Design and synthesis of molecular umbrellas

Article abstract of DOI:10.1021/ja962405i

This paper describes the design and synthesis of a series of conjugates derived from cholic acid, spermidine, and 5-(dimethylamino)-1-naphthalenesulfonyl (dansyl), which effectively shield the dansyl moiety from water. Direct coupling of cholic acid to both terminal amino groups of spermidine, and attachment of the environmentally-sensitive dansyl moiety to the remaining secondary amine, yields a 'molecular umbrella' (Ia) whose fluorescent properties (λ(max) and emission intensity) reflect a nonpolar microenvironment in water and one that is relatively polar in intermediate dimethoxyethane/water mixtures. Comparison of Ia with analogous 'single-walled' (II) and 'no-walled' (III) umbrellas further indicates that a minimum of two walls is necessary in order to have 'umbrella-like' properties. Examination of the fluorescent properties of a related double-walled umbrella, bearing a flexible (2-hydroxyethyl)carbamate moiety at the C-3 position of the sterol (Ib), reveals that 'umbrella-like' properties are present even when facial amphiphilicity is not rigorously maintained; however, the molecule's ability to shield the fluorophore, as judged by its relative emission intensity, is diminshed. 'Methyl-capping' of the (2-hydroxyethyl)carbamate (i.e., Ic) enhances the umbrella's ability to provide a hydrophobic shelter in water. A tetra-walled analogue of Ia, bearing four cholic acid units (i.e., IV), has been synthesized and its dansyl group found to have reduced exposure toward water. The potential utility of molecular umbrellas in the area of drug delivery is briefly discussed.

Full text of DOI:10.1021/ja962405i

640
J. Am. Chem. Soc. 1997, 119, 640-647
Design and Synthesis of Molecular Umbrellas
Vaclav Janout, Marion Lanier, and Steven L. Regen*
Contribution from the Department of Chemistry and Zettlemoyer Center for Surface Studies,
Lehigh UniVersity, Bethlehem, PennsylVania 18015
X
ReceiVed July 12, 1996. ReVised Manuscript ReceiVed October 8, 1996
Abstract: This paper describes the design and synthesis of a series of conjugates derived from cholic acid, spermidine,
and 5-(dimethylamino)-1-naphthalenesulfonyl (dansyl), which effectively shield the dansyl moiety from water. Direct
coupling of cholic acid to both terminal amino groups of spermidine, and attachment of the environmentally-sensitive
dansyl moiety to the remaining secondary amine, yields a “molecular umbrella” (Ia) whose fluorescent properties
(
λmax and emission intensity) reflect a nonpolar microenvironment in water and one that is relatively polar in
intermediate dimethoxyethane/water mixtures. Comparison of Ia with analogous “single-walled” (II) and “no-walled”
III) umbrellas further indicates that a minimum of two walls is necessary in order to have “umbrella-like” properties.
(
Examination of the fluorescent properties of a related double-walled umbrella, bearing a flexible (2-hydroxyethyl)-
carbamate moiety at the C-3 position of the sterol (Ib), reveals that “umbrella-like” properties are present even when
facial amphiphilicity is not rigorously maintained; however, the molecule’s ability to shield the fluorophore, as judged
by its relative emission intensity, is diminished. “Methyl-capping” of the (2-hydroxyethyl)carbamate (i.e., Ic) enhances
the umbrella’s ability to provide a hydrophobic shelter in water. A tetra-walled analogue of Ia, bearing four cholic
acid units (i.e., IV), has been synthesized and its dansyl group found to have reduced exposure toward water. The
potential utility of molecular umbrellas in the area of drug delivery is briefly discussed.
Introduction
Scheme 1
Biological membranes play an essential role in cells by
serving as selective barriers for transport.1 Although they
readily permit the passage of those molecules and ions that are
necessary for maintaining the living state, they inhibit the entry
of many classes of biologically-active molecules that have
therapeutic potential, especially those that are highly hydrophilic
and/or charged, e.g., antisense oligonucleotides, DNA, proteins,
and certain peptides.2
-7
Finding ways to promote the passive
transport of such agents across cellular membranes represents
a formidable challenge, and one that has significant practical
implications.
maximized and the external face of each wall is hydrated. When
the agent is immersed in a hydrocarbon environment, the
umbrella then favors a shielded conformation such that intramo-
lecular dipole-dipole and hydrogen-bonding interactions are
maximized and the hydrophobic faces are effectively solvated.
Our working hypothesis is that such a molecule will permeate
across a lipid membrane via the following sequence of events:
With this challenge in mind, we have conceived of a novel
class of surfactant molecules that mimic the structure and
function of umbrellas, i.e., molecules that can cover an attached
8
agent and shield it from an incompatible environment. In
essence, our “molecular umbrella” concept may be summarized
as follows: two or more “amphiphilic walls” (i.e., rigid hydro-
carbon units that maintain a hydrophobic and a hydrophilic face)
are coupled to a suitable scaffold that bears a biologically-active
agent (Scheme 1, where the “darkened face” is hydrophobic
and the agent is hydrophilic). When the agent is immersed in
an aqueous environment, a fully-exposed conformation is
favored such that intramolecular hydrophobic interactions are
(i) diffusion of the conjugate to the biomembrane surface in a
fully exposed state, (ii) insertion into the outer monolayer leaflet
by flipping into a shielded state, (iii) diffusion to the inner
monolayer leaflet, and (iv) entry into the cytoplasm via the
sequential reversal of steps ii and i.
In addition to their potential for promoting the transport of
polar agents across cellular membranes, we also envisioned that
molecular umbrellas could improve the water solubility and
stability of hydrophobic drugs of therapeutic interest. Specif-
ically, attachment of a hydrophobic agent to an umbrella should
result in a fully exposed conformation in hydrocarbon medium
and a shielded conformation in an aqueous environment
X
Abstract published in AdVance ACS Abstracts, January 1, 1997.
(1) Gennis, R. B. Biomembranes: Molecular Structure and Function;
Springer-Verlag: New York, 1989.
(
2) Akhtar, S.; Juliano, R. L. Trends Cell Biol. 1992, 2, 139.
(3) Akhtar, S.; Basu, S.; Wickstrom, E.; Juliano, R. L. Nucleic Acids
Res. 1991, 19, 5551.
4) De Mesmaeker, A.; Haner, R.; Martin, P.; Moser, H. E., Acc. Chem.
Res. 1995, 28, 366.
(
(
5) Uhlmann, E.; Peyman, A., Chem. ReV. 1990, 90, 543.
(Scheme 1, where the “darkened face” is hydrophilic and the
(6) Lewis, J. G.; Lin, K. Y.; Kothavale, A.; Flanagan, W. M.; Matteucci,
agent is hydrophobic), i.e., conformational preferences that are
opposite to those of an umbrella-polar agent conjugate. In
principle, one can imagine that a nonpolar cross-linking agent
that is susceptible toward hydrolysis (e.g., chlorambucil) might
exhibit improved stability and solubility in water when seques-
M. D.; DePrince, R. B.; Mook, R. A., Jr.; Hendren, R. W., Wagner, R. W.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3176.
(
7) Burton, P. S.; Conradi, R. A.; Hilgers, A. R. AdV. Drug DeliV. 1991,
, 365.
8) A preliminary report of this work has previously appeared: Janout,
V.; Lanier, M.; Regen, S. L. J. Am. Chem. Soc. 1996, 118, 1573.
7
(
S0002-7863(96)02405-5 CCC: $14.00 © 1997 American Chemical Society

Design and Synthesis of Molecular Umbrellas
J. Am. Chem. Soc., Vol. 119, No. 4, 1997 641
tered within an umbrella.9 Subsequent binding to a biological
membrane would then be expected to release the active agent
by “flipping” from a shielded to a fully exposed state.
A primary aim of the work that is described herein was to
with 10% aqueous HCl. Concentration under reduced pressure and
3 3
purification by column chromatography (silica, CHCl /CH OH, 10/1,
v/v) afforded 489 mg (80%) of the methyl ester of 3-(2-hydroxyethyl)-
1
cholic acid carbamate as a white solid: mp 99-100 °C; R
NMR (CDCl ) δ 0.65 (s, 3 H), 0.76 (s, 3 H), 0.86 (d, 3 H), 0.94-1.87
m, 19 H), 2.15-2.34 (m, 5 H), 3.26 (bs, 3 H), 3.39-3.41 (m, 4 H),
f
) 0.5; H
3
8
demonstrate the feasibility of constructing a molecular umbrella.
(
In addition, we sought to define how the shielding effects of
an umbrella depend upon the number of walls that are present
and also on the wall’s ability to maintain “facial amphiphilic-
3
1
.63 (s, 3 H), 3.81 (br s, 1 H), 3.94 (br s, 1 H), 4.42(m, 1 H), 6.00 (bs,
H); HRMS for (C28H O NNa) calcd 532.3250, found 532.3223.
47 7
+
Selective methyl ester hydrolysis was carried out by dissolving the sterol
(489 mg, 0.9 mmol) in 15 mL of methanol plus 3 mL of a 10% aqueous
1
0,11
ity”.
the environmentally-sensitive fluorophore 5-(dimethylamino)-
-naphthalenesulfonyl (dansyl) as a “mock” agent. Previous
In order to test for umbrella action, we have selected
Na
temperature, concentration under reduced pressure, dissolution in 20
mL of CHCl , washing with aqueous HCl, drying over anhydrous Na
SO , and concentration under reduced pressure afforded crude 1, which
was purified by column chromatography (silica, CHCl /CH OH, 8/1,
v/v). The desired product [0.375 g (79%)] was obtained as a white
2 3
CO and refluxing the mixture for 12 h. Sequential cooling to room
1
3
2
-
studies have demonstrated that increased exposure of the dansyl
group to an aqueous environment leads to a longer λmax and to
a decrease in fluorescence intensity.12 Since the dansyl moiety
is hydrophobic, derivative umbrellas were expected to favor
exposed or fully exposed conformations in solvents of low
polarity (i.e., dimethoxyethane and aqueous solutions that
contain high concentrations of dimethoxyethane) and a shielded
conformation in highly polar solvents (i.e., pure water).
Dimethoxyethane was specifically chosen for this study because
of its high miscibility with water and its low polarity.
4
3
3
1
solid: mp 243-247 °C; R
f
) 0.3; H NMR (CDCl ) δ 0.67 (s, 3 H),
3
0
.89 (s, 3 H), 0.97 (d, 3 H), 1.00-1.95 (m, 22 H), 2.15-2.44 (m, 5
H), 3.62-3.68 (m, 4 H), 3.83 (br s, 1 H), 3.96 (br s, 1 H), 4.43 (m, 1
H), 5.67 (m, 1 H).
3-(2-Methoxyethyl)cholic Acid Carbamate (2). Using procedures
similar to those used to prepare 1, the methyl ester of 2 was first
synthesized (using 2-methoxyethylamine) in 82% yield: mp 74-76
1
°
0
2
C; R
f
) 0.80 (silica, CHCl
3
/CH
3
OH, 9/1, v/v); H NMR (CDCl
3
) δ
.66 (s, 3 H), 0.87 (s, 3 H), 0.96 (d, 3 H), 1.01-1.95 (m, 21 H), 2.15-
Experimental Section
.35 (m, 5 H), 3.30-3.41 (m, 7 H), 3.62 (s, 3 H), 3.78 (br s, 1 H),
+
General Methods. Unless stated otherwise, all reagents and
chemicals were obtained from commercial sources and used without
further purification. Cholic acid and cholic acid methyl ester were
purchased from Sigma Chemical Co. Dansylglycine, monodansylca-
daverine, and acetic acid N-hydroxysuccinimide ester (A-NHS) were
commercially available (Sigma Chem. Co.) and used as obtained. O-(N-
succinimidyl)-N,N,N′N′-tetramethyluronium tetrafluoroborate (TSU),
50 7
3.94 (br s, 1 H), 4.39 (m, 1 H), 5.08 (m, 1 H); HRMS for (C29H O N)
calcd 524.3587, found 524.3558. Mild ester hydrolysis yielded 2
(80%): mp 134-137 °C; R ) 0.55 (silica, CHCl /CH OH, 9/1, v/v);
) δ 0.69 (s, 3 H), 0.89 (s, 3 H), 1.01 (d, 3 H), 1.01-
f
3
3
1
H NMR (CDCl
3
1.95 (m, 21 H), 2.18-2.43 (m, 5 H), 3.32-3.45 (m, 7 H), 3.84 (br s,
1 H), 3.99 (br s, 1 H), 4.46 (m, 1 H), 5.19 (m, 1 H).
1
3
N ,N -Spermidinebis[3-(2-hydroxyethyl)cholic acid carbamate
amide] (3b). To a solution of 0.4 mmol of 2 in 5 mL of anhydrous
DMF was added 0.44 mmol of DIPEA (dropwise addition), followed
by the addition of 0.44 mmol of O-(N-succinimidyl)-N,N,N′N′-
tetramethyluronium tetrafluoroborate (TSU) in one portion. The
resulting solution was then stirred at room temperature for 3 h and
then added directly to a separate solution that was made from 0.19
mmol of spermidine, 0.5 mmol of DIPEA, and 0.5 mL of dry DMF.
After stirring for 12 h at room temperature, the product mixture was
concentrated under reduced pressure and purified by preparative TLC
(silica, CHCl /CH OH/NH OH, 65/25/4, v/v/v) to give 53 mg (27%)
1
-hydroxybenzotriazole (HOBt), and diisopropylethylamine (DIPEA)
were obtained from Aldrich Chemical Co. N-(O-Succinimidyl)choleate
1
3
was prepared using literature procedures. EMS silica Gel 60 was
used for column chromatrography; preparative thin layer chromatograpy
employed EM Science silica gel 60 F-254. Detection by TLC was
2
made by a combination of sulfuric acid 10% in water, I , and UV (254
and 365 nm). House-deionized water was purified using a Millipore
Milli-Q filtering system containing one carbon and two ion-exchange
1
stages. All H NMR spectra were recorded on a Bruker 360 MHz
instrument; chemical shifts are reported in parts per million and were
referenced to residual solvents. High-resolution mass spectra (fast atom
bombardment) were obtained at the Mass Spectrometry Facility of the
University of California, Riverside. Surface tension measurements (23
3
3
4
1
of 3b: mp 195-198 °C; R ) 0.3; H NMR (CD OD) δ 0.68 (s, 6 H),
f
3
0.86 (s, 6 H), 0.97 (d, 6 H), 1.00-2.47 (m, 54 H), 2.63 (mult., 4H),
3.05-3.20 (m, 8 H), 3.52-3.56 (t, 4 H), 3.79 (br s, 2 H), 3.95 (br s,
+
°
C) were made using a tensiometer/microbalance (NIMA, Model
2 H), 4.37 (m, 2 H); HRMS for (C H O N ) calcd 1100.7838, found
6
1
106 12
5
ST9000). Unless noted otherwise, all fluorescence measurements were
made using a Perkin Elmer LS 50 luminescence spectrometer using
1100.7900.
1
3
N ,N -Spermidinebis[3-(2-methoxyethyl)cholic acid carbamate
amide] (3c). Using procedures similar to those used for the preparation
of 3b, 3c was synthesized from 2 in 42% yield: mp 138-139 °C; R
) 0.1 (silica, CHCl /CH OH/NH OH, 65/25/10); H NMR (CD OD)
0
.5 µM solutions of fluorophore. The excitation wavelength and
spectral band width used in all cases were 330 and 7 nm, respectively.
-(2-Hydroxyethyl)cholic Acid Carbamate (1). To a cooled
solution (0 °C) of 500 mg (1.2 mmol) of cholic acid methyl ester in 15
mL of anhydrous CH
Cl was added 4.3 mL of 1.9 M phosgene in
f
1
3
3
3
4
3
δ 0.72 (s, 6 H), 0.94 (s, 6 H), 1.03 (d, 6 H), 1.08-2.26 (m, 54 H), 2.80
(m, 4 H), 3.09-3.45 (m, 18 H), 3.80 (br s, 2 H), 3.96 (br s, 2 H), 4.38
2
2
+
toluene. The mixture was then warmed to room temperature and stirred
for 3 h (selective chloroformylation of the C-3 hydroxyl group shifts
its methine proton from 3.45 to 4.62 ppm). Concentration under
(m, 2 H); HRMS for (C H O N ) calcd 1128.8151, found 1128.8105.
63
110 12
5
1
3
N ,N -Spermidinebis[cholic acid amide] (3a). The N-hydroxysuc-
13
cinimide ester of cholic acid (NHS-DCC method) was isolated prior
to its coupling with spemidine. Reaction of this ester with spermidine
reduced pressure afforded the monochloroformate as a white solid: 1
H
NMR (CDCl
m, 27 H), 3.65 (s, 3 H), 3.85 (br s, 1 H), 3.98 (br s, 1 H), 4.62 (m, 1
H). Dissolution of this sterol in 5 mL of anhydrous CH Cl , followed
3
) δ 0.68 (s, 3 H), 0.89 (s, 3 H), 0.97 (d, 3 H), 1.1-2.4
was carried out in CHCl
to those used for the preparation of 3b, 3a was obtained from cholic
acid in 50% yield: mp 164-166 °C; R 0.69 (silica, CHCl /CH OH/
OD) δ 0.65 (s, 6 H), 0.78 (s,
3 3
/CH CN. Using purification procedures similar
(
2
2
f
3
3
by dropwise addition of dry ethanolamine (8 mmol) and stirring for 3
h at room temperature, afforded a product mixture that was then washed
1
4 3
NH OH, 65/25/10, v/v/v); H NMR (CD
6
H), 0.87 (d, 6 H), 0.85-2.20 (m, 54 H), 2.50 (m, 4 H), 3.09-3.18
(m, 4 H), 3.30 (m, 2 H), 3.70 (br s, 2 H), 3.83 (br s, 2 H); HRMS for
(
9) Cullis, P. M.; Davies, L. M.; Weaver, R. J. Am. Chem. Soc. 1995,
17, 8033.
10) (a) Cheng, Y.; Ho, D. M.; Gottlieb, C. R.; Kahne, D.; Bruck, M. A.
+
(C
55
H
96
O
8
N
3
)
calcd 926.7197, found 926.7163.
1
2
1
3
N -(Dansylglycinamido)-N ,N -Spermidinebis[3-(2-hydroxyethyl)-
(
J. Am. Chem. Soc. 1992, 114, 7319. (b) Venkatesan, P.; Cheng, Y.; Kahne,
D. Ibid. 1994, 116, 6955.
cholic acid carbamate amide] (Ib). To a stirred solution of 92.5 mg
(0.3 mmol) of dansylglycine and 34 mg (0.25 mmol) of HOBt in 2
mL of anhydrous DMF were added 72 mg (0.35 mmol) of DCC at 0
(
11) (a) Stein, T. M.; Gellman, S. H. J. Am. Chem. Soc. 1992, 114, 3943.
b) McQuade, D. T.; Barrett, D. G.; Desper, J. M.; Hayashi, R. K.; Gellman,
S. H. Ibid. 1995, 117, 4862.
(
°
C and, after 15 min, 88 mg (0.08 mmol) of 3b. After stirring for an
additional 20 h at room temperature, the product mixture was
concentrated under reduced pressure, dissolved in 1 mL of methanol,
(
(
12) MacGregor, R. B.; Weber, G. Nature 1986, 319, 70.
13) Okahata, Y.; Ando, R.; Kunitake, T., Bull. Chem. Soc. Jpn. 1979,
5
2, 3647.
and precipitated in 20 mL of a saturated aqueous solution of NaHCO
3
.

642 J. Am. Chem. Soc., Vol. 119, No. 4, 1997
Janout et al.
The precipitate was redissolved in methanol and then precipitated in
pure water. This last step was repeated another time. Purification by
(3/1, v/v) afforded 400 mg (56%) of N-dansyliminodiacetic acid: mp
1
134-137 °C; H NMR (CDCl
3
) δ 2.76 (s, 6 H), 4.16 (s, 4 H), 7.14-
+
preparative TLC (silica, CHCl
.1 mg (9%) of Ib: mp 210-212 °C; R
δ 0.67 (s, 6 H), 0.88 (s, 6 H), 0.99 (d, 6 H), 1.05-2.20 (m, 54 H), 2.85
br s, 6 H), 3.09-3.24 (m, 12 H), 3.55 (t, 4 H), 3.65 (s, 2 H), 3.78 (br
3
/CH
3
OH/H
2
0, 65/25/3, v/v/v) afforded
7.47 (m, 3 H), 8.16-8.45 (m, 3 H); HRMS for (C16
H
18
O
6
N
2
S) calcd
1
8
f
) 0.72; H NMR (CD
3
OD)
366.0886, found 366.0873. To a solution of 18.3 mg (54 µmol) of
N-dansyliminodiacetic acid in 0.5 mL of dry DMF was added 18.7 µL
(110 µmol) of DIPEA and 36 mg (110 µmol) of TSU. After 2 h at
room temperature (the solution becomes bright yellow), 180 µL of
DIPEA and 88 mg (95 µmol) of 3a were added. The mixture was
then stirred for 22 h at room temperature, 15 min at 50 °C, concentrated
under reduced pressure, and purified by preparative TLC (silica gel,
(
s, 2 H), 3.93 (br s, 2 H), 4.32 (m, 2 H), 7.19 (d, 1H), 7.47-7.59 (m,
2
H), 8.16 (d, 1H), 8.30 (d, 1H), 8.51 (d, 1H); HRMS for (C75
H
119
O
15
N
7
-
+
SNa) calcd 1412.8383, found 1412.8458.
2
1
3
N -(Dansylglycinamido)-N ,N -Spermidinebis[cholic acid amide]
Ia). Using procedures similar to those used for the preparation of Ib,
(
CHCl
3 3 2
/CH OH/H O, 105/27/4, v/v/v) to give 51 mg (25%) of IV: mp
1
Ia was synthesized from 3a in 14% yield: mp 228-231 °C; R
f
0.60
213 °C; R 0.65; H NMR (CDCl /CD OD 10/1, C:2.8 mM) δ 0.59
f
3
3
1
(m, 12 H), 0.79-2.06 (m, 130 H), 2.78 (s, 6 H), 3.09-3.31 (m, 16 H),
(
(
(
silica, CHCl /CH OH/H O, 65/25/4, v/v/v); H NMR (CDCl ) δ 0.70
3
3
2
3
s, 6 H), 0.90-2.20 (m, 66 H), 2.88 (s, 6 H), 3.00-3.25 (m, 8 H), 3.47
m, 2 H), 3.81 (m, 2 H), 3.93 (m, 2 H), 4.40 (d, 2 H), 6.20 (t, 1 H),
3.34 (m, 4 H), 3.72 (br s, 4 H), 3.85 (br s, 4 H), 4.38 (s, 4H), 7.10-
+
7.45 (m, 3H), 8.50-8.86 (m, 3H); HRMS for (C126
H
204
O
20
N
N
8
SNa)
8
.
6
.90 (m, 1 H), 7.15-7.66 (m, 3 H), 8.28-8.62 (m, 3H); HRMS for
calcd 2204.4810, found 2204.4954. Anal. Calcd for C126
H
204
O20S 4
+
2
H O: C, 67.11; H, 9.48; N, 4.92. Found: C, 66.91; H, 9.58; N, 4.92.
(C
69
H
110
O
11
N
5
S) calcd 1216.7923, found 1216.7983. Anal. Calcd
S‚2 H O: C, 66.11; H, 9.17; N, 5.59. Found: C,
6.14; H, 8.96; N, 5.65.
for C69
H
109
O
11
N
5
2
Results
6
2
1
3
N -(Dansylglycinamido)-N ,N -Spermidinebis[3-(2-methoxyeth-
Selection of Starting Materials. Starting materials that were
of particular interest to us for constructing molecular umbrellas
were those that are present in mammalian cells, i.e., we sought
materials that would furnish umbrellas that are potentially
biocompatible and biodegradable. Thus, cholic acid and sper-
midine were selected as amphiphilic wall and scaffold material,
respectively. Cholic acid, a major component of bile acids, has
a sterol nucleus that presents a hydroxylated, “water-loving”
(W) face and also a face that is all-hydrocarbon and “oil-loving”
yl)cholic acid carbamate amide] (Ic). Using procedures similar to
those used for the preparation of Ib, Ic was synthesized from 3c in
2
9% yield: mp 210-2 °C; R
f
) 0.83 (silica, CHCl
3 3
/CH OH, 8/2, v/v);
1
H NMR (CD
3
OD) δ 0.65 (s, 6 H), 0.87 (s, 6 H), 0.97 (d, 6 H), 1.08-
2
3
.32 (m, 54 H), 2.78 (s, 6 H), 3.09-3.45 (m, 24 H), 3.79 (br s, 2 H),
.92 (br s, 2 H), 4.40 (m, 2 H), 7.19 (d, 1H), 7.47-7.59 (m, 2H), 8.16
+
(d, 1H), 8.30 (d, 1H), 8.51 (d, 1H); HRMS for (C77
H
123
O
H
15
N
7
SNa)
calcd 1440.8696, found 1440.8770. Anal. Calcd for C77
123
O N S:
15 7
C, 65.18; H, 8.74; N, 6.91. Found: C, 65.42; H, 8.86; N, 6.81.
N-Dansyl, N′-Cholic Acid Amide Cadaverine (II). To a stirred
solution of 67.0 mg (2.0 mmol) of dansylcadaverine in 7 mL of CH
Cl was added 102 mg (2.0 mmol) of N-(O-succinimidyl)choleate in
10,14,15
(O).
Spermidine, which is also found in mammalian cells,
2
-
has terminal amino groups that can be used for wall attachment,
and a secondary amino group can serve as a “handle” for binding
an agent.
2
three equal portions at room temperature. After being stirred for 3 h,
the product mixture was concentrated under reduced pressure and
3 3
purified by preparative TLC (silica, CHCl /CH OH, 8/1, v/v) to give
1
8
0
(
1.0 mg (56%) of II: mp 126-8 °C; R
f
) 0.48; H NMR (CD OD) δ
3
.68 (s, 3 H), 0.88 (s, 3 H), 1.19 (d, 3 H), 1.20-2.22 (m, 33 H), 2.90
s, 6 H), 2.91 (t, 2 H), 3.09 (m, 2 H), 3.41 (br m, 1 H), 3.82 (br s, 1 H),
3
8
7
.95 (br s, 1 H), 5.37 (t, 1 H), 6.05 (t, 1 H), 7.16-7.56 (m, 3 H),
+
.19-8.53 (m, 3 H); HRMS for (C41
H
64
O
6
N
3
S) calcd 726.4516, found
26.4497.
2
1
3
N -(Dansylglycinamido)-N ,N -Spermidinebis(acetamide) (III).
To a stirred solution of 0.25 g (1.72 mmol) of spermidine in 100 mL
of CH Cl was added, dropwise, a solution of 0.55 g (3.18 mmol) of
acetic acid N-hydroxysuccinimide ester (A-NHS) in 30 mL of CH Cl
at 0 °C. After the reaction mixture was stirred for 1 h at 0 °C and for
2 h at room temperature, the solvent was removed under reduced
pressure. Purification by column chromatography (silica, CH OH, NH
OH, 100/8, v/v) afforded 0.10 g (27%) of N ,N -spermidinebis-
Target Molecules. Specific target molecules that were
chosen for this study included three double-walled umbrellas
(Ia,b,c), one single-walled analogue (II), a dansylated spermi-
dine derivative that was devoid of sterol (III), and one tetra-
walled umbrella (IV). A comparison of the fluorescence
properties of Ia, II, and III was expected to verify our
hypothesis that a minimum of two amphiphilic walls is necessary
for effective shielding. A comparison of Ia with Ib was
expected to provide insight into the importance of rigorously
maintaining facial amphilicity with respect to the umbrella’s
ability to provide an effective “cover” for the attached agent.
Thus, in contrast to Ia, which has all three of its hydroxyl groups
firmly positioned on the W face, the hydroxyl group that is
tethered to the C-3 position in Ib can lie on either face of the
2
2
2
2
2
3
4
-
1
3
1
(
2
6
acetamide): H NMR (CDCl
3
) δ 1.48-1.65 (m, 7 H), 1.93 (d, 6 H),
.59 (t, 2 H), 2.66 (t, 2 H), 3.24 (t, 2 H), 3.30 (t, 2 H), 5.91 (br s, 1 H),
.51 (m, 1 H). To a stirred solution of 67 mg (0.35 mmol) of 1,3-
dicyclohexylcarbodiimide in 2 mL of anhydrous THF (cooled to 0 °C)
was added 100 mg (0.32 mmol) of dansylglycine. After the mixture
1
3
was stirred for 15 min, a solution of 70 mg (0.31 mmol) of N ,N -
spermidinebis[acetamide] in 6 mL of THF/ethyl acetate/CHCl (1/1/1,
3
v/v/v) and 2 mg of DMAP was added. After the resulting solution
was stirred at room temperature for 36 h, the solvents were removed
under reduced pressure and the residue then dissolved in 5 mL of
(
(
14) Burrows, C. J.; Saute, R. A. J. Inclusion Phenom. 1987, 5, 117.
15) Hjelmeland, L. M.; Nebert, D. W.; Osborne, J. C. Anal. Biochem,
CHCl
8 mg (31%) of III: R
NMR (CDCl ) δ 1.31-1.52 (m, 6 H), 1.87-1.91 (d, 6 H), 2.84 (s, 6
3
, washed with water, and purified by preparative TLC to afford
1
983, 130, 72.
16) We have found that the fluorescence emission intensities are sensitive
to the onset of micelle formation, but the corresponding λ values are
1
4
f 3 3
0.40 (silica, CHCl /CH OH, 10/1, v/v); H
(
3
max
H), 2.80-2.85 (m, 2 H), 2.90-3.10 (m, 2 H), 3.13-3.31 (m, 4 H), 3.71
not. Thus, a 2-fold increase in the concentration of Ia from 0.5 to 1.0 µM,
which places it just below its cmc of 1.1 µM, results in a doubling of its
fluorescence intensity. In contrast, a 4-fold increase to 2.0 µM leads to a
(
d, 2 H), 5.84-6.30 (m, 3 H), 7.15-7.59 (m, 3 H), 8.19-8.53 (m, 3
+
H); HRMS for (C25
H
38
O
5
N
5
S) calcd 520.2594, found 520.2582.
6.4-fold increase in intensity. The λmax values that were observed at 0.5
Tetra-Walled Umbrella IV. To a stirred solution of 260 mg (1.95
mmol) of iminodiacetic acid in 11 mL of water was added 530 mg (5
mmol) of Na CO , followed by 30 mL of acetone. To the resulting
2 3
solution was added 530 mg (1.97 mmol) of dansylchloride in three
equal portions, and the mixture then stirred at room temperature for
and 2.0 µM, however, were unchanged, i.e., 495.2 ( 0.6 and 495.6 ( 0.3
nm, respectively. Similarly, as the cmc of Ib is exceeded (cmc ) 0.8 µM),
its emission intensity increases disproportionally, i.e., on going from 0.5
µM to 1.0 and 2.0 µM, the fluorescence intensity increased by factors of
4
.3 and 12.7, respectively; the λmax values that were observed at 0.5 and
.0 µM were 493.5 ( 0.5 and 493.5 ( 0.3 nm, respectively. Although we
2
1
4 h. After the solvents were removed under reduced pressure, the
residue was dissolved in 6 mL of 1 M HCl. Subsequent removal of
water under reduced pressure and recrystallization from H O/CH OH
do not yet understand this phenomenon, we suspect that changes in
fluorescent lifetimes and/or aggregation (turbidity) are primary factors that
are responsible for this behavior.
2
3

Design and Synthesis of Molecular Umbrellas
J. Am. Chem. Soc., Vol. 119, No. 4, 1997 643
sterol. A methyl-capped analogue of Ib (i.e., Ic) was also of
interest since it bears the same ethylcarbamate spacer at the
C-3 position but lacks the strongly hydrophilic hydroxyl group;
an increased level of facial amphiphilicity was, therefore,
expected. Finally, the tetra-walled umbrella (IV) was considered
to be a worthy target since the dansyl group may now, in
principle, be enclosed from three and/or four sides; a situation
that could result in even greater shielding efficiency.
Umbrella Synthesis. Schemes 2 and 3 summarize the
synthetic routes that were used to prepare Ia-c, II, and III. In
brief, selective functionalization of cholic acid methyl ester at
the C-3 position using an excess of phosgene, followed by
condensation with 2-hydroxyethylamine and mild ester hydroly-
sis afforded 1; alternative conjugation with 2-methoxyethylamine
and hydrolysis yielded 2. Conversion of 1 and 2 to their
N-hydroxysuccinimide esters, followed by condensation with
the primary amino groups of spermidine (to give 3b and 3c,
respectively) and coupling with dansylglycine, afforded the
requisite double-walled umbrellas Ib,Ic; similar transformations
that were carried out using unmodified cholic acid afforded Ia.
The analogous single-walled umbrella, II, was prepared by
similar methods using monodansylcadaverine as starting mate-
rial. Compound III was synthesized by acetylation of spermi-
dine with acetic acid N-hydroxysuccinimide ester (A-NHS),
followed by condensation with dansylglycine.
The synthetic sequence that was used to prepare the tetra-
walled umbrella is shown in Scheme 4. Thus, dansylation of
iminodiacetic acid, followed by activation with O-(N-succin-
imidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate and cou-
pling with 3a, afforded IV.
Critical Micelle Concentrations. In order to ensure that
shielding effects in pure water reflect the monomeric state of
each umbrella, critical micelle concentrations (cmc’s) were
determined by use of standard surface tension methods. Thus,
plots of the surface tension versus amphiphilic concentrations
yielded cmc values of 1.1, 0.8, 2.1, and 3.3 µM for Ia, Ib, Ic,
and IV, respectively (Figure 1); the cmc of II was 3.0 µM (data
not shown).
Umbrella Properties. The fluorescence emission spectra that
were measured for 0.5 µM solutions of Ia in varying dimethox-
yethane/water mixtures are shown in Figure 2. Incremental
replacement of DME with water resulted in a continuous
decrease in fluorescence intensity until a composition of ca. 10/
90 (DME/water, v/v) was reached (Figure 2A). Further
increases in water content resulted in a significant increase in
emission intensity (Figure 2B). A plot of the relative emission
intensity at λmax for Ia as a function of water content (v/v) is
presented in Figure 2C. The fluorescence intensity that was
measured in pure DME was similar to that which was found in
pure water. The specific value of λmax was also sensitive to

644 J. Am. Chem. Soc., Vol. 119, No. 4, 1997
Janout et al.
Figure 1. Surface tension as a function of umbrella concentration for
Ia (0), Ib (9), Ic (O), and IV (b), where each data point represents
an average of ca. 10 independent measurements.
Scheme 2
Scheme 3
Scheme 4
the solvent compostion that was used. Thus, a maximum value
of 516.1.6 ( 1.6 nm was reached at ca. 20/80 (DME/water,
v/v). In pure water, the λmax sharply decreased to 495.5 ( 0.4
nm; a value that is essentially the same as that which has been
found in hexane (496 nm). In sharp contrast, an incremental
increase in water content for the single-walled analogue, II,
produced a continuous shift in λmax to longer wavelengths and
a continuous decrease in fluorescence intensity oVer the entire
range of solVent mixtures used (Figure 3A); exactly analogous
behavior was observed for III (Figure 3B).
Figure 2. (A) Fluorescence emission spectra of 0.5 µM solutions of
Ia in DME/water (v/v) mixtures: (A) (a) 100/0 (pure DME), (b) 50/
5
0, (c) 20/80, (d)10/90. (B) (a) 10/90, (b) 5/95, (c) 0/100 (pure water).
Introduction of a (hydroxyethyl)carbamate group at the C-3
position of cholic acid component of the umbrella significantly
(C) Plot of relative emission intensity at λmax for Ia as a function of
water content (v/v).

Design and Synthesis of Molecular Umbrellas
J. Am. Chem. Soc., Vol. 119, No. 4, 1997 645
Figure 4. (A) Fluorescence emission spectra of 0.5 µM solutions of
Ib in DME/water (v/v) mixtures: (a) 100/0 (pure DME), (b) 60/40,
(c) 0/100 (pure water), (d) 10/90. For purposes of clarity, not all of the
data are shown. (B) Plot of relative emission intensity at λmax for Ib as
a function of water content (v/v).
water, v/v) was reached (Figure 4); further increases in water
content resulted in only a slight increase in intensity. In pure
water, the emission intensity of Ib was ca. 15% of that which
Figure 3. Fluorescence emission spectra of 0.5 µM solutions in DME/
water (v/v) mixtures of (A) II: (a) 100/0 (pure DME), (b) 80/20, (c)
1
7
was measured in pure DME; values of λmax for Ib that were
measured in pure DME and pure water are listed in Table 1,
along with a maximum value in a 40/60 mixture of DME/water.
The methyl-capped analogue, Ic, also showed a minimum in
fluorescence intensity and a maximum in λmax at intermediate
DME/water mixtures. In pure water, the emission intensity was
significantly greater than that of Ib (Figure 5); also, the λmax
was significantly lower (Table 1).
5
0/50, (d) 20/80, (e) 0/100 (pure water) and (B) III: (a) 100/0 (pure
DME), (b) 90/10, (c) 80/20, (d) 50/50, (e) 20/80, (f) 10/90, (g) 0/100
pure water).
(
Table 1. Values of λmax for Molecular Umbrellas in DME and
Water
umbrella
DME
DME/watera
water
Ia
Ib
Ic
501.0 ( 0.6
497.9 ( 0.4
501.4 ( 0.4
507.1 ( 1.0
516.6 ( 1.6
511.1 ( 0.9
507.5 ( 2.5
516.5 ( 0.8
495.5 (0.4
493.8 ( 0.9
490.7 ( 0.7
492.8 ( 0.2
The fluorescence properties of the tetra-walled umbrella (IV)
were qualitatively similar to those of its double-walled analogue
IV
(Ia), except that its minimum intensity extended over a much
a
Maximum λmax for Ia,b,c and IV occurring at ca. 20/80, 30/70,
(
17) A widely held view in the drug delivery field is that molecular
4
0/60, and 50/50 DME/water (v/v), respectively.
weight is an important determinant of a molecule’s ability to cross a
membrane (e.g., Pidgeon, C.; Ong, S.; Liu, H.; Qiu, X.; Pidgeon, M.;
Dantzig, A. H.; Munroe, J.; Hornback, W. J.; Kasher, J. S.; Glunz, L.;
Szczerba, T. J. Med. Chem. 1995, 38, 590. A comparison of the permeation
properties of single-, double-, and tetra-walled umbrellaconjugates should
either lend support or dispel this notion.
affected its fluorescence properties. Thus, the fluorescence
intensity of Ib was found to continuously decrease with
increasing water content, until a composition of ca. 10/90 (DME/

646 J. Am. Chem. Soc., Vol. 119, No. 4, 1997
Janout et al.
Figure 5. (A) Fluorescence emission spectra of 0.5 µM solutions of
Ic in DME/water (v/v) mixtures: (a) 100/0 (pure DME), (b) 0/100 (pure
water), (c) 60/40, (d) 20/80. For purposes of clarity, not all of the data
are shown. (B) Plot of relative emission intensity at λmax for Ic as a
function of water content (v/v).
Figure 6. (A) Fluorescence emission spectra of 0.5 µM solutions of
IV in DME/water (v/v) mixtures: (a) 0/100 (pure water), (b) 100/0
(pure DME), (c) 22/78, (d) 50/50. For purposes of clarity, not all of
the data are shown. (B) Plot of relative emission intensity at λmax for
IV as a function of water content (v/v).
broader range (Figures 6). In addition, its relative intensity in
pure water was significantly greater, and its λmax was substan-
tially lower than that of Ia (Table 1).
Although Ib also provides a shield for the dansyl group, as
judged by its relatively low λmax value in pure water, its low
emission intensity implies that it is less effective than Ia. The
improvement in shielding efficiency that results from methyl
capping (i.e., Ic), as judged by both its λmax and emission
intensity, is a likely consequence of enhanced facial amphiphi-
licity due to the removal of the highly polar hydroxyl group in
the C-3 position. Finally, a comparison of the λmax and relative
emission intensity that are associated with Ia and IV in pure
water indicates that the tetra-walled umbrella provides a much
more hydrophobic shelter for the fluorophore. The simplest
explanation for this result, we believe, is that IV encloses the
dansyl group from three and/or four sides, thereby further
reducing the exposure of the dansyl group toward water.
Discussion
The fluorescence properties of Ia in varying mixtures of
water/DME provide strong evidence that it acts like a molecular
umbrella. Specifically, the changes in its fluorescence intensity
and λmax, on going from pure DME to intermediate DME/water
mixtures, clearly reflect an increase in the polarity surrounding
the dansyl moiety and the presence of exposed and/or fully-
exposed conformations. The sharp reversal in these spectral
changes that accompanies further increases in water content
demonstrates that Ia functions like an umbrella by providing a
hydrophobic shield over the dansyl moiety. The fact that a
single-walled analogue (II) and a dansylated bis-acetylated
spermidine (III) that is devoid of amphiphilic walls do not show
such a reversal provides strong support for our hypothesis that
a minimum of two umbrella walls is necessary for effectiVe
shielding of an attached agent.
Conclusions
The feasibility of creating molecules that can cover an
attached agent and shield it from an incompatible environment
has been demonstrated through the use of double- and tetra-
walled “molecular umbrellas”. These umbrellas have been

Design and Synthesis of Molecular Umbrellas
J. Am. Chem. Soc., Vol. 119, No. 4, 1997 647
constructed from cholic acid, spermidine, and dansyl chloride.
A minimum of two walls has been found necessary in order to
provide an effective cover for the dansyl moiety; increasing the
number of walls to four enhances its shielding efficiency.
Although the rigorous maintenance of facial amphiphilicity is
not essential in order to observe molecular umbrella-like
properties, its presence does appear to improve its effectiveness.
Studies that are currently in progress are aimed at (i) defining
the interactions of molecular umbrellas with lipid bilayers, (ii)
synthesizing umbrella-drug conjugates of therapeutic interest,
exploring the therapeutic potential of umbrella-drug conjugates
in Vitro. The results of these studies will be reported in due
course.
1
7
Acknowledgment. We are grateful the National Institutes
of Health (PHS Grant GM51814) for support of this research.
Supporting Information Available: 1H NMR spectra for
Ia,b,c and II-IV and a complete set of surface tension
measurements (9 pages). See any current masthead page for
ordering and Internet access instructions.
(
iii) defining the permeation properties of umbrella-drug
conjugates with respect to phospholipid bilayers, and (iv)
JA962405I