Spacer-arm modulated gene delivery efficacy of novel cationic glycolipids: Design, synthesis, and in vitro transfection biology

Article abstract of DOI:10.1021/jm030464i

Design, syntheses and relative in vitro gene delivery efficacies of six novel cationic glycolipids 1-6 containing open-form galactosyl units in CHO, COS-1, MCF-7 and A549 cells are described. The results of the present structure-activity investigation con

Full text of DOI:10.1021/jm030464i

3938
J . Med. Chem. 2004, 47, 3938-3948
Articles
Sp a cer -Ar m Mod u la ted Gen e Deliver y Effica cy of Novel Ca tion ic Glycolip id s:
Design , Syn th esis, a n d in Vitr o Tr a n sfection Biology
Yenugonda Venkata Mahidhar, Mukthavaram Rajesh, and Arabinda Chaudhuri*
Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad-500 007, India
Received September 18, 2003
Design, syntheses and relative in vitro gene delivery efficacies of six novel cationic glycolipids
1-6 containing open-form galactosyl units in CHO, COS-1, MCF-7 and A549 cells are described.
The results of the present structure-activity investigation convincingly demonstrate that the
in vitro gene delivery efficacies of galactosylated cationic glycolipids are strikingly dependent
on the absence of a spacer-arm between the open-form galactose and the positively charged
nitrogen atom in their headgroup region. While the cationic glycolipids 1-3 with no headgroup
spacer unit between the positively charged nitrogen and galactose showed high in vitro gene
transfer efficacies in all four cells (lipids 1 and 2 with myristyl and palmityl tails, respectively,
being the most efficacious), lipids 4-6 with five-carbon spacer units between the quaternized
nitrogen and galactose heads were essentially transfection incompetent. The transfection
inhibiting role of the five-carbon spacer unit in the headgroup region of the present novel class
of cationic lipids was demonstrated by both â-galactosidase reporter gene expression and
histochemical X-gal staining assays. Results of MTT assay-based cell viability measurements
in representative MCF7 cells show that cell viabilities of lipoplexes (lipid:DNA complexes)
prepared from all the lipids 1-6 are remarkably high. Thus, possibilities of differential cellular
cytotoxicities playing any key role behind the strikingly contrasting transfection properties of
lipids 1-3 with no spacer and lipids 4-6 with a spacer unit in the headgroup regions was
ruled out. Electrophoresis gel patterns in DNase I sensitivity assays are consistent with more
free DNA (accessible to DNase I) being present in lipoplexes of lipids 4-6 than in lipoplexes
of lipids 1-3. Thus, the results of our DNase I protection experiments support the notion that
enhanced degradation of DNA associated with lipoplexes of lipids 4-6 may play an important
role in abolishing their in vitro gene transfer efficacies.
In tr od u ction
cationic transfection lipids,25-33 we recently demon-
strated the potential of nonglycerol-based cationic gly-
colipids containing open-arabinose and -xylose sugar-
heads for use in liposomal gene delivery. In the molecular
structures of these novel cationic glycolipids, both the
arabinose and xylose sugar-heads were directly linked
to the positively charged nitrogen atoms of the cationic
glycolipids without using any spacer in between.32 With
a view to probe the role of spacer-arm between the
positively charged nitrogen atom and the sugar-head
functionality in cationic glycolipid-mediated gene de-
livery, in the present investigation, we have designed
and synthesized six novel cationic glycolipids 1-6
(Chart 1) with either having five-carbon spacer arms
between the positively charged nitrogen and the galac-
tose-heads (lipids 4-6) or having no spacer-arms (lipids
1-3). As described below, lipids 1-3 with no spacer unit
in the headgroup region were found to be remarkably
more efficient in transfecting multiple cells including
CHO, COS-1, MCF-7 and A549 than lipids 4-6 contain-
ing five-carbon spacer arms. Herein, we report on the
design, synthesis, physicochemical characterizations
and the strikingly spacer-arm-dependent in vitro trans-
fection biology of the novel cationic glycolipids 1-6.
Gene therapy is gaining increasing importance as a
therapeutic modality to combat myriads of inherited
diseases, dreadful viral infections and cancer. A key
challenge in the gene therapy approach is to develop
safe and efficient vehicles for delivering therapeutic
genes into body cells. Recombinant viral vectors, al-
though remarkably efficient in transfecting body cells,
suffer from numerous biosafety related disadvantages.1-3
Viral vectors have low insert-size limit (for the thera-
peutic genes they can pack inside) and are capable of
generating: (a) imflammatory and immunogenic re-
sponses against their structural components and (b)
replication-competent viruses through recombination
events with the host genome.4-8 Conversely, cationic
transfection lipids, because of their least immunogenic
nature, robust manufacture, ability to deliver large
pieces of DNA, and ease of handling and preparation
techniques, are increasingly becoming the alternative
nonviral vectors of choice for delivering genes into body
cells.9-24
In our ongoing program on designing efficient novel
* Corresponding author. Tel: 91-40-27193201, fax: 91-40-27160757,
e-mail: arabinda@iict.res.in.
10.1021/jm030464i CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/23/2004

Gene Delivery Efficacy of Cationic Glycolipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3939
1
Ch a r t 1. Structures of the New Cationic Glycolipids
1-6
confirmed by H NMR, LSIMS, and HRMS. However,
the presence of multiple exchangeable hydroxyl func-
tions in the headgroup regions of the final lipids (1-6)
caused severe line broadening of the peaks in the 1H
NMR spectra, particularly in the range δ 3-5 ppm.
Thus, the final lipids were characterized by the molec-
ular ion peaks in their LSIMS.
Tr a n sfection Biology. Figure 1 summarizes the
relative in vitro gene delivery efficacies of lipids 1-6 in
transfecting CHO, COS-1, MCF7 and A549 cells. In
these in vitro transfection biology experiments, the
cationic liposomes of lipids 1-6 were prepared in
combination with an equimolar amount of cholesterol
as the colipid and pCMV-SPORT-â-gal plasmid DNA
was used as the reporter gene across the lipid:DNA
charge ratios of 9:1 to 0.1:1. Lipids 1-3 with no spacer
arms between the quaternized nitrogen atom and the
galactose unit were found to be dramatically more
efficient in transfecting all four cells than lipids 4-6
containing five-carbon headgroup spacer-arms. While
lipids 1 and 2 were the most efficacious among the
transfection-efficient glycolipids 1-3 with no headgroup
spacer units, all three lipids 4-6 with a five-carbon
spacer-arm in the headgroup region were essentially
incompetent in transfecting any of the four cells across
the entire lipid:DNA charge ratios of 0.1:1 to 9:1 (Figure
1). The optimal gene delivery efficiencies of lipids 1 and
2, except in COS-1 cells, were comparable to that of
DMRIE-C, a popular commercially available cationic
transfection lipid (Figure 1). Lipids 1 and 2 showed their
maximum cell transfection efficacies around lipid:DNA
charge ratios of 0.3:1 to 3:1 (Figure 1). Such strikingly
contrasting transfection profiles of glycolipids 1-3 and
4-6 were also observed in histochemical whole cell
staining experiments with X-gal using the representa-
Resu lts a n d Discu ssion
Ch em istr y. Cationic glycolipids 1-3 were synthe-
sized by reacting the common intermediate 2,3:5,6-
diisopropylidene-4-(tert-butyldimethylsilyl)-D-galac-
tose (V, prepared conventionally in four steps from
D-galactose as outlined in Scheme 1) with appropriate
N,N-di-n-alkylamine followed by reduction with sodium
cyanoborohydride (Scheme 1). The resulting tertiary
amine intermediates VI (Scheme 1) upon quaternization
with excess methyl iodide followed by acid deprotection
and chloride ion exchange afforded lipids 1-3 (Scheme
1). Lipids 4-6 with a five-carbon spacer arm between
the galactose functionality and the quaternized nitrogen
atom were prepared by sequentially reacting the com-
mon primary amine intermediate X (prepared conven-
tionally from intermediate III, the 2,3:5,6-diisoprop-
ylidene-D-galactose-thioacetal, in eight steps as depicted
schematically in Scheme 2) with 2 equiv of the ap-
propriate chain-length alkyl bromide, excess methyl
iodide, acid deprotection and chloride exchange over
Amberlyst A-26 (Scheme 2). Structures of all the
synthetic intermediates shown in Schemes 1 and 2 were
Sch em e 1. Synthesis of Lipids 1-3a
a
Reaction conditions: (a) C2H5SH/concd HCl; (b) 2,2-DMP/PTSA/acetone; (c) (CH3)3Si(CH3)2C1/imidazole/dry DCM; (d) HgCl2-HgO/
acetone:water (8:2), 55 °C; (e) (R)2NH/Na3BH3CN/dry DCM; (f) CH3I/DCM; (g) trifluoroacetic acid/chloride ion exchange with Amberlyst
A-26.

3940 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Sch em e 2. Synthesis of Lipids 4-6a
Mahidhar et al.
a
Reaction conditions: (a) MOM-Cl/(iPr)2N C2H5/dry DCM; (b) HgCl2/CdCO3/acetone:water (8:2), 55 °C; (c) NaBH4, methanol, glacial
acetic acid; (d)BnO(CH2)5Br, NaH, dry DMF, 80 °C (e) 10% Pd/C, H2 (2 atm), ethyl acetate; (f) CBr4/Ph3P/imidazole/dry DCM; (g and h)
NaN3/dryDMF/80 °C; Ph3P/THF/H2O; (i) RBr, K2CO3, ethyl acetate, 70 °C, (j) CH3I/DCM; (k) 6 N HCl/aqueous THF, Cl- exchange with
Amberlyst A-26; (l) BnBr, Ag2O, DCM; (m) CBr4/Ph3P/imidazole/dry DCM.
tive CHO cells. Results of such histochemical staining
experiments, once again, revealed that the percent of
CHO cells transfected with lipids 1 and 2 are dramati-
cally higher than that of CHO cells transfected with
lipids 4-6 at both lipid:DNA charge ratios of 1:1 and
3:1 (Figure 2). Results of the histochemical whole cell
staining experiments also demonstrated that lipid 2 is
the most efficacious lipid among the three transfection
efficient lipids 1-3 (Figure 2). Taken together, the
results summarized in Figures 1 and 2 convincingly
demonstrate that covalent grafting of spacer units
between the quaternized nitrogen atom and the sugar
functionalities in the headgroup region may seriously
impede the in vitro gene delivery efficacies of cationic
glycolipids.
MTT-based cell viability assays were performed in
representative MCF7 cells across the entire range of
lipid:DNA charge ratios used in the actual transfection
experiments. Percent cell viabilities of both the glyco-
lipids 1-3 and 4-6 were found to be remarkably high
particularly up to lipid:DNA charge ratios of 3:1 in
MCF7 cells (Figure 3). Thus, the dramatically opposite
in vitro gene transfer efficacies of lipids 1-3 and 4-6
(Figures 1 and 2) are unlikely to originate from varying
cell cytotoxicities of the present lipids. The global
surface charge and sizes of the lipoplexes 1-3 and 4-6
across the varying lipid:DNA charge ratios (in the
presence of DMEM) were measured using dynamic laser
light scattering instrument equipped with zeta-sizing
capacity. Interestingly, the lipoplex sizes and surface
potentials of the most transfection efficient cationic
glycolipid 2 were significantly larger and more negative
compared to the rest of the glycolipids (Table 1).
However, such lipoplex size and surface potentials
measurement did not present any mechanistic insight
to the possible origin behind their phenomenally op-
posite transfection profiles.
Lip id :DNA Bin d in g In ter a ction s a n d Lip op lex
Sen sitivities to DNa se I. Finally, with a view to
characterizing the electrostatic binding interactions
between the plasmid DNA and the present cationic
liposomes as a function of the lipid:DNA charge ratios,
we performed both the conventional electrophoretic gel
retardation assay and DNase I sensitivity assays.
Results of the simple gel retardation assay revealed
several interesting features. All the lipids (1-6) were
capable of completely inhibiting the electrophoretic
mobility of plasmid DNA from lipoplexes prepared at
high lipid:DNA charge ratios of 9:1 (Figure 4, Parts A,
C and E). However, at lower lipid:DNA charge ratios,
more free DNA was found to be present in lipoplexes of
lipids 4 and 5 compared to that in the lipoplexes
prepared from lipids 1 and 2 (Figure 4, Parts A and C).
Such gel patterns are consistent with the notion that

Gene Delivery Efficacy of Cationic Glycolipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3941
F igu r e 1. In vitro transfection efficiencies of lipids 1-6 in CHO (A), COS-1 (B), MCF-7 (C) and A549 (D) cells using cholesterol
as colipid (at lipid:cholesterol mole ratio of 1:1). Units of â-galactosidase activity were plotted against the varying lipid to DNA
(+/-) charge ratios. The o-nitrophenol formation (micromoles of o-nitrophenol produced per 10 min) was converted to â-galactosidase
activity units using the standard curve obtained with pure (commercial) â-galactosidase. The transfection efficiencies of
commercially available DMRIE are shown for comparison sake. The transfection values shown are average of triplicate experiments
performed on the same day.
suboptimal lipid:DNA binding interactions could play
an important role in abolishing the in vitro gene transfer
efficacies of lipids 4 and 5 containing five-carbon spacer
arms in their headgroup region. In agreement with this
notion, a significant amount of free DNA was also found
to be present at lower lipid:DNA charge ratios for
lipoplexes made with lipid 3, the weakest among the
transfection efficient lipids 1-3 (Figure 4, Part E).
The presence of relatively higher amounts of free
DNA in the lipoplexes prepared with transfection inef-
ficient lipids 4-6 (compared to those in lipoplexes 1-3)
was more convincingly demonstrated by monitoring the
sensitivities of the lipoplexes upon treatment with
DNase I. After the free DNA digestion by DNase I, the
total DNA (both digested and inaccessible DNA) was
separated from the lipid and DNase I (by extracting
with organic solvent) and loaded onto a 1% agarose gel.
Resulting electrophoretic gel pattens in such DNase I
protection experiments are summarized in Figure 4
(Parts B, D and F). Band intensities of inaccessible and
therefore undigested DNA associated with transfection-
incompetent lipoplexes 4-6 were significantly less than
those associated with transfection efficient lipoplexes
1-3 essentially across the entire range of lipid:DNA
charge ratios of 9:1 to 0:3:1 (Figure 4, Parts B, D and
F). Such gel patterns in DNase I sensitivity assays
indicate that the plasmid DNA associated with lipids
4-6 are likely to be more susceptible to degradation by
cellular DNase I than the DNA complexed to lipids 1-3.
Thus, one of the factors abolishing the in vitro trans-
fection efficacies of lipids 4-6 with five-carbon spacer
arm units in the headgroup region could be higher
degradation rates of the associated DNA by DNase I
within the cell.
In conclusion, the results of the present structure-
activity investigation involving the use of six novel
cationic glycolipids (1-6) demonstrate that the in vitro
gene delivery efficacies of galactosylated cationic gly-
colipids could be strikingly dependent on the presence
or absence of a spacer-arm between the carbohydrate
functionality and the positively charged nitrogen atom
in the headgroup region. In general, while the glycolip-
ids 1-3 with no-spacer arms in the headgroup region
were highly efficacious in transfecting CHO, COS-1,
MCF7 and A549 cells (lipids 1 and 2 with myristyl and
palmityl tails, respectively, being the most efficient
lipids), lipids 4-6 with a five-carbon spacer arm in the
headgroup region were essentially incompetent in trans-

3942 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Mahidhar et al.
F igu r e 2. Histochemical whole cell staining of transfected CHO cells with X-gal with lipids 1-6 at lipid:DNA charge ratios of
3:1 and 1:1. Cells expressing â-galactosidase were stained with X-gal as described in the text.
fecting all the four cells. Thus, the results of our DNase
I sensitivity assays indicate that relatively poor lipid:
DNA interactions and therefore possibly more DNA
degradation by DNase I within the cell may play an

Gene Delivery Efficacy of Cationic Glycolipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3943
Ta ble 1.
lipids
lipid:DNA (() 0.1:1
lipid:DNA (() 0.3:1
lipid:DNA (() 1:1
lipid:DNA (() 3:1
lipid:DNA (() 9:1
A. Sizes of Lipoplexes (presence of DMEM)a
1
2
3
4
5
6
259.5 ( 1.6
549.3 ( 1.5
282 ( 1.1
324.3 ( 6.8
259.5 ( 15.9
236.4 ( 14.8
302.8 ( 1.6
737.4 ( 45
286.2 ( 2.8
370.3 ( 3.9
242.4 ( 3.5
254.7 ( 9.8
258.9 ( 1.8
1083 ( 44.6
288.1 ( 3.4
296.8 ( 7.9
248.2 ( 6.8
275.3 ( 7.4
879.5 ( 66.3
1403 ( 121.1
1067.9 ( 47.8
562 ( 16.7
1205.4 ( 83.6
1972 ( 169
1514.5 ( 70.1
1056.9 ( 61.9
832.9 ( 102
1381.9 ( 77
1074 ( 160
1311 ( 42.3
B. Zeta Potentials (ê) of Lipoplexesa (presence of DMEM)
1
2
3
4
5
6
-1.6 ( 2.7
-34.4 ( 1.1
-5.4 ( 4.5
-3.4 ( 2
-20.7 ( 5.0
2.2 ( 2
-3.2 ( 2.2
-34.4 ( 1.1
-4.6 ( 2.6
-8.0 ( 3.0
-21.6 ( 3.1
-6.1 ( 4.1
-6.6 ( 1.6
-33.4 ( 1.1
-15.5 ( 5.9
-5.9 ( 2.3
-32.2 (0.9
-5.6 ( 3.1
-9.3 ( 3.9
-27.8 ( 5.6
-8.2 ( 3.1
-12.9 ( 3.4
-35.8 ( 1.2
-13 ( 3.6
17.6 ( 3
10.6 ( 4.9
6.2 ( 4.4
8.2 ( 1.4
-15.8 ( 3.9
8.3 ( 2
a
Sizes and ê potentials were measured by laser light scattering technique using Zetasizer 3000A (Malvern Instruments, UK). Values
shown are the averages obtained from three (size) and 10 (zeta potential) measurements.
bon tetrabromide and ethanethiol were purchased from Fluka
(Switzerland) and N,N-diisopropylethylamine were procured
from Aldrich (Milwaukee, WI). Unless otherwise stated, all
other reagents were purchased from local commercial suppliers
and were used without further purification. Column chroma-
tography was performed with silica gel (Acme Synthetic
Chemicals, India, 60-120 mesh). p-CMV-SPORT-â-gal plas-
mid was a generous gift from Dr. Nalam Madhusudhana Rao
of Centre for Cellular and Molecular Biology, Hyderabad,
India. Cell culture media, fetal bovine serum, 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly-
(ethylene glycol) 8000, o-nitrophenyl-â-D-galactopyranoside
and cholesterol were purchased from Sigma (St. Louis, MO).
NP-40, antibiotics and agarose were purchased from Hi-media,
India. COS-1 (SV 40 transformed african green monkey kidney
cell), and CHO (Chinese hamster ovarian cell), MCF-7 (human
brest adenocarcinoma cell) and A549 (human pulmonary
carcinoma cell) cell lines were procured from the National
Centre for Cell Sciences (NCCS), Pune, India. Cells were grown
at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with
10% FBS in a humidified atomosphere containing 5% CO2/
95% air. Purity of lipids 2 and 5 was confirmed by elemental
F igu r e 3. MTT assay-based cellular cytotoxicities of lipid 1-6
analyses (C, H, N), and lipids 1, 3, 4 and 6 were found to be
against representative MCF-7 cells. The percent cell viability
more than 95% pure by reversed phase HPLC analysis
values shown are average of triplicate experiments performed
(Shimadzu Model LC10A) using two different mobile phases
on the same day.
(A: pure methanol and B: 95:5, v/v, methanol/water), PAR-
TISIL 5 ODS-3 WCS analytical column (4.6 × 250 mm,
important role behind the seriously compromised trans-
fection efficacies of glycolipids 4-6 with a five-carbon
spacer unit in the headgroup region. Clearly, further
cell biology experiments, including cellular uptake stud-
ies and intracellular trafficking experiments aimed at
understanding subcellular localizations of the lipid:DNA
complexes, etc., need to be carried out in the future for
gaining better mechanistic insight into the origin of the
diametrically opposite transfection profiles of lipids 1-3
and 4-6. Investigations toward this end are currently
in progress in our laboratories.
Whatman Inc., Clifton, NJ ) and a flow rate of 1.6-1.8 mL/
min. Typical retention times in mobile phase A were 1.09 min
(lipid 1); 0.87 min (lipid 3); 1.21 min (lipid 4) and 1.00 min
(lipid 6).
Syn t h eses of Lip id s 1-6. As representative details,
syntheses and spectral characterization of lipids 2 and 5 and
all their synthetic intermediates shown in Schemes 1 and 2
are provided below. Lipids 1 and 3 and lipids 4 and 6 were
synthesized following essentially the same protocols adopted
for preparing lipids 2 and 5, respectively.
Syn th esis of Lip id 2 (Sch em e 1). Step a : Syn th esis of
D-Ga la ctose-d ieth yl-d ith ioa ceta l (II). In a 250 mL round-
bottomed flask, D-galactose I (20 g, 111 mmol) was dissolved
in concentrated HCl (50 mL). The reaction mixture was stirred
for 10 min in ice-salt bath. Ethanethiol (16.4 mL, 222 mmol)
was added dropwise to the cold solution. The reaction mixture
was stirred in ice-salt bath for 1 h, stirred at 0 °C for 5 h and
kept at 4 °C for 12 h. The mixture was dissolved in methanol
(1000 mL), and the solution was neutralized by addition of
lead carbonate. The salts were filtered, and the filtrate was
concentrated. The resulting solid upon drying under vacuum
afforded pure compound II (24 g, 75% yield). mp: 138-140
°C (lit: 140-142 °C).34
Exp er im en ta l Section
Gen er a l P r oced u r es a n d Ma ter ia ls. High-resolution
mass spectometric (HRMS) analysis was performed on a
Micromass AUTOSPEC-M mass spectrometer (Manchester,
UK) with OPUS V 3.1X data system. Data were acquired by
liquid secondary ion mass spectrometry (LSIMS) using m-
nitrobenzyl alcohol as the matrix. 1H NMR spectra were
recorded on a Varian FT 200 MHz, AV 300 MHz or Varian
Unity 500 MHz instrument. Elemental analyses (C, H, N) were
performed on Perkin-Elmer 240c in the microanalytical labo-
ratory of the University of Hyderabad. 1-Bromohexadecane,
n-hexadecylamine, boron trifluoride etherate, sodium cy-
anoborohydride, tert-butyldimethylsilyl chloride, tetra-n-bu-
tylammonium fluoride (1 M solution in THF) and Amberlyst
A-26 were purchased from Lancaster (Morecambe, UK). Car-
Step b: Syn th esis of 2,3:5,6-Diisop r op ylid en e-D-ga la c-
tose-d ieth yl-d ith ioa ceta l (III). In a 250 mL two necked
round-bottom flask, 2,2-dimethoxypropane (24 mL, 209 mmol)
was added dropwise at 0 °C to a solution of II (10 g, 34.9 mmol)
and PTSA (2.63 g, 13.8 mmol) in dry acetone (50 mL) under

3944 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Mahidhar et al.
F igu r e 4. Electrophoretic gel patterns for lipoplex-associated DNA in gel retardation (Parts A, C and E) and DNase I sensitivity
assays (Parts B, D and F). The lipid:DNA charge ratios are indicated at the top of each lane. The details of treatment are as
described in the text.
nitrogen atmosphere. The reaction mixture was stirred for 2
h, neutralized with NaHCO3 and filtered. The filtrate was
dried over anhydrous Na2SO4 and concentrated in a rotary
evaporator. The residue upon 60-120 mesh silica gel column
using 4% ethyl acetate in hexane as eluent afforded the pure
liquid title compound III (6.5 g, 58% yield, Rf ) 0.6, 30% ethyl
acetate/hexane). 1H NMR (300 MHz, CDCl3): δ (ppm) ) 1.20-
1.29 (m,6H,-S-CH2-CH3),1.30-1.42 (4s,12H,-2{COC(CH3)2-
OC}, 2.00-2.20 (brs, 1H, OH), 2.70 (q, 4H, SCH2CH3), 3.70-
3.80 (m, 3H, H-5, H-6 and H-6′), 4.00 (d, H-1), 4.05-4.10 (m,
2H, H-2 and H-3), 4.29-4.35 (dd, 1H, H-1)
Step c: Syn th esis of 2,3:5,6-Diisop r op ylid en e-4-(ter t-
bu tyl-d im eth ylsilyloxy)ga la ctose-d ith iok eta l (IV). In a 50
mL two neck round-bottom flask, a mixture of compound III
(2.23 g, 6.80 mmol) and imidazole (2.78 g, 40.8 mmol) was
dissolved in dry dichloromethane (10 mL) and stirred at 0 °C.
TBDMSCl (3.0 g, 20.4 mmol) dissolved in dichloromethane was
added to the cold reaction mixture. A white precipitate
appeared after addition of TBDMSCl was completed. The
temperature of the reaction mixture was slowly raised to room
temperature, and the stirring was continued for 1 h. The
mixture was concentrated on a rotary evaporator, and the
residue upon column chromatographic purification (using 60-
120 mesh size silica gel and 1% ethyl acetate/hexane, v/v, as
eluent) afforded the title compound IV as a colorless liquid
(1.94 g, 66% yield, Rf ) 0.8, 20:80 ethylacetae:hexane). 1H
NMR (200 MHz, CDCl3): 0.00-0.10 (6H, Si(CH3)2), 0.90-1.00
(m, 9H, (CH3)3Si), 1.20-1.30 (m, 6H, 2SCH2CH3), 1.35-1.50
(m, 12H, 2{COC(CH3)2OC}, 2.60-2.80 (m, 4H, 2SCH2CH3),
3.65-3.90 (m, 3H, H-5, H-6 and H-6′), 4.00-4.20 (m, 3H,
H-1,H-2, and H-3), 4.30 (dd, 1H, H-4).
St ep d : Syn t h esis of 1-Ald eh yd o-2,3:5,6-d iisop r op -
ylid en e-4-(ter t-b u t yld im et h ylsilyloxy)-D-ga la ct ose (V).
Compound IV (0.99 g, 2.23 mmol) was deprotected by refluxing
in 50 mL of 80/20 acetone/water (v/v) with mercuric chloride
(1.8 g, 6.6 mmol) and red mercuric oxide (1.7 g, 8.24 mmol)
for exactly 1.5 h. The reaction mixture was cooled and filtered.
The filtrate was concentrated, the resulting residue was
dissolved in CHCl3 and the chloroform extract was washed
with 1 N KI (3 × 50 mL) followed by brine solution (2 × 50
mL). The organic layer was separated, dried over anhydrous
Na2SO4 and filtered. The filtrate upon concentration in a
rotavapor afforded the crude aldehyde (V) as a liquid (0.6 g,
85%, Rf ) 0.6, 30:70 ethyl acetate:hexane).
Step e: Syn th esis of 1-Deoxy-1-[di-n -h exadecyl(am in o)]-
2,3:5,6-d iisop r op ylid en e-4-(ter t-bu tyld im eth ylsilyloxy)-
D-ga la ctose (VI). The crude aldehyde V prepared above (0.3
g, 1.0 mmol) and N,N-di-n-hexadecylamine (0.4 g 1.1 mmol)
were dissolved in 10 mL of dichloromethane and was stirred
for 0.5 h under nitrogen in room temperature. Sodium cy-
anoborohydride (0.1 g, 1.6 mmol) was added to the reaction
mixture, and the stirring was continued further for 24 h under
nitrogen atmosphere. The reaction mixture was concentrated,
and the residue upon column chromatographic purification
(using 60-120 mesh size silica gel and 1.8-2% ethyl acetate
in hexane as eluent) afforded the title compound VI as a
semisolid (0.19 g, 24% yield, Rf ) 0.8, 20:80 ethyl acetate:
hexane). 1H NMR (500 MHz, CDCl3): δ (ppm) 0.10 (S, 6H,
2SCH3), 0.90-1.00 (m, 15H), 1.15-1.35 (m, 56H), 1.36-1.40
(4S, 12H, 2{COC(CH3)2OC}), 2.40-2.65 (m, 5H, CH1H2N(CH2-
CH2), 2.70-2.90 (m, 1H, CH1H2N(CH2CH2), 3.58-3.72 (m, 2H,
H-5 and H-6′), 3.83-3.97 (m, 3H, H-3, H-4 and H-6), 4.00-
4.20 (m, 1H, H-2).
Step f: Syn th esis of 1-Deoxy-1-[d i-n -h exa d ecyl(m eth -
yl)a m in o]-2,3:5,6-d iisop r op ylid en e-4-(ter t-b u t yld im et h -
ylsilyloxy)-D-ga la ctose (VII). Compound VI (0.12 g, 0.14
mmol) was treated with excess methyl iodide (3 mL) and
stirred for 3 h at room temperature. The reaction mixture was
concentrated, and the residue upon column chromatographic
purification (using 60-120 mesh size silica gel and 2.5%
methanol in chloroform as eluent) afforded the title compound
VII as a white solid (0.073 g, 60% yield, Rf ) 0.5, 5% methanol/
chloroform). 1H NMR (200 MHz, CDCl3): δ (ppm) 0.10 (S, 6H,
Si(CH3)2), 0.80-1.00 (m, 15H, SiC(CH3)3 and CH3(CH2)14, 1.10-
1.50 (m, 64H), 1.60-1.90 (m, 4H), 3.50 (S, 3H, CH3N+), 3.58-
4.40 (m, 12H, N(CH2)3, H-2, H-3, H- 4, H-5, H-6 and H-6′).
FABMS (LSIMS): m/z: 839 [M]+ + 1 for C51H10405NSi.
St ep g: Syn t h esis of 1-Deoxy-1-[d i-n -h exa d ecyl-
(m eth yl)a m in o])-D-ga la ctitol (lip id 2, Sch em e 1). Com-
pound VII (0.073 g, 0.086 mmol) was dissolved in neat
trifluoroacetic acid (2 mL) and stirred at room temperature
for 48 h. Excess trifluoroacetic acid was removed with nitrogen
flow, and the residue upon column chromatographic purifica-
tion (using 60-120 mesh size silica gel and 6-8% methanol
in chloroform as eluent) followed by chloride ion exchange in
Amberlyst A-26 (using methanol as eluent) afforded target
lipid 1 as a white solid (0.025 g, 45% yield, Rf ) 0.1, 1:9
methanol;chloroform). 1H NMR (200 MHz,CDCl3): δ (ppm)

Gene Delivery Efficacy of Cationic Glycolipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3945
0.80 (t,6H, (CH3)2(CH2)n) 1.10-1.80 (m, 56H, 2 (CH3)(CH2)14-
CH2N); 3.10 (S, 3H, N+CH3); 3.20-5.20 (m, 12H, N(CH2)2,
N-H-1, H-1′, H-2, H-3, H- 4, H-5, H-6 and H-6′). FABMS
(LSIMS) m/z: 645 [M]+ + 1 for C39H8205N. HRMS (LSIMS)
m/z: calcd (for C39H82O5N the quaternary ammonium ion,
100%) 644.6193, found 644.6172.
Step b: Syn th esis of 2,3:5,6-Diisop r op ylid en e-4-m eth -
oxym eth yl-D-ga la cta l (V). Compound IV (4.50 g, 10.97
mmol) and CdCO3 (34.07 g, 197.5 mmol) were suspended in
60 mL of a mixture of acetone and water (8:2, v/v), and the
reaction mixture was heated at 50 °C. HgCl2 (15.9 g, 58.8
mmol) dissolved in 10 mL of acetone was added over a period
of 5 min. The reaction mixture was stirred for 2 h at 50 °C.
The reaction mixture was cooled and filtered through fresh
CdCO3. The filtrate was concentrated, and the residue ob-
tained was dissolved in CHCl3 (100 mL) and washed first with
1 N aqueous KI (500 mL) and finally with brine solution (100
mL). The organic layer was separated and dried over anhy-
drous Na2SO4 and concentrated to afford aldehyde V as a
liquid (2.66 g, 85% yield, Rf ) 0.7,1:1 ethyl acetate:hexane).
Syn th esis of 1-Deoxy-1-[di-n -tetr adecyl(m eth yl)am in o]-
D-ga la ctitol (lip id 1, Sch em e 1). The title lipid 1 (white solid)
was synthesized following the same synthetic procedure as
described above for preparing lipid 2 using N,N-di-n-tetradec-
ylamine in step e with an overall yield of 44%. All the isolated
intermediates gave spectroscopic data in agreement with their
1
assigned structures shown in Scheme 1. H NMR (200 MHz,-
CDCl3) of intermediate VI (Scheme 1 where R ) n-C14H29): δ
(ppm) 0.10 (S, 6H, 2SiCH3), 0.90-1.00 (m, 15H), 1.10-1.30
(m, 48H), 1.36-1.50 (4S, 12H, 2{COC(CH3)2OC}), 2.40-2.65
(m, 5H, CH1H2N(CH2CH2), 2.70-2.90 (m, 1H, CH1H2N(CH2-
CH2), 3.60-4.10 (m, 6H, H-2, H-3, H-4, H-5, H-6 and H-6′).
1H NMR (200 MHz,CDCl3) of intermediate VII (Scheme 1
where R ) C14H29): δ (ppm) 0.10 (S, 6H, Si(CH3)2, 0.80-1.00
(m, 15H, SiC(CH3)3 and CH3(CH2)14, 1.18-1.32 (m, 56H), 1.65-
1.90 (m, 4H), 3.50 (S, 3H, CH3N+), 3.60-3.80 (m, 6H, N(CH2)3),
3.81-4.40 (m, 6H, H-2, H-3, H- 4, H-5, H-6 and H-6′). FABMS
(LSIMS): m/z: 783 [M]+ + 1 for C47H9605NSi. 1H NMR (200
MHz, CDCl3+DMSO) of lipid 1: δ (ppm) 0.90 (t, 6H, (CH3)2-
(CH2)n) 1.15-1.40 (m, 44H), 1.50-1.90 (m, 4H), 3.20-3.90 (m,
9H, N+CH3, N(CH2)2, N-H-1, H-1′), 4.30-5.40 (m, 6H, H-2, H-3,
Step c: Syn th esis of 2,3:5,6-Di-O-isop r op ylid en e-4-
m eth oxym eth yl-ga la ctitol (VI). Crude aldehyde V (2.60 g,
14.1 mmol) was dissolved in 10 mL of methanol, and the
solution was cooled to 0 °C. NaBH4 (0.33 g, 8.51 mmol) was
added in portions to the cold solution. The reaction mixture
was stirred at 0 °C for 1 h and stirred at room temperature
for 3 h. The solvent was evaporated, and the residue was
dissolved in 10 mL water, neutralized with glacial acetic acid
and extracted with ethyl acetate (2 × 10 mL). The combined
organic extract was washed with brine solution, dried over
anhydrous Na2SO4 and concentrated on rotary evaparator. The
residue upon chromatography over 60-120 mesh silica gel
column using 20% ethyl acetate/hexane as eluent afforded pure
compound VI as a colorless liquid (1.57 g, 60% yield, Rf ) 0.5,
40:60 ethyl acetate/hexane). 1H NMR (200 MHz, CDCl3): δ
(ppm) ) 1.20-1.40 (4s, 12H, 2{COC(CH3)2OC})), 2.39-2.50
(brs, 1H, OH), 3.30 (s, 3H, OCH3), 3.40-3.80 (m, 6H, H-2, H-3,
H-4, H-5, H-6 and H-6′), 3.85-4.10 (m, 2H, H-1, H-1′), 4.60 (s,
2H, OCH2O). FABMS (LSIMS): m/z: 307 [M]+ + 1 for
H- 4, H-5, H-6 and H-6′). FABMS (LSIMS) m/z: 588 [M]+
+
1 for C35H7405N.
Syn th esis of 1-Deoxy-1-[di-n -octadecyl(m eth yl)am in o]-
D-ga la ctitol (lip id 3, Sch em e 1). The title lipid 3 (white solid)
was synthesized following the same synthetic synthetic pro-
cedure as described above for preparing lipid 2 using N,N-di-
n-octadecylamine with an overall yield of 44%. All the isolated
intermediates gave spectroscopic data in agreement with their
C
14H26O7.
Step d : Syn th esis of 5-Ben zyloxyp en tyl-2,3:5,6-d i-O-
isop r op ylid en e-4-m eth oxym eth y-D-ga la ctosid e (VII). In
a 50 mL two neck round-bottom flask, compound VI (1.54 g, 5
mmol) dissolved in 10 mL of dry DMF was added at 0 °C to a
suspension of NaH (0.6 g, 12.6 mmol) in 10 mL of dry DMF.
The reaction mixture was stirred at 0 °C for 1 h. 5-O-benzyl-
1-bromo-pentane (1.55 g, 6 mmol) was added to the reaction
mixture and kept under stirring initially for 1 h at 0 °C and
then at room temperature for 6 h and finally at at 80 °C for
48 h. The reaction mixture was then diluted with CHCl3 (100
mL) and washed with H2O (3 × 50 mL). The organic layer
was separated, dried over anhydrous Na2SO4 and concentrated
by rotary evaporator. The residue upon chromatographic
purification on a 60-120 mesh size silica gel column using
3.5-5% ethyl acetate/hexane as the eluent afforded compound
VII as a colorless liquid (1.3 g, 53.6% yield, Rf ) 0.6, 30:70
ethyl acetate/hexane). 1H NMR (200 MHz, CDCl3): δ (ppm) )
1.30-1.48 (4s, 12H, 2{COC(CH3)2OC}, 1.50-1.89 (m, 6H), 3.40
(s, 3H, OCH3), 3.41-3.58 (m, 6H), 3.60-3.80 (m, 4H, H-3, H-5,
H-6, H-6′), 4.00-4.02 (m, 2H, H-2 and H-4), 4.50 (s, 2H, OCH2-
Ph) 4.70 (s, 2H, OCH2O), 7.20-7.40 (m, 5H, aromatic). FABMS
(LSIMS): m/z: 467 [loss of CH3] for C26H42O8.
Step e: Syn th esis of 5-Hyd r oxyp en tyl-2,3:5,6-d i-O-
isop r op ylid en e-4-m et h oxym et h yl-D-ga la ct osid e (VIII).
Compound VII (0.9 g, 1.87 mmol) dissolved in ethyl acetate
(10 mL) was added to the 10% Pd/C (200 mg) suspended in
ethyl acetate (5 mL), and the reaction mixture was stirred at
room temp for 20 h under H2 (2 atm). The mixture was filtered
using Celite, and the filtrate was dried over anhydrous Na2-
SO4 and concentrated. The crude product upon column chro-
matograpphy over 60-120 mesh size silica gel using 20:80
ethyl acetate:hexane as eluent afforeded pure compound VIII
as a colorless liquid (0.71 g, 97% yield, Rf ) 0.6, 1:1 ethyl
acetate/hexane. 1H NMR (300 MHz, CDCl3): δ (ppm) ) 1.30-
1.40 (m, 12H, 2{COC(CH3)2OC}, 1.45-1.60 (m, 6H), 1.65-1.80
(brs, 1H, OH), 3.30 (s, 3H, OCH3), 3.40-3.80 (m, 10H), 3.90-
4.10 (m, 2H, H-2 and4), 4.60 (s, 2H, OCH2).
1
assigned structures shown in Scheme 1. H NMR (300 MHz,-
CDCl3) of intermediate VI (Scheme 1 where R ) n-C18H37): δ
(ppm) 0.10 (s, 6H, 2SCH3), 0.80-1.00 (m, 15H), 1.10-1.30 (m,
64H), 1.36-1.50 (4S, 12H, 2{COC(CH3)2OC}), 2.40-2.65 (m,
5H, CH1H2N(CH2CH2), 2.70-2.90 (m, 1H, CH1H2N(CH2CH2),
3.55-4.10 (m, 6H, H-2, H-3, H-4, H-5, H-6 and H-6′). FABMS
(LSIMS) m/z: 879 [M]+ + 1 for C54H10905NSi. 1H NMR (200
MHz,CDCl3) of intermediate VII (Scheme 1 where R )
C
18H37): δ (ppm) 0.10 (S, 6H, Si(CH3)2), 0.80-1.00 (m, 15H,
SiC(CH3)3 and 2CH3(CH2)14), 1.20-1.50 (m, 72H), 1.55-1.90
(m, 4H), 3.50 (S, 3H, CH3N+), 3.60-4.40 (m, 12H, N(CH2)3),
H-2, H-3, H- 4, H-5, H-6 and H-6′). FABMS (LSIMS) m/z: 895
[M]+ + 1 for C55H112O5NSi. 1H NMR (200 MHz,CDCl3) of lipid
3: δ (ppm) 0.90 (t, 6H, (CH3)2(CH2)n) 1.10-1.80 (m, 64H) 3.10
(S, 3H, N+CH3), 3.20-4.60 (m, 12H, N(CH2)2, N-H-1, H-1′, H-2,
H-3, H- 4, H-5, H-6 and H-6′). FABMS (LSIMS) m/z: 701 [M]+
+ 1 for C43H9005N.
Syn th esis of Lip id 5 (Sch em e 2). Step a : Syn th esis of
2,3:5,6-Diisop r op ylid en e-4-m eth oxym eth yl-D-ga la ctose-
d ieth yl-d ith ioa ceta l (IV). In a 50 mL two-necked round-
bottom flask, compound III (6.30 g, 17.24 mmol) was dissolved
in dry dichloromethane (15 mL) at 0 °C. Diisopropylethylamine
(6.0 mL, 34.2 mmol) was added dropwise to the solution, the
mixture stirred for 1 h at 0 °C and methoxymethyl cholride
(1.56 mL, 54.6 mmol) added dropwise to the reaction mixture
under stirring. The reaction mixture was stirred for 6 h at
room temparature, diluted with chloroform (100 mL) and
washed with H2O (50 mL). The organic layer was separated
and dried over anhydrous Na2SO4 and concentrated in a rotary
evaporator to afford the crude product which upon purification
on 60-120 mesh size silica gel column using 2% ethyl acetate
in hexane as eluent afforded pure compound IV as a pale
yellow liquid (4.92 g, 79% yield, Rf ) 0.8, 30% ethyl acetate/
1
hexane). H NMR (200 MHz, CDCl3): δ (ppm) ) 1.25 (m, 6H,
SCH2CH3), 1.30-1.42 (4s, 12H, 2{COC(CH3)2OC}, 2.70 (q, 4H,
2SCH2CH3), 3.39 (s, 3H, OCH3), 3.60 (dd, 1H, H-5), 3.65-3.80
(m, 2H, H-6, 6′), 4.00 (d, 1H, H-1), 4.02-4.39 (m, 3H, H-2, H-3
and H-4), 4.60 (s, 2H, OCH2O).
Step f: Syn th esis of 5-Br om op en tyl-2,3:5,6-d i-O-iso-
p r op ylid en e-4-m eth oxym eth yl-D-ga la ctosid e (IX). In a 50
mL two-neck round-bottom flask, imidazole (0.24 g, 3.54 mmol)

3946 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Mahidhar et al.
and triphenylphosphine (0.93 g, 3.55 mmol) were dissolved in
dry dichloromethane under nitrogen atmosphere. Compound
VIII (0.7 g, 1.78 mmol), dissolved in dry dichloromethane, was
added dropwise followed by addition of CBr4 (1.17 g, 3.54
mmol) to the reaction mixture. The reaction mixture was
stirred at 0 °C for 1 h, the temperature gradually raised to
room temp and the reaction mixture was further stirred at
room temperature for 12 h. The reaction mixture was concen-
trated, and the residue upon column chromatographic purifi-
cation (using 60-120 mesh size silica gel and 10:90 ethyl
acetate:hexane as eluent) afforded compound IX as a colorless
liquid (0.60 g, 75% yield, Rf ) 0.4, 20:80 ethyl acetate/hexane).
1H NMR (200 MHz, CDCl3): δ (ppm) ) 1.30-1.45 (4s, 12H,
2{COC(CH3)2OC}, 1.45-1.70 (m, 4H),1.80-2.00 (m, 2H), 3.39
(s, 3H, O-CH3), 3.40-3.80 (m, 10H), 4.00-4.20 (m, 2H, H-2
and H-4), 4.70 (s, 2H, OCH2O). FABMS (LSIMS): m/z: 455
[M]+ for C19H35O7Br.
reaction mixture was stirred at room temp for 3 h. The reaction
mixture was concentrated, and the residue upon column
chromatographic purification (using 60-120 mesh size silica
gel and 20:80 methanol:chloroform as eluent) afforded com-
pound XII as an yellowish solid (0.3 g, 98% yield, Rf ) 0.6,1:1
acetone/hexane). 1H NMR (200 MHz, CDCl3) δ (ppm) ) 0.8 (t,
6H, CH3(CH2)14), 1.10-1.80 (m, 74H), 3.30 (s, 3H, CH3N+-
(CH2)3), 3.34 (s, 3H, OCH3) 3.40-3.78 (m, 14H, H-1, H-1′, H-3,
H-5, H-6, H-6′, OCH2, N+ (CH2)3), 4.00-4.10 (m, 2H, H-2 and
H-4), 4.60 (s, OCH2O). FABMS (LSIMS): m/z: 855 [M]+ for
C
54H104O7N.
Step k : Syn th esis of 5-[(N,N-Di-n -h exa d ecyl-N-m eth -
yl)a m in o]-1-O-p en t yl-D-ga la ct ose (lip id 5, Sch em e 2).
Compound XII (0.3 g, 0.35 mmol) was dissolved in tetrahy-
drofuran (5 mL) containing 6 N HCl (2 mL). The mixture was
stirred at 50 °C for 5-6 h. The excess tetrahydrofuran was
removed on rotary evaporator, and the residue upon column
chromatographic purification (using a 60-120 silica gel mesh
size, 8-9% methanol:chloroform as eluent) followed by chloride
ion exchange (using Amberlyst A-26 with methanol as the
eluent) afforded the pure title lipid 5 as a colorless solid (0. 11
Step s g a n d h : Syn th esis of 5-Am in op en tyl-2,3:5,6-d i-
O-isop r op ylid en e-4-m eth oxym eth yl-D-ga la ctosid e (X). In
a 25 mL round-bottomed flask, sodium azide (0.25 g, 3.97
mmmol) was added to a solution of compound IX (0.600 g, 1.31
mmol) in dry DMF under nitrogen atmosphere. The reaction
mixture was stirred at 70 °C for 16 h. The reaction mixture
was diluted with CHCl3 (50 mL) and washed with water (3 ×
50 mL). The organic layer was separated, dried over anhydrous
Na2SO4 and filtered. Removal of the solvent from the filtrate
on rotary evaporator afforded pure compound X as a colorless
liquid (0.41 g, 73% yield, Rf ) 0.4, 20:80 ethyl acetate/hexane).
1H NMR (200 MHz, CDCl3): δ (ppm) ) 1.30-1.45 (4s, 12H,
2{COC(CH3)2OC}, 1.45-1.70 (m, 4H), 1.80-2.00 (m, 2H), 3.20
(t, 2H CH2N3), 3.39 (s, 3H, OCH3), 3.40-3.80 (m, 8H), 4.00-
4.20 (m, 2H, H-2 and H-4),4.70 (s, 2H, OCH2). FABMS
1
g, 42% yield, Rf ) 0.2, 10:90 methanol/chloroform). H NMR
(200 MHz, CDCl3): 0.90 (t, 6H, -CH2-CH3), 1.00-1.60 (m,
62H), 3.40-5.20 (m, 19H). FABMS (LSIMS): m/z: 731 [M]+
+ 1 for C44H92O6N HRMS (LSIMS) m/z: Calcd (for C44H92O6N
the 4° ammonium ion,100%) 730.6924, found 730.6889.
Syn th esis of O-Ben zyl-5-br om o-1-p en ta n ol. Step l: 5-O-
Ben zyl-1-p en ta n ol. The precursor to the title reagent used
in step d was prepared from the corresponding 1,5-pentanediol
(2.0 mL) following a previously described monobenzylation
procedure of symmetric diol.35 The residue upon purification
by column chromatography (using 60-120 silica gel mesh size,
20:80 ethyl acetate/hexane as a elutent) afforded pure 5-O-
benzyl-1-pentanol, as a colorless liquid (1.41 g, 75% yield, Rf
(LSIMS): m/z: 440 [M]+ + Na for C19H35O7N3. IR: Vmax
)
2097.3 cm-1 (N3).
1
In a 25 mL round-bottom flask, a mixture of the azide
intermediate obtained above in step g (0.4 g, 0.97 mmol) and
triphenylphosphine (0.38 g, 1.43 mmol) dissolved in tetra-
hydrofuran (5 mL) was stirred at room temparature. After 10
min, 3 drops of water was added and the reaction mixture was
kept under stirring for 16 h. The reaction mixture was then
concentrated on rotary evaporator, and column chromato-
graphic purification (using 60-120 mesh size silica gel and
8-9% methanol in chloroform as the eluent) of the residue
afforded the title compound X as a colorless liquid (0.32 g, 84%
yield, Rf ) 0.4, 10:90 methanol/chloroform). 1H NMR (200
MHz, CDCl3):δ (ppm) )1.20-1.40 (4s, 12H, 2{COC(CH3)2OC},
1.40-1.80 (m, 6H), 2.80-3.00 (m, 2H, CH2NH2), 3.30 (s, 3H,
OCH3), 3.39-3.90 (m, 8H, CH2O, H-1, H-1′, H-3, H-5, H-6,
H-6′), 3.90-4.19 (m, 2H, H-2, H-4), 4.60 (s, 2H, OCH2O).
Step i: Syn th esis of 5-(N,N-Di-n -h exa d ecyla m in o)-
p en t yl-2,3:5,6-d i-O-isop r op ylid en e-4-m et h oxym et h yl-D-
ga la ctosid e (XI). In a 50 mL round-bottom flask, a mixture
of n-hexadecyl bromide (0.6 g, 2.00 mmol), compound X (0.31
g, 0.79 mmol) and K2CO3 (0.17 g, 1.19 mmol) in ethyl acetate
(30 mL) was stirred at 70 °C for 24 h. Another 0.5 mL of
n-hexadecyl bromide and 0.05 g of K2CO3 were added to the
reaction mixture, and stirring was continued for additional 48
h at 70 °C. The reaction mixture was diluted with 60 mL of
ethyl acetate and washed with water (2 × 30 mL). The organic
layer was separated, dried over Na2SO4, filtered and concen-
trated. The residue upon column chromatographic purification
(using 60-120 mesh size silica gel and 5:95 acetone:hexane
as eluent) afforded pure compound XI as a liquid (0.45 g, 66%
yield, Rf ) 0.6, 30:70 acetone/hexane). 1H NMR (200 MHz,
CDCl3): δ (ppm) ) 0.90 (t, 6H, CH3(CH2)14), 1.10-1.90 (m,
74H) 2.20-2.40 (m, 6H (CH2)3N), 3.39 (S, 3H, OCH3), 3.40-
3.80 (m, 8H, H-1, H-1′, H-3, H-5, H-6 and H-6′, OCH2), 4.00-
4.20 (m, 2H, H-2 and H-4), 4.70 (s, OCH2O). FABMS
(LSIMS): m/z: 841 [M]+ + 1 for C51H101O7N.
) 0.4,3:7 ethyl acetate/hexane). H NMR (200 MHz, CDCl3):
δ (ppm)) 1.28-1.7 (m, 6H, OCH2(CH2)3CH2OH), 1.9 (s,1H,
OH), 3.4 (t, 2H, HOCH2CH2), 3.48 (t, 2H, CH2OBn), 4.4 (s, 2H,
OCH2Ph), 7.2-7.3 (m, 5H, aromatic).
Step m . In a 50 mL two-neck round-bottom flask, imidazole
(0.68 g, 10 mmol) and triphenylphosphine (2.63 g, 10 mmol)
were dissolved in dry dichloromethane under nitrogen atmo-
sphere. To the resulting solution was added dropwise the
compound obtained in step l (1.30 g, 6.7 mmol) dissolved in
dry dichloromethane followed by addition of CBr4 (3.32 g, 32
mmol). The reaction mixture was stirred at 0 °C for 1 h, and
the temperature was gradually raised to room temperature.
The reaction mixture was kept under stirring at room tem-
perature for 6 h. The reaction mixture was then concentrated,
and the residue upon column chromatographic purification
using 60-120 mesh silica gel and 3% ethyl acetate in hexane
as eluent afforded the title reagent (O-benzyl-5-bromo-1-
pentanol used in step f) as a colorless liquid (0.95 g, 55.2%
yield, Rf ) 0.7 with 15:85 ethyl acetate: hexane). 1H NMR
(200 MHz, CDCl3): δ (ppm) ) 1.2-1.4 (m, 6H, OCH2(CH2)3-
CH2Br), 3.3-3.5 (m, 4H, OCH2(CH2)3CH2Br), 4.45 (s, OCH2-
Ph), 7.2-7.4 (m, 5H, aromatic).
Step k : Syn th esis of 5-(N,N-Di-n -tetr a d ecyl-N-m eth -
yla m in o)-1-O-p en tyl-D-ga la ctose (lip id 4, Sch em e 2). The
title lipid 4 (white solid) was synthesized following the same
synthetic procedure as described above for preparing lipid 5
using n-tetradecyl bromide in step i with an overall yield of
34%. All the isolated intermediates gave spectroscopic data
in agreement with their assigned structures shown in Scheme
1
2. H NMR (200 MHz, CDCl3) of intermediate XI (Scheme 2,
where R ) n-C14H29). δ (ppm) ) 0.90 (t, 6H, CH3(CH2)12), 1.10-
1.50 (m, 62H), 1.55-1.70 (m, 4H), 2.20-2.40 (m, 6H (CH2)3N),
3.39 (S, 3H, OCH3), 3.40-3.80 (m, 8H, H-1,H-1′, H-3, H-5, H-6
and H-6′, OCH2), 4.00-4.20 (m, 2H, H-2 and H-4), 4.70 (s, 2H,
OCH2O). FABMS (LSIMS): m/z: 783 [M]+ + 1 for C47H93O7N.
1H NMR (200 MHz, CDCl3) of intermediate XII (Scheme 2,
where R ) n-C14H29: δ (ppm) ) 0.90 (t, 6H, CH3(CH2)14), 1.20-
1.30 (m, 44H), 1.35-1.45 (m, 12H), 1.60-1.80 (m, 4H), 3.35
(s, 3H, CH3N+(CH2)3), 3.40 (s, 3H, OCH3) 3.45-3.80 (m, 14H,
H-1, H-1′, H-3, H-5, H-6, H-6′, OCH2, N+(CH2)3), 4.00-4.10
Step j: Syn th esis of 5-[(N,N-Di-n -h exa d ecyl-N-m eth -
yl)a m in o]p e n t yl-2,3:5,6-d i-O-isop r op ylid e n e -4-m e t h -
oxym eth yl-D-ga la ctosid e (XII). In a 25 mL round-bottom
flask, methyl iodide (3 mL) was added to compound XI (0.3 g,
0.35 mmol) dissolved in dichloromethane (5 mL), and the

Gene Delivery Efficacy of Cationic Glycolipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3947
(m, 2H, H-2 and H-4), 4.60 (s,2H OCH2O). FABMS (LSIMS):
m/z: 799 [M]+ + 1 for C48H96O7N. 1H NMR (200 MHz,
CDCl3+DMSO): (lipid 4) δ (ppm) ) 0.90 (t, 6H, CH2CH3),
1.00-1.80 (m, 54H), 3.20-3.90 (m, 13H), 4.32-5.30 (m, 6H).
FABMS (LSIMS): m/z: 675 [M]+ + 1 for C40H84O6N.
tetrazolium bromide (MTT) reduction assay as described
earlier.32 The cytotoxicity assay was performed in 96-well
plates by maintaining the same ratio of number of cells to
amount of cationic lipid, as used in the transfection experi-
ments. MTT was added 3 h after addition of cationic lipid to
the cells. Results were expressed as percent viability )
[A540(treated cells) - background/A540(untreated cells) - back-
ground] × 100.
St ep k : Syn t h esis of 5-(N,N-Di-n -oct a d ecyl-N-m et h -
yla m in o)-1-O-p en tyl-D-ga la ctose (lip id 6, Sch em e 2). The
title lipid 6 (white solid) was synthesized following the same
synthetic synthetic procedure as described above for lipid 5
using n-octadecyl bromide with an overall yield of 35%. All
the isolated intermediates gave spectroscopic data in agree-
ment with their assigned structures shown in Scheme 2. 1H
NMR (200 MHz, CDCl3) of intermediate XI (Scheme 2, where
R ) n-C18H37): δ (ppm): ) 0.90 (t, 6H, CH3(CH2)12), 1.10-
1.60 (m, 82H), 2.20-2.50 (m, 6H (CH2)3N), 3.39 (s, 3H, OCH3),
3.40-3.90 (m, 8H, H-1,H-1′, H-3, H-5, H-6 and H-6′, OCH2),
4.00-4.20 (m, 2H, H-2andH-4), 4.70 (s, 2H, OCH2O). FABMS
(LSIMS): m/z: 897 [M]+ + 1 for C55H109O7N. 1H NMR (200
MHz, CDCl3) of intermediate XII in Scheme 2, where R )
C18H37, δ (ppm) ) 0.90 (t, 6H, CH3(CH2)14), 1.10-1.90 (m,82H),
3.30 (S, 3H,CH3N+(CH2)3), 3.39 (3H, OCH3) -3.80 (m, 14H,
H-1, H-1′, H-3, H-5, H-6, H-6′, OCH2, N+(CH2)3), 4.00-4.10
(m, 2H, H-2 and H-4), 4.60 (s, 2H OCH2O). FABMS (LSIMS):
m/z: 911 [M]+ + 1 for C56H112O7N. 1H NMR (200 MHz,
CDCl3): (lipid 6) δ (ppm) ) 0.90 (t, 6H, CH2CH3), 1.00-2.00
(m, 70H), 3.00-5.00 (m, 19H). FABMS (LSIMS): m/z: 787
[M]+ + 1 for C48H100O6N.
P r ep a r a tion of Lip osom es. The cationic lipid and cho-
lesterol in 1:1 mole ratio was dissolved in a mixture of
chloroform and methanol (3:1, v/v) in a glass vial. The solvent
was removed with a thin flow of moisture-free nitrogen gas,
and the dried lipid film was then kept under high vacuum for
8 h. Sterile deionized water (5 mL) was added to the vacuum-
dried lipid film, and the mixture was allowed to swell
overnight. The vial was then vortexed for 2-3 min at room
temperature to produce multilamellar vesicles (MLVs). MLVs
were then sonicated in an ice bath until clarity using a
Branson 450 sonifier at 100% duty cycle and 25 W output
power to produce small unilamellar vesicles (SUVs).
Zeta P oten tia l (ê) a n d Size Mea su r em en ts. The sizes
and the surface charges (zeta potentials) of liposomes and
lipoplexes were measured by photon correlation spectroscopy
and electrophoretic mobility on a Zeta sizer 3000HSA (Malvern
UK). The sizes were measured in deionized water with a
sample refractive index of 1.59 and a viscosity of 0.89. The
system was calibrated by using the 200 nm ( 5 nm polystyrene
polymer (Duke Scientific Corps., Palo Alto, CA). The diameters
of liposomes and lipoplexes were calculated by using the
automatic mode. The zeta potential was measured using the
following parameters: viscosity, 0.89 cP; dielectric constant,
79; temperature, 25 °C; F(Ka), 1.50 (Smoluchowski); maximum
voltage of the current, V. The system was calibrated by using
DTS0050 standard from Malvern, UK. Measurements were
performed 10 times with the zero field correction. The poten-
tials were calculated by using the Smoluchowski approxima-
tion.
DNA Bin d in g Assa y. The DNA binding ability of the
cationic lipids 1 and 6 (Figure 4, Parts A, C, and E) were
assessed by their gel retardation assay on a 1% agarose gel
(prestained with ethidium bromide) across the varying lipid:
DNA charger ratios of 0.1:1 to 9:1. pCMV-â-gal (0.30 µg) was
complexed with the varying amount of cationic lipids in a total
volume of 20 µL in Hepes buffer, pH 7.40 and incubated at
room temperature for 20-25 min. 4 µL of 6X loading buffer
(0.25% bromophenol blue in 40% (w/v) sucrose in H2O) was
added to it, and the resulting solution (24 µL) was loaded on
each well. The samples were electrophoresed at 80 V for 45
min, and the DNA bands were visualized in the Gel documen-
tation unit.
DNa se 1 Sen sit ivit y Assa y. Briefly, in a typical assay,
pCMV-â-gal (1000 ng) was complexed with the varying amount
of cationic lipids (using indicated lipid:DNA charge ratios in
Figure 4, Parts B, D and F) in a total volume of 30 µL in Hepes
buffer, pH 7.40, and incubated at room temperature for 30 min
on a rotary shaker. Subsequently, the complexes were treated
with 10 µL of DNase I (at a final concentration of 1 µg/mL) in
the presence of 20 mM MgCl2 and incubated for 20 min at 37
°C. The reactions were then halted by adding EDTA (to a final
concentration of 50 mM) and incubated at 60 °C for 10 min in
a water bath. The aqueous layer was washed with 50 µL of
phenol:chloroform:isoamyl alcohol (25:24:1mixture, v/v) and
centrifuged at 10 000g for 5 min. The aqueous supernatants
were separated, loaded (15 µL for lipids 1, 2, 4 and 5 and 30
µL for lipids 3 and 6) on a 1% agarose gel (prestained with
ethidium bromide) and electrophoresed at 100 V for 1 h.
X-Ga l Sta in in g. Cells expressing â-galactosidase were
histochemically stained with the substrate 5-bromo-4-chloro-
3-indolyl-â-D-galactopyranoside (X-gal) as described previ-
ously.37 Briefly, 48 h after transfection with lipoplexes in 96-
well plates, the cells were washed two times (2 × 100 µL) with
phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and fixed with 0.5%
glutaraldehyde in PBS (225 µL). After 15 min incubation at
room temperature, the cells were washed again with PBS three
times (3 × 250 µL) and subsequently were stained with 1.0
mg/mL X-gal in PBS containing 5.0 mM K3[Fe(CN)6], 5.0 mM
K4[Fe(CN)6] and 1 mM MgSO4 for 2-4 h at 37 °C. Blue-colored
cells were identified by light microscope (Leica, Germany).
P r ep a r a tion of P la sm id DNA. pCMV-SPORT-â-gal plas-
mid DNA was prepared by alkaline lysis procedure and
purified by PEG-8000 precipitation according to the protocol
described by Maniatis and co-workers.36 The plasmid prepara-
tions showing a value of OD260/OD280 more than 1.8 were used.
Tr a n sfection Biology. Cells were seeded at a density of
20000 cells (for CHO, MCF-7, A549) and 15000 cells (for COS-
1) per well in a 96-well plate 18-24 h before the transfection.
0.3 µg of plasmid DNA was complexed with varying amounts
of lipids (0.09-8.1 nmol) in plain DMEM medium (total volume
made up to 100 µL) for 30 min. The charge ratios were varied
from 0.1:1 to 9:1 (+/-) over these ranges of the lipids. The
complexes were then added to the cells. After 3 h of incubation,
100 µL of DMEM with 20% FBS was added to the cells. The
medium was changed to 10% complete medium after 24 h, and
the reporter gene activity was estimated after 48 h. The cells
were washed twice with PBS (100 µL each) and lysed in 50
µL of lysis buffer [0.25 M Tris-HCl pH 8.0, 0.5% NP40]. Care
was taken to ensure complete lysis. The â-galactosidase
activity per well was estimated by adding 50 µL of 2X-
substrate solution [1.33 mg/mL of ONPG, 0.2 M sodium
phosphate (pH 7.3) and 2 mM magnesium chloride] to the
lysate in a 96-well plate. Adsoption at 405 nm was converted
to â-galactosidase units using a calibration curve constructed
with pure commercial â-galactosidase enzyme. The values of
â-galactosidase units in triplicate experiments assayed on the
same day varied by less than 20%. The transfection experiment
was carried in duplicate and the transfection efficiency values
shown in Figure 1 are the average of triplicate experiments
performed on the same day. Each transfection experiment was
repeated two times and the day to day variation in average
tansfection efficiency was found to be within 2-fold. The
transfection profiles obtained on different days were identical.
Abbr evia tion s
MOM-Cl, methoxymethyl chloride; PTSA, p-toluene-
sulfonic acid; TBAF, tetra-n-butylammonium fluoride;
TBDMSCl, tert-butyldimethylsilyl chloride; DMP, 2,2-
dimethoxypropane; Chol, cholesterol; CBr4, carbon tet-
Toxicity Assa y. Cytotoxicities of the lipids 1-6 were
assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

3948 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Mahidhar et al.
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rabromide DCM, dichloromethane; DMAP, 4-(N,N-
dimethylamino)pyridine; DMEM, Dulbecco’s Modified
Eagles Medium; DMF, N,N-dimethylformamide; DOPE,
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; FBS, fe-
tal bovine serum; ONPG, o-nitrophenyl-â-D-galacto-
pyranoside; PBS, phosphate-buffered saline; TFA, tri-
fluoroacetic acid.
Ack n ow led gm en t. Financial support received from
the Department of Biotechnology, Government of India
(to A.C.), is gratefully acknowledged. Y.V.M. and M.R.
thank the Council of Scientific and Industrial Research
(CSIR), Government of India, for their doctoral research
fellowship. Technical help from E. Subbaiah for taking
the gel picture and T.K.Prasad for helping us in his-
tochemical cell staining experiments are gratefully
acknowledged.
Su ppor tin g In for m ation Available: Reverse phase HPLC
chromatograms for the lipids 1, 3, 4 and 6 in two mobile phases
A and B with details of used HPLC parameters and actual
elemental analysis results (C, H, N) for lipids 2 and 5. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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