64 (s, 3H, CH3-18); 13C NMR (50 MHz):
d 170.30, 169.52 and 169.30 (3 C(O)CH3), 140.64 (C-5), 122.22
(C-6), 99.80 (C-1 Glc), 80.10 (C-3), 73.45 (C-3 Glc), 71.75 (C-2
Glc), 72.05 (C-4 Glc), 70.99 (C-5 Glc), 67.05 (CH2NCH2), 59.37 (C-
6 Glc), 57.02 (C-14), 56.51 (C-17), 54.71 (CH2OCH2), 50.52 (C-9),
42.62 (C-13), 40.04 (C-12), 39.74 (C-24), 39.17 (C-4), 37.54 (C-1),
36.96 (C-10), 36.44 (C-22), 35.93 (C-20), 32.17 (C-7, 8), 29.82 (C-
2), 28.29 (C-16), 28.17 (C-25), 24.46 (C-15), 24.05 (C-23), 22.91
and 21.69 (C-26, 27), 21.30 (C-11), 20.87, 20.75 and 20.67 (3
C(O)CH3), 19.49 (C-19), 18.95 (C-21), 12.04 (C-18); C43H69NO9
(744.01, 743.4972), FABMS: m/z 744.5 [M+H]+; Anal. Calcd for
C(O)CH3), 19.24 (C-19), 18.87 (C-21), 12.00 (C-18); C45H69NO11
S
(832.10, 831.4591), MALDI-TOFMS: m/z 737.5 [MꢀMsO+H]+; HRE-
SIMS: found 736.4775, calcd for [MꢀMsO]+ 736.4793.
C
43H69NO9ꢄH2O: C, 67.78; H, 9.39; N, 1.84. Found: C, 67.77; H,
9.49; N, 1.40.
3.6. Cholest-5-en-3b-yl 2,3,4-tri-O-acetyl-6-deoxy-6-(N-
methylimidazolio)-b-
D-glucopyranoside methanesulfonate (5b)
3.7.2. Quaternarization of amine 6a
A mixture of 6a (0.050 g, 0.067 mmol) and freshly distilled MeI
(2 mL) was kept for 3 h at 40 °C. The excess of MeI was removed
under diminished pressure, and the residue was recrystallized
from dry Et2O to give 5c as a yellow amorphous solid (0.049 g,
A mixture of 4 (0.075 g, 0.10 mmol) and N-methylimidazole
(0.4 mL, 5 mmol) in dry ethyl methyl ketone (2 mL) was kept for
19 h at 100 °C. The reaction soln was concentrated under dimin-
ished pressure. The crude product was purified by FC (40/100
l,
82%); ½a 2D5
ꢃ
ꢀ13.72 (c 1.0, CHCl3); 1H NMR (500 MHz): d 5.48 (d,
7:3 CHCl3–MeOH) to give 5b as an amorphous solid (0.066 g,
1H, J1,2 8.1 Hz, H-1 Glc), 5.41–5.45 (m, 1H, H-6), 5.34 (t, 1H, J3,2,
80%); ½a 2D5
ꢃ
ꢀ30.03 (c 1.0, CHCl3); 1H NMR (500 MHz): d 10.47 (s,
J3,4 9.6 Hz, H-3 Glc), 4.98–5.04 (m, 1H, H-5 Glc), 4.96 (dd, 1H, J2,1
8.1 Hz, J2,3 9.6 Hz, H-2 Glc), 4.90 (t, 1H, J4,3, J4,5 9.6 Hz, H-4 Glc),
3.95–4.17 (m, 7H, CH2OCH2, CH2N+, Hb-6 Glc), 3.87 (s, 3H,
CH3N+), 3.76–3.85 (m, 2H, CH2N+), 3.68 (dd, 1H, J6a,5 9.4 Hz,
1H), 7.44 (s, 1H) and 7.19 (s, 1H, H imidazole), 5.33–5.37 (m, 1
H, H-6), 5.24 (t, 1 H, J3,2, J3,4 9.6 Hz, H-3 Glc), 4.85 (t, 1H, J4,3, J4,5
9.6 Hz, H-4 Glc), 4.68–4.79 (m, 4H, H-1 Glc, H-2 Glc, Ha,b-6 Glc),

2446
M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
J6a,6b13.9 Hz, Ha-6 Glc), 3.51–3.58 (m, 1H, H-3), 2.43–2.48 (m, 1H,
Hb-4), 2.30 (s, 3H, COCH3), 2.10–2.18 (m, 1H, Ha-4), 2.07 (s, 3H)
and 2.00 (s, 3H, 2 COCH3), 1.92–1.99 (m, 3H, Hb-7, Ha,b-12), 1.04–
1.84 (m, 23H, H cholesterol), 1.00 (s, 3H, CH3-19), 0.97 (m, 1H,
H-9), 0.95 (d, 3H, J21,20 6.5 Hz, CH3-21), 0.87 (d, 6H, J26,25, J27,25
6.6 Hz, CH3-26, 27), 0.67 (s, 3H, CH3-18); 13C NMR (125 MHz,
APT): d 170.66 (+), 170.17 (+) and 169.54 (+, 3 C(O)CH3), 139.63
(+, C-5), 123.14 (–, C-6), 98.67 (–, Glc-1), 80.29 (–, C-3), 72.10 (–,
C-3Glc), 71.07 (–, C-2 Glc), 69.99 (–, C-4 Glc), 67.70 (–, C-5 Glc),
64.20 (+, C-6 Glc), 62.70 (+) and 61.59 (+, CH2N+CH2), 60.84 (+,
CH2OCH2), 56.88 (–, C-14), 56.25 (–, C-17), 50.11 (–, N+CH3),
49.98 (–, C-9), 42.51 (+, C-13), 39.92 (+, C-12), 39.72 (+, C-24),
39.01 (+, C-4), 37.24 (+, C-1), 36.79 (+, C-10), 36.39 (+, C-22),
35.97 (–, C-20), 32.13 (+, C-7), 32.07 (–, C-8), 29.77 (+, C-2),
28.50 (+, C-16), 28.20 (–, C-25), 24.47 (+, C-15), 24.03 (+, C-23),
22.99 and 21.74 (–, C-26, 27), 22.56 (–, C(O)CH3), 21.25 (+, C-11),
20.87 (–) and 20.78 (–, 2 C(O)CH3), 19.51 (–, C-19), 18.91 (–, C-
21), 12.04 (–, C-18); C44H72INO9 (885.96, 885.4252), HRESIMS:
found 758.5197, calcd for [MꢀI]+ 758.5201.
overlapped with s at 1.99, 9H, cholesterol, CH2CH2CH2), 0.99–
1.84 (m, 25H, H cholesterol, CH2CH2CH2), 0.97 (s, 3H, CH3-19),
0.95 (m, 1H, H-9), 0.92 (d, 3H, J21,20 6.5 Hz, CH3-21), 0.86 (d, 6H,
J26,25, J27,25 6.6 Hz, CH3-26, 27), 0.67 (s, 3H, CH3-18); 13C NMR
(125 MHz, APT):
d 169.57 (+), 170.04 (+) and 170.56 (+, 3
C(O)CH3), 139.64 (+, C-5), 123.04 (–, C-6), 98.64 (–, C-1 Glc),
80.23 (–, C-3), 72.12 (–, C-3 Glc), 71.03 (–, C-2 Glc), 69.95 (–, C-4
Glc), 67.79 (–, C-5 Glc), 63.66 (+, C-6 Glc), 62.59 (+, CH2N+CH2),
56.84 (–, C-14), 56.26 (–, C-17), 50.83 (–, C-9), 50.00 (–, N+CH3),
42.46 (+, C-13), 40.87 (+, C-12), 39.69 (+, C-24), 38.95 (+, C-4),
37.20 (+, C-1), 36.75 (+, C-10), 36.34 (+, C-22), 35.96 (–, C-20),
32.09 (+, C-7), 32.00 (–, C-8), 29.70 (+, C-2), 28.41 (+, C-16), 28.20
(–, C-25), 24.47 (+, C-15), 24.01 (+, C-23), 23.01 (–) and 22.75 (–,
C-26, 27), 22.34 (–, C(O)CH3), 21.23 (+, C-11), 20.92 (–) and 20.80
(–, 2 C(O)CH3), 20.43 (+, CH2CH2CH2), 19.50 (–, C-19), 18.89 (–, C-
21), 12.03 (–, C-18); C45H74INO8 (883.98, 883.4459), HRESIMS:
found 756.5395, calcd for [MꢀI]+ 756.5409.
3.9. General procedure for the removal of acetyl groups
3.8. Cholest-5-en-3b-yl 2,3,4-tri-O-acetyl-6-deoxy-6-(N-
methylpiperidinio)-b-D-glucopyranoside iodide (5d)
Freshly prepared sodium methylate (0.1 N, 1.4 mL) in MeOH
was added to the protected glucosides 5a–d (0.141 mmol) in
MeOH (3.5 mL). The reaction mixture was kept at 20 °C until no
starting material was detectable (TLC) and then treated with Dow-
ex 50w ꢂ 8 (H+) ion-exchange resin. The resin was filtered off,
washed with MeOH, the combined filtrate was evaporated and
the crude product was purified by the FC on RP-18 silica gel Lichro-
prepÒ (Merck). The respective starting glucosides, reaction time,
chromatographic eluent, yield, and the physical and spectroscopic
data for 7a–d are as follows.
3.8.1. Cholest-5-en-3b-yl 2,3,4-tri-O-acetyl-6-deoxy-6-piperidi-
no-b- -glucopyranoside (6b)
D
Methanesulfonate 4 (0.07 g, 0.093 mmol) was treated with
piperidine (1 mL) at 60 °C for 3 h and Ac2O (0.05 mL, 0.53 mmol)
as described for 6a. The FC on silica gel (40/63 lm, CHCl3) gave
6b (0.04 g, 58%) as a white solid: mp 198–200 °C, ½a D25
ꢀ24.25 (c
ꢃ
1.0, CHCl3); 1H NMR (200 MHz): d 5.27–5.34 (m, 1H, H-6), 5.12
(t, 1H, J3,2, J3,4 9.4 Hz, H-3 Glc), 5.01 (dd, 1H, J4,3 9.4 Hz, J4,5
9.8 Hz, H-4 Glc), 4.86 (dd, 1H, J2,1 8.1 Hz, J2,3 9.4 Hz, H-2 Glc),
4.49 (d, 1H, J1,2 8.1 Hz, H-1 Glc), 3.58 (dt, 1H, J5,6 4.7 Hz, J5,4
9.8 Hz, H-5 Glc), 3.31–3.48 (m, 1H, H-3), 2.43 (d, 2H, J6,5 4.7 Hz,
H-6 Glc), 2.26–2.40 (m, 4H, CH2NCH2), 2.02–2.21 (m, 2H, Ha,b-4),
2.00 (s, 3H), 1.98 (s, 3H) and 1.96 (s, 3H, 3 COCH3), 1.00–1.94 (m,
32H, H cholesterol, (CH2)3), 0.93 (s, 3H, CH3-19), 0.86 (d, 3H,
J21,20 6.4 Hz, CH3-21), 0.81 (d, 6H, J26,25, J27,25 6.8 Hz, CH3-26,27),
0.62 (s, 3H, CH3-18); 13C NMR (50 MHz): d 169.34, 169.63 and
170.67 (3 C(O)CH3), 140.74 (C-5), 122.14 (C-6), 99.73 (C-1 Glc),
80.07 (C-3), 73.52 (C-3 Glc), 72.94 (C-2 Glc), 72.13 (C-4 Glc),
71.28 (C-5 Glc), 60.03 (C-6 Glc), 57.06 (C-14), 56.51 (C-17), 55.59
(CH2NCH2), 50.52 (C-9), 42.61 (C-13), 40.04 (C-12), 39.75 (C-24),
39.23 (C-4), 37.54 (C-1), 36.96 (C-10), 36.44 (C-22), 35.93 (C-20),
32.17 (C-7, 8), 29.79 (C-2), 28.39 (C-16), 28.17 (C-25), 26.26
(CH2CH2CH2), 24.50 (C-15), 24.34 (CH2CH2CH2), 24.05 (C-23),
22.91 and 22.69 (C-26, 27), 21.30 (C-11), 20.89, 20.78, 20.71 (3
C(O)CH3), 19.50 (C-19), 18.95 (C-21), 12.04 (C-18); C44H71NO8
(742.04, 741.5180), FABMS: m/z 742.5 [M+H]+; Anal. Calcd for
C44H71NO8: C, 71.22; H, 9.64; N, 1.89. Found: C, 71.37; H, 9.80;
N, 1.81.
3.9.1. Cholest-5-en-3b-yl 6-deoxy-6-pyridinio-b-D-glucopyrano-
side methanesulfonate (7a)
Compound 5a (0.117 g, 0.141 mmol), 1 h, 10:100:1 CHCl3–
MeOH–water; 7a as a beige amorphous solid (0.078 g, 78%); ½a D25
ꢃ
ꢀ14.2 (c 1.0, CHCl3–CH3OH, 2:1); 1H NMR (400 MHz, 2:1 CDCl3–
CD3OD): d 8.87 (m, 2H), 8.50 (m, 1H) and 8.01 (m, 2H, C5H5N+),
5.17–5.21 (m, 1H, H-6), 4.94 (dd, 1H, J6b,5 3.0, J6b,6a 13.5, Hb-6
Glc), 4.69 (dd, 1H, J6a,5 7.6, J6a,6b 13.5, Ha-6 Glc), 4.26 (d, 1 H, J1,2
7.8, H-1 Glc), 3.66–3.73 (m, 1H, H-5 Glc), 3.39 (br t, 1H, J3,2, J3,4
9.0, H-3 Glc), 3.15–3.25 (m, 1H, H-3), 3.00–3.11 (m, 2H, H-4 Glc,
H-2 Glc), 2.66 (s, 3H, SCH3), 2.07–2.22 (m, 2H, H-4a,b), 1.87–1.98
(m, 3H, Hb-7, Ha,b-12), 1.66–1.82 (m, 3H, Hb-1, Hb-2, Hb-16),
0.95–1.59 (m, 20H, H cholesterol), 0.90 (s, 3H, H-19), 0.84 (d, 3H,
J21,20 6.5, CH3-21), 0.78 (d, 6H, J26,25, J27,25 6.6, CH3-26, 27), 0.60
(s, 3H, CH3-18); 13C NMR (50 MHz): d 145.84, 145.71, 139.98,
127.91, 122.13, 101.74, 79.73, 75.84, 73.67, 73.30, 70.91, 62.51,
56.73, 56.19, 50.21, 42.26, 39.73, 39.43, 38.81, 38.48, 37.15,
36.58, 36.11, 35.70, 31.80, 29.60, 28.10, 27.88, 24.14, 23.73,
22.49, 22.25, 21.01, 19.05, 18.51, 11.65; C39H63NO6S (705.99,
705.4274), MALDI-TOFMS: m/z 610.492 [M-MsO]+; HRESIMS:
found 610.4472, calcd for [MꢀMsO]+ 610.4471.
3.8.2. Quaternization of amine 6b
Cholest-5-en-3b-yl 2,3,4-tri-O-acetyl-6-deoxy-6-(N-methylpipe-
3.9.2. Cholest-5-en-3b-yl 6-deoxy-6-(N-methylimidazolio)-b-D-
ridinio)-b-
D-glucopyranoside iodide (5d) was prepared by the pro-
glucopyranoside methanesulfonate (7b)
cedure described for compound 5c using 6b (0.030 g, 0.04 mmol)
and MeI (0.6 mL). The product was purified by the FC on silica
Compound 5b (0.095 g, 0.11 mmol), 3 h, 100:1 MeOH–water;
7b as a beige amorphous solid (0.037 g, 45%); ½a D25
ꢀ44 (c 1, 1:2
ꢃ
gel (40/63
l
, 10:1 CHCl3–MeOH) to yield 5d as a yellow solid
CHCl3–MeOH); 1H NMR (400 MHz, 2:1 CDCl3–CD3OD): d 9.02 (s,
1H), 7.43 (s, 1H) and 7.45 (s, 1H, imidazole), 5.30–5.35 (m, 1 H,
H-6), 4.39–4.55 (m, 3H, H-1 Glc, Ha,b-6 Glc), 3.94 (s, 3H, CH3N+),
3.57–3.64 (m, 1H, H-5 Glc), 3.39–3.49 (m, 2H, H-3, H-3 Glc),
3.09–3.17 (m, 1H, H-4 Glc), 2.97–3.04 (m, 1H, H-2 Glc), 2.69 (s,
3H, SCH3), 0.94–2.30 (m overlapped with s at 0.98, 28H, H choles-
terol), 0.99 (s, 3H, CH3-19), 0.89 (d, 3H, J21,20 6.5, CH3-21), 0.84 (d,
6H, J26,25, J27,25 6.6, CH3-26, 27), 0.66 (s, 3H, CH3-18); 13C NMR
(0.022 g, 72%): mp 142–144 °C ½a D25
ꢃ
ꢀ15.67 (c 1.0, CHCl3); 1H
NMR (500 MHz) d 5.42–5.45 (m, 1H, H-6), 5.40 (d, 1H, J1,2 8.1 Hz,
H-1 Glc), 5.31 (br t, 1H, J3,2, J3,4 9.3 Hz, H-3 Glc), 4.95 (dd, 1H, J2,1
8.1 Hz, J2,3 9.7 Hz, H-2 Glc), 4.82–4.89 (m, 2H, H-4 Glc, H-5 Glc),
3.96–4.09 (m, 2H, CH2N+), 3.73–3.88 (m, 2H, CH2N+), 3.68–3.70
(m, 1H, Hb-6 Glc), 3.66 (s, 3H, CH3N+), 3.51–3.58 (m, 2H, H-3, Ha-
6 Glc), 2.38–2.43(m, 1H, Hb-4), 2.28 (s, 3H, COCH3), 2.09–2.17 (m,
1H, Ha-4), 2.06 (s, 3H) and 1.99 (s, 3H, 2 COCH3), 1.79–2.02 (m
(100 MHz):
d 139.82, 124.02, 123.51, 122.45, 101.86, 79.98,

M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
2447
76.22, 73.70, 73.58, 70.65, 57.07, 56.51, 50.73, 50.56, 42.61, 40.07,
39.77, 38.87, 37.58, 36.97, 36.45, 36.33, 36.03, 32.17, 29.93, 28.43,
28.21, 24.49, 24.06, 22.84, 22.60, 21.35, 19.42, 18.86, 11.99;
HPLC (produced at the Institute of Chemical Biology and Funda-
mental Medicine, Siberian Branch, Russian Academy of Sciences,
Russia). The purity of oligonucleotide, as analyzed by electrophore-
sis in 20% PAAm/8 M urea gel, was 95–98%. FITC-labeling of the oli-
gonucleotide was performed as previously described.54 FITC-ODN
was isolated by conventional HPLC (Alliance, Waters, USA) using
a Waters XTerra column and a linear acetonitrile concentration
gradient. The purity of FITC-ODN samples as analyzed by electro-
phoresis in 20% PAAm/8 M urea gel was 95–98%. Concentrations
of oligonucleotides were measured in a BioMate 3 (Termo Electron
Corporation, USA) spectrophotometer. FITC-ODN soln was stored
at ꢀ20 °C until used.
C
38H64N2O8S (708.99, 708.4383), MALDI-TOFMS: m/z 613.111
[MꢀMsO]+; HRESIMS: found 613.4580, calcd for [MꢀMsO]+
613.4581.
3.9.3. Cholest-5-en-3b-yl 6-deoxy-6-(N-methylmorpholinio)-b-D-
glucopyranoside iodide (7c)
Compound 5c (0.102 g, 0.115 mmol), 3 h, 100:1 MeOH–water;
7c as a slightly yellow amorphous solid (0.045 g, 52%); ½a D25
ꢀ31
ꢃ
(c 1, 1:2 CHCl3–MeOH); 1H NMR (400 MHz, Py-d5): d 5.07–5.19
(m, 2H, H-6, H-1 Glc), 4.58–4.66 (m, 1H, H-5 Glc), 4.45–4.52 (m,
1H, Hb-6 Glc), 3.95–4.26 (m, 1H, H-3 Glc), 3.73–4.14 (m, 9H,
CH2OCH2, CH2N+CH2, Ha-6 Glc), 3.56–3.72 (m, 6H, H-3, H-2 Glc,
H-4 Glc, CH3N+), 2.52–2.61 (m, 1H, Hb-4), 2.15–2.28 (m, 1H, Ha-
4), 0.76–1.90 (m, 28H, H cholesterol), 0.73 (d, 3 H, J21,20 6.5, CH3-
21), 0.69 (s, 3H, CH3-19), 0.64 (d, 6 H, J26,25, J27,25 6.6, CH3-26,
27), 0.41 (s, 3H, CH3-18); 13C NMR (100 MHz): d 140.37, 122.53,
100.66, 79.57, 75.71, 73.37, 71.30, 69.94, 66.07, 65.13, 61.43,
60.84, 57.01, 56.44, 50.45, 50.30, 42.51, 39.98, 39.71, 38.76,
37.54, 36.94, 36.40, 35.96, 32.11, 29.82, 28.39, 28.18, 24.45,
24.03, 22.88, 22.64, 21.27, 19.40, 18.86, 15.16, 12.00; C38H66INO6
(759.85, 759.3935), MALDI-TOFMS: m/z 632.161 [MꢀI]+; HRESIMS:
found 632.4891, calcd for [MꢀI]+ 632.4890.
3.12. siRNA duplex
The siRNA targeted to EGFP mRNA (encoding enhanced green
fluorescent protein) with the following sense and antisense strand
sequences was used: 5’-GAA CGG CAU CAA GGU GAA CTT-3’
(sense) and 5’-GUU CAC CUU GAU GCC GUU CTT-3’ (antisense).55
The chosen siRNA-EGFP was able to inhibit the EGFP expression
in transgenic cells. The sense and the antisense strands of siRNA
synthesized by the solid-phase phosphoramidite method were
kindly provided by Dr. Venyaminova (Institute of Chemical Biology
and Fundamental Medicine, Siberian Branch, Russian Academy of
Sciences, Russia). To obtain the siRNA duplexes, equal amounts of
oligoribonucleotides were suspended in hybridization buffer
(50 mM potassium acetate, 1 mM magnesium acetate, and
15 mM HEPES/KOH pH 7.4) and annealed by heating to 90 °C for
5 min with subsequent slow cooling. The resulting siRNA duplexes
were analyzed by electrophoresis in 15% PAAm/8 M urea gel. The
stock solutions were stored at ꢀ20 °C.
3.9.4. 5-Cholest-5-en-3b-yl 6-deoxy-6-(N-methylpiperidinio)-b-
D
-glucopyranoside iodide (7d)
Compound 5d (0.041 g, 0.046 mmol), 3 h, 10:100:1 CHCl3–
MeOH–water?4:10 chloroform–methanol; 7d as an amorphous
solid (0.032 g, 89%); ½a D25
ꢃ
ꢀ24 (c 1, 1:2 CHCl3–MeOH); 1H NMR
(400 MHz, CDCl3–CD3OD, 4:1) d 5.26–5.31 (m, 1H, H-6), 4.45 (d,
1 H, J1,2 7.8, H-1 Glc), 3.77–3.85 (m, 1H, H-5 Glc), 3.67–3.74 (m,
1H, Hb-6 Glc), 3.28–3.49 (m, 10H, H-3, CH3N+, CH2N+CH2, H-3
Glc, Ha-6 Glc), 3.05–3.18 (m, 2H, H-2 Glc, H-4 Glc), 2.17–2.38 (m,
2H, Ha,b-4), 1.79–2.02 (m, 9H, cholesterol, CH2CH2CH2), 0.95–1.84
(m, 26H, H cholesterol, CH2CH2CH2), 0.94 (s, 3H, CH3-19), 0.84 (d,
3H, J21,20 6.5, CH3-21), 0.79 (d, 6H, J26,25, J27,25 6.6, CH3-26,27),
0.61 (s, 3H, CH3-18); 13C NMR (100 MHz): 139.73, 123.09, 100.56,
79.71, 75.15, 73.20, 71.03, 69.92, 63.84, 62.36, 56.98, 56.38,
50.76, 50.28, 42.50, 40.07, 39.71, 38.85, 37.36, 36.81, 36.34,
35.92, 32.14, 32.00, 29.74, 28.36, 28.18, 24.45, 24.06, 22.89,
22.68, 21.23, 20.49, 19.45, 18.90, 11.98; C39H68INO5 (757.88,
757.4142), MALDI-TOF MS: m/z 630.174 [MꢀI]+.
3.13. Preparation of the complexes of cationic glycolipids (or
liposomes) and nucleic acids
Prior to use, the complexes of cationic glycolipids (or liposomes)
and nucleic acids were formed in a serum-free Opti-MEM medium
(Invitrogen, USA) by vigorously mixing 25
lL of glycolipid or lipo-
somal solution and 25 L of oligonucleotide or siRNA taken at an
l
appropriate concentration; the resulting mixtures were incubated
for 20 min at a room temperature.
3.14. Biological methods
3.14.1. Cell line and cultivation conditions
3.10. Lipid formulations
The genetically modified BHK IR780 cell line containing con-
stantly expressed EGFP gene insert in the genomic DNA, obtained
from the parental cell line of BHK (hamster kidney) cells, was
kindly provided by Prof. Prasolov (Engelhardt Institute of Molecu-
lar Biology, Russian Academy of Sciences, Moscow, Russia). The ini-
tial BHK and the transgenic BHK IR780 cell lines were maintained
in the DMEM supplemented with 10% fetal bovine serum (FBS),
Solns of cationic glycolipids 7a–d in Me2SO were prepared as
follows: the weighed samples of the lipids were supplemented
with Me2SO, and the mixtures were intensively vortexed and son-
icated in an ultrasonic bath (Cole-Parmer, USA) until soln clarifica-
tion. The stock solns of the lipids in Me2SO (1 mM) were stored at
ꢀ20 °C.
100 U/ml penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL
For producing the liposomes, lipids 7a–d were supplemented
with the DOPE soln in CHCl3 (Fluka) and the solvent was carefully
removed under an argon flow. The obtained lipid film was dried
under a vacuum (0.1 Torr) for 1 h. Water was added, and the lipid
film was hydrated at 50 °C by intensive vortexing. The samples
were flushed with argon and sonicated for 20 min in a bath-type
sonicator. Liposomes were stored at 4 °C under argon atmosphere.
amphotericin in a humidified atmosphere containing 5% CO2/95%
air at 37 °C and were regularly passaged to keep the exponential
growth.
3.14.2. Cell viability test (MTT assay)
The cytotoxicities of glycolipids and liposome formulations
were assessed using a colorimetric assay based on the reduction
of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) by living cells.44 Prior to the cytotoxicity assay, the condi-
tions for cells growth were optimized to achieve 90% of cell mono-
layer after 48 h incubation. The cells were seeded into 96-well
3.11. Oligonucleotide
The 25-mer oligodeoxyribonucleotide with an aminohexyl lin-
ker at the 3’-end (5’-TAC AGT GGA ATT GTA TGC CTA TTA T-3’)
was synthesized by a phosphoramidite method and purified by
plates (104 cells per well) and cultivated in 100
supplemented with 10% FBS at 37 °C in a humidified atmosphere
lL of the DMEM

2448
M. A. Maslov et al. / Carbohydrate Research 345 (2010) 2438–2449
with 5% CO2 for 24 h to adhere. Then the medium in wells was
replaced with 150 L of DMEM without antibiotics (with or with-
out 10% FBS) containing different concentrations of the cationic
glycolipids or liposome formulations (1–120 M). After 24 h incu-
bation in the presence of glycolipids or liposomes, MTT was added
to the cells to a final concentration of 0.5 g/mL. The optical den-
FITC-positive cells was calculated by determining their fluores-
cence emission at a PMT equipped with a 502 LP dichroic mirror
and a wavelength optical filter 530/30 nm. All experimental points
were run in duplicate for statistical analysis; the standard devia-
tion did not exceed 5%.
l
l
l
sity of the samples was measured 3 h after MTT addition at
570 nm in a Multiscan RC (Labsystems). All experimental points
were run in four replicates. The results were represented as a per-
centage of living cells versus the control (untreated cells, 100%).
S.D. was below 5%. For each glycolipid (liposome formulation),
the IC50 value, that is, the concentration of a compound providing
for 50% cell lethality was determined.
3.15. Physicochemical characterizations of liposomes and
lipoplexes
Particle size and f potential were measured using a Delsa Nano
dynamic light scattering instrument with f-sizing capacity
(Beckman Coulter). For measuring complexes characteristics ODN
in 450 mL of distilled water was mixed with an equal vol of
liposomal formulation (in distilled water) at the ratio 0.8:20 mol
and incubated for 15 min at room temperature.
3.14.3. Cellular delivery of FITC-labeled oligonucleotide and
siRNA
The delivery efficiency for the nucleic acids complexed with
glycolipids was evaluated in transfection experiments with the
25-mer FITC-labeled oligonucleotide and anti-EGFP siRNA.
For FITC-ODN transfection experiments, BHK cells were seeded
into 24-well plates (3 ꢂ 104cells/well) in the DMEM containing
10% FBS and antibiotics 24 h before transfection and grown in an
incubator at 3 °C in the presence of 5% CO2. Immediately before
transfection, the cells were rinsed with PBS supplemented with
3.15.1. Transmission electron microscopy: characterization of
liposomes and ODN/liposome complexes by negative staining
The liposomal formulations or their complexes with ODN were
prepared as described above. The samples were absorbed on cop-
per grids covered with formvar film during 20 s, then dried by
touch with filter paper. For contrasting, the grids were put on a
drop of 2% phosphotungstic acid (pH 7.2) for 15 s. Excess stain
was removed by filter paper. The grids were examined in Jem
1400 transmission electron microscope (JEOL, Japan) at 80 kV
accelerating voltage. Digital images were collected by Veleta CCD
camera (Olympus SIS, Germany).
200
taining 10% FBS. The complexes of cationic lipids (or liposomes)
(10 M or 20 M) and FITC-ODN (0.8 or 5 M) were formed in
50 L of serum-free Opti-MEM medium as described above. Cells
lL of fresh serum-free culture medium or the medium con-
l
l
l
l
were supplemented with these complexes and incubated for 4 h.
FITC-ODN delivery with Lipofectamine2000 was done according
to manufacture protocol (www.invitrogen.com).
For siRNA delivery, BHK IR780 cells were seeded into 24-well
plates (2 ꢂ 104 cells/well) in the DMEM medium containing 10%
FBS and antibiotics 24 h before transfection and grown in an incu-
bator at 37 °C in the presence of 5% CO2. Immediately before trans-
fection, the cells were rinsed with PBS and supplemented with
200 lL of fresh serum-free culture medium or the medium con-
taining 10% FBS. The complexes of siRNA and cationic glycolipids
(liposomes) were preformed using 50 nM siRNA and glycolipids
at concentrations of 5, 10, 20, or 40 lM as described above and
Acknowledgments
The authors are grateful to Professor V. Prasolov (Engelhardt
Institute of Molecular Biology, Russian Academy of Sciences,
Moscow, Russia), who produced and kindly provided the geneti-
cally modified BHK IR780 cell line used in this study; to Dr. Ya.
Efremov (Center of Microscopic Analysis, Institute of Cytology
and Genetics, Siberian Branch, Russian Academy of Sciences,
Novosibirsk Russia), who performed FACS analysis of our speci-
men; and to A. Vladimirova (Institute of Chemical Biology and
Fundamental Medicine, Siberian Branch, Russian Academy of
Sciences), who maintained all cell cultures used in this work.
This work was supported by the Russian Academy of Sciences
under the programs ‘Molecular and Cell Biology’ and ‘Science for
Medicine’; Russian Foundation for Basic Research (Grants Nos.
08-04-00753 and 09-03-00874), Ministry of Science and Education
of the Russian Federation (state contract no, 02.512.11.2200),
President’s program in support of leading scientific schools
SS-7101.2010.4.
were added to the cells; 4 h after transfection, the culture medium
was replaced with the DMEM containing 10% FBS. siRNA delivery
with Lipofectamine2000 was done according to manufacture pro-
tocol (www.invitrogen.com).
3.14.4. FACS analysis
Flow cytometry studies were used to characterize the efficiency
of cell transfection with FITC-labeled oligonucleotide and the
EGFP-targeted siRNA mediated by glycolipids (liposome formula-
tions). In the case of oligonucleotide delivery, the cells were
analyzed immediately after transfection (4 h after addition of gly-
colipid/oligonucleotide complexes). In the case of siRNA delivery,
cells were analyzed 72 h after transfection (this time is necessary
for the already synthesized protein to be degraded, because the
half-life time for EGFP is considered to be 48 h). The cells were
rinsed twice with PBS and detached from the plate by trypsin treat-
ment (0.5 mg/mL in PBS) at 37 °C for 2 min. The trypsinized cells
were resuspended in the culture medium and collected by centri-
fugation (a Contron T42K centrifuge, Centricon Instruments) at
1000 rpm for 10 min at 4 °C. After centrifugation, the medium
was removed and the cells were washed with PBS and fixed with
2% formaldehyde in PBS. The resulting samples assayed by cytoflu-
orometry using a fluorescence-activated cell sorter (BD FACSAria,
Becton Dickinson). Fluorescence intensity of individual cells was
measured in relative fluorescence units (RFU). The fluorescence
was collected from 3 ꢂ 104 individual cells. The percentage of
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