aid of a redox reaction involving ceric ammonium nitrate. The
resulting primary alcohol 4 was then subjected to a Swern
oxidation and converted into a product that gave two spots when
developed with rhodanine. Both products showed the presence
of an aldehyde proton in the NMR (one at 9.70 ppm and the
other at 9.28 ppm in a ratio of 37 and 63 respectively).
Purification of the mixture by chromatography gave the latter
aldehyde whose NMR spectrum indicated the occurrence of a
E1cb-type elimination of a benzoate during the Swern oxida-
tion. In the course of the actual synthesis, however, the aldehyde
mixture (5 and 5A) was used without separation in a Wittig–
Horner reaction to produce two phosphonates (6 and 6A) in 25%
and 40% yields, respectively. At this point, phosphonate 6 was
isolated by chromatography and hydrogenated to 7, a reduction
that both removed the unsaturation and converted the nitro
group into an amino group. Meanwhile, in this convergent
synthesis, chemistry was being performed on the steroidal
portion of the molecule starting with cholesterol 8. Thus,
cholesterol was transformed into a-cholesterylamine 10 using a
literature procedure with only slight modification.8 We now had
in hand the necessary two amines which were joined together
via a succinoyl unit by first acetylating 10 with succinic
anhydride to give 11. Carbodiimide coupling of 11 with 7 then
gave 12 which needed only to be deprotected to produce the
final compound.
Fig. 1 (a) A giant vesicle containing the phosphate counterpart of 14 has
attached itself to a cluster of cancer cells. (b) The vesicle is slowly pulled
away from the cells with a micropipet. A fiber (arrow) forms between the
vesicle and cells. Bar = 20 mm.
Deprotection was accomplished by first debenzoylating 12
with methanolic ammonia. The phosphonate diester 13 was
used to obtain the disodium phosphonate salt with the aid of
trimethylsilylbromide; purification of 14 was performed by
chromatography on silica gel. An overall yield of only 2.6% is
indicative, in part, of the difficulties in preparing and purifying
this large and bipolar molecule.
Fig. 2 A giant vesicle containing adhesive 14 and attached to a cancer cell
showing a sphere-to-ellipsoid distortion upon application of a lateral force.
Bar = 20 mm.
Microscopically visible giant vesicles, comprised of 1-palmi-
toyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), com-
pound 14 or its phosphate counterpart,9 and 1-palmitoyl-
2-oleoyl-3-phosphoglycerol (POPG) were prepared in Hepes
solution (1 mM) by the hydration method,7 using ratios from
87+5+8 to 75+17+8, respectively. An aliquot of the vesicle
suspension was added to a petri dish containing MCF-7 breast
cancer cells (half-confluent) in Dulbecco’s phosphate buffer
solution (DPBS, Sigma). The petri dish was gently swirled and
placed under a light microscope. Giant vesicles adjacent to
cancer cells were grasped with a micropipet under suction and
slowly withdrawn from the cells. The majority of the giant
vesicles containing 14 or its phosphate counterpart failed to
release the cells but continued to stick to them. When such
vesicles were pulled away from the cancer cells, either the cell
(Fig. 1) or the vesicle (Fig. 2) was substantially distorted. Fig. 1
shows how a withdrawn vesicle retains its contact with the
cancer cell via an elongated fiber originating from the cancer
cell. Fig. 2 shows how a spherical giant vesicle, attached to a
cancer cell via 14, deforms into an ellipsoid upon application of
a lateral force. Control runs, carried out with identical vesicles
except that their bilayers possessed a non-adhesive steroid, the
succinate derivative of cholesterylamine, failed to produce a
similar effect in the majority of cases. These experiments
suggest that 14 does indeed serve as a chemical adhesive.
Submicroscopic vesicles (100 nm in diameter) were prepared
by hydration of a film of POPC–14–POPG mixture with a 0.96
mM solution of a water-soluble fluorescent dye (Lucifer Yellow
CH, dipotassium salt), followed by 19 extrusions through a 100
nm polycarbonate filter. Elution through a Sephadex G-50
column removed all dye not bound in or on the small vesicles.
The fluorescent vesicles were then added to a cancer cell culture
in medium and allowed to sit for 40 min. At the end of this
period, medium and unbound vesicles were washed away with
DPBS, and epifluorescence microscope pictures were taken of
the cancer cells (Fig. 3). The cancer cells are seen to display a
fluorescence consistent with surface binding of the fluorescent
vesicles. As a control experiment, the chemical adhesive 14 was
omitted from the vesicles, and the cancer cells failed to display
fluorescence. This again suggests the efficacy of the chemical
linkage between vesicle and cell. Definite experiments includ-
Fig. 3 (a) Phase contrast image of MCF-7 cells that have been incubated
with submicroscopic vesicles. The vesicles contain adhesive 14 within their
membranes and Lucifer Yellow CH in their aqueous interior. (b)
Fluorescence image of the same cluster showing fluorescence-labeled cells.
Bar = 25 mm.
ing flow cytometry are being planned. Yet even at this stage of
our work the steroidal phosphonate appears to be a promising
tool in selective targeting of breast cancer cells.
This work was supported by grant GM21457 from the
National Institutes of Health to F. M. Menger.
Notes and references
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86
Chem. Commun., 2001, 85–86