Design, Syntheses and In Vitro Gene Delivery Efficacies of Novel Mono-, Di- and Trilysinated Cationic Lipids: A Structure-Activity Investigation

Article abstract of DOI:10.1021/jm030541+

Structure-activity investigation including design, syntheses, and evaluation of relative in vitro gene delivery efficacies of a novel series of cationic amphiphiles (1-10) containing mono-, di-, and trilysine headgroups are described in CHO, COS-1, and He

Full text of DOI:10.1021/jm030541+

J . Med. Chem. 2004, 47, 2123-2132
2123
Design , Syn th eses a n d In Vitr o Gen e Deliver y Effica cies of Novel Mon o-,
Di- a n d Tr ilysin a ted Ca tion ic Lip id s: A Str u ctu r e-Activity In vestiga tion
Priya P. Karmali,† Valluripalli V. Kumar,† and Arabinda Chaudhuri*
Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad-500 007, India
Received October 24, 2003
Structure-activity investigation including design, syntheses, and evaluation of relative in vitro
gene delivery efficacies of a novel series of cationic amphiphiles (1-10) containing mono-, di-,
and trilysine headgroups are described in CHO, COS-1, and HepG2 cells. Several interesting
and rather unexpected transfection profiles were observed. In general, lipid 1 with the myristyl
tail used in combination with DOPE as colipid exhibited superior transfection properties
compared to (a) the monolysinated analogues with longer hydrocarbon tails (lipids 2-4), (b)
the dilysine (lipids 5-7) and the trilysine headgroup analogues (lipids 8-10), and (c)
commercially available LipofectAmine with multiple positive charges in its polar region. As a
preliminary estimate of the relative DNA-compacting efficacies of these new lysinated cationic
lipids, the hydrodynamic diameters of representative lipoplexes were measured using dynamic
laser light scattering technique. Our lipoplex size data are consistent with the notion that
covalent grafting of an increasing number of positively charged functional groups in the
headgroup region of cationic lipids need not necessarily result in more compacted lipoplexes.
Both gel retardation and DNase I sensitivity assays indicated similar lipid/DNA binding
interactions for all the novel mono-, di-, and trilysinated cationic lipids. MTT-assay-based cell
viability results clearly demonstrate that the overall lower transfection properties of trilysine
analogues (8-10) compared to their mono- (1-4) and dilysinated (5-7) counterparts are unlikely
to originate from differential toxicity related effects. Taken together, the present findings
support the notion that caution needs to be exercised in ensuring enhanced gene delivery
efficacies of cationic lipids through covalent grafting of multiple lysine functionalities in the
headgroup region.
In tr od u ction
tions of lipoplexes made from cationic lipids with
different net cationic charges have been reported.25 An
obvious way of enhancing electrostatic interactions
between cationic lipid and DNA is to use cationic lipids
with more than one positively charged functional groups
in their headgroup regions. This is why the headgroup
functionalities of the some of the most widely used
commercially available cationic transfection lipids con-
tain more than four positive charges (such as Lipo-
fectAmine). However, the transfection efficiencies of a
number of monocationic lipids,26-28 including our
own,1,2,4,6,12,29,30 have been demonstrated to be superior
to that of LipofectAmine. Although investigations de-
lineating transfection efficiencies of either purely trily-
sinated cationic lipids5,31 or exclusively monolysinated
cationic lipids3,8,32 have been reported, systematic struc-
ture-activity investigations using a library of cationic
lipids with an increasing number of positively charged
headgroups have hardly been undertaken. Toward this
end, using a series of novel cationic lipids with mono-,
di-, and trilysine headgroups and a common hydropho-
bic anchor skeleton (lipids 1-10, Schemes 1-3), we
demonstrate, for the first time, that the monolysine lipid
with a myristyl tail (lipid 1) exhibits superior transfec-
tion efficacies compared to its di- and trilysine analogues
in CHO, COS-1, and HepG2 cells. In addition, toward
evaluating relative DNA-compacting efficacies of these
new lysinated cationic lipids, the hydrodynamic diam-
eters of representative lipoplexes were measured using
Design of efficient cationic lipid based gene delivery
reagents as alternatives to potentially immunogenic
viral vectors continues to be an intensely pursued area
of contemporary research in nonviral gene therapy.1-17
Cationic transfection lipids (also known as cytofectins)
have been amply demonstrated in recent years to be
capable of delivering functional genes (lipofection) to
murine lungs,18 rat pulmonary epithelium,19 and the
nasal epithelium of patients with cystic fibrosis.20
Although details of lipofection pathways are still in-
completely understood, the crucial steps include (a)
formation of electrostatic complexes (popularly known
as “lipoplexes”) between polyanionic macromolecular
DNA (genes) and the positively charged lipids, (b)
endocytotic internalization of the resulting lipoplexes,
(c) escape of the DNA from the endosome compartment
to the cell cytoplasm, and (d) nuclear trafficking of the
endosomally released DNA to access the nuclear tran-
scription apparatus before the final transgene expres-
sion in cytosol.21-24
Broadly speaking, gene delivery efficacies of cationic
lipids depend on their ability to electrostatically compact
naked DNA such that the endocytotic cellular uptake
of the resulting compacted DNA (lipid/DNA complex)
is ensured. Studies aimed at biophysical characteriza-
* To whom correspondence should be addressed. Phone:
914027193201. Fax: 914027160757. E-mail: arabinda@iict.res.in.
† P.P.K. and V.V.K. contributed equally to this work.
10.1021/jm030541+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/19/2004

2124 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Karmali et al.
Sch em e 1. Synthesis of Lipids 1-4 with Monolysine Headgroupsa
a
Reagents: (a) DCC, HOSu, DMAP; (b) MeI (excess); (c) TFA, DCM; (d) Cl- ion exchange (AmberlystA-26 resin).
Sch em e 2. Synthesis of Lipids 5-7 with Dilysine
region need not necessarily impart superior transfection
efficacies and enhanced DNA-compacting properties to
cationic amphiphiles.
Headgroupsa
Resu lts a n d Discu ssion
Ch em istr y. Toward probing relative in vitro gene
transfer efficiencies of cationic lipids with increasing
headgroup positive charges, we have designed and
synthesized a series of novel mono- (1-4), di- (5-7), and
trilysinated (8-10) cationic lipids. Lipids 1-10 were
synthesized by DCC coupling of the appropriate tertiary-
primary mixed amine intermediates (prepared conven-
tionally by reacting the corresponding N,N-di-n-alkyl-
amine with N-tert-butyloxycarbonyl protected 2-bromo-
ethylamine in ethyl acetate in the presence of anhydrous
potassium carbonate followed by deprotection and neu-
tralization2) with appropriately protected lysine deriva-
tives followed by acid deprotection and chloride ion
exchange chromatography as outlined in Schemes 1-3.
Structures of all the synthetic intermediates shown in
Schemes 1-3 were confirmed by 1H NMR. However, the
presence of highly exchangeable protonated primary
amine functions in the headgroup regions of the final
lipids (1-10) caused severe line broadening of the peaks
a
Reagents: (a) DCC, HOSu, DMAP; (b) HCOONH4, 10% Pd/
C; (c) BOC(Lys)BOC OH, DCC, HOSu, DMAP; (d) MeI (excess);
(e) TFA, DCM; (f) Cl- ion exchange (Amberlyst resin).
Sch em e 3. Synthesis of Lipids 8-10 with Trilysine
Headgroupsa
1
in the H NMR spectra, particularly in the range δ 3-5
ppm. Thus, the final lipids were characterized by the
molecular ion peaks in their LSIMS as well as by high-
resolution mass spectrometry (HRMS-LSIMS). Unfor-
tunately, because of their relatively high molecular
weights, HRMS (LSIMS) could not be performed for
lipids 8-10 containing trilysine headgroups. Purity of
all the final lipids (1-10) was confirmed by reverse-
phase analytical HPLC using two different mobile
phases. As representative details, synthesis and spectral
characterizations of lipids 1, 5, and 8 and all their
intermediates (Schemes 1-3) are delineated below in
the Experimental Section. Lipids 2-4, 6, 7, 9, and 10
were synthesized following essentially the same proto-
cols adopted for the synthesis of lipids 1, 5, and 8,
respectively.
Tr a n sfection Biology. Figures 1, 2, and 3 sum-
marize the relative in vitro gene delivery efficacies of
lipids 1-10 in CHO, COS-1, and HepG2 cells, respec-
tively, across the lipid/DNA charge ratios 9:1 to 0.1:1
using both DOPE and cholesterol as colipid. Several
interesting and rather unexpected transfection profiles
were observed. In general, lipid 1 with the myristyl tail
used in combination with DOPE as colipid exhibited
superior transfection properties compared to (a) the
monolysinated analogues with longer hydrocarbon tails
(lipids 2-4), (b) the dilysine (lipids 5-7) and the
trilysine headgroup analogues (lipids 8-10), and (c)
commercially available LipofectAmine with multiple
a
Reagents: (c) DCC, HOSu, DMAP; (d) HCOONH4, 10% Pd/C;
(e) BOC(Lys)BOC OH, DCC, HOSu, DMAP; (f) MeI (excess); (g)
TFA, DCM; (h) Cl- ion exchange (Amberlyst resin).
dynamic laser light scattering technique. Taken to-
gether, results of the present structure-activity inves-
tigation indicate that covalent grafting of multiple
positively charged functionalities in the headgroup

Lysinated Cationic Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2125
F igu r e 2. Transfection efficiencies of lipids 1-10 in COS-1
cells with DOPE (A) and cholesterol (B) as colipid (at 1:1 mole
ratio of lipid to colipid). The transfection efficiencies of the
lipids were compared to that of commercially available Lipo-
fectAmine. Transfection experiments were performed as de-
scribed in the text. All the lipids were tested on the same day,
and the data presented are the average of three experiments
performed on three different days.
F igu r e 1. Transfection efficiencies of lipids 1-10 in CHO cells
with DOPE (A) and cholesterol (B) as colipid (at 1:1 mole ratio
of lipid to colipid). The transfection efficiencies of the lipids
were compared to that of commercially available Lipo-
fectAmine. Transfection experiments were performed as de-
scribed in the text. All the lipids were tested on the same day,
and the data presented are the average of three experiments
performed on three different days.
An important point worth emphasizing here is that
the lipofection efficacies are likely to be more influenced
by the overall structure of the cationic lipids than by
any specific structural features such as headgroup
charge, anchor chain lengths, nature of linker group
connecting polar heads and hydrophobic tails, etc.
Perhaps this is why different alkyl chain lengths are
required for optimal lipofection mediated by cationic
lipids with varying headgroup structures.3-5,11,30 Simi-
larly, in the present investigation, although the optimal
transfection properties for cationic lipids with monolysine
headgroups (1-4) were obtained with myristyl tails,
those for dilysinylated (5-7) and trilysinylated lipids
(8-10) were observed with higher alkyl chain lengths
(Figures 1-3). Such transfection profiles most likely owe
their origin to the optimal balance of headgroup charges
and the anchor hydrophobicities. A limitation of the
present transfection assay with the pCMV-SPORT-â-
gal plasmid carrying a â-galactosidase reporter gene is
that it does not provide any information on the percent
of cells transfected. In other words, an observed trans-
fection efficiency may result either from an overall low
percent of cells transfected, with few cells receiving large
numbers of reporter gene, or from overall low levels of
gene insertion in many cells. Taken together, the
transfection results summarized in Figures 1-3 con-
positive charges in its polar region (Figures 1A, 2A, and
3A). However, the transfection efficacy of lipid 1 in
combination with cholesterol was seriously compromised
in HepG2 cells (Figure 3B). Interestingly, monolysinated
lipids with longer than myristyl tails (lipids 2 and 4) in
combination with cholesterol were found to be promising
in transfecting COS-1 cells (Figure 2B). Although mono-
(1-4) and dilysinated lipids (5-7) were mostly trans-
fection-efficient at either 3:1 or 1:1 charge ratios, their
transfection efficacies were comparatively less at 9:1
lipid/DNA charge ratio (Figures 1-3). In contrast, the
trilysines analogues (8-10) showed their optimal gene
delivery efficiencies mostly at 9:1 lipid/DNA charge
ratios (Figures 1-3). All the lipids (1-10) became
virtually transfection-inefficient at lipid/DNA charge
ratios less than 1:1 (Figures 1-3). Similar high positive
charges of the polycation/DNA complexes (thereby
ensuring small and positively charged polyplexes least
susceptible to possible DNase degradation) are also
required for efficient cellular gene transfection mediated
by cationic polymers such as polylysines.33 However, at
high polycation/DNA charge ratios, cationic polymers
are, in general, toxic to cells.

2126 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Karmali et al.
F igu r e 4. Representative percent cell viabilities of lipids
1-10 in CHO cells using MTT assay. The absorption obtained
with reduced formazan with cells in the absence of lipids was
taken to be 100. The toxicity assays were performed as
described in the text. The data presented are the average
values of three independent experiments (n ) 3): 1 (filled
square), 2 (open circle), 3 (filled triangle), 4 (open inverted
triangle), 5 (filled diamond), 6 (open square), 7 (filled circle),
8 (open triangle), 9 (filled inverted triangle), 10 (open dia-
mond).
Ta ble 1. Representative Hydrodynamic Diameters (as
Approximate Indicators of the DNA-Compacting Efficacies) of
Lipoplexes Measured by Laser Light Scattering Techniquea
hydrodynamic diameters (nm)
for the indicated lipid/DNA charge ratios
lipid
1:1
3:1
9:1
F igu r e 3. Transfection efficiencies of lipids 1-10 in HepG2
cells with DOPE (A) and cholesterol (B) as the colipid (at 1:1
mole ratio of lipid to colipid). The transfection efficiencies of
the lipids were compared to that of commercially available
LipofectAmine. Transfection experiments were performed as
described in the text. All the lipids were tested on the same
day, and the data presented are the average of three experi-
ments performed on three different days.
1
5
8
107.5 ( 1.8
129.7 ( 4.5
134.7 ( 6.7
134.2 ( 0.7
116.9 ( 2.9
135.1 ( 1.3
89.8 ( 1.2
111.1 ( 7.7
90.4 ( 0.5
a
All size measurements were done in deionized water contain-
ing 1 mM NaCl. The size of pure pDNA (without lipid) was
measured to be 243.2 ( 1.2 nm. The values shown are an average
of three independent measurements (n ) 3).
vincingly demonstrate that covalent grafting of multiple
positively charged functionalities in the headgroup
region need not necessarily impart enhanced gene
delivery efficacies to cationic lipids.
originate from any differential toxicity related effects
and indirectly hint at some other transfection-inhibiting
parameter for lipids with trilysine headgroups (8-10).
Lip op lex Sizes a n d ú P oten tia ls. On the basis of
purely electrostatic interactions, the trilysinated lipids
(8-10) were anticipated to be better DNA-condensing
cationic amphiphiles than their mono- (1-4) and dil-
ysine analogues (5-7). In other words, the hydrody-
namic diameters for lipoplexes prepared from lipids
8-10 with trilysine headgroups were expected to be
smaller than lipoplexes prepared from mono- (1-4) and
dilysine (5-7) counterparts. Toward obtaining ap-
proximate estimates of relative DNA-compacting effica-
cies of the present lipids, we measured the hydrody-
namic diameters of pure plasmid DNA and some
representative lipid/DNA complexes (lipoplexes) across
the lipid/DNA charge ratios 9:1 to 1:1 using dynamic
laser light scattering technique. Contrary to our expec-
tation, the sizes of the lipoplexes prepared from monoly-
sinated lipid 1 were found to be equal or smaller than
the lipoplexes of the trilysine analogue (lipid 8) across
the entire range of lipid/DNA charge ratios (Table 1).
At 1:1 lipid/DNA charge ratio, the hydrodynamic diam-
eter of the lipid 1/DNA complex (107.5 ( 1.8 nm) was
found to be somewhat lower than that of lipoplexes
Cell Via bilities. With a view to gaining insight into
whether possible higher cytotoxicities of trilysinated
lipids (8-10) are at the root of their relatively lower
transfection efficacies compared to their mono- (1-4)
and di- (5-7) lysinated lipids, we performed the MTT-
based cell viability assays for all the lipids in the
representative CHO cells. The cell viability results
depicted in Figure 4 clearly demonstrate that the
trilysinated lipids are virtually nontoxic in the entire
range of lipid/DNA charge ratios of 9:1 to 0.1:1. Cell
viability of all the mono- and dicationic lipids (lipids
1-7) was found to be close to 100% in the lipid/DNA
charge ratio change 0.1:1 to 3:1. However, lipids 1-7
were found to be somewhat toxic at 9:1 lipid/DNA charge
ratio (Figure 4). Such relatively higher cytotoxic nature
of lipids 1-7 at 9:1 lipid/DNA charge ratios might play
some role in reducing the efficacies of all the mono- and
dilysinated lipids at 9:1 lipid/DNA charge ratios in
transfecting all three cells (Figures 1-3). Taken to-
gether, the overall lower transfection properties of
trilysine analogues (8-10) compared to their mono- (1-
4) and dilysinated (5-7) counterparts are unlikely to

Lysinated Cationic Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2127
amounts of free DNA were present that showed the
usual electrophoretic migration into 1% agarose gel
(Figure 5A). Interestingly, the same gel retardation
pattern as depicted in Figure 5A for lipoplexs of dily-
sinated lipid 5 was also observed for representative
mono- (1) and trilysinated (8) lipids (data not shown).
Such similar electrophoretic mobilities of mono-, di-, and
trilysinated lipids demonstrated that lipid/DNA elec-
trostatic binding interactions are similar irrespective of
the number of lysine functionalities in the headgroup
region of the present cationic lipids.
The existence of substantial amounts of free DNA in
the lipoplexes of all the mono-, di-, and trilysinated
lipids was further confirmed by monitoring the sensi-
tivities of the lipoplexes upon treatment with DNase I.
After the free DNA digestion by DNase I, the total DNA
(both the digested and inaccessible DNA) was separated
from the lipid (by extracting with organic solvents) and
loaded onto a 1% agarose gel. Figure 5B summarizes
the results of such DNase I protection experiments for
lipoplexex prepared from the representative dilysinated
cationic lipid 5 across the entire lipid/DNA charge ratios
of 9:1 to 0.1:1. While at high lipid/DNA charge ratios of
9:1 and 3:1, the lipoplex-associated DNA was very
strongly bound to lipids and was completely inaccessible
to Dnase I; increasing amounts of free DNA accessible
to DNase I digestion were observed to be present in the
lipoplexes as the lipid/DNA charge ratios decreased from
1:1 to 0.1:1 (Figure 5B). Interestingly, although in the
gel retardation assay no free DNA was detected even
at a lipid/DNA charge ratio of 1:1 (Figure 5A), signifi-
cant free DNA was detected in DNase I protection
experiments for the same lipid/DNA charge ratio (Figure
5B). This implies that although the lipid/DNA interac-
tion at a 1:1 lipid/DNA charge ratio is strong enough to
prevent the DNA from entering the gel, some fraction
of lipoplex-associated DNA is still accessible to DNase
I at the same lipid/DNA charge ratio. Once again, very
similar gel patterns in DNase I protection experiments
were also observed for lipoplexes of representative
mono- (1) and trilysinated (8) lipids (data not shown).
Similar lipid/DNA binding interactions for all the mono-,
di-, and trilysinated lipids were additionally supported
by the lipoplex global surface charge estimates using a
dynamic laser light scattering instrument equipped with
ú-sizing capacity. Clearly, issues such as (1) the origin
of the overall lower transfection efficiencies of trilysi-
nated lipids 8-10 compared with those of their monoly-
sinated counterparts (1-4) in all the three cells (Figures
1-3) despite trilysine analogues being least cytotoxic
and equally DNA compacting, (2) factors leading to the
dramatic fall of transfection efficacy of lipid 1 in HepG2
cells when cholesterol (and not DOPE) is used as the
colipid (Figure 3), and (3) why in COS-1 cells lipids 2
and 4 are transfection-efficient only with cholesterol
(and not DOPE) as colipid (Figure 2) remain elusive at
this stage of investigation.
F igu r e 5. Gel retardation (A) and DNase I sensitivities (B)
of lipoplex-associated DNA for representative lipid 5. The lipid/
DNA charge ratios are indicated at the top of each lane. The
details of the treatment are as described in the text.
made from di- (5) and trilysine (8) lipids (Table 1).
Strikingly, the sizes of lipid 1/DNA complexes were
essentially same as those of lipid 8/DNA complexes at
both higher lipid/DNA charge ratios of 9:1 and 3:1 (Table
1). Since the apparent sizes of all the lipoplexes were
found to be similar (Table 1), lipoplex sizes are unlikely
to play any key role in imparting superior transfection
properties to the monolysinylated lipids. The global
surface potentials of lipoplexes made from representa-
tive mono-, di-, and trilysinated lipids (1, 5, and 8,
respectively) were found to be similarly negative (around
-35 mV) in the presence of DMEM. Thus, surface
potentials of lipid/DNA complexes are also unlikely to
play any key role in modulating transfection efficacies
of the presently described lysinated cationic lipids.
Lip id /DNA Bin d in g a n d Lip op lex Sen sitivities
to DNa se I. Toward initial characterization of the
present lipoplexes, the electrostatic interactions between
the plasmid DNA and cationic liposomes as a function
of lipid/DNA charge ratios were determined by conven-
tional electrophoretic gel retardation assay. The repre-
sentative results summarized in Figure 5A demonstrate
that the dilysinated cationic lipid 5 strongly binds to
the plasmid DNA and completely inhibits its electro-
phoretic mobility at lipid/DNA charge ratio g 1:1. At
lipid/DNA charge ratios 0.3:1 and 0.1:1, significant
In summary, evaluating the in vitro gene delivery
efficacies of a series of novel cationic lipids with mono-,
di-, and trilysine headgroups and a common hydropho-
bic anchor skeleton (lipids 1-10, Schemes 1-3), we have
demonstrated, for the first time, that the monolysine
lipid with a myristyl tail exhibits superior transfection
efficacies compared to its di- and trilysine analogues in

2128 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Karmali et al.
protected immediate precursors for all the final lipids (provided
below) were in complete agreement with their molecular
structures (Schemes 1-3).
CHO, COS-1, and HepG2 cells. Contrary to popular
belief, our lipoplex size data indicate that singly lysi-
nated cationic amphiphiles can also be better or equally
DNA-compacting than their di- and trilysine counter-
parts. Both gel retardation and DNase I sensitivity
assays indicated similar lipid/DNA binding interactions
for all the novel mono-, di-, and trilysinated cationic
lipids. Taken together, the present findings support the
notion that caution needs to be exercised in ensuring
enhanced gene delivery efficacies of cationic lipids
through covalent grafting of multiple lysine function-
alities in the headgroup region.
Syn th esis of DTMLAC (Lip id 1, Sch em e 1). Step a .
Solid HOSu (0.20 g, 1.73 mmol) and DCC (0.36 g, 1.73 mmol)
were added sequentially to an ice-cold and stirred solution of
NR,Nꢀ-di-tert-butyloxycarbonyl-L-lysine (0.6 g, 1.73 mmol, pre-
pared from L-lysine and di-tert-butyldicarbonate as described
previously34) in dry DCM/dry DMF (9:1, v/v). After half an
hour, N-aminoethyl-N,N-di-n-tetradecylamine (I, 0.65 g, 1.44
mmol, prepared as described previously31) and DMAP (cata-
lytic) dissolved in dry DCM were added to the reaction mixture.
The resulting solution was left stirred at room temperature
for 24 h, solid DCU was filtered, and the solvent from the
filtrate was evaporated. The residue was taken in ethyl acetate
(100 mL) and washed sequentially with ice-cooled 1 N HCl
(3 × 100 mL), saturated sodium bicarbonate (3 × 100 mL),
and water (3 × 100 mL). The organic layer was dried over
anhydrous sodium sulfate and filtered, and the solvent from
the filtrate was removed by rotary evaporation. The residue
upon column chromatographic purification with 60-120 mesh
silica gel using 1-2% methanol/dichloromethane (v/v) as eluent
afforded 0.8 g (71.4%) of the pure intermediate N-2-[N′-(NR,Nꢀ-
di-BOC-L-Lysyl)]aminoethyl-N,N-di-n-tetradecylamine (Rf )
0.55, 5% methanol/dichloromethane, v/v). 1H NMR (200 MHz,
CDCl3): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)11-], 1.2-1.4 [bs, 44H,
-(CH2)11-], 1.3-1.6 [m, 4H, LysCγH2 + LysCδH2], 1.4 [s, 9H,-
Exp er im en ta l Section
Gen er a l P r oced u r es a n d Ma ter ia ls. The high-resolution
mass spectrometric (HRMS) analyses were performed on a
Micromass AUTOSPEC-M mass spectrometer (Manchester,
U.K.) with an OPUS V3 1X data system. Data were acquired
by the liquid secondary ion mass spectrometry (LSIMS)
technique using m-nitrobenzyl alcohol as the matrix. LSIMS
analysis was performed in the scan range 100-1000 amu at
a rate of 3 scans/s. 1H NMR spectra were recorded on a Varian
FT 200 MHz, AV 300 MHz, or Varian Unity 400 MHz
spectrometer. 1-Bromotetradecane, 1-bromohexadecane, 1-bro-
mooctadecane, n-tetradecylamine, n-hexadecylamine, and n-
octadecylamine were procured from Lancaster (Morecambe,
England). Oleylamine was purchased from Fluka (Switzer-
land), and L-lysine and NR-Z-L-lysine were purchased from
Aldrich. The progress of the reactions was monitored by thin-
layer chromatography on 0.25 mm silica gel plates. Column
chromatography was performed with silica gel (Acme Synthetic
Chemicals, India, finer than 200 and 60-120 mesh). p-CMV-
SPORT-â-gal plasmid was a generous gift from Dr. Nalam
Madhusudhana Rao. LipofectAmine was purchased from In-
vitrogen life technologies. Cell culture media, fetal bovine
serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (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 pur-
chased from Hi-media, India. DOPE was purchased from
Fluka (Switzerland). Unless otherwise stated, all the other
reagents purchased from local commercial suppliers were of
analytical grade and were used without further purification.
COS-1 (SV 40 transformed African green monkey kidney cells),
CHO (Chinese hamster ovary), and HepG2 (human hepato-
carcinoma) 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.
The purity of all the final lipids (1-10) was determined by
analytical HPLC (Shimadzu model LC10A) using a PARTISIL
5 ODS-3 WCS analytical column (4.6 mm × 250 mm, What-
man Inc., Clifton, NJ ) in two different mobile phases. One
solvent system (A) was methanol/acetonitrile/water/trifluoro-
acetic acid in the ratio 65:10:25:0.05 (v/v) for 15 min with a
flow rate of 0.8 mL/min. The other solvent system (B) was
methanol:water:trifluoroacetic acid in the ratio 75:25:0.05 for
15 min with a flow rate of 0.8 mL/min. Peaks were detected
by UV absorption at 219 nm. All the target lipids (1-10)
showed more than 95% purity. Typical retention times in
mobile phase B were 3.54 min (lipid 1), 3.60 min (lipid 2), 3.59
min (lipid 3), 3.58 min (lipid 4), 3.65 min (lipid 5), 3.57 min
(lipid 6), 3.86 min (lipid 7), 3.55 mins (lipid 8), 3.65 min (lipid
9), and 3.56 min (lipid 10).
CO-O-C(CH ) ], 1.45 [m, 4H, -N(-CH2-CH2-)2 ], 1.7 [m,
3
2H, LysCâH2],32.4 [t, 4H, -N(-CH2-CH2-)2], 2.5 [t, 2H, -N-
CH2-CH2- NH-CO], 3.1 [m, 2H, LysωCH2], 3.3 [m,2H, -N-
CH2-CH2-NH-CO-O-], 4.0 [m, 1H, LysCRH], 4.6 [m, 1H,
BOC -NH], 5.1 (m, 1H, Boc-NH), 6.8 [m, 1H, -CH2-CH2-
NH-CO-O-].
Step b. The intermediate obtained in step a was dissolved
in 3 mL of dichloromethane/methanol (2:1, v/v), and 3 mL of
methyl iodide was added. The solution was stirred at room
temperature overnight. Solvent was removed on a rotary
evaporator. The residue upon column chromatographic puri-
fication with 60-120 mesh size silica gel and 2-3% methanol
in dichloromethane (v/v) as eluent afforded 0.7 g (88.7% yield)
of the intermediate N-2-[N′-(NR,Nꢀ-di-BOC-L-Lysyl)]amino-
ethyl-N,N-di-n-tetradecyl-N-methylammonium iodide (Rf
)
0.45, 5% methanol in dichloromethane, v/v). 1H NMR (200
MHz, CDCl3): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)11-], 1.2-1.4
[bs, 44H, -(CH2)11-], 1.3-1.6 [m, 4H, LysCγH2 + LysCδH2],
1.4 [s, 9H, -CO-O-C(CH3)3], 1.6-1.8 [m, 4H, -N+(-CH2-
CH2-)2 ], 1.7 [m, 2H, LysCâH2], 3.1 [m, 2H, LysωCH2], 3.3 [s,
3H,-N+ -CH3], 3.4-3.5 [m, 4H, -N+(-CH2-CH2-)2], 3.7 [m,
4H, -N+-CH2-CH2-NH-CO-O], 4.1 [m, 1H, LysCRH], 4.8
[m, 1H, BOC -NH], 5.5 [m, 1H, BOC-NH], 8.2 [m, 1H,
-CH2-CH2-NH-CO-O-].
Step s c a n d d . The intermediate obtained in step b (0.7 g,
0.76 mmol) was dissolved in dry DCM (2 mL), and TFA (2 mL)
was added at 0 °C. The resulting solution was left stirred at
room temperature overnight to ensure complete deprotection.
Excess TFA was removed by flushing nitrogen to give the title
compound as a trifluoroacetate salt. Column chromatographic
purification using 60-120 mesh size silica gel and 10-12%
(v/v) methanol/chloroform as eluent followed by chloride ion
exchange chromatography (using amberlyst A-26 chloride ion
exchange resin) afforded 0.26 g (36.5% yield) of the pure title
compound (DTMLAC, Rf ) 0.2, 20% methanol in chloroform,
v/v). LSIMS (lipid 1): m/z 596 [M+] (calcd forC37H79ON4, 100%).
HRMS (LSIMS, lipid 1): m/z (for C37H79ON4, 100%) 595.6254;
found 595.6332.
DHMLAC (Lip id 2). 1H NMR (200 MHz, CDCl3) of N-2-
[N′-(NR,Nꢀ-di-BOC-L-Lysyl)]aminoethyl-N,N-di-n-hexadecyl-N-
methylammonium iodide (immediate precursor of lipid 2):
δ/ppm ) 0.9 [t, 6H, CH3-(CH2)13-], 1.2-1.4 [bs, 52H, -(CH2)13-
], 1.3-1.6 [m, 4H, LysCγH2 + LysCδH2], 1.4 [s, 9H, -CO-O-
C(CH3)3], 1.6-1.9 [m, 6H, -N+(-CH2-CH2-)2 + LysCâH2], 3.1
[m, 2H, LysωCH2], 3.3 [s, 3H, -N+-CH3], 3.4-3.5 [m, 4H, -N+-
(-CH2-CH2-)2], 3.8 [m,4H, -N+-CH2-CH2-NH-CO-O],
4.1 [m, 1H, LysCRH], 4.8 [m, 1H, BOC-NH], 5.45 [m, 1H,
Syn th esis. As representative examples, details of the
synthetic procedures are provided below for lipids 1, 5, and 8.
Lipids 2-4, 6 and 7, and 9 and 10 were synthesized following
the same protocol as those for lipids 1, 5, and 8, respectively.
Because of extensive line broadening in 1H NMR spectra of
all the final deprotected lipids (particularly in the region δ/ppm
) 3-5), all the final lipids were characterized by LSIMS and
HRMS (LSIMS). However, 1H NMR spectra of the BOC-

Lysinated Cationic Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2129
BOC-NH], 8.3 [m, 1H, -CH2-CH2-NH-CO-O-]. LSIMS:
m/z 651 [M+] (calcd for C41H87ON4 40 ammonium ion, 100%).
HRMS (LSIMS): m/z (for C41H87ON4, 40 ammonium ion, 100%)
651.6880; found 651.6841.
3.2-3.3 [m, 3H, -N-CH2-CH2-NH-CO and LysCRH], 4.5
[m, 1H, BOC-NH], 7.5 [m, 1H, -CH2-CH2-NH-CO-].
Step c. NR,Nꢀ-di-tert-Butyloxycarbonyl-L-lysine (0.38 g, 1.10
mmol) was coupled with intermediate II (0.63 g, 0.92 mmol,
prepared above in step b) in the presence of solid HOSu (0.13
g, 1.10 mmol), DCC (0.23 g, 1.10 mmol), and DMAP (catalytic)
following essentially the same protocol as described above in
step a of the DTMLAC synthesis. The resulting crude product
upon column chromatographic purification with 60-120 mesh
silica gel and 10% acetone in petroleum ether (v/v) as eluent
afforded 0.74 g (79.7% yield) of the pure compound N-2-[N′-
(NR,Nꢀ-di-BOC-L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-di-n-tet-
radecylamine as a white gummy solid. (Rf ) 0.42, 30% acetone
in pet-ether, v/v). 1H NMR (200 MHz, CDCl3): δ/ppm ) 0.9 [t,
6H, CH3-(CH2)11-], 1.2-1.4 [bs, 44H, -(CH2)11-], 1.3-1.9 [m,
16H, LysCâH2 + LysCâ′H2 + LysCγH2 + LysCγ′H2 + LysCδH2
+ LysCδ′H2 + -N(-CH2-CH2-)2], 1.4 [s, 27H, CO-O-
C(CH3)3], 2.4 [t, 4H, -N(-CH2-CH2-)2], 2.5 (t, 2H,-N-CH2-
CH2-NH-CO], 3.1 [m, 4H, LysωCH2 + Lysω′CH2], 3.3 [m, 2H,-
N-CH2-CH2-NH-CO-], 4.1 [m, 1H, LysCRH], 4.3 [m, 1H,
LysCR′H], 4.8 [m, 2H, BOC-NH], 5.4 [m, 1H, BOC-NH], 6.6
[m, 1H, CH2-CH2-NH-CO-], 6.8 [m, 1H, -CO-CH-NH-
CO-].
DOMLAC (Lip id 3). 1H NMR (200 MHz, CDCl3) of N-2-
[N′-(NR,Nꢀ-di-BOC-L-Lysyl)]aminoethyl-N,N-di-n-octadecyl-N-
methylammonium iodide (immediate precursor of lipid 3):
δ/ppm ) 0.9 [t, 6H, CH3-(CH2)15-], 1.2-1.4 [bs, 60H, -(CH2)15-
], 1.3-1.6 [m, 4H, LysCγH2 + LysCδH2], 1.4 [s, 9H, -CO-O-
C(CH3)3], 1.6-1.85 [m, 6H, -N+(-CH2-CH2-)2 + LysCâH2],
3.1 [m, 2H, LysωCH2], 3.3 [s, 3H, -N+-CH3], 3.4-3.5 [m, 4H,
-N+(-CH2-CH2-)2], 3.7 [m,4H, -N+-CH2-CH2-NH-CO-
O], 4.1 [m, 1H, LysCRH], 4.85 [m, 1H, BOC -NH], 5.45 [m,
1H, BOC-NH], 8.25 [m, 1H, -CH2-CH2-NH-CO-O-].
LSIMS: m/z 708 [M+] (calcd for C45H97ON4, 40 ammonium ion,
40%). HRMS (LSIMS): m/z (for C45H97ON4, 40 ammonium ion
100%) 707.7506; found 707.7466.
1
OOLAC (Lip id 4). H NMR (300 MHz, CDCl3) of N-2-[N′-
(NR,Nꢀ-di-BOC-L-Lysyl)]-N-n-octadecyl-N-oleyl-N-methylam-
monium iodide (immediate precursor of lipid 4): δ/ppm ) 0.9
[t, 6H, CH3-(CH2)17-], 1.2-1.4 [bs, 52H, -(CH2)13-], 1.3-1.6
[m, 4H, LysCγH2 + LysCδH2], 1.4 [s, 9H, -CO-O-C(CH3)3],
1.6-1.85 [m, 6H, -N+(-CH2-CH2-)2 + LysCâH2], 1.90-2.20-
[m, 4H, -CH2-CHdCH-CH2-], 3.1 [m, 2H, LysωCH2], 3.3 [s,
3H, -N+-CH3], 3.4-3.5 [m, 4H, -N+(-CH2-CH2-)2], 3.8 [m,
4H, -N+-CH2-CH2-NH-CO-O], 4.1 [m, 1H, LysCRH], 4.8
[m, 1H, BOC-NH], 5.30 [t, 2H, -CH2-CHdCH-CH2-], 5.45
[m, 1H, BOC-NH], 8.3 [m, 1H, -CH2-CH2-NH-CO-O-].
LSIMS: m/z 705 [M+] (calcd for C45H95ON4, 40 ammonium ion,
100%). HRMS (LSIMS): m/z (for C45H95ON4, 40 ammonium
ion, 100%) 705.7349; found 705.7347.
Step d . The intermediate obtained in step c (0.74 g, 0.73
mmol) was dissolved in 3 mL of dichloromethane/methanol (2:
1, v/v), and 3 mL of methyl iodide was added. The solution
was stirred at room temperature overnight, and the solvent
was removed on a rotary evaporator. The residue upon column
chromatographic purification with 60-120 mesh size silica gel
and 32% acetone in petroleum ether (v/v) as eluent afforded
0.37
g
(57.2% yield) of intermediate N-2-[N′-(NR,Nꢀ-di-
BOC-L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-di-n-tetradecyl-N-meth-
ylammonium iodide as a white gummy solid (Rf ) 0.24,
30% acetone in pet ether, v/v). 1H NMR (200 MHz, CDCl3):
δ/ppm ) 0.9 [t, 6H, CH3-(CH2)11-], 1.2-1.4 [bs, 44H, -(CH2)11-
], 1.3-2.0 [m, 16H, LysCâH2 + LysCâ′H2 + LysCγH2 + LysCγ′H2
+ LysCδH2 + LysCδ′H2 + -N+(-CH2-CH2-)2], 1.4 [s, 27H,
CO-O-C(CH3)3], 3.1 [m, 4H, LysωCH2 + Lysω′CH2], 3.3 [s, 3H,
-N+-CH3 ], 3.4 [m, 4H, -N+(-CH2-CH2-)2], 3.7 [m, 4H,
-N+-CH2-CH2-NH-CO], 4.1 [m, 1H, LysCRH], 4.3 [m, 1H,
LysCR′H], 4.9 [m, 2H, BOC-NH], 5.4 (m, 1H, BOC-NH), 7.2
[m, 1H, CH2-CH2-NH-CO-], 8.2 [m, 1H, -CO-CH-NH-
CO-].
Syn th esis of DTML2AC (Lip id 5, Sch em e 2). Step a . Nꢀ-
tert-Butyloxycarbonyl-NR-benzyloxycarbonyl-L-lysine (3.63 g,
9.53 mmol, prepared from NR-benzyloxycarbonyl-L-lysine and
ditertiarybutyl dicarbonate as described previously34) and
N-aminoethyl-N,N-di-n-tetradecylamine (I, 3.59 g, 7.94 mmol)
were coupled in the presence of DCC (1.96 g, 9.53 mmol), solid
HOSu (1.1 g, 9.53 mmol), DMAP (catalytic) following es-
sentially the same protocol as described above in step a of the
DTMLAC synthesis. The resulting crude product upon column
chromatographic purification with 60-120 mesh silica gel and
7% acetone in petroleum ether (v/v) as eluent afforded 4.43 g
(76.8%) of the pure intermediate N-2-[N′-(NR-Z-Nꢀ-BOC-L-
Lysyl)]aminoethyl-N,N-di-n-tetradecylamine as a white solid.
(Rf ) 0.43, 30% acetone in petroleum ether, v/v). 1H NMR (200
MHz, CDCl3): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)11-], 1.2-1.4
[bs, 44H, -(CH2)11-], 1.3-1.6 [m, 4H, LysCγH2 + LysCδH2],
1.4 [s, 9H, -CO-O-C(CH3)3], 1.45 [m, 4H, -N(-CH2-CH2-
)2 ], 1.7[m, 2H, LysCâH2], 2.4 [t, 4H, -N(-CH2-CH2-)2], 2.5
[t, 2 H,-N-CH2-CH2-NH-CO-O-], 3.1 [m, 2H, LysωCH2],
3.3 [m, 2H, -N-CH2-CH2-NH-CO-O-], 4.1 [m, 1H,
LysCRH], 4.6 [m, 1H, BOC-NH], 5.1 (s, 2H, Z-CH2), 5.5 (m,
1H, Z-NH), 6.8 [m, 1H, -CH2-CH2-NH-CO-], 7.3 (s, 5H,
Ar-H).
Step s e a n d f. The compound obtained in step d (0.36 g,
0.32 mmol) was dissolved in dry DCM (2 mL), and TFA (2 mL)
was added at 0 °C. The resulting solution was left stirred at
room temperature overnight to ensure complete deprotection.
Excess TFA was removed by flushing nitrogen to give the title
compound as a trifluoroacetate salt. Repeated crystallization
from diethyl ether/methanol (4:1) followed by chloride ion
exchange chromatography (using Amberlyst A-26 chloride ion
exchange resin) afforded 0.21 g (55.5% yield) of the pure title
compound DTML2AC (Rf ) 0.15, 20% methanol/dichloro-
methane, v/v). LSIMS (lipid 5): m/z 723 [M+] (calcd for
C
43H91O2N6, 40 ammonium ion, 10%). HRMS (LSIMS, lipid
Step b. The intermediate (1 g, 1.23 mmol) obtained in step
a was dissolved in dry methanol (10 mL) containing a few
drops of glacial acetic acid. Ammonium formate (0.39 g, 6.14
mmol) and 10% Pd/C (0.25 g) were added, and the reaction
mixture was allowed to stir under nitrogen atmosphere for 4
h. The catalyst was removed by filtration through Celite, and
the filtrate was evaporated to dryness. The residue was taken
in ethyl acetate (50 mL) and washed sequentially with 30%
aqueous potassium carbonate (3 × 50 mL), saturated sodium
chloride (3 × 50 mL), and water (3 × 50 mL). The organic layer
was dried over anhydrous sodium sulfate and filtered, and
rotatory evaporation of the solvent from the filtrate afforded
0.7 g (83.8%) of the intermediate N-2-[N′-(Nꢀ-BOC-L-Lysyl)]-
aminoethyl-N,N-di-n-tetradecylamine (II, Scheme 2) as a pure
white solid (Rf ) 0.2, 5% methanol in dichloromethane, v/v).
1H NMR (200 MHz, CDCl3): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)11-
5): m/z (for C43H91O2N6, 40 ammonium ion, 100%) 723.7184;
found 723.7204.
1
DHML2AC (Lip id 6). H NMR (200 MHz, CDCl3) of N-2-
[N′-(NR,Nꢀ-di-BOC-L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-di-n-
hexadecyl-N-methylammonium iodide (immediate precursor
of lipid 6): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)13-], 1.2-1.4 [bs,
52H, -(CH2)13-], 1.3-1.6 [m, 8H, LysCγH2 + LysCγ′H2
+
LysCδH2 + LysCδ′H2], 1.4 [s, 27H, CO-O-C(CH3)3], 1.6-1.8
[m, 4H, -N+(-CH2-CH2-)2], 2.0-2.2 [m, 4H, LysCâH2
+
LysCâ′H2], 3.1 [m, 4H, LysωCH2 + Lysω′CH2], 3.3 [s, 3H, -N+-
CH3 ], 3.4 [m, 4H, -N+(-CH2-CH2-)2], 3.7 [m, 4H, -N+-
CH2-CH2-NH-CO], 4.1 [m, 1H, LysCRH], 4.3 [m, 1H,
LysCR′H], 5.0 [m, 2H, BOC-NH], 5.65 (m, 1H, BOC-NH), 7.35
[m, 1H, CH2-CH2-NH-CO-], 8.25 [m, 1H, -CO-CH-NH-
CO-]. LSIMS: m/z 780 [M+] (calcd for C47H99O2N6, 40 am-
monium ion, 70%). HRMS (LSIMS): m/z (for for C47H99O2N6,
40 ammonium ion, 100%) 779.7830; found 779.7893.
], 1.2-1.4 [bs, 44H, -(CH2)11-], 1.3-1.6 (m, 4 H, LysCγH2
+
LysCδH2), 1.4 (s, 9H, CO-O-C(CH3)3, 1.45 [m, 4H, -N(-CH2-
CH2-)2 ], 1.7 (m, 2H, LysCâH2), 2.4 [t, 4H, -N(-CH2-CH2-
)2], 2.5 (t, 2 H,-N-CH2-CH2- NH-CO], 3.1 (m, 2H, LysωCH2),
1
DOML2AC (Lip id 7). H NMR (200 MHz, CDCl3) of N-2-
[N′-(NR,Nꢀ-di-BOC-L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-di-n-

2130 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Karmali et al.
+ LysCγ′H2 + LysCγ′′H2 + LysCδH2 + LysCδ′H2 + LysCδ′′H2
-N(-CH2-CH2-)2], 1.4 [s, 36H, -CO-O-C(CH3)3], 2.4 [t, 4H,
-N(-CH2- CH2-)2], 2.5 [t, 2H, -N-CH2-CH2-NH-CO-],
3.1 [m, 6H, LysωCH2 + Lysω′CH2 + Lysω′′CH2], 3.3 [m, 2H, -N-
CH2-CH2-NH-CO], 4.1 [m, 1H, LysCRH], 4.2-4.4 [m, 2H,
LysCR′H and LysCR′′H], 5.0-5.1 [m, 3H, BOC-NH], 5.8 [m, 1
H, BOC-NH], 7.4-7.7 [m, 3H, NH-CO ].
Step f. The compound prepared in step e (0.98 g, 0.78 mmol)
was dissolved in 3 mL of dichloromethane/methanol (2:1, v/v),
and 3 mL of methyl iodide was added to the solution. The
reaction mixture was stirred at room temperature overnight,
and the solvent was removed on a rotary evaporator. The
residue upon column chromatographic purification with silica
gel (60-120 mesh size) and 35% acetone in petroleum ether
(v/v) as eluent afforded 0.66 g (61% yield) of N-2-[N′-(NR,Nꢀ-
di-BOC-L-Lys-Nꢀ-BOC-L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-
di-n-tetradecyl-N-methylammonium iodide as a colorless gummy
solid (Rf ) 0.19, 30% acetone in petroleum ether, v/v). 1H NMR
(300 MHz, CDCl3): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)11-], 1.2-
1.4 [bs, 44H, -(CH2)11-], 1.3-1.6 [m, 12H, LysCγH2 + LysCγ′H2
+ LysCγ′′H2 + LysCδH2 + LysCδ′H2 + LysCδ′′H2], 1.4 [s, 36H,
octadecyl-N-methylammonium iodide (immediate precursor of
lipid 7): δ/ppm ) 0.9 [t, 6H, CH3-(CH2) 15-], 1.2-1.4 [bs, 60H,
-(CH2)15-], 1.3-2.0 [m, 16H, LysCâH2 + LysCâ′H2 + LysCγH2
+ LysCγ′H2 + LysCδH2 + LysCδ′H2 + -N+(-CH2-CH2-)2], 1.4
[s, 27H, CO-O-C(CH3)3], 3.1 [m, 4H, LysωCH2 + Lysω′CH2],
3.3 [s, 3H, -N+-CH3 ], 3.4 [m, 4H, -N+(-CH2-CH2-)2], 3.7
[m, 4H, -N+-CH2-CH2-NH-CO], 4.1 [m, 1H, LysCRH], 4.3
[m, 1H, LysCR′H], 5.0 [m, 2H, BOC NH], 5.65 (m, 1H, BOC
NH), 7.35 [m, 1H, CH2-CH2-NH-CO-], 8.25 [m, 1H, -CO-
CH-NH-CO-]. LSIMS: m/z 836 [M+] (calcd for C51H107O2N6,
40 ammonium ion, 75%). HRMS (LSIMS): m/z (for C51H107O2N6,
40 ammonium ion, 100%) 835.8581; found 835.8576.
+
Syn th esis of DTML3AC (Lip id 8, Sch em e 3). Step s a
a n d b. The synthesis is the same as steps a and b described
above for preparing intermediate II in DTML2AC synthesis.
Step c. Nꢀ-tert-Butyloxycarbonyl-NR-benzyloxycarbonyl-L-
lysine (0.87 g, 2.29 mmol) was coupled with intermediate II
(1.3 g, 1.91 mmol) in the presence of solid HOSu (0.27 g, 2.29
mmol), DCC (0.47 g, 2.29 mmol), and DMAP (catalytic)
following essentially the same protocol as described above in
step a of the DTMLAC synthesis. The resulting crude product
upon column chromatographic purification with 60-120 mesh
silica gel using 2.2% methanol in dichloromethane (v/v) as
eluent afforded 1.55 g (77.9% yield) of N-2-[N′-(NR-Z,Nꢀ-BOC-
L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-di-n-tetradecylamine as
a gummy solid (Rf ) 0.37, 30% acetone in petroleum ether,
-CO-O-C(CH3)3], 1.7 [m, 6H, LysCâH2 + LysCâ′H2
+
LysCâ′′H2], 1.8-1.95 [m, 4H, -N+(-CH2-CH2-)2 ], 3.1 [m, 6H,
LysωCH2 + Lysω′CH2 + Lysω′′CH2], 3.3 [s, 3H, -N+CH3], 3.4-
3.5 [m, 4H, -N+(-CH2-CH2-)2], 3.7-3.8 [m, 4H, -N+-CH2-
CH2-NH-CO-], 4.1 [m, 1H, LysCRH], 4.3 [m, 2H, LysCR′H
and LysCR′′H], 5.0-5.2 [bm, 3H, BOC-NH], 6.0 [m, 1H, BOC-
NH], 7.6 [m, 1H, -CH2-CH2-NH-CO-], 7.8 [m, 1H, -CO-
CH-NH-CO-], 7.9 [m, 1H, -CO-CH-NH-CO-].
1
v/v). H NMR (200 MHz, CDCl3): δ/ppm ) 0.9 [t, 6H, CH3-
(CH2)11-], 1.2-1.4 [bs, 44H, -(CH2)11-], 1.3-1.9 [m, 16H,
LysCâH2 + LysCâ′H2 + LysCγH2 + LysCγ′H2 + LysCδH2
+
LysCδ′H2+ -N(-CH2-CH2-)2], 1.4 [s, 18H, CO-O-C(CH3)3],
2.4 [t, 4H, -N(-CH2-CH2-)2, 2.5 [t, 2H, -N-CH2-CH2-
NH-CO-], 3.1 [m, 4 H, LysωCH2 + Lysω′CH2], 3.3 [t, 2 H, -N-
CH2-CH2-NH-CO-], 4.2 [m, 1H, LysCRH], 4.4 [m, 1H,
LysCR′H], 4.9 [m, 2H, BOC NH], 5.1 (s, 2H, Z-CH2), 5.9 [m,
1H, Z-NH], 6.7 [m, 1H, -CH2-CH2-NH-CO-], 7.0 [m, 1H,
-CO-CH-NH-CO-], 7.3 [s, 5H, Ar-H].
Step s g a n d h . The quarternized intermediate prepared
in step f (0.66 g, 0.47 mmol) was dissolved in dry DCM (3 mL),
and TFA (3 mL) was added to the solution at 0 °C. The
resulting solution was left stirred at room temperature over-
night. Excess TFA was removed by flushing nitrogen to give
the title compound as a trifluoroacetate salt. Repeated crystal-
lization from diethyl ether/methanol (4:1, v/v) followed by
chloride ion exchange chromatography (using Amberlyst A-26
chloride ion exchange resin) afforded 0.4 g (52.7% yield) the
pure title compound DTML3AC (Rf ) 0.1, 30% methanol in
dichlomethane, v/v). LSIMS (lipid 8): m/z 852 [M+] (calcd for
Step d . The compound prepared in step c (1.53 g, 1.47
mmol) was dissolved in dry methanol (10 mL) containing a
few drops of glacial acetic acid. Ammonium formate (0.46 g,
7.34 mmol) and 10% Pd/C (0.38 g) were added to the solution,
and the reaction mixture was allowed to stir under a nitrogen
atmosphere for 4 h to ensure complete hydrogenolysis. The
catalyst was removed by filtration through Celite, and the
filtrate was evaporated to dryness. The residue was taken in
ethyl acetate (50 mL) and washed sequentially with 30%
aqueous potassium carbonate (3 × 50 mL), saturated sodium
chloride (3 × 50 mL), and water (3 × 50 mL). The organic layer
was dried over anhydrous sodium sulfate, and the filtrate was
evaporated in a vacuum to afford 0.1.09 g (81.9%) of pure N-2-
[N′-(Nꢀ-BOC-L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-di-n-tet-
radecylamine as a white gummy solid (Rf ) 0.14, 5% methanol
C
49H103O3N8, 60%).
DHML3AC (Lip id 9). H NMR (400 MHz, CDCl3) of N-2-
1
[N′-(NR,Nꢀ-di-BOC-L-Lys-Nꢀ-BOC-L-Lys-Nꢀ-BOC-L-Lys)]amino-
ethyl-N,N-di-n-hexadecyl-N-methylammonium iodide (im m e-
d ia te p r ecu r sor of lipid 9): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)
13-], 1.2-1.4 [bs, 52H, -(CH2) 13-], 1.3-1.6 [m, 12H, LysCγH2
+ LysCγ′H2 + LysCγ′′H2 + LysCδH2 + LysCδ′H2 + LysCδ′′H2],
1.4 [s, 36H, -CO-O-C(CH3)3], 1.7 [m, 6H, LysCâH2
+
LysCâ′H2 + LysCâ′′H2], 1.8-1.95 [m, 4H, -N+(-CH2-CH2-)2
], 3.1 [m, 6H, LysωCH2 + Lysω′CH2 + Lysω′′CH2], 3.3 [s, 3H,
-N+CH3], 3.35-3.45 [m, 4H, -N+(-CH2-CH2-)2], 3.65-3.8
[m, 4H, -N+-CH2-CH2-NH-CO-], 4.1 [m, 1H, LysCRH], 4.3
[m, 2H, LysCR′H and LysCR′′H], 5.0-5.2 [bm, 3H, BOC NH],
6.0 [m, 1H, BOC NH], 7.6 [m, 1H, -CH2-CH2-NH-CO-],
7.8 [m, 1H, -CO-CH-NH-CO-], 7.9 [m, 1H, -CO-CH-
NH-CO-]. LSIMS: m/z 908 [M+] (calcd for C53H111O3N8, 40
ammonium ion, 14%).
1
in dichloromethane, v/v). H NMR (200 MHz, CDCl3): δ/ppm
) 0.9 [t, 6H, CH3-(CH2)11-], 1.2-1.4 [bs, 44H, -(CH2)11-],
1.3-1.9 [m, 16H, LysCâH2 + LysCâ′H2 + LysCγH2 + LysCγ′H2
+ LysCδH2 + LysCδ′H2 + N(-CH2-CH2-)2], 1.4 [s, 18H, -CO-
O-C(CH3)3], 2.4 [t, 4H, -N(-CH2-CH2-)2,], 2.5 (t, 2H, -N-
CH2-CH2-NH-CO-], 3.1 [m, 4H, LysωCH2 + Lysω′CH2], 3.2-
3.4 [m, 3H, -N-CH2-CH2-NH-CO- + LysCRH], 4.3 [m, 1H,
LysCR′H], 4.7 [m, 2H, BOC NH], 6.6 [m, 1H, -CH2-CH2-NH-
CO-], 7.7 [m, 1H, -CO-CH-NH-CO-].
1
DOML3AC (Lip id 10). H NMR (200 MHz, CDCl3) of N-2-
[N′-(NR,Nꢀ-di-BOC-L-Lys-Nꢀ-BOC-L-Lys-Nꢀ-BOC-L-Lys)]amino-
ethyl-N,N-di-n-octadecyl-N-methylammonium iodide (imme-
diate precursor of lipid 10): δ/ppm ) 0.9 [t, 6H, CH3-(CH2)
15-], 1.2-1.4 [bs, 44H, -(CH2)15-], 1.3-1.6 [m, 12H, LysCγH2
+ LysCγ′H2 + LysCγ′′H2 + LysCδH2 + LysCδ′H2 + LysCδ′′H2],
1.4 [s, 36H, -CO-O-C(CH3)3], 1.6-1.95 [m, 10H, LysCâH2 +
LysCâ′H2 + LysCâ′′H2 + -N+(-CH2-CH2-)2 ], 3.1 [m, 6H,
LysωCH2 + Lysω′CH2 + Lysω′′CH2], 3.3 [s, 3H, -N+CH3], 3.4-
3.5 [m, 4H, -N+(-CH2-CH2-)2], 3.7-3.8 [m, 4H, -N+-CH2-
CH2-NH-CO-], 4.1 [m, 1H, LysCRH], 4.3[m, 2H, LysCR′H and
LysCR′′H], 5.0-5.2 [bm, 3H, BOC NH], 6.0 [m, 1H, BOC NH],
7.6-7.8 [m, 2H, -CH2-CH2-NH-CO- + -CO-CH-NH-
CO-], 7.95 [m, 1H, -CO-CH-NH-CO-]. LSIMS: m/z 964
[M+] (calcd for C57H123O3N8, 40 ammonium ion, 12%).
Step e. NR,Nꢀ-Di-tert-butyloxycarbonyl-L-lysine (0.5 g, 1.44
mmol) was coupled with the intermediate prepared above in
step d (1.09 g, 1.2 mmol) in the presence of solid HOSu (0.17
g, 1.44 mmol), DCC (0.3 g, 1.44 mmol), and DMAP (catalytic)
following essentially the same protocol as described above in
step a of the DTMLAC synthesis. The resulting crude product
upon column chromatographic purification with 60-120 mesh
silica gel using 4% methanol in dichloromethane (v/v) as eluent
afforded 1.0 g (67.4% yield) of N-2-[N′-(NR,Nꢀ-di-BOC-L-Lys-
Nꢀ-BOC-L-Lys-Nꢀ-BOC-L-Lys)]aminoethyl-N,N-di-n-tetradecy-
lamine interemediate as a gummy solid (Rf ) 0.35, 30%
1
acetone in petroleum ether, v/v). H NMR (200 MHz, CDCl3):
δ/ppm ) 0.9 [t, 6H, CH3-(CH2)11-], 1.2-1.4 [bs, 44H, -(CH2)11-
Cell Cu ltu r e. COS-1 (SV 40 transformed African green
monkey kidney cells), CHO (Chinese hamster ovary), and
], 1.4-1.9 (m, 22H, LysCâH2 + LysCâ′H2 + LysCâ′′H2 + LysCγH2

Lysinated Cationic Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2131
HepG2 (human hepatocarcinoma) cell lines were procured from
the National Centre for Cell Sciences (NCCS), Pune, India.
Cells were cultured at 37 °C in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% fetal bovine serum, 50 µg/mL
penicillin, 50 µg/mL streptomycin, and 20 µg/mL kanamycin
in a humidified atmosphere containing 5% CO2.
P r ep a r a tion of Lip osom es. The cationic lipid and the
colipid (Chol or DOPE) in 1:1 mole ratio were 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. An amount of 5 mL of sterile deionized water
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 and occasionally sonicated
in a bath sonicator to produce multilamellar vesicles (MLVs).
MLVs were then sonicated in an ice bath until clear using a
Branson 450 sonifier at 100% duty cycle and 25 W output
power. The resulting clear aqueous liposomes were used in
forming lipoplexes.
phoretic mobility on a Zeta sizer 3000HSA (Malvern, U.K.).
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 a 200 ( 5 nm polystyrene polymer
(Duke Scientific Corps., Palo Alto, CA). The diameters of
liposomes and lipoplexes were calculated by using the auto-
matic mode. The ú potential was measured using the following
parameters: viscosity, 0.89 cP; dielectric constant, 79; tem-
perature, 25 °C; F(Ka), 1.50 (Smoluchowski); maximum voltage
of the current, V. The system was calibrated by using the
DTS0050 standard from Malvern. Measurements were done
10 times with the zero-field correction. The potentials were
calculated by using the Smoluchowski approximation.
Gel Reta r d a tion Assa y. DNA/lipid complexes were formed
by mixing 5 µL of plasmid DNA (0.1 µg/µL in 10 mM Hepes
buffer, pH 7.4) with varying amounts of cationic lipids so that
the final lipid/DNA charge ratios were mainted at 0.1:1 to 9:1
in a total volume of 50 µL. Complexes were incubated for 30
min at room temperature, after which 15 µL of each lipoplex
was loaded on a 1% agarose gel and electrophoresed (100 V, 2
h). The bands were visualized with ethidium bromide staining.
DNa se 1 Sen sitivity Assa ys. Briefly, in a typical assay,
1.5 nmol of DNA (500 ng) was complexed with lipid using the
indicated lipid/DNA charge ratios (Figure 5B) in 10 mM Hepes
buffer (pH 7.4) in a volume of 40 µL, and the mixture was
incubated at room temperature for 30 min on a rotary shaker.
Subsequently, the complexes were treated with DNase I (at a
final concentration of 1 ng/1.5 nmol of DNA) in the presence
of 10 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 mixture (1:1, v/v) and centrifuged at 10000g
for 5 min. The aqueous supernatants were separated, loaded
(15 µL) on a 1% agarose gel, and electrophoresed at 100 V for
2 h. The bands were visualized with ethidium bromide
staining.
P la sm id DNA. pCMV-SPORT-â-gal plasmids were a gen-
erous gift from Dr. Nalam Madhusudhana Rao (Centre for
Cellular and Molecular Biology, Hyderabad, India) and were
amplified in DH5R strain of Escherichia coli, isolated by
alkaline lysis procedure, and finally purified by PEG-8000
precipitation as described previously.35 The purity of plasmid
was checked by A260/A280 ratio (around 1.9) and 1% agarose
gel electrophoresis.
Tr a n sfection of Cells. Cells were seeded at a density of
15 000 (for COS-1) and 20 000 cells (for CHO and HepG2) per
well in a 96-well plate 18-24 h before the transfection. An
amount of 0.3 µg of plasmid DNA was complexed with varying
amounts of lipids (0.033-2.7 nmol for the monolysine series,
0.025-2.025 nmol for dilysine series, and 0.02-1.62 nmol for
trilysine series) 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. Immediately prior
to transfection, cells plated in the 96-well plate were washed
with PBS (2 × 100 µL) followed by the addition of lipoplexes.
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) and 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. Absor-
bance of the product o-nitrophenol at 405 nm was converted
to â-galactosidase units by using a calibration curve con-
structed using pure commercial â-galactosidase enzyme. Each
transfection experiment was repeated three times on three
different days. The transfection values reported were average
values from three replicate transfection plates assayed on
three different days. The values of â-galactosidase units in
replicate plates assayed on the same day varied by less than
20%. The day to day variation in transfection efficiency values
for identically treated transfection plates was mostly within
2- to 3-fold and was dependent on the cell density and condition
of the cells.
Ack n ow led gm en t. Financial support (to A.C.) re-
ceived from the Department of Biotechnology, Govern-
ment of India, New Delhi, is gratefully acknowledged.
P.P.K. and V.V.K. acknowledge the Senior Research
Fellowships received from the University Grant Com-
mision and Council of Scientific and Industrial Re-
search, Government of India, respectively.
Ap p en d ix
Ab b r evia t ion s: Boc, tert-butyloxycarbonyl; Chol,
cholesterol; DCC, dicyclohexylcarbodiimide; DCM, dichlo-
romethane; DMAP, 4-(dimethylamino)pyridine; DMEM,
Dulbecco’s modified Eagle’s medium; DMF, N,N-di-
methylformamide; DOPE, 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine; FBS, fetal bovine serum; HOSu,
N-hydroxysuccinimide; DTMLAC, N,N-di-n-tetradecyl-
N-m et h yl-N-2-[N′-(L-Lysyl)]a m in oet h yla m m on iu m
chloride; DHMLAC, N,N-di-n-hexadecyl-N-methyl-N-2-
[N′-(L-Lysyl)]aminoethylammonium chloride; DOMLAC,
N,N-di-n-octadecyl-N-methyl-N-2-[N′-(L-Lysyl)]amino-
ethylammonium chloride; OOMLAC, N-n-octadecyl-N-
oleyl-N-methyl-N-2-[N′-(L-Lysyl)]aminoethylammoni-
um chloride; DTML2AC, N,N-di-n-tetradecyl-N-meth-
yl-N-2-[N′-(L-Lysyl)2]aminoethylammonium chloride;
DHML2AC, N,N-di-n-hexadecyl-N-methyl-N-2-[N′-(L-
Lysyl)2]aminoethylammonium chloride; DOML2AC,
N,N-di-n-octadecyl-N-methyl-N-2-[N′-(L-Lysyl)2]amino-
ethylammonium chloride; DTML3AC, N,N-di-n-tetra-
decyl-N-methyl-N-2-[N′-(L-Lysyl)3]aminoethylammon-
ium chloride; DHML3AC, N,N-di-n-hexadecyl-N-
methyl-N-2-[N′-(L-Lysyl)3]aminoethylammonium chlo-
ride; DOML3AC, N,N-di-n-octadecyl-N-methyl-N-2-[N′-
Toxicity Assa y. Cytotoxicities of the lipids 1-10 were
assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) reduction assay as described earlier.4
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 experiments. 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) - background]} ×
100.
ú P oten tia l a n d Size Mea su r em en ts. The sizes and the
surface charges (ú potentials) of liposomes and lipoplexes were
measured by photon correlation spectroscopy and electro-

2132 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Karmali et al.
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