Polycations. 18. The synthesis of polycationic lipid materials based on the diamine 1,4-diazabicyclo[2.2.2]octane

Article abstract of DOI:10.1016/j.chemphyslip.2008.12.003

Herein is reported the preparation of several series of polyammonium salts that serve as cationic lipids or precursors thereof, and are structurally based on the parent diamine 1,4-diazabicyclo[2.2.2]octane (dabco). Through selective alkylation of dabco a variety of di- and tetracationic lipid species and precursors thereof have been prepared. The resultant materials are of significant interest for a variety of purposes, including serving as antimicrobial agents and antihydrophobic species, the details of which are provided in separate reports.

Full text of DOI:10.1016/j.chemphyslip.2008.12.003

Chemistry and Physics of Lipids 158 (2009) 61–69
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Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Polycations. 18. The synthesis of polycationic lipid materials based on the
diamine 1,4-diazabicyclo[2.2.2]octane
Robert Engela,∗, JaimeLee Iolani Rizzob, Christina Riverab, Mariah Ramireza, Mia Lace Huanga,
Diego Montenegroa, Craig Copodiferroa, Valbona Behaja, Marie Thomasa,c
Barbara Klaritch-vranaa, Jeanne Fabian Engela
,
a Department of Chemistry and Biochemistry, Queens College of CUNY, 65-30 Kissena Boulevard, Flushing, NY 11367, USA
b Department of Chemistry and Physical Sciences, Pace University, 1 Pace Plaza, New York, NY 10038, USA
c Doctoral Program in Chemistry, The Graduate Center of CUNY, 365 5th Avenue, New York, NY 10016, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein is reported the preparation of several series of polyammonium salts that serve as cationic lipids
or precursors thereof, and are structurally based on the parent diamine 1,4-diazabicyclo[2.2.2]octane
(dabco). Through selective alkylation of dabco a variety of di- and tetracationic lipid species and precursors
thereof have been prepared. The resultant materials are of significant interest for a variety of purposes,
including serving as antimicrobial agents and antihydrophobic species, the details of which are provided
in separate reports.
Received 14 November 2008
Received in revised form 11 December 2008
Accepted 15 December 2008
Available online 25 December 2008
Keywords:
Cationic lipid
© 2008 Elsevier Ireland Ltd. All rights reserved.
Dabco
Antimicrobial
Polycation
Quaternary ammonium salt
Antihydrophobic effect
1. Introduction
ing cationic lipids with variable positive charge depending on the
pH of the medium. Further, gemini lipids may be constructed read-
The di-tertiary amine 1,4-diazabicyclo[2.2.2]octane (dabco)
constitutes a most useful species for the construction of intrigu-
ing series of cationic lipids. The two tertiary amine sites, initially
identical and highly reactive as nucleophiles, are readily differen-
tiated in their reactivities upon alkylation of one through judicious
choice of solvent and electrophilic substrate so as to allow selective
alkylation at each of the two sites. Judicious choice of solvent and
electrophilic reagents used provides a wide range of cationic lipids
and other useful materials that can be prepared from dabco.
ily using dabco bearing these characteristics at each end of the
structure.
Prior efforts of these laboratories have been reported in which a
variety of polycationic salts have been constructed from dabco and
used for the preparation of room temperature ionic liquids (Lall et
al., 2000, 2002a,b; Engel et al., 2002; Cohen et al., 2002; Wishart et
al., 2005), as well as for their activity as specific ion binders (Cohen
et al., 2000), antimicrobial agents (Abel et al., 2002, 2004; Cohen et
al., 2004; Fabian et al., 1997), modifiers for chromatographic appli-
cations (Behaj et al., 2002), modifiers of DNA interactions (Strekas
et al., 1999), antihydrophobic agents for modification of solubility
(Cohen et al., 1998) and ion channel regulators (Gordon et al., 2006).
Additional summaries of these efforts have also been published
(Cohen and Engel, 1998, 2002).
The antimicrobial (antibacterial and antifungal) activity of the
cationic lipids and related cationic large molecular species has been
investigated and is understood in some instances (bacteria and
biofilms) as involving invasion and distortion of the cell wall leading
to lysis of the bacterial cell (Russell and Chopra, 1996; Cabral, 1992;
Kugler et al., 2005; Endo et al., 1987; Fidal et al., 1997; Friedrich et al.,
2000; Isquith et al., 1972; Tapias et al., 1994; Sicchierolli et al., 1995;
Campanhã et al., 2001; Lincopan et al., 2003; Lincopan et al., 2005;
One particularly intriguing aspect of cationic lipids prepared
using dabco is that a polar head group can be generated in which
two positive charges are present, as well as the possibility of prepar-
∗
Corresponding author. Fax: +1 728 997 5531.
E-mail address: robert.engel@qc.cuny.edu (R. Engel).
0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2008.12.003

62
R. Engel et al. / Chemistry and Physics of Lipids 158 (2009) 61–69
Table 1
Newly synthesized 1-alkyl-1-azonia-4-azabicyclo[2.2.2]octane salts.
.
Compound
number
R–X
Solvent
EtOAc
Yield
76%
1H NMR (solvent) (ı)
13 C NMR (ı)
Analysis
1
2
3
4
n-C3H7Br
(D2O) 0.83 (3H) t, 1.71
(2H) br, 3.10 (6H) br,
3.15 (2H) br, 3.29 (6H), br
13.2, 21.7, 44.2,
52.1, 64.7
Calcd: C9H19 N2Br(H2O)
C: 42.70%; H: 8.36%
Found: C: 42.57%; H: 8.50%
i-C3H7Br
i-C4H9Br
n-C5H11 Br
EtOAc
EtOAc
EtOAc
54%
61%
53%
(D2O) 0.94 (6H) d, 3.11
(6H) br, 3.28 (6H) br,
3.45 (1H) m
14.4, 45.1, 53.0,
66.3
Calcd: C9H19 N2Br(H2O)
C: 42.70%; H: 8.36%
Found: C: 42.63%; H: 8.42%
(D2O) 0.77 (6H) d, 1.65
(1H) br, 3.11, (6H) br,
3.45 (1H) m
13.1, 22.0, 44.6,
52.4, 64.4
Calcd: C10 H21 N2Br(H2O)
C: 44.95%; H: 8.68%
Found: C: 44.88%; H: 8.83%
(D2O) 0.79 (3H) t, 1.24
(4H) br, 1.66 (2H) br
3.11 (6H) br, 3.17 (2H), br,
3.23 (6H) br
13.0, 20.8, 21.5,
22.2, 42.0, 44.2
64.6
Calcd: C11 H23N2Br (2H2O)
C: 44.15%; H: 9.09%
Found: C: 44.22%; H: 9.00%
5
6
EtOAc
EtOAc
41%
64%
(D2O) 0.68 (3H) t, 0.91
12.7, 13.6, 22.7,
Calcd: C18 H30N2O3S
(3H) d, 1.22 (2H) br,
28.1, 44.5, 51.8,
64.4
C: 60.98%; H: 8.53%
Found: C: 60.79%; 8.67%
1.63 (1H) br, 3.12 (6H), br,
3.18 (2H) br, 3.33, (6H) br
(D2O) 0.78 (3H) t, 1.28
(6H) br, 1.67 (2H) br,
3.10 (6H) br, 3.17 (2H), br,
3.31 (6H) br
n-C6H13 Br
13.2, 21.0, 21.7,
25.2, 30.4, 44.2,
52.0, 64.7
Calcd: C12 H25N2Br(H2O)
C: 48.81%; H: 9.22%
Found: C: 48.71%; H: 9.23%
1.63 (1H) br, 3.12 (6H), br,
3.18 (2H) br, 3.33, (6H) br
(D2O) 0.79 (3H) t, 1.28
(8H) br, 1.69 (2H) br,
3.13 (6H) br, 3.18 (2H), br,
3.33 (6H) br
(D2O) 0.80 (3H) t, 1.13-
1.32 (14H) br, 1.72 (2H)
br, 3.14 (6H) m, 3.25, (2H)
m, 3.43 (6H) m
64.4
Found: C: 60.79%; 8.67%
7
8
9
n-C7H15
I
EtOAc
EtOAc
EtOAc
49%
96%
53%
13.4, 21.2, 21.9,
25.6, 27.8, 30.8,
44.3, 52.1, 64.8
Calcd: C13 H27N2I
C: 46.02%; H: 8.04%
Found: C: 46.02%; H: 8.15%
n-C10 H21 Br
n-C18 H37Br
13.9, 21.7, 22.7,
26.3, 29.1, 29.4,
29.51, 29.55, 31.9, 44.2, 52.1,
64.4
14.1, 22.2, 22.7,
Calcd: C16 H33N2Br
C: 57.65%; H: 9.98%
Found: C: 57.60%; H: 10.02%
(CDCl3) 0.81 (3H) t,
Calcd: C24H49N2Br
1.19–1.27 (30H) br, 1.76
(2H) br, 3.21 (6H) br, 3.44
(2H) br, 3.59 (6H), br
26.4, 28.9, 29.36,
C: 64.69%; H: 11.08%
Found: C: 64.73%; H: 11.00%
29.41, 29.43, 29.47, 29.51, 29.59,
29.63, 29.68, 29.72, 29.77,
29.85, 29.94, 31.93, 42.2, 45.4
13.0, 22.3, 22.8,
10
n-C22H45Br
EtOAc
58%
(CDCl3) 0.80 (3H) t,
Calcd: C28H57N2Br
1.17–1.30 (38H) br, 1.75
(2H) br, 3.20 (6H) br, 3.42
(2H) br, 3.60 (6H), br
26.3, 28.66, 28.93,
C: 67.04%; H: 11.48%
Found: C: 66.95%; H: 11.48%
29.34, 29.40, 29.44, 29.47,
29.50, 29.52, 29.58, 29.61,
29.67, 29.70, 29.73, 29.75,
29.84, 29.90, 29.95, 32.00, 45.3,
52.4
11
12
EtOAc
EtOAc
73%
44%
(D2O) 3.05 (6H) br, 3.27
44.1, 51.9, 66.5,
123.6, 129.0
Calcd: C9H17 N2Cl(2H2O)
(6H) br, 3.75 (2H) br,
5.57 (2H) m, 5.83 (1H), m
C: 48.10%; H: 9.42%
Found: C: 46.17%; H: 9.38%
(D2O) 0.91 (3H) d, 1.27
13.7, 21.6, 22.3,
Calcd: C11 H23N2OCl(H2O)
(2H) br, 1.63 (2H) br,
3.25 (6H) br, 3.71 (9H), br
44.7, 52.1, 63.3,
68.2
C: 52.27%; H: 9.99%
Found: C: 51.98%; H: 10.10%
13
14
EtOAc
EtOAc
61%
73%
(D2O) 1.85 (2H) m, 2.01
17.3, 31.1, 46.0,
Calcd: C11 H21 N2OCl
(3H) s, 2.55 (2H) br,
3.10 (8H) br, 3.34 (6H) br
53.9, 64.4, 65.1,
165.0
C: 56.76%; H: 9.09%
Found: C: 56.61%; H: 9.21%
(D2O) 3.09 (6H) t, 3.28
44.1, 53.2, 63.6,
65.2, 66.6
Calcd: C9H19 N2O2Cl
(2H) m, 3.40–3.52 (8H)
m, 4.23–4.29 (1H) m
C: 48.54%; H: 8.60%
Found: C: 48.14%; H: 8.36%

R. Engel et al. / Chemistry and Physics of Lipids 158 (2009) 61–69
63
Table 1 (Continued )
Compound
number
R–X
Solvent
EtOAc
Yield
65%
1H NMR (solvent) (ı)
13 C NMR (ı)
Analysis
15
16
(D2O) 1.99 (2H) br, 2.35
16.8, 30.0, 44.1,
Calcd: C11 H21 N2O2Cl(H2O)
(2H) br, 3.10 (6H) br,
3.19 (2H) br, 3.35 (6H), br,
3.71 (3H) s
52.0, 52.3, 63.2,
175.9
C: 49.53%; H: 8.69%
Found: C: 49.71%; H: 8.72%
EtOAc
86%
(D2O) 3.13 (3H) s, 3.59
(12H) m, 3.73 (2H) s
47.8, 51.6, 52.1,
60.2, 164.0
Calcd: C9H17 N2O2Cl
C: 45.91%; H: 10.70%
Found: C: 46.02%; H: 10.85%
Thome et al., 2003; Kanazawa et al., 1993; Popa et al., 2003), and
is at least one instance with fungi (C. albicans) has been shown to
be the result of changing the charge nature of the cell wall without
lysis, but leading to cell death (Vieira and Carmona-Ribeiro, 2006).
Such investigations have been performed using cationic ionene
polymeric species bearing monocationic regions or simple mono-
cationic lipid species. The incorporation of higher charge densities
within the polar portions of the cationic lipids would be anticipated
to provide corresponding effects at lower concentrations owing to
increased electrostatic disruption of the normal cell wall structure.
Indeed, when incorporated into surfaces, such polycationic species
exhibit very efficient cell lysis as determined with the Gram posi-
tive bacterium S. aureus (Abel et al., 2002, 2004; Cohen et al., 2004;
Melkonian, 2008). Specifically, S. aureus cells stained with Gram
stain when placed on such treated surfaces immediately release
the stain to the surroundings, the change in color and dispersal of
dye indicating immediate cell lysis.
Numerous additional cationic lipids and related salts based on
the dabco molecule have more recently been synthesized. These
have found use in a variety of biological systems for specific bio-
logical regulatory purposes. Among these purposes are their uses
as antiviral agents (Melkonian, 2008), antiparasitic agents (Yarlett,
2008), cardiac ion channel regulators (Abbott, 2008), and neuronal
ion channel regulators (Brumberg, 2008). The particular biolog-
ical characteristics of these polycationic lipids in these systems
will be discussed in reports concerning the specific biological phe-
nomena. The current report is concerned with the syntheses of
new polycationic lipids based on dabco and related salts that are
used therein. Continuing efforts will be concerned with the use of
such materials for generating artificial membranes with character-
istics quite different from those commonly found with biological
lipids.
is shown in Eq. (1).
(1)
The materials prepared in this manner can serve as simple
cationic lipids (with a wide range in chain lengths and atten-
dant functionality) and as precursors to structurally more complex
species. The full range of compounds (1–16) thus synthesized is
shown in Table 1 with data concerning the reaction yield, reaction
solvent, analytical data, and NMR (1H and 13C) spectra. Along with
other monoalkylated dabco salts previously reported (Cohen et al.,
2000), species 7–10 are of particular interest for direct binding to
pre-existing surfaces to render them antimicrobial, and are of inter-
est in establishing specific structural relationships with bacteria
and fungi studied. These materials by themselves are not observed
to have antiviral effects, although other types of dabco derivatives
(vide infra) have exhibited such effects (Melkonian, 2008). Other
species are of particular use for further structural elaboration.
The principal difficulty in accomplishing these syntheses in good
yield is related to the proper choice of solvent that allows reac-
tion to be limited to monoalkylation with minimal formation of
the dialkylated product. As anticipated many of these species are
quitehydrophilic andbecome hydratedupon standingforevenbrief
times open to the surrounding atmosphere. Anhydrous materials
can be obtained by drying under high vacuum with immediate use,
although this is generally not necessary for continuing syntheses
involving them as discussed here.
While ethyl acetate is the most common solvent used for effi-
cient monoalkylation of dabco, other solvent systems can be used,
including methylene chloride and simple alcohols. The use of ethyl
acetate facilitates the precipitation of the monoalkylated product
and thus prevents extensive dialkylation. With methylene chlo-
ride, while there is some solubility of the monoalkylated material,
the reactivity difference between dabco and the monoalkylated
species is such that selectivity can be attained simply by limiting
the amount of alkylating reagent used. Acetonitrile, on the other
hand, should be avoided for the solvent to allow maximization of
monoalkylation; the monoalkylated salts exhibit significant solu-
bility and reactivity in acetonitrile.
2. Results and discussion
Syntheses and descriptions of quaternary ammonium poly-
cationic lipids and their precursors, as well as derived species,
are organized herein according to structural types. The funda-
mental structural building blocks are presented first, with further
constructed materials following according to detailed structural
type.
2.1. 1-Alkyl-1-azonia-4-azabicyclo[2.2.2]octane salts
2.2. 4-(␻ꢀ-{1ꢀꢀ-Azonia-4ꢀꢀ-azabicyclo[2.2.2]octyl}-˛ꢀ-
alkyl)azonia-1-azabicyclo[2.2.2]octane
salts
These materials bearing a single quaternary ammonium site
within the dabco framework, along with a free tertiary amine site
on the same dabco unit, are produced by simple nucleophilic sub-
stitution reaction involving one of the two reactive dabco tertiary
amine sites on an alkyl unit bearing a reasonable leaving group, as
These materials constitute a building block both for more com-
plex salts derived from dabco, as well as gemini lipids via simple

64
R. Engel et al. / Chemistry and Physics of Lipids 158 (2009) 61–69
Table 2
Newly synthesized 4-(␻ꢀ-{1ꢀꢀ-azonia-4ꢀꢀ-azabicyclo[2.2.2]octyl)-␣ꢀ-alkyl)azonia-1-azabicyclo[2.2.2]octane salts.
.
Compound number
X–X
Solvent
CH3CN
Yield
74%
1H NMR (solvent) (ı)
13 C NMR (ı)
Analyses
17
Br(CH2)3Br
(D2O) 2.32 (2H) m,
3.19 (12H) br, 3.35
(4H) br, 3.45 (12H), br
14.0, 42.8, 51.1,
59.2
Calcd: C15 H30N4Br2
C: 42.27%; H: 7.09%
Found: C: 42.51%; H: 7.23% 17
18
19
20
21
22
Cl(CH2)4Cl
Cl(CH2)6Cl
ClCH2)8Cl
Br(CH2)9Br
Cl(CH2)10 Cl
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
71%
82%
77%
92%
92%
(D2O) 1.34 (4H) br,
3.18 (16H) br, 3.38
(12H) br
17.9, 45.5, 53.2,
65.7
Calcd: C16 H32N4Cl2
C: 54.69%; H: 9.18%
Found: C: 54.50%; H: 9.01%
(D2O) 1.29 (4H) br,
1.66 (4H) br, 3.00–3.16
(16H) br, 3.26 (12H), br
27.8, 31.9, 50.8,
58.7, 71.0
Calcd: C18 H36N4Cl2(H2O)
C: 54.40%; H: 9.64%
Found: C: 54.31%; H: 9.72%
(D2O) 1.31 (8H) br,
1.71 (4H) br, 3.14
(16H) br, 3.35 (12H), br
21.4, 25.7, 28.1,
31.2, 44.4, 64.7
Calcd: C20H40N4Cl2
C: 58.95%; H: 9.90%
Found: C: 58.79%; H: 10.03%
(D2O) 1.25 (10H) br,
1.63–1.65 (4H) br,
3.12–3.17 (16H) br, 3.27–3.31 (12H) br
19.3, 23.6, 26.2,
26.3, 42.4, 50.2,
62.8
Calcd: C21 H42N4Br2
C: 49.42%; H: 8.29%
Found: C49.21%; H: 8.45%
(D2O) 1.25 (12H) br,
1.65 (4H) br, 3.05–3.10
(16H) br, 3.25–3.31, (12H) br
22.7, 27.1, 29.7,
29.9, 45.7, 53.6,
66.2
Calcd: C22H44N4Cl2(1.5H2O)
C: 57.13%; H: 10.24%
Found: C: 57.22%; H: 10.51%
23
CH3CN
82%
(D2O) 3.14 (12H) br,
3.44 (16H) m, 3.66
(4H) s, 3.93 (4H) br
46.2, 55.1, 65.3,
65.4, 71.6
Calcd: C18 H36N4O2Cl2(H2O)
C: 50.34%; H: 8.92%
Found: C: 50.08%; H: 9.04%
alkylation of the remaining tertiary amine sites. Each of these
salts bears two positively charged sites (quaternary ammonium)
as well as two tertiary amine sites at opposite ends of the molecule.
Preparations are generally performed as shown in Eq. (2), with
acetonitrile as the solvent, which allows the initially formed inter-
mediate mono-salt to remain substantially in solution while the
second alkylation occurs.
Preparation of this category of species is best accomplished
through monoalkylation of dabco using an ␻-halo-1-alkanol fol-
lowed by conversion of the remaining residual primary alcohol site
to a halide by standard alcohol-to-halide conversion processes (vide
infra). This halide site then can be utilized in reaction with another
mono-alkylated dabco species to generate unsymmetrical di-dabco
species.
2.3. 1-Alkyl-4-alkyl’-1,4-diazoniabicyclo[2.2.2]octane salts
These unsymmetrical dicationic derivatives of dabco are an
intriguing set of species that can serve a in a variety of roles depend-
ing on the specific natures of the attached alkyl groups. With one
attached alkyl group relatively small, they can represent a relatively
simple group of ordinary cationic lipids with a highly charged unit
as the polar head group. With two large attached groups which
have the capability of folding toward each other they can serve as
a unique type of cationic lipid, again with a highly charged polar
head group.
They are readily constructed by the alkylation of the previously
noted 1-alkyl-1-azonia-4-azabicyclo[2.2.2]octane salts with an
additional electrophilic reagent (providing alkyl’). As the precursor
mono-quaternary salts exhibit significant solubility in acetonitrile,
while the dicationic target species exhibits only much more lim-
ited solubility in that solvent, isolation of the pure target species
is relatively simple. Following the occurrence of the alkylation,
the product precipitates from the acetonitrile solution. In some
instances other solvent systems were used that allow control of
the two separate alkylation procedures. The fundamental reaction
system is shown in Eq. (3).
(2)
Dicationic species of this type generally are insoluble in ethyl
acetate, but with slight solubility in acetonitrile. This character
allows the isolation of the materials in good yield, and the slight
solubility in acetonitrile, while not preventing their isolation, also
allows further reactions to be performed upon them. An excess of
dabco is used in these syntheses, the substrate bearing two displa-
cable units being added dropwise to facilitate optimal formation of
the desired dicationic salt.
The new compounds of this type presently synthesized for use
in further preparation of more complex species, including gemini
lipids, are listed in Table 2 with yields, 1H and 13C NMR data, and
elemental analyses.
Numerous attempts have been made to perform monoalkylation
reactions on substrates of the ␣, ␻-dihaloalkane type used herein
for attachment of dabco units at each end of the chain. These were
universally unsuccessful for the efficient preparation of species of
the type shown below.
(3)

R. Engel et al. / Chemistry and Physics of Lipids 158 (2009) 61–69
65
Table 3
Newly synthesized 1-alkyl-4-alkyl’-1,4-diazoniabicyclo[2.2.2]octane salts.
.
Compound
number
RꢀY
Yield
1H NMR
13 C NMR (ı)
Analyses
(solvent) (ı)
a
24
25
72
%
(D2O) 1.25 (2H) m, 2.71
26.2, 51.3, 52.0,
Calcd: C12 H24N2OclBr
C: 43.98%; H: 7.38%
(2H) br, 2.88 (2H) br,
3.52–4.11 (14H) br, 5.24,
(2H) m, 5.76 (1H) br
(D2O) 2.77 (2H) br,
55.0, 65.4, 66.4,
119.5, 131.4
Found: C: 44.11%; H: 7.53%
C6H5CH2Cl 11
63
%
44.0, 50.8, 67.1,
Calcd: C16 H24N2Cl2
3.79–3.95 (12H) br,
68.9, 122.3,
C: 60.95%; H: 7.91%
4.77 (2H) s, 5.68 (2H), m,
5.91 (1H) m, 7.51, (5H) br
122.5, 129.7, 131.1, 131.4,
131.1, 131.4,
Found: C: 60.78%; H: 7.91%
a
26
C6H5CH2Cl
79
%
(D2O) 0.81 (3H) t,
1.15–1.33 (16H)
13.2, 21.2, 21.9
23.4, 25.0, 26.2
Calcd: C24H44N2BrCl
br, 1.75 (2H) br, 3.05–3.51
(6H) m
C: 60.41%; H: 9.40%
3,90-3.99 (12H) m,
7.45–7.81 (5H) m
27.9, 28.2, 28.3, 28.4,
28.5, 28.6, 29.0, 29.7,
Found: C: 60.56%; H: 9.32%
31.0, 44.0, 50.6, 51.0, 65.1,
124.7, 129.5, 131.5, 132.7
b
27
C6H5CH2Cl
81
%
(D2O) 1.13–1.36 (16H)
21.3, 21.8, 25.1,
28.0, 28.3, 28.4,
Calcd: C23H40N2OCl2
br, 1.77 (2H) br, 3.06–3.49
(4H) br, 3.88–4.00
(12H) m, 7.46–7.88 (5H),
m
C: 64.02%; H: 9.34%
28.7, 31.1, 34.6, 44.1, 50.7,
50.9, 65.2, 124.9, 129.4,
131.6, 132.9
Found: C: 60.28%; H: 8.03%
b
28
29
ClC12 H25
81
%
(D2O) 0.84 (3H) t,
1.14–1.38 (20H) m,
1.77–2.08
(4H) m, 3.72 (6H) m,
4.03 (12H) m
13.9, 21.9, 22.6,
Calcd: C22H46N2OCl2
24.6, 25.8, 28.8,
C: 62.10%; H: 10.90%
Found: C: 61.96%; H: 11.01%
29.3, 29.4, 29.6, 29.7,
31.9, 34.2, 41.9, 51.2, 57.7,
58.4, 62.9, 65.1
b
ClC16 H33
72
%
(D2O) 0.85 (3H) t,
1.28–1.41 (28H) br,
1.85–2.26
13.9, 22.0, 22.7,
Calcd: C26H54N2OCl2
(4H) br, 3.65 (6H) m,
4.12, (12H) m
24.6, 26.0, 28.9,
C: 64.84%; H: 11.30%
Found: C: 64.58%; H: 11.42%
29.0, 29.1, 29.3, 29.6, 29.7,
29.9, 30.0, 30.2, 32.0,
34.1, 41.8, 51.2, 57.7, 58.4,
62.9, 65.1
b
30
ClC16 H33
63
%
(D2O) 0.79 (3H) t,
1.14–1.63 (30H) br,
1.70–1.85
13.8, 21.5, 22.0,
Calcd: C28H58N2OCl2
(6H) m, 3.51 (6H) m,
5.00 (12H) m
22.6, 24.5, 24.7,
C: 65.98%; H: 11.47%
Found: C: 65.83%; H: 11.52%
25.0, 26.1, 26.2, 29.1, 29.6,
29.8, 29.9, 30.1, 32.2, 45.4,
51.1, 61.4, 61.6, 65.1, 65.2

66
R. Engel et al. / Chemistry and Physics of Lipids 158 (2009) 61–69
Table 3 (Continued )
Compound
number
RꢀY
Yield
1H NMR
13 C NMR (ı)
Analyses
(solvent) (ı)
b
31
32
ClC12 H25
77
%
(D2O) 0.81 (3H) t,
1.12–1.33 (20H) br,
1.50–1.92
(2H) m, 3.34–3.82 (18H)
br
13.6, 16.9, 18.4,
Calcd: C21 H44N2OCl2
18.9, 27.2, 27.4,
C: 61.30%; H: 10.78%
Found: C: 61.25%; H: 10.84%
29.2, 32.4, 33.0, 33.2,
33.4, 35.1, 44.0, 51.1, 58.0,
61.3, 61.4
b
C6H5CH2Cl
83
%
(D2O) 1.38 (2H) m, 1.90
26.2, 34.2, 44.9,
Calcd: C16 H26N2OCl2
(2H) br, 3.02–3.51 (4H)
br, 3.91–4.07 (12H) br,
7.44–7.81 (5H) m
50.8, 51.2, 65.3,
125.4, 132.7, 131.3, 132.7
C: 57.66%; H: 7.86%
Found: C: 57.50%; H: 7.99%
33
34
15c
80
%
(D2O) 3.78 (6H) s, 4.30
(12H) br, 4.63 (4H) s
51.3, 52.5, 60.3,
163.2
Calcd: C12 H22N2O4Cl2
C: 45.98%; H: 10.77%
Found: C: 46.11%; H: 10.91%
a,c
76
%
(D2O) 1.23–1.45 (8H)
21.5, 25.6, 28.7,
Calcd: C15 H30N2O3Cl2
br, 3.31 (4H) br, 3.71
(3H) s, 4.11 (6H) br, 4.20
(6H) br, 4.49 (2H), br
28.8, 28.9, 32.2,
52.4, 54.0, 62.2, 65.8,
164.0
C: 45.70%; H: 10.87%
Found: C: 45.81%; H: 10.99%
c,d
35
36
68
%
(D2O) 1.20–1.41 (16H)
21.7, 25.3, 25.5,
Calcd: C19 H38N2O3Cl2
br, 3.29 (4H) br, 3.76
(3H) s, 4.07 (6H) br, 4.16
(6H) br, 4.53 (2H), br
28.4, 28.6, 28.7,
28.8, 31.6, 44.3, 51.7, 52.4,
54.0, 62.2, 65.8, 165.0
C: 55.20%; H: 9.26%
Found: C: 55.08%; H: 9.37%
a
C6H5CH2Cl
82
%
(D2O) 0.78 (3H) t,
1.08–1.32 (20H) br, 1.70
(2H)
m, 3.41 (2H) br. 3.82–3.97
(12H) br, 7.59 (5H)
m
13.6, 21.5, 22.2,
25.4, 26.2, 28.3,
Calcd: C25H44N2BrCl
C: 61.53%; H: 9.09%
28.6, 28.7, 28.8, 29.0,
Found: C: 61.44%; H: 9.25%
31.4, 50.7, 51.1, 65.2, 68.8,
124.8, 129.6, 131.6, 132.9
c,d
37
C6H5CH2Cl
71
%
(D2O) 1.29 (4H) br, 1.38
24.5, 24.9, 25.0,
Calcd: C19 H32N2OCl2
(2H) br, 1.73 (2H) br,
3.31–3.57 (6H) br,
3.78–3.90 (12H) br, 7.44
(5H) m
30.8, 50.7, 51.0,
61.6, 65.2, 68.9, 124.8,
129.6, 131.6, 132.9
C: 60.79%; H: 8.59%
Found: C: 60.70%; H: 8.64%
a
Cohen et al. (2000).
Fabian et al. (1997).
Methylene chloride was used as the solvent in this reaction.
Cohen and Engel (2002).
b
c
d
The characteristics and reaction data for these species, both for
example of this extension process is shown with the species
listed in Table 4. This specific derivatization involved the con-
version of 32 to the chloride 36 by reaction under commonly
used conditions (Fabian et al., 1997) with thionyl chloride. The
resultant 36 is then extended (unsymmetrically) by reaction with
1-(3ꢀ-hydroxypropyl)-1-azonia-4-azabicyclo[2.2.2] octane chloride
to generate 37. This two-step general approach can provide access
to the class of polycationic “string” species that are unsymmetrical
(have unlike termini).
structures within which R = Rꢀ and R =/ Rꢀ, are shown in Table 3.
References for reagent species previously reported are indicated as
well in Table 3.
Further derivatization of the products of these reactions has
been performed to provide multifunctional species that have par-
ticular capabilities for specific binding. Of particular note is the
conversion of a hydroxyl group to a halide, allowing continued
alkylation and extension of the polycationic “string” species. An

R. Engel et al. / Chemistry and Physics of Lipids 158 (2009) 61–69
67
Table 4
Newly synthesized derivatives of fundamental dabco salts.
Compound number
Precursor
Reactant
SOCl2
Yield
87%
1H NMR (solvent) (␦)
13 C NMR (ı)
Analysis
38
32
(D2O) 1.43 (2H) m, 1.88
(2H) br, 3.45–4.07 (16H)
br, 7.43–7.82 (5H) m
23.5, 41.1, 63.4,
69.4, 51.2, 51.9,
125.5, 128.9, 131.1, 132.7
Calcd: C16 H26N2OCl2
C: 54.63%; H: 7.16%
Found: C: 54.59%; H: 7.33%
a
39
38
72%
(D2O) 1.32 (2H) m, 1.45
23.5, 23.6, 49.8,
Calcd: C25H44N4OCl4
50.3, 50.4, 50.7,
51.1, 56.8, 59.8,
(2H) m, 1.91 (2H) br,
3.22–3.41 (8H) br, 3.51–4.11
(24H) br, 7.40–7.79 (5H) m
62.1, 68.1,
123.9, 128.8, 130.8, 132.0
C: 53.77%; H: 7.94%
Found: C: 53.82%; H: 7.83%
a
Cohen et al. (2000).
Symmetrical extension of termini for derivatives of
4-(␻ꢀ-{1ꢀꢀ-azonia-4ꢀꢀ-azabicyclo[2,2,2]octyl}-␣ꢀ-alkyl)azonia-1-
azabicyclo[2.2.2]octane salts These symmetrical derivatives of the
cursors is illustrated in Eq. (4), and new compounds of this type
presently synthesized are illustrated in Table 5 with their particular
preparation, NMR, and analytical data.
(4)
4-(␻ꢀ-{1ꢀꢀ-azonia-4ꢀꢀ-azabicyclo[2,2,2]octyl}-␣ꢀ-alkyl)azonia-1-
azabicyclo[2.2.2]octane salts have been prepared to generate
cationic lipid species and their precursors that incorporate not
only additional cationic sites within a defined structure, but at
the same time provide a variety of terminal functionality. This
process for construction of tetracationic lipids and their pre-
3. Experimental
3.1. General
All chemicals and solvents used in these syntheses and purifi-
cations were of commercial reagent quality and used without
Table 5
Newly synthesized symmetrical tetracationic derivatives of dabco.
Compound
number
Precursor
Reactant
Yield 1H NMR (solvent) (ı)
13 C NMR (ı)
Analysis
a
40
41
51%
81%
(D2O) 1.15–1.31 (28H)
21.3, 24.9, 25.1,
Calcd: C40H74 N4O2Br2Cl2
br, 1.35 (4H) m, 1.67
(4H) br, 3.36 (8H) br, 3.82
(12H) br, 3.94, (12H) br,
7.68 (4H) s
28.0, 28.2, 28.3,
28.4, 31.2, 44.1, 50.9, 51.1,
61.8, 65.4, 128.2, 134.1
C: 54.98%; H: 8.54%
Found: C: 54.87%; 8.59%
b
(D2O) 0.91 (6H) t, 0.95–1.38 14.0, 20.7, 21.0,
(56H) br, 2.11 (6H)
Calcd: C61H110 N4O7S2Cl2
s, 3.38–3.71 (33H) br,
22.8, 26.5, 27.5,
C: 63.90%; H: 9.67%
7.30 (8H) AA’BB’
28.2, 29.1, 29.6, 29.8, 29.9,
30.1, 30.2, 30.4, 31.2, 32.2,
43.4, 43.8, 50.6, 62.4, 72.0,
125.6, 129.0, 140.1, 141.3
21.4, 24.7, 51.3,
Found: C: 63.75%; H: 9.91%
42
43
44
HO(CH2)2Cl
HO(CH2)3Cl
TsO(CH2)13 CH3
19
19
19
68%
64%
63%
(D2O) 1.38 (4H) br,
1.82 (4H) br, 3.31–3.79
(12H) br, 3.85–4.15, (24H)
br
Calcd: C22H46N4O2Cl4
C: 48.89%; H: 8.58%
Found: C: 48.60%; H: 8.77%
52.0, 54.9, 64.9,
66.3
(D2O) 1.34 (4H) br,
1.77 (4H) br, 1.98 (4H)
br, 3.41–3.69 (12H) br, 3.98
(24H) br
(D2O) 0.81 (6H) t, 0.91-
1.36 (56H) br, 2.14 (6H)
s, 3.02 (4H) br, 3.37–3.76
(28H) br, 7.40 (8H), AA’BB’
21.3, 24.4, 24.7,
51.1, 51.2, 57.7,
62.8, 64.8
Calcd: C24H50N4O2Cl4
C: 50.71%; H: 6.67%
Found: C: 50.66%; H: 8.92%
13.8, 20.8, 21.6,
22.7, 26.2, 28.8,
29.0, 29.5, 29.6, 29.7, 29.8,
29.9, 30.0, 32.0, 32.2, 43.9,
50.8, 63.1, 69.1, 70.7, 125.7,
129.0, 140.1, 141.2
Calcd: C60H108 N4O6S2Cl2
C: 64.54%; H: 9.75%
Found: C: 64.38%; H: 9.81%
45
HO(CH2)10 Cl
21
83%
(D2O) 1.15–1.30 (36H)
br, 1.39 (4H) br, 1.71,
(8H) br, 3.38 (12H) br, 3.85
(24H) br
21.3, 21.4, 24.9,
25.1, 25.2, 28.0,
28.1, 28.2, 28.3, 28.4, 28.5,
31.2, 43.9, 50.8, 51.0
Calcd: C42H86N4O2Cl4
C: 61.44%; H: 10.56%
Found: C: 61.22%; H: 10.73%
a
Cohen et al. (2000).
Fabian et al. (1997).
b

68
R. Engel et al. / Chemistry and Physics of Lipids 158 (2009) 61–69
further purification. All 1H and 13C NMR spectra were measured
with samples in commercial deuterated solvents using a Brüker
400 MHz DPX400 instrument. Elemental analyses were performed
by Schwarzkopf Microanalytical Services of Woodside, NY.
ethyl acetate (3× 50 mL) and the solid material was dried under
high vacuum. Yield and analytical data are shown in Table 4.
3.6. Preparation of
1-benzyl-4-(3ꢀ-{4ꢀꢀ-[3ꢀꢀꢀ-hydroxypropyl]-1ꢀꢀ,4ꢀꢀ-
diazoniabicyclo[2.2.2]octane}-1,4-diazoniabicyclo[2.2.2]octane
tetrachloride (39)
3.2. Preparation of 1-alkyl-1-azonia-4-azabicyclo[2.2.2]octane
salts—general procedure (1–16)
The dicationic salt 1-benzyl-4-(3ꢀ-chloropropyl)-1,4-diazoni-
abicyclo[2.2.2]octane dichloride (38) (2.0 g; 5.6 mmol) along with
4-(3ꢀ-hydroxypropyl)-1-aza-4-azoniabicyclo[2.2.2]octane chloride
(1.1 g; 5.6 mmol) were mixed in acetonitrile (50 mL) and heated
at reflux with stirring for 3 days. After cooling, the solvent was
evaporated and the solid residue was washed with ethyl acetate
(3× 30 mL) and anhydrous ether (3× 30 mL) and dried under high
vacuum. Yield and analytical data are shown in Table 4.
The diamine dabco (1 equivalent amount) and the appropriate
haloalkane (0.5 equivalent amount) were dissolved in ethyl acetate
(4 x the combined volumes of the reagents) in a round-bottomed
flask and stirred at ambient temperature with a magnetic stirrer
for 24 h. After this time the white precipitate was collected by suc-
tion filtration through sintered glass, washed with ethyl acetate
(3× 35 mL) and anhydrous ether (3× 30 mL), and dried under high
vacuum. Excess dabco and any unreacted haloalkane were washed
away in the purification process. Typical yields of products and
analytical data are given in Table 1. In the instance of compound
5, rather than the haloalkane the alcohol, 2-methyl-1-butanol was
converted to its tosylate ester by reaction with p-toluenesulfonyl
chloride in pyridine using a standard procedure (Cohen et al., 2000),
and the resultant tosylate ester used as the electrophile.
3.7. Preparation of 1-alkyl-4-(␻ꢀ-{4ꢀꢀ-alkyl-1ꢀꢀ,4ꢀꢀ-diazonia
bicyclo[2.2.2]octane}alkyl’)-1,4-diazoniabicyclo[2.2.2]-octane
tetraanion salts—general procedure by reaction of a
1-alkyl-1-azonia-4-azabicyclo[2.2.2]octane salt with an
˛,␻-disubstituted alkylidene (40–41)
3.3. Preparation of 4-(␻ꢀ-{1ꢀꢀ-azonia-4ꢀꢀ-azabicyclo[2.2.2]octyl}-
˛ꢀ-alkyl)azonia-1-azabicyclo[2.2.2]octane salts (17–23)—general
procedure
The ␣,␻-ditosylalkane (or corresponding dihalo xylylidene) (1
equivalent amount) was dissolved in acetonitrile (4 × the volume
of the combined reagents) and to it was added the appropri-
ate 1-alkyl-1-azonia-4-azabicyclo[2.2.2]octane salt (2 equivalent
amounts) and the mixture was stirred with heating at reflux for
3 days. After this time the reaction mixture was cooled, the sol-
vent evaporated under reduced pressure, and the residual solid was
washed with ethyl acetate (3× 30 mL) and anhydrous ether (3×
30 mL) and dried under high vacuum. Yields and analytical data are
shown in Table 5.
The diamine dabco (1 equivalent amount) was dissolved in
acetonitrile (4 × the combined volumes of the reagents) in a
round-bottomed flask and the appropriate ␣, ␻-dihaloalkane (0.25
equivalent amount) dissolved in acetonitrile (2 × the combined
volumes of the reagents) was added dropwise with stirring. The
reaction was stirred at ambient temperature for 24 h after which
time the resultant white precipitate was collected by suction fil-
tration through sintered glass and was washed with ethyl acetate
(3× 30 mL) and anhydrous ether (3× 30 mL), and dried under high
vacuum. Typical yields of products and analytical data are shown
in Table 2.
3.8. Preparation of 1-alkul-4-(␻ꢀ-{4ꢀꢀ-alkyl-1ꢀꢀ,4ꢀꢀ-
diazoniabicyclo[2.2.2]octane}alkyl’)-1,4-
diazoniabicyclo[2.2.2]octane}alkyl’)-1,4-
diazoniabicyclo[2.2.2]octane tetraanion salts - general procedure
by reaction of a 4-(␻ꢀ-{1ꢀꢀ-azonia-4ꢀꢀ-azabicyclo[2.2.2]octyl}-˛ꢀ-
alkyl)azonia-1-azabicyclo[2.2.2]octane salt with a 1-haloalkane
or 1-alkane tosylate (42–45)
3.4. Preparation of
1-alkyl-4-alkyl’-1,4-diazoniabicyclo[2.2.2]octane salts
(24–36)—general procedure
The 1-alkyl-4-(␻ꢀ-{4ꢀꢀ-alkyl-1ꢀꢀ,4ꢀꢀ-diazoniabicyclo[2.2.2]octane
salt (1 equivalent amount) was dissolved in acetonitrile (4 × the
combined volume of reagents) and to it was added the 1-haloalkane
(or 1-alkane tosylate) (2 equivalent amounts) and the reaction mix-
ture stirred with heating at reflux for 3 days. After this time the
reaction mixture was cooled, the solvent evaporated under reduced
pressure, and the residual solid washed with ethyl acetate (3×
30 mL) and anhydrous ether (3× 30 mL) and dried under high vac-
uum. Yields and analytical data are shown in Table 5.
The appropriate 1-alkyl-1-azonia-4-azabicyclo[2.2.2]octane
salt (1 equivalent amount) was dissolved in acetonitrile (4 × the
combined volume of the reagents) and to it was added the appro-
priate haloalkane (1 equivalent amount). The reaction mixture was
stirred for 3 days at ambient temperature after which time the
white precipitate was collected by suction filtration through sin-
tered glass, washed with ethyl acetate (3× 30 mL) and anhydrous
ether (3× 30 mL), and dried under high vacuum. Typical yields of
products and analytical data are shown in Table 3.
Acknowledgements
3.5. Preparation of
1-benzyl-4-(3ꢀ-chloropropyl)-1,4-diazoniabicyclo[2.2.2]octane
We are grateful to the Magnet Program at the CUNY Graduate
Center and the NYC LSAMP for financial support to one of us (MT), as
well as the Howard Hughes Medical Institute for financial support
of the pre-college research experience, as well as Pace University
Scholarly Research Award (JR and CR), and finally the PSC-CUNY
Research Award program for general financial support of this effort.
dichloride (38)
The
dicationic
salt
1-benzyl-4-(3ꢀ-hydroxypropyl)-1,4-
diazoniabicyclo[2.2.2]octane dichloride (32) (3.0 g; 9.0 mmol)
was added to CHCl3 (25 mL) in a round bottomed flask (50 mL)
fitted with magnetic stirrer, oil bath and reflux condenser. To
it was added an excess of thionyl chloride (6.6 g; 56 mmol) and
the reaction mixture was stirred with heating at reflux for 16 h.
After this time ethanol (8 mL) was added to destroy the excess
thionyl chloride. After 2 h stirring the solvent was evaporated
under reduced pressure and the solid residue was washed with
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