Design and synthesis of asymmetric acyclic phospholipid bolaamphiphiles

Article abstract of DOI:10.1021/jo048167o

(Chemical Equation Presented) A synthetic route was devised for the generation of asymmetric lipid bolaamphiphiles through the sequential esterification of an alkyldioic acid, bearing distinct terminal protecting groups, with propanylamine and lyso-phosph

Full text of DOI:10.1021/jo048167o

Design and Synthesis of Asymmetric Acyclic Phospholipid
Bolaamphiphiles
Toshitsugu Kai,† Xue-Long Sun,*,† Keith M. Faucher,† Robert P. Apkarian,‡ and
Elliot L. Chaikof*,†,§
Departments of Surgery and Biomedical Engineering, Emory University School of Medicine, Integrated
Microscopy & Microanalytical Facility, Emory University, Atlanta, Georgia 30322, and School of
Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
xsun@emory.edu; echaiko@emory.edu
Received October 16, 2004
A synthetic route was devised for the generation of asymmetric lipid bolaamphiphiles through the
sequential esterification of an alkyldioic acid, bearing distinct terminal protecting groups, with
propanylamine and lyso-phosphatidylcholine headgroups. Bolaamphiphile self-assembly was
investigated in solvent mixes of varying polarity by nuclear magnetic resonance (NMR) and Fourier
transform-infrared (FT-IR) spectroscopy, as well as in water by cryo-high-resolution scanning
electron microscopy (cryo-HRSEM). We anticipate that asymmetric lipid bolaamphiphiles will
provide facile building blocks for engineering a variety of unique membrane-mimetic structures.
Introduction
assembly of lipid membranes or membrane mimics on
solid supports are relatively weak hydrophobic interac-
tions, film instability remains a major obstacle for many
long-term applications of this technology. Prior studies
from our group and others have demonstrated that the
polymerization of a planar lipid assembly provides a
feasible strategy for enhancing membrane stability.7-17
Nonetheless, a limitation of polymerization approaches
includes the potential of the initiating species or growing
polymer chain to inactivate or otherwise alter membrane-
The fabrication of supported bilayer lipid membranes
(sBLMs) that mimic cell surfaces have attracted consid-
erable attention due to their potential application as tools
to probe cellular and molecular interactions and as
bioactive coatings for biosensor or medical implant ap-
plications.1,2 In most studies, phospholipids differing in
chemical composition, degree of saturation, and size have
been utilized as the primary building blocks of lipid
membrane-based structures because of their capacity to
self-assemble into lamellar systems of high packing
density that exhibit limited nonspecific protein adsorp-
tion.3-6 However, since the major driving forces for the
(7) Kusumi, A.; Singh, M.; Tirrell, D. A.; Oehme, G.; Singh, A.;
Samuel, N. K. P.; Hyde, J. S.; Regen, S. L. J. Am. Chem. Soc. 1983,
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* To whom correspondence should be addressed. Tel:
(404) 727-8027; (404) 727-8413. Fax: (404) 727-3660.
† Emory University School of Medicine.
(10) Lamparski, H.; Lee, Y. S.; Sells, T. D.; O’Brien, D. F. J. Am.
Chem. Soc. 193, 115, 8096-102.
‡ Emory University.
(11) Sells, T. D.; O’Brien, D. F. Macromolecules 1994, 27, 226-33.
(12) Sisson, T. M.; Lamparski, H. G.; Koelchens, S.; Elayadi, A.;
O’Brien, D. F. Macromolecules 1996, 29, 8321-8329.
(13) Lamparski, H. G.; Oblinger, E.; O’Brien, D. F. Macromolecules
1999, 32, 5450-5452.
§ Georgia Institute of Technology.
(1) Fuhrhop, J.-H.; Koning, J. Membranes and Molecular As-
semblies: The Synkinetic Approach; The Royal Society of Chemistry:
London, 1994.
(2) Sackmann, E.; Tanaka, M. Trans Biotechnol. 2000, 18, 58-64.
(3) Lamparski, H.; Liman, U.; Frankel, D. A.; Ramaswami, V.;
Brown, M. F.; O’Brien, D. F. Biochemistry 1992, 31, 685-694.
(4) Plant, A. L. Langmuir 1993, 9, 2764-2767.
(5) Plant, A. L.; Brigham-Burke, M.; Petrella, E. C.; O’Shannessy,
D. J. Anal. Biochem. 1995, 226, 342-348.
(6) O’Brien, D. F.; Armitage, A. B.; Bennett, D. E.; Lamparski, H.
G.; Lee, Y.-S.; Srisiri, W.; Sisson, T. M. Acc. Chem. Res. 1998, 31, 861-
868.
(14) Marra, K. G.; Winger, T. M.; Hanson, S. R.; Chaikof, E. L.
Macromolecules 1997, 30, 6483-6488.
(15) Orban, J. M.; Faucher, K. M.; Dluhy, R. A.; Chaikof, E. L.
Macromolecules 2000, 33, 4205-4212.
(16) Sun, X.-L.; Liu, H.; Orban, J. M.; Sun. L.; Chaikof, E. L.
Bioconjugate Chem. 2001, 12, 673-677.
(17) Liu, H.; Faucher, K. M.; Sun, X.-L.; Feng, J.; Johnson, T. L.;
Orban, J. M.; Apkarian, R. D.; Dluhy, R. A.; Chaikof, E. L. Langmuir
2002, 18, 1332-1339.
10.1021/jo048167o CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/23/2005
2606
J. Org. Chem. 2005, 70, 2606-2615

Synthesis of Asymmetric Acyclic Phospholipid Bolaamphiphiles
FIGURE 1. Design of phospholipid-based asymmetric bolaamphiphile.
associated bioactive proteins or carbohydrates. Further,
the cost of an increase in membrane stability is a
concomitant loss of local molecular mobility. That is, the
creation of surface composed of either multiple lipid-
based polymer chains or a cross-linked networks of chains
restricts the movement of nonlipid components within
the film.
and initial investigations of the self-assembly of these
compounds in both organic and aqueous media.
Results and Discussion
A scheme was devised to synthesize asymmetric dialkyl
bolaamphiphiles based upon the sequential esterification
of an alkyldioic acid with propanylamine and lyso-
phosphatidylcholine (PC) headgroups. The initial syn-
thesis of an alkyl diester bearing unique terminal pro-
tecting groups provided a key intermediate for this
strategy (Scheme 1).
The asymmetrically protected C32-diester 8a was
synthesized by C-C bond coupling of hexadecanoic acid
to an ortho-protected iodoester (Scheme 2). Briefly,
transformation of lactone 1a using trimethylsilyl chloride
and sodium iodide afforded 16-iodo acid 2a, which was
further converted to a benzyl protected iodo ester 3a and
an ortho-protected iodo ester 6a. The benzyl ester can
be cleaved either by using basic conditions or catalytic
hydrogenation, while the ortho ester can be cleaved under
acidic conditions. In this regard, the benzyl-protected iodo
ester 3a was oxidized to the aldehyde 4a and the ortho-
protected iodo ester 6 was converted to the phosphonium
salt 7a. The Wittig reaction of aldehyde 4a and phos-
phonium salt 7a was then performed to provide dotria-
contan-16-ene dioic acid ester 8a bearing distinct termi-
Archaebacterial lipids are naturally occurring double-
chain lipid bolaamphiphiles, which are members of a
family of branched hydrocarbons that contain isoprenoid
phytanol groups connected to glycerol backbones via an
ether linkage. These lipid constituents allow archae-
bacteria and related synthetic systems to maintain
membrane integrity at high temperature (∼90 °C) and
low pH (1-1.5).18,19 Although several structural features
of archaebacterial lipids are responsible for enhanced
membrane stability, the principle of tail-to-tail lipid
coupling has been recognized as a critical attribute.20 As
a result of these observations, we have postulated that
membrane-spanning phospholipids bolaamphiphiles, ei-
ther alone or as a constituent of a multicomponent lipid
membrane would provide a starting point for generating
membrane-mimetic films that exhibit long-term stability
while facilitating lateral mobility of membrane associated
guest species (Figure 1).
As an initial step toward this goal, we designed
asymmetric bolaamphiphiles that contain ester linked
phosphatidylcholine and amine functionalities at opposite
chain ends. It is noteworthy that while a large variety of
single- or double-chain lipid-based bolaamphiphiles have
been synthesized, nearly all double-chain bolaamphiphiles
are symmetric compounds with identical headgroups that
are coupled to a lipophilic core through ether linkages.21-31
Significantly, we believe that the presence of the amine
group provides a convenient means for introducing a
probe group or surface linker; the latter facilitating the
formation of a substrate bound assembly of bolaam-
phiphiles. In this paper, we describe the synthesis of a
new class of asymmetric phospholipid bolaamphiphiles
(21) Wang, G.; Hollingsworth, R. I. J. Org. Chem. 1999, 64, 4140-
4147.
(22) Moss, R. A.; Fujita, T.; Okumura, Y. Langmuir 1991, 7, 2415-
2418.
(23) Kim, J.-M.; Thompson, D. H. Langmuir 1992, 8, 637-644.
(24) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.;
Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600-6608.
(25) Menger, F. M.; Littau, C. A. J. Am. Chem. Soc. 1993, 115,
10083-10090.
(26) Heiser, U. F.; Dobner, B. J. Chem. Soc., Perkin Trans. 1 1997,
809-815.
(27) Eguchi, T.; Arakawa, K.; Terachi, T.; Kakinuma, K. J. Org.
Chem. 1997, 62, 1924-1933.
(28) Svenson, S.; Thompson, D. H. J. Org. Chem. 1998, 63, 7180-
7182.
(29) Lecollinet, G.; Auze´ly-Velty, R.; Daniel, M.; Benvegnu, T.;
Mackenzie, G.; Goodby, J. W.; Plusquellec, D. J. Org. Chem. 1999, 64,
3139-3150.
(18) De Rosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. Rev. 1986,
50, 70-80.
(19) Setter, K. O. FEMS Microbiol. Rev. 1996, 18, 149-158.
(20) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman,
P. D. J.; Ragase, B.; Wieczoreck, L.; Pace, R. J. Nature 1997, 387, 580-
583.
(30) Miyawaki, K.; Takagi, T.; Shibakami, M. Synlett 2002, 1326-
1328.
(31) Collier, J. H.; Messersmith, P. B. Annu. Rev. Mater. Res. 2001,
31, 237-263.
J. Org. Chem, Vol. 70, No. 7, 2005 2607

Kai et al.
SCHEME 1. Retrosynthetic Analysis of
Phospholipid-Based Bolaamphiphile
group by either catalytic hydrogenation or via use of
potassium hydroxide led to the unintended decomposition
of the ortho ester group (Scheme 3). However, selective
deprotection of the ortho ester group could be achieved
by adding pyridinium p-toluenesulfonate to a solution of
8a in methanol followed by treatment with 1 equiv of
lithium hydroxide to give 10a/b.
The propanylamine intermediate 17a/17b was synthe-
sized from 1,2-dihydroxypropanylamine (15) by initial
Boc protection of the amino group, followed by acylation
of the primary hydroxyl group using 1 equiv of palmitic
acid (Scheme 4). A trace byproduct of 1,2-diacylated
propanylamine was noted. DCC-catalyzed esterification
of compound 10a with 17a produced the semi-bolaam-
phiphile 18a in 51% yield. Catalytic hydrogenation of 18a
afforded the carboxylic acid 19a that was subsequently
coupled with lyso-phosphatidylcholine to generate the
Boc-protected asymmetric bolaamphiphile 20a in 22%
yield. Removal of the Boc group was performed quanti-
tatively with trifluoroacetic acid to yield the desired
compound 21a. In a similar manner, a smaller bolaam-
phaphile 21b was also synthesized from 10b. All inter-
mediates and the final compounds were characterized by
1H NMR and mass spectroscopy. Of note, the presence
of two distinctive multiplet peaks at 5.10 and 5.20 ppm
in the spectra of bolaamphiphiles 20a and 21a indicate
successful esterification of the C-2 hydroxyl groups of the
two glycerol backbones.33 In sonicated dispersions of 21a
and 21b in pure water, endothermic transitions were
observed at 82.8 and 55.5 °C, respectively, which are
consistent with relative differences in bolaamphiphile
size.
SCHEME 2. Synthesis of 32/22-Ene Dioic Acid
Derivative 8a
Solvent-Mediated Behavior of an Asymmetric
Bolaamphiphile. 1H NMR spectroscopy provided in-
sight into the influence of solvent polarity on conforma-
tion and self-assembly of phospholipid bolaamphiphile
21a. Characteristically, the motion of polar groups
increases in solvents of increasing polarity, which is
represented by relative line sharpening of the corre-
sponding 1H NMR signal.34 While somewhat speculative,
1H NMR spectroscopy suggests that micelle-like struc-
tures are formed in polar solvents, while reverse micelles
are generated in apolar solvents. As shown in Figure 2,
the downfield shift of the water peak δ ) 1.65 ppm in
CDCl3, 4.00 ppm in CDCl3-CD3OD (9:1), 4.12 ppm in
CDCl3-CD3OD (3:1), 4.62 ppm in CDCl3-CD3OD (1:1)
and 4.80 ppm in CDCl3-CD3OD (1:3) referenced to TMS,
is directly indicative of the increasing polarity of the
solvent. Signals corresponding to N+Me3 protons
(δ 3.20 ppm) and the C-2 protons of both glycerol
backbones were quite broad in CDCl3 but became sharper
in solvent mixes of increasing polarity. In contrast, an
opposite trend was noted for the signal attributed to the
terminal methyl groups of both alkyl chains (δ 0.80 ppm).
Of interest, a distinct resonance band at 4.50 ppm that
is consistent with bound water was observed when the
bolaamphiphile was dissolved in CDCl3-CD3OD (1:3).
Surprisingly, the high-field proton Hb-2 assigned to the
propanyl moiety appeared downfield in CDCl3-CD3OD
(3:1-1:1) but shifted upfield with a lower integration
a Reagents and conditions: (a) TMSCl, NaI/CH3CN, reflux
(2a: 96%, 2b: 92%); (b) BnOH, DCC/Ch2Cl2 (3a: quant, 3b:
quant); (c) DMSO, NaHCO3, 150 °C (4a: 72%, 4b: 60%);
(d) 3-methyl-3-oxetanemethanol, DCC/CH2Cl2 (5a: 91%, 5b: 80%);
(e) BF2‚Et2O/CH2Cl2, -15 to 0 °C (6a: 68%, 6b: 60%); (f) Ph3P/
benzene-toluene, reflux (7a: quant, 7b: 73%); (g) n-BuLi, THF,
-78 °C (8a: 52%, 8b: 61%).
nal protecting groups. C22-Diester 8b was synthesized
in a similar manner from lactone 1b in good yield.
In performing the Wittig reaction, sodium methoxide
was initially investigated as a base.21 However, the
desired product was not observed despite increasing base
concentration (1.2-6.0 equiv), reaction temperature, or
time. As a consequence, the effectiveness of n-butyl-
lithium was evaluated in accord with prior reports.32 The
desired compound 8a was obtained in 52% yield after
detailed optimization of the reaction conditions (Table 1).
1H NMR spectrum of 8a demonstrated that the coupling
constant of the two olefin protons was 9.2 Hz, which was
consistent with a cis-isomer of the CdC double bond.
In evaluating optimal conditions for selective depro-
tection of 8a/8b, it was noted that removal of the benzyl
(33) Hebert, N.; Beck. A.; Lennox, R. B.; Just, Q. J. Org. Chem. 1992,
57, 1777-1783.
(32) Paulain, S.; Noiret, N.; Fauconnot, L.; Nugier-Chauvin, C.;
Patin, H. Tetrahedron 1999, 55, 3595-3604.
(34) Winnik, F. M.; Miyazawa, K. Macromolecules 2002, 35, 2440-
2444.
2608 J. Org. Chem., Vol. 70, No. 7, 2005

Synthesis of Asymmetric Acyclic Phospholipid Bolaamphiphiles
TABLE 1. Wittig Reaction of Aldehyde 4a and Phosphonium Salt 7a
base
run
aldehyde (equiv)
phosphonium salt (equiv)
MeONa (equiv)
n-BuLi (equiv)
solvent
time (h)
yield (%)
a
1
2
3
4
5
1.0
1.0
1.0
1.0
1.0
1.2-6.0
6.0
1.0-6.0
DMF
THF
THF
THF
THF
12-44
-
7.2
4.1
3.6
1.3
2
2
2
2
9
21
29
52
6.0
3.0
1.2
a -, no reaction.
SCHEME 3. Selective Deprotection of 32/22-Ene Dioic Acid Derivative 8a/8ba
a Reagents and conditions: (a) PPTS/MeOH; (b) LiOH/THF; (c) KOH/MeOH-THF; (d) H2, Pd-C/EtOH-AcOEt.
value in CDCl3-CD3OD (1:3). Hydration of the sn-2 ester
carbonyl with small amounts of adventitious water
provides a possible explanation for this observation. The
different integration value indicates that the conforma-
tions of the two C-2 protons are different in the micellar
domain in CDCl3-CD3OD (1:3), which is consistent with
FTIR spectral data reported below. Further studies will
need to be performed in order to address this phenom-
enon in greater detail.
It is noteworthy that both the trimethylammonium and
terminal methyl groups of both alkyl chains present
sharp signals in solvent mixes of intermediate polarity
(CDCl3-CD3OD 3:1-1:1). Thus, under these conditions
micelle formation does not occur with both hydrophilic
and hydrophobic moieties of the bolaamphiphile having
unrestricted motion.
nol (1:1) or water were notable for adsorption bands
consistent with the acyl chain region at 2900-2800 cm-1
,
the interface between the hydrophobic and hydrophilic
zones at 1780-1660 cm-1, and the polar headgroup
region at 1130-1150 cm-1 (Figure 3). Of note, two ester
carbonyl stretching regions at 1770 and 1680 cm-1 were
observed. The high-frequency component (1770 cm-1) is
mainly associated with free carbonyl groups, while the
low frequency (1680 cm-1) feature is typically assigned
to carbonyl groups hydrogen-bonded to water. The in-
tensity of the low-frequency carbonyl (1680 cm-1) in-
creased in the presence of water, which further supports
this assignment.
To determine which ester carbonyl hydrogen bonded
with water, FTIR studies were performed on two related
building block components of bolaamphiphile 21a, DPPC,
and 1,2-dipalmitoylpropanylamine (23) (Figure 4).
1,2-Dipalmitoylpropanylamine (23) was synthesized, as
shown in Scheme 5. A low-frequency carbonyl adsorption
band (1680 cm-1) was observed in the spectrum of 23,
the intensity of which increased when the compound was
cast from water. Consistent with these data, prior reports
indicate a greater preference of water to hydrogen bond
to the CdO group of the sn-2 ester than that of the sn-1
ester.35 Therefore, the low-frequency carbonyl band
FTIR Properties of Aymmetric Bolaamphiphiles.
Fourier transform infrared spectroscopy provides a con-
venient tool for elucidating unique structural features of
phospholipids in water or when in contact with ions and
membrane proteins.35,36 Infrared spectra for the asym-
metric bolaamphiphile 21a cast from cholorofom/metha-
(35) Blume, A.; Hu¨bner, W.; Messner, G. Biochemistry 1988, 27,
8239-8249.
(36) Domingo, J. C.; Madariaga, M. A. D. Collo. Surf. A: Physichem.
Eng. Aspects 1996, 115, 97-105.
J. Org. Chem, Vol. 70, No. 7, 2005 2609

Kai et al.
SCHEME 4. Synthesis of Asymmetric Bolaamphiphile 21a
a Reagents and conditions: (a) (Boc)2O, DMAP/CHCl3-MeOH (59%); (b) a: palmitic acid, b: undecanoic acid, DCC/CH2Cl2 (17a: 63%,
17b: 63%); (c) 10, DCC/CH2Cl2 (18a: 70%, 18b: 51%); (d) H2, 10% Pd-C/EtOH (19a: 51%, 19b: 81%); (e) a: 1-palmitoyl-2-hydroxy-
sn-glycero-3-phosphocholine, b: 1-undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, DCC/CH2Cl2 (20a: 9%, 20b: 22%); (f) TFA/CH2Cl2
(21a: quant, 21b: quant).
(1680 cm-1) of 21a is likely attributable to the sn-2 ester
carbonyl of the propanylamine backbone.
alkyldioic acid, bearing distinct terminal protecting
groups, with propanylamine and lyso-phosphatidylcholine
headgroups. High thermal stability was verified by DSC
and bolaamphiphile self-assembly investigated in solvent
mixes of varying polarity by nuclear magnetic resonance
(NMR) and Fourier transform-infrared (FT-IR) spectros-
copy. Cryo-high-resolution scanning electron microscopy
demonstrated that bolaamphiphiles in water could be
processed to form multilamellar vesicles. We anticipate
that asymmetric lipid bolaamphiphiles will provide facile
building blocks for engineering a variety of unique
membrane-mimetic structures with potential applications
in drug delivery, biosensors, and biofunctional prosthetic
devices.
Cryo-High-Resolution SEM of Phospholipid Bo-
laamphiphiles in Water. The self-assembling proper-
ties of bolaamphiphilic lids 21a and 21b in water were
evaluated using cryo-HRSEM, which provided direct
visulization of the bolaamphiphile lipid membrane. As
shown in Figure 5A,B, the C32 bolaamphiphile 21a
preferred to self-assembled as uniform microparticles of
1400-1500 nm diameter, which formed a homogeneous
suspension in water and were stable for more than 3
months. Detailed imaging also revealed occasional for-
mation of “onion” shape, giant multilamellar vesicles of
1500-2000 nm diameter (Figure 5C,D). The smaller C22
bolaamphiphile 21b exhibited a preference for assem-
bling as uniform, “onion” shape, giant multilamellar
vesicles of 2500-3000 nm diameter (Figure 5E-H).
Experimental Section
16-Iodohexadecanoic Acid (2a). 16-Hexadecanolide
(20.0 g, 78.7 mmol), sodium iodide (35.4 g, 235.8 mmol), and
acetonitrile (250 mL) were added to a 500 mL round-bottom
flask. Chlorotrimethylsilane (29.9 mL, 235.8 mmol) in aceto-
nitrile (20 mL) was subsequently added to this solution, and
the reaction mixture refluxed under argon atmosphere for
16 h. The reaction mixture was then cooled to room temper-
Conclusions
A synthetic route was devised for the generation of a
new class of asymmetric phospholipid-based bolaam-
phiphiles through the sequential esterification of an
2610 J. Org. Chem., Vol. 70, No. 7, 2005

Synthesis of Asymmetric Acyclic Phospholipid Bolaamphiphiles
FIGURE 2. 1H NMR spectra of the asymmetric bolaamphiphile in CDCl3-CD3OD (9:1), (3:1), (1:1), and (1:3) mixtures, respectively.
ature, and the product silyl ester was hydrolyzed to the
corresponding carboxylic acid by adding water (200 mL). The
reaction mixture was taken up in ether (500 mL) and washed
successively with water (200 mL × 3), 10% sodium thiosulfate
solution (200 mL), and brine (200 mL) to remove inorganic
salts and iodine. The ether layer was dried over sodium sulfate.
Removal of ether afforded crude carboxylic acid 2a, which was
further purified by silica gel column chromatography
(n-hexane/ethyl acetate ) 5:1) to obtain a colorless solid
(27.71 g, 92%). Similarly, 2b (16.3 g) was synthesized from
1b (10.0 g, 54.3 mmmol) in 96% yield
(20 mL) was added dropwise at 0 °C to a solution of carboxylic
acid 2a (20.98 g, 54.9 mmol), benzyl alcohol (28.4 mL,
274.6 mmol), and 4-(dimethylamino)pyridine (0.67 g,
5.49 mmol) in dichloromethane (250 mL). The reaction mixture
was stirred for 20 h at room temperature under argon
atmosphere. Dicyclohexylurea was removed by filtering through
Celite, and the filtrate was evaporated to provide a residue,
which was purified by silica gel column chromatography
(n-hexane/ethyl acetate ) 10:1) to afford 3a as a colorless solid
(27.9 g, quant). Similarly, 3b (7.73 g) was synthesized from
2b (6.0 g, 19.2 mmol) quantitatively.
2a: 1H NMR (CDCl3) δ 3.18 (t, 2H, J ) 7.0), 2.34 (t, 2H,
J ) 7.4 Hz), 1.82 (q, 2H, J ) 7.0 Hz), 1.63 (q, 2H, J ) 7.4 Hz),
1.42-1.20 (br s, 22H); HR-MS (FAB) calcd for C16H31O2LiI
389.1529, obsd 389.1526 [M + Li]+.
3a: 1H NMR (CDCl3) δ 7.36 (s, 5H), 5.11 (s, 2H), 3.19
(t, 2H, J ) 7.7 Hz), 2.35 (t, 2H, J ) 7.7 Hz), 1.82 (m, 2H), 1.63
(m, 2H), 1.40-1.20 (br s, 22H); HR-MS (FAB) calcd for
C23H37O2LiI 479.1998, obsd 479.1982 [M + Li]+.
2b: 1H NMR (CDCl3) δ 3.18 (t, 2H, J ) 7.1), 2.34 (t, 2H,
J ) 7.4 Hz), 1.81 (q, 2H, J ) 7.1 Hz), 1.62 (q, 2H, J ) 7.4 Hz),
1.39-1.20 (br s, 12H); HR-MS (FAB) calcd for C11H22O2I
313.0665, obsd 313.0660 [M + H]+.
3b: 1H NMR (CDCl3) δ 7.34 (s, 5H), 5.10 (s, 2H), 3.17
(t, 2H, J ) 7.1 Hz), 2.34 (t, 2H, J ) 7.4 Hz), 1.82 (m, 2H), 1.63
(m, 2H), 1.36-1.18 (br s, 12H); HR-MS (FAB) calcd for
C18H28O2I 403.1134, obsd 403.1142 [M + H]+.
Benzyl 16-Iodohexadecanoate (3a). A solution of dicyclo-
hexylcarbodiimide (22.6 g, 109.8 mmol) in dichloromethane
Benzyl 16-Oxohexadecanoate (4a). A mixture of sodium
hydrogen carbonate (4 g) and anhydrous methyl sulfoxide
J. Org. Chem, Vol. 70, No. 7, 2005 2611

Kai et al.
10H); HR-MS (FAB) calcd for C18H26O3Li 297.2042, obsd
297.2054 [M + Li]+.
(3-Methyloxetan-3-yl)methyl 16-iodohexadecanoate
(5a). A solution of dicyclohexylcarbodiimide (5.40 g, 26.2 mmol)
in dichloromethane (20 mL) was added dropwise at 0 °C to a
solution of carboxylic acid (2a) (5.00 g, 13.1 mmol), 3-methyl-
3-oxetanemethanol (6.68 g, 65.4 mmol), and 4-(dimethyl-
amino)pyridine (0.160 g, 1.31 mmol) in dichloromethane
(200 mL). The reaction mixture was stirred for 20 h at room
temperature under argon atmosphere. Dicyclohexylurea was
removed by filtering through Celite, and the filtrate was
evaporated to provide a residue, which was purified by silica
gel column chromatography (n-hexane/ethyl acetate ) 7:1) to
afford 5a as a colorless solid (4.83 g, 80%). Similarly, 5b
(9.38 g) was synthesized from 2b (8.10 g, 25.9 mmmol) in 91%
yield.
5a: 1H NMR (CDCl3) δ 4.49 (d, 2H, J ) 5.5 Hz), 4.35 (d, 2H,
J ) 5.5 Hz), 4.13 (s, 3H), 3.15 (t, 2H, J ) 6.8 Hz), 2.32 (t, 2H,
J ) 7.3 Hz), 1.96-1.54 (m, 6H), 1.35-1.22 (br s, 22H); HR-
MS (FAB) calcd for C21H40O3I 467.2022, obsd 467.2027
[M + H]+.
5b: 1H NMR (CDCl3) δ 4.50 (d, 2H, J ) 5.4 Hz), 4.36 (d, 2H,
J ) 5.4 Hz), 4.13 (s, 3H), 3.17 (t, 2H, J ) 6.7 Hz), 2.34 (t, 2H,
J ) 7.6 Hz), 1.80 (q, 2H, J ) 6.8 Hz), 1.62 (q, 2H, J ) 7.6 Hz),
1.38-1.26 (br s, 12H); HR-MS (FAB) calcd for C16H29O3ILi
403.1322, obsd 403.1323 [M + Li]+.
1-(15-Iodopentadecanyl)-4-methyl-2,6,7-trioxabicyclo-
[2.2.2]octane (6a). Boron trifluoride etherate (0.745 mL,
5.89 mmol) was added with continuous stirring to a solution
of oxetane ester (10.97 g, 23.5 mmol) in anhydrous dichlo-
romethane (23.5 mL) at -15 °C. After stirring at 0 °C under
argon atmosphere for 16 h, the reaction mixture was quenched
by the addition of triethylamine (3.28 mL, 23.5 mmol), diluted
with ether (25 mL), and filtered to remove the amine-BF3
complex. The filtrate was concentrated and the residue was
purified by silica gel column chromatography (n-hexane/ethyl
acetate/triethylamine ) 60:10:1) to afford 6a as a colorless
solid (6.58 g, 60%). Similarly, 6b (6.40 g) was synthesized from
5b (9.38 g, 23.7 mmmol) in 68% yield.
6a: 1H NMR (CDCl3) δ 3.78 (s, 6H), 3.36 (t, 2H, J )
7.0 Hz), 1.81 (q, 2H, J ) 7.0 Hz), 1.64 (q, 2H, J ) 7.0 Hz),
1.43-1.36 (m, 2H), 1.23 (br s, 22H), 0.79 (s, 3H); HR-MS (FAB)
calcd for C21H40O3I 467.2022, obsd 467.2017 [M + H]+.
6b: 1H NMR (CDCl3) δ 3.89 (s, 6H), 3.17 (t, 2H, J )
6.8 Hz), 1.80 (q, 2H, J ) 6.8 Hz), 1.67-1.62 (m, 2H), 1.40-
1.33 (m, 2H), 1.30-1.25 (br s, 12H), 0.79 (s, 3H); HR-MS (FAB)
calcd for C16H30O3I 397.1240, obsd 397.1225 [M + H]+.
1-[(31-Benzyloxycarbonyl)hentriacont-15-en-1-yl]-4-
methyl-2,6,7-trioxabicyclo[2.2.2] octane (8a). The ortho
ester 6a (5.02 g, 10.7 mmol) and triphenylphosphine (5.65 g,
21.5 mmol) were stirred in anhydrous benzene and anhydrous
toluene (2:1, 75 mL) under refluxing conditions for 24 h in an
argon atmosphere. After the solvent was removed, the residue
was taken up in ether (100 mL) and the solution stirred
vigorously for 1 h. The mixture was filtered to remove the
triphenylphosphine in ether, and the white solid was collected
and washed with ether to ensure that all excess triphenyl-
phosphine was removed. The product (phosphonium salt) (7a)
was dried with a vacuum pump for 24 h with a resultant yield
of 5.68 g (73%). The salt (7) (5.68 g, 7.80 mmol) and anhydrous
tetrahydrofuran (18.6 mL) were mixed in a round-bottom flask
at -20 °C under argon atmosphere. n-Butyllithium (1.6 M) in
hexane (5.28 mL, 8.45 mmol) was dropwise added to the
solution. After 30 min at -20 °C, the mixture was cooled to
-78 °C, followed by the addition of DMPU (3.93 mL,
32.5 mmol) and aldehyde 4a (2.34 g, 6.50 mmol). After 1 h at
-78 °C, the mixture was allowed to warm to room tempera-
ture. The mixture was extracted with ether (200 mL) and then
washed with water (100 mL). The ether layer was dried over
anhydrous sodium sulfate. Removal of ether afforded crude
product 8a, which was further purified by silica gel column
chromatography (n-hexane/ethyl acetate/triethylamine ) 80:
FIGURE 3. FTIR spectra of bolaamphiphile 21a cast from
(A) 1:1 chloroform-methanol, (B) water at 60 °C, and (C) water
at 90 °C.
(100 mL) was heated to 150 °C under argon atmosphere. To
this mixture was added compound 3a (3.90 g, 8.262 mmol) in
anhydrous methyl sulfoxide (10 mL), with continued heating
and stirring for 10 min. The flask was cooled, and the mixture
was poured into ice-water, which was stirred for another 1 h
after TLC (n-hexane/ethyl acetate ) 10:1) confirmation of the
starting material disappearance. The white solid that formed
was recovered by extracting with ether (100 mL × 4) and the
ether layer dried over sodium sulfate. Removal of ether
afforded crude aldehyde 4a, which was further purified by
silica gel column chromatography (n-hexane/ethyl acetate )
8:1) to obtain a colorless solid (1.79 g, 60%). Similarly, 4b
(2.413 g) was synthesized from 3b (4.63 g, 11.5 mmmol) in
96% yield.
4a: 1H NMR (CDCl3) δ 9.75 (t, 1H, J ) 1.9 Hz), 7.36-7.33
(m, 5H), 5.10 (s, 2H), 2.41 (dt, 2H, J ) 1.9, 7.2 Hz), 2.34
(t, 2H, J ) 7.6 Hz), 1.66-1.59 (m, 4H), 1.34-1.22 (br s, 20H);
HR-MS (FAB) calcd for C23H36O3Li 367.2824, obsd 367.2821
[M + Li]+.
4b: 1H NMR (CDCl3) δ 9.75 (t, 1H, J ) 2.2 Hz), 7.36-7.33
(m, 5H), 5.10 (s, 2H), 2.41 (dt, 2H, J ) 2.2, 7.5 Hz), 2.35
(t, 2H, J ) 7.5 Hz), 1.62 (q, 4H, J ) 7.5 Hz), 1.34-1.22 (br s,
2612 J. Org. Chem., Vol. 70, No. 7, 2005

Synthesis of Asymmetric Acyclic Phospholipid Bolaamphiphiles
FIGURE 4. FTIR spectra of two structural building blocks of bolaamphiphile 21a: (A) DPPC cast from 1:1 chloroform-methanol,
(B) DPPC cast from water at 50 °C, (C) 23 cast from 1:1 chloroform-methanol, and (D) 23 cast from water at 50 °C.
SCHEME 5. Synthesis of 1,2-Dipalmitoylpropanyl Amine 23a
a Reagents and conditions: (a) palmitic acid, DCC/CH2Cl2 (95%); (b) TFA/CH2Cl2 (quant).
10:1) to obtain 8a as a white solid (2.30 g, 52%). Similarly, 8b
(5.08 g) was synthesized from 6b (6.12 g, 15.4 mmmol) and
4b (10.1 g, 15.4 mmmol) in 61% yield.
9a: 1H NMR (CDCl3) δ 7.36-7.33 (m, 5H), 5.34 (dt, J ) 4.6,
9.2 Hz), 5.10 (s, 2H), 4.19 (s, 2H), 3.54 (q, 4H, J ) 11.5 Hz),
2.72 (br s, 2H), 2.35 (t, 2H, J ) 7.7 Hz), 2.34 (t, 2H, J ) 7.7
Hz), 2.00 (q, 4H, J ) 4.6 Hz), 1.63 (q, 4H, J ) 7.4 Hz), 1.25
(s, 44H), 0.83 (s, 3H); HR-MS (FAB) calcd for C44H76O6Li
707.5802, obsd 707.5786 [M + Li]+.
8a: 1H NMR (CDCl3) δ 7.34-7.32 (m, 5H), 5.33 (dt, J ) 4.6,
9.2 Hz), 5.09 (s, 2H), 3.87 (s, 6H), 2.33 (t, 2H, J ) 7.7 Hz),
2.01-1.97 (m, 4H), 1.66-1.60 (m, 4H), 1.23 (br s, 46H), 0.77
(s, 3H); HR-MS (FAB) calcd for C44H74O5Li 689.5696, obsd
689.5696 [M + Li]+.
9b: 1H NMR (CDCl3) δ 7.36-7.32 (m, 5H), 5.34 (dt, J ) 4.6,
9.2 Hz), 5.10 (s, 2H), 4.17 (s, 2H), 3.54 (q, 4H, J ) 11.5 Hz),
2.93 (br s, 2H), 2.34 (t, 4H, J ) 7.2 Hz), 2.00 (q, 4H, J ) 4.7
Hz), 1.65-1.60 (m, 4H), 1.38-1.22 (br s, 24H), 0.83 (s, 3H);
HR-MS (FAB) calcd for C34H56O6Li 567.4237, obsd 567.4248
[M + Li]+.
8b: 1H NMR (CDCl3) δ 7.36-7.32 (m, 5H), 5.34 (dt, J ) 4.8,
9.6 Hz), 5.11 (s, 2H), 3.89 (s, 6H), 2.35 (t, 2H, J ) 7.6 Hz),
2.00 (q, 4H, J ) 4.8 Hz), 1.68-1.61 (m, 4H), 1.44-1.39
(m, 2H), 1.32-1.24 (br s, 24H), 0.79 (s, 3H); HR-MS (FAB)
calcd for C34H55O5 543.4050, obsd 543.4027 [M + H]+.
Benzyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropyl-
16-dotriacontenedioate (9a). p-Toluenesulfonate (18.0 mg,
0.0716 mmol) was added to a solution of 8a (1.00 g, 1.47 mmol)
in methanol and chloroform (2:1) (15 mL). The reaction
mixture was stirred for 2 h at room temperature. Ethyl acetate
(40 mL) was then added to the solution and the mixture was
washed with saturated sodium hydrogen carbonate solution
(20 mL). The organic layer was dried over anhydrous sodium
sulfate. Removal of ether afforded crude product 9a, which was
further purified by silica gel column chromatography (chloro-
form/methanol ) 50:1) to obtain 9a as a colorless solid
(0.972 g, 94%). Similarly, 9b (1.62 g) was synthesized from
8b (2.49 g, 4.59 mmmol) in 63% yield.
Benzyl Hydrogen 16-Dotriacontenedioate (10a). Lithium
hydroxide (33.3 mg, 1.39 mmol) was added to a solution of 9a
(0.972 g, 1.39 mmol) in tetrahydrofuran (15 mL) and water
(5 mL). The mixture was stirred for 4 h at room temperature.
Hydrochloric acid solution (1 M, 20 mL) was added to the
solution, and the mixture was extracted with ethyl acetate
(40 mL × 3). The ethyl acetate layer was dried over anhydrous
sodium sulfate. Removal of ethyl acetate afforded crude
product 10a, which was further purified by silica gel column
chromatography (chloroform/methanol ) 50:1) to obtain 10a
as a colorless solid (0.508 g, 61%). Similarly, 10b (164 mg) was
synthesized from 9b (400 mg, 0.715 mmmol) in 50% yield.
10a: 1H NMR (CDCl3) δ 7.34 (s, 5H), 5.34 (dt, J ) 4.6,
9.2 Hz), 5.11 (s, 2H), 2.34 (t, 4H, J ) 7.7 Hz), 2.02-1.96
J. Org. Chem, Vol. 70, No. 7, 2005 2613

Kai et al.
mmol), palmitic acid (700 mg, 2.73 mmol), and 4-(dimethyl-
amino)pyridine (31.9 mg, 0.261 mmol) in dichloromethane
(10 mL). The reaction mixture was stirred for 20 h at room
temperature under argon atmosphere. Dicyclohexylurea was
removed by filtering through Celite and the filtrate was
evaporated to give a residue, which was purified by silica gel
column chromatography (chloroform/methanol ) 50:1) to afford
17a as a cololess solid (0.706 g, 63%). Similarly, 17b (2.46 g)
was synthesized from 16 (2.0 g, 0.715 mmmol) in 59% yield.
17a: 1H NMR (CDCl3) δ 5.00 (br s, 1H), 4.16-4.03 (m, 2H),
3.91 (m, 1H), 3.19-3.12 (m, 2H), 2.32 (t, 2H, J ) 6.8 Hz), 1.60
(t, 2H, J ) 6.8 Hz), 1.43 (s, 9H), 1.24 (br s, 24H), 0.86 (t, 3H,
J ) 6.2 Hz); HR-MS (FAB) calcd for C24H47O5NLi 436.3614,
obsd 436.3598 [M + Li]+.
17b: 1H NMR (CDCl3) δ 5.51 (br s, 1H), 4.19 (br s, 2H), 3.69
(m, 1H), 3.58-3.45 (m, 2H), 3.23-3.14 (m, 2H), 1.39 (s, 9H);
HR-MS (FAB) calcd for C8H17O4NLi 198.1318, obsd 198.1319.
(S)-3-N-(tert-Butoxycarbonyl)amino-1,2-propanediol
1-Palmitate 2-(31-Benzyloxycarbonyl-16-hentriaconten)-
oate (18a). A solution of dicyclohexylcarbodiimide (360 mg,
1.74 mmol) in dichloromethane (5 mL) was added dropwise
at room temperature to a solution of 17a (500 mg, 1.16 mmol),
10a (836 mg, 1.39 mmol), and 4-(dimethylamino)pyridine
(14.2 mg, 0.116 mmol) in dichloromethane (30 mL). The
reaction mixture was stirred for 44 h at room temperature
under argon atmosphere. Dicyclohexylurea was removed by
filtering through Celite and the filtrate was evaporated to give
a residue, which was purified by silica gel column chromatog-
raphy (n-hexane/ethyl acetate ) 4:1) to afford 18a as a
colorless solid (599 mg, 51%). Similarly, 18b (2.46 g) was
synthesized from 17b (419 mg, 0.913 mmmol) and 10b
(492 mg, 1.369 mmmol) in 70% yield.
18a: 1H NMR (CDCl3) δ 7.34 (s, 5H), 5.33 (dt, 2H, J ) 4.8,
9.6 Hz), 5.10 (s, 2H), 5.08 (q, 1H, J ) 5.5 Hz), 4.75 (t, 1H,
J ) 5.5 Hz), 4.26 (dd, 1H, J ) 5.5, 12.2 Hz), 4.03 (dd, 1H,
J ) 5.5, 12.2 Hz), 3.35 (dd, 2H, J ) 5.5, 12.2 Hz), 2.36-2.28
(m, 6H), 2.00 (dd, 4H, J ) 6.7, 12.5 Hz), 1.67-1.60 (m, 6H),
1.42 (s, 9H), 1.24 (br s, 68H), 0.87 (t, 3H, J ) 6.7 Hz); HR-MS
(FAB) calcd for C63H111O8NLi 1016.8470, obsd 1016.8485
[M + Li]+.
18b: 1H NMR (CDCl3) δ 7.38-7.31 (m, 5H), 5.34 (dt, 2H,
J ) 4.6, 9.2 Hz), 5.11 (s, 2H), 5.08 (q, 1H, J ) 5.3 Hz), 4.75
(t, 1H, J ) 5.3 Hz), 4.26 (dd, 1H, J ) 4.2, 12.0 Hz), 4.11
(dd, 1H, J ) 5.3, 12.0 Hz), 3.35 (dd, 2H, J ) 5.3, 12.0 Hz),
2.36-2.27 (m, 6H), 2.00 (dd, 4H, J ) 6.4, 11.4 Hz), 1.65-1.58
(m, 6H), 1.43 (s, 9H), 1.36-1.19 (br s, 38H), 0.87 (t, 3H,
J ) 6.6 Hz); HR-MS (FAB) calcd for C48H81O8NLi 806.6122,
obsd 806.6098 [M + Li]+.
(S)-3-N-(tert-Butoxycarbonyl)amino-1,2-propanediol
1-palmitate 2-(Hentriacontane-31-carboxyl)ate (19a). To
a solution of 18a (392 mg, 0.387 mmol) in ethanol (20 mL)
was added 10% palladium carbon (270 mg). Hydrogen gas was
added to the solution, which was then stirred at room tem-
perature for 16 h. The mixture was filtered, washed with
chloroform, and evaporated to give a residue, which was
purified by silica gel column chromatography (n-hexane/ethyl
acetate ) 4:1, and then 3:1) to obtain 19a as a colorless solid
(290 mg, 81%). Similarly, 19b (95 mg) was synthesized from
18b (209 mg, 0.261 mmmol) in 51% yield.
19a: 1H NMR (CDCl3) δ 5.08 (q, 1H, J ) 5.7 Hz), 4.75
(t, 1H, J ) 5.7 Hz), 4.26 (dd, 1H, J ) 5.7, 12.5 Hz), 4.11 (dd,
1H, J ) 5.7, 12.5 Hz), 3.33 (dd, 2H, J ) 5.7, 12.5 Hz), 2.35-
2.27 (m, 6H), 1.63-1.58 (m, 6H), 1.42 (s, 9H), 1.24 (br s, 76H),
0.87 (t, 3H, J ) 6.7 Hz); HR-MS (FAB) calcd for C56H107O8NLi
928.8157, obsd 928.8176 [M + Li]+.
FIGURE 5. Cryo-high-resolution SEM of sonicated bola-
amphiphile 21a (A-D) and 21b (E-H) in water (5 mg/mL).
(m, 4H), 1.66-1.60 (m, 4H), 1.25 (br s, 44H); HR-MS (FAB)
calcd for C39H65O4 597.4883, obsd 597.4891 [M]+.
10b: 1H NMR (CDCl3) δ 7.37-7.33 (m, 5H), 5.34 (dt, J )
4.6, 9.2 Hz), 5.11 (s, 2H), 2.35 (t, 2H, J ) 7.5 Hz), 2.34 (t, 2H,
J ) 7.5 Hz), 2.00 (q, 4H, J ) 4.8 Hz), 1.65-1.60 (m, 4H), 1.38-
1.22 (br s, 24H); HR-MS (FAB) calcd for C29H46O4Li 465.3556,
obsd 465.3535 [M + Li]+.
(S)-3-N-(tert-Butoxycarbonyl)-amino-1,2-pro-
panediol (16). Di-tert-butyl dicarbonate (7.18 g, 32.9 mmol),
4-(dimethylamino)pyridine (670 mg, 5.49 mmol), triethylamine
(4.58 mL, 32.9 mmol) were added to a solution of (S)-3-amino-
1,2-propanediol (15) (2.0 g, 22.0 mmol) in chloroform and
methanol (1:1) (40 mL) at 0 °C under argon atmosphere. The
reaction mixture was stirred for 16 h at room temperature.
The solvent was evaporated and the residue was dissolved in
ethyl acetate (100 mL), washed with 0.1 M hydrochloric acid
solution (60 mL), saturated sodium hydrogen carbonate solu-
tion (60 mL), and brine (60 mL). The chloroform layer was
dried over anhydrous sodium sulfate and the solvent was
evaporated to give a residue, which was purified by silica gel
column chromatography (chloroform/methanol ) 15:1) to afford
16a as an oil (2.46 g, 59%). 1H NMR (CDCl3) δ 5.51 (br s, 1H),
4.19 (br s, 2H), 3.69 (m, 1H), 3.58-3.45 (m, 2H), 3.23-3.14
(m, 2H), 1.39 (s, 9H); HR-MS (FAB) calcd for C8H17O4NLi
198.1318, obsd 198.1319 [M + Li]+.
19b: 1H NMR (CDCl3) δ 5.08 (q, 1H, J ) 5.5 Hz), 4.76
(t, 1H, J ) 5.5 Hz), 4.26 (dd, 1H, J ) 4.1, 11.5 Hz), 4.11 (dd,
1H, J ) 5.5, 11.5 Hz), 3.35 (dd, 2H, J ) 5.5, 11.5 Hz), 2.35-
2.27 (m, 6H), 1.63-1.58 (m, 6H), 1.43 (s, 9H), 1.36-1.20 (br s,
46H), 0.87 (t, 3H, J ) 6.8 Hz); HR-MS (FAB) calcd for
C41H77O8NLi 718.5809, obsd 718.5788 [M + Li]+.
(S)-3-N-(tert-Butoxycarbonyl)-amino-1,2-pro-
panediol 1-palmitate (17a). A solution of dicyclohexyl-
carbodiimide (0.647 g, 3.13 mmol) in dichloromethane (5 mL)
was added dropwise at 0 °C to a solution of 16 (0.500 g, 2.61
2614 J. Org. Chem., Vol. 70, No. 7, 2005

Synthesis of Asymmetric Acyclic Phospholipid Bolaamphiphiles
Boc-Protected Amine Bearing Phosphocholine Lipid
(20a). A solution of dicyclohexylcarbodiimide (97.4 mg,
0.471 mmol) in dichloromethane (2 mL) was added dropwise
at room temperature to a solution of 19a (290 mg, 0.314 mmol),
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (312 mg,
0.629 mmol), and 4-(dimethylamino)pyridine (3.84 mg,
0.0314 mmol) in dichloromethane (20 mL). The reaction
mixture was stirred for 7 d at room temperature under argon
atmosphere. Dicyclohexylurea was removed by filtering through
Celite and the filtrate was evaporated to give a residue, which
was purified by silica gel column chromatography (chloroform/
methanol/water ) 65:25:3), and then freeze-dried to afford 20a
as a colorless powder (94.9 mg, 22%). Similarly, 20b (12 mg)
was synthesized from 19b (95 mg, 0.134 mmmol) in 9% yield.
(S)-1,2-Dipalmitoylpropanylamine (23). Trifluoroacetic
acid (2 mL) was added to a solution of 22 (30 mg, 0.0449 mmol)
in dichloromethane (2 mL). This solution was stirred at room
temperature for 16 h, and the solvent was evaporated and
coevaporated with ether to afford 23 as a colorless solid
(90 mg, quant): 1H NMR (CDCl3) δ 5.26 (m, 1H,), 4.32 (m. s,
1H), 4.13 (m, 1H), 3.49 (dd, 1H, J ) 5 0.4, 12.2 Hz), 2.32
(m, 4H), 1.23 (br. s, 48H), 0.87 (t, 6H, J ) 6.8 Hz).
DSC, FTIR, and SEM Sample Preparation. Lipid sample
21a (10 mg) was dissolved in 2 mL of 1:1 chloroform/methanol
in a round-bottom flask and evaporated with a stream of
nitrogen gas. Pure water (2 mL) was then added and the
suspension hydrated by heating above the phase transition
(Tm) with vortexing for 30 s. The resultant emulsion was
sonicated for 30 min at 60 °C and remained stable for several
months.
1
20a: H NMR (CDCl3) δ 5.19 (m, 1H), 5.08 (q, 1H, J ) 5.2
Hz), 4.74 (t, 1H, J ) 5.2 Hz), 4.26 (dd, 1H, J ) 4.2, 12.2 Hz),
4.11 (dd, 1H, J ) 5.8, 12.2 Hz), 3.93-3.83 (m, 2H), 3.35 (br s,
9H), 2.32-2.23 (m, 8H), 1.62-1.54 (m, 8H), 1.42 (s, 9H), 1.23
(br s, 100H), 0.86 (t, 6H, J ) 6.5 Hz); HR-MS (FAB) calcd for
C80H156O14N2P 1400.1294, obsd 1400.1229 [M + H]+.
Differential Scanning Calorimetry (DSC). Phase-
transition temperatures and enthalpies were determined by
heating and cooling aqueous samples at a rate of 10 °C/h
between 10 and 90 °C.
20b: 1H NMR (CDCl3) δ 5.19 (br s, 1H), 5.09 (q, 1H, J ) 5.2
Hz), 4.76 (t, 1H, J ) 5.2 Hz), 4.26 (dd, 1H, J ) 3.8, 11.7 Hz),
4.11 (dd, 1H, J ) 5.2, 11.7 Hz), 3.37-3.32 (m, 2H), 3.34 br s,
9H), 2.32-2.24 (m, 8H), 1.62-1.54 (m, 8H), 1.43 (s, 9H), 1.36-
1.20 (br s, 60H), 0.87 (t, 3H, J ) 6.8 Hz); HR-MS (FAB) calcd
for C60H116O14N2P 1119.8164, obsd 1119.7532 [M + H]+
Amine Bearing Phosphocholine Lipid (21a). Trifluoro-
acetic acid (3 mL) was added to a solution of 20 (94.9 mg,
0.0677 mmol) in dichloromethane (3 mL). This solution was
stirred at room temperature for 16 h and the solvent was
evaporated and coevaporated with ether to afford 21a as a
colorless solid (90 mg, quant.). Similarly, 21b (10 mg) was
synthesized from 20b (12 mg, 0.011 mmmol) quantitatively.
Cryo-High-Resolution SEM. Aliquots (∼10 µL) of the
sonicated suspensions placed on gold planchets were plunge-
frozen into liquid ethane at its melting point (-183.2 °C) and
subsequently stored under liquid nitrogen. Ethane-encrusted
samples were transferred into a precooled (-170 °C) cryo-
preparation chamber under a positive flow of nitrogen gas
(∼5 psi) and secured onto a cold stage. Specimens were
fractured with the flick of a chilled razor blade and rinsed with
liquid nitrogen. The shutters on the cold stage were closed to
avoid frost contamination and the stage was quickly trans-
ferred into a Cr coater against a positive pressure of nitrogen
gas. The Cr coater was evacuated to 2 × 10-7 Torr and then
back-filled to 5 × 10-3 Torr with Ar gas. The shutters were
opened and the frozen specimen, which was held below
-150 °C, was sputter-coated with 1 nm of Cr after which the
shutters were closed and the vacuum again brought to
2 × 10-7 Torr. The chamber of the Cr coater was flushed with
dry nitrogen gas, returning it to atmospheric pressure, and
the cold stage was removed and transferred to the upper stage
of a field emission SEM. The temperature of the sample was
increased from about -160 to -110 °C in order to allow any
nanometer-sized frost, which had condensed on the surface of
the Cr film to sublime in the microscope prior to imaging. The
specimens were imaged at 25 kV accelerating voltage, digitally
recorded, and processed.
1
21a: H NMR (CDCl3) δ 5.24 (m, 1H), 5.15 (m, 1H), 4.09-
4.05 (m, 2H), 3.64-3.58 (m, 2H), 3.15 (br s, 9H), 2.32-2.23
(m, 8H), 1.60-1.52 (m, 8H), 1.21 (br s, 100H), 0.83 (t, 6H,
J ) 6.5 Hz HR-MS (FAB) calcd for C75H148O12N2P 1301.9655,
obsd 1301.7448 [M + H]+.
21b: 1H NMR (CDCl3) δ 5.19 (br. m, 2H), 4.40-4.05
(m, 9H), 3.90 (m, 2H), 3.55 (m, 2H), 3.15 (br s, 9H), 2.38-2.23
(m, 8H), 1.60-1.52 (m, 8H), 1.21 (br s, 80H), 0.85 (t, 6H,
J ) 6.5 Hz, 6H); HR-MS (FAB) calcd for C55H108O12N2P
1019.7640, obsd 1019.8171 [M + H]+
(S)-3-N-(tert-Butoxycarbonyl)amino-1,2-propanediol 1,2-
Dipalmitate (22). A solution of dicyclohexylcarbodiimide
(0.807 g, 3.91 mmol) in dichloromethane (5 mL) was added
dropwise at 0 °C to a solution of 16 (0.500 g, 2.61 mmol),
palmitic acid (2.67 g, 10.4 mmol), and 4-(dimethylamino)-
pyridine (3.91 mg, 0.261 mmol) in dichloromethane (10 mL).
The reaction mixture was stirred for 20 h at room temperature
under argon atmosphere. Dicyclohexylurea was removed by
filtering through Celite and the filtrate evaporated to give a
residue, which was purified by silica gel column chromatog-
raphy (chloroform/methanol ) 50:1) to afford 22 as a colorless
solid (1.65 g, 95%): 1H NMR (CDCl3) δ 5.08 (q, 1H, J ) 4.9
Hz), 4.73 (br. s, 1H), 4.26 (dd, 1H, J ) 3.3, 12.4 Hz), 4.12 (dd,
1H, J ) 5 0.5, 12.4 Hz), 3.55 (dd, 1H, J ) 5.5, 12.4 Hz), 3.18
(dd, 1H, J ) 3.3, 12.4 Hz), (m, 2H), 2.30 (t, 4H, J ) 6.8 Hz),
1.60 (m, 4H), 1.24 (br s, 48H), 0.86 (t, 6H, J ) 6.2 Hz).
Acknowledgment. This work was supported by
grants from the NIH. We acknowledge the Emory
University NMR and Mass Spectrometry Centers for
their facilities.
Supporting Information Available: General methods
and 1H NMR spectra for key intermediates and final com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO048167O
J. Org. Chem, Vol. 70, No. 7, 2005 2615