Determining the binding and intracellular transporting abilities of a host-[3]rotaxane

Article abstract of DOI:10.1021/jo0623641

(Chemical Equation Presented) The cellular permeability of compounds can be enhanced in the presence of a host-[2]rotaxane (HR). The effective concentration of an HR is limited by the stoichiometry of the complex formation of the HR and the delivered comp

Full text of DOI:10.1021/jo0623641

Determining the Binding and Intracellular Transporting Abilities of
a Host-[3]Rotaxane
Xiaofeng Bao,† Idit Isaacsohn,‡ Angela F. Drew,‡ and David B. Smithrud*,†
Department of Chemistry, UniVersity of Cincinnati, Cincinnati, Ohio 45221, and the Department of
Genome Science, UniVersity of Cincinnati, Cincinnati, Ohio 45237
daVid.smithrud@uc.edu
ReceiVed NoVember 15, 2006
The cellular permeability of compounds can be enhanced in the presence of a host-[2]rotaxane (HR).
The effective concentration of an HR is limited by the stoichiometry of the complex formation of the
HR and the delivered compound. We speculate that a complex forms between the HR and a guest during
membrane passage. To further explore the relationship between guest binding and guest delivery and to
obtain more efficient delivery devices, we present, in this report, the first example of a cyclophane-[3]-
rotaxane (Cy3R), which has two wheels and a cyclophane as a blocking group. The properties of Cy3R
were compared to a new cyclophane-[2]rotaxane (Cy2R) that has the same cyclophane pocket as Cy3R
but only a single wheel. The second wheel of Cy3R can form additional noncovalent bonds, e.g., salt
bridges, cation-π interactions or aromatic-aromatic interactions, with appropriately functionalized guests.
We show by flow cytometric analysis that Cy3R transfers Fl-AVWAL (76%) and to a lesser degree
Fl-QEAVD (26%) into live cells. The level of Fl-peptide within a cell is concentration dependent and
largely temperature and ATP independent, suggesting that a Cy3R‚Fl-peptide complex passes through
the cellular membrane without requiring active cell-mediated processes. Cy2R, on the other hand, forms
weaker complexes and requires a higher concentration to transfer materials into cells. These results
demonstrate that the addition of a second wheel on a rotaxane can improve guest binding in various
solvents and hence delivery through cellular membranes.
Introduction
can result from alterations in solvent, pH, or temperature. To
overcome these shortcomings, mimetic proteins are being
extensively pursued. A conceptually simple solution would be
to replace the unstable parts of a protein with a synthetic
scaffold. These mimetics would be cheap to produce, stable to
changes in the environment, and their function could be readily
modified by attaching different amino acids. Amino acids, their
side chains, or short peptides have been attached to cyclodex-
trins,1-5 cyclophanes,6-8 sugars,9,10 or steroids and bile acids.11-17
Proteins rely on highly organized recognition domains to
perform the various functions necessary for cell survival.
Modifying proteins to perform other functions or to operate in
non-native environments, however, can be problematic because
function requires the protein to be correctly folded. Denaturation
† Department of Chemistry.
‡ Department of Genome Science.
10.1021/jo0623641 CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/04/2007
3988
J. Org. Chem. 2007, 72, 3988-4000

Binding and Transporting Abilities of Host-[3]Rotaxanes
FIGURE 1. The host-rotaxanes used in the studies. Cy2R 2 and Cy3R differ from Cy2R 1 by having a larger pocket and an additional piperidinyl
ring (indicated by an arrow) that give the pockets greater rigidity, but moves the wheel further away from the pocket.
Synthetic scaffolds, being formed of covalent bonds, are
incapable of aligning the recognition elements in the same spatial
arrangement as proteins, which use a myriad of noncovalent
bonds to obtain their unique structures. Newer protein mimetics,
such as the template assembled synthetic proteins (TASPs),18-25
are more protein-like, relying on oligo- or polypeptides attached
to scaffolds to form secondary structures. As mimetics become
more similar to proteins, however, they will develop the same
problems as proteins, such as incorrect folding, instability to
changes in the environment, and high production costs. Thus,
one option available for mimetic construction is the use of
covalent bonds, which are stable, but give a nonoptimal
orientation of functional groups. Another possibility is the
creation of assemblages through noncovalent bonds, which give
a greater variation of functional group orientation, but are less
stable. A third choice is now available with the successful
demonstration of protein mimetics that are formed from
interlocked molecules.
Rotaxanes comprise a class of interlocked molecules contain-
ing a wheel threaded onto an axle with blocking groups on the
ends to keep the wheel from unthreading.26-33 We converted
rotaxanes into protein mimetics by swapping a blocking group
with an aromatic rich pocket (cleft,34 cyclophane,34 or calix[4]-
arene35). The binding domain of these host-rotaxanes (HRs) is
divided between the pocket and the wheel (Figure 1), which is
a dibenzyl-24-crown-8 ring derivatized with arginines. Tight
complexes are formed with guests of various sizes and polarities
in DMSO, water, CHCl3, and mixed solvent systems. The HRs
form different conformations in different solvents.36 In an
aqueous environment, the aromatic moieties of the wheel and
the pocket tend to assemble. This “closed” conformation
produces an apolar binding domain that forms strong interactions
with apolar moieties of a guest. In an apolar environment, the
dominant “open” conformation has the wheel residing over the
ammonium ion of the axle.
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In the presence of HRs, some impermeable materials can be
brought into cells.34,36,37 We have shown that efficient transfer
of material depends on the rotaxane forming the different
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J. Org. Chem, Vol. 72, No. 11, 2007 3989

Bao et al.
conformations in response to changes in the environment that
occur during the transfer process.36 The concentrations of the
components have to be at least in the low micromolar range to
obtain an observable amount of intracellular material. This
minimal concentration is consistent with magnitude of the KA’s
for the complexes in buffered water (pH 7.3), DMSO, and
CHCl3, which range from 2 × 104 to 9 × 105 M-1. These
solutions were used to represent the polarities of the aqueous
domain, cellular surface, and lipid portion of cells, respectively.38
We also observed a slight concentration dependence for the
amount of material that entered the cells.36 These results suggest
that the HRs are acting as transporters, whereby an HR forms
a noncovalent complex with an impermeable material and carries
it across the membrane. Such a transport process would depend
on the ability of an HR to form a complex in the environments
that impede delivery.
Herein, we compare the abilities of Cy3R and Cy2R 2 to
bind highly charged and apolar guests in various solvents
(buffered water, DMSO, and MeCN) and determine whether
an observed binding selectivity correlates to more material being
brought into COS 7 cells. Fl-AVWAL and Fl-QEAVD were
initially chosen to test transfer. Fl-AVWAL has apolar side
chains. The Cy3R‚Fl-AVWAL complex should be more stable
than the Cy2R 2‚Fl-AVWAL complex in water because of the
additional aromatic surfaces of Cy3R. Fl-QEAVD contains
negatively charged side chains. The additional arginine moieties
of Cy3R should make additional salt bridges with the negatively
charged side chains. Thus, the Cy3R‚Fl-QEAVD complex
should be more stable than the Cy2R 2‚Fl-QEAVD complex in
apolar solvents or environments. Buffered water, DMSO, and
MeCN were chosen to represent the environments outside the
cells, at the cell surface, and within the lipid membrane,
respectively. MeCN was chosen instead of CHCl3, which was
used in earlier studies, to increase the solubility of some of the
highly charged guests. The interaction strength for charged or
polar groups diminishes from water to DMSO to MeCN. Thus,
we predicted that apolar guests would be bound the strongest
in buffered water and charged guests would be bound the
strongest in MeCN. A comparison is also made between the
new HRs and Cy2R 1.
We discovered that Cy3R can form substantially more
favorable complexes than Cy2R 2. Cy3R efficiently transports
both Fl-peptides into cells, whereas Cy2R 2, surprisingly, does
not. We initially thought that the properties of Cy2R 2 and Cy2R
1 would be similar because Cy2R 2 is a modified version of
Cy2R 1. Cy2R 2’s pocket is slightly larger and contains an
additional piperidinyl ring (Figure 1). Also contrary to our
expectations, we found that more stable complexes exist for
some of the charged and polar guests in DMSO than in MeCN.
The results of these experiments suggest that the open and closed
conformations are important features of Cy3R and Cy2R 2.
Having two wheels, Cy3R can exist in an open conformation
for one wheel and a closed conformation for the other (an open-
closed conformation). These dominant conformations are most
likely responsible for the unexpected binding energy and,
consequently, responsible for the different transporting abilities
of Cy3R and Cy2R 2. We found that for HRs that form a very
stable complex with a guest in DMSO, which has a similar
polarity as the cell surface, significantly enhanced amounts of
guest peptide enters into cells in the presence of the HR.
Complex formation depends on the intrinsic attraction
between an HR and a guest and the concentration of the
components. To more fully explore the necessity of complex
formation for delivery, a host-[3]rotaxane (Cy3R, Figure 1) and
Cy2R 2 were created. The HRs contain a larger, more rigid
cyclophane pocket than for Cy2R 134 to potentially produce
more stable complexes. Cy3R contains a second wheel. We
envisioned that the additional aromatic rings of the second wheel
would stabilize guests in an aqueous environment, and that the
additional arginine moieties would more efficiently cover polar
or charged groups of a guest as it passes through the membrane.
The arginines could also enhance the effective molarity of the
host-rotaxane at the cell surface through their attraction to the
phosphates on the cell surfaces. These HRs should form
complexes with substantially different stabilities, and these
differences should be reflected in the amount of material brought
into cells if transport occurs.
(21) Causton, A. S.; Sherman, J. C. Bioorg. Med. Chem. 1999, 7, 23-
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Results
Design of the Host-Rotaxanes and Experimental Methods.
Of proteins, host-rotaxanes most closely mimic antibodies.
Antibodies can contain small binding domains and use a few
to all of six hypervariable peptide loops (complementary
determining regions) to bind a ligand.39-41 We envisioned the
pocket of the host-rotaxane being a mimic of the shallow groove
and a functionalized dibenzyl-24-crown-8 ring mimicking a
hypervariable loop. Fluorescein was chosen as the initial guest
since its association with a host can be monitored by fluores-
cence quenching assays, and cellular delivery can be monitored
(30) Clemente-Leon, M.; Marchioni, F.; Silvi, S.; Credi, A. Synth. Met.
2003, 139, 773-777.
(31) Armaroli, N.; Balzani, V.; Collin, J. P.; Gavina, P.; Sauvage, J. P.;
Ventura, B. J. Am. Chem. Soc. 1999, 121, 4397-4408.
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31, 405-414.
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the World of Molecular Topology; Sauvage, J.-P., Dietrich-Buchecker, C.
O., Eds., Wiley-VCH: Weinheim, Germany, 1999.
(34) Dvornikovs, V.; House, B. E.; Kaetzel, M.; Dedman, J. R.; Smithrud,
D. B. J. Am. Chem. Soc. 2003, 125, 8290-8301.
(35) Smukste, I.; House, B. E.; Smithrud, D. B. J. Org. Chem. 2003,
68, 2559-2571.
(36) Bao, X.; Isaacsohn, I.; Drew, A. F.; Smithrud, D. B. J. Am. Chem.
Soc. 2006, 128, 12229-12238.
(37) Wang, X.; Bao, X.; McFarland-Mancini, M.; Isaacsohn, I.; Drew,
A. F.; Smithrud, D. B. J. Am. Chem. Soc. In press.
(38) Avdeef, A. Absorption and Drug DeVelopment: Solubility, Perme-
ability, and Charge State; Wiley-Interscience: Hoboken, NJ, 2003: Chapter
5.
(39) Padlan, E. A. Antibody-Antigen Complexes; R.G. Landes Com-
pany: Austin, TX, 1994.
(40) Getzoff, E. D.; Geysen, H. M.; Rodda, S. J.; Alexander, H.; Tainer,
J. A.; Lerner, R. A. Science 1987, 235, 1191-1196.
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Sci. U.S.A. 1985, 82, 178-182.
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Binding and Transporting Abilities of Host-[3]Rotaxanes
SCHEME 1
via fluorescence microscopy. Therefore, the host-rotaxanes
contain recognition groups that are similar to the side chains
found in antibodies that bind fluorescein. For example, fluo-
rescein is bound by antibody 4-4-20 in an aromatic slot and
makes contact with three tyrosines and two tryptophan side
chains.42 Arginine residues contact the negatively charged
enolate oxygen atom and the carboxylate of fluorescein or
fluorescein derivatives.42,43
assays. Fl-peptides and fluorescein were also chosen to test the
ability of the host-rotaxanes to deliver materials into cells.
Synthesis of the Host-Rotaxanes. The construction of the
cyclophane pocket was based on the known cyclophane
chemistry45 (Scheme 1). The DCC-rotaxane method48 was used
to construct the rotaxane. â-Alanine was attached to the
piperidinyl ring to provide a primary amine, which was coupled
to DCC-rotaxane 6 after cyclophane 5 was formed. A mixture
of [2]rotaxane 7 and [3]rotaxane 11 (30 and 55% yields,
respectively) was obtained by adding 2 equiv of DCC-rotaxane
6 to cyclophane 5. The primary amine of the [2]rotaxane was
protected with trichloroacetate. This protecting group is stable
to the reagents needed to attach and subsequently deprotect the
Boc3-arginines. It also can be selectively removed to enable the
conversion of Cy2R 2 to additional host-[3]rotaxanes using the
DCC-rotaxane method.
The Complexation of Highly Charged Guests. Ac-Trp-
CO2H, Ac-Glu-Trp-Glu-CONH2, Ac-Glu-Trp-Arg-CONH2, and
Ac-Arg-Trp-Arg-CONH2 were used as guests to determine the
advantage the additional arginine moieties of Cy3R have on
guest recognition. Since the hydrogen bonding ability of the
solvents used in the assays increases from MeCN to DMSO to
H2O, the strength of a salt bridge between the glutamic acid
residue of these guests and a guanidinium moiety of a wheel
should be the strongest in MeCN and the weakest in H2O.
Theoretical calculations have shown that the strength of
cation-π interactions is solvent independent.49 Thus, similarly
stabilizing cation-π interactions could exist between the
arginine side chains and the aromatic rings of the HRs and the
guests in the solvents used in this study. The indole side chain
of the guests will form favorable aromatic-aromatic interactions
with the HRs. In water, desolvation of the indole ring of the
guests and the apolar surfaces of an HR will drive association
in accordance with the hydrophobic effect.50,51
Considering that one face of Cy2R 1 interacts with a guest,
there are five aromatic rings (four in the cyclophane and one in
the crown ether) and one arginine residue available for guest
recognition. The methoxy derivatized rings of Cy2R 1 give it a
deep, polarizable aromatic pocket.44,45 Benzylic rings were used
on the axle side of the pocket to enable the recognition groups
of the wheel and the pocket to form a tight pocket to bind small
guests. The pocket of Cy2R 1 is not rigid, however, and thus it
is not optimized for guest association. The benzylic rings could
collapse, blocking a guest from the pocket. Cy3R and Cy2R 2
contain octamethoxy cyclophanes with piperidinyl rings on both
ends to obtain more open, rigid pockets (Figure 1). The size of
the pockets was also increased by two -CH2- units. Diederich
showed that these cyclophanes are ideal for binding aromatic
guests.45-47 A series of guests were chosen to determine the
extent to which the additional aromatic surfaces and arginine
moieties of the Cy3R can contribute to a host-guest complex.
Because most host-guest complexes form in the low micro-
molar range, fluorescent guests were used to enable association
constants (KA’s) to be obtained through fluorescence quenching
(42) Herron, J. N.; Terry, A. H.; Johnston, S.; He, X.-M.; Guddat, L.
W.; Voss, E. W., Jr.; Edmundson, A. B. Biophys. J. 1994, 67, 2167-2183.
(43) Honegger, A.; Spinelli, S.; Cambillau, C.; Plu¨ckthun, A. Protein
Sci. 2005, 14, 2537-2549.
(44) Diederich, F. In Cyclophanes; Stoddart, J. F., Ed.; Monographs in
Supramolcular Chemistry; The Royal Society of Chemistry: London, 1991.
(45) Ferguson, S. B.; Seward, E. M.; Diederich, F.; Sanford, E. M.; Chou,
A.; Inocencio-Szweda, P.; Knobler, C. B. J. Org. Chem. 1988, 53, 5593-
5595.
(48) Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 3, 2485-2486.
(49) Gallivan, J. P.; Dougherty, D. A. J. Am. Chem. Soc. 2000, 122,
870-874.
(50) Xu, H. F.; Dill, K. A. J. Phys. Chem. B 2005, 109, 23611-23617.
(51) Dill, K. Biochemistry 1990, 29, 7133-7155.
(46) Diederich, F. Angew. Chem., Int. Ed. Engl. 1988, 27, 362-386.
(47) Krieger, C.; Diederich, F. Chem. Ber. 1985, 118, 3620-3631.
J. Org. Chem, Vol. 72, No. 11, 2007 3991

Bao et al.
TABLE 1. Association Constants (KA, M-1)a for HR-guest
water molecules is actually slighter larger for Ac-Glu-Trp-Glu-
CONH2 than for Ac-Arg-Trp-Arg-CONH2. Apparently, salt-
bridge formation with the HR is strong enough to overcome
this penalty. Because the KA’s for the HRs bound to the
carboxylic guests are approximately equivalent, the second
wheel of Cy3R does not contribute to the binding free energy
of the complexes. On the other hand, there is at least a 3-fold
larger KA for the association of Ac-Arg-Trp-Arp-CONH2 with
Cy3R as compared to Cy2R 2. The second wheel of Cy3R
stabilizes the complex, most likely through an additional
cation-π interaction between a guanidinium moiety of the guest
and an aromatic ring of the wheel (see Supporting Information
for molecular modeling picture of this complex).
Complexes Obtained through Fluorescence Quenching Assays
KA,Cy3R
/
b
c
guest
Ac-Trp-CO2H
solvent KA,Cy3R KA,Cy2R 2 KA,Cy2R 2
waterd
19
18
7
1.0
2.3
1.7
1.1
1.3
15
DMSO 17
MeCNe 55
32
16
16
33
15
11
25
ND
ND
ND
17
17
13
16
18
5
Ac-Glu-Trp-Glu-CONH2
Ac-Glu-Trp-Arg-CONH2
Ac-Arg-Trp-Arg-CONH2
Ac-Trp-CO2Me
water
DMSO 21
MeCN
water
DMSO 18
MeCN
water
DMSO 11
MeCN
water
DMSO 44
MeCN
water
DMSO 18
MeCN
water
DMSO 14
18
490
17
1.1
1.6
1.7
> 3
>11
>1
1.5
2.6
1.6
1.4
1.0
4.3
2.2
3.4
1.9
3.2
2.5
42
3
1
26
To determine whether salt bridges would form between the
guests and both wheels of Cy3R in apolar environments, such
as cell surfaces and lipid layers, complexation was investigated
in DMSO and MeCN. The properties of DMSO and MeCN are
similar except that DMSO is a moderately strong H-bond
acceptor and MeCN is a poor H-bond acceptor.53,54 DMSO is
also known to interact strongly with proteins.55 The guest’s
carboxylates were presented as tetramethyl ammonium salts to
enhance their solubilities. Most KA’s derived for complexes in
DMSO are similar to the ones obtained in the aqueous solution.
Cy3R is more selective for Ac-Arg-Trp-Arg-CONH2, as com-
pared to Cy2R 2, in DMSO than in water (11-fold vs 3-fold,
respectively). This selectivity could arise through the formation
of more stable cation-π interactions in DMSO than in water
or because Cy3R is able to form a better binding conformation
(see Discussion). As expected, the largest KA’s and greatest
binding selectivity are observed for the association of the
carboxylic guests in MeCN, which interacts weakly with charged
groups. More importantly, a 15-fold larger KA is seen for the
Cy3R‚Ac-Glu-Trp-Glu-CONH2 complex as compared to Cy2R
2‚Ac-Glu-Trp-Glu-CONH2 complex (Figure 2A; see Supporting
Information for a molecular modeling picture). This result is
consistent with the second wheel of Cy3R forming an additional
salt bridge with a Glu side chain of Ac-Glu-Trp-Glu-CONH2.
The Complexation of Aromatic Guests. A series of
compounds with extensive aromatic surfaces were used as guests
to determine the extent to which the second wheel of Cy3R
contributes to binding of aromatic compounds. For the series
Ac-Trp-OMe, Ac-Trp-Phe-OMe, Ac-Trp-Phe-Phe-OMe in the
buffered solution, only the Cy3R‚Ac-Trp-Phe-Phe-OMe com-
plex shows a significantly larger KA in water (Table 1). This
result is consistent with the hydrophobic effect.50,51 Removing
water molecules from aromatic surfaces and back to the bulk
provides a large favorable entropic term for the association of
aromatic rings. The fact that the tripeptide Ac-Trp-Phe-Phe-
OMe is necessary to produce an uniquely favorable complex
indicates that the hydrophobic pocket of Cy3R is large. A large
guest maximizes the contact surface area with the hydrophobic
groups of Cy3R, resulting in a greater number of released water
molecules. The observed drop in the KA’s for the HRs bound
to Ac-Trp-Phe-Phe-OMe in DMSO and MeCN demonstrates
the importance of the hydrophobic effect for the large KA’s
observed in buffered water. In the three solvent systems, both
wheels of Cy3R contribute to the binding event.
21
21
Ac-Trp-Phe-OMe
22
49
Ac-Trp-Phe-Phe-OMe
22
4
MeCN
water
13
51
7
Fl-Ala-Val-Trp-
Ala-Leu-CONH2
16
150
(38)
24
13
92
(35)
35
DMSO 410
(100)f
97
26
MeCN
water
4.0
2.0
4.3
Fl-Gln-Glu-Ala-
Val-Asp-CONH2
DMSO 400
(20)
MeCN
>300
(70)
26
>9
H2N-Ser-Tyr-Ser-Met-Glu-
water
15
1.4
4.2
1.8
0.8
2.3
-
1.5
1.0
1.5
1.0
2.0
2.4
His-Phe-Arg-Trp-Gly-CO2H DMSO 43
10
(hormone 1)
MeCN
water
DMSO 23
MeCN
water
880
14
483
17
10
INS
29
CO2H-Glu-His-Trp-Ser-Tyr-
Gly-Leu-Arg-Pro-Gly-NH2
(hormone 2)
INS
42
fluorescein
DMSO 40
37
MeCN
water
DMSO 24
MeCN 92
41
283
27
270
12
pyrene
39
a The assays were performed at room temperature, the standard deviation
is less than 10% for each KA, and the tabulated KA values have been divided
by 1 × 103. b KA for Cy3R complexes. c KA for Cy2R 2 complexes. d 98%
water (phosphates 1 mM, pH 7.0)/2% DMSO. e 98% MeCN/2% DMSO.
f Parenthetical values are the KA’s for two HRs bound to a guest.
In a buffered solution (1 mM phosphate, pH 7.0), similar
KA’s are observed for these guests bound to Cy3R and Cy2R 2
except for Ac-Arg-Trp-Arg-CONH2, which has a significantly
lower KA (Table 1). The more stable complexes for Ac-Trp-
CO2H, Ac-Glu-Trp-Glu-CONH2, and Ac-Glu-Trp-Arg-CONH2
could be a result of a salt bridge that forms between a
guanidinium moiety of an HR and a guest’s carboxylate, which
does not exist for Ac-Arg-Trp-Arg-CONH2. Another possibility
is that greater energy is required to desolvate the arginine side
chains of Ac-Arg-Trp-Arg-CONH2 as compared to the car-
boxylates of the other guests that occurs during the binding
event. Host-guest complexation is similar to the transport of
amino acids from an aqueous phase into octanol. Water/octanol
distribution parameters of amino acids are available and can be
combined to calculate the distribution parameters of the trip-
eptides.52 Calculated log D values show that the tripeptides are
highly hydrophilic and that the desolvation penalty for removing
(53) Barton, A. F. M. CRC Handbook of Solubility Parameters and other
Cohesion Parameters; CRC Press: Boca Raton, FL, 1991.
(54) Gokel, G. W. Dean’s Handbook of Organic Chemistry, 2nd ed.;
McGraw-Hill: New York, 2004
(55) Jackson, M.; Mantsch, H. H. Biochim. Biophys. Acta 1991, 1078,
231-235.
(52) Tao, P.; Wang, R.; Lai, L. J. Mol. Model. 1999, 5, 189-195.
3992 J. Org. Chem., Vol. 72, No. 11, 2007

Binding and Transporting Abilities of Host-[3]Rotaxanes
aromatic-aromatic interactions. The unexpected KA’s can be
explained by a better binding geometry existing for the HRs in
DMSO than in MeCN (see Discussion).
To further explore aromatic-aromatic interaction energies
in HR complexes, pyrene was chosen as a guest. It has an
extensive aromatic surface without the ability to form salt
bridges and H-bonds, which are possible with the peptidic
guests. KA’s for the HRs bound to pyrene are large in the
aqueous solution (2.7 × 105 M-1). The octamethoxy cyclophane
performed as expected by providing tighter complexes of
aromatic compounds in water than Cy2R 134 (KA for the Cy2R
1‚pyrene complex is 8 × 104 M-1). Because the KA’s of the
HRs bound to pyrene are approximately equal, a binding
advantage is not provided by the second wheel of Cy3R.
Desolvation of water molecules provides for the driving force
for the association of pyrene since the complexes in DMSO
and MeCN are substantially weaker. The second ring of Cy3R
appears to be involved in the complex of pyrene in DMSO and
MeCN, giving the observed 2-fold larger KA than observed for
Cy2R 2 binding pyrene.
The Complexation of Fluoresceinated Peptides and Large
Peptides. The fluoresceinated peptides Fl-AVWAL and Fl-
QEAVD were used as guests to determine the abilities of the
HRs to bind larger peptides with aliphatic and aromatic side
chains and with negatively charged side chains, respectively.
Combined with fluorescein, these peptides were also used to
determine the abilities of the HRs to transfer materials into cells.
Fluorescein has an extensive aromatic surface like pyrene, but
it also has two negatively charged groups in pH 7.0 water. The
strong interactions between water molecules and the charged
groups significantly weaken the attraction for fluorescein by
either HR as compared to pyrene (Table 1). Fl-AVWAL is
bound by the HRs in water with KA values that are similar to
those observed for the complexes with Ac-Trp-Phe-Phe-OMe.
This is expected since both guests have multiple apolar side
chains. Cy3R shows a slight selectivity for Fl-AVWAL over
fluorescein. Apparently, the larger Fl-AVWAL and Ac-Trp-Phe-
Phe-OMe make more favorable contact with the second wheel
of Cy3R than smaller guests. Fl-QEAVD is more weakly bound
than Fl-AVWAL by either HR in buffered water. Because of
its negatively charged side chains, Fl-QEAVD has a large
desolvation penalty for host-guest association.
Even though fluorescein forms essentially the same stable
complex in the three solvent systems, the complex strength of
the Fl-peptides shows a dramatic solvent dependency (Figure
2B). Cy2R 2 shows a 4-fold greater selectivity for the binding
of the Fl-peptides in DMSO over the other solvents. Cy3R has
an impressive 10-fold preference for the Fl-peptides in DMSO
as compared to the same complex in buffered water. The
complex of Cy3R‚Fl-QEAVD in MeCN shows a 10-fold larger
KA than the Cy2R 2‚Fl-QEAVD complex. This binding selec-
tivity is also seen for the association of Cy3R with the other
highly negatively charged guest Ac-Glu-Trp-Glu-CONH2. A
greater preference is observed for the binding of Cy3R with
the larger Fl-peptides (2-fold to 10-fold) than fluorescein (1.5
fold), as compared to Cy2R 2. Thus, the larger Fl-peptides make
greater contact with the second wheel of Cy3R than the smaller
fluorescein. The same phenomenon was observed in the aqueous
solution.
FIGURE 2. A comparison of the association constants (KA’s) shows
that Cy3R forms more stable complexes than Cy2R. Cy3R is selective
for (A) Ac-Glu-Trp-Glu-CONH2 in MeCN and (B) the Fl-pentapeptides
in DMSO. A large KA is also observed for Fl-Gln-Glu-Ala-Val-Asp-
CONH2 bound to Cy3R in MeCN.
Two interesting trends are observed for the binding of the
aromatic peptides in DMSO and MeCN. First, equivalently
stable or more stable complexes are formed in DMSO than
MeCN. Because greater energy is required to remove DMSO
from the guanidinium moieties than MeCN, the arginines of
the HRs appear not be involved in the complexes. This would
leave aromatic-aromatic interactions as the driving force for
association. DMSO and MeCN, however, should produce similar
desolvation energies with aromatic surfaces since they have
similar ET(30) values.56 The ET(30) parameters are derived from
the absorbance wavelength of the Reichard’s dye in various
solvents and have been shown to linearly correlate with the free
energy of complexes formed between pyrene and an aromatic
rich cyclophane in various solvents.57 Furthermore, the second
interesting trend is that as the number of aromatic rings within
the guests increases the corresponding KA’s decrease. An
opposite trend would be expected if complexation is driven by
(56) Reichardt, C. SolVents and SolVent Effects in Organic Chemistry;
Wiley-VCH: Weinheim, Germany, 2003.
(57) Smithrud, D. B.; Diederich, F. J. Am. Chem. Soc. 1990, 112, 339-
343.
To more fully explore the size requirement for strong
complexes, commercially available hormone fragments (hor-
mone 1 and hormone 2, Table 1) were investigated as guests.
J. Org. Chem, Vol. 72, No. 11, 2007 3993

Bao et al.
FIGURE 3. Fluorescence microscopy of COS 7 cells incubated with host-rotaxanes. Cells were viewed for Fl-peptide fluorescence (Fl-AVWAL
or Fl-QEAVD uptake; left panels), white light (middle panels), and calcein blue fluorescence (viability; right panels). (A) Cy3R (10 µM) and
Fl-AVWAL (10 µM), (B) Cy3R (10 µM) and Fl-QEAVD (10 µM), (C) Cy2R 2 (10 µM) and Fl-AVWAL (10 µM), (D) Cy2R 2 (50 µM) and
Fl-AVWAL (20 µM), (E) Cy2R 2 (10 µM) and Fl-QEAVD (10 µM), (F) Cy2R 2 (50 µM) and Fl-QEAVD (20 µM), and (G) a typical example of
a Fl-peptide (10 µM) only. Cell viability is similar for each combination of an HR and Fl-peptide (original magnification: 100×).
These 10-mer peptides contain the same amino acids as Ac-
Arg-Trp-Glu-CONH2, but the amino acids are not sequentially
linked together. Hormone 1 and hormone 2 have different
sequences, and three of the amino acids are different (Ser, Met,
and Phe for hormone 1 and Gly, Pro, and Leu for hormone 2).
Considering these differences, we did not expect that the KA’s
for the hormone fragments and for Ac-Arg-Trp-Glu-CONH2
bound to the HRs would be very similar in buffered water and
DMSO. Cy3R binds the hormones better than Cy2R 2, except
for hormone 2 in buffered water, and prefers hormone 1. In
MeCN, the strengths of the complexes are dramatically greater.
The KA’s of the Cy3R‚hormone 1 and Cy3R‚Ac-Glu-Trp-Glu-
CONH2 complexes are similar in MeCN. This suggests that
multiple salt bridges are formed with hormone 1. Interestingly,
a 15-fold larger KA exists for the Cy2R 2‚hormone 1 complex
than the Cy2R 2‚Ac-Glu-Trp-Glu complex. Most likely, the 10-
mer peptide is large enough to interact with both guanidinium
moieties of the single wheel of Cy2R 2. The poor solubility of
hormone 2 precluded KA determination in MeCN.
Cellular Transport Efficiencies of the Host-Rotaxanes. We
recently showed that HRs aid in the delivery of fluorescein and
fluoresceinated peptides into COS 7 cells.34,36,37 Of the Fl-
peptides used previously,37 Fl-AVWAL and Fl-QEAVD were
chosen to test Cy3R and Cy2R 2 as cellular delivery agents. A
comparison of the delivery levels of these guests will demon-
strate any advantage provided by the aromatic and arginine
moieties of the second wheel of Cy3R for transport. Cy3R
displayed a high affinity for Fl-AVWAL (buffer KA ) 5.1 ×
104 M-1, DMSO KA ) 4.1 × 105 M-1) and for Fl-QEAVD
(buffer KA ) 2.6 × 104 M-1, DMSO KA ) 4.0 × 105 M-1).
The magnitudes of these KA’s are similar to the values obtained
for the association of Cy2R 1 with the same Fl-peptides. Cy2R
3994 J. Org. Chem., Vol. 72, No. 11, 2007

Binding and Transporting Abilities of Host-[3]Rotaxanes
FIGURE 4. Relative fluorescence (FITC) of COS 7 cells exposed to Fl-AVWAL (10 µM) or Fl-QEAVD (10 µM) alone, with Cy3R (10 or 5 µM),
or with Cy2R 2 (10 µM) as determined by flow cytometry.
2, on the other hand, forms weaker complexes for these guests,
with generally a 2-fold to 4-fold reduction in KA. If complex
strength in these solvent systems provides a measure of the
delivery levels, we would expect that Cy3R would deliver the
peptides to a similar level as Cy2R 1. Cy2R 2, at the same
concentration, would show a lower level of transport than the
other HRs. Furthermore, raising the concentration of Cy2R 2
to compensate for the weaker complexes formed with the guests
should result in intracellular delivery if host-guest complexation
is necessary for membrane passage.
We used fluorescence microscopy analysis to determine
whether CyR3 and Cy2R 2 delivered the fluoresceinated
peptides at a concentration range consistent with the KA values
for the corresponding complexes. The assay procedure has
previously been described in detail.36 Briefly, COS 7 cells were
incubated with an HR in a phosphate-buffered saline solution
(PBS), pH 7.4, for 30 min before the addition of a Fl-peptide.
After an additional hour, the cells were washed thoroughly, and
calcein blue AM was added to each well to demonstrate cell
viability or potential toxicity after incubation with rotaxanes
J. Org. Chem, Vol. 72, No. 11, 2007 3995

Bao et al.
TABLE 2. Quantification of Fl-Peptide Uptake in COS 7 Cells by Flow Cytometrya
% high
Fl-peptide
cells
% low
Fl-peptide
cells
% total
Fl-peptide
cells
% cbAMc
cells
condition
rt
compound(s)b
Cy3R (10 µM) + Fl-AVWAL
Cy3R (5 µM) + Fl-AVWAL
Cy3R (0.5 µM) + Fl-AVWAL
Cy2R 2 (50 µM) + Fl-AVWAL (20 µM)
Cy2R 2 (10 µM) + Fl-AVWAL
Fl-AVWAL
Cy3R (10 µM) + Fl-QEAVD
Cy3R (5 µM) + Fl-QEAVD
Cy3R (0.5 µM) + Fl-QEAVD
Cy2R 2 (50 µM) + Fl-QEAVD (20 µM)
Cy2R 2 (10 µM) + Fl-QEAVD
Fl-QEAVD
15
5
1
60
53
10
36
8.2
4.9
25
16
8.3
24
75
58
11
39
8
80
76
96
97
84
86
91
77
99
98
86
89
94
3
-
0.3
1
-
0.2
1
1
-
-
5
26
16
8
25
6
5.0
4.6
1.0
5
1
none
4 °C
Cy3R (10 µM) + Fl-AVWAL
Cy3R (5 µM) + Fl-AVWAL
Cy2R 2 (10 µM) + Fl-AVWAL
Cy3R (10 µM) + Fl-QEAVD
Cy3R (5 µM) + Fl-QEAVD
Cy2R 2 (10 µM) + Fl-QEAVD
5
2
3
5
6
1
29
17
14
35
30
10
34
19
17
40
36
11
76
71
76
84
80
77
depleted
ATPd
Cy3R (10 µM) + Fl-AVWAL
Cy3R (5 µM) + Fl-AVWAL
Cy3R (10 µM) + Fl-QEAVD
Cy3R (5 µM) + Fl-QEAVD
2
-
-
1
62
24
21
20
64
24
21
21
81
83
85
68
a All transport assays were performed in PBS (pH 7.3) for 1 h. b [Fl-peptide] ) 10 µM, unless indicated otherwise. c [Calcein blue AM] ) 1 µM. d The
assay solutions contained 2-deoxyglucose and NaN3.
and peptides. Cells were examined via inverted fluorescence
microscopy at 100× magnification (Zeiss Axiovert 200M). High
to moderate fluorescence was observed in cells that were
exposed to Cy3R (10 µM) and Fl-AVWAL (10 µM) (Figure
3A). The majority of cells exposed to Fl-QEAVD (10 µM) and
Cy3R (10 µM) were moderately to weakly fluorescent (Figure
3B). Cells exposed to Cy2R 2 (10 µM) and Fl-FAVWAL (10
µM) or Fl-QEAVD (10 µM) (Figure 3C,E) were only weakly
fluorescent, similar to the background fluorescence obtained
from exposure to the Fl-peptides alone (Figure 3G). Raising
the concentration of Cy2R 2 to 20 µM did not increase the
observed fluorescence (data not shown). Raising the concentra-
tion of Cy2R 2 to 50 µM and Fl-AVWAL or Fl-QEAVD to 20
µM resulted in moderately and weakly fluorescent cells (Figure
3D,F). Strong calcein blue fluorescence was observed in the
majority of cells regardless of treatment, which demonstrates
cell viability.
and Fl-AVWAL from 50 and 20 µM to 10 and 10 µM,
respectively, also resulted in a lowering of the number of
fluorescent cells (39 to 8%). The same concentration dependency
is observed for the transport of Fl-QEAVD. The total fluores-
cence of cells exposed to Fl-QEAVD and an HR, however, is
less than that for cells exposed to Fl-AVWAL and an HR. This
reduction is reasonable considering that QEAVD has charged
side chains and AVWAL does not. The observed concentration
dependency for cellular fluorescence suggests that a noncovalent
complex is formed between a Fl-peptide and HRs during the
penetration process. Cells exposed to Fl-peptides alone resulted
in a small percentage of weakly fluorescent cells, which was
set at 5%. A high proportion of calcein blue positive cells
was observed in most assays (see also Supporting Information),
demonstrating that the HRs and Fl-peptides are only minimally
toxic at these concentrations. Calcein blue and peptide fluores-
cence were independent variables.
Flow cytometry was used to quantify the relative levels of
Fl-AVWAL, Fl-QEAVD, and calcein blue AM within the cells.
After incubating cells with an assay solution, trypsin digestion
was used to lift the cells from the wells and to remove any
residual Fl-peptide that may be nonspecifically attached to the
outer cellular membrane.58 The relative fluorescence intensity
of these cells was quantified by multicolor flow cytometry (BD
FacsAria). Thresholds and gates were set as previously de-
scribed36,37 to enable a direct comparison between the host-
rotaxanes. Cy3R efficiently delivered Fl-AVWAL into a large
percentage of the cells (75% above background; Figure 4 and
Table 2). A small percentage of cells (15%) were highly
fluorescent. A lower total percentage of fluorescent cells existed
when the concentration of Cy3R was lowered to 5 µM (58%)
and to 0.5 µM (11%). Lowering the concentrations of Cy2R 2
Fl-peptide transfer depends on the concentration of an HR
and a guest (Table 2 and Figure 5). The minimal concentration
necessary to observe fluorescent cells matches the minimal
concentration needed to form complexes in the solvents used
in this study. These observations suggest that a host-guest
complex forms during delivery. Cy3R forms tighter complexes
with the Fl-peptides than Cy2R 2 in the solvents used to mimic
the cellular environments. To determine which environment (as
represented by a solvent) is crucial for complex formation, the
ability of Cy3R and Cy2R 1 to deliver fluorescein into cells
was measured. Cy2R 1 binds fluorescein strongly in DMSO
(KA ) 8.6 × 105 M-1 34
) and delivers it into cells. On the other
hand, Cy3R binds fluorescein less favorably in DMSO by a
factor of 20 (KA ) 4.0 × 104 M-1). The KA’s for these
complexes in water only differ by a factor of 1.5. The same
KA’s (within experimental error) are seen for these complexes
in MeCN (4.1 × 104 and 4.3 × 104 M-1). Flow cytometric
results show that fluorescein at 0.5 µM is delivered into a small
(58) Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.;
Gait, M. J.; Chernomordik, L. V.; Lebleu, B. J. Biol. Chem. 2003, 278,
585-590.
3996 J. Org. Chem., Vol. 72, No. 11, 2007

Binding and Transporting Abilities of Host-[3]Rotaxanes
FIGURE 5. Plot of the total fluorescence of cells against the concentration of Cy3R that was used in the assay. The concentration of the Fl-
peptides was 10 µM (Fl-FAVWAL diamonds and Fl-QEAVD squares).
percentage of cells (19%) when combined with Cy2R 1, but
not when combined with Cy3R (Table 3). These results are
consistent with complexation being required for intracellular
delivery and show that strong complexation in DMSO is
required for these HRs to be delivery agents.
TABLE 3. Quantification of Fluorescein Uptake in COS 7 Cells by
Flow Cytometry
total %
Fl-peptide
cells
% cbAM
cells
compound(s)a
Determining the Cellular Delivery Mechanism. Because
some highly argininated peptides enter cells through endocy-
tosis,58 we needed to determine whether Cy3R, which contains
four arginine moieties, uses this pathway to deliver materials.
Energy-dependent pathways were restricted by performing the
delivery assays at 4 °C or by using the classic energy-depleting
cocktail of 2-deoxyglucose and NaN3.58-69 Since some cell death
is caused by these procedures (assessed by cell detachment and
loss of calcein blue fluorescence), further analysis was restricted
to live cells that remained attached to the plates. For the energy-
poisoned assay, cells were preincubated with the cocktail for 1
h prior to the addition of transporter/peptides to this solution.
Cells with a depleted level of ATP showed a similar level of
transport as seen for cells in PBS. For example, a minor
reduction from 75 to 64% fluorescent cells was observed for
cells exposed to 10 µM Cy3R and Fl-AVWAL after ATP
depletion (Table 2). Similarly, 26 and 21% fluorescent cells
were found for cells exposed to 10 µM Cy2R and Fl-QEAVD
after ATP depletion.
Cy2R 1 + fluorescein
Cy3R + fluorescein
fluorescein
19
5.2
4.6
99
94
86
a [HR] ) 5 µM, [fluorescein] ) 0.5 µM.
exposed to Cy3R and Fl-AVWAL. Examination of the cells,
however, showed that precipitation of Fl-AVWAL occurs at
some of the cells (Figure 6A). This precipitation was not
observed for cells exposed to Cy3R and Fl-QEAVD or for cells
exposed to Fl-AVWAL alone. For cyclophanes, lowering the
temperature generally increases the stability of their complexes44
and reduces their water solubility. The precipitated material is
most likely caused by the presence of Cy3R since precipitated
material is not observed for Fl-AVWAL alone at 4 °C. Cy3R
covers some of the hydrophilic moieties of Fl-AVWAL at the
cells surface, which could reduce its solubility and reduce the
level of delivered material. Other cells at 4 °C, however,
fluoresced brightly with well-defined nuclei (Figure 6B). Fl-
QEAVD shows an enhancement in the fluorescent intensity of
cells at 4 °C (Figure 6C) as compared to rt (40 to 26%,
respectively, for 10 µM Cy3r, Table 2). A slightly greater
number of fluorescent cells is also observed for cells exposed
to Cy2R 2 (10 µM) and Fl-QEAVD (10 µM) or Fl-AVWAL
(10 µM) at 4 °C. The greater amount of material present within
live cells at 4 °C is expected for a delivery mechanism that
relies upon complex formation, which can be promoted with a
lowering of the temperature. The similar delivery level observed
at rt and at 4 °C suggests that holes are not formed in the
membrane. Membrane integrity for cells exposed to the HRs at
a concentration to obtain delivery was also verified by the low
percentage of cells containing propidium iodide (8 and 12% of
cells exposed to Cy3R (10 µM) and Cy2r2 (50 µM), respec-
tively, untreated cells: 5%). Similarly, the level of lactate
dehydrogenase released from the cells was not different than
that of untreated cells (Supporting Information).
Performing the assay at 4 °C resulted in a significant reduction
in the number of fluorescent cells from 75 to 34% for cells
(59) Vassiliou, G.; McPherson, R. J. Lipid Res. 2004, 45, 1683-1693.
(60) Iwamoto, M.; Allen, R. D. J. Histochem. Cytochem. 2004, 52, 557-
565.
(61) Panyam, J.; Labhasetwar, V. Pharm. Res. 2003, 20, 212-220.
(62) Langel, U. Cell Penetrating Peptides: Processes and Applications;
CRC Press: Boca Raton, FL, 2002.
(63) Suzuki, T.; Futaki, S.; Niwa, M.; Tanaka, S.; Ueda, K.; Sugiura, Y.
J. Biol. Chem. 2002, 277, 2437-2443.
(64) Futaki, S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda,
K.; Sugiura, Y. J. Biol. Chem. 2001, 276, 5836-5840.
(65) Skiba, P. J.; Zha, X. H.; Maxfield, F. R.; Schissel, S. L.; Tabas, I.
J. Biol. Chem. 1996, 271, 13392-13400.
(66) Pooga, M.; Hallbrink, M.; Zorko, M.; Langel, U. FASEB J. 1998,
12, 67-77.
(67) Afonina, E.; Stauber, R.; Pavlakis, G. N. J. Biol. Chem. 1998, 273,
13015-13021.
(68) Vives, E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 16010-
16017.
The energy-depleted cells were examined under 400×
(69) Derossi, D.; Calvet, S.; Trembleau, A.; Brunissen, A.; Chassaing,
G.; Prochiantz, A. J. Biol. Chem. 1996, 271, 18188-18193.
magnification to further explore the possibility that endocytosis
J. Org. Chem, Vol. 72, No. 11, 2007 3997

Bao et al.
FIGURE 7. Fluorescence photomicrographs showing representative
examples of COS 7 cells exposed to Cy3R (10 µM) and Fl-AVWAL
(10 µM) (A-C) and Fl-QEAVD (10 µM) (D-F) at rt (A and D),
depleted ATP (B and E), and 4 °C (C and F) (original magnification:
400×).
is necessary for the other HRs, as well, since they are built from
similar components (pocket, wheel, axle, and blocking group).
In apolar environments, such as lipids, the open conformation
can exist in Cy3R and Cy2R 2 with the wheel(s) residing at
the axle’s ammonium ion. The geometry of the closed confor-
mation was determined by molecular modeling to be at the two-
carbon chain between the amide moieties of the axle. The
distances between the wheel and the pocket for both conforma-
tions, however, are larger in CyR2 2 and CyR3 as compared to
CyR2 1 because piperidinyl rings separate the wheel(s) from
the pocket (Figure 1). Thus, the closed-closed conformation
of Cy3R (Figure 8) and the closed conformation of Cy2R 2 do
not form a tight aromatic pocket for small guests.
The separation of the wheel from the pocket is most likely
largely responsible for the poor performance of Cy2R 2 to
transfer the pentapeptides and diminishes the ability of Cy3R,
as well. Although Ac-Trp-Phe-Phe-CO2Me and the larger
peptides are large enough to make simultaneous contact with
the cyclophane pocket and aromatic rings of both wheels of
Cy3R, the guanidinium moieties are forced away from the guests
because of steric crowding. The steric clash between the
guanidiniums over the pocket also keeps the smaller guests from
simultaneously interacting with the pocket and both guanidini-
ums simultaneously. Thus, the closed-closed conformation of
Cy3R most likely is not the dominant binding conformation in
aqueous solutions. The optimum conformation for most guests
is the closed-open conformation. It enables a guest to be
embedded with the pocket and interact with both wheels. For
Cy2R 2, the preferred conformation for most guests is the closed
conformation. Because of the piperidinyl ring, the binding of
the guanidinium moiety to the guest forces the wheel near the
pocket.
FIGURE 6. Fluorescence microscopy of COS 7 cells incubated with
Cy3R and Fl-peptides at 4 °C. Cells were viewed for FITC fluorescence
(Fl-AVWAL or Fl-QEAVD uptake; left panels), white light (middle
panels), and calcein blue fluorescence (viability; right panels). Pre-
cipitated material appears to exist for some cells exposed to Cy3R and
Fl-AVWAL (10 µM) (A), whereas other cells are highly fluorescent
(B). Fl-QEAVD (10 µM) shows highly fluorescent cells. Cell viability
is similar for each combination of a Cy3R and Fl-peptide (original
magnification: 100×).
is the entry mechanism for the complexes. COS 7 cells were
grown on microscope slides. After exposing cells to the various
reagents, the unfixed cells were examined by fluorescence
microscopy (Zeiss Axioplan 2). Fl-AVWAL was observed
strongly in the nucleus and less in the cytoplasm (Figure 7A-
C). Fl-QEAVD was more equally distributed throughout the
cellular compartments (Figure 7D-F). The distribution and the
fluorescence intensity of the Fl-peptides are not changed for
cells lacking energy-dependent pathways. This holds for cells
exposed to Cy3R and Fl-AVWAL at 4 °C. These observations
are consistent with the flow cytometric results that suggest that
endocytosis is not the major pathway for the delivery of
materials into cells. Similar results were obtained for other
HRs.36,37
Discussion
The additional argininesprovided by its second wheelsgives
Cy3R a binding advantage over Cy2R 2. For the series Ac-
Trp-CO2Me, Ac-Trp-Phe-CO2Me, and Ac-Trp-Phe-Phe-CO2Me
bound to Cy3R, the indole ring resides in the pocket and one
guanidinium can interact with the carbonyl oxygen atom of the
ester and the other can form a cation-π interaction with a
Host-rotaxanes adjust their binding domains with a change
in environment to maintain stable complexes with a guest. Two
dominant conformations exist (open and closed), depending on
the environment. This feature is necessary for the ability of a
cleft-rotaxane to transfer materials into cells36 and most likely
3998 J. Org. Chem., Vol. 72, No. 11, 2007

Binding and Transporting Abilities of Host-[3]Rotaxanes
FIGURE 8. Conformations of Cy3R that are available in the host-guest complexes. (A) The closed-closed conformation brings the aliphatic and
aromatic surfaces together, but the arginine moieties are moved away from the combining site because of steric crowding. (B) The open-closed
conformation appears to be the optimal conformation for the binding of most guests in this study and forms readily in DMSO. (C) The open-open
conformation is dominant in MeCN, and it is best suited for the binding of large guests.
guest’s aromatic ring. Representative modeling pictures for the
various complexes are available in Supporting Information. For
the larger di- and tripeptides, both guanidiniums can form
cation-π interactions. Similarly aligned complexes can exist
with Ac-Trp-CO2H, Ac-Glu-Trp-Glu-CONH2, and Ac-Glu-Trp-
Arg-CONH2 except that one guanidinium makes a salt bridge
with a guest’s carboxylate. For Ac-Glu-Trp-Glu-CONH2 as a
guest, it can stretch across the pocket to form two salt bridges
with a guanidinium from each wheel. Ac-Arg-Trp-Arg-CONH2
also stretches across the pocket, but in this complex, it is the
guest’s guanidinium groups that make contact with the aromatic
rings of opposing wheels. For Fl-AVWAL and Fl-QEAVD, all
four guanidinium ions of Cy3R can form noncovalent bonds,
whereas Cy2R 2 can use a maximum of two guanidinium ions.
The additional guanidinium ions available for Cy3R readily
account for its stable complexes with the Fl-peptides and the
10-mer peptides.
The modeling results (Supporting Information) and previous
studies36 indicate that complex strength depends on the con-
formation of the host-rotaxane. We also know that the dominant
conformation of the HRs depends on the solvent. These facts
combined provide an explanation for the unexpected discoveries
that some complexes are more stable in DMSO than MeCN
even though there should be a greater desolvation penalty for
DMSO. DMSO interacts favorably with the ammonium ion of
the axle. This enables the wheel to slide more freely along the
axle to adopt stable conformations, such as the open-closed
conformation of Cy3R. In MeCN, a strong noncovalent bond
exists between the axle’s ammonium ion and the wheel with a
bond energy on the order of 1-2 kcal/mol.48 Therefore in
MeCN, the dominant conformation of Cy3R is the open-open
conformation. To form the open-closed conformation in MeCN,
one bond between the ammonium ion and the wheel needs to
be broken. Breaking this bond detracts from the overall binding
free energy.
of Cy3R to interact with the Fl-peptides. Only a single wheel
is close enough to make contact with a Fl-peptide in the open-
open conformation. The large, 10-mer hormone 1 shows a
significantly larger KA in MeCN, as compared to that in DMSO
for both HRs. The open-open conformation for Cy3R and the
open conformation for Cy2R are dominant in MeCN. These
larger binding domains of the HRs are a better fit for the large
peptide. These studies show that guest selectivity for host-
rotaxanes requires a matching of a host-rotaxane with a guest
in the environment in which the complex would be formed.
These selectivity rules are also reflected in the transferring
abilities of the HRs. At the same concentration of 10 µM, Cy3R
transfers Fl-AVWAL and Fl-QEAVD, whereas Cy2R 2 does
not. A comparison of the KA’s shows that Cy3R forms more
stable complexes with the Fl-peptides than Cy2R 2. The weakest
complexes occur between Cy2R 2 and Fl-peptides in buffered
water. This would suggest that it is the lack of complex
formation in the extracellular domain that keeps Cy2R 2 from
being a transporter. On the other hand, Cy3R is a better binding
agent than Cy2R 2 in all three solvents. Therefore, we cannot
determine whether it is the weaker association at the extracellular
domain, the cell surface, the lipid portion of the cells, or all
environments that reduce the delivery efficiency of Cy2R 2.
The importance of complex formation at the cell surface, as
represented by DMSO, is seen by comparing the properties of
Cy2R 1 and Cy3R. Both HRs transfer the Fl-peptides into cells,
and similarly large KA’s exist for the complexes in the three
solvent systems. Fluorescein, however, is observed within cells
for cells exposed to fluorescein and Cy2R 1, but not for cells
exposed to fluorescein and Cy3R (both HRs at 5 µM).
Fluorescein is bound by Cy2R 1 and Cy3R with approximately
equivalent stabilities in buffered water (pH 7.0) and MeCN. In
DMSO, however, Cy2R 1 binds fluorescein with a very large
KA on the order of the Fl-peptides. Cy3R forms a 22-fold less
stable complex with fluorescein in DMSO. The stability of the
Cy3R-fluorescein complex in DMSO (KA ) 4 × 104 M-1) is
on the same order of magnitude as that for complexes with other
HRs, which transfer material into cells, in buffered water,
MeCN, and chloroform (KA 2.5 × 104 to 1 × 105 M-1).34,36,37
These results suggest that the tightest complexes need to form
at the cell surface, and future delivery devices should be
designed to bind guests strongly in DMSO.
Conformation selectivity appears to occur for the complexes
of Ac-Arg-Trp-Arg-CONH2 and the Fl-peptides in DMSO and
hormone 1 in MeCN. Ac-Arg-Trp-Arg-CONH2 fits best in the
open-closed conformation of Cy3R. Its indole ring can reside
in the pocket with both arginines in contact with each wheel.
In the open-open conformation, only a single wheel can interact
with Ac-Arg-Trp-Arg-CONH2 if its indole ring resides in the
pocket. Since the strength of the cation-π interaction is
essentially solvent independent, a more stable complex is formed
in DMSO because the open-closed conformation can more
readily form in this solvent than in MeCN. In a similar manner,
the open-closed conformation enables all four guanidiniums
Conclusion
To create more efficient delivery devices that operate at lower
concentrations, a host-[3]rotaxane was constructed with a larger
J. Org. Chem, Vol. 72, No. 11, 2007 3999

Bao et al.
culture plates in DME with 10% fetal calf serum until they had
reached 50-70% confluency (approximately 1 × 105 cells per well).
After 24 h, the growth medium was removed from the wells, and
the cells were washed twice with phosphate-buffered saline (PBS,
11.9 mM phosphates, 137 mM NaCl, 2.7 mM KCl, pH 7.4). For
the assays performed at room temperature, PBS buffer (1 mL) was
added to each well. HRs and Fl-peptides were added from DMSO
stock solutions to keep the amount of DMSO at less than 0.4%
(v/v) of the total volume. A baseline level of fluorescence was
obtained from wells containing Fl-peptide or fluorescein, and other
wells contained no reagents except DMSO (referred to as untreated
cells). An HR was added to a well followed by the addition of a
Fl-peptide or fluorescein. After rocking the wells at rt for 1 h in
the dark, the assay solutions were removed, and the cells were
thoroughly washed. A calcein blue AM solution (1 µM in PBS)
was added to each well. For cells exposed to the HRs, 2 µL of a
50 µg/mL propidium iodide solution was added to the wells. After
10 min, cells were observed by inverted fluorescence microscopy
(Zeiss Axiovert 200M). The cells were then harvested with trypsin
versene (0.4 mL of a solution containing 0.25% trypsin, 0.01%
EDTA incubated at 37 °C for 10 min) for flow cytometry. The
cells were pelleted via centrifugation (5 min at 600g). After
removing the PBS solution, the cells were resuspended in FACS
buffer (1% fetal bovine serum in PBS), and the proportion of
fluorescent cells was measured using 2 color flow cytometry (BD
FACSAria). The thresholds for Fl-peptides alone were set at 5%,
which was used as in the previous studies.36,37 To determine the
level of PI, all events were analyzed.
For cells assayed at 4 °C, the plates were stored at 4 °C for 15
min before the addition of an HR, and the above procedure was
followed with the plates maintained at 4 °C, except for the final
wash step, which was performed at room temperature. For depletion
of cellular ATP, the cells were preincubated with 2-deoxy-D-glucose
(6 mM) and sodium azide (10 mM) in PBS for 1 h, as described.57
HRs and guests were added to the solution containing 2-deoxy-D-
glucose and sodium azide, and the above assay procedure was
followed.
Determination of fluoresceinated peptide localization in cells was
achieved by high-magnification microscopy. COS 7 cells were
grown on 11 × 11 mm glass coverslips (Corning) in a 6-well culture
plate with one coverslip per well. Cells were exposed to HRs and
guest in the culture plate, as described above. The coverslips were
then inverted and mounted onto microscope slides with Permafluor
aqueous mounting medium (ThermoShandon). The unfixed cells
were examined at 400× magnification by fluorescence microscopy
(Zeiss Axioplan 2).
more rigid cyclophane pocket and two wheels that contain two
arginine moieties per wheel. A host-[2]rotaxane, which contains
the same cyclophane pocket but only a single arginine-
derivatized wheel, was constructed to determine whether the
presence of a second wheel on a host-rotaxane would enhance
guest association and intracellular delivery. A comparison
between Cy2R 2 and the transporter Cy2R 1 shows that a greater
separation of the wheel from the pocket results in a poorer
binding agent for some guests and, more importantly, hampers
the transport of materials into cells. The second wheel available
in Cy3R can partially overcome these deficiencies. Its four
guanidinium moieties can strengthen guest association and,
apparently, enhances cellular delivery. Adding more arginines
to an HR, however, did not result in greater Fl-peptide transport
into cells or in a more selective delivery device for negatively
charged peptides. At 5 µM, Cy2R 1 delivers Fl-AVWAL and
Fl-QEAVD into 82 and 37% of the cells, respectively, whereas
Cy3R delivers Fl-AVWAL and Fl-QEAVD into 58 to 16% of
the cells, respectively. The size of KA for complexes in DMSO
appears to be a key indicator as to whether an HR will transfer
the corresponding guest into cells. We are currently investiga-
ting the permeabilities of the HRs and guests using solid
supported membranes. A better understanding of the properties
of host-rotaxanes will facilitate the development of highly
specific transporters for delivering therapeutic agents across cell
membranes.
Experimental Section
Determination of Association Constants. Fluorescence quench-
ing assays were performed to obtain the association constants.
DMSO and MeCN were freshly distilled and stored over molecular
sieves (3 Å) prior to making the stock solutions and performing
the assays. The water solution was buffered with phosphate (1 mM)
at pH 7.0. A quantity of 2.7 mL of these solutions was placed into
a 3.5 mL cuvette. Guests were added to these solutions from a
DMSO stock solution to give final concentrations for most guests
as 3.0 × 10-6 M. For complexes with larger KA values, a 1 × 10-7
M solution of guest was used. For complexes with smaller KA
values, a 1 × 10-5 M solution of guest was used. Multiple aliquots
of an HR stock solution in DMSO were added to the cuvette to
give HR concentrations starting at approximately 0.3-fold lower
than that of a guest’s concentration and ending at approximately
40-fold higher concentration than that of a guest’s concentration.
The total change in volume caused by addition of the guest and a
rotaxane was less than 2%. The fluorescence spectrum was recorded
and analyzed after each addition of a rotaxane. Plots of the changes
observed in the quenching assays were fitted using a nonlinear least-
squares procedure to derive KA and ∆Fmax values.70 The assays were
duplicated, giving a standard deviation of less than 10% of the value
obtained for the association constant. Benesi-Hildebrand analysis71
was used to obtain the KA2 values for the HR2‚guest complexes.
Intracellular Transport Assays. We followed the procedures
described in detail previously.36 Briefly, cells were grown in 6-well
Acknowledgment. This material is based upon work sup-
ported by the National Science Foundation under Grant No.
CHE-0400539 and the American Cancer Society, Ohio Division,
(AFD). We thank Dr. David R. Plas for assistance in performing
and analyzing the FACS experiments.
Supporting Information Available: General experimental
1
procedures, synthetic procedure for Cy3R and Cy2R 2, H NMR
spectra, molecular modeling of Cy3R, photographs of cells exposed
to Cy3R and Cy2R 2. This material is available free of charge via
the Internet at http://pubs.acs.org.
(70) Binding assays: Conners, K. A. Binding Constants, The Measure-
ment of Molecular Complex Stability; Wiley: New York, 1987.
(71) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703-
2707.
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