Host-[2]rotaxanes as cellular transport agents

Article abstract of DOI:10.1021/ja034918c

Host-[2]rotaxanes, containing a diarginine-derivatized dibenzo-24-crown-8 (DB24C8) ether as the ring and a cyclophane pocket or an aromatic cleft as one blocking group, are cell transport agents. These hosts strongly associate with a variety of amino acid

Full text of DOI:10.1021/ja034918c

Published on Web 06/17/2003
Host-[2]Rotaxanes as Cellular Transport Agents
Vadims Dvornikovs,† Brian E. House,† Marcia Kaetzel,‡ John R. Dedman,‡ and
David B. Smithrud*,†
Contribution from the Department of Chemistry, UniVersity of Cincinnati,
Cincinnati, Ohio 45221, and the Department of Genome Science,
UniVersity of Cincinnati, Cincinnati, Ohio 45267
Received February 28, 2003; E-mail: david.smithrud@uc.edu
Abstract: Host-[2]rotaxanes, containing a diarginine-derivatized dibenzo-24-crown-8 (DB24C8) ether as
the ring and a cyclophane pocket or an aromatic cleft as one blocking group, are cell transport agents.
These hosts strongly associate with a variety of amino acids, dipeptides, and fluorophores in water (1 mM
phosphate buffer, pH 7.0), DMSO, and a 75/25 (v/v) buffer to DMSO solution. All peptidic guests in all
solvent systems have association constants (KA’s) in the range of 1 × 104 to 5 × 104 M-1, whereas the KA
range for the fluorophores is 1 × 104 to 9 × 105 M-1. Association constants for the cyclophane itself,
cyclophane 3, are smaller. These values are in the 1 × 103 to 5 × 103 M-1 range, which shows that the
rotaxane architecture is advantageous for guest binding. Cyclophane-[2]rotaxane 1 efficiently transports
fluorescein and a fluorescein-protein kinase C (PKC) inhibitor into eukaryotic COS-7 cells, including the
nucleus. Interestingly, cleft-[2]rotaxane 2 does not transport fluorescein as efficiently, even though the
results from the fluorescence assays show that both [2]rotaxanes bind fluorescein with the same ability.
Introduction
polymeric pockets are sprinkled with various functional groups,
which are locked in a programmed position to strongly associate
The goals of protein mimicry include identifying the structural
elements that give proteins their function and incorporating their
essential features into synthetic systems to obtain novel recep-
tors. Extensive research in molecular recognition chemistry has
produced compounds that resemble the structures of protein
binding domains by positioning amino acids or peptides around
a hydrophobic pocket.1-5 The attachment of peptide loops onto
synthetic scaffolds has impressively provided mimetics that
recognize large patches on protein surfaces.6-8 Molecularly
imprinted polymers have demonstrated the importance of a
convergent arrangement of recognition elements.9-15 These
with the template used to form the pocket or other guests with
similar physical properties. Our approach to creating mimics
of protein binding domains is to use the rotaxane architecture
to arrange multiple functional groups in a convergent fashion.
Now that several methods can be used to construct mimetic
pockets, the next challenge facing chemists is the creation of
synthetic compounds that provide protein-like function. One
important function, highlighted in this paper, is cell transport.16
Cell transport agents need to bind a guest strongly in the
different environments experienced in cells and be soluble in
these environments. The agent needs to “cover” any feature of
a guest, such as an anionic charge, that prevents membrane
passage. In this report we show that host-[2]rotaxanes can
satisfy these requirements. The long-term goal of this research
is to obtain mimetics that operate in the various biological
milieus, including those found within cells.
We have shown that a host-[2]rotaxane, containing an
arginine-derivatized DB24C8 ring and a calix[4]arene, binds a
variety of guests (with a preference for aromatic carboxylates)
in DMSO, water, and mixed solvent systems with large
association constants.17 One unique feature of the rotaxane
architecture for pocket formation is that the ring, which carries
the recognition elements, can adjust to a guest by changing its
position on the axle to maximize the favorable binding free
energy.18 The aromatic rings and polar recognition elements of
the DB24C8 ring can both contribute to guest recognition, and
* To whom correspondence should be addressed.
† Department of Chemistry.
‡ Department of Genome Science.
(1) Grawe, T.; Schrader, T.; Finocchiaro, P.; Consiglio, G.; Failla, S. Org.
Lett. 2001, 3, 1597-1600.
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2000, 19, 3155-3184.
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Schmidt; Mutter, M. J. Pept. Sci. 1999, 5, 313-322.
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J. AM. CHEM. SOC. 2003, 125, 8290-8301
10.1021/ja034918c CCC: $25.00 © 2003 American Chemical Society

Host−[2]Rotaxanes as Cellular Transport Agents
A R T I C L E S
the importance of an interaction depends on the nature of the
guest and the environment. Considering these features, we
predicted that host-rotaxanes would be able to transport guests
through cellular membranes. We envisioned that in an aqueous
environment the pocket would contract, bringing the aromatic
moieties of the ring closer to an encapsulated guest. In apolar
environments, such as a membrane, the pocket would expand,
allowing salt bridges and H-bonds to form between the guest
and the recognition elements of the ring. In this paper, we
compare the ability of cyclophane-[2]rotaxane 1 and cleft-
[2]rotaxane 2 to bind various guests and transport fluorescein
and show that, although there was not a demonstrated advantage
for guest recognition in the fluorescence assays, there is an
advantage afforded a cyclophane pocket for fluorescein transport
into a cell. A comparison is also made to cyclophane 3 to show
that hosts built on the [2]rotaxane architecture can be better for
guest association. Cyclophane 3 could not be tested as a transport
agent because of its poor water solubility.
contains half of the cyclophane of [2]rotaxane 1 with additional
benzylic groups to give it a greater aromatic surface area (Chart
1). The DB24C8 ring used in these studies is similar to the one
used in previous studies,17,18 except that the arginine recognition
elements are fully deprotected.
Construction of host-[2]rotaxanes 1 and 2 is based on the
DCC-[2]rotaxane method, which involves a reaction between
an amine with a DCC-[2]rotaxane.35 The piperidinylamine of
the Diederich cyclophane initially appeared to be a convenient
site for the attachment of an alkylamine chain to react with the
DCC-[2]rotaxane. However, molecular modeling results sug-
gested that the depth of the ring could prevent the arginine
residue on the ring from reaching the pocket or cleft. Therefore,
one piperidine ring was replaced with an alkylamine. The
methoxy groups were also eliminated from the same side of
the hosts to reduce possible steric congestion with the ring’s
arginine residues.
Synthesis of the [2]Rotaxanes. Because synthetic routes have
already been developed to construct the cyclophane portion of
the host-[2]rotaxanes,36 new methods were only needed to
attach the axle to the cyclophane and cleft. 4,4′-Dihydroxyben-
zophenone 4 was chosen as the commercially available precursor
of the derivatized end of the hosts (Scheme 1). The phenolic
oxygen atoms were protected as benzyl ethers, and the carbonyl
group was reduced to give the diphenylcarbinol 5. Formation
of a carbocation, through dehydration of the secondary dibenzyl
alcohol, was accomplished upon exposure to trifluoroacetic acid
(TFA). Allyltrimethylsilane was added to react with the car-
bocation to form a C-C bond; however, the initial product is
ether 6, which exists in equilibrium with the carbocation. After
extended reaction times (24 h), the carbocation is fully consumed
through the irreversible reaction with allyltrimethylsilane to give
cleft 7. Conventional deprotection of the benzylic groups with
H2/Pd was not used, because the olefin would be reduced as
well. Instead, the single-electron reducing agent lithium di-tert-
butylbiphenylide was used to remove the benzylic groups in
high yields. However, complete deprotection required as many
as 16 equiv of this expensive reagent. Later, we found that
employing boron trichloride is a more economical way to
deprotect cleft 7, albeit in a slightly lower yield. Bisphenol 8
was coupled with dibromide 9 to furnish cyclophane 10 in a
16-20% yield, which is typical for this type of macrocycliza-
tion.20 Hydroboration of the olefin gave alcohol 11, which was
brominated by treating the alcohol with carbon tetrabromide
and triphenylphosphine. The resulting bromide 12 was displaced
with azide to give cyclophane 13, and the azidoalkane was
reduced with H2/Pd on carbon to give aminocyclophane 14. A
similar synthetic route was used to construct the cleft framework
(compounds 4-8, 15, 17-19, Scheme 1).
Results
Design of the Host-[2]Rotaxanes. To further test rotaxanes
as hosts and transporters, two new host-[2]rotaxanes were
created by combining a cyclophane or an aromatic cleft with
an arginine-derivatized DB24C8 ring. Association constants
were derived for the hosts bound to amino acids, dipeptides,
and various fluorophores in water, DMSO, and a 75/25 (v/v)
water to DMSO mixture. These solvents were chosen to
represent the various biological milieus. The guests presented
a wide range of functional groups for recognition, including
mono- and dicarboxylates, amides, aromatic rings, and aliphatic
moieties. Guests containing dicarboxylates were tested to
determine whether both positively charged groups of the ring’s
recognition elements (guanidinium and ammonium ions) could
form noncovalent bonds. To measure [2]rotaxanes’ abilities to
associate with aliphatic moieties, dipeptides containing Ala, Leu,
and Ile were used as guests. Cellular transport was investigated
with fluorescein and a fluorescein-PKC inhibitor conjugate19
as the guests, because fluorescein is strongly bound by the [2]-
rotaxanes and its location within a cell can be easily monitored
through fluorescence microscopy.
A cyclophane-pocket was chosen as a blocking group for one
[2]rotaxane, because cyclophanes usually interact strongly with
aromatic guests, especially in aqueous solutions through the
hydrophobic effect.20 Moreover, their rigid, preformed pockets
contribute favorable binding energy by reducing the desolvation
and entropic penalty that can occur upon complexation.21-25
The chosen cyclophane is similar to ones developed by
Diederich, which bind mono and para-disubstituted aromatic
rings in water with KA values in the 103 M-1 range.20 Clefts
have also been used as hosts,26-34 and cleft-[2]rotaxane 2
(19) Chen, C. S.; Poenie, M. J. Biol. Chem. 1993, 268, 15812-15822.
(20) Diederich, F. In Cyclophanes, Monographs in Supramolecular Chemistry;
Stoddart, J. F., Ed.; The Royal Society of Chemistry: London, 1991.
(21) Cram, D. J. Angew. Chem., Int. Ed. 1986, 25, 1039-1134.
(22) Diederich, F.; Smithrud, D. B.; Sanford, E. M.; Wyman, T. B.; Ferguson,
S. B.; Carcanague, D. R.; Chao, I.; Houk, K. N. Acta Chem. Scand. 1992,
46, 205-215.
(27) Tamaru, S.; Shinkai, S.; Khasanov, A. B.; Bell, T. W. Proc. Natl. Acad.,
U.S.A. 2002, 99, 4972-4976.
(28) Shizuma, M.; Kadoya, Y.; Takai, Y.; Imamura, H.; Yamada, H.; Takeda,
T.; Arakawa, R.; Takahashi, S.; Sawada, M. J. Org. Chem. 2002, 67, 4795-
4807.
(29) Jansen, R. J.; de Gelder, R.; Rowan, A. E.; Scheeren, H. W.; Nolte, R. J.
M. J. Org. Chem. 2001, 66, 2643-2653.
(23) Smithrud, D. B.; Wyman, T. B.; Diederich, F. J. Am. Chem. Soc. 1991,
113, 5420-5426.
(30) Isaacs, L.; Witt, D.; Lagona, J. Org. Lett. 2001, 3, 3221-3224.
(31) Sebo, L.; Schweizer, B.; Diederich, F. HelV. Chim. Acta 2000, 83, 80-92.
(32) Potluri, V. K.; Maitra, U. J. Org. Chem. 2000, 65, 7764-7769.
(33) Hernandez, J. V.; Almaraz, M.; Raposo, C.; Martin, M.; Lithgow, A.; Crego,
M.; Caballero, C.; Moran, J. R. Tetrahedron Lett. 1998, 39, 7401-7404.
(34) Murray, B. A.; Whelan, G. S. Pure Appl. Chem. 1996, 68, 1561-1567.
(35) Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486.
(36) Krieger, C.; Diederich, F. Chem. Ber. 1985, 118, 3620-3631.
(24) Smithrud, D. B.; Sanford, E. M.; Chao, I.; Ferguson, S. B.; Carcanague,
D. R.; Evanseck, J. D.; Houk, K. N.; Diederich, F. Pure Appl. Chem. 1990,
62, 2227-2236.
(25) Smithrud, D. B.; Diederich, F. J. Am. Chem. Soc. 1990, 112, 339-343.
(26) Goshe, A. J.; Steele, I. M.; Ceccarelli, C.; Rheingold, A. L.; Bosnich, B.
Proc. Natl. Acad., U.S.A. 2002, 99, 4823-4829.
9
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A R T I C L E S
Dvornikovs et al.
Chart 1
An alternative route was developed that gave higher yields
of cyclophane. The primary alcohol was created on the cleft
(15) prior to macrocyclization. This approach allows the more
routine H2/Pd reduction method to be used to remove the benzyl
groups, which gives bisphenol 16. By using bisphenol 16, the
yield of macrocyclization was improved as well from 16-20%
to 25-30%. These reactions were performed several times.
Formation of the [2]rotaxanes was accomplished using DCC-
[2]rotaxane 2018 (Scheme 2), which exists as a mixture of syn
and anti constitutional isomers; the syn isomer is shown
throughout the text. The addition of DCC-[2]rotaxane 20 to
cleft 19 and to cyclophane 14 resulted in good yields of [2]-
rotaxanes 24 and 21, respectively. Formation of the [2]rotaxanes
removed readily using a 1:1 ratio of trifluoroacetic and acetic
acids (or a 1:1:1 TFA/CH3CO2H/CH2Cl2 solution). The desired
[2]rotaxanes 1 and 2, each bearing five positive charges, were
purified on silica gel (CH2Cl2/MeOH/TFA).
Determining Acidity Constants. An accurate comparison
of association constants of molecular complexes requires
knowledge of the ionization states of the components, which
requires deriving the acidity constants (pKa’s) of acidic func-
tional groups. This is especially important in DMSO and DMSO/
water mixtures, wherein carboxylic acids can become less acidic
than alkylammonium ions. The pKa values were derived through
potentiometric titrations and used to set the pH of the buffered
assay solutions to a value that minimized the amount of proton
exchange.
1
can be conveniently followed by H NMR spectroscopy. The
key signal is the approximately 0.4 ppm downfield shift of the
benzylic protons of the axle, which occurs upon threading
(Figure 1). Removal of the ring’s Boc protecting groups
proceeded smoothly with TFA in CH2Cl2. Fully Boc protected
arginines were added to the ring through DCC coupling with a
catalytic amount of HOBt. In a previous paper,18 we mentioned
that (Boc)3-Arg should not be used in the construction of [2]-
rotaxanes, because removing all six protecting groups is difficult.
However, we subsequently found that the Boc groups can be
The pKa values of host-[2]rotaxane 1 were derived, and we
assume that the pKa values of host-rotaxanes 1 and 2 are equal.
For potentiometric titrations in DMSO, the plot of [2]rotaxane
1 showed only two end points with the addition of over 3 equiv
of base. The first pKa (8.1 ( 0.2) was assigned to both
ammonium ions of the ring’s side chains, and pKa2 (9.5 ( 0.2)
was assigned to the ammonium ion in the axle. We have
previously found that the axle’s ammonium ion generally has a
large pKa value.18 This reduction in acidity is apparently caused
9
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Host−[2]Rotaxanes as Cellular Transport Agents
A R T I C L E S
Scheme 1
by favorable interactions between the oxygen atoms of the ring
and the ammonium ion. Because of the limited solubility of
[2]rotaxane 1 (and [2]rotaxane 2) in a 75/25 (v/v) water to
DMSO solution, (the assay solution was equal to or greater than
1 mM), a direct measure of their pKa values could not be easily
obtained. Thus, a suitable compound was needed to obtain
representative pKa’s. Di-Arg-DB24C8 was not chosen, because
intermolecular or intramolecular interactions could exist between
the ammonium ion and the electron-rich cavity of the crown
ether, resulting in an unrepresentative pKa. Instead, arginine
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A R T I C L E S
Dvornikovs et al.
Scheme 2
amide was chosen to represent the [2]rotaxanes, which has a
pKa of 7.3 ( 0.1. As discussed above, the pKa of the axle’s
ammonium ion is usually greater than that of a free amine, so
it is assigned a pKa value greater than 7.3.
a guest is shown in Figure 2. The cyclophane pocket is large
enough to accommodate a single side chain (in Figure 2, Leu
is positioned within the pocket) and one Arg moiety of the ring
forms a salt bridge with the carboxyl terminus of the dipeptide.
We should note that the same complex energies were obtained
for rotaxanes having either isomer of the ring, i.e., the arginine
moieties positioned syn or anti to each other.
For studies in DMSO, amino acids containing carboxylic
acids were converted to carboxylates by adding an equal molar
amount of Me4NOH to the stock solutions. Fluorescein and the
fluorescein-PKC conjugate were used as the disodium salts,
and 1,5-DNS was investigated as the sodium chloride salt. [2]-
Rotaxanes 1,2 and cyclophane 3 were onium salts with
trifluoroacetate counterions. The pKa values of guests and hosts
are close in DMSO, and thus, a small percentage of the
components may have undergone proton exchange upon mixing.
Association constants were also derived for complexes formed
in aqueous solutions. These solutions were buffered at pH values
that ensured that the carboxylic acids remain deprotonated and
the [2]rotaxane-amines remain protonated during association.
Thus, according to the derived pKa values, the 75/25 (v/v) water
N-Ac-Trp-Asp was chosen as the representative guest for pKa
determination. Their carboxylates, being close together, may
interact in DMSO, giving them a second pKa value that is larger
than the other carboxylic guests. Thus, we expect that the pKa
values of the other carboxylic guests to lie within the pKa range
of N-Ac-Trp-Asp. For N-Ac-Trp-Asp, a single end point was
observed in the potentiometric titration plot with the addition
of over 2 equiv of base. Apparently, the carboxylates do not
interact significantly, resulting in only a single pKa of 7.5 (
0.1. A single end point was also observed for N-Ac-Trp-Asp in
a 75/25 (v/v) water to DMSO solution, which resulted in a pKa
of 4.8 ( 0.1. For both N-Ac-Trp-Asp and arginine amide in
98% water, pKa values were assumed to be equal to their
corresponding values obtained in 100% water.
Determining Association Constants. Association constants
were derived for complexes formed between various rotaxanes
and guests (Table 1). Fluorescence quenching assays were
performed in the DMSO, 75% water, and 98% water solutions.37
A typical calculated structure of a [2]rotaxane 1 associated with
(37) For a representative titration plot, see ref 17, Figure 3 for cyclophane 2.
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Host−[2]Rotaxanes as Cellular Transport Agents
A R T I C L E S
Table 1. Association Constants of Various Host-Guest
Complexes Measured at 25 °Ca
%H Oina
2
KA × 10-4 (M-1) for host−guest complexes
H O/DMSO
2
guest
Ac-Trp
solution
1
2
3
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
98
75
0
2.9
2.1
2.5
2.1
2.4
1.6
2.3
3.0
1.7
2.1
2.0
2.3
1.2
1.8
2.0
1.3
2.0
1.8
2.1
2.0
2.9
2.2
1.9
3.5
2.0
2.2
2.7
2.2
2.0
2.6
6.6
3.1
2.4
2.1
2.2
4.8
1.8
1.7
2.2
2.3
1.6
1.5
1.5
2.0
1.3
1.6
2.4
1.5
1.9
2.0
2.6
2.2
2.5
2.2
1.8
2.5
2.8
1.8
2.2
2.2
1.9
2.4
5.3
2.8
NSb
0.6
0.1
NS
0.4
0.5
NS
0.6
0.5
NS
0.5
0.5
NS
0.5
0.4
NS
0.5
0.5
NS
0.4
0.4
NS
0.4
0.5
NS
0.5
0.6
NS
0.4
0.5
NS
0.9
0.5
NS
0.1
0.4
NS
0.6
0.4
Ac-Trp-NH2
indole
In(CH2)2CO2Hc
Ac-Trp-Gly
Ac-Trp-Glu
Ac-Trp-Asp
Ac-Trp-Ala
Ac-Trp-Leu
Ac-Trp-Ile
fluorescein
pyrene
86
77
8.0
2.7
2.2
3.4
2.3
2.0
4.1
1.8
1.7
5.8
3.9
1.5
1,5-DNSd
98
75
0
a Determined from analysis of fluorescence spectra, water solutions were
buffered with 1 mM phosphate (pH 7.0) for the 98% water studies and 0.1
mM phosphate (pH 6.5) for the 75% water studies. Standard deviations are
less than 5% of the reported values, except for the fluorescein study in 0%
buffer and studies of host 3, where the standard deviations are less than
10%. b NS means not soluble. c 3-Indolepropionic acid. d 1-(Dimethyl-
amino)-5-naphthalenesulfonate.
compared to other large cyclophanes, is an important feature
for cell transport agents.
Figure 1. Stacked 1H NMR plots demonstrate the large downfield shift of
the benzylic protons of the axle (indicated by arrow) that occurs when the
crown ether is threaded onto the axle.
Cellular Transport Studies. The demonstrated ability of the
[2]rotaxanes to form tight complexes in a variety of solvents
suggested to us that they could act as artificial transporters by
carrying a guest through the many environments encountered
during cell penetration. Fluorescein was chosen as the guest to
test this hypothesis, because it is not cell permeable in the
anionic state and its strong fluorescence makes monitoring its
cellular location convenient. COS-7 cells were plated on slides,
placed in 1 mL of buffer (1.0 mM phosphate, pH 7.0), and
exposed to fluorescein or mixtures of fluorescein and [2]-
rotaxanes 1 or 2. As expected, fluorescein does not noticeably
penetrate the cells (Figure 3A). Fluorescein, however, is
noticeably transported when the cells are exposed for ca. 30
min to the same concentration of fluorescein (0.4 µM) in the
presence of 6 µM of [2]rotaxane 1 (Figure 3B). [2]rotaxane 2
at 6 µM did not noticeably transport fluorescein; however,
increasing its concentration to 60 µM did result in observable
to DMSO solution was buffered with 0.1 mM phosphate at pH
6.5. In this solution, a small amount of precipitate formed at
the highest concentration of cyclophane 3 used in the assays.
The titration curves, however, look consistent throughout the
concentration range, and the derived association constants have
reasonably low standard deviations. For binding assays per-
formed in the 98% water solution, the water was buffered with
1.0 mM phosphate at pH 7.0. Besides minimizing proton
exchange, this pH value was chosen because it is similar to the
pH generally found in biological solutions. The poor solubility
of cyclophane 3 in water prevented us from testing its ability
to form complexes, whereas the [2]rotaxanes were soluble in
the 98% water solution to at least a 0.1 mM concentration. This
relatively high water solubility of the host-[2]rotaxanes, as
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A R T I C L E S
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[2]rotaxanes can efficiently deliver fluorescein and some
fluorescein-derivatized agents through the various membrane
barriers into the cytoplasm and the nucleus and uniformly deliver
these agents throughout the entire population of cells indepen-
dent of cellular differentiation.
Discussion
The association constants for complexes formed with cyclo-
phane-[2]rotaxane 1 and cleft-[2]rotaxane 2 are quite large
in the 98% aqueous solution (Table 1). KA values in the 104
M-1 range compare well with current hosts designed to bind
small peptides.39-43 A comparison to cyclophane 3 cannot be
obtained, because this host is insoluble in the 98% aqueous
solution at the concentration needed to obtain complexes. With
an increase in DMSO from 2% to 25%, cyclophane 3 is
sufficiently soluble to observe association. A 4-6-fold enhance-
ment in KA is observed for the hosts built on the [2]rotaxane
architecture, as compared to cyclophane 3. One exception is
the complexes formed with pyrene, which are preferred by the
[2]rotaxanes by 10-20-fold. This result suggests that the
aromatic surfaces supplied by the rotaxane ring contribute
significantly to the binding free energy. Favorable binding
energy could arise through dispersion interactions, aromatic
stacking interactions, or the hydrophobic effect. Having a large
hydrophobic pocket that is reasonably soluble in water is an
important feature for compounds that mimic protein function.
Host-[2]rotaxanes 1 and 2 also form more stable complexes
in DMSO than cyclophane 3. In this solvent, interaction energies
caused by aromatic and aliphatic groups are diminished in
importance, whereas interactions between polar and charged
groups become more important. For cyclophane 3, there is very
little change in the association constants going from 25% to
100% DMSO. The weak association of N-Ac-Trp in DMSO is
an anomaly. The degree of association of the similar guest N-Ac-
Trp-NH2, which lacks the negative charge, is consistent with
the other guests. Possibly burying the indole ring of N-Ac-Trp
into the pocket may position the carboxylate too close to the
apolar host. There is a 3-30-fold increase in KA for complexes
formed with [2]rotaxanes in DMSO as compared to cyclophane
3, except for fluorescein. The strong preference of the [2]-
rotaxanes for fluorescein (ca. 200-fold larger association
constants) is consistent with our previous rotaxane study.17 The
fact that all guests investigated (with apolar and polar residues)
are bound more strongly by the [2]rotaxanes than cyclophane
3 shows that the ring of the rotaxane contributes favorable
binding free energy through a variety contacts, e.g., salt bridges,
H-bonds, and dispersion interactions, with the guests. The
strongest evidence for participation of the ring’s functional
groups is the 40-fold preference of the [2]rotaxanes for
fluorescein, which has a carboxylate available for salt bridge
formation, as compared to pyrene in DMSO.
Figure 2. A low-energy conformation of the complex between [2]rotaxane
1 and N-Ac-Trp-Leu (molecular mechanics calculations,18 MM+ force field
as presented by Hyperchem38). Several components of the [2]rotaxane and
guest are labeled and the H-atoms are removed for clarity.
transport (Figure 3E). Note that a longer film exposure time
was needed to observe the fluorescence (15 s for the assays
containing 60 µM [2]rotaxane 2 and 6 µM [2]rotaxane 1 versus
3 s for assays with 60 µM of [2]rotaxane 1). Increasing the
concentration of [2]rotaxane 1 to 60 µM produced strongly
fluorescent cells, showing clearly the various organelles, includ-
ing the nucleus (Figure 3C,D). The cells appeared to be healthy,
and we observed at least one cell undergoing mitosis (Figure
3D). A single outer membrane contained two nuclei, which is
consistent with the cell being in the telophase of mitosis. These
results show that [2]rotaxane 1 and fluorescein are not im-
mediately toxic to the cells.
To further investigate the potential of rotaxanes as transport
agents, the ability of [2]rotaxane 1 to transport fluorescein-
PKC inhibitor conjugate 27 into COS-7 cells was determined.
Host-[2]rotaxanes 1 and 2 bind the guests with similar
affinities in the solvent systems investigated. Apparently, the
Conjugate 27 is transported into acidic solutions (pH < 6.5)
and used to locate PKC within cells.19 [2]Rotaxane 1 strongly
binds conjugate 27 in the 98% buffer solution (KA ) 1.2 ( 0.2
× 105 M-1) and in DMSO (KA ) 2.0 ( 0.2 × 104 M-1).
Conjugate 27 is transported into COS-7 cells by [2]rotaxane 1
in a pH 7.5 solution using the same experimental conditions as
discussed above (0.4 µM conjugate exposed to 60 µM [2]-
rotaxane 1 for 30 min, Figure 3F). Without the rotaxane, no
fluorescence was observed. These results demonstrate that host-
(38) Hypercube, Inc., Gainesville, FL.
(39) Hortala, M. A.; Fabbrizzi, L.; Marcotte, N.; Stomeo, F.; Taglietti, A. J.
Am. Chem. Soc. 2003, 125, 20-21.
(40) Tsubaki, K.; Kusumoto, T.; Hayashi, N.; Nuruzzaman, M.; Fuji, K. Org.
Lett. 2002, 4, 2313-2316.
(41) Rensing, S.; Schrader, T. Org. Lett. 2002, 4, 2161-2164.
(42) Ait-Haddou, H.; Wiskur, S. L.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem.
Soc. 2001, 123, 11296-11297.
(43) Mizutani, T.; Wada, K.; Kitagawa, S. J. Am. Chem. Soc. 1999, 121, 11425-
11431.
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Host−[2]Rotaxanes as Cellular Transport Agents
A R T I C L E S
Figure 3. Fluorescence microscopy experiments showing the transport of fluorescein (0.4 µM) or a fluorescein-PKC inhibitor (0.4 µM) into COS-7 cells
by the host-[2]rotaxanes. The cells were grown on slides, placed in 1 mL of phosphate buffer (pH 7), and exposed to the agents for ca. 30 min. (A)
Fluorescein does not penetrate the cell membranes (30 s film exposure time). (B) Fluorescein is transported into the cells in the presence of 6 µM [2]rotaxane
1 (40× magnification, 15 s film exposure time). (C) In the presence of 60 µM of [2]rotaxane 1 the cells are highly fluorescent (20× magnification, 3 s film
exposure time). This picture also shows that fluorescein is uniformly delivered throughout the entire population of cells. (D) An example of the cells
undergoing mitosis (indicated by the arrow), which shows that [2]rotaxane 1 and fluorescein are not immediately toxic to the cells. Interestingly, transport
of fluorescein was not observed with 6 µM of [2]rotaxane 2. (E) At a higher concentration of [2]rotaxane 2 (60 µM), transport was observable (40×
magnification, 15 s film exposure time). F. [2]Rotaxane 1 also efficiently transports fluorescein-PKC inhibitor 27 (40× magnification, 3 s film exposure
time).
pocket of [2]rotaxane 1 does not provide a large advantage for
association compared to the cleft. We thought that the aliphatic
dipeptides would be more favorably sequestered into the pocket
of [2]rotaxane 2 (Figure 2). There is a slight preference for the
aliphatic dipeptides associating with [2]rotaxane 1 in DMSO,
but this preference is lost with the addition of water to the
solution. A partial collapse of the cyclophane pocket in an
aqueous environment could explain the loss in guest affinity.
For larger guests [1-(dimethylamino)-5-naphthalenesulfonate
(1,5-DNS), pyrene, and fluorescein], the slight preference for
the pocket maintains throughout the solvent systems. The larger
preorganized aromatic surface provided by [2]rotaxane 1 may
be responsible for the greater affinity, as compared to the open
cleft of [2]rotaxane 2. Larger association constants are observed
for complexes formed with the large aromatic guests than for
the amino acids and dipeptides in the aqueous solutions. The
additional binding energy, most likely, stems from a greater
hydrophobic effect.
Interactions between the aromatic and aliphatic surfaces
appear to be the dominant source of favorable binding free
energy for the smaller guests as well. Other interactions, such
as a salt bridge, seem to provide a small amount of favorable
binding energy. For example, [2]rotaxane 1 binds N-Ac-Trp
more favorably than N-Ac-Trp-NH2 and indole in DMSO and
in the 98% buffered solution. Among the negatively charged
peptidic guests, there is a slight preference for smaller guests.
By comparing N-Ac-Trp to N-Ac-Trp-Glu, one can see that
extending the carboxylate further from the aromatic ring
weakens association. This effect is also observed by comparing
the association constants of the dicarboxylate guests N-Ac-Trp-
Asp and N-Ac-Trp-Glu in DMSO and the 98% buffer solution.
The fact that N-Ac-Trp-Asp has a larger KA than N-Ac-Trp-
Gly in all solvent systems, and especially in the 98% buffered
solution, suggests that the two cationic groups of the ring
(ammonium and guanidinium ions) can favorably interact with
guests having multiple anionic charges.
9
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A R T I C L E S
Dvornikovs et al.
Another important consideration when comparing KA’s is the
differences in the solvent-guest interactions. In this study,
complexation describes a guest being removed from bulk solvent
into an apolar pocket of a host. Although changing the solvent
pocket is beneficial for cell transport. Successful transport of
the fluorescein-PKC inhibitor conjugate suggests that other
fluorescein-linked agents can also be transported. These studies
are currently being pursued.
can have a profound effect on the degree of complexation,25
a
Experimental Section
somewhat surprising, small solvent effect is observed. For
example, fluorescein and pyrene have similar interaction ener-
gies with the rotaxanes in the aqueous solutions, even though
fluorescein is highly water soluble and pyrene is not. The close
similarity in the magnitude of the KA values of most guest
complexes (pyrene, 1,5-DNS, amino acids, and dipeptides)
suggests that the combining sites of the [2]rotaxanes can
accommodate all the functional groups that interact weakly with
solvent molecules. Furthermore, since similar KA values were
obtained for guests that have dissimilar properties, the combining
sites appear to adjust to maximize the number of favorable
contacts between the various functional groups. The ability of
the host-[2]rotaxanes to change their conformations to maintain
a maximum in the favorable binding free energy can explain
the efficient transport of fluorescein and fluorescein-PKC
conjugate 27. In aqueous environments, the hydrophobic effect
keeps the complex together, and within membranes, the com-
bining sites adjust, making salt bridges and H-bonds more
important sources of favorable binding free energy.
General. Solvents, reagents, and starting materials for the hosts were
purchased from Aldrich. The derivatized amino acids are available
commercially (Sigma or CHEM-IMPEX). 1H NMR and 13C NMR
spectra were obtained in CDCl3 using a Bruker AMX400 spectrometer
operating at 400.14 MHz for proton and 100.23 MHz for carbon nuclei
or a Bruker AC250 operating at 62.9 MHz for carbon nuclei and 250
MHz for proton. Chemical shifts are in ppm and are referenced using
an internal TMS standard. A MEL-TEMP (Laboratory Devices)
apparatus was used to determine the melting points of the compounds;
these values are given uncorrected. HPLC analysis was performed on
a Shimadzu 10A series HPLC. Solvents were purified by drying over
a suitable drying agent and then distilled. Water for potentiometric
titrations and HPLC assays was purified on a Millipore water
purification system.
Potentiometric Titrations. 5.0 mM solutions of the templates were
prepared from freshly distilled DMSO and boiled water. These solutions
were titrated under Ar with solutions containing 0.10 M of t-BuOK
for experiments performed in DMSO and 0.105 M of KOH for the
experiments performed in 75% and 100% water. The pKa values of
the hosts and amino acids were calculated using the BEST program.44
Monitoring Association. Fluorescence binding assays were per-
formed on the hosts and fluorescent guests. A known amount of guest,
dissolved in a particular solution, was added to a 3.5 mL cuvette
(maintained at 25 °C), and aliquots from a concentrated host stock
solution in DMSO were added. The change in volume caused by the
addition of a host was less than 2% for [2]rotaxanes 1 and 2 and less
than 10% for cyclophane 3. A fluorescence spectrum was recorded
and analyzed after each addition of host. The amino acids and dipeptides
were held at 1 × 10-5 M, and the concentration of [2]rotaxanes 1 and
2 was increased in a 10-5 M increment from 1 × 10-5 to 8 × 10-5 M.
The fluorophores were held at 3 × 10-6 M and the hosts were added
at various concentrations from 1 × 10-6 to 3 × 10-5 M. For studies of
cyclophane 3, the host concentration was increased from 1 × 10-5 to
5 × 10-4 M, and the guests were held at a constant concentration of 3
× 10-5 M. Hosts were added to the assay solutions until saturation
began to be observable, giving an experimental maximum change in
fluorescence equal to 65-90% of the calculated value (∆Fmax). Plots
of the changes observed in the binding titrations were fitted using a
nonlinear least-squares procedure to derive KA and ∆Fmax values.45 Most
assays were repeated using different experimentalists with different
stock solutions. The deviation in the results of these repeated assays
was equal to or below the standard deviation (given in Table 1) obtained
from the mathematical analysis of the data.
These studies demonstrate that knowledge gained from
binding assays performed in different solvents can indicate the
likelihood of cell transport. The observed differences in the
transport efficiencies of [2]rotaxane 1 with a change in its
concentration is consistent with the KA values obtained from
the fluorescence assays. Assuming that the association constants
for cellular and fluorescence assays are the same and with
fluorescein at 0.4 µM, a change of concentration of [2]rotaxane
1 from 6 to 60 µM increases the percentage of fluorescein in a
complex to free in solution from approximately 25% to 75%.
Results obtained from the fluorescence assays also suggest that
[2]rotaxane 2 should transport fluorescein as well as [2]rotaxane
1. However, [2]rotaxane 2 is a less efficient transporter than
[2]rotaxane 1, requiring the higher concentration of rotaxane
(60 µM) to produce noticeable transport. The cyclophane pocket
of [2]rotaxane 1 is not beneficial for complex formation in
DMSO and buffered solutions, but it is apparently beneficial
for cell transport. Another interesting observation is that [2]-
rotaxane 1 transports fluorescein to a greater extent into the
nucleus, whereas [2]rotaxane 2 delivers fluorescein more
uniformly throughout a cell.
Compound Synthesis. Dipeptides were constructed by mixing an
amino acid with N-Ac-Trp-CO2H, which was activated with ethylchlo-
roformate. Pure product was obtained through HPLC separation, and
Conclusion. Two new host-[2]rotaxanes were readily as-
sembled through the key step of combining a DCC-[2]rotaxane
with a cyclophane or an aromatic cleft. These hosts form tighter
complexes with amino acids, dipeptides, and fluorophores than
a more traditional cyclophane in water, DMSO, and 75/25 (v/
v) water to DMSO solution. The similarity in the KA values of
the complexes formed with cyclophane-[2]rotaxane 1 and
cleft-[2]rotaxane 2 suggests that the unique assembly of the
recognition elementssheld together through threadingsallows
them to adjust to maintain a maximum in the favorable binding
free energy. Although both [2]rotaxanes transport fluorescein
into COS-7 cells, the transport ability of cyclophane-[2]-
rotaxane 1 is substantially better. This result is surprising
considering that the [2]rotaxanes bind fluorescein to the same
extent in the fluorescence assays. Apparently, the cyclophane
1
its authenticity was verified via H NMR and mass spectral analysis.
Bis(4-benzyloxyphenyl)methanol (5). A 25.0 g (117 mmol) sample
of 4,4′-dihydroxybenzophenone 4 was dissolved in the mixture of 300
mL of chloroform and 300 mL of methanol. Benzyl bromide (36.3
mL, 304 mmol), followed by 48.3 g (350 mmol) of potassium carbonate,
was added. The reaction mixture was refluxed for 2 days. Solvents
were evaporated, and the residue was partitioned between 2.0 L of
chloroform and 0.5 L of water. The organic layer was dried over sodium
sulfate. The solvent was evaporated and the residue was washed with
3 × 50 mL of hexane in order to remove the residual benzyl bromide.
(44) Martell, A. E.; Motekaitis, R. J. The Determination and Use of Stability
Constants; VCH: New York, 1988.
(45) Conners, K. A. Binding Constants, The Measurement of Molecular Complex
Stability; Wiley: New York, 1987.
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Host−[2]Rotaxanes as Cellular Transport Agents
A R T I C L E S
White crystals were produced (44.6 g, 11.3 mmol, 97%). The spectral
data and the melting point matched the published information about
the compound.46 A 35.6 g (90.3 mmol) sample of the resulting 4,4′-
dibenzyloxybenzophenone was dissolved in 400 mL of a 50/50 CH2-
Cl2/EtOH solution, and 3.43 g (90.3 mmol) of NaBH4 was added. After
stirring of the reaction mixture at room temperature for 24 h, the
solvents were removed in vacuo. The residue was partitioned between
200 mL of methylene chloride and 200 mL of water. The aqueous layer
was extracted twice with 50 mL of methylene chloride, and the
combined organic extracts were dried over sodium sulfate. Removal
of the solvent in vacuo gave 35.5 g (89.4 mmol) of alcohol 5 in a 99%
25 mL of CH2Cl2, and the organic extracts were combined and dried
over Na2SO4. After the solvent was removed under reduced pressure,
the crude material was purified by column chromatography (95/5 CH2-
Cl2/EtOH) to give 0.33 g (0.44 mmol) of host 10 in a 28% yield, as a
1
white foam (mp ) 95-97 °C). H NMR (CDCl3): 2.10-2.14 (7H,
m), 2.25 (4H, m), 2.79 (2H, t, J ) 7.2 Hz), 3.42 (12H, s), 3.48 (2H,
m), 3.66 (2H, m), 3.84 (1H, t, J ) 5.1 Hz), 4.11 (4H, t, J ) 5.2 Hz),
4.21 (2H, t, J ) 5.2 Hz), 4.93-5.07 (2H, m), 5.6-5.8 (1H, m), 6.24
(4H, s), 6.74 (4H, d, J ) 8.4 Hz), 7.06 (4H, d, J ) 8.4 Hz). 13C NMR
(CDCl3): 21.1, 30.2, 36.1, 36.5, 37.1, 37.6, 38.4, 38.6, 49.5, 55.4, 64.0,
69.5, 104.8, 114.2, 115.8, 127.8, 135.7, 136.7, 137.1, 140.6, 152.5,
157.4, 168.6. MS: found 752.3693, calcd for C45H54NO9 752.3799.
Cyclophane Alcohol (11). A 200 mg (0.26 mmol) sample of
cyclophane 10 was dissolved in 4 mL of THF, and a 0.16 mL (0.32
mmol) sample of 2 M borane-dimethyl sulfide complex in THF was
added dropwise. The mixture was stirred for 4 h followed by the slow
addition (exothermic!) of 0.3 mL of 30% H2O2 and 0.3 mL of 2 M
NaOH. After removal of the solvent under reduced pressure, and the
residue was partitioned between 10 mL of CH2Cl2 and 10 mL of water.
The aqueous layer was washed with 2 × 10 mL of CH2Cl2, and the
organic extracts were combined and dried over Na2SO4. After the
solvent was removed, the crude material was purified by column
chromatography (EtOAc) to give 180 mg (0.23 mmol) of alcohol 11
in a 88% yield, as a white foam (mp ) 83-85 °C). 1H NMR (CDCl3):
1.57 (2H, m), 2.10-2.12 (9H, m), 2.24 (4H, m), 3.41 (12H, s), 3.48
(2H, m), 3.64-3.67 (4H, m), 3.75 (1H, t, J ) 6.1 Hz), 4.11 (4H, t, J
) 5.0 Hz), 4.21 (4H, t, J ) 5.1 Hz), 6.25 (4H,s), 6.74 (4H, d, J ) 8.3
Hz), 7.01 (4H, d, J ) 8.3 Hz). 13C NMR (CDCl3): 21.3, 30.4, 30.7,
31.2, 36.1, 36.3, 36.7, 37.1, 37.3, 37.7, 38.7, 43.7, 55.6, 62.6, 64.2,
69.7, 104.9, 114.4, 127.9, 135.8, 137.8, 140.7, 152.7, 157.6, 169.0.
MS: found 792.3754, calcd for C45H55NO10Na [M + Na] 792.3724.
An alternative route for the formation of cyclophane 11 involves
mixing a 4.8 g (7.1 mmol) sample of dibromide 9 and a 1.83 g (7.1
mmol) sample of 3,3-bis(4-hydroxyphenyl)propanol 16 (vide infra) in
708 mL of anhydrous DMF. The reaction mixture was degassed three
times by keeping it under vacuum for ∼2 min and then introducing
dry Ar. To this mixture was added 9.24 g (28.4 mmol) of finely
powdered CsCO3. The reaction mixture was stirred under Ar at 80-
100 °C for 72 h. Following the same purification procedure as used
above yielded 32% of cyclophane 11.
1
yield, as a white solid (mp ) 115-117 °C). H NMR (CDCl3): 5.05
(4H, s), 5.77 (1H, s), 6.94 (4H, d, J ) 8.3 Hz), 7.28 (4H, d, J ) 8.4
Hz), 7.32-7.43 (10H, m). 13C NMR (CDCl3): 69.9, 75.2, 114.7, 127.4,
127.7, 127.9, 128.5, 136.6, 136.9, 158.1. MS: found 419.1648, calcd
for C27H24O3Na [M + Na] 419.1623.
3,3-Bis(4-benzyloxyphenyl)propene (7). A solution of trifluoro-
acetic acid (0.72 g, 6.3 mmol) in 1 mL of CH2Cl2 was added dropwise
to a 30 mL solution of CH2Cl2 containing 5.00 g (12.6 mmol) of alcohol
5 and 2.89 g (25.3 mmol) of allyltrimethylsilane. The reaction mixture
was stirred at room temperature for 24 h. The solvent and volatile
byproducts were evaporated under reduced pressure to give 5.09 g (12.1
mmol) of alkene 7 in a 96% yield, as an off-white solid (mp ) 93-95
°C). 1H NMR (CDCl3): 2.75 (2H, t, J ) 7.3 Hz), 3.91 (1H, t, J ) 7.7
Hz), 4.91-5.05 (6H, m), 5.69 (1H, m), 6.89 (4H, d, J ) 8.5 Hz), 7.13
(4H, d, J ) 8.5 Hz), 7.29-7.43 (5H, m). 13C NMR (CDCl3): 40.4,
49.6, 70.0, 114.8, 116.2, 127.5, 127.9, 128.6, 128.9, 137.1, 137.3, 137.4,
157.2. MS: found 443.2013, calcd for C30H28O2Na [M + Na] 443.1987.
3,3-Bis(4-hydroxyphenyl)propene (8). A 154 mg (22.0 mmol)
sample of lithium metal was added in small pieces to a solution of
5.32 g (20.0 mmol) of 4,4′-di-tert-butylbiphenylene dissolved in 44
mL of anhydrous THF. The reaction mixture was stirred under argon
for 24 h until all of lithium was consumed. Alkene 7 (700 mg, 1.7
mmol) was dissolved in 2 mL of THF and added dropwise to the freshly
prepared solution of LDDB. To this solution was added 2 mL of
methanol followed by 1 M of HCl until the pH ) 3. The mixture was
extracted with 3 × 50 mL of CH2Cl2. The combined organic layers
were washed with brine and dried over Na2SO4. Evaporation of the
solvents under reduced pressure gave a yellow solid. Purification of
the crude material by column chromatography (5:100 of EtOH/CH2-
Cl2) yielded 370 mg (1.5 mmol) of alkene 8 in a 92% yield, as pink
crystals (mp ) 111-113 °C).1H NMR (CDCl3): 2.72 (2H, t, J ) 7.0
Hz), 3.88 (1H, t, J ) 7.3 Hz), 4.93-5.04 (2H, m), 5.65-5.71 (1H, m),
6.74 (4H, d, J ) 8.4 Hz), 7.06 (4H, d, J ) 8.4 Hz). 13C NMR
(CDCl3): 40.3, 49.5, 115.2, 116.0, 128.8, 136.8, 137.1, 154.1. MS:
found 263.1058, calcd for C16H16O2Na [M + Na] 263.1048.
Cyclophane Bromide (12). A 660 mg (0.86 mmol) sample of
alcohol 11 and 298 mg (1.14 mmol) of triphenylphosphine were
dissolved in 5 mL of CH3CN. To this solution was added 377 mg (1.14
mmol) of CBr4, dissolved in 1.6 mL of CH3CN. The reaction mixture
was stirred for 4 h, and the solvent was removed in vacuo. The residue
was separated by column chromatography (gradient elution with EtOAc
to 5:100 MeOH/EtOAc) to give 620 mg (0.74 mmol) of bromide 12 in
An alternative deprotection procedure involves mixing 10.0 g (23.8
mmol) of alkene 7, dissolved in 200 mL of CH2Cl2, and 50 mL (50
mmol) of 1 M boron trichloride, which was introduced via a syringe.
After stirring the reaction mixture for 1 h, the solution was washed
with water and dried over Na2SO4, and solvent was removed under
reduced pressure. The crude material was washed with hexane to
remove benzyl chloride and then subjected to purification by column
chromatography (10:90 EtOH/CH2Cl2) to give 4.48 g (18.7 mmol) of
alkene 8 in a 78% yield.
1
an 87% yield, as a white foam (mp ) 95-97 °C). H NMR (CDCl3):
1.86 (2H, quintet, J ) 7.6 Hz), 2.10-2.26 (13H, m), 3.41 (14H, m),
3.48 (2H, m), 3.66 (2H, m), 3.76 (1H, m), 4.11 (4H, m), 4.20 (4H, m),
6.25 (4H, s), 6.75 (4H, d, J ) 8.4 Hz), 7.06 (4H, d, J ) 8.4 Hz). 13C
NMR (CDCl3): 21.3, 30.4, 31.4, 33.0, 33.8, 36.1, 36.3, 36.7, 37.1,
37.4, 37.8, 38.7, 43.7, 55.7, 64.3, 69.7, 105.0, 114.5, 127.8, 135.9, 137.3,
140.8, 152.7, 157.7, 168.9. MS: found 832.3093, calcd for C45H55-
BrNO9 [M + 1] 832.3055.
Cyclophane Alkene (10). A 1.04 g (1.54 mmol) sample of dibromide
936 and a 1.83 g (7.1 mmol) sample of 3,3-bis(4-hydroxyphenyl)propene
8 were dissolved in 150 mL of anhydrous DMF. The reaction mixture
was degassed three times by keeping it under vacuum for ca. 2 min
and then introducing dry Ar. To this mixture was added 2.00 g (6.16
mmol) of finely powdered CsCO3. The reaction mixture was stirred
under Ar at 80-100 °C for 24 h. After removal of the solvent under
reduced pressure, and the residue was partitioned between 50 mL of
CH2Cl2 and 50 mL of water. The aqueous layer was washed with 2 ×
Cyclophane Azide (13). A 312 mg (4.80 mmol) sample of NaN3
was added to 500 mg (0.60 mmol) of bromide 12, dissolved in 10 mL
of CH3CN. The reaction mixture was refluxed for 72 h, cooled to room
temperature, and the salts were removed by filtration. After removal
of the solvent under reduced pressure, the residue was dissolved in 10
mL of CH2Cl2. Insoluble materials were removed by filtration.
Removing the solvent in vacuo resulted in 488 mg (0.60 mmol) of
azide 13 in a quantitative yield, as a white foam (mp ) 89-91 °C). 1H
NMR (CDCl3): 1.58 (2H, quintet, J ) 7.2 Hz), 2.01-2.15 (9H, m),
2.15-2.30 (4H, m), 3.27 (2H, t, J ) 6.4 Hz), 3.41 (12H, s), 3.48 (2H,
m), 3.66 (2H, m), 3.76 (1H, m), 4.11 (4H, m), 4.20 (4H, m), 6.26 (4H,
(46) Tadros, W. J. Chem. Soc. 1949, 442-444.
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A R T I C L E S
Dvornikovs et al.
s), 6.75 (4H, d, J ) 7.8 Hz), 7.06 (4H, d, J ) 7.8 Hz). 13C NMR
(CDCl3): 21.2, 27.2, 30.2, 31.4, 35.9, 36.2, 36.6, 36.9, 37.2, 37.6, 38.5,
43.5, 51.2, 55.5, 64.1, 69.6, 104.7, 114.3, 127.7, 135.7, 137.2, 140.6,
152.5, 157.5, 168.7. MS: found 817.3813, calcd for C45H54N4O9Na
[M + Na] 817.3788.
through filtration. Removal of the solvent in vacuo resulted in 4.66 g
(10.1 mmol) of azide 18 in a quantitative yield, as a pale yellow oil.
1H NMR (CDCl3): 1.55 (2H, quintet, J ) 7.1 Hz), 2.05 (2H, q, J )
8.0 Hz), 3.26 (2H, t, J ) 6.5 Hz), 3.80 (1H, t, J ) 7.8 Hz), 5.02 (4H,
s), 6.89 (4H, d, J ) 8.6 Hz), 7.12 (4H, d, J ) 8.5 Hz), 7.30-7.42 (5H,
m). 13C NMR (CDCl3): 27.1, 32.6, 48.9, 50.9, 69.5, 114.5, 127.1, 127.5,
128.1, 128.3, 136.8, 137.0, 156.9. MS: the azide is unstable to mass
spectral analysis.
Cyclophane Amine (14). A 1.09 g (1.37 mmol) sample of azide 13
was dissolved in 20 mL of freshly distilled DMF. To this solution was
added 146 mg (0.14 mmol) of 10% Pd/C, and the reaction mixture
was hydrogenated under 50 psi of H2 overnight. The suspension was
removed by passing the solution through a Celite pad to remove the
catalyst. After the solvent was removed under reduced pressure, the
residue was separated by column chromatography (CH2Cl2 to 5:100
Et3N/CH2Cl2) to give 660 mg (0.82 mmol) of amine 14 in a 60% yield,
as an amorphous glass. 1H NMR (CDCl3): 1.44 (2H, quintet, J ) 7.1
Hz), 1.99-2.13 (9H, m), 2.24 (4H, m), 2.72 (2H, t, J ) 6.5 Hz), 3.41
(12H, s), 3.48 (2H, m), 3.66 (2H, m), 3.72 (1H, t, J ) 7.5 Hz), 4.10
(4H, t, J ) 5.2 Hz), 4.20 (4H, t, J ) 5.1 Hz), 6.24 (4H, s), 6.73 (4H,
d, J ) 8.3 Hz), 7.05 (4H, d, J ) 8.3 Hz). 13C NMR (CDCl3): 21.3,
29.2, 30.3, 31.8, 36.3, 36.7, 37.3, 37.7, 38.6, 49.6, 53.3, 55.6, 64.3,
69.8, 104.8, 114.3, 127.8, 135.8, 137.8, 140.7, 152.6, 157.5, 168.8.
MS: found 769.4062, calcd for C45H57N2O9 [M + 1] 769.4064.
Benzylcleft Alcohol (15). A 21.0 mL (42.5 mmol) sample of 2 M
borane-dimethyl sulfide complex in THF was added dropwise to 16.8
g (40.1 mmol) of alkene 8 dissolved in 40 mL of THF. The mixture
was stirred for 4 h followed by the slow addition (exothermic!) of 10
mL of 30% H2O2 and 10 mL of 2 M NaOH. After extracting the
solution with 3 × 70 mL of CH2Cl2, the combined extracts were washed
with brine and dried over Na2SO4. Evaporation of solvents under
reduced pressure gave pale yellow oil, which was purified by column
chromatography (5:100 EtOH/CH2Cl2) to give 12.68 g (28.9 mmol) of
alcohol 15 in a 78% yield, as an oil, which crystallizes upon standing
(mp ) 60-62 °C). 1H NMR (CDCl3): 1.53 (2H, quintet, J ) 7.6 Hz),
2.05 (2H, q, J ) 7.2 Hz), 3.63 (2H, t, J ) 6.3 Hz), 3.81 (1H, t, J ) 7.6
Hz), 5.01 (4H, s), 6.89 (4H, d, J ) 8.5 Hz), 7.14 (4H, d, J ) 8.4 Hz),
7.30-7.43 (5H, m). 13C NMR (CDCl3): 31.2, 32.1, 49.4, 62.7, 69.9,
114.7, 127.4, 127.8, 128.5, 128.6, 137.1, 137.7, 157.1. MS: found
461.2054, calcd for C30H30O3Na [M + Na] 461.2093.
Cleft Amine (19). A 400 mg (1.6 mmol) sample of triphenylphos-
phine and 42 mg (2.3 mmol) of water were added to 720 mg (1.6 mmol)
of azide 18 in 5 mL of CH3CN. The reaction mixture was stirred for
4 h, and the solvent was removed in vacuo. The residue was separated
by column chromatography (successive elutions with 100 mL 1:1
hexane/CH2Cl2, 100 mL of CH2Cl2, 100 mL of 4:100 EtOH/CH2Cl2,
100 mL of 5:10:100 EtOH/Et3N/CH2Cl2) to give 600 mg (1.4 mmol)
1
of amine 19 in a 89% yield, as a yellow solid (mp ) 78-80 °C). H
NMR (CDCl3): 1.43 (2H, quintet, J ) 7.3 Hz), 1.66 (2H, m), 2.00
(2H, q, J ) 7.6 Hz), 2.70 (2H, t, J ) 7.0 Hz), 3.79 (1H, t, J ) 7.6 Hz),
5.00 (4H, s), 6.88 (4H, d, J ) 8.3 Hz), 7.12 (4H, d, J ) 8.4 Hz),
7.30-7.42 (5H, m). 13C NMR (CDCl3): 31.2, 32.7, 41.2, 48.9, 69.3,
114.1, 126.8, 127.2, 127.9, 128.0, 136.6, 137.2, 156.5. MS: found
438.2398, calcd for C30H32NO2 [M + 1] 438.2433.
BocNH-cyclophane-[2]Rotaxane (21). A DCC-[2]rotaxane 2018
solution (0.64 mmol, 1 mL CHCl3) was cooled to 0 °C, and 270 mg
(0.35 mmol) of amine 14 in 0.2 mL of chloroform was rapidly added.
The reaction was brought to a room temperature and stirred overnight.
Then 20 mL of CH3CN was added to the solution, and the resulting
precipitated dicyclohexylurea was removed by filtration. After removal
of the solvent under reduced pressure, the residue was dissolved in 1
mL of CH2Cl2 and approximately 20 mL of diethyl ether was added to
remove the N-(3,5-di-tert-butylbenzyl)valerolactam byproduct as a
precipitate. The solution sat for 4 h until all of the insoluble material
precipitated. After decanting the ether layer, the precipitation procedure
was repeated. The ether layer was collected, and the solvent was
removed in vacuo. Separation of the crude material was achieved using
radial centrifugal chromatography (CH2Cl2 to 5:100 MeOH/CH2Cl2)
to give 400 mg (0.21 mmol) of [2]rotaxane 1 in a 60% yield, as an
1
off-white foam (mp ) 138-140 °C). H NMR (CDCl3): 1.27 (18H,
Cleft Alcohol (16). A 440 mg (1.0 mmol) sample of alcohol 15
was dissolved in 10 mL of a 50:50 MeOH/THF solution, and 100 mg
(0.1 mmol) of 10% Pd/C was added. The mixture was stirred overnight
under 50 psi of H2. After the reaction mixture was filtered through a
Celite pad and the solvent removed in vacuo, 300 mg (1.0 mmol) of
alcohol 16 was obtained in a quantitative yield, as a pale yellow glass.
1H NMR (CDCl3): 1.29 (2H, quintet, J ) 7.6 Hz), 1.89 (2H, q, J )
7.4 Hz), 3.35 (2H, m), 3.65 (1H, t, J ) 7.7 Hz), 4.33 (1H, t, J ) 5.1
Hz OH), 6.64 (4H, d, J ) 8.3 Hz), 7.01 (4H, d, J ) 8.5 Hz), 9.11
(2H,s OH). 13C NMR (CDCl3): 31.4, 32.2, 49.0, 60.9, 115.2, 128.5,
136.4, 155.5. MS: found 281.1101, calcd for C16H18O3Na [M + Na]
281.1154.
s), 1.49-1.51 (18H, 2 × s), 1.74 (4H, m), 1.92 (2H, m), 2.10 (3H, s),
2.25 (10H, m), 2.8 (2H, m), 3.1-3.3 (4H, m), 3.40 (12H, s), 3.48 (2H,
m), 3.5-3.8 (18H, m), 4.0-4.3 (17H, m), 4.6 (2H, m), 6.25 (4H, s),
6.71-7.08 (12H, m), 7.20 (2H, m), 7.39 (3H, m). 13C NMR (CDCl3):
172.5, 169.0, 157.5, 153.1, 152.7, 151.1, 147.4, 143.1, 141.0, 138.0,
135.8, 133.1, 128, 124.4, 123.1, 114.5, 113.2, 113.1, 111.8, 105.2, 104.9,
80.1, 70.4, 69.8, 68.2, 64.4, 55.7, 49.5, 48.8, 46.3, 43.7, 39.3, 37.6,
36.6, 35.7, 34.7, 31.3, 30.4, 28.2, 27.9, 25.8, 22.4, 21.3. MS: found
1748.9751, calcd for C99H138N5O22 [M + 1] 1748.9833.
BocNH-cleft-[2]Rotaxane (24). Construction of [2]rotaxane 24
followed the same procedure used to synthesize and purify [2]rotaxane
23 with 0.21 mmol of DCC-[2]rotaxane 2018 and 0.14 mmol of amine
19 in 0.5 mL of CDCl3. Purification using radial centrifugal chroma-
tography (CH2Cl2 to 5:100 EtOH/CH2Cl2) gave 150 mg (0.105 mmol)
of [2]rotaxane 24 in a 75% yield, as a yellow foam (mp ) 115-117
Cleft Bromide (17). A 4.87 g (11.1 mmol) sample of 3,3-bis(4-
benzyloxyphenyl)propanol 15 and 3.88 g (14.8 mmol) of triphenylphos-
phine were dissolved in 30 mL of CH3CN. To this solution was added
dropwise 4.91 g (14.8 mmol) of carbon tetrabromide, dissolved in 2
mL of CH3CN. The reaction mixture was stirred for 4 h. The solvent
was removed under vaccum, and the residue was purified by column
chromatography (4:1 hexane/ethyl acetate) to give 5.07 g (10.1 mmol)
1
°C). H NMR (CDCl3): 1.26-1.28 (18H, 2 × s), 1.49-1.51 (18H, 2
× s), 1.60 (4H, m), 1.86-2.02 (4H, m), 2.72-2.89 (2H, m), 3.08-
3.25 (2H, m), 3.40-3.57 (2H, m), 3.57-3.90 (13H, m), 3.90-4.13
(8H, m), 4.13-4.37 (4H, m), 4.57 (2H, m), 4.98-5.03 (4H, 2 × s),
6.47 (2H, br s), 6.74 (2H, s), 6.79-6.91 (6H, m), 7.04-7.22 (6H, m),
7.28-7.43 (13H, m). 13C NMR (CDCl3): 172.3, 156.9, 153.0, 148.5,
147.9, 143.2, 143.1, 137.8, 137.0, 134.6, 134.3, 132.9, 128.5, 128.3,
127.6, 127.2, 124.9, 114.6, 113.0, 106.5, 80.2, 70.3, 69.8, 68.4, 68.1,
52.5, 48.6, 39.2, 35.0, 34.6, 33.0, 31.2, 31.2, 28.1, 25.5, 25.4, 22.3.
MS: found 1417.8268, calcd for C84H113N4O15 [M + 1] 1417.8202.
NH2-cleft-[2]Rotaxane (25). A 490 mg (0.31 mmol) sample of
BocNH-cleft-[2]rotaxane 24 was dissolved in 3.0 mL of CH2Cl2, To
this solution was added 1.5 mL of trifluoroacetic acid, dissolved in 1.0
1
of bromide 17 in a 91% yield, as pale yellow oil. H NMR (CDCl3):
1.81 (2H, quintet, J ) 7.2 Hz), 2.14 (2H, q, J ) 7.6 Hz), 3.40 (2H, t,
J ) 6.4 Hz), 3.81 (1H, t, J ) 7.6 Hz), 5.02 (4H, s), 6.89 (4H, d, J )
8.8 Hz), 7.13 (4H, d, J ) 8.4 Hz), 7.31-7.42 (5H, m). 13C NMR
(CDCl3): 31.0, 33.7, 34.2, 48.7, 69.8, 114.7, 127.2, 127.7, 128.3, 128.4,
137.0, 137.1, 157.0. MS: found 523.1310, calcd for C30H29BrO2Na
[M + Na] 523.1249.
Cleft Azide (18). A 3.94 g (60.6 mmol) sample of NaN3 was added
to 5.07 g (10.1 mmol) of bromide 17, dissolved in 20 mL of CH3CN.
The suspension was refluxed for 72 h, and the salts were removed
9
8300 J. AM. CHEM. SOC. VOL. 125, NO. 27, 2003

Host−[2]Rotaxanes as Cellular Transport Agents
A R T I C L E S
mL of CH2Cl2. The reaction mixture was stirred for 2 h, washed with
2 M KOH, and dried over Na2SO4. The organic phase was removed in
vacuo to give 410 mg (0.31 mmol) of [2]rotaxane 25 in a quantitative
152.5, 151.0, 146.9, 143.5, 143.4, 140.6, 137.7, 135.7, 127.8, 124.3,
122.8, 122.5, 114.2, 104.8, 84.8, 79.0, 78.7, 70.6, 69.7,68.4, 64.0, 55.5,
53.2, 49.3, 48.7, 48.4, 46.1, 44.0, 43.5, 39.1, 38.4, 37.1, 36.1, 34.7,
34.6, 33.6, 31.1, 30.2, 28.1, 27.9, 27.7, 25.1, 24.7, 24.2, 21.0, 19.5.
MS: found 2461.3928, calcd for C131H194N13O32 [M + 1] 2461.3953.
Cleft-[2]Rotaxane (2). A 300 mg (0.20 mmol) sample of (Boc)3Arg-
cleft-[2]rotaxane 26 was dissolved in 18 mL of CH2Cl2 and 18 mL of
acetic acid. Then 18 mL of TFA was added dropwise to this solution.
After stirring for 48 h, volatile materials were removed under reduced
pressure. The residue was separated using column chromatography
(CH2Cl2/TFA 100:1 to CH2Cl2/TFA/MeOH 50:1:50) to give 210 mg
(0.14 mmol) of rotaxane 2 in a 71% yield, as a brown glass. 1H NMR
(CDCl3): 1.19 (18H, s), 1.22-2.03 (18H, m), 3.15 (2H, m), 3.40-
3.54 (2H, m), 3.65-3.77 (16H, m), 3.99-4.24 (15H, m), 4.61 (2H,
m), 5.02 (4H, m), 6.80-6.90 (8H, m), 7.07-7.22 (6H, m), 7.22-7.46
(13H, m). 13C NMR (CDCl3): 175.1, 167.5, 160.9 (q, J ) 40 Hz),
158.6, 158.3, 152.4, 148.6, 146.3, 145.8, 139.2, 138.7, 133.4, 129.7,
129.0, 128.7, 125.1, 124.7, 118.4 (q, J ) 290 Hz), 117.1, 116.0, 114.0,
113.9, 107.0, 71.7, 71.1, 70.4, 69.4, 55.7, 54.9, 50.2, 49.7, 41.7, 40.5,
36.0, 35.6, 31.8, 29.3, 28, 26.9, 24.6, 24.4, 23.6. MS: found 1529.9103,
calcd for C86H120N12O13 [M + 1] 1529.9176.
1
yield, as a brown glass. H NMR (CDCl3): 1.31 (18H, s), 1.25-1.50
(4H, m), 1.85-2.00 (4H, m), 2.48-2.70 (2H, m), 3.36 (4H, m), 3.53-
3.58 (2H, m), 3.73-3.86 (16H, m), 3.92-4.13 (9H, m), 4.98-5.01
(4H, 2 × s), 6.18 (2H, m), 6.30 (2H, m), 6.71 (2H, m), 6.88 (4H, m),
7.13 (4H, m), 7.30-7.43 (13H, m). 13C NMR (CDCl3): 157.0, 149.9,
148.1, 142.1, 141.1, 137.7, 137.0, 130.9, 128.6, 128.5, 128.3, 127.6,
127.2, 118.0,117.5,114.6,113.8, 107.3, 102.7, 70.8, 70.3, 67.9, 49.4,
41.8, 34.6, 33.6, 33.2, 32.1, 31.3, 29.4, 24.9, 22.4, 18.9. MS: found
1217.7141, calcd for C74H97N4O11 [M + 1] 1217.7154.
NH2-cyclophane-[2]Rotaxane (22). A 100 mg (0.064 mmol)
sample of BocNH-cyclophane-[2]rotaxane 21 was dissolved in 1.0
mL of CH2Cl2. To this solution was added 0.3 mL of trifluoroacetic
acid, dissolved in 0.5 mL of CH2Cl2. The reaction mixture was stirred
for 5 h, washed with 2 M KOH, and dried over Na2SO4. The organic
phase was removed in vacuo to give 75 mg (0.062 mmol) of [2]rotaxane
1
22 in a 98% yield, as a pink glass. H NMR (CDCl3): 1.31 (18H, s),
1.39-1.58 (4H, m), 1.81-2.02 (2H, m), 2.10 (3H, s), 2.02-2.15 (6H,
m), 2.17-2.32 (6H, m), 2.45-2.61 (2H, m), 3.39 (12H, s), 3.33-3.93
(24H, m), 3.93-4.23 (17H, m), 6.13-6.30 (8H, m), 6.50-6.86 (6H,
m), 7.04-7.19 (7H, m). 13C NMR (CDCl3): 172.6, 168.8, 157.4, 152.7,
150.9, 148.1, 142.3, 140.8, 139.7, 137.9, 135.9, 127.9, 127.5, 124.7,
122.9, 114.6, 114.3, 107.2, 105.0, 101.8, 70.1, 69.6, 68.1, 64.3, 55.6,
53.3, 49.4, 48.8, 46.2, 43.6, 39.1, 38.6, 37.5, 36.5, 34.7, 31.2, 30.4,
29.7, 27.9, 25.9, 22.3, 21.1. MS: found 1548.8770, calcd for C89H122N5O18
[M + 1] 1548.8785.
(Boc)3Arg-cleft-[2]Rotaxane (26). A 410 mg (0.31 mmol) sample
of the NH2-cleft-rotaxane 25 and 300 mg (0.64 mmol) of (Boc)3ArgOH
were dissolved in 3.5 mL of CH2Cl2. To this solution was added 9 mg
(0.06 mmol) of 1-hydroxybenzotriazole (HOBt), followed by a rapid
injection of 132 mg (0.64 mmol) of DCC, dissolved in 0.5 mL of CH2-
Cl2. The reaction mixture was stirred for 2 h and diluted with 40 mL
of CH3CN, and the precipitated DCU was removed by filtration. After
collection and removal of the solvent in vacuo, the crude material was
separated by means of radial centrifugal chromatography (5:100 MeOH/
CH2Cl2 to 20:100 MeOH/CH2Cl2) to give 609 mg (0.28 mmol) of
rotaxane 26 in a 91% yield, as a pink foam (mp ) 96-99 °C dec). 1H
NMR (CDCl3): 1.26 (18H, s), 1.40-1.50 (54H, m), 1.56-2.06 (18H,
m), 3.09-3.31 (2H, m), 3,34-3.56 (2H, m), 3.56-3.93 (8H, m), 3.93-
4.26 (16H, m), 4.41-4.53 (2H, m), 4.96-5.00 (4H, 2 × s), 5.83-6.10
(2H, m), 6.68-6.93 (8H, m), 6.94-7.15 (6H, m), 7.22-7.45 (13H,
m), 7.63 (2H, m), 7.77 (2H, m). 13C NMR (CDCl3): 171.8, 171.2, 170.4,
163.1, 163.0, 160.6, 160.5, 157.1, 156.9, 156.1, 155.5, 154.6, 143.4,
137.3, 137.0, 131.5, 131.1, 130.1, 128.5, 128.4, 127.8, 127.7, 127.6,
127.3, 114.5, 114.1, 113.7, 107.0, 84.0, 84.0, 79.7, 71.1, 69.8, 69.7,
68.4, 55.6, 53.4, 49.1, 48.6, 43.8, 41.5, 39.2, 34.8, 34.7, 33.7, 31.4,
30.9, 28.3, 28.2, 27.9, 25.3, 24.9, 24.3. MS: found 2130.2234, calcd
for C116H169N12O25 [M + 1] 2130.2322.
Cyclophane-[2]Rotaxane (1). A 140 mg (0.055 mmol) sample of
(Boc)3Arg-cyclophane-[2]rotaxane 23 was dissolved in 3 mL of CH2-
Cl2 and 3 mL of acetic acid. Then 3 mL of TFA was added dropwise
to this solution. After stirring for 24 h, volatile materials were removed
under reduced pressure. The residue was separated using column
chromatography (CH2Cl2/TFA 100:1 to CH2Cl2/TFA/MeOH 50:1:50)
to give 87 mg (0.047 mmol) of rotaxane 1 in a 85% yield, as a brown
1
glass. H NMR (CDCl3): 1.16 (18H, s), 1.35-1.56 (4H, m), 1.63-
1.79 (8H, m), 1.90-2.14 (10H, m), 2.18 (3H, s), 2.25-2.52 (4H, m),
3.10-3.30 (4H, m), 3.34 (12H, s), 3.40-3.90 (18H, m), 3.90-4.30
(23H, m), 4.63 (2H, m), 6.37 (4H, s), 6.74 (4H, d, J ) 8.5 Hz), 6.82
(4H, m), 6.95-7.15 (6H, m), 7.15-7.60 (3H, m). 13C NMR (CDCl3):
175.5, 167.4, 160.7 (q, J ) 40 Hz), 158.9, 158.5, 154.1, 152.5, 148.6,
145.8, 142.4, 139.2, 137, 132.9, 132.6, 129.3, 125.1, 124.7, 118.9, 117.1
(q, J ) 290 Hz), 114.0, 113.9, 107.0, 106.4, 71.8, 71.2, 71.0, 70.8,
69.6, 69.4, 65.6, 56.7, 54.8, 53.9, 50.3, 49.7, 47.3, 41.7, 40.6, 36.0,
35.6, 32.8, 31.8, 31.4, 29.2, 28.8, 26.9, 24.4, 23.8, 20.6. MS: found
1861.0824, calcd for C101H146N13O20 [M + 1] 1861.0807.
Cell Studies. COS-7 African green monkey kidney cells (ATCC
CRL 1651) were propagated in Dulbecco’s modified Eagle’s medium
with 4 mM glutamine adjusted to contain 1.5 g/L sodium bicarbonate
and 4.5 g/L glucose (90%) and fetal bovine serum (10%) (GIBCO/In
Vitrogen). Cells were grown at 37 °C in a 5% CO2 humidified incubator.
Cells were subcultured 1:8 twice per week. For analytical studies, cells
were plated for 24 h on 11 × 11 mm glass coverslips (Corning) in
12-well culture plates (Fisher). Individual slides were placed in
individual wells containing 1 mL phosphate buffer (50 mM, pH 7.0)
and exposed to either (i) fluorescein (0.4 µM), (ii) fluorescein (0.4 µM)
and cleft-[2]rotaxane 2 (6 µM or 60 µM), (iii) fluorescein (0.4 µM)
and cyclophane-[2]rotaxane 1 (6 µM or 60 µM), or (iv) fluorescein-
PKC inhibitor conjugate (0.4 µM) and cyclophane-[2]rotaxane 1 (60
µM). After ca. 30 min, the fluorescence intensity of the cells was
analyzed via fluorescence microscopy. For weakly fluorescent cells,
the film was exposed for 15-30 s, and for strongly fluorescent cells,
the film was exposed for 3-5 s. These studies were repeated twice,
producing similar results.
(Boc)3Arg-cyclophane-[2]Rotaxane (23). [2]Rotaxane 23 was
created using the same synthetic and purification procedures used to
construct [2]rotaxane 26, except that 255 mg (0.145 mmol) of
[2]rotaxane 22 and 143 mg (0.30 mmol) of (Boc)3ArgOH were
dissolved in 0.2 mL. To this solution was added 10 mg (0.075 mmol)
of HOBt and 25 mg (0.31 mmol) of DCC dissolved in 1 mL.
Purification of the crude material gave 221 mg (0.089 mmol) of rotaxane
1
23 in a 62% yield, as a pink form (mp )128-130 °C dec). H NMR
(CDCl3): 1.29 (18H, s), 1.42-1.49 (58H, m), 1.58-2.03 (10H, m),
2.11 (3H, s), 2.20-2.35 (12H, m), 2.74-2.92 (2H, m), 3.12-3.31 (2H,
m), 3.42 (12H, s), 3.47-3.56 (2H, m), 3.56-3.90 (20H, m), 3.97-
4.38 (21H, m), 4.37-4.54 (2H, m), 6.27 (4H, s), 6.68-6.86 (8H, m),
7.00-7.28 (6H, m), 7.35-7.51 (3H, m). 13C NMR (CDCl3): 175.7,
172.2, 171.2, 170.5, 168.6, 163.1, 160.5, 159.4, 157.2, 155.4, 154.6,
Acknowledgment. Acknowledgment is made to the donors
of the Petroleum Research Fund, administered by the ACS, for
partial support of this research. We also thank the University
of Cincinnati for partial funding.
JA034918C
9
J. AM. CHEM. SOC. VOL. 125, NO. 27, 2003 8301