Synthesis and fluorescence properties of new fluorescent, polymerizable, metal-chelating lipids

Article abstract of DOI:10.1021/jo991836r

Liposomes incorporating fluorescent, metal-chelating lipids find applications in molecular recognition of peptides, 2D protein recystallization, protein targeting, and biological sensing. It would be advantageous to combine the usefulness of polymerizable, metal-chelating lipids and fluorescent lipids. Herein, we report the synthesis and fluorescence properties of several fluorescent, polymerizable, metal- chelating lipids. They have been successfully incorporated into liposomes and then polymerized. These lipids can be used as membrane probes to study the polymerizable liposomes in the unpolymerized state and to investigate lipid redistribution during polymerization. In addition, if a luminescent metal ion (e.g., Eu³⁺, Tb³⁺, etc.) is used to complex the headgroup, the lipids can probe the membrane interior and exterior simultaneously.

Full text of DOI:10.1021/jo991836r

3644
J . Org. Chem. 2000, 65, 3644-3651
Syn th esis a n d F lu or escen ce P r op er ties of New F lu or escen t,
P olym er iza ble, Meta l-Ch ela tin g Lip id s
Bidhan C. Roy, Rachel Peterson, Sanku Mallik,* and Andres D. Campiglia
Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105
Received November 30, 1999
Liposomes incorporating fluorescent, metal-chelating lipids find applications in molecular recognition
of peptides, 2D protein recystallization, protein targeting, and biological sensing. It would be
advantageous to combine the usefulness of polymerizable, metal-chelating lipids and fluorescent
lipids. Herein, we report the synthesis and fluorescence properties of several fluorescent, polymer-
izable, metal-chelating lipids. They have been successfully incorporated into liposomes and then
polymerized. These lipids can be used as membrane probes to study the polymerizable liposomes
in the unpolymerized state and to investigate lipid redistribution during polymerization. In addition,
if a luminescent metal ion (e.g., Eu3+, Tb3+, etc.) is used to complex the headgroup, the lipids can
probe the membrane interior and exterior simultaneously.
Liposomes are spherically closed lipid bilayers with
The need for increased stability and controlled perme-
ability has led to the synthesis of polymerizable lipids
and polymerizable liposomes.9 Polymerized liposomes are
appreciably more stable compared to the nonpolymeriz-
able counterparts. Though a variety of polymerizable
lipids have been reported in the literature,9 reports for
the synthesis of polymerizable metal-chelating lipids are
relatively few.10
aqueous interiors.1 Due to the ease of preparation, they
are widely used in supramolecular chemistry, e.g., as cell
models, as separation agents, as catalysts, etc.2 Lipo-
somes are also widely used in pharmacology and medi-
cine (as drug carriers, immunoadjuvants, and diagonistic
agents) and in genetic engineering (for gene delivery).3
For these applications, it is very important to study the
interaction of the liposomes with their targets.
It would be advantageous to combine the usefulness
of polymerizable, metal-chelating lipids and fluorescent
lipids. Herein, we report the synthesis and fluorescence
properties of several fluorescent, polymerizable, metal-
chelating lipids. The metal-chelating lipids were prepared
incorporating pyrene, rhodamine B, coumarin, (trifluo-
romethyl)coumarin, and disperse orange as the fluores-
cent groups. To our knowledge, only pyrene- and
rhodamine B-based nonpolymerizable lipids have been
used as membrane probes in the literature. The lipids
were successfully incorporated into liposomes and then
polymerized. Our studies demonstrate that the polymer-
izable, metal-chelating, pyrene-based lipid can be used
as a membrane probe to study the polymerizable lipo-
somes in the unpolymerized state and to investigate lipid
redistribution during polymerization employing the or-
ganic fluorophore. In addition (not demonstrated in these
studies), if a lanthanide ion12 (e.g., Eu3+, Tb3+, etc.) is
used to complex the headgroup, the luminescence prop-
erty of the metal ions can be used to detect protein
binding to the liposomes.
Fluorescence spectroscopy (employing fluorescent lip-
ids) is an important technique to probe the binding of
liposomes to proteins, DNA, and other biomolecules.4
Fluorescent lipids with metal-chelating headgroups have
been reported in the literature. The resultant liposomes
find applications in molecular recognition of peptides,5
2D protein recystallization,6 protein targeting,7 and
biological sensing.8 Usually, for these applications, the
fluorophore is positioned within the lipid bilayer of the
liposomes. When a metal ion binds to the headgroups,
fluorescence spectra change due to lipid redistribution
in the bilayer. The resultant assembly can be imaged
using epifluorescence microscopy.6,7
* To whom correspondence should be addressed. Phone: (701) 231-
8829. Fax: (701) 231-8831. E-mail: mallik@badlands.nodak.edu.
(1) Lasic, D. D. Liposomes: from Physics to Applications; Elsevier:
New York, 1993.
(2) Lasic, D. D. Handbook of Nonmedical Applications of Liposomes;
CRC Press, Boca Raton, FL, 1996.
(3) Gregoriadis, G., Ed. Liposome Technology; CRC Press: Boca
Raton, FL, 1993; Vol. 3.
(4) (a) Bhattacharya, S.; De, S. Langmuir 1999, 15, 3400-3410. (b)
Kachel, K.; Asuncion-Punzalan, E.; London, E. Biochim. Biophys. Acta
1998, 1374, 63-76. (c) Polozova, A.; Winnik, F. M. Langmuir 1999,
15, 4222-4229. (d) Einaga, Y.; Sato, O.; Iyoda, T.; Fujishima, A.;
Hashimoto, K. J . Am. Chem. Soc. 1999, 121, 3745-3750. (e) Owen, D.
J .; Alexandrov, K.; Rostkova, E.; Scheidig, A. J .; Goody, R. S.; Waldman,
H. Angew. Chem., Int. Ed. Engl. 1999, 38, 509-512. (e) Daugherty, D.
L.; Gellman, S. H. J . Am. Chem. Soc. 1999, 121, 4325-4333.
(5) Dorn, I. T.; Neumaier, K. R.; Tampe, R. J . Am. Chem. Soc. 1998,
120, 2753-2763.
(9) Ceve, G., Ed. Phospholipids Handbook; Marcel Dekker Inc.: New
York, 1993; pp 233-292.
(10) (a) Dorn, I. T.; Hofmann, U. G.; Pltonen, J . P.; Tampe, R.
Langmuir 1998, 14, 4836-4842. (b) Markowitz, M.; Puranik, D. B.;
Singh, A. Chem. Phys. Lipids 1995, 76, 63-71. (c) Singh, A.; Tsao, L.
I.; Puranik, D. B. Synth. Commun. 1995, 25, 573-586. (d) Storrs, R.
W.; Tropper, F, D.; Li, H. Y.; Song, C. K.; Kuniyoshi, J . K.; Sipkins, D.
A.; Li, K. C. P.; Berdnarski, M. K. J . Am. Chem. Soc. 1995, 117, 7301-
7306. (e) Roy, B. C.; Mallik, S. J . Org. Chem. 1999, 64, 2969-2974.
(11) Krapcho, A. P.; Kuell, C. S. Synth. Commun. 1990, 20, 2559-
2564.
(6) Pack, D. W.; Chen, G.; Maloney, K. M.; Chen, C. T.; Arnold, F.
H. J . Am. Chem. Soc. 1997, 119, 2479-2487.
(7) (a) Ng, K.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir
1995, 11, 4048-4055. (b) Dietrich, C.; Boscheinen, O.; Scharf, K. D.;
Schmitt, L.; Tampe, R. Biochemistry 1996, 35, 1100-1105.
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Langmuir 1997, 13, 5049-5053. (b) Malony, K. M.; Shnek, D. R.;
Sasaki. D. Y.; Arnold, F. H. Chem. Biol. 1996, 3, 185-192.
(12) (a) Lemieux, G. A.; Yarema, K. J .; J acobs, C. L.; Bertozzi, C. R.
J . Am. Chem. Soc. 1999, 121, 4278-4279. (b) Dutton, P. J .; Conte, L.
Langmuir 1999, 15, 613-617. (c) Okabayashi, Y.; Ikeuchi, I. Analyst
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10.1021/jo991836r CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/16/2000

Synthesis of New Fluorescent Metal-Chelating Lipids
J . Org. Chem., Vol. 65, No. 12, 2000 3645
F igu r e 1. Structures of the fluorescent, polymerizable, metal-chelating lipids.
Resu lts a n d Discu ssion
BOP reagent and triethylamine afforded 8 in 61% yield
after chromatographic purification. For the synthesis of
the lipids 1, 2, and 3, compound 8 was reacted with the
corresponding amines (pyrenemethylamine, amino(tri-
fluoromethyl)coumarin, and disperse orange yellow, re-
spectively). For lipids 4 and 5, the fluorophores (carboxy-
coumarin and rhodamine B) contained carboxylic acids.
The carboxylic acids were reacted with mono-N-Boc-
ethylenediamine11 followed by deprotection to generate
amines 11a and 11b. These compounds were then
coupled with 8. After mild basic hydrolysis (LiOH),
lowering the pH of the reaction medium with dilute HCl
(pH ∼ 3.0) precipitated the lipids. Once isolated, the
Syn th esis of th e Lip id s. The structures of the lipids
synthesized are shown in Figure 1. These lipids have the
iminodiacetate (IDA) as the metal-chelating headgroup.
IDA has strong affinity (>1010 M-1) for various transition-
metal ions (e.g., Cu2+, Ni2+, Co2+, etc.).13 IDA has been
successfully used to study metal-ion-mediated liposome-
protein interactions for nonpolymerizable fluorescent
lipids.6-8 For the studies reported here, a conjugated
dialkyne was chosen as the polymerizable group. Di-
alkyne lipids have been reported in the literature to form
liposomes.9 A diethylene glycol spacer was introduced
between the polymerizable moiety and the metal-chelat-
ing headgroup. The liposome can be easily polymerized
under UV irradiation (254 nm) to form stable struc-
tures.14 Widely used fluorophores (pyrene, coumarin,
rhodamine B, (trifluoromethyl)coumarin, and disperse
orange)15 have been used for our studies.
It is reported that pyrene and rhodamine, when
attached to the hydrophobic alkyl groups of nonpolym-
erizable lipids, are embedded into lipid bilayers of lipo-
somes.7,8,16 Thus, the flourophores of lipids 1 and 5 should
be in the bilayer region of the vesicles. In addition, the
fluorophores of lipids 2, 3, and 4 have hydrophobic
aromatic rings. The IDA headgroups of all of the lipids
will be charged at pH 7.0. These lipids, when incorpo-
rated into liposomes, are expected to present the charged
IDA groups on the surface and position the fluorophores
inside the lipid bilayers.
1
lipids were found to be pure (by H and 13C NMR and
elemental analysis). The pure lipids were prepared in
140-280 mg quantity. They were stored at low temper-
ature (-20 °C), under nitrogen, in the dark.
Vesicle P r ep a r a tion . Vesicles were prepared (50 mM
HEPES buffer, pH 8.0) following a literature procedure17
incorporating polymerizable phosphocholine 1218 (85%),
amine lipid 1319 (5%), cationic lipid 1420 (5%), and the
fluorescent lipid (5%). The structures of lipids 12, 13, and
14 are shown in Figure 2. Experimental conditions
(sonication time, power, rate of cooling, and temperature)
were optimized (detailed procedures are given in the
Experimental Section) for liposome formation. The re-
sultant liposomes were polymerized by UV irradiation
(450 W, 15 min) at 25 °C. Liposome formation was
confirmed by transmission electron microscopy (TEM).
When cupric chloride was added prior to probe sonication,
Syntheses of the lipids (1, 2, 3, 4, and 5) are shown in
Scheme 1. Commercially available polymerizable diacid
6 (available from GFS Chemicals Inc., Columbus, OH)
was combined with the known amine 710e to afford 8. Slow
addition of a solution of 7 (over a 10 h period using a
syringe pump) to a solution of the diacid 6 containing
the metal-chelating headgroups coordinated to Cu2+
.
Representative transmission electron micrographs of
polymerized liposomes incorporating the rhodamine lipid
5 (polymerized in the absence and presence of Cu2+) are
shown in Figure 2. All of the lipids synthesized were
successfully incorporated into liposomes and then po-
lymerized.
(13) Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum
Press: New York, 1975; Vol. 2.
(14) (a) Ulrich, J .; Shah, K.; Norvez, S.; Charych, D. H. J . Am. Chem.
Soc. 1999, 121, 4580-4588. (b) Cheng, Q.; Stueens, R. C. Langmuir
1998, 14, 1974-1976. (c) J elinek, R.; Okada, S.; Norvez, S.; Charych,
D. Chem. Biol. 1998, 5, 619-629.
(15) Haugland, R. P. Handbook of Fluorescent Probes & Research
Chemicals, 6th ed.; Molecular Probes Inc.: Eugene, OR, 1996.
(16) Pramanik, A.; Thyberg, P.; Rigler, R. Chem. Phys. Lipids 2000,
104, 35-47.
(17) Peek, B. M.; Callahan, J . H.; Namboodiri, K.; Singh, A.; Gaber,
B. P. Macromolecules 1994, 27, 292-297.
(18) Available from Avanti Polar Lipids, Alabaster, AL.
(19) The detailed procedure for the synthesis of this lipid is provided
in the Experimental Section.
(20) (a) Hub, H. H.; Hupfer, B. Koch, H.; Ringsdorf, H. Angew.
Chem., Int. Ed. Engl. 1980, 11, 938-940. (b) Ringsdorf, H.; Schlarb,
B.; Venzmer, J . Angew. Chem., Int. Ed. Engl. 1988, 27, 114-158.

3646 J . Org. Chem., Vol. 65, No. 12, 2000
Roy et al.
Sch em e 1
F lu or escen ce P r op er ties. Fluorescence spectra of
the lipids and the polymerized liposomes were recorded
with a continuous source (450 W xenon lamp) for sample
excitation. The sample compartment of the spectrofluo-
rimeter was constantly purged with dry nitrogen to
minimize possible quenching by oxygen. Fluorescence
spectra for pyrene-based polymerizable lipid 1 and the
corresponding polymerized liposomes are shown in Fig-
ure 3 (spectra for other lipids are available as Supporting
Information). Liposomes incorporating the other poly-
merizable lipids (2, 3, 4, and 5) were found to be
unsuitable as membrane probes. The fluorescence spectra
in the absence and presence of cupric ions (during
polymerization of the liposomes) did not show significant
changes (fluorescence spectra are available as Supporting
Information).
aggregated, the excimer emission peak (480 nm) in-
creases in intensity compared to the monomer peak (390
nm). The intensity ratio of the excimer and monomer
peaks (Iex/IM) has been used for sensing metal ions and
biological molecules.8,22 Pyrene-based metal-chelating
lipids also display similar properties.8
To investigate the ability of lipid 1 to detect lipid
redistribution, liposomes were polymerized in the pres-
ence and absence of cupric ions. The Cu2+ ions coordi-
nated to the IDA headgroup of lipid 1. It has been
reported (for nonpolymerizable lipids) that the coordina-
tion leads to the dispersion of IDA-Cu2+ lipids in
(21) (a) Allevi, P.; Ananstasia, M.; Cajone, F. Chem. Phys. Lipids
1999, 100, 89-99. (b) Huang, G.; Hollingsworth, R. I. Tetrahedron
1998, 54, 1355-1360. (c) Pap, E. H. W.; Houbiers, M. C.; Santema, J .
S.; Van Hoek, A.; Visser, A. J . W. G. Eur. Biophys. J . 1996, 24, 223-
231.
(22) Sasaki, D. Y.; Shenk, D. R.; Pack, D. W.; Arnold, F, H. Angew.
Chem., Int. Ed. Engl. 1995, 34, 905-907.
Pyrene-based nonpolymerizable lipids have been widely
used as membrane probes.21,15 When the lipids are

Synthesis of New Fluorescent Metal-Chelating Lipids
J . Org. Chem., Vol. 65, No. 12, 2000 3647
5 nm (280 nm peak). The two well-resolved emission
peaks at 380 and 400 nm that are observed in Figure 3B
are not present in the emission spectrum of the polym-
erized liposomes. Instead, a broad peak with maximum
emission at 395 nm is observed. Since no significant
modifications were observed in the spectral characteris-
tics of pyrenemethylamine when an appropriate volume
of its DMSO solution was mixed with the buffer solution,
the spectral modifications shown in Figure 3 can be
attributed to the different chemical environments sur-
rounding the fluorescence probe.
Table 1 shows the fluorescence intensities and the
intensity ratios of the pyrene excimer and monomer in
different chemical environments. It is important to note
that intercolumn comparison of fluorescence intensities
is inappropriate because different concentrations were
used to measure the reported values. However, the
intensity ratios serve as an indication of the extent of
excimer formation. The results indicate that when py-
renemethylamine is incorporated into the lipid (1), ex-
cimer formation is observed probably due to the proximity
among pyrene moieties. When the liposomes incorporat-
ing lipid 1 are polymerized in the absence of Cu2+, an
increase in the excimer-to-monomer ratio is observed (Iex/
IM ) 0.39), and that can be attributed to a higher degree
of aggregation. When Cu2+ ions are added before polym-
erization of the liposomes, the lipids are dispersed and
the excimer-to-monomer ratio is reduced (Iex/IM ) 0.24).
The lipid 1 reports its aggregation state inside the lipid
bilayer of the liposomes. Similar behavior has been
reported for nonpolymerizable, metal-chelating lipids
with the pyrene group as the fluorophore.8,22
In conclusion, several fluorescent, polymerizable, metal-
chelating lipids have been synthesized. The lipids were
successfully incorporated into liposomes and then po-
lymerized by UV light. The pyrene-containing lipid 1 can
be used as a membrane probe for lipid aggregation inside
the bilayer of the liposomes. The fluorescence property
of the lipid changes in response to cupric ions added
during the polymerization process. Current studies in our
laboratories include the potential application of this lipid
for the detection of metal ion binding to the headgroup.
F igu r e 2. TEM pictures of polymerized liposomes (incorpo-
rating fluorescent lipid 5) without (A) and with (B) Cu2+ ions.
Structures of polymerizable lipids used (in addition to the
lipids synthesized) are also shown.
liposomes.22 The IDA-Cu2+ headgroup is negatively
charged under the experimental conditions (pH 8.0) due
to the deprotonation of the water molecules bound to the
cupric ions.23 The proximity of the negative charges is
likely to contribute to the redistribution of lipid 1 during
the polymerization process.
Exp er im en ta l Deta ils
Figure 3 shows the fluorescence spectra of lipid 1 (B),
and the polymerized liposomes with lipid 1 in the absence
(C) and presence (D) of Cu2+. The spectrum of pyrene-
methylamine is included as a reference. Pyrenemethyl-
amine in DMSO showed three excitation peaks at 280,
335, and 350 nm. Three fluorescence peaks were observed
with maximum emissions at 380, 400, and 420 nm. The
comparison of parts A and B of Figure 3 show the
appearance of a relatively broad fluorescence band in the
pyrene lipid 1 spectrum with maximum emission at 485
nm. Since the excitation spectrum followed at the emis-
sion maximum of this band corresponds to the excitation
spectrum of pyrenemethylamine (data not shown), the
emission at 485 nm can be attributed to the pyrene
excimer. The comparison of parts B and C of Figure 3
shows significant changes in the spectral characteristics
of lipid 1 after incorporation into liposomes and subse-
quent polymerization. Maximum excitation wavelengths
were red-shifted by 10 nm (350 and 335 nm peaks) and
Commercially available reagents were purchased from
either Aldrich or Acros Chemical Co. and used as supplied
unless stated otherwise. Polymerizable diacid (10,12-docosa-
diynedioic acid) was used as obtained from GFS Chemicals.
Organic solvents were of spectroscopy grade from Fisher
Scientific. All aqueous solutions were prepared from Nanopure
water (Millipore). Experiments were conducted under an
atmosphere of dry nitrogen. For workup, the organic layer was
dried on anhydrous Na2SO4 and concentrated in vacuo. Melting
points were recorded on a micro-melting-point apparatus, and
all melting points are uncorrected. Elemental analyses were
performed by an in-house materials characterization labora-
tory. TLC was performed with Absorsil Plus 1P, 20 × 20 cm
plate, 0.25 µm (Alltech Associates, Inc.). Chromatography
plates were visualized either with UV light or in an iodine
chamber. 1H, 13C, and 19F NMR spectra are recorded using 400
and 500 MHz spectrometers in one of the following solvents:
CDCl3, D2O, CD3OD, DMSO-d6, C6D6, and CD3COCD3 with
TMS as internal standard. 13C NMR spectral data have been
reported with two digits after the decimal to distinguish
between close resonances. The polymerizable lipids were stored
in the absence of light at -20 °C in chloroform solution under
nitrogen. Melting points are reported for the compounds which
have sharp melting points.
(23) Zachariou, M.; Hearn, M. T. W. Biochemistry 1996, 35, 202-
211.

3648 J . Org. Chem., Vol. 65, No. 12, 2000
Roy et al.
F igu r e 3. Fluorescence spectra of pyrenemethylamine (A), lipid 1 (B), and polymerized liposomes incorporating this lipid without
(C) or with (D) Cu2+ added during liposome fabrication.
Syn th esis. P olym er iza ble Acid 8. Amine ester 710e (1.3
g, 4.39 mmol) in 50 mL of acetonitrile was added to a solution
of 10,12-docosadiynedioic acid (6; 1.59 g, 4.39 mmol), BOP
reagent (1.94 g, 4.39 mmol), and Et3N (2.1 mL, 3.5 equiv) in
70 mL of acetonitrile at room temperature over a period of 10
h (5 mL/h) by syringe pump. Thereafter, the reaction was
continued for another 12 h at room temperature and then
quenched with a saturated solution of NaCl. The compound
was extracted by ethyl acetate. The combined organic layer
was successively washed with 4% citric acid, water, 4%

Synthesis of New Fluorescent Metal-Chelating Lipids
J . Org. Chem., Vol. 65, No. 12, 2000 3649
Ta ble 1. F lu or escen ce In ten sitiesa a n d In ten sity Ra tios of P yr en e Mon om er (380 n m ) a n d Excim er (485 n m ) P ea k s
liposome with 1,
liposome with 1,
wavelength (nm)
pyrenemethylamine
pyrene lipid 1
polymerized, no Cu2+
polymerized, with Cu2+
380
5212087
2939947
b
b
395
485
485/380
485/395
b
c
c
b
b
26994
10461
b
69804
16900
b
546699
0.186
b
0.388
0.242
a
Fluorescence intensities correspond to background-subtracted intensities (net analyte signals). Net analyte signals are reported in
counts per second (cps). The relative standard deviations for three measurements (N ) 3) of fluorescence intensities and blank intensities
b
are 3% and 5%, respectively. Deoxygenated solutions (N2 bubbling) were used for fluorescence measurements. No measurement was
performed. c No net analyte signal was observed.
NaHCO3 solution, and water. The organic layer was dried over
anhydrous Na2SO4, and solvent was removed in vacuo at 40
°C to provide crude 8. Purification was achieved by silica gel
column chromatography with 3% methanol in CHCl3 as the
elutant (Rf ) 0.4). Yield: 1.62 (61%). Semisolid. 1H NMR (400
MHz, CDCl3-D2O): δ 1.19-1.35 (m, 22H), 1.44-1.51 (m, 4H),
1.56-1.62 (m, 4H), 2.14-2.23 (m, 6H), 2.28-2.33 (m, 2H), 2.96
(t, 2H, J ) 5.4 Hz), 3.40-3.45 (m, 2H), 3.49-3.52 (m, 2H),
3.55 (s, 4H), 3.59-3.62 (m, 6H), 4.13 (q, 4H, J ) 7.1 Hz). 13C
NMR (100 MHz, CDCl3): δ 14.34, 19.24, 24.76, 25.76, 28.28,
28.32, 28.34, 28.76, 28.83, 28.88, 28.91, 28.94, 29.03, 29.13,
29.28, 29.32, 29.35, 34.07, 36.71, 39.27, 53.52, 55.94, 60.64,
65.31, 65.35, 65.38, 70.03, 70.24, 70.36, 77.33, 77.55, 77.57,
173.74, 178.54, 179.01.
Com p ou n d s 10a a n d 10b. Coumarin or Rhodamine B (5.26
mmol) and BOP reagent (2.33 g, 5.26 mmol) were dissolved in
CH3CN (50 mL) followed by the addition of Et3N (1.9 mL, 14
mmol). A solution of mono-N-Boc-ethylenediamine11 (0.84 g,
5.26 mmol) in CHCl3 (15 mL) was added, and the solution was
stirred at room temperature for 10 h. The workup procedure
was the same as described for 8.
9.5 Hz), 6.34 (d, 2H, J ) 2.4 Hz), 6.40 (s, 1H), 6.42 (s, 1H),
7.06-7.08 (m, 1H), 7.44-7.47 (m, 2H), 7.85-7.87 (m, 1H). 13
NMR (125 MHz, CDCl3): δ 12.81, 40.81, 41.18, 44.57, 97.88,
104.24, 108.61, 123.23, 124.09, 128.51, 128.61, 130.32, 133.21,
149.18, 153.45, 153.88, 170.25.
C
P yr en e Lip id 1. 1-Pyrenemethylamine hydrochloride salt
(0.22 g, 0.83 mmol) was suspended in 15 mL of DMF in the
presence of Et3N (0.5 mL, 3.2 mmol), and BOP reagent (0.366
g, 0.83 mmol) was added. Polymerizable acid 8 (0.55 g, 0.83
mmol) was dissolved in CHCl3 (20 mL) and added dropwise
at room temperature. Stirring was continued for 20 h. CHCl3
was removed in vacuo. The DMF solution was added to a
saturated aqueous NaCl solution. A white precipitate ap-
peared. The precipitate was filtered and successively washed
with water, 4% aqueous citric acid, 4% aqueous NaHCO3 and
water. It was dried under vacuum at room temperature to yield
1
the product diester. Yield: 0.63 g (87%). H NMR (400 MHz,
C6D6-CDCl3): δ 0.93-0.96 (m, 6H), 1.15-1.29 (m, 20H), 1.55-
1.69 (m, 4H), 1.91-2.07 (m, 8H), 2.91 (q, 2H, J ) 5.1 Hz), 3.20-
3.28 (m, 6H), 3.36-3.39 (m, 2H), 3.40-3.45 (q, 2H, J ) 5.4
Hz), 3.58 (s, 2H), 3.59 (s, 2H), 3.88-3.94 (m, 4H), 5.08 (d, 2H,
J ) 5.1 Hz), 6.25 (br s, 1H), 6.45 (br s, 1H), 7.75-7.83 (m,
4H), 7.90-7.96 (m, 4H), 8.32-8.35 (d, 1H, J ) 9.4 Hz). 13C
NMR (100 MHz, C6D6-DMSO-d6): δ 14.16, 18.95, 25.86, 25.99,
28.31, 28.69, 28.72, 28.94, 29.25, 29.30, 36.09, 39.16, 39.32,
39.53, 39.74, 39.95, 40.37, 40.57, 41.20, 53.70, 55.69, 59.95,
66.15, 70.04, 70.09, 70.16, 70.53, 77.76, 123.89, 124.83, 124.87,
124.97, 125.29, 129.03, 130.77, 130.99, 131.42, 133.75, 171.11,
172.38, 172.59.
Com p ou n d 10a . 10a was purified by silica gel column
chromatography with 2% MeOH in CHCl3 as the eluant (Rf )
0.4) to yield a red solid. Yield: 1.65 g (94%). Mp: 142-143
1
°C. H NMR (400 MHz, CDCl3): δ 1.41 (s, 9H), 3.33-3.39 (q,
2H, J ) 9.0 Hz), 3.46 (q, 2H, J ) 9.0 Hz), 4.97 (br s, 1H), 7.33-
7.40 (m, 2H), 7.63-7.69 (m, 2H), 8.88 (s, 1H), 8.97 (br s, 1H).
13C NMR (125 MHz, CDCl3): δ 28.60, 40.24, 40.79, 46.75,
116.86, 118.38, 118.70, 125.55, 130.06, 134.39, 148.71, 154.65,
161.49, 162.50.
The ester (0.25 g, 0.28 mmol) was dissolved in benzene (10
mL), and a solution of LiOH (0.05 g, 1.2 mmol) in water (5
mL) was added. The stirring was continued for 12 h at room
temperature. Benzene was removed in vacuo, and insoluble
starting ester (0.025 g) was filtered off. The aqueous solution
was acidified by 1 N HCl (pH 3.0), and a white solid
precipitated. The solid was filtered and washed with water to
Com p ou n d 10b. Product was purified by silica gel column
chromatography with 2% methanol in CHCl3 (Rf ) 0.4) to
afford the pure product as a pink solid. Yield: 2.64 g (81%).
Mp: 62-64 °C. 1H NMR (400 MHz, CDCl3-D2O): δ 1.15 (t,
12H, J ) 7.1 Hz), 1.38 (s, 9H), 2.89-2.92 (m, 2H), 3.27-3.35
(m, 10H), 6.25 (dd, 2H, J ) 2.4, 8.8 Hz), 6.36 (d, 2H, J ) 2.4
Hz), 6.42 (s, 1H), 6.44 (s, 1H), 7.05 (dd. 1H, J ) 2.5, 6.2 Hz),
7.42 (dd, 2H, J ) 3.0, 5.9 Hz), 7.89 (dd, 1H, J ) 3.0, 5.9 Hz).
13C NMR (125 MHz, CDCl3): δ 12.81, 28.65, 38.56, 40.64,
41.17, 44.56, 97.93, 105.18, 108.41, 123.05, 124.02, 128.27,
128.72, 130.73, 132.83, 149.02, 153.38, 154.11, 155.99, 169.63.
Am in es 11a a n d 11b. The Boc-protected amine (1.18 mmol)
was dissolved in cold trifluoroacetic acid (5 mL) and stirred
at room temperature for 3 h. Excess TFA was removed in
vacuo, and crude product was repeatedly treated with dry CH2-
Cl2; the CH2Cl2 was removed under vacuum to remove residual
TFA. The free amine was regenerated from TFA salt by
passing through the weakly basic ion exchange column (Dowex
66) and eluting with methanol. Eluants were collected until
pH 7.0. The free amine was obtained by evaporating the
solvent in vacuo.
1
afford the gray white lipid 1. Yield: 0.165 g (78%). H NMR
(400 MHz, DMSO-d6): δ 1.15-1.24 (m, 14H), 1.33-146 (m,
6H), 1.52-1.56 (m, 2H), 2.04 (t, 2H, J ) 7.4 Hz), 2.14-2.26
(m, 8H), 2.83 (t, 2H, J ) 5.6 Hz), 3.18 (q, 2H, J ) 7.0 Hz),
3.39 (t, 2H, J ) 5.9 Hz), 3.46-3.51 (m, 10H), 5.01 (d, 2H, J )
5.4 Hz), 7.68 (t, 1H, J ) 5.4 Hz), 8.01-8.09 (m, 2H), 8.14 (s,
1H), 8.15 (s, 1H), 8.21-8.31 (m, 4H), 8.36-8.41 (m, 2H). 13C
NMR (100 MHz, DMSO-d6): δ 18.83, 18.87, 25.76, 25.88, 28.27,
28.30, 28.70, 28.76, 28.89, 29.12, 29.14, 29.17, 35.89, 35.93,
39.04, 40.72, 40.95, 53.81, 55.91, 65.93, 69.77, 69.88, 70.06,
70.10, 78.54, 78.56, 123.86, 124.52, 124.63, 125.19, 125.64,
125.73, 126.74, 130.67, 130.88, 131.38, 133.76, 172.56, 172.76,
173.00. Anal. Calcd for C49H63N3O8.C6H6: C, 73.56; H, 7.52;
N, 4.68. Found: C, 73.37; H, 7.61; N, 4.62.
(Tr iflu or om eth yl)cou m a r in Lip id 2. The coupling of
polymerizable acid 8 (0.5 g, 0.75 mmol) and 7-amino-4-
(fluoromethyl)coumarin (0.17 g, 0.75 mmol) was achieved with
BOP reagent (0.33 g, 0.75 mmol) and Et3N (0.4 mL, 2.87 mmol)
using CH3CN-CHCl3 as the solvent mixture (10 mL/10 mL).
The reaction mixture was stirred at room temperature for 15
h. The workup procedure was the same as described for 8.
Crude product was purified by silica gel column chromatog-
raphy (Rf ) 0.4, 2% MeOH in CHCl3) to give a yellow
fluorescent solid. Yield: 0.58 g (87%). 1H NMR (400 MHz,
DMSO-d6): δ 1.14-1.31 (m, 20H), 1.41-1.46 (m, 10H), 2.04
Cou m a r in Am in e 11a . Yellow solid. Yield: 0.38 g (91%).
1H NMR (400 MHz, D2O): δ 3.21 (t, 2H, J ) 8.7 Hz), 3.69 (t,
2H, J ) 8.7 Hz), 7.33-7.41 (m, 2H), 7.67-7.73 (m, 2H), 8.68
(s, 1H). 13C NMR (125 MHz, CD3OD-D2O): δ 41.35, 43.41,
115.08, 118.81, 121.38, 124.68, 129.67, 130.86, 132.70, 133.66,
168.38, 170.04.
1
Rh od a m in e Am in e 11b. Red solid. Yield: 0.9 g (95%). H
NMR (400 MHz, CDCl3-D2O): δ 1.15 (t, 12H, J ) 7.0 Hz),
2.70 (t, 2H, J ) 5.4 Hz), 3.26 (t, 2H, J ) 5.4 Hz), 3.32 (q, 8H,
J ) 7.2 Hz), 6.24 (d, 1H, J ) 2.7 Hz), 6.27 (dd, 1H, J ) 2.7,

3650 J . Org. Chem., Vol. 65, No. 12, 2000
Roy et al.
(t, 2H, J ) 7.6 Hz), 2.25-2.28 (m, 6H), 2.81 (t, 2H, J ) 5.5
Hz), 3.16 (q, 2H, J ) 5.9 Hz), 3.34-3.41 (m, 2H), 3.44-3.49
(m, 6H), 3.53 (s, 4H), 4.05 (q, 4H, J ) 7.1 Hz), 6.45 (s, 1H),
6.50 (s, 1H), 6.54 (s, 1H), 6.65 (d, 1H, J ) 7.6 Hz), 7.36 (d, 1H,
J ) 7.8 Hz), 7.82 (br s, 1H). 13C NMR (100 MHz, CDCl3-CD3-
OD): δ 13.59, 18.65, 24.75, 24.80, 25.76, 28.23, 28.47, 28.55,
28.59, 28.73, 28.77, 28.83, 28.86, 28.94, 29.02, 29.05, 33.69,
33.88, 35.94, 39.10, 50.99, 53.55, 55.60, 65.22, 69.46, 70.00,
70.02, 70.05, 76.73, 99.62, 103.09, 107.16 (q, J ) 6.1 Hz),
123.35 (q, J ) 273.6 Hz), 126.11 (q, J ) 2.4 Hz), 142.09 (q, J
) 31.7 Hz), 154.08, 157.03, 161.19, 171.72.
temperature. Solvent was evaporated in vacauo, and 30 mL
of saturated solution of NaCl was added. A red solid precipi-
tated. The rest of the workup was the same as for 1. The crude
product was purified by silica gel column chromatography with
1
2% MeOH in CHCl3 (Rf ) 0.3). Yield: 0.87 g (83%). H NMR
(400 MHz, CDCl3): δ 1.16-1.35 (m, 22H), 1.42-1.51 (m, 4H),
1.54-1.65 (m, 4H), 2.14-2.24 (m, 8H), 2.97 (t, 2H, J ) 5.5
Hz), 3.41-3.45 (m, 2H), 3.47-3.53 (m, 4H), 3.57 (s, 4H), 3.59-
3.63 (m, 8H), 4.15 (q, 4H, J ) 7.0 Hz), 6.09 (br s, 1H), 6.24 (br
s, 1H), 7.37-7.42 (m, 2H), 7.66-7.71 (m, 2H), 8.89 (s, 1H),
9.05 (br s, 1H). 13C NMR (100 MHz, CDCl3): δ 14.36, 19.26,
19.28, 25.79, 25.97, 28.33, 28.35, 28.38, 28.84, 28.86, 28.88,
29.00, 29.04, 29.28, 29.30, 29.31, 29.36, 36.74, 36.85, 39.22,
39.53, 40.49, 53.61, 56.04, 60.61, 65.33, 70.07, 70.28, 70.54,
77.34, 77.58, 77.60, 116.79, 118.15, 118.64, 125.51, 129.94,
134.46, 154.51, 161.39, 162.92, 171.53, 173.35, 173.72.
The diester (0.36 g, 0.21 mmol) was dissolved in THF (10
mL), and LiOH (0.06 g, 1.44 mmol) in MeOH/H2O (10 mL/2
mL) was added (color changed from yellow to red; again with
stirring, it turned back to yellow). The resulting mixture was
stirred at room temperature for 15 h. After standard workup
(same as lipid 1), a yellow fluorescent solid of lipid 2 was
Selective hydrolysis of the ethyl esters (0.25 g, 0.28 mmol)
was achieved in LiOH solution (0.04 g, 0.95 mmol) in a CH2-
Cl2-MeOH-H2O mixture (5 mL/5 mL/2 mL). Stirring was
continued for 12 h at room temperature. The workup procedure
was the same as that described for 1. Crude product was dried
in vacuo. Product was suspended in water and filtered off to
1
obtained. Yield: 0.28 g (83%). H NMR (400 MHz, DMSO-d6-
D2O): δ 1.15-1.32 (m, 16 H), 1.38-1.51 (m, 8H), 2.05 (t, 2H,
J ) 7.4 Hz), 2.18 (t, 2H, J ) 7.4 Hz), 2.24-2.30 (m, 4H), 3.01
(t, 2H, J ) 4.8 Hz), 3.16 (t, 2H, J ) 5.4 Hz), 3.38 (t, 2H, J )
5.8 Hz), 3.43-3.48 (m, 6H), 3.54 (s, 4H), 6.41(s, 1H), 6.51 (d,
1H, J ) 1.6 Hz), 6.64 (d, 1H, J ) 8.9 Hz), 7.34 (d, 1H, J ) 8.9
Hz). 19F NMR (376 MHz, DMSO-d6): δ 200.15. 13C NMR (100
MHz, CDCl3-CD3OD): δ 18.35, 24.66, 24.77, 25.67, 28.18,
28.50, 28.64, 28.69, 28.74, 28.78, 28.86, 28.89, 28.95, 33.44,
33.58, 35.69, 39.00, 50.64, 54.70, 65.05, 65.92, 69.38, 69.88,
70.03, 76.48, 76.51, 99.32, 103.20, 108.30 (q, J ) 6.1 Hz),
123.48 (q, J ) 264.0 Hz), 126.09 (q, J ) 2.4 Hz), 140.93 (q, J
) 31.7 Hz), 153.57, 157.12, 159.55, 172.34, 173.44. Anal. Calcd
For C42H56F3N3O10: C, 61.53; H, 6.88; N, 5.13. Found: C, 61.37;
H, 6.89; N, 4.88.
1
provide a gray solid. Yield: 0.15 g (64%). H NMR (400 MHz,
CDCl3-CD3OD): δ 1.18-1.32 (m, 16 H), 1.36-1.50 (m, 4H),
1.54-1.60 (m, 4H), 2.11-2.21 (m, 8H), 3.35 (t, 2H, J ) 5.5
Hz), 3.39-3.45 (m, 4H), 3.50-3.57 (m, 4H), 3.58-3.62 (m, 4H),
3.69 (t, 2H, J ) 5.1 Hz), 3.78 (s, 4H), 7.40-7.44 (m, 2H), 7.69-
7.74 (m, 1H), 7.78-7.81 (m, 1H), 8.84 (br s, 1H), 8,82 (s, 1H),
9.13 (br s, 1H). 13C NMR (100 MHz, CDCl3-CD3OD): δ 18.77.
25.75, 28.23, 28.25, 28.65, 28.81, 28.84, 29.03, 29.10, 36.02,
36.14, 38.78, 39.08, 39.17, 39.30, 54.78, 55.93, 65.27, 66.54,
69.60, 69.93, 70.21, 76.94, 76.98, 116.42, 118.07, 118.56,
125.42, 130.02, 134.46, 148.47, 154.50, 161.12, 162.78, 169.38,
175.16, 175.43. Anal. Calcd for C44H62N4O11: C, 64.21; H, 7.59;
N, 6.81. Found: C, 64.06; H, 7.57; N, 6.61.
Rh od a m in e Lip id 5. The coupling reaction between amine
11b (0.34 g, 0.65 mmol) and polymerizable acid 8 (0.43 g, 0.64
mmol) was carried out with BOP reagent (0.29 g, 0.65 mmol)
using Et3N (0.4 mL, 2.8 mmol) as a base and CHCl3 (25 mL)
as the solvent. The reaction mixture was stirred at room
temperature for 20 h. The workup procedure was the same as
that for 8. The crude product was purified with column
chromatography (silica gel, 4% MeOH/CHCl3, Rf ) 0.2) to
afford the pure compound as a pink solid. Yield: 0.58 g (80%).
1H NMR (400 MHz, CDCl3): δ 1.16 (t, 12 H, J ) 7.1 Hz), 1.23-
1.37 (m, 22 H), 1.44-1.51 (m, 4H), 1.53-1.62 (m, 4H), 2.05 (t,
2H, J ) 7.6 Hz), 2.14-2.24 (m, 6H), 2.97 (t, 2H, J ) 5.4 Hz),
3.01-3.05 (m, 2H), 3.27-3.35 (m, 10 H), 3.41-3.45 (m, 2H),
3.52 (t, 2H, J ) 5. 0 Hz), 3.57 (s, 4 H), 3.61-3.65 (m, 6H), 4.15
(q, 4H, J ) 7.0 Hz), 4.71 (br s, 1H), 6.13 (br s, 1H), 6.25 (d,
1H, J ) 2.4 Hz), 6.27 (d, 1H, J ) 2.4 Hz), 6.36 (d, 2H, J ) 2.4
Hz), 6.40 (s, 1H), 6.42 (s, 1H), 6.80 (br s, 1H), 7.05-7.08 (m,
1H), 7.43-7.46 (m, 2H), 7.88-7.90 (m, 1H). 13C NMR (100
MHz, CDCl3): δ 12.67, 14.32, 19.26, 25.75, 25.79, 28.41, 28.88,
28.90, 29.03, 29.29, 29.36, 36.63, 36.68, 36.74, 36.77, 39.09,
39.22, 40.22, 40.51, 40.63, 44.41, 53.64, 55.76, 60.64, 65.32,
65.35, 65.74, 70.17, 70.21, 70.32, 77.30, 77.54, 77.60, 97.85,
104.81, 108.31, 122.84, 123.94, 128.24, 128.46, 130.49, 133.82,
149.00, 153.33, 154.15, 169.99, 171.94, 173.20, 173.42.
Disp er se Or a n ge Lip id 3. Disperse orange (0.36 g, 1.49
mmol) and polymerizable acid 8 (0.8 g, 1.20 mmol) were
dissolved in CHCl3/DMF (20 mL/20 mL) (a portion of the dye
was insoluble), and then BOP reagent (0.53 g, 1.20 mmol) and
Et3N (0.5 mL, 3.6 mmol) were added. The resulting deep red
solution was stirred for 15 h at room temperature. The
insoluble part was filtered off, and the workup procedure was
the same as described for 8. Pure product was obtained by
silica gel column chromatography (2% MeOH/CHCl3, Rf ) 0.6)
to afford the diester as a red semisolid. Yield: 0.46 g (50%).
1H NMR (400 MHz, CDCl3): δ 1.22-1.38 (m, 22H), 1.45-1.54
(m, 6H), 1.58-1.62 (m, 2H), 2.16 (t, 2H, J ) 7.8 Hz), 2.20-
2.24 (m, 6H), 2.97 (t, 2H, J ) 5.4 Hz), 3.41-3.46 (m, 2H), 3.52
(t, 2H, J ) 4.7 Hz), 3.56 (s, 4H), 3.61-3.63 (m, 6H), 4.15 (q,
4H, J ) 7.2 Hz), 6.73-6.76 (m, 2H), 7.84-7.86 (m, 2H), 7.92-
7.95 (m, 2H), 8.05 (s, 1H), 8.07 (s, 1H), 8.31-8.35 (m, 2H). 13
C
NMR (125 MHz, CDCl3): δ 19.41, 25.13, 25.95, 28.51, 28.53,
28.97, 29.02, 29.11, 29.19, 29.29, 29.45, 29.51, 34.30, 36.87,
39.35, 51.71, 51.86, 51.88, 53.87, 55.93, 55.97, 65.44, 65.46,
70.25, 70.38, 70.44, 70.51, 77.72, 77.45, 114.76, 123.04, 124.91,
126.42, 172.08, 173.58, 174.53.
Hydrolysis of the diester (0.18 g, 0.2 mmol) was carried out
with LiOH (0.08 g, 1.9 mmol) in THF/MeOH (3 mL/6 mL) at
room temperature for 12 h as described for lipid 1. Lipid 3
1
was obtained as a deep red semisolid. Yield: 0.14 g (83%). H
NMR (400 MHz, CDCl3-D2O): δ 1.23-1.36 (m, 16H), 1.45-
1.52 (m, 4H), 1.56-1.63 (m, 4H), 2.16-2.24 (m, 6H), 2.29 (t,
2H, J ) 7.5 Hz), 2.97 (t, 2H, J ) 5.2 Hz), 3.41-3.45 (m, 2H),
3.51-3.55 (m 2H), 3.60-3.65 (m, 6H), 3.69 (s, 4H), 6.76 (d,
2H, J ) 8.6 Hz), 7.86 (d, 2H, J ) 8.6 Hz), 7.94 (d, 2H, J ) 8.8
Hz), 8.33 (d, 2 H, J ) 9.2 Hz). 13C NMR (100 MHz, CDCl3-
CD3OD): δ 18.91, 24.70, 25.67, 28.12, 28.14, 28.56, 28.61,
28.70, 28.81, 28.86, 28.89, 29.06, 29.09, 33.91, 36.17, 38.97,
39.00, 51.37, 51.74, 53.79, 55.32, 65.06, 69.64, 69.84, 70.01,
70.19, 77.42, 114.68, 119.67, 122.34, 123.10, 124.48, 124.58,
126.56, 144.59, 147.26, 174.04, 174.69. Anal. Calcd. For
Selective hydrolysis of the ethyl esters (0.35 g, 0.31 mmol)
was performed with LiOH (0.04 g, 0.95 mmol) in THF-MeOH
(4 mL/8 mL). The reaction time and workup were the same as
for lipid 1. Lipid 5 was obtained as a pink solid. Yield: 0.26 g
1
(79%). H NMR (400 MHz, DMSO-d6-D2O): δ 1.05 (t, 12H, J
) 7.0 Hz), 1.14-1.27 (m, 16H), 1.32-1.45 (m, 8H), 1.82 (t, 2H,
J ) 7.4 Hz), 2.03 (t, 2H, J ) 7.3 Hz), 2.15-0.2.25 (m, 4H),
2.81-2.89 (m, 4H), 3.01 (t, 2H, J ) 7.1 Hz), 3.13-3.3.17 (m,
2H), 3.26-3.32 (m, 8H), 3.36 (t, 2H, J ) 5.7 Hz), 3.45 (s, 4H),
3.49-3.53 (m, 6H), 6.32-6.37 (m, 6H), 6.96-7.00 (m, 1H),
7.47-7.52 (m, 2H), 7.76-7.79 (m, 1H). 13C NMR (100 MHz,
CDCl3): δ 11.59, 18.39, 25.44, 25.69, 28.17, 28.22, 28.48, 28.55,
28.71, 28.77, 28.83, 28.91, 29.02, 33.46, 33.63, 35.70, 35.72,
38.16, 39.03, 39.56, 44.19, 44.24, 54.54, 56.23, 65.16, 65.82,
69.42, 69.88, 70.07, 76.58, 97.90, 97.98, 104.77, 104.86, 108.34,
108.41, 122.24, 123.80, 128.23, 128.30, 130.47, 132.91, 148.76,
C
44H60N6O10‚HCl: C, 60.72; H, 7.02; N, 9.66. Found: C, 60.60;
H, 6.85; N, 9.85.
Cou m a r in Lip id 4. To a solution of amine 11a (0.43 g, 1.44
mmol) and polymerizable acid 8 (0.8 g, 1.22 mmol) in CHCl3/
CH3CN (20 mL/20 mL) was added BOP reagent (0.53 g, 1.22
mmol) followed by the addition of Et3N (0.8 mL, 5.7 mmol).
The resulting clear red solution was stirred for 12 h at room

Synthesis of New Fluorescent Metal-Chelating Lipids
J . Org. Chem., Vol. 65, No. 12, 2000 3651
148.82, 148.87, 153.37, 153.41, 153.96, 169.48, 174.67, 174.72,
175.14. Anal. Calcd for C62H87N6O10: C, 69.18; H, 8.15; N, 7.81.
Found: C, 69.06; H, 7.98: N, 7.58.
After extrusion, the clear liposome solution was cooled to
25 °C at 0.5 °C/min. After equilibrating at 25 °C, both solutions
(liposomes with and without metal ions) were centrifuged for
10 min at 3200 rpm. The clear solution was decanted from
sedimented solid and transferred into a quartz reaction
chamber. The gently stirring vesicle solution was polymerized
under a slow stream of nitrogen (15 min of UV irradiation,
450 W) and then stored at room temperature.
Am in e Lip id 13. A solution of the diamine 2,2-(ethylene-
dioxy)bis(ethylamine) (3.25 g, 21.98 mmol) and Et3N (0.3 mL,
2.19 mmol) in CHCl3 was cooled to -15 °C. Another solution
of polymerizable acid 8,10-henecicosadiynedioic acid (0.7 g,
2.19 mmol) and BOP reagent (0.97 g, 2.19 mmol) in CH3CN/
CHCl3 (30 mL/10 mL) was added dropwise over 5 h through a
syringe pump under nitrogen. The resulting solution was
stirred at -15 °C for 6 h and then at room temperature for 12
h. A white precipitate formed. The precipitate was filtered,
and the workup procedure was the same as for lipid 1. Crude
product was purified by silica gel column chromatography (15%
MeOH in CHCl3, Rf ) 0.1). Yield: 0.75 g (76%). Waxy white
F lu or escen ce Sp ectr a . Fluorescence measurements were
performed with a Fluorolog-3 spectrofluorimeter (ISA, J obin-
Yvon-Spex, model FL3-11) equipped with a continuous source
(450 W xenon) for sample excitation. Two single-grating (1200
grooves/mm) spectrometers were used for wavelength selec-
tion. The detector was a photomultiplier tube (Hamamatsu,
model R928) operating at room temperature in a photon-
counting mode. The appropriate software (dM 3000, Spex
Industries, Inc.) was used for automated scanning and lumi-
nescence data acquisition. All luminescence spectra were
uncorrected for instrumental response.
1
solid. H NMR (500 MHz, CDCl3): δ 0.84 (t, 3H, J ) 7.0 Hz),
1.20-1.30 (m, 16H), 1.32-1.39 (m, 4H), 1.45-1.49 (m, 4H),
1.57-1.63 (m, 2H), 1.69 (bs, 2H), 2.14 (t, 2H, J ) 7.5 Hz), 2.19-
2.22 (m, 4H), 3.40-3.44 (m, 2H), 3.49-3.54 (m, 4H), 3.57-
3.61 (m, 4H), 6.30 (s, 1H). 13C NMR (125 MHz, CDCl3): δ 14.33,
19.33, 19.40, 22.88, 25.75, 28.32, 28.54, 28.71, 28.39, 29.06,
29.31, 29.51, 29.68, 29.77, 32.09, 36.76, 39.29, 41.84, 65.37,
65.58, 70.18, 70.29, 70.45, 73.43, 77.47, 77.84, 173.18. Anal.
Calcd for C22H48N2O3: C, 72.28; H, 10.78; N, 6.24. Found: C,
72.30; H, 10.47; N, 6.16.
Quartz cuvettes, 10 mm × 10 mm × 45 mm (Fisher
Scientific Co.), were used in all fluorescence measurements.
No attempts were made to remove oxygen from the analyte
solutions. A flow of dry nitrogenspreviously passed through
a CaSO4 bedswas constantly purged through the sample
compartment of the spectrofluorimeter to minimize possible
oxygen quenching. Before measurement, each sample was
exposed to nitrogen purging in the sample compartment for 2
min. Sample signals were collected at 90° using long pass
filters to eliminate second-order emission from the excitation
monochromator.
Vesicle P r ep a r a tion . Solid phosphocholine 12 (42.5 mg,
85%), amine 13 (2.5 mg, 5%), cationic lipid 14 (2.5 mg, 5%),
and fluorescent lipid (2.5 mg, 5%) were dissolved in 25 mL of
HPLC grade chloroform in a clean 250 mL round-bottom flask.
A thin lipid film was formed inside the flask by slowly
evaporating the solvent in vacuo on the rotary evaporator. The
film was then dried under vacuum for 20 h. This film was
hydrated by 20 mL of HEPES buffer (50 mM, pH 8.0, filtered
through a 0.1 µm filter). The suspension was warmed to 55
°C in a water bath and then frozen in a cooling bath (cooled
with ethylene glycol/dry ice). This freeze-thaw cycle was
repeated six times. For metal loading, after the suspension
was warmed to room temperature, 130 µL of CuCl2 solution
(11.7 mM) was added dropwise in 10 mL of lipid suspension
with continuous mixing in a vortex mixer (∼70% of the total
Cu2+ compared to the metal-chelating lipids). This step was
omitted for liposomes without any metal ions. It was sonicated
by a probe sonicator (power 50 W) under nitrogen for 30 min
at 55 °C, then cooled to 25 °C at a rate of ∼0.25 °C/min, and
equilibrated at room temperature for 1 h. Liposomes were
passed through a 0.5 µm polycarbonate filter (Nucleopore) at
55 °C using an extruder (Lipex Biomembranes Inc., Vancouver,
British Columbia, Canada) under nitrogen pressure at 60 psi
(six times). The temperature of the extruder was maintained
at 55 °C with a circulating water bath, and the collector
temperature was kept at 42 °C.
TEM Sa m p le P r ep a r a tion . One drop of the liposome
solution was placed on a poly-L-lysine-coated (0.01%) grid and
stained with phosphotungstic acid (negative staining). The
liposomes were examined using a J EOL J EM 100CX trans-
mission electron microscope and photographed with Kodak
4489 film (black and white).
Ack n ow led gm en t. This research was supported by
an NSF CAREER award (Grant CHE-9896083) and an
AREA award from NIGMS (NIH, Grant 1R15 59594-
01) to S.M.
Su p p or tin g In for m a tion Ava ila ble: Fluorescence spec-
tra for the lipids 2, 3, 4, and 5, in solution and incorporated
into liposomes, and then polymerized in the absence and
presence of cupric ions. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O991836R