Rational approaches to the design of cationic gemini surfactants for gene delivery

Article abstract of DOI:10.1021/ja005681c

We report a new class of amphiphilic gemini surfactants as vehicles for gene delivery into cells, and the beginnings of a systematic structure-activity study. Preliminary results suggest that combining gemini surfactants with dioleoylphosphatidylethanolamine (DOPE) should allow the preparation of liposomes of various sizes and lipid compositions. Control of such colloidal changes could be as significant as the changes in the molecular composition of the gemini surfactants in delivering optimum gene expression in animal models.

Full text of DOI:10.1021/ja005681c

VOLUME 123, NUMBER 26
J ULY 4, 2001
© Copyright 2001 by the
American Chemical Society
Rational Approaches to the Design of Cationic Gemini Surfactants for
Gene Delivery
Caroline McGregor,† Christele Perrin,‡ Myrna Monck,§ Patrick Camilleri,*,‡ and
Anthony J. Kirby*,†
Contribution from the UniVersity Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, U.K.,
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North) Third AVenue,
Harlow, Essex CM19 5AW, U.K., and SmithKline Beecham Pharmaceuticals,
1250 South CollegeVille Road, P.O. Box 5089, CollegeVille, PennsylVania 19426-0989
ReceiVed October 10, 2000
Abstract: We report a new class of amphiphilic gemini surfactants as vehicles for gene delivery into cells,
and the beginnings of a systematic structure-activity study. Preliminary results suggest that combining gemini
surfactants with dioleoylphosphatidylethanolamine (DOPE) should allow the preparation of liposomes of various
sizes and lipid compositions. Control of such colloidal changes could be as significant as the changes in the
molecular composition of the gemini surfactants in delivering optimum gene expression in animal models.
Introduction
Although entry into a targeted cell using a viral vector is
generally very efficient, the use of such vectors is not without
the risk of adverse or immunogenic reaction, or replication,
depending on the virus being used. As a result, delivery of DNA
mediated by cationic lipids has in many cases become a
preferred alternative to viral gene delivery into eukaryotic cells,
although levels of gene expression are usually lower than those
achieved by the use of viral vectors.5
Cationic lipids possess many features of an “ideal” vector.
In contrast to most viral systems, the size of the plasmid that
can be delivered is unrestricted. Moreover, the physicochemical
properties of cationic lipids can be varied to facilitate formula-
tion and adaptation to Good Manufacturing Practice. Com-
mercially attractive cationic lipids can in principle be designed
to combine good transfection efficiency with other desirable
features such as lower levels of toxicity and immunogenicity
as well as ease of manufacture.
A range of techniques and vehicles have been developed for
the transfer of genes into cells to induce protein expression.
Earlier methods included the direct injection of DNA in the
presence of calcium phosphate,1 and electroporation, where
DNA is driven across cell membranes by the application of an
electric current.2 More recent approaches involve the use of a
carrier molecule (the vehicle, or “vector”) to deliver the engi-
neered or foreign gene into target cells. Vehicles for the delivery
of genetic material are usually divided into two groups: those
that use viruses (e.g., retrovirus, adenovirus)3 and those using
cationic lipids, either alone or in formulation with a “helper”
lipid, such as dioleoylphosphatidylethanolamine (DOPE).4
† University Chemical Laboratory.
‡ SmithKline Beecham Pharmaceuticals, U.K.
§ SmithKline Beecham Pharmaceuticals, Pennsylvania.
(1) Graham, F. L.; Van der Eb, A. J. Virology 1973, 54, 536-539.
(2) Potter, H. Anal. Biochem. 1988, 174, 361-373.
(3) Galipeau, J.; Benaim, E.; Spencer, H. T.; Blakely, R.; Sorrentino, B.
P. Hum. Gene Ther. 1997, 8, 1773-1783.
Although a large number of cationic lipids have been
synthesized as potential gene delivery vehicles, we are not aware
of published attempts to generate quantitative structure-activity
(4) Felgner, P. L.; Tsai, Y. J.; Sukhu, L.; Wheeler, C. J.; Manthorpe,
M.; Marshall, J.; Cheng, S. H. Ann. N.Y. Acad. Sci. 1995, 772, 126-
139.
(5) Ledley, F. D. Curr. Opin. Biotechnol. 1994, 5, 626-636.
10.1021/ja005681c CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/07/2001

6216 J. Am. Chem. Soc., Vol. 123, No. 26, 2001
McGregor et al.
Figure 1. Structure-activity dependence on hydrocarbon tail length and peptide head section (see Table 1 for compound structures). CHO-DG44
cells were incubated with DNA and five different concentrations (4, 8, 10, 20, and 30 µM) of gemini surfactant, and luciferase activity (in counts
per second) averaged over four measurements. (a) The full set of data is shown for compound GS11 as a function of increasing gemini concentration;
highest activity was generally observed at 4 µM, and data for other compounds (panels b and c) represent measurements in the presence of 4 µM
surfactant. Data for L2000 (used under the recomended conditions) are included for comparison. (b) Effect of varying chain length (group R) for
the series with ꢀ, ꢀ-linked trilysine. (c) Effect of varying trilysine linkage; data are shown for compounds with the oleyl (C18) side-chain and (inset)
for the corresponding C12 derivatives (GS14, 4, 3, and 2).
Table 1. Structures of Selected Peptide-Based Gemini Surfactantsa
Scheme 1. General Route for the Synthesis of Gemini
Surfactants GS8
GS
(AA)n
R
GS
(AA)n
R
1
2
3
4
5
6
7
(Lys)2Lys-
C12
C12
8
9
Lys-R-Lys-R-Lys- C16
Lys-R-Lys-ꢀ-Lys- C18:1
∆9
∆9
∆9
∆9
∆9
Lys-ꢀ-Lys-ꢀ-Lys-
Lys-ꢀ-Lys-R-Lys- C12 10 (Lys)2Lys-
C18:1
C18:1
Lys-R-Lys-ꢀ-Lys- C12 11 Lys-ꢀ-Lys-ꢀ-Lys-
Lys-ꢀ-Lys-ꢀ-Lys-
C14 12 Lys-R-Lys-R-Lys- C18:1
Lys-R-Lys-R-Lys- C14 13 Lys-ꢀ-Lys-R-Lys- C18:1
Lys-ꢀ-Lys-ꢀ-Lys- C16 14 Lys-R-Lys-R-Lys- C12
a Lys-R-Lys indicates a normal peptide linkage: Lys-ꢀ-Lys-ꢀ-Lys-
is linked through the side-chain ꢀ-amino-groups (see Scheme 2), and
(Lys)2Lys- has lysines linked by amide bonds to both R- and ꢀ-amino
groups of the first lysine.
relationships (QSAR), essential for the optimization of drug
activity.6 We described recently the synthesis of several peptide-
based gemini surfactants which showed significant activity for
gene transfection.7 We now report the synthesis of a series of
novel multivalent gemini surfactants GS (Table 1), where the
nature of both the “head” peptide (AA)n and the “tail” group
(R, unbranched) has been systematically varied. The generic
structure and the general synthesis of these surfactants are shown
in Scheme 1.
more, these levels can be raised severalfold by appropriate
formulation. Control experiments (data not shown) showed that
single-chain analogues based on S-methyl cysteine were inactive.
The capabilities of compounds GS 1-14 to mediate the
transfer of a luciferase reporter gene across Chinese hamster
ovary (CHO-DG44) cell membranes were compared to that of
LipofectAMINE 2000 (L2000), a potent nonviral vehicle
recently commercialized by Life Technologies. Transfection
activity was determined by assay for luciferase activity (Figure
1a).
Increasing the length of the hydrocarbon “tail” leads to a
substantial increase in transfection activity (Figure 1b). Gene
expression was also found to depend on the nature of the amide
linkages between the three lysine residues in the headgroup.
Thus, three lysines linked through their ꢀ-amino groups rather
than through partial or total R-linkage appear to provide optimal
interaction of these dimeric cationic lipids with DNA (Figure
1c). The interaction must be at least partially electrostatic and
dependent on the spacing of the ammonium groups of the
Results and Discussion
We observe levels of transfection at least an order of
magnitude higher than for the original GS systems.7 Further-
(6) Miller, A. J. Angew. Chem., Int. Ed. 1998, 37, 1768-1785.
(7) Camilleri, P.; Kremer, A.; Jennings, K. H.; Jenkins, O.; Marshall, I.;
McGregor, C.; Neville, W.; Rice, S. Q.; Smith, R. J.; Wilkinson, M. J.;
Kirby, A. J. J. Chem. Soc., Chem. Commun. 2000, 1253-1254.
(8) Conditions. (i) BrCH2CH2Br, NaHCO3, 88% yield. (ii) Di-tert-
butyldicarbonate, NaOH, 66% yield. (iii) N-hydroxysuccinimide, dicyclo-
hexylcarbodiimide, quantitative. (iv) L-Serine, K2CO3, 90% yield. (v)
N-Hydroxysuccinimide, dicyclohexylcarbodiimide, quantitative. (vi) RNH2
(dodecylamine, tetradecylamine, hexadecylamine, natural oleylamine (85/
15 cis/trans, oleylamine, and elaidylamine), triethylamine, 50-90% yield.
(vii) concentrated HCl, CHCl3, 70-90% yield. (viii) N-hydroxysuccinimide
ester of BOC-protected peptide (N.B. all peptides were prepared by
traditional methods of peptide bond formation, NaOH, 50-70% yield. (ix)
concentrated HCl, CH3OH, 50-70% yield.

Design of Gemini Surfactants for Gene DeliVery
J. Am. Chem. Soc., Vol. 123, No. 26, 2001 6217
1 are at least doubled in the presence of the PLUS reagent, a
basic polypeptide available commercially from Life Technolo-
gies (proprietary composition). Replacement of the PLUS
reagent by the considerably less expensive polylysine had the
same enhancing effect, as shown in Figure 2, which compares
the transfection efficiencies of GS11 in C2C12 mouse muscle
cells in the presence of these additives. The addition of either
the pure enantiomeric form of polylysine (molecular weight
range 1000-4000) to the plasmid gave the same result as the
racemic mixture; the addition of polylysine of a higher-
molecular weight range produced a much smaller effect (data
not shown).
The good gene expression efficiency found for cationic
peptide-based lipids of the type shown in Table 1 is found in a
variety of eukaryotic cells other than CHO-DG44 and C2C12.
We are studying cells of various types (human cell lines of
kidney or neuronal origin, mouse cell lines, etc.) and measuring,
as described above, the activity of luciferase following trans-
fection with plasmid DNA. We have obtained satisfactory levels
of gene expression in such cell lines as SHSY-5Y or 1321N1,
of neuronal origin, normally considered difficult to transfect.
Similar or higher levels of gene-transfer activity were observed
using GS11 in combination with polylysine, compared with
results using LipofectAMINE 2000; increases of 2-5-fold were
observed in human neuronal SHSY-5Y and mouse MOPC-315
cells.
Figure 2. Effects of basic polypeptides on the gene expression
efficiency of GS11 in C2C12 mouse muscle cells. The luciferase
reporter gene (0.5 µg) was complexed with either PLUS reagent (1.5
µL) or poly-D,L-lysine (molecular weight range 1000 to 4000, 1 and 2
µL of stock solution) prior to the addition of GS11. The L2000:DNA
ratio was 1:3 (w:w), as recommended by Life Technologies. Stock
solutions contained 1 mg/mL of PLUS reagent or polylysine.
Nonviral approaches to in vitro gene transfer and expression
mediated by cationic lipids have used helper lipids to improve
efficiency, giving rise to so-called lipoplex systems. (This
formulation approach has led to the commercialization of
transfection reagents such as Lipofectamine). An appropriate
lipoplex formulation can have substantial advantages for a
chosen route of administration: lipoplexes can preserve the
structural integrity of plasmid DNA and have the potential to
achieve targeted delivery. As part of a planned series of in vivo
studies of the use of gemini surfactants, we have investigated a
similar formulation approach, combining a gemini surfactant,
GS11, with DOPE. We have carried out preliminary structural
characterizations of these formulations and tested their ability
to effect gene transfer and expression of luciferase in a CHO
vehicle; thus, the two (Lys)2Lys derivatives (GS1 and 10) also
showed low transfection activities. High transfection activity
requires both the right cationic headgroup and the optimal chain
length; although the activities of the R,R-linked trilysyl systems
(GS14, 6, 8, and 12) do increase with increasing chain length,
the most effective (oleyl) derivative GS12, is still less efficient
than the least effective compound with an ꢀ,ꢀ-linkage.
It is well-known that the gene expression efficiency of
cationic lipids can be improved in the presence of basic
peptides,9 which are thought to act as DNA “packaging” agents.
This effect is also observed for our gemini surfactants,
notwithstanding the basic tripeptide units built in. Thus, the gene
expression efficiencies of all of the gemini surfactants in Table
Figure 3. (A) Transfection experiment in CHO-DG44. The cells were incubated with the transfection mixtures overnight. GS11 was used alone
or in combination with DOPE. (B) Negative stained TEM micrograph of 70 mol % DOPE and 30 mol % GS11 in water. Bar ) 0.10 µm.

6218 J. Am. Chem. Soc., Vol. 123, No. 26, 2001
McGregor et al.
DX400 (400 MHz) instruments, using the solvent as an internal
deuterium lock. 13C NMR spectra were recorded on Bruker AC200
(50 MHz), Bruker AC250F (62.9 MHz), or Bruker AM400 (100 MHz)
instruments using internal lock and proton decoupling. Moisture-
sensitive reactions were performed under an inert argon atmosphere,
and all glassware was either flame- or oven-dried before use. Yields
are approximate because assessing purity is a problem; the compounds
are difficult to separate by TLC, while the hydrophobic tails make for
strong affinity with reverse phase HPLC columns. FAB (LSIMS) mass
spectra were obtained on a Micromass Autospec high-resolution double-
focusing instrument. Accurate masses were performed using poly-
(ethylene glycol) as a mass reference. Electrospray analysis was
performed on a Micromass Quattro II low-resolution triple quadrupole
mass spectrometer or Finnigan MAT 900 XLT high-resolution double
focusing instrument. Nebulization was pneumatically assisted by a flow
of nitrogen through a sheath around the capillary. Mass spectroscopic
analysis was performed by the EPSRC National Mass Spectrometry
Service Centre, University of Wales, Swansea.
The Central Unit. S,S′-Ethanediyl-bis-L-cysteine (1, R ) R′ ) H)10
was protected as the N,N′-bis-tert-butoxycarbonyl derivative and
converted to the bis-N-hydroxysuccinimide (NHS) ester, as follows.
1, R ) R′ ) H (2.68 g, 0.01 mol) was suspended in 30 mL of H2O
and 0.96 g (0.024 mol) of NaOH was added. After 5 min a clear solution
had formed, and the mixture was cooled to <10 °C. A solution of 4.36
g (0.02 mol) of (BOC)2O in 30 mL of THF was added dropwise over
30 min and the mixture stirred at room temperature overnight. After
further addition of a solution of 0.25 g (0.006 mol) of NaOH in 2.5
mL of H2O and 1.5 g (0.007 mol) of (BOC)2O in 7.5 mL of THF, the
mixture was stirred for a further 18 h.
The mixture was then acidified to pH 2 with 2 M HCl, 30 mL of
brine was added, and the solution was extracted with 3 × 40 mL of
THF and then 2 × 30 mL of ethyl acetate. The combined organic layers
were dried over MgSO4, and the solvent was removed under reduced
pressure. The resulting solid was recrystallized from MEK/pentane
(stirred at room temperature for 2 h and then kept at -20 °C for 2 h)
to give 3.1 g (65.9%) of 1 (R ) H, R′ ) COOBu-t) as white crystals,
mp 124-126 °C. 1H NMR (d6-DMSO, 400 MHz) δ 1.35 (s, 18H,
t-BOC group), 2.70 (s, 4H, (CH2)2, ethylene linker), 2.74 and 2.88 (Two
dd, 4H, J ) 4.5, 15.05 Hz and 7.14, 15.05 Hz, CH2 groups from cysteine
fragment), 4.05 (dt, 2H, J ) 4.5, 10 Hz, CHNH-t BOC group), 7.00
(d, 2H, J ) 8.41 Hz, NH group) and 12.35 (s, 2H, carboxyl group).
m/z 491.6432 [M + Na]+. C18H32O8N2S2 requires 468.5588.
cell line in vitro. Suspensions of DOPE/GS11 mixtures in water
give rise to a mixture of lipid vesicles and more complex
structures corresponding to particles e500 nm in diameter (see
Figure 3b). DOPE/GS11 formulations in molar ratios of 50/50,
60/40, and 70/30 were found to effect luciferase expression in
CHO cells at levels comparable to those attained using GS11
alone (see Figure 3a) and to Lipofectamine. Finally, we note
that the cytotoxicity of these systems is low. Cell death after
overnight incubations with GS11, either alone or mixed with
DOPE, was no more than that observed after 3 h incubations
with lipofectAMINE 2000.
Conclusions
We have systematically varied the structure of peptide-based
gemini surfactants GS (Scheme 1), leading to an increase in
levels of gene expression in vitro compared to well-established
nonviral reagents. These cationic lipids are relatively simple to
synthesize using standard peptide chemistry, and their chemical
structures can readily be tailored for effective interaction with
DNA. We have shown also that they are incorporated into
liposome formulations, providing further dimension in the
optimization of these molecules for in vivo studies.
We are currently introducing peptide and carbohydrate cell-
recognition sequences into the “head” groups of these systems.
These structural changes are designed to confer a degree of
selectivity in transfection by targeting specific tissues, perhaps
via cell surface receptors.
Experimental Section
Synthesis of Gemini Surfactants. Synthesis (Scheme 2) involved
the construction of the protected dimeric central unit (1), activation of
the cysteine carboxyl groups, followed by coupling with serine to give
2, and then similar activation of the serine carboxyl groups to allow
acylation of the long chain primary amine tail. Deprotection and
Scheme 2
To a solution of 2.96 g (6.33 mmol) of 1 (R ) H, R′ ) COOBu-t)
in 100 mL of THF (still-dried over lithium aluminum hydride) were
added 1.46 g (12.67 mmol) of N-hydroxysuccinimide and 2.61 g (12.67
mmol) of dicyclohexyl carbodiimide (DCC). The mixture was stirred
at room temperature for 18 h and then filtered through 1 cm of Celite,
the residue was washed with 100 mL of THF, and the filtrate was
evaporated to yield 1 (R ) N-succinimido, R′ ) COOBu-t) as an
off-white solid (4.09 g, 97.69), mp 140 °C (dec). 1H NMR (d6-DMSO,
400 MHz) 1.35 (s, 18H, t-BOC group), 2.85 (s, 4H, CH2 on linker),
2.74 (s, 4H, succinimide CH2). 3.00 (two dd, 4H, J ) 4.5, 15.1 Hz and
7.14, 15.1 Hz, CH2 groups from cysteine fragment), 4.5 (dt, 2H, J )
4.5, 10 Hz, CHNH-t BOC group), and 7.7 (d, 2H, J ) 8.21 Hz, NH
group). m/z 685.6914 [M + Na]+. C26H38O12N4S2 requires 662.7342.
Coupling of the Central Unit to L-Serine. L-Serine (2.10 g, 20
mmol) and K2CO3 (2.76 g, 20 mmol) were dissolved in 150 mL of
H2O. A suspension of 6.29 g (9.5 mmol) of 1 (R ) N-succinimido,
R′ ) COOBu-t) in 150 mL of THF was added. The cloudy mixture
was stirred at room temperature for 72 h. Most of the THF was removed
from the yellowish solution by evaporation, and the aqueous solution
was acidified to pH 1 with 1 M HCl. The mixture was extracted with
CH2Cl2 + 15% methanol (125, 50, and 50 mL) and the combined
organic layers were dried over MgSO4, filtered, and then evaporated.
2 (5.5 g, 90.22%) (R ) H, R′ ) COOBu-t) was collected as a colorless
solid foam. 1H NMR (d6-DMSO, 400 MHz) 1.35 (s, 18H, t-BOC group),
2.50 (s, 4H, linker CH2), 3.00 (two dd, 4H, J ) 4, 15 Hz and 7, 15 Hz,
CH2 group from cysteine fragment), 3.70 (dd, 4H, J ) 4, 12 Hz, serine
CH2), 4.15 (dt, 2H, J ) 4, 10 Hz, CH-NH-t-BOC), 4.25 (m, 2H, serine
acylation of the cysteine amino groups with the protected, activated
peptide gave the protected target compound. The procedure is described
for the preparation of GS11. The other surfactants were prepared by
straightforward modification of this route, which allows for independent
variation of all of the component parts of the surfactant.
1H NMR spectra were recorded at ambient probe temperature on
Bruker AC250F (250 MHz), Bruker WM250 (250 MHz), or Bruker
(9) Gao, X.; Huang, L. Biochemistry 1996, 35, 1027-1036.
(10) Zahn, H.; Traumann, K. Ann. Chem. 1953, 581, 168-191.

Design of Gemini Surfactants for Gene DeliVery
J. Am. Chem. Soc., Vol. 123, No. 26, 2001 6219
CH-NH), 6.9 (d, 2H, J ) 9 Hz, serine NH), 8.0 (d, 2H, J ) 8 Hz,
t-BOC protected NH) and 12.25 (s, 2H, serine carboxyl group). m/z
665.1879 [M + Na]+. C26H38O12N4S2 requires 642.7438.
7.52 (d, 1H, J ) 8 Hz, t-BOC-protected NH attached to R-CH), 7.75
(t, 1H, J ) 6 Hz, peptide bond NH attached to CH2). m/z 694.8951 [M
+ Na]+. C31H53O11N5 requires 671.7837.
Attachment of the Long-Chain Amine Tail. Compound 2 (2.02
g,3.15 mmol) (R ) H, R′ ) COOBu-t) prepared above was dissolved
in 47.5 mL of THF (still-dried over lithium aluminum hydride) under
argon, 0.82 g (7.13 mmol) of N-hydroxysuccinimide and 1.47 g (7.13
mmol) of DCC were added, and the mixture was stirred at room
temperature for 24 h. The reaction mixture was then filtered through 1
cm Celite, the filter was rinsed with 12 mL of THF, and the filtrate
was evaporated to give 2.75 g (>100%) of off-white solid. This solid
was dissolved in refluxing diisopropyl ether/THF 50/50 v/v and
precipitated by adding diisopropyl ether to yield 2 (R ) N-succinimido,
R′ ) COOBu-t) as an off-white powder (1.49 g, 56.6%), mp 52 °C
(dec). 1H NMR (d6-DMSO, 400 MHz) 1.5 (s, 18H, t-BOC group), 2.70
(s, 4H, linker CH2), 2.72 (s, 4H, succinimide CH2),3.02 (m, 4H, cysteine
CH2), 3.90 (t, 4H, J ) 6 Hz, serine CH2), 4.30 (m, 2H, CH-NH-t-
BOC), 4.81 (dd, 2H, J ) 6, 8 Hz, serine CH-NH), 5.35 (t, 2H, J ) 6
Hz, serine OH), 7.01 (d, 2H, J ) 8 Hz, t-BOC protected NH), 8.70 (d,
2H, J ) 8 Hz, serine NH), and 7.70 (d, 2H, J ) 8.21 Hz, NH group).
m/z 859.9234 [M + Na]+ C32H48O16N6S2 requires 836.8892.
4 (2.40 g, max. 3.47 mmol) (n ) 2, R′′ ) N-succinimido) prepared
above was dissolved in 17 mL of THF, and a solution of 0.94 g (3.82
m mol) N-R-t-BOC-L-lysine and 0.53 g (3.82 mmol) K2CO3 in 17 mL
of H2O was added immediately. The mixture was stirred at room
temperature for 72 h, and then most of the THF was removed by
evaporation and the remaining slurry acidified to pH 2 with 1 M HCl.
This mixture was then extracted with CH2Cl2 (2 × 50 mL), and the
combined organic layers were dried over Na2SO4 and evaporated to
yield 4 (n ) 3, R′′ ) H) as a white solid foam (2.74 g, 98.56%).1H
NMR (d6-DMSO, 400 MHz) 1.25 (s, 36H, t-BOC group), 1.42-1.62
(m, 18H, lysine CH2), 2.85 (m, 2H, lysine CH2NH-t-BOC), 2.91 (m,
4H, lysine CH2NH (peptide)), 3.79 (m, 3H, CH-NH-t-BOC), 6.61 (d,
2H, J ) 8 Hz, t-BOC-protected NH-CH), 6.72 (t, 1H, J ) 6 Hz, t-BOC-
protected NH-CH2), 7.02 (d, 1H, J ) 8 Hz, t-BOC-protected NH-CH),
7.75 (t, 2H, J ) 6 Hz, peptide bond NH attached to CH2) and 12.15 (s,
1H, carboxyl group). m/z 826.4496 [M + Na]+. C38H70O12N6 requires
803.0010.
Final Coupling Reaction. Activation of the Peptide Fragment. 4
(2.74 g, 3.42 mmol) (n ) 3, R′′ ) H), prepared above, was dissolved
under argon in 57 mL of THF (still-dried over lithium aluminum
hydride) containing 0.39 g (3.42 mmol) of N-hydroxysuccinimide and
0.71 g (3.42 m mol) of DCC. The mixture was stirred at room
temperature for 24 h and then filtered through 1 cm Celite, and the
residue was washed with THF. The filtrate was evaporated to yield
3.11 g (>100%) of 4 (n ) 3, R′′ ) N-succinimido) as a white solid
2 (3.59 g, 4.29 mmol) (R ) N-succinimido, R′ ) COOBu-t)
prepared above was dissolved in 65 mL of THF and stirred with
oleylamine (Aldrich, natural mixture: 2.35 g, 8.77 mmol) and 0.89 g
(1.22 mL, 8.77 mmol) of NEt3 at room temperature for 48 h. The THF
was removed by evaporation, and the off-white residue was redissolved
in 86 mL of chloroform (CHCl3) and extracted with 2 × 49 mL of
brine. The combined brine layers were extracted with 17 mL of CHCl3
The combined CHCl3 layers were then dried over MgSO4, and the
solvent was removed by evaporation. The residue was purified on a
silica column by flash chromatography using dichloromethane/ethyl
acetate/methanol 4/5/1 v/v/v. This produced 3.1 g (63.4%) of 3 (R )
1
foam. H NMR (d6-DMSO, 400 MHz) 1.30 (s, 36H, t-BOC), 1.41-
1.63 (m, 18H, lysine CH2), 2.78 (s, 4H, N-hydroxysuccinimide CH2),
2.90 (m, 2H, lysine CH2NH-t-BOC), 3.01 (m, 4H, lysine CH2NH
(peptide)), 3.79 (m, 2H, CH-NH-t-BOC), 4.27 (m, 1H, CH-NH-t-BOC),
6.6 (d, 2H, J ) 8 Hz, t-BOC-protected NH-CH), 6.70 (t, 1H, J ) 6
Hz, t-BOC-protected NH-CH2), 7.55 (d, 1H, J ) 8 Hz, t-BOC-protected
NH-CH), 7.71 (t, 2H, J ) 6 Hz, peptide bond NH attached to CH2).
m/z 922.4095 [M + Na]+. C42H73O14N7 requires 899.5215.
1
oleyl, R′ ) COOBu-t) as an off-white solid. H NMR (CDCl3, 400
MHz) 0.9 (t, 6H, J ) 6 Hz, terminal CH3 of oleyl chain), 1.2 (bs, 44H,
CH2 on long chain), 1.4 (s, 18H, t-BOC group), 1.95 (s, 8H, CH2CHd
CH), 2.73 (s, 4H, linker CH2), 3.01 (m, 8H, cysteine CH2 and tail NCH2
groups), 3.60 (m, 4H, serine CH2), 4.15 (m, 4H, CH-NH-t-BOC and
serine R-CH-NH), 4.90 (m, 2H, serine OH) and 5.21 (m, 4H, alkene
CH). In d6-DMSO amide NH peaks were observed at 7.01 (d, 2H, J )
9 Hz, t-BOC-protected NH), 7.70 (t, 2H, J ) 5 Hz, on oleylamine
NH) and 7.95 (d, 2H, J ) 8 Hz, serine NH). m/z: 1164.6111 [M +
Na]+. C60H112O10N6S2 requires 1141.7088.
Deprotection of the central unit. 3.1 g (2.72 mmol) of compound 3
(R ) oleyl, R′ ) COOBu-t) was dissolved in 35 mL of CH2Cl2 and
cooled in ice while 35 mL of HCl was added. The solution was stirred
until the reaction mixture reached room temperature. The CH2Cl2 was
then removed by evaporation, ethanol was added to form an azeotrope,
and the remaining solvent was removed slowly under vacuum. The
yellow solid was washed with several portions of diethyl ether to give
Assembly of the Protected Peptide Unit. BOC2LysOSuc (1.64 g,
3.69 mmol) was dissolved in 18 mL of THF, and a solution of 1 g (4.1
mmol) of N-R-t-BOC-L-lysine and 0.57 g (4.1 mmol) of K2CO3 in 18
mL of H2O was added immediately. The mixture was stirred at room
temperature for 72 h and then most of the THF was removed by
evaporation and the remaining slurry acidified to pH 2 with 1 M HCl.
This mixture was extracted with CH2Cl2 (2 × 54 mL), and the combined
organic layers were dried over Na2SO4 and evaporated to yield tri-t-
BOC-ꢀ-linked lysyl-lysine 4 (n ) 2, R′′ ) H) as a white solid foam
(1.99 g, 93.91%). 1H NMR (d6-DMSO, 400 MHz) 1.20 (s, 27H, t-BOC
group), 1.4-1.6 (m, 12H, lysine CH2), 2.85 (m, 2H, lysine CH2NH-
t-BOC), 2.90 (m, 2H, lysine CH2NH (peptide)), 3.78 (m, 2H, CH-NH-
t-BOC), 6.6 (d, 1H, J ) 8 Hz, t-BOC-protected NH-CH), 6.73 (t, 1H,
J ) 6 Hz, t-BOC protected NH-CH2), 7.00 (d, 1H, J ) 8 Hz, t-BOC-
protected NH attached to R-CH), 7.75 (t, 1H, J ) 6 Hz, peptide bond
NH attached to CH2) and 12.15 (s, 1H, COOH). m/z 597.1729 [M +
Na]+. C27H50O9N4 requires 574.7110.
1
2.6 g (92.9%) of 3 (R ) oleyl, R′ ) H), mp 59-61 °C (dec). H
NMR (CD3OD, 400 MHz) 0.90 (t, 6H, J ) 6 Hz, terminal CH3 of
oleyl chains), 1.21 (s, 44H, CH2 of oleyl chains), 1.95 (s, 8H,
CH2CHdCH), 2.71 (s, 4H, linker CH2), 3.00 (m, 8H, cysteine CH2
and tail NCH2 groups), 3.61 (m, 4H, serine CH2), 4.15 (m, CH-NH-
t-BOC and serine CH-NH) and 5.21 (s, 4H, alkene CH). In d6-DMSO
additional (NH) peaks were observed at 7.65 (t, 2H, J ) 6 Hz,
oleylamine NH on tail), 7.64 (m, 4H, cysteine CH-NH2) and 8.75 (d,
2H, J ) 8 Hz, serine NH). m/z 941.8432 [M]+: C50H96O6N6S2 requires
941.6832.
Final Coupling. Compound 3 (R ) oleyl, R′ ) H) 425 mg (0.42
mmol) prepared above was dissolved in 13 mL of H2O, and 37 mg
(0.93 mmol) of NaOH was added, followed by enough THF (25 mL)
to give a clear solution. A solution of activated protected peptide 4 (n
) 3, R′′ ) N-succinimido, 875 mg, g0.97 mmol) dissolved in 13 mL
of THF was added immediately, and the reaction mixture was stirred
at room temperature for 48 h. Most of the THF was removed by
evaporation, and another 13 mL of H2O was added; stirring continued
for 1 h. The solid precipitate was collected by filtration, rinsed with
water, and dried to yield the BOC-protected intermediate (1.03 g, 98%).
1H NMR (CD3OD, 400 MHz) 0.90 (t, 6H, J ) 6 Hz, terminal CH3 on
oleyl chains), 1.30 (s, 44H, oleyl chain CH2), 1.43 (s, 72H, t-BOC-
CH3),1.49-1.63 (m, 16H, lysine and oleyl CH2), 1.65-1.74 and 1.8-
1.87 (m, 24H, lysine CH2), 2.00 (m, 8H, CH2-CHdCH), 2.95 and
3.1-3.25 (m, 24H, CH2 groups from cysteine fragment, hydrocarbon
chain, lysine and linker CH2), 3.79-3.89 (s, 4H, serine CH2), 3.91-
3.99 (s, 6H, lysine R-CH-NH2), 4.39-4.44 (s, 4H, serine and cysteine
R-CH-NH), 5.33 (cis) and 5.38 (trans) (t, 4H, J ) 6 Hz, alkene CH).
4 (1.99 g, 3.47 mmol) (n ) 2, R′′ ) H) prepared above was
dissolved under argon in 60 mL of THF (still-dried over lithium
aluminum hydride) containing N-hydroxysuccinimide (0.40 g, 3.47
mmol) and DCC (0.72 g, 3.47 mmol). The mixture was stirred at room
temperature for 24 h and then filtered through 1 cm Celite, and the
residue was washed with THF. The filtrate was evaporated to yield 4
(n ) 2, R′′ ) N-succinimido, 2.40 g) as a white solid foam (>100%
1
yield). H NMR (d6-DMSO, 400 MHz) 1.30 (s, 27H, t-BOC groups),
1.41-1.60 (m, 12H, lysine CH2, 2.75 (s, 4H, N-hydroxysuccinimide
CH2), 2.85 (m, 2H, lysine CH2NH-t-BOC), 2.91 (m, 2H, lysine CH2NH
(peptide)), 3.78 (m, 1H, CH-NH-t-BOC), 4.24 (m, 1H, CH-NH-t-BOC),
6.81 (m, 2H, t-BOC-protected NH-CH and t-BOC-protected NH-CH2),

6220 J. Am. Chem. Soc., Vol. 123, No. 26, 2001
McGregor et al.
Final Deprotection. The solid BOC-protected intermediate (1.03
g, 0.49 mmol) prepared above was dissolved in 20 mL of CH3OH, and
the solution was cooled in ice while 20 mL of HCl was added. This
mixture was stirred (ca.1 h) until it reached room temperature. Ethanol
was added to form an azeotrope, and the solvent was removed slowly
under reduced pressure. The solid residue was redissolved in CH3OH;
addition of diethyl ether precipitated GS11 (0.56 g) as an off-white
camera system were calibrated using a MAG*I*CAL Si Ge lattice
(Norrox Scientific).
Cell Culture and DNA Transfection. Transfection studies were
performed using the adherent Chinese hamster ovary cell line, CHO-
DG44, and mouse muscle cell line, C2C12. Complete medium for CHO-
DG44 consisted of MEM alpha medium supplemented with 10% v/v
fetal bovine serum and 1× L-glutamine. C2C12 cells were grown in
DMEM medium supplemented with 10% v/v fetal bovine serum. All
media and supplements were obtained from Life Technologies.
In Vitro Gene Transfection. Cells were seeded into Biocat poly-
D-lysine 96 well/plates (Becton Dickinson) 16-18 h prior to transfection
at an approximate density of 2-4 × 104 cells per well. For transfection,
0.5 µg of the luciferase reporter gene plasmid, pGL3-Control Vector
(Promega), was incubated with various concentrations of the gemini
surfactants and complexing agents in a final volume of 100 µL. After
30 min incubation at rt, 0.4 mL of Opti-MEM medium (Life
Technologies) was added to the transfection mixture and the solution
placed on the cells (100 µL/well). Following 3 h or overnight incubation
at 37 °C, the transfection solution was replaced with complete medium,
and the cells were incubated at 37 °C. Control transfections were carried
out using lipofectAMINE 2000 (Life Technologies) according to the
manufacturer’s guidelines. Reporter gene assays were performed
according to the manufacturer’s guidelines (Roche Diagnostics) ap-
proximately 48 h post-transfection. Luminescence was measured in a
Packard TopCount NXT Microplate Scintillation and Luminescence
Counter.
1
powder (68%). H NMR (CD3OD, 400 MHz) 0.87 (t, 6H, J ) 6 Hz,
terminal CH3 on oleyl chains), 1.30 (s, 44H, oleyl chain CH2), 1.4-
1.55 (m, 16H, lysine and oleyl CH2), 1.56-1.67 and 1.7-1.8 (m, 24H,
lysine CH2), 1.93-1.99 (m, 8H, CH2-CHdCH), 2.91-3.00 and 3.15-
3.28 (m, 24H, CH2 groups of cysteine fragment, hydrocarbon chain,
lysine, and linker group), 3.72-3.86 (m, 4H, serine CH2), 3.89-4.09
(m, 6H, lysine R-CH-NH2), 4.4-4.55 (m, 4H, serine and cysteine R-CH-
NH), 5.33 (cis) and 5.38 (trans) (t, 4H, J ) 6 Hz, alkene CH). m/z
1710.2579 [M]+. C86H168O12N18S2 requires 1710.5192.
Preparation of DOPE/GS11 Lipid Mixtures. DOPE and GS11
were prepared individually as ethanol stock solutions. The stock
solutions were warmed briefly to approximately 50 °C, and aliquots
of each were taken to prepare 5 mg (total lipid) quantities of DOPE/
GS11 mixtures at 50/50, 60/40, and 70/30 molar ratios. The ethanol
was evaporated under a stream of nitrogen, leaving a thin lipid film
from which trace ethanol was removed under high vacuum overnight.
The DOPE/GS11 dried films were resuspended in Nanopure-filtered
water using one cycle of vortex mixing, bath sonication, and vortex
mixing. Samples were stored at approximately 4 °C.
TEM Negative Staining. Specimens (2 µL) were adsorbed on
carbon/Formvar-coated grids (Agar Scientific) and stained with 2.5%
w/v ammonium molybdate, containing 0.5% w/v trehalose, pH 7.0,
for 15 s and blotted dry. Samples were examined in a Hitachi H7100
microscope fitted with a Gatan MSC791 digital camera controlled by
Gatan Digital micrograph V.2.5 software (Gatan). The microscope and
Acknowledgment. This work is a contribution from the
European Network on Gemini Surfactants, supported by the
TMR Program of the European Commission.
JA005681C