Synthesis of pyridinium amphiphiles used for transfection and some characteristics of amphiphile/DNA complex formation

Article abstract of DOI:10.1002/(SICI)1099-0690(200002)2000:4<665::AID-EJOC665>3.0.CO;2-A

Pyridinium amphiphiles have found practical use for the delivery of DNA into cells. Starting from 4-methylpyridine, a general synthesis has been devised for the production of pyridinium amphiphiles which allows variation in both the hydrophobic part and in the headgroup area of the compounds. By means of differential scanning microcalorimetry, zeta potential, particle size measurements and cryo electron microscopy, some characteristics of the pyridinium amphiphile/DNA complexes have been determined.

Full text of DOI:10.1002/(SICI)1099-0690(200002)2000:4<665::AID-EJOC665>3.0.CO;2-A

FULL PAPER
Synthesis of Pyridinium Amphiphiles Used for Transfection and Some
Characteristics of Amphiphile/DNA Complex Formation
[a]
[a]
[a]
[a]
ˇ
´
Arthur A. P. Meekel, Anno Wagenaar, Jarmila Smisterova, Jessica E. Kroeze,
Peter Haadsma,[a] Bouke Bosgraaf,[a] Marc C. A. Stuart,[b] Alain Brisson,[b]
Marcel H. J. Ruiters,[c] Dick Hoekstra,[d] and Jan B. F. N. Engberts*[a]
Keywords: Cationic amphiphiles / Transfection / Pyridinium salts / Vesicles / Lipoplex
Pyridinium amphiphiles have found practical use for the de-
livery of DNA into cells. Starting from 4-methylpyridine, a
By means of differential scanning microcalorimetry, zeta po-
tential, particle size measurements and cryo electron micro-
general synthesis has been devised for the production of pyr- scopy, some characteristics of the pyridinium amphiphile/
idinium amphiphiles which allows variation in both the hy- DNA complexes have been determined.
drophobic part and in the headgroup area of the compounds.
Introduction
called SAINT (Synthetic Amphiphiles INTerdisciplinary),
to transfect efficiently eukaryotic cells.[4] Here we describe
in detail the synthesis of these pyridinium amphiphiles. In
addition, we report some physiochemical properties of the
SAINT amphiphiles and amphiphile/DNA complexes
which may be relevant for influencing the efficiency by
which these amphiphiles deliver DNA into the cell.
In recent times cationic amphiphiles have established a
firm position as reliable (re)agents for the delivery of DNA,
RNA, and oligonucleotides into eukaryotic cells. Although
the mechanism of action is not yet clear, in several struc-
ture–activity studies an appreciable number of cationic am-
phiphiles which promote the delivery of exogenous DNA
into mammalian cells have been reported.[1] In addition,
with respect to the in vivo delivery of polynucleotides, cat-
ionic amphiphiles are regarded as a viable alternative to
viral-based methods.[2] The latter are associated with risks
attendant with the uncontrolled recombination of viral
DNA with the host DNA, and the immunological reaction
generally related to the use of viruses. With the use of cat-
ionic amphiphiles these risks are avoided, although their
use in vitro is generally accompanied by cytotoxicity to a
variable extent. Compared to viral-mediated gene delivery,
the cationic amphiphiles are simple to use and display much
greater versatility in the type of material which can be de-
livered. Ultimately these developments may lead to accept-
able protocols for gene therapy.
Results and Discussion
Synthesis
The alkylpyridinium amphiphiles were synthesized by
double alkylation of 4-methylpyridine (1) by deprotonation
with 2 equivalents of LDA followed by the addition of 2
equivalents of the appropriate alkyl bromide or alkyl iodide
2 (Scheme 1). For the preparation of 3e, two separate al-
kylation steps were carried out successively. By using this
general procedure a representative series of 4-alkylated pyr-
idines 3 were synthesized. Quaternisation of the nitrogen of
the resulting alkylated pyridine with methyl iodide and
In general, mixing of the cationic amphiphile with the
polynucleotide of choice is sufficient for the formation of
the active complex or so-called lipoplex.[3] Previously, we
have reported on the capability of pyridinium amphiphiles,
[a]
University of Groningen, Stratingh Institute,
Nijenborgh 4, NL-9747 AG Groningen, The Netherlands
Fax: (internat.) ϩ 31-50/363-4296
E-mail: j.b.f.n.engberts@chem.rug.nl
[b]
University of Groningen, Department of Biophysical
Chemistry,
Nijenborgh 4, NL-9747 AG Groningen, The Netherlands
[c]
University of Groningen, Department of Pathology and
Laboratory Medicine,
Oostersingel 59, NL-9713 EZ Groningen, The Netherlands
[d]
University of Groningen, Department of Physiological
Chemistry,
Antonius Deusinglaan 1, NL-9713 AV Groningen, The
Netherlands
Scheme 1. Synthesis of SAINT amphiphiles
Eur. J. Org. Chem. 2000, 665Ϫ673
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J. B. F. N. Engberts et al.
FULL PAPER
subsequent ion-exchange gave the desired pyridinium am-
For satisfactory yields of the desired cationic amphiphiles
phiphiles 4 as the chloride salts.
7, the alkylation step was performed in neat pyridine or 4-
Whilst the saturated alkyl halides 2a–2d are commercially methyl pyridine (Scheme 5).
available, unsaturated alkyl halides had to be prepared.
Technical grade oleyl alcohol, 85% cis-isomer, was used for
the preparation of oleyl iodide. Mesylation of the alcohol,
followed by reaction with sodium iodide yielded the desired
iodide 2f. For the preparation of the alkyl halides having
either 100% cis (2g) or trans (2h) orientation, the corre-
sponding commercially available methyl esters were used as
Scheme 5. Synthesis of SAINT amphiphiles with inverted head-
precursors. Reduction with lithium aluminium hydride af- groups
forded the corresponding alcohols, which were then con-
Besides variation in the hydrophobic part of the am-
verted into the desired iodides as described above
(Scheme 2). The ratio of cis to trans double bond in the
final products was determined by double resonance NMR
experiments.
phiphile, variation in the headgroup was also pursued in
attempts to improve the efficiency of transfection. In par-
ticular, introduction of additional cationic charge could re-
sult in a more profitable interaction with the DNA to be
delivered into the cells. As described above for the ‘‘inverted
headgroup’’ pyridinium amphiphiles, pyridines can be read-
ily alkylated on the nitrogen with a variety of alkyl halides.
Quaternary amines are the cationic functionality of choice
to be incorporated into the headgroup. A series of bromoal-
kyltrimethylammonium bromides 8 with different (short)
alkyl chain lengths was synthesized following a known pro-
cedure.[6] Reaction of a dibromoalkane in ether with a solu-
tion of trimethylamine in ethanol results in substitution of
one bromine by a trimethylamine group with precipitation
of the desired product. Disubstitution is thus prevented. Al-
kylation of the 4-substituted pyridines in refluxing ethanol
with the bromoalkyltrimethylammonium salts results in
amphiphiles having two positive charges separated by a
short spacer (9) (Scheme 6). A twofold excess of a 4-substi-
tuted pyridine, which, after complete conversion of the bro-
mide could be easily recovered by chromatography, was
used in these reactions.
Scheme 2. Synthesis of alkenyl iodides
For the preparation of internal alkynes a common strat-
egy based on bromination of internal alkenes followed by
di-dehydrobromination proved troublesome. Alternatively,
the desired alkynyl bromides 2i, 2j, and 2k were synthesized
by deprotonation of terminal alkynes followed by alkylation
with an excess of an α,ω-dibromo alkane (Scheme 3). This
approach has the advantage that long-chain alkene brom-
ides with the double bond at different positions may be pre-
pared by partial hydrogenation of the internal triple bond.
Scheme 3. Synthesis of alkynyl bromides
In order to investigate if the position of the positive
charge in the pyridinium ring has an influence on the trans-
fection efficiency, amphiphiles with an ‘‘inverted head-
group’’ were synthesized. For the preparation of the re-
quired amphiphiles, a straightforward approach (which an-
ticipated direct alkylation of the pyridine nitrogen with the
hydrophobic moiety of interest) was adopted. The com-
pounds 6, which contain alkyl chains with a leaving group
at the central carbon atom, were obtained by a double
Grignard reaction with ethyl formate,[5] followed by mesyl-
ation of the resulting alcohol 5 (Scheme 4).
Scheme 6. Synthesis of double-charged SAINT amphiphiles
A primary amino group was introduced by a slightly dif-
ferent route. The alkylating agent 4-bromobutyl-phtalimide
10 (commercially available or readily prepared from 1,4-di-
bromobutane and potassium phthalimide) was used. Again
an excess of 4-substituted pyridine was employed in order
to allow the reaction to proceed at an appreciable rate and
to give a satisfactory yield. It was observed that after chro-
matography over Al2O3 (neutral, activity III) some of the
phthalimide product was hydrolysed to leave an acylated
amino group. Treatment of the resulting mixture of com-
pounds with hydrogen bromide in acetic acid afforded the
Scheme 4. Synthesis of double-chained mesylates
666
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Synthesis of Pyridinium Amphiphiles
FULL PAPER
pyridinium amphiphile bearing a free amino group 11 icles of cationic amphiphiles. It has been suggested that
(Scheme 7).
DOPE plays a role in destabilising the target membrane
through which the DNA has to be delivered.[8] For transfec-
tion studies in vivo, vesicles are generally prepared from
mixtures of cationic amphiphiles and cholesterol. The cho-
lesterol serves to stabilise the bilayer, consequently resulting
in a longer circulation time. We have determined the phase
transition temperatures of 1:1 mixtures of cholesterol and
the pyridinium amphiphiles and the data presented in
Figure 1.
Scheme 7. Synthesis of ammonium and guanidinium substituted
pyridinium salts
The amino group in amphiphile 11 was converted into a
guanidinium group with 12 (prepared from pyrazole and
cyanamide[7]).
Another class of interesting amphiphiles are the so-called
gemini compounds 14 in which the headgroups of two am-
phiphiles are linked via a carbon-chain spacer of variable
length. In order to achieve disubstitution of α,ω-dibromo
alkanes by 3c, f more than two equivalents of pyridine are
required and prolonged reaction times are necessary to
achieve complete disubstitution. It is important here not to
employ too high reaction temperatures to avoid degradation
of the 4-substituted pyridines. Acetone is the solvent of
choice (Scheme 8).
Figure 1. Phase transition temperatures of amphiphiles 4a–d pure
(circles), as a 1:1 mixture with cholesterol (squares) and as a 1:1
mixture with DOPE (triangles)
As expected, a linear relationship between Tm and the
chain length is observed for the pure compounds. Previ-
ously it has been shown that in mixtures there is a deviation
of this linearity.[9] There is a substantial influence on the
stability of the bilayers by DOPE as well as cholesterol. All
amphiphile/DOPE bilayers are in a liquid crystalline phase
at the temperature at which transfections are performed and
in the presence of cholesterol packing in the bilayer is much
tighter. Whether or not this has (a) an influence on the time
the complex of DNA and cationic amphiphile/cholesterol
will circulate in an organism and (b) an influence on the
efficiency of DNA delivery has yet to be established. Inter-
estingly, in vitro the cholesterol-containing complexes trans-
fect cells as effectively as DOPE-containing complexes, al-
though compared to the latter a two-fold higher concentra-
tion is usually required to obtain the same transfection effi-
ciency (unpublished observations). Finally we note that
amphiphiles with unsaturation in the chain have phase
Scheme 8. Synthesis of gemini SAINT amphiphiles
Some Characteristics for Amphiphile/DNA Complex
Formation
As evidenced by transmission electron microscopy, all transition temperatures below 0 °C.
amphiphiles used in this study form bilayer structures (data
The overall charge of the active amphiphile/DNA com-
not shown). In the complex interactions between such ves- plex is of importance for the efficiency of cationic delivery
icles and DNA, the fluidity of the bilayer plays an impor- systems.[10] Apart from interaction with the negatively
tant role. It should be noted that in vitro transfections on charged phosphate ester moieties of the DNA backbone, a
cell-cultures are normally performed at 37 °C. For the com- surplus of positive charge is needed for interaction with the
pounds 4a–d the main phase transition temperatures were net negatively charged cell membranes. By determining the
determined using differential scanning microcalorimetry. electrokinetic potential (zeta potential), a measure for the
This was carried out for the pure compounds as well as for net positive charge of the amphiphile/DNA complex can be
mixtures of the pyridinium amphiphiles with dioleoylphos- obtained. These zeta potentials were determined for a series
phatidylethanolamine (DOPE), a phospholipid commonly of complexes of 4f/DOPE (1:1) vesicles and DNA. The
used as co- or helper-lipid in transfections employing ves- DNA concentration was kept constant while the concentra-
Eur. J. Org. Chem. 2000, 665Ϫ673
667

J. B. F. N. Engberts et al.
FULL PAPER
tion of 4f/DOPE vesicles was varied. With equimolar dominating factor in determining whether a complex will
amounts of both DNA and 4f/DOPE vesicles, a strongly transfect a cell efficiently. These data do suggest however,
negative zeta potential, of ca. –20 mV, was measured. This that the bilayer remains intact on complex formation. At
value corresponds with those obtained when there is an ex- equivalent charge concentration not all negative charge is
cess of DNA. From approximately a ϩ/– charge ratio of 1.5 neutralized, resulting in an overall negatively charged com-
the zeta potential turned strongly positive, ca. 35 mV, and plex. However, at less then 2 equivalents of positive charge,
remained at this value up to a ϩ/– ratio of 7. Optimal trans- the bilayer, while remaining intact, has rearranged thus
fection is found at a ϩ/– charge ratio of 2.5.[4b] Interestingly, compacting the DNA.[11] This notion could not be substan-
the ‘‘inverted headgroup’’ amphiphile 7d gives similar tiated by particle size measurements, employing dynamic
values. Obviously the value of the zeta potential is not a light scattering, due to the polydispersity of the samples.
Typically particles of 120 to 220 nm in diameter were ob-
served. At ϩ/– charge ratios greater then 5, larger aggre-
gates, 600–800 nm, were formed.
Direct information on the structure of the amphiphile/
DNA complex was obtained by cryo-electron microscopy
(EM). Vesicles of 4f/DOPE 1:1 were prepared by sonication
in water and were analysed by cryo-EM. A large population
of vesicles of 30–40 nm in size was observed. On addition
of plasmid DNA, 4700 base pairs in size, resulting in a ϩ/–
charge ratio of 2.5, aggregates of approximately 150–
200 nm in diameter were found. All vesicles appeared to be
recruited by the DNA, and no ‘‘empty’’ or small vesicles
could be observed. Extruded vesicles of 100 nm size, as
shown in Figure 2a, were also prepared. When DNA was
added, only the dense structures (represented in Figure 2b,
c) were found. Again a rather uniform aggregate size was
observed. In some areas spikes, protruding at the surface of
the aggregates, can clearly be seen (Figure 2d arrows). It
would be of interest to investigate to what extent the size
of the DNA dictates the size of its aggregate with cationic
amphiphiles. The amphiphile/DNA aggregates clearly re-
veal a specific association which is tentatively recognized as
a bilayer sandwich. Lammelar structures were found at a
repeat distance of 6.5 nm, similar to structures reported in
the literature.[12]
Transfection Experiments
Transfections were carried out using a β-galactosidase as-
say.[13] Briefly, vesicles of cationic amphiphiles and DOPE
(1:1) were mixed with plasmid DNA which contained an
encoded reporter protein. The resulting aggregate was put
onto cultured cells (COS-7) and after 2 days the cells were
analysed for the presence of the reporter protein. A selec-
tion of the results is listed in Table 1. The transfection effici-
encies are given in arbitrary units and compared to the effi-
ciency of Lipofectin [a mixture of dioleyloxyproyl trimeth-
ylammonium chloride (DOTMA) and DOPE], which is one
of the most widely used transfection reagents.[14]
It is evident from these results that introduction of unsat-
uration in the alkyl chains increases the transfection effici-
ency. The orientation, cis or trans, and in particular, the
bond multiplicity i.e. double or triple of the unsaturated
group have a significant influence. Furthermore, the intro-
duction of an additional positive charge in the headgroup
strongly affects the transfection efficiency, depending upon
the nature of the second cationic moiety. For example, an
additional trimethylammonium group 9a–9d has a large
decremental effect on the transfection efficiency. The gemini
Figure 2. Cryo-TEM micrographs. Top: vesicles of 4f/DOPE (1:1).
Bottom: complex of vesicles of 4f/DOPE (1:1) with DNA. Bar rep-
resents 100 nm
668
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Synthesis of Pyridinium Amphiphiles
FULL PAPER
elemental analyses were difficult to obtain due to the presence of
shorter and longer carbon chain homologues in the alkyl-chain
starting materials, as was evidenced by electrospray mass spectro-
metry. – Differential scanning calorimetry (DSC) measurements
were carried out on a Perkin–Elmer DSC-7 apparatus. Sample con-
centration was 10–40 m. Scan rate was 1 °C/min. From 5 °C to –
5 °C, the scan rate was 0.5 °C/min and at 0 °C an equilibration
time of 30 min was employed. – The zeta potential measurements
were performed on a MALVERN 200 apparatus, equipped with a
He/Ne laser. The concentration of DNA used was 15 mg/mL in
20 m Hepes buffer, pH 7.4. An average molecular weight for a
nucleic acid monomer of 330 was taken to calculate the concentra-
tion of negative charge. Dynamic light scattering was performed on
a Neyvis instrument equipped with an Ar laser. Threefold diluted
samples used for the zeta potential determinations were em-
ployed. – Cryo-electron microscopy was performed on holey car-
bon films. Images were recorded under low dose conditions on a
Philips (CM 120) electron microscope operating at 120 kV,
equipped with a Gatan model 626 cryo stage. Samples were 2.4 m
in DNA concentration and were prepared in water. Transfections
were performed as previously described.[4]
Table 1. Transfection efficiencies (TE) of SAINT amphiphiles, in
arbitrary units
Compound
TE
Lipofectin
4a
1
R ϭ C12H25
0.5
1
4b
R ϭ C14H29
4c
R ϭ C16H33
R ϭ C18H37
1
4d
0.5
1.6
4.8
5.9
2.1
1
4e
R ϭ C18H35; R ϭ C18H37
R ϭ C18H35 (85% cis)
R ϭ C18H35 (100% cis)
R ϭ C18H35 (100% trans)
R ϭ C16H29
4f
4g
4h
4i
4k
R ϭ C18H33
2
7b
R ϭ C16H33; R’ ϭ CH3
R ϭ C16H33; n ϭ 3, 4, 5
R ϭ C18H35; n ϭ 4
R ϭ C16H33; n ϭ 3, 4, 5
R ϭ C18H35; n ϭ 4
0.1
0
9a–c
9d
0
14a–c
14d
1
0.1
SAINTS 14a–14c have a performance similar to that of
lipofectin, but in the case of octadecenyl chains 14a, the
transfection ability is reduced. We note, however, that the
effects of unsaturation and additional charge separately are
not additive. Interestingly, inversion of the headgroup 7
leads to a large decrease in the ability to deliver DNA.
Synthesis of Oleyl Iodides 2f–h: A solution of mesyl chloride
(6.5 mL, 84 mmol) in dichloromethane (50 mL) was added drop-
wise to an ice-cold solution of oleyl alcohol (20.5 g, 76.3 mmol)
and triethylamine (15.5 mL, 1.5 equiv.) in dichloromethane
(350 mL). The mixture was stirred at room temperature. The reac-
tion was monitored with NMR spectroscopy and quenched as soon
as the oleyl alcohol had disappeared (after 15–60 min) by the addi-
tion of water. The organic phase was washed successively with HCl,
NaHCO3, and brine then dried (Na2SO4), and the solvent evapo-
rated to give the desired mesylate as a viscous oil (25.5 g,
73.6 mmol, 96%). The precursor for 2f is a solid at 0 °C. The pre-
cursors 2g–h were crystallized from ethanol: m.p. cis-mesylate 9 °C,
m.p. trans-mesylate 32 °C. – 1H NMR (200 MHz, CDCl3): δ ϭ
0.87 (t, 3 H, CH3), 1.26 (m, 22 H, CH2), 1.67–1.78 (m, 2 H,
CH2CH2OMs), 1.97–2.05 (m, 4 H, CH2CHϭCHCH2), 3.00 (s, 3
H, OMs), 4.20 (t, J ϭ 6.6 Hz, 2 H, CH2OMs), 5.32–5.37 (m, 2 H,
CH). Sodium iodide (5.1 g, 34 mmol) was added to a solution of
the mesylate (9.26 g, 26.7 mmol) in acetone (100 mL) and the reac-
tion mixture heated under reflux for 2 h. After cooling to room
temperature, the mixture was filtered, the filtrate evaporated and
then taken up in hexane and again filtered through Al2O3. On evap-
oration of the solvent a clear oil was obtained (8.9 g, 23.5 mmol,
88%). The cis- to-trans ratio was determined with double resonance
NMR experiments and irradiation at 2.00 ppm. Two singlets ap-
peared at 5.34 for the cis and at 5.38 for the trans orientation.
Integration of these signals gave the relative amounts of the iso-
Conclusions
A number of cationic pyridinium amphiphiles have been
synthesized in order to identify structural parameters that
influence the efficiency of these amphiphiles to deliver
DNA into eukaryotic cells. Unsaturation in the hydro-
phobic moiety of the amphiphile has the most marked ef-
fect. An additional cationic charge in the headgroup also
affects the transfection efficiency. Both effects are not syner-
gistic. Mixed bilayers of pyridinium amphiphile and DOPE
are in a fluid state under the conditions for transfection. As
evidenced by cryo-EM, well-defined SAINT/DNA com-
plexes are formed. Atomic force microscopic studies of the
structure of the amphiphile/DNA complexes are currently
in progress.
1
Experimental Section
mers. – H NMR (200 MHz, CDCl3): δ ϭ 0.87 (t, 3 H, CH3), 1.26
(m, 22 H, CH2), 1.67–1.86 (m, 2 H, CH2CH2I), 1.97–2.05 (m, 4 H,
CH2CHϭCHCH2), 3.19 (t, J ϭ7.1 Hz, 2 H, CH2I), 5.32–5.40 (m,
2 H, CH).
General: All reactions were carried out in dried solvents under a
nitrogen atmosphere using oven-dried glassware. – For column
chromatography Al2O3 (activity II–III), prepared by adding the in-
dicated amount of water to Merck aluminium oxide 90 active, neu-
tral (activity I), and Merck silica gel 60, 230–400 mesh were used
with the indicated eluents. – Melting points (uncorrected) were de-
termined using a Koffler melting point microscope. Several com-
pounds displayed liquid crystalline behaviour, in these cases no
Synthesis of Alkynyl Bromides 2i–k. – Synthesis of 1-Bromooctadec-
9-yne (2k): A solution of butyllithium (2.5  in hexane; 17 mL,
43 mmol) was added to a solution of 1-decyne (7.0 mL, 38 mmol)
in THF (40 mL) cooled in an ice/salt bath. After stirring for 30 min
a solution of 1,8-dibromo octane (22 mL, 110 mmol) in THF
melting points are reported. – NMR: Varian Gemini 200, Varian (20 mL) was added and the reaction mixture was heated under re-
VXR 300. – MS: NERMAG R 3010 triple quadrupole mass spec-
trometer equipped with an in house built atmospheric pressure
ionisation source and ionspray interface. – Elemental analyses:
Analytical Department of the University of Groningen, carried out
by Mr. J. Ebels, Mr. J. Hommes, and Mr. H. Draaijer. Accurate
flux for 3 h. After cooling the THF was evaporated and the residue
was partitioned between water and pentane. The organic phase was
dried (MgSO4) and evaporated. The excess dibromo octane (re-
usable) was recovered by kugelrohr distillation (80 °C; 0.01 mbar)
to leave a colorless residue (9.2 g, 28 mmol, 74%). – 1H NMR
Eur. J. Org. Chem. 2000, 665Ϫ673
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J. B. F. N. Engberts et al.
FULL PAPER
(200 MHz, CDCl3): δ ϭ 0.88 (t, 3 H, CH3), 1.35–1.60 (m, 22 H,
on Al2O3, gave 3e as a colorless oil in 67% yield. – 1H NMR
CH2), 1.78–1.92 (m, 2 H, CH2CH2Br), 2.15 (m, 4 H, CH2CCCH2), (300 MHz, CDCl3): δ ϭ 0.87 (t, J 6.6, 6 H, CH3), 1.25 (m, 56 H,
3.40 (t, J ϭ 6.8 Hz, 2 H, CH2Br). The same procedure was used to
synthesize 2i and 2j. From 1-nonyne and 1,7-dibromoheptane, 1-
CH2), 1.45–1.65 [m, 4 H, CH(CH2)2], 1.97–2.00 (m, 4 H, CH2CHϭ
CHCH2), 2.43–2.49 (m, 1 H, CH), 5.31–5.81 (m, 2 H, CH), 7.05
bromohexadec-8-yne (2i) was obtained in 52% yield. – 1H NMR (d, J ϭ 5.9, 2 H, CHar), 8.48 (d, J ϭ 5.5, 2 H, CHar). – 13C NMR
(200 MHz, CDCl3): δ ϭ 0.88 (t, 3 H, CH3), 1.27–1.60 (m, 18 H, (75.5 MHz, CDCl3): δ ϭ 13.8 (p), 22.4, 26.9, 27.2, 29.1, 29.2, 29.4,
CH2), 1.78–1.92 (m, 2 H, CH2CH2Br), 2.15 (m, 4 H, CH2CCCH2), 31.7, 36.0 (s), 45.4 (t), 123.1 (t), 129.7, 129.8 (t, Ccis), 130.2, 130.2
3.40 (t, J ϭ 6.8 Hz, 2 H, CH2Br); from 1-octyne and 1,8-dibro-
mooctane, 1-bromohexadec-9-yne (2j) was obtained in 30% yield. –
1H NMR (200 MHz, CDCl3): δ ϭ 0.88 (t, 3 H, CH3), 1.35–1.50
(m, 18 H, CH2), 1.79–1.91 (m, 2 H, CH2CH2Br), 2.15 (m, 4 H,
CH2CCCH2), 3.40 (t, J ϭ 6.8 Hz, 2 H, CH2Br).
(t, Ctrans), 149.5 (t), 155.4 (q).
4-(Dioleylmethyl)pyridines 3f–h: All purified by chromatography on
Al2O3. Compound 3f was obtained as a colorless oil, in 73%
yield. – 1H NMR (200 MHz, CDCl3): δ ϭ 0.89 (t, J 6.2, 6 H, CH3),
1.27 (m, 48 H, CH2), 1.50 [m, 4 H, CH(CH2)2], 2.00 (m, 8 H,
CH2CHϭCHCH2), 2.43 (m, 1 H, CH), 5.34 (m, 4 H, CH), 7.06 (d,
J ϭ 4.6, 2 H, CHar), 8.49 (d, J ϭ 4.6, 2 H, CHar). – 13C NMR
(75.5 MHz, CDCl3): δ ϭ 14.0 (p), 22.6, 27.1, 27.3, 29.1, 29.2, 29.4,
29.5, 29.6, 29.7, 31.8, 36.1 (s), 45.5 (t), 123.1 (t), 129.7, 129.8 (t,
Ccis), 130.2 (t, Ctrans), 149.5 (t), 155.3 (q). Spectral data for 3g–h
were identical. Compound 3g was obtained in 75% yield as a color-
less oil. Compound 3h, was recrystallized from acetone and ob-
tained as white crystals, m.p. 25–26 °C in 60% yield.
4-(Dihexadec-8-ynylmethyl)pyridine (3i): Purified by chromato-
graphy on Al2O3 and obtained as a light yellow oil in 58% yield. –
1H NMR (200 MHz, CDCl3): δ ϭ 0.88 (t, J ϭ 6.6 Hz, 6 H, CH3),
1.20–1.73 (m, 44 H, CH2), 2.10 (m, 8 H, CH2CCCH2), 2.44 (m, 1
H, CH), 7.04 (d, J ϭ 5.9 Hz, 2 H, CHar), 8.48 (d, J ϭ 5.5 Hz, 2
H, CHar).
4-(Dihexadec-9-ynylmethyl)pyridine (3j): Purified by chromatog-
raphy on Al2O3 and obtained as a light yellow oil in 54% yield. –
1H NMR (200 MHz, CDCl3): δ ϭ 0.87 (t, J ϭ 6.6 Hz, 6 H, CH3),
1.20–1.74 (m, 44 H, CH2), 2.10 (m, 8 H, CH2CCCH2), 2.44 (m, 1
H, CH), 7.04 (d, J ϭ 5.9 Hz, 2 H, CHar), 8.48 (d, J ϭ 5.5 Hz, 2
H, CHar).
4-(Dioctadec-9-ynylmethyl)pyridine (3k): Purified by chromatog-
raphy on Al2O3 and obtained as a light yellow oil in 60% yield. –
1H NMR (200 MHz, CDCl3): δ ϭ 0.88 (t, J ϭ 6.6 Hz, 6 H, CH3),
1.20–1.73 (m, 52 H, CH2), 2.10 (m, 8 H, CH2CCCH2), 2.44 (m, 1
H, CH), 7.04 (d, J ϭ 5.9 Hz, 2 H, CHar), 8.48 (d, J ϭ 5.5 Hz, 2
H, CHar).
General Procedure for the Synthesis of 4-Alkylpyridines: A solution
of butyllithium (1.6  in hexane; 2.05 equiv.) was added to a solu-
tion of diisopropylamine (0.2 ; 2.1 equiv.) in dry ether at –15 °C.
After stirring for 30 min, freshly distilled 4-methylpyridine (1
equiv.) was added dropwise. The resulting red solution was stirred
for 15 min at –15 °C and then a solution of alkyl halide (1 ; 2.05
equiv.) in dry ether was added in one portion. The mixture was
stirred overnight at room temperature. Ether was added and the
reaction mixture washed twice with 1  NH4Cl solution, dried with
Na2SO4 and evaporated to dryness. The product was either crystal-
lized from acetone or purified by chromatography on Al2O3 (neu-
tral, activity II–III), gradient elution with hexane and finally hex-
ane/ether (5:1) gave the products in 60–90%.
4-(Didodecylmethyl)pyridine (3a): White crystals from acetone (82%
1
yield), m.p. 48 °C. – H NMR (200 MHz, CDCl3): δ ϭ 0.87 (t, 6
H, CH3), 1.23 (m, 40 H, CH2), 1.56 [m, 4 H, CH(CH2)2], 2.47 (m,
1 H, CH), 7.17 (d, J ϭ 5.9 Hz, 2 H, CHar), 8.49 (d, J ϭ 5.9 Hz, 2
H, CHar). – 13C NMR (50.3 MHz, CDCl3): 13.9 (p), 24.4, 26.1,
29.2, 29.3, 29.5, 31.7, 36.1 (s), 45.5 (t), 123.2 (t), 149.6 (t), 155.5
(q). – C30H55N (429.77): calcd. C 83.84, H 12.90, N 3.26; found C
83.58, H 12.75, N 3.13.
4-(Ditetradecylmethyl)pyridine (3b): White crystals from acetone,
m.p. 57 °C. – 1H NMR (200 MHz, CDCl3): δ ϭ 0.87 (t, 6 H, CH3),
1.24 (m, 48 H, CH2), 1.50–1.61 [m, 4 H, CH(CH2)2], 2.22 (m, 1 H,
CH), 7.00 (d, J ϭ 5.9 Hz, 2 H, CHar), 8.49 (d, J ϭ 5.9 Hz, 2 H,
CHar). – 13C NMR (50.3 MHz, CDCl3): δ ϭ 13.8 (p), 22.4, 27.2,
29.1, 29.2, 29.4, 31.7, 36.0 (s), 45.4 (t), 123.1 (t), 149.5 (t), 155.4 (q).
General Procedure for the Quaternization of 4-Alkylpyridines with
Methyl Iodide: The 4-substituted alkylpyridines were quaternized
with an excess (ca. 5 equiv.) of MeI (0.3 ) in acetone for 1 h at
room temperature to give the corresponding 1-methylpyridinium
iodide salts in quantitative yields. The iodide salts were converted
into the corresponding chloride salts by ion-exchange chromatog-
raphy with Dowex 1ϫ8 200–400 mesh (Cl– form) with MeOH as
eluent.
4-(Dihexadecylmethyl)pyridine (3c): White crystals from acetone
1
(90% yield), m.p. 63 °C. – H NMR (200 MHz, CDCl3): δ ϭ 0.87
(t, 6 H, CH3), 1.23 (m, 56 H, CH2), 1.56 [m, 4 H, CH(CH2)2], 2.47
(m, 1 H, CH), 7.17 (d, J ϭ 6.1 Hz, 2 H, CHar), 8.49 (d, J ϭ 6.1 Hz,
2 H, CHar). – 13C NMR (50.3 MHz, CDCl3): δ ϭ 13.9 (p), 24.4,
26.1, 29.2, 29.3, 29.5, 31.7, 36.1 (s), 45.5 (t), 123.2 (t), 149.6 (t),
155.5 (q).
4-(Dioctadecylmethyl)pyridine (3d): White powder from acetone,
m.p. 65 °C, in 87% yield. – 1H NMR (200 MHz, CDCl3): δ ϭ 0.87
(t, J ϭ 6.2, 6 H, CH3), 1.24 (m, 64 H, CH2), 1.50 [m, 4 H,
CH(CH2)2], 2.47 (m, 1 H, CH), 7.05 (d, J ϭ 4.6 Hz, 2 H, CHar),
8.48 (d, J ϭ 4.6 Hz, 2 H, CHar). – 13C NMR (50.3 MHz, CDCl3):
δ ϭ 13.8 (p), 22.4, 27.2, 29.1, 29.2, 29.4, 31.7, 36.0 (s), 45.4 (t),
123.1 (t), 149.5 (t), 155.4 (q).
4-(Didodecylmethyl)-1-methylpyridinium Chloride (4a): White crys-
tals from acetone. – MS (70 V); m/z: 444.8 [C31H33Nϩ]. – 1H NMR
(200 MHz, CDCl3): δ ϭ 0.87 (t, J ϭ 6.5, 6 H, CH3), 1.23 (m, 40
H, CH2), 1.55–1.72 [m, 4 H, CH(CH2)2], 2.78 (m, 1 H, CH), 4.75
(s, 3 H, NCH3), 7.71 (d, J ϭ 6.6, 2 H, CHar), 9.45 (d, J ϭ 6.6, 2
H, CHar). – 13C NMR (50.3 MHz, CDCl3): δ ϭ 14.0 (p), 22.6,
27.3, 29.2, 29.5, 31.8, 35.7 (s), 46.5 (t), 47.9 (p), 126.9 (t), 145.3 (t),
167.0 (q).
4-(Ditetradecylmethyl)-1-methylpyridinium Chloride (4b): White
crystals from acetone, m.p. 57–60 °C. – 1H NMR (200 MHz,
CDCl3): δ ϭ 0.87 (t, J ϭ 6.5, 6 H, CH3), 1.23 (m, 48 H, CH2),
1.55–1.72 [m, 4 H, CH(CH2)2], 2.78 (m, 1 H, CH), 4.75 (s, 3 H,
4-(Nonadec-10-enyl)pyridine and 4-(Octadecyl, oleylmethyl)pyridine
(3e): This was prepared by the initial introduction of one oleyl
group from a slight excess of 4-methylpyridine. Purification by
chromatography on Al2O3 gave a light yellow oil in 40% yield. –
1H NMR (200 MHz, CDCl3): δ ϭ 0.87 (t, J 6.2, 3 H, CH3), 1.27
(m, 24 H, CH2), 1.54–1.65 [m, 2 H, CH2CH2Ar)], 1.96–2.02 (m, 4 NCH3), 7.71 (d, J ϭ 6.6, 2 H, CHar), 9.45 (d, J ϭ 6.6, 2 H, CHar). –
H, CH2CHϭCHCH2), 2.59 (t, J 7.6, 2 H, CH2Ar), 5.32–5.37 (m, 13C NMR (50.3 MHz, CDCl3): δ ϭ 14.0 (p), 22.6, 27.3, 29.2, 29.5,
2 H, CH), 7.10 (d, J ϭ 5.4 Hz, 2 H, CHar), 8.48 (d, J ϭ 5.1, 2 H, 31.8, 35.7 (s), 46.5 (t), 47.9 (p), 126.9 (t), 145.3 (t), 167.0 (q). –
CHar). Treatment of this product with 1.1 equivalents of LDA and C35H66ClN (536.37): calcd. C 78.38, H 12.40, Cl 6.61, N 2.61;
1.05 equivalents of octadecyl bromide, followed by chromatography
found C 78.39, H 12.51, Cl 6.56, N 2.67.
670
Eur. J. Org. Chem. 2000, 665Ϫ673

Synthesis of Pyridinium Amphiphiles
FULL PAPER
4-(Dihexadecylmethyl)-1-methylpyridinium Chloride (4c): White
4.69 (s, 3 H, NCH3), 7.70 (d, J ϭ 6.6, 2 H, CHar), 9.50 (d, J ϭ 6.6,
1
crystals from acetone m.p. 64 °C. – H NMR (200 MHz, CDCl3): 2 H, CHar). – 13C NMR (50.3 MHz, CDCl3): δ ϭ 13.8 (p), 18.5,
δ ϭ 0.87 (t, J ϭ 6.5, 6 H, CH3), 1.24 (m, 56 H, CH2), 1.48–1.78
22.4, 27.1, 28.5, 28.6, 28.8, 28.9, 29.2, 31.6, 35.5 (s), 46.4 (t), 47.7
[m, 4 H, CH(CH2)2], 2.81 (m, 1 H, CH), 4.75 (s, 3 H, NCH3), 7.72 (p), 79.9 (q), 80.1 (q), 126.8 (t), 145.4 (t), 166.8 (q).
(d, J ϭ 6.7, 2 H, CHar), 9.48 (d, J ϭ 6.5, 2 H, CHar). – 13C NMR
Dialkyl Carbinols 5a–b: These were synthesized according to the
literature procedure.[5] Dihexadecyl carbinol 5a (78% yield) was ob-
(50.3 MHz, CDCl3): δ ϭ 13.8 (p), 22.4, 27.2, 29.1, 29.4, 31.7, 35.4
1
(s), 46.4 (t), 48.4 (p), 126.9 (t), 144.9 (t), 167.3 (q).
–
tained as a white powder from CHCl3, m.p. 90–92 °C. – H NMR
(200 MHz, CDCl3): δ ϭ 0.90 (t, 6 H, CH3), 1.30 (m, 60 H, CH2),
3.60 (m, 1 H, CHOH). For the synthesis of dioleyl carbinol 5b,
oleyl bromide was synthesized from oleyl mesylate (see the prepara-
tion of 2f). A mixture of oleyl mesylate (15 g, 43.3 mmol) and LiBr
(12 g, 138 mmol) in acetone (500 mL) was refluxed for 3 h. After
cooling, the resulting suspension was filtered and the filtrate evap-
orated. Ether (250 mL) was added and the precipitate removed by
filtration. The filtrate was evaporated and the resulting crude prod-
uct purified by chromatography on Al2O3 with hexane as eluent.
The product was a colorless viscous oil (11.7 g, 35.3 mmol, 81%). –
1H NMR (200 MHz, CDCl3): δ ϭ 0.87 (t, 3 H, CH3), 1.26 (m,
22 H, CH2), 1.78–1.92 (m, 2 H, CH2CH2Br), 1.97–2.05 (m, 4 H,
CH2CHϭCHCH2), 3.40 (t, J ϭ 6.8 Hz, 2 H, CH2Br), 5.32–5.40
(m, 2 H, CH). Dioleyl carbinol 5b was purified by chromatography
on Al2O3. Gradient elution with CHCl3/Et2O gave a colorless oil
(91% yield). – 1H NMR (200 MHz, CDCl3): δ ϭ 0.90 (t, 6 H, CH3),
1.3 (m, 52 H, CH2), 2.05 (m, 8 H, CH2CHϭCHCH2), 3.60 (m, 1
H, CHOH), 5.32–5.40 (m, 4 H, CH).
C39H74ClN H2O (610.49): calcd. C 76.73, H 12.55, Cl 5.81; found
C 76.53, H 12.42, Cl 5.78.
4-(Dioctadecylmethyl)-1-methylpyridinium Chloride (4d): An off-
white powder from acetone. – 1H NMR (300 MHz, CDCl3): δ ϭ
0.87 (t, J ϭ 6.5, 6 H, CH3), 1.24 (m, 64 H, CH2), 1.48–1.78 [m, 4
H, CH(CH2)2], 2.78 (m, 1 H, CH), 4.74 (s, 3 H, NCH3), 7.72 (d,
J ϭ 6.8, 2 H, CHar), 9.43 (d, J ϭ 6.4, 2 H, CHar). – 13C NMR
(75.5 MHz, CDCl3): δ ϭ 13.8 (p), 22.4, 27.2, 29.1, 29.3, 29.4, 31.7,
35.4 (s), 46.4 (t), 48.4 (p), 126.9 (t), 144.9 (t), 167.3 (q). –
C43H82ClN H2O (666.60): calcd. C 77.48, H 12.70, N 2.10; found:
C 76.88, H 12.84, N 2.11.
4-(Oleyl, octadecylmethyl)-1-methylpyridinium Chloride (4e):
A
white solid from acetone/acetonitrile (10:1), m.p. 59–61 °C. – 1H
NMR (300 MHz, CDCl3): δ ϭ 0.86 (t, J ϭ 6.5, 6 H, CH3), 1.24
(m, 56 H, CH2), 1.40–1.80 [m, 4 H, CH(CH2)2], 1.93–2.06 (m, 4 H,
CH2CHϭCHCH2), 2.74–2.83 (m, 1 H, CH), 4.70 (s, 3 H, NCH3),
5.31–5.38 (m, 2 H, CH), 7.75 (d, J ϭ 6.6, 2 H, CHar), 9.26 (d, J ϭ
6.6, 2 H, CHar). – 13C NMR (75.5 MHz, CDCl3): δ ϭ 13.8 (p),
22.4, 26.4, 26.9, 27.1, 28.7, 28.9, 29.1, 29.2, 29.4, 31.6, 35.4 (s), 46.2
(t), 47.8 (p), 126.8 (t), 129.5, 129.7 (t, Ccis), 130.1, 130.3 (t, Ctrans),
145.3 (t), 166.6 (q). – C43H80ClN H2O (666.60): calcd. C 77.71, H
12.44, N 2.11; found C 76.98, H 12.60, N 2.10.
Bis(alkyl)methyl Mesylates 6a–b: Mesylation was carried out as de-
scribed for the preparation of 2f. Bis(hexadecyl)methyl mesylate 6a
was obtained as a white powder from methanol (66% yield), m.p.
1
51–53 °C. – H NMR (200 MHz, CDCl3): δ ϭ 0.90 (t, 6 H, CH3),
1.30 (m, 56 H, CH2), 1.6 [m, 4 H, (CH2)2CHOMs], 3.0 (s, 3 H,
OMs), 4.7 (m, 1 H, CHOMs). Bis(oleyl)methyl mesylate 6b was
obtained as a light yellow oil in 82% yield. – H NMR (200 MHz,
CDCl3): δ ϭ 0.90 (t, 6 H, CH3), 1.3 (m, 48 H, CH2), 1.6 [m, 4 H,
(CH2)2CHOMs], 2.0 (m, 8 H, CH2CHϭCHCH2), 3.0 (s, 3 H,
OMs), 4.7 (m, 1 H, CHOMs), 5.35 (m, 4 H, CH).
4-(Dioleylmethyl)-1-methylpyridinium Chlorides 4f–g and 4-(Dielai-
dylmethyl)-1-methylpyridinium Chloride 4h: These were recrystal-
lized from acetone/acetonitrile (10:1) and obtained as white solids.
The ratio of cis to trans double bonds in the products was deter-
mined by double resonance NMR experiments. Irradiation of the
protons adjacent to the double bond at 2.00 ppm resulted in two
singlets for the olefinic protons at 5.34 ppm for the cis and at 5.38
1
General Procedure for the Preparation of 1-Alkylpyridinium Salts
7a–d: A stirred solution of the mesylates 6a–b, approximately 10 ,
in either pyridine or 4-methyl pyridine was kept at 85 °C for 24 h.
Then the excess pyridine was evaporated and the product chroma-
tographed on Al2O3. Gradient elution with CHCl3/MeOH followed
by ion-exchange chromatography with Dowex 1ϫ8 200–400 mesh
(Cl– form) with MeOH as eluent gave the chloride salts.
1
for the trans orientation. – MS (70 V); m/z: 608 [C43H78Nϩ]. – H
NMR (300 MHz, CDCl3): δ ϭ 0.87 (t, J 6.5, 6 H, CH3), 1.26 (m,
44 H, CH2), 1.53 (m, 4 H, CH2CH3), 1.73 [m, 4 H, CH(CH2)2],
2.00 (m, 8 H, CH2CHϭCHCH2), 2.77 (m, 1 H, CH), 4.76 (s, 3 H,
NCH3), 5.35–5.37 (m, 4 H, CH), 7.72 (d, J ϭ 6.2, 2 H, CHar), 9.50
(d, J ϭ 6.2, 2 H, CHar). – 13C NMR (75.5 MHz, CDCl3): δ ϭ 13.9
(p), 22.4, 26.9, 27.2, 28.9, 29.1, 29.3, 29.4, 29.5, 31.6, 35.4 (s), 46.4
(t), 48.3 (p), 126.8 (t), 129.5, 129.7 (t, Ccis), 130.1, 130.3 (t, Ctrans),
144.9 (t), 167.1 (q). – C43H78ClN (644.55): calcd. C 80.13, H 12.20,
Cl 5.50, N 2.17; found for 4f: C 79.77, H 12.00, Cl 5.45, N 2.24;
found for 4g: C 79.53, H 12.18, Cl 5.42, N 2.24; found for 4h: C
79.97, H 12.21, Cl 5.40, N 2.17.
1-(Dihexadecylmethyl)pyridinium Chloride (7a): A yellow powder
from CHCl3, (48% yield). – MS (70 V); m/z: 542.6 [C38H72Nϩ]. –
1H NMR (300 MHz, CDCl3): δ ϭ 0.95 (t, 6 H, CH3), 1.30 (m, 48
H, CH2), 2.0 [m, 4 H, (CH2)2CHN], 4.8 (m, 1 H, CHN), 8.25 (m,
2 H, CHar), 8.50 (m, 1 H, CHar), 9.50 (m, 2 H, CHar). –
C38H72ClN H2O (596.46): calcd. C 76.52, H 12.50, N 2.42; found
C 77.62, H 12.40, N 2.60. – 13C NMR (50.3 MHz, CDCl3): δ ϭ
13.9 (p), 22.4, 25.6, 28.9, 29.0, 29.1, 29.2, 29.4, 31.7, 35.6 (s), 74.2
(t), 128.7 (t), 143.8 (t), 145.1 (q).
4-(Dihexadec-8-ynylmethyl)-1-methylpyridinium Chloride (4i) and 4-
(Dihexadec-9-ynylmethyl)-1-methylpyridinium Chloride (4j): These
compounds were obtained as colorless oils. Compound 4i: MS
(70 V); m/z: 548.5 [C39H66Nϩ]. – 4j: MS (70 V); m/z 548.5
1-(Dihexadecylmethyl)-4-methylpyridinium Chloride (7b): A slightly
red powder from CHCl3, (60% yield). – MS (70 V); m/z: 556.6
[C39H74Nϩ]. –1H NMR (300 MHz, CDCl3): δ ϭ 0.95 (t, 6 H, CH3),
1.30 (m, 48 H, CH2), 2.0 [m, 4 H, (CH2)2CHN], 2.7 (s, 3 H,
CH3Ar), 4.8 (m, 1 H, CHN), 8.0 (d, 2 H, CHar), 9.40 (d, 2 H,
CHar). – C39H74ClN H2O (610.49): calcd. C 76.73, H 12.55, N
2.29; found C 76.52, H 12.51, N 2.34. – 13C NMR (50.3 MHz,
CDCl3): δ ϭ 13.9 (p), 21.9 (p), 22.4, 25.6, 28.9, 29.0, 29.1, 29.2,
29.4, 31.7, 35.5 (s), 73.2 (t), 129.1 (t), 142.8 (t), 159.0 (q).
1
[C39H66Nϩ] – H NMR (200 MHz, CDCl3): δ ϭ 0.86 (t, J ϭ 6.5,
6 H, CH3), 1.20–1.74 (m, 44 H, H2), 2.10 (t, 8 H, CH2CCCH2),
2.74 (m, 1 H, CH), 4.70 (s, 3 H, NCH3), 7.70 (d, J ϭ 6.6, 2 H,
CHar), 9.50 (d, J ϭ 6.6, 2 H, CHar). – 13C NMR (50.3 MHz,
CDCl3): δ ϭ 13.8 (p), 18.5, 22.4, 27.1, 28.5, 28.6, 28.8, 29.0, 29.2,
31.6, 35.5 (s), 46.4 (t), 47.7 (p), 79.9 (q), 80.1 (q), 126.8 (t), 145.4
(t), 166.8 (q).
4-(Dioctadec-9-ynylmethyl)-1-methylpyridinium Chloride (4k): Ob-
tained as a colorless oil. – MS (70 V); m/z: 604.4 [C43H74Nϩ]. – 1H
1-(Dioleylmethyl)pyridinium Chloride (7c): Purified by chromatog-
NMR (200 MHz, CDCl3): δ ϭ 0.86 (t, J ϭ 6.5, 6 H, CH3), 1.20– raphy on Al2O3. Gradient elution with CHCl3/MeOH gave a yellow
1
1.73 (m, 52 H, CH2), 2.10 (t, 8 H, CH2CCCH2), 2.80 (m, 1 H, CH), oil in 71% yield. – H NMR (200 MHz, CDCl3): δ ϭ 0.95 (t, 6 H,
Eur. J. Org. Chem. 2000, 665Ϫ673
671

J. B. F. N. Engberts et al.
FULL PAPER
CH3), 1.3 (m, 48 H, CH2), 2.0 [m, 12 H, (CH2)2CHN and (CH2)2CH], 1.93 (m, 8 H, CH2CHϭCHCH2), 2.15–2.25 [m, 4 H,
CH2CHϭCHCH2)], 4.8 (m, 1 H, CHN), 5.35 (m, 4 H, CH), 8.25 (CH2)2CH2N(CH3)3], 2.71 (m, 2 H, CH), 3.39 [s, 9 H, N(CH3)3],
(m, 2 H, CHar), 8.50 (m, 1 H, CHar), 9.50 (m, 2 H, CHar). – 13C 3.98 [m, 2 H, CH2N(CH3)3], 5.25 (m, 2 H, NarCH2), 5.28 (m, 4 H,
NMR (50.3 MHz, CDCl3): δ ϭ 13.9 (p), 22.4, 25.6, 26.9, 28.9, 29.1, CH), 7.66 (d, J ϭ 6.3 Hz, 2 H, CHar), 9.83 (d, J ϭ 6.3 Hz, 4 H,
29.3, 29.4, 31.6, 32.3, 35.6 (s), 74.1 (t), 128.7 (t), 129.5, 129.8 (t),
143.8 (t), 145.1 (t).
CHar). – 13C NMR (75.5 MHz, CDCl3): δ ϭ 13.8 (p), 19.7, 22.4,
26.9, 27.2, 27.8, 29.0, 29.2, 29.4, 29.5, 31.6, 32., 32.3, 35.4 (s), 46.3
(t), 53.3 (p), 59.0 (s), 65.0 (s), 127.0 (t), 129.6 (t), 129.8 (t), 145.0
(t), 166.9 (q).
1-(Dioleylmethyl)-4-methylpyridinium Chloride (7d): Purified by
chromatography on Al2O3. Gradient elution with CHCl3/MeOH
gave a red oil in 76% yield. – MS (70 V); m/z: 608.7 [C43H78Nϩ]. –
1H NMR (300 MHz, CDCl3): δ ϭ 0.95 (t, 6 H, CH3), 1.30 (m, 48
H, CH2), 2.0 [m, 12 H, (CH2)2CHN and CH2CHϭCHCH2)], 2.7
(s, 3 H, CH3Ar), 4.8 (m, 1 H, CHN), 5.35 (m, 4 H, CH), 8.0 (d, 2
H, CHar), 9.40 (d, 2 H, CHar). – 13C NMR (50.3 MHz, CDCl3):
δ ϭ 13.9 (p), 22.0 (p), 22.4, 25.6, 26.9, 28.9, 29.1, 29.3, 29.5, 31.7,
32.4, 35.5 (s), 73.3 (t), 129.2 (t), 129.6, 129.9 (t), 142.8 (t), 159.0 (q).
1-(4-Ammoniobutyl)-4-(dihexadecylmethyl)pyridinium
Dichloride
(11): A solution of pyridine 3c (1.62 g, 3.0 mmol) and phthalimide
10 (564 mg, 2.0 mmol) in acetone (10 mL) was refluxed for 21 days.
After evaporation of the solvent, the red product was purified on
Al2O3. Elution with hexane/CH2Cl2 (1:1) afforded the excess pyrid-
ine 3 and subsequent elution with CH2Cl2/10% MeOH gave the
product as a solid (1.05 g, 1.26 mmol, 63%). The resulting phthali-
mide (412 mg, 0.5 mmol) was heated overnight in HBr/HOAc
(4 mL, 1:1 v/v). After cooling the crystalline compound obtained
was filtered, washed with 50% HOAc and dried over P2O5. The
(Bromoalkyl)trimethylammonium Bromides 8a–c: Compounds 8a–c
were prepared according to a literature procedure.[6] All the com-
pounds were isolated as white crystalline solids; propyl compound
8a, m.p. 209–212 °C; butyl compound 8b, m.p. 132–133 °C; pentyl product was recrystallized from THF/EtOH abs. (5:1). The dibro-
compound 8c, m.p. 141–142 °C.
mide salt was then converted into the dichloride salt 11 (69% yield)
by ion-exchange chromatography with Dowex 1ϫ8 200–400 mesh
(Cl– form) with MeOH as eluent. – MS (70 V); m/z: 613.6
[C42H82Nϩ]. – 1H NMR (300 MHz, CDCl3): δ ϭ 0.87 (t, 6 H,
CH3), 1.25 (m, 56 H, CH2), 1.56–1.69 [m, 4 H, (CH2)2CH], 2.14 [t,
2 H, CH2(CH2)2NH3], 2.35 (t, 2 H, CH2CH2NH3), 2.75 (m, 1 H,
CH), 3.30 (t, 2 H, CH2NH3), 5.14 (t, 2 H, NarCH2), 7.71 (d, J ϭ
6.6 Hz, 2 H, CHar), 9.64 (d, J ϭ 6.6 Hz, 2 H, CHar). – 13C NMR
(75.5 MHz, CDCl3): δ ϭ 13.9 (p), 22.5, 23.9, 27.2, 28.8, 29.2, 29.4,
29.6, 31.8, 35.3, 38.8 (s), 46.3 (t), 59.8 (s), 127.0 (t), 144.9 (t),
166.6 (q).
General Procedure for the Preparation of 1-Alkylpyridinium Salts
9a–d: A 0.1  solution of ammonium bromide 8a–c and 2 equiva-
lents of pyridine 3c, f in absolute ethanol was heated under reflux.
The reaction was monitored with NMR and upon completion,
after 3–6 days, the solvent was evaporated and the resulting product
chromatographed on Al2O3. Excess pyridine 3 was recovered by
elution with CHCl3. The product was eluted with CHCl3/10%
MeOH. The resulting dibromide salt was converted into the
dichloride salts by ion-exchange chromatography with Dowex 1ϫ8
200–400 mesh (Cl– form) and elution with MeOH. Compound 9a
was recrystallized from THF and obtained as white crystals in 84%
yield m.p. 170°C (dec.). – MS (70 V); m/z: 321.3 [C44H86Nϩ2 ϩ]. –
1H NMR (300 MHz, CDCl3): δ ϭ 0.86 (t, 6 H, CH3), 1.23 (m, 56
H, CH2), 1.51–1.68 [m, 4 H, (CH2)2CH], 2.75 (m, 1 H, CH), 2.98
[m, 2 H, CH2CH2N(CH3)3], 3.39 [s, 9 H, N(CH3)3], 4.05 [t, 2 H,
CH2N(CH3)3], 5.13 (t, 2 H, NarCH2), 7.70 (d, J ϭ 5.8 Hz, 2 H,
CHar), 9.87 (d, J ϭ 5.8 Hz, 2 H, CHar). – 13C NMR (75.5 MHz,
CDCl3): δ ϭ 13.9 (p), 22.5, 26.2, 27.2, 29.2, 29.3, 29.5, 31.7, 35.3
(s), 46.3 (t), 53.9 (p), 56.9 (s), 62.4 (s), 127.1 (t), 145.1 (t), 167.3 (q).
4-(Dihexadecylmethyl)-1-(4-guanidiniobutyl)pyridinium Dichloride
(13): A mixture of amine 11 (775 mg, 1.0 mmol), pyrazole carbox-
amidine 12[7] (437 mg, 3.0 mmol), and diisopropylethylamine
(387 mg, 3.0 mmol) in DMF (8 mL) was heated at 65 °C for 24 h.
After cooling, ether was added (12 mL) and the precipitate, which
was produced by vigorous stirring, was filtered off, washed with
ether and dried. Ion exchange using Dowex 1ϫ8 200–400 mesh
(Cl– form) with MeOH as eluent followed by recrystallization from
CH3CN afforded white crystals, m.p. 125 °C (dec.) (420 mg,
1
Compound 9b was recrystallized from CH3CN and obtained as
white crystals in 75% yield m.p. 170°C (dec.). – MS (70 V); m/z:
0.58 mmol, 58%). – H NMR (300 MHz, CDCl3, 55 °C): δ ϭ 0.89
(t, 6 H, CH3), 1.27 (m, 56 H, CH2), 1.55–1.74 [m, 4 H, (CH2)2CH],
1.86 (m, 2 H, CH2CH2NH), 2.29 [m, 2 H, CH2(CH2)2NH3], 2.80
1
328.4 [C45H88Nϩ2 ϩ]. – H NMR (300 MHz, CDCl3): δ ϭ 0.87 (t, 6
H, CH3), 1.23 (m, 56 H, CH2), 1.55–1.58 [m, 4 H, (CH2)2CH], 2.30 (m, 1 H, CH), 3.58 (m, 2 H, CH2NH), 5.06 (m, 2 H, NarCH2), 7.41
[m, 4 H, (CH2)2CH2N(CH3)3], 2.75 (m, 1 H, CH), 3.39 [s, 9 H, [br. s, 4 H, NHC(NH2)2], 7.73 (d, J ϭ 6.6 Hz, 2 H, CHar), 8.34 (br.
N(CH3)3], 4.06 [t, 2 H, CH2N(CH3)3], 5.14 (t, 2 H, NarCH2), 7.69 s, 1 H, NH), 9.46 (d, J ϭ 6.6 Hz, 2 H, CHar). – 13C NMR
(d, J ϭ 6.5 Hz, 2 H, CHar), 9.87 (d, J ϭ 6.5 Hz, 2 H, CHar). – 13C
NMR (75.5 MHz, CDCl3): δ ϭ 13.9 (p), 22.5, 27.2, 28.6, 29.2, 29.4,
(75.5 MHz, CDCl3): δ ϭ 14.0 (p), 22.6, 27.3, 29.3, 29.4, 29.5, 29.6,
31.8, 35.4, 40.8 (s), 46.4 (t), 59.8 (s), 127.0 (t), 144.3 (t), 167.1 (q). –
29.5, 29.6, 31.7, 35.1 (s), 46.1 (t), 53.2 (p), 59.7 (s), 65.3 (s), 127.0 C43H84Cl2N4 (728.09): calcd. C 70.94, H 11.63, Cl 9.74, N 7.70;
(t), 144.9 (t), 166.5 (q).
found C 70.94, H 11.66, Cl 9.60, N 7.85.
Compound 9c was crystallized from THF and obtained as white
crystals in 90% yield m.p. 150°C (dec.). – MS (70 V); m/z: 335.3
[C46H90Nϩ2 ϩ]. – 1H NMR (300 MHz, CDCl3): δ ϭ 0.87 (t, 6 H,
CH3), 1.03 (m, 2 H, CH2) 1.23 (m, 54 H, CH2), 1.55–1.73 [m, 6 H,
General Procedure for the Preparation of the Gemini Pyridinium
Amphiphiles 14a–d: A solution of α,ω-dibromo alkane, 0.1 , and
3 equivalents of pyridine 3c, f in acetone was heated under reflux
until all alkyl dibromide had reacted (1–3 weeks). The solvent was
evaporated and the product was purified on Al2O3. Excess pyridine
3 was recovered by elution with hexane/CHCl3 (1:3), whilst the
product was eluted with CHCl3/6% MeOH. The resulting dibro-
mide salt was converted into the dichloride salt by ion-exchange
chromatography with Dowex 1ϫ8 200–400 mesh (Cl– form) with
MeOH as eluent. Compound 14a was obtained as a waxy solid. –
MS (70 V); m/z: 562.5 [C79H148N2ϩϩ]. – 1H NMR (200 MHz,
CDCl3): δ ϭ 0.87 (t, 12 H, CH3), 1.23 (m, 104 H, CH2), 1.34–1.56
(CH2)2CH
and CH2(CH2)2N(CH3)3], 2.14 [t,
2
H,
CH2(CH2)2N(CH3)3], 2.27 [t, 2 H, CH2CH2N(CH3)3], 2.73 (m, 1
H, CH), 3.41 [s, 9 H, N(CH3)3], 3.92 [t, 2 H, CH2N(CH3)3], 5.09
(t, 2 H, NarCH2), 7.69 (d, J ϭ 5.8 Hz, 2 H, CHar), 9.85 (d, J ϭ
5.8 Hz, 2 H, CHar). – 13C NMR (75.5 MHz, CDCl3): δ ϭ 14.0 (p),
22.4, 22.6, 27.3, 29.3, 29.4, 29.6, 31.8, 35.6 (s), 46.4 (t), 53.5 (p),
59.2 (s), 65.7 (s), 126.8 (t), 145.0 (t), 166.5 (q).
Compound 9d was obtained as a white solid in 59% yield. – MS
(70 V); m/z: 354 [C49H92N2ϩϩ]. – 1H NMR (300 MHz, CDCl3): (m, 8 H, CH2), 2.01 [m, 8 H, (CH2)2CH], 2.75 (m, 2 H, CH), 3.23
δ ϭ 0.81 (t, 6 H, CH3), 1.20 (m, 48 H, CH2), 1.50–1.62 [m, 4 H, (m, 2 H, NCH2CH2CH2N), 5.14–5.21 (m, 4 H, NCH2CH2CH2N),
672
Eur. J. Org. Chem. 2000, 665Ϫ673

Synthesis of Pyridinium Amphiphiles
FULL PAPER
7.66 (d, J ϭ 6.6 Hz, 4 H, CHar), 9.79 (d, J ϭ 6.4 Hz, 4 H, CHar). –
An E. coli β-galactosidase standard was used for conversion of ab-
13C NMR (50.3 MHz, CDCl3): δ ϭ 13.9 (p), 22.4, 27.3, 29.1, 29.4, sorbance data to β-galactosidase units.
29.5, 30.2, 31.7, 35.5 (s), 46.4 (t), 59.3 (s), 126.9 (t), 145.1 (t),
Acknowledgments
166.6 (q).
Compound 14b was obtained as a white powder. – MS (70 V); m/z:
This work was supported by the Netherlands Foundation for
Chemical Research (SON)/Netherlands Technology Foundation
(STW). Zeta potential and particle size measurements were per-
formed at the University of Utrecht, Department of Pharmaceutics.
The group of Prof. W. E. Hennink is thanked for their hospitality.
Mrs. C. M. Jeronimus-Stratingh, University Centre for Pharmacy
is thanked for recording the electrospray mass spectra.
569.6 [C80H150N2ϩϩ]. – H NMR (200 MHz, CDCl3): δ ϭ 0.88 (t,
1
12 H, CH3), 1.04 (m, 4 H, CH2), 1.25 (m, 108 H, CH2), 1.53–1.92
[m, 8 H, (CH2)2CH], 2.50 [m, 4 H, NCH2(CH2)2CH2N], 2.76 (m,
2 H, CH), 5.14 [m, 4 H, NCH2(CH2)2CH2N], 7.69 (d, J ϭ 6.4 Hz, 4
H, CHar), 9.78 (d, J ϭ 6.4 Hz, 4 H, CHar). – 13C NMR (75.5 MHz,
CDCl3): δ ϭ 13.9 (p), 22.5, 27.2, 28.2, 29.2, 29.3, 29.4, 29.5, 31.7,
35.6 (s), 46.4 (t), 59.4 (s), 127.0 (t), 145.0 (t), 166.9 (q).
Compound 14c was obtained as a white powder. – 1H NMR
(200 MHz, CDCl3): δ ϭ 0.86 (t, 12 H, CH3), 1.23 (m, 114 H, CH2),
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[O99460]
Eur. J. Org. Chem. 2000, 665Ϫ673
673