Transbilayer MoVement of Molecular Umbrellas
J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5861
aqeuous NaHCO3 and 5 mL of dioxane was added 9-fluorenylmeth-
ylcarbonyl chloride (Fmoc-Cl) (0.90 g, 3.67 mmol) portionwise at room
temperature. The reaction mixture was stirred in a closed flask at room
temperature for 20 min, and the solvent then removed under reduced
pressure (40 °C). The resulting oil was acidified via addition of 20 mL
of 1 M HCl. The colorless solid product was then dissolved in 20 mL
of CHCl3 and washed, sequentially, with 10 mL 1 M HCl (twice) and
10 mL of water (three times). Removal of CHCl3 under reduced pressure
and recrystallization (twice) from 30 mL of CH3OH afforded 0.75 g
In this paper, we present chemical evidence that a tetra-walled
molecular umbrella having a molecular weight of 2146 Da can
move across phospholipid bilayers with a half-life that is <20
min. Such a rate far exceeds that associated with the transbilayer
movement of phospholipids themselves, which have half-lives
that are on the order of days to weeks.6 In a broader context,
our results reported herein cast serious doubt on the notion of
a ca. 750 Da molecular weight cutoff for rapid transbilayer
movementsa notion that has, until now, discouraged the
exploration of large molecules as carriers of drugs for cellular
delivery via passive transport.
1
(58%) of the Fmoc carbamate of iminodiacetic acid, having H NMR
[CD3OD/CDCl3, 2/1 (v/v)] δ 4.10 (d, 4 H). 4.20 (t, 1 H), 4.34 (d, 2 H),
7.27 (t, 2 H), 7.31 (t, 2 H), 7.58 (d, 2 H), 7.74 (d, 2 H).
Fmoc-Protected Tetra-walled Umbrella. To a solution that was
composed of FmocN(CH2CO2H)2 (0.0192 g, 0.054 mmol), diisopro-
pylethylamine (0.0156 g, 0.12 mmol), and 0.3 mL of anhydrous DMF
was added the tetrafluoroborate salt of tetramethyluronium succinimide
(TSU, 0.036 g, 0.19 mmol). The reaction mixture was stirred at room
temperature for 75 min and then diluted with 0.2 mL of DMF and 0.1
mL of diisopropylethylamine, followed by direct addition of N1,N3-
spermidinebis[cholic acid amide] (98.5 g, 0.106 mmol). After the
mixture was stirred for 24 h, the solvents were removed under reduced
pressure (23 °C, 0.5 mmHg). The crude product was dissolved in 1
mL of CH3OH and then precipitated by pouring into 20 mL of saturated
NaHCO3. The precipitate was washed with 10 mL of water (twice)
and purified by preparative thin-layer chromatography [1 mm EM
Science silica, CHCl3/CH3OH/H2O, 65/25/3 (v/v/v)] to give 0.072 g
(69%) of the Fmoc-protected tetra-walled umbrella having Rf 0.75
Experimental Section
General Methods. Unless stated otherwise, all reagents and
chemicals were obtained from commercial sources and used without
1
further purification. All H NMR spectra were recorded on a Bruker
360 MHz instrument; chemical shifts are reported in ppm and were
referenced to residual solvents. The preparation of N1,N3-spermidinebis-
[cholic acid amide], which was required for the synthesis of di- and
tetra-walled umbrellas, has previously been described.1b
N-[O-1,2,3-Benzotriazin-4(3H)one-yl]-3-(2-pyridyldithio)propi-
onate [BPDP]. Procedures that were used for the synthesis of BPDP
were similar to those that have previously been described for the
preparation of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).7
To a solution of 2-carboxyethyl-2-pyridyl disulfide (0.50 g, 2.3 mmol)
in 10 mL of CH2Cl2 was added N-hydroxybenzotriazinone (0.40 g, 2.5
mmol) as a powder. After addition of dicyclohexylcarbodiimide (0.53
g, 2.5 mmol), the resulting solution was stirred for 3 h at room
temperature. Removal of the resulting urea by filtration, followed by
concentration under reduced pressure and recrystallization (two times)
from ethanol afforded 0.3 g (61%) of BPDP having mp 59-61 °C and
1H NMR (CDCl3) δ 3.2 (m, 4 H), 7.1-8.5 (m, 8 H). Anal. Calcd for
C15H12N4O3S2: C, 49.98; H, 3.36; N, 15.56. Found: C, 49.83; H, 3.45;
N, 15.54.
Di-walled Umbrella (1). To a heterogeneous mixture that was
composed of N1,N3-spermidinebis[cholic acid amide] (0.08 g, 0.086
mmol), 2 mL of CH2Cl2, and diisopropylethylamine (0.044 g, 0.34
mmol) was added BPDP (0.032 g, 0.089 mmol). The mixture became
homogeneous within 1 min. After the mixture was stirred for 1 h at
room temperature, the solvent was removed under reduced pressure
and the residue then dissolved in 2 mL of CH3OH. The organic product
was then precipitated by addition to an aqeuous solution that was made
from 20 mL of saturated NaHCO3 and 1 mL of 10% Na2CO3. The
solid was then triturated with 2 × 5 mL of water and purified by
preparative thin-layer chromatography [silica, CHCl3/CH3OH/H2O, 103/
27/3 (v/v/v)] to give 71 mg (73%) of the corresponding di-walled
umbrella bearing a reactive “tag” (i.e.,1), which was characterized by
Rf 0.68 and 1H NMR [CDCl3/CD3OD, 2/1 (v/v)] δ 0.66 (s, 6 H), 0.80-
2.30 (m, 60 H), 2.36 (t, 2 H), 2.80 (t, 2 H), 2.96-3.30 (m, 8 H), 3.35
(s, 2 H), 3.78 (bs, 2 H), 3.92 (bs, 2 H), 7.18 (m, 1 H), 7.75 (m, 2 H),
8.38 (d, 1 H). HRMS for (C63H103N4O9S2)+: calcd, 1123.7167; found,
1123.7165.
1
(Note: the Rf is extremely sensitive to solvent polarity) and H NMR
(CD3OD) δ 0.60 (d, 12 H), 0.63-2.19 (m, 132 H), 2.95-3.25 (m, 16
H), 3.35 (m, 4 H), 3.73 (s, 4 H), 3.86 (s, 4 H), 4.00 (bs, 4 H), 4.14 (d,
2 H), 4.35 (t, 1 H), 7.22-7.71 (m, 8 H).
Tetra-walled Umbrella 2. A solution of the Fmoc-protected tetra-
walled umbrella (150 mg, 0.07 mmol) in 2 mL of CH3OH plus 0.10
mL of piperidine was stirred at 40 °C for 5 h. Removal of solvent
under reduced pressure and purification by column chromatography
[silica, CHCl3/CH3OH/H2O, 103/27/3 (v/v/v)] afforded the correspond-
ing deprotected umbrella (4) having Rf 0.38 (the protected umbrella
has an Rf of 0.70 under undentical TLC conditions) and 1H NMR (CD3-
OD) δ 0.70 (s, 12 H), 0.91-2.30 (m, 132 H), 3.17 (m, 8 H), 3.35 (m,
12 H), 3.54 (bd, 4 H), 3.78 (bs, 4 H), 3.94 (bs, 4 H).
To a heterogeneous mixture of 4 (0.082 g, 0.042 mmol) in 1 mL of
CH2Cl2 and 0.044 g (0.34 mmol) of N,N-diisopropylethylamine was
added a solution of BPDP (0.0152 g, 0.042 mmol) in 0.3 mL of CH2-
Cl2. After addition of 0.15 mL of CH3OH, the reaction mixture became
homogeneous and was stirred for 24 h at room temperature. The solvent
was then removed under reduced pressure, and the residue dissolved
in 2 mL of CH3OH. Subsequent precipitation by addition of 20 mL of
water plus 1 mL of saturated NaHCO3, followed by washing with 5
mL of water (twice) and purification by preparative TLC [silica, CHCl3/
CH3OH/H2O, 65/25/3 (v/v/v)] afforded 62 mg (69%) of the tetrawalled
umbrella bearing a reactive “tag” (i.e., 2), having Rf 0.71 and 1H NMR
(CD3OD) δ 0.69 (s, 12 H), 0.90-2.29 (m, 132 H), 2.70 (t, 2 H), 3.04
(t, 2 H), 3.10-3.30 (m, 20 H), 3.78 (bs, 4 H), 3.94 (s, 4 H), 4.22-4.36
(m, 4 H), 7.21 (m, 1 H), 7.81 (m, 2 H), 8.39 (d, 1 H).
Di-walled Umbrella-Glutathione Conjugate. The di-walled um-
brella 1 (0.071 g, 0.063 mmol) was dissolved in 2.0 mL of CH3OH
and then added to a solution of glutathione (0.0193 g, 0.063 mmol) in
0.4 mL of water. After the mixture was stirred at room temperature
for 24 h under a nitrogen atmosphere, the solvent was then removed
under reduced pressure, and the desired conjugate purified by column
chromatography [silica, CHCl3/CH3OH/H2O, 65/40/10 (v/v/v)] to give
52 mg (63%) of the double-walled umbrella-glutathione conjugate
having Rf 0.48 and 1H NMR (CD3OD) δ 0.67 (s, 6 H), 0.87-2.24 (m,
62 H), 2.73 (m, 2 H), 2.96 (m, 2 H), 3.20 (m, 8 H), 3.30-3.60 (m, 7
H), 3.78 (s, 2 H), 3.93 (s, 2 H), 4.57 (m, 1 H). HRMS for
(C68H115N6O15S2)+: calcd, 1319.7862; found, 1319.7876.
Tetra-walled Umbrella-Glutathione Conjugate. A solution of 2
(0.052 g, 0.024 mmol) in 1 mL of CH3OH was added to a solution of
glutathione (0.0075 g, 0.024 mmol) in 0.1 mL of water, and the mixture
allowed to stir at room temperature for 24 h under a nitrogen
atmosphere. An additional 0.005 g of glutathione was then added, and
the mixture stirred for an additional 48 h. Removal of solvent under
reduced pressure, followed by purification via column chromatography
[silica, CHCl3/CH3OH/H2O, 65/25/3 (v/v/v)] afforded 11 mg (20%) of
the tetra-walled umbrella-glutathione conjugate having Rf 0.32 and
1H NMR (CD3OD) δ 0.70 (s, 12 H), 0.90-2.24 (m, 120 H), 2.58 (m,
2 H), 2.96 (m, 2 H), 3.18 (m, 8 H), 3.30-3.60 (m, 17 H), 3.78 (s, 4
H), 3.92 (s, 4 H), 4.17 (d, 4 H), 4.69 (m, 1 H). HRMS for
(C127H211N10O25S2Na2)+: calcd, 2386.4784; found, 2386.4879.
Fmoc Carbamate of Iminodiacetic Acid. To a solution of imino-
diacetic acid (0.49 g, 3.68 mmol) in a mixture of 4 mL of saturated
Thioether Analogue of 2. To a stirred solution of 4 (80 mg, 0.041
mmol) in 0.5 mL of 2-methyl-1-pyrrolidone was added 3.7 mg of
acryloyl chloride (0.041 mmol). The mixture was stirred at room
(6) Ganong, B. R.; Bell, R. M. Biochemistry 1984, 23, 4977.
(7) Janout, V.; Lanier, M.; Regen, S. L. Tetrahedron Lett. 1999, 40, 1107.