Host-[2]rotaxane: Advantage of converging functional groups for guest recognition

Article abstract of DOI:10.1021/jo026522+

A host-[2]rotaxane was constructed by converting a diaminophenylcalix[4]arene into a [2]rotaxane using the DCC-rotaxane method (Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486). N-Ac-Arg groups were attached to the dibenzo-24-crown-8 ring of t

Full text of DOI:10.1021/jo026522+

Host-[2]Rota xa n e: Ad va n ta ge of Con ver gin g F u n ction a l Gr ou p s
for Gu est Recogn ition
Inese Smukste, Brian E. House, and David B. Smithrud*
Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172
david.smithrud@uc.edu
Received October 4, 2002
A host-[2]rotaxane was constructed by converting a diaminophenylcalix[4]arene into a [2]rotaxane
using the DCC-rotaxane method (Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486).
N-Ac-Arg groups were attached to the dibenzo-24-crown-8 ring of the rotaxane to provide a
convergent functional group. To demonstrate the advantage provided by the rotaxane architecture
for recognition of guests that contain a variety of functional groups, association constants (KA) for
N-Ac-Trp, indole, N-Ac-Gly, fluorescein, 1-(dimethylamino)-5-naphthalenesulfonate, and pyrene
1
bound to the [2]rotaxane were determined by performing H NMR and fluorescence spectroscopic
experiments. The host-[2]rotaxane had the highest affinity for fluorescein with a KA ) 4.6 × 106
M-1 in a 98/2 buffer (1 mM phosphate, pH 7)/DMSO solution. A comparison of KA values
demonstrates that both the aromatic pocket and ring of the host-[2]rotaxane contribute binding
free energy for complexation. Association constants were also derived for the same guests bound
to the diaminophenylcalix[4]arene and to a diphenylcalix[4]arene that contained arginine residues
displayed in a nonconvergent fashion. The host-[2]rotaxane provides higher affinity and specificity
for most guests than the host with divergent N-Ac-Arg groups of the one that only has an aromatic
pocket. For example, the KA for the complex of the host-[2]rotaxane and fluorescein in the DMSO/
water mixture is more than 2 orders of magnitude greater than association constants derived for
the other hosts.
In tr od u ction
Attaching functional groups to such synthetic pockets
does provide for enhanced recognition and function.7-12
However, in most cases, these groups are not truly
convergent; they do not point toward the pocket. Con-
vergent functional groups, such as those found in capped
crown ethers or cyclophanes, do enhance association.13,14
A new powerful method for constructing convergent
functional groups is found in the molecular imprint
polymers, which have become an intriguing source of
artificial receptors and catalysts.15-17
Mimicry of protein binding domains continues to be an
important goal of modern chemistry. Antibodies provide
the paradigm for molecular recognition in biology and for
the development of synthetic hosts.2 Small molecules are
bound within hydrophobic grooves or pockets on antibody
surfaces and make contact with multiple polar side
chains displayed on hypervariable loops to give highly
specific recognition.3-5 In theory, mimetic antibodies
should be possible if molecules could be made that have
a hydrophobic pocket with convergent functional groups.
However, developing synthetic hosts that contain this
arrangement is not a trivial endeavor. Cyclophanes
demonstrated early on the importance of a preformed,
rigid, aromatic pocket for strong complex formation.6
(7) Rowan, S. J .; Sanders, J . K. M. Curr. Opin. Chem. Biol. 1997, 1,
483-490.
(8) Casnati, A.; Pirondini, L.; Pelizzi, N.; Ungaro, R. Supramol.
Chem. 2000, 12, 53-65.
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(10) Fitzmaurice, R. J .; Kyne, G. M.; Douheret, D.; Kilburn, J . D. J .
Chem. Soc., Perkin Trans. 1 2002, 841-864 and references therein.
(11) Miyaji, H.; Dudic, M.; Tucker, J . H. R.; Prokes, I.; Light, M. E.;
Hursthouse, M. B.; Stibor, I.; Lhotak, P. Tetrahedron Lett. 2002, 43,
873-878.
* To whom correspondence should be addressed. Fax: (513) 556-
9239.
(1) Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486.
(2) (a) Park H. S.; Lin, Q.; Hamilton A. D. J . Am. Chem. Soc. 1999,
121, 8-13. (b) Hamuro, Y.; Calama, M. C.; Park H. S.; Hamilton A. D.
Angew. Chem., Int. Ed. Engl. 1997, 36, 2680-2683.
(3) Nezlin, R. S. The Immunoglobulins: Structure and Function;
Academic Press: San Diego, 1998.
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Gahmen, U.; Fremont, D. H.; Stura, E. Structural Aspects of Antibodies
and Antibody-Antigen Complexes. Ciba Found. Symp. 1991, 159, 13-
39.
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473.
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London, 1991.
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482.
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2002, 41, 1757-1759.
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2001, 123, 5847-5848.
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202, 1105-1108.
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478.
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Imprinted Materials for Recognition and Catalysis in Template Organic
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heim, Germany, 2000.
10.1021/jo026522+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/05/2003
J . Org. Chem. 2003, 68, 2559-2571
2559

Smukste et al.
In the preceding paper,18 we showed that templates
based on the rotaxane architecture19-22 can support
convergent functional groups. Amino acid-[2]rotaxanes
with opposing guanidinium and carboxylate groups form
a strong intramolecular salt bridge, and ones with
converging carboxylates associate with N-Ac-Arg in
DMSO/water mixtures and methanol. As the percentage
of water increased in these experiments, the strength of
association dropped dramatically.
To improve the performance of [2]rotaxanes as mimics
of protein binding domains, one blocking group was
replaced with a calix[4]arene. The calix[4]arene provides
the necessary hydrophobic pocket for strong association
in aqueous solutions, and we envision the rotaxane ring
as a crude mimic of an antibody hypervariable loop.
Calix[4]arenes were chosen to provide the framework for
the hosts because they are readily synthesized in large
quantities and derivatized with a variety of functional
groups on their upper and lower rims. Acetyl-protected
arginines were used as the recognition elements because
arginines are a key residue in protein binding domains23
and contain a chiral center that could potentially provide
diastereomeric complexes with chiral guests.
calix[4]arenes form capsules around guests,33 diphenyl-
phosphacalix[4]arenes act as metal catalysts,34 and a
dibenzoate-derivatized calix[5]arene binds two molecules
of imidazole.35
For our studies, three novel hosts were constructed:
host-[2]rotaxane 1, calix[4]arene 2, and calix[4]arene 3.
Association constants for complexes of these hosts with
guests, which contain a variety of functional groups and
aromatic surfaces, were derived and compared to deter-
mine whether convergent functional groups do enhance
guest recognition.
Although some calix[4]arenes form solid-phase inclu-
sion complexes with aromatic compounds,24,25 simple
calix[4]arenes in the solution phase form weak com-
plexes.26,27 Calix[4]arenes underivatized at the upper
rims fail to form even solid-phase inclusion complexes.25
Therefore, rigid calix[4]arenes with an extended hydro-
phobic cavity are necessary for effective binding of
organic molecules in the solution phase.28 Several “deep-
cavity”, aryl-substituted calix[4]arenes have been pre-
pared,29-40 and some have shown the ability to form
complexes. As examples, tetraamidophenyl-substituted
(18) Smukste, I.; Smithrud, D. B. J . Org. Chem. 2003, 68, 2547-
2558.
(19) Nepolgodiev, S. A.; Stoddart, J . F. Chem. Rev. 1998, 98, 1959-
1976.
(20) Gibson, H. W. Rotaxanes. In Large Ring Molecules; Semlyen,
J . A., Ed.; J ohn Wiley and Sons: New York, 1996; Chapter 6.
(21) Amablilino, D. B.; Stoddart, J . F. Chem. Rev. 1995, 95, 2725-
2828.
(22) Stoddart, J . F. Acc. Chem. Res. 2001, 34, 410-411.
(23) Bogan, A. A.; Thorn, K. S. J . Mol. Biol. 1998, 280, 1-9.
(24) Andreetti, G. D.; Ungaro, R.; Pochini, A. J . Chem. Soc., Chem.
Commun. 1979, 1005-1007.
(25) Calix[4]arenes a Versatile Class of Macrocyclic Compounds;
Vincens, J ., Bohmer, V., Eds.; Topics in Inclusion Science; Kluwer
Academic Publishers: Dordrecht, The Netherlands, 1991; pp 87-123.
(26) Gutsche, C., D.; Bauer, L., J . J . Am. Chem. Soc. 1985, 107,
6059-6063.
(27) Bohmer, V. Angew. Chem., Int. Ed. Engl. 1995, 34, 713-745.
(28) Rudkevich, D. M.; Rebek, J ., J r. Eur. J . Org. Chem. 1999, 1991-
2005.
(29) (a) Gutsche, C. D.; No, K. H. J . Org. Chem. 1982, 47, 2708-
2712. (b) No, K. H.; Gutsche, C. D. J . Org. Chem. 1982, 47, 2713-
2719.
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Tetrahedron Lett. 1990, 31, 4653-4656.
(31) Makha, M.; Raston, C. L. Tetrahedron Lett. 2001, 42, 6215-
6217.
(32) Miyaura, N.; Yanagi, T.; Suzuki, A. Synth. Commun. 1981, 11,
513-519.
(33) Cho, Y. L.; Rudkevich, D. M.; Rebek, J . J . Am. Chem. Soc. 2000,
122, 9868-9869.
(34) Shimizu, S.; Shirakawa, S.; Sasaki, Y.; Hirai, C. Angew. Chem.,
Int. Ed. 2000, 39, 1256-1259.
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Tetrahedron Lett. 1998, 39, 9171-9174.
Resu lts
(36) Dondoni, A.; Ghiglione, C.; Marra, A.; Scoponi, M. J . Org. Chem.
1998, 63, 9535-9539.
Design of th e Hosts. [2]Rotaxane 1 has arginine
residues as recognition elements, which can point toward
the pocket. We should note that rotation around the
(37) J aime, C.; de Mendoza, J .; Prados, P.; Nieto, P. M.; Sa´nchez,
C. J . Org. Chem. 1991, 56, 3372-3376.
2560 J . Org. Chem., Vol. 68, No. 7, 2003

Guest Recognition by Host-[2]Rotaxane
F IGURE 1. Lowest energy structures of [2]rotaxane 1 bound to N-Ac-Trp and fluorescein obtained using Monte Carlo simulations
at 300 K (molecular mechanics calculations, MM+ force field). H-atoms are removed for clarity.
biaryl bond can give syn and anti isomers of the calix-
[4]arenes; the syn isomer is shown throughout the text.
The arginine residues are not connected to the pocket of
[2]rotaxane 1, but the two domains are held together in
a threaded manner. Their participation in the binding
event depends on the energy gained through noncovalent
bond formation balanced against the energy lost through
a reduction in the motion of the arginine moiety and ring.
In the preceding paper, we showed that the reduction in
ring motion with complex formation does not detract
significantly from the binding free energy.18 Lowest
energy structures of [2]rotaxane 1 bound to N-Ac-Trp and
fluorescein (Figure 1), as determined by Monte Carlo
simulations, indicate that the host is appropriately
designed to recognize small and large compounds that
have aromatic rings and carboxylates.
Calix[4]arene 2 also has arginine residues as recogni-
tion elements. However, these groups are held in a more
divergent arrangement. A simultaneous interaction of the
guest with both the hydrophobic pocket and the arginine
side chains of calix[4]arene 2 is unlikely. A large portion
of the binding free energy would be used to pay for the
loss of entropic energy required to force the arginine side
chains to bend toward the pocket. There may also be a
substantial loss of enthalpic energy due to geometrical
constraints placed on the side chains.
in CHCl343 gave calix[4]arene 5 in high yield, which likely
locks it in the cone conformation.44 m-Nitrophenyl rings
were successfully attached using a Suzuki coupling
reaction of m-nitrophenylboronic acidscommercially
availablesand calix[4]arene 5. Unfortunately, the aque-
ous conditions (Na2CO3, H2O, toluene, and methanol)
hydrolyzed the ethyl esters. Pd(PPh3)4-catalyzed coupling
reaction between dibromocalix[4]arene 5 and m-nitro-
phenylboronic acid under anhydrous conditions (Cs2CO3,
DMF) resulted in a mixture of mono- and di-p-phenylcalix-
[4]arenes in low yields. Numerous attempts to employ
other Pd(0) catalysts (Pd(OAc)2‚2P(otol)345 or the Negishi
method46) resulted in no reaction or a complicated
mixture of products. Therefore, the reaction was run
under the basic aqueous conditions, and the acids were
re-esterified. Product formation was monitored by ob-
serving the loss of the bromophenol’s aromatic signal at
7.04 ppm and the growth of the m-nitrophenyl signal at
1
7.36 ppm in H NMR spectra.
The coupling reaction and hydrolysis provided calix-
[4]arene 6 in 70-80% yield. Calix[4]arene 6 is poorly
soluble in most organic solvents, and thus, it was purified
by trituration with ethyl ether to remove excess m-
nitrophenylboronic acid and then recrystallized from
methanol. Refluxing calix[4]arene 6 in a MeOH/CHCl3
solution with a catalytic amount of acid provided calix-
[4]arene 7 in 70-80% yield. Methyl esters were chosen
Calix[4]arene 3 contains the same aromatic pocket as
[2]rotaxane 1 without the arginine residues. Larger
association constants determined for guests complexed
to [2]rotaxane 1 as compared to calix[4]arene 3 would
show that the arginine residue or the ring of the rotaxane
contributes to the binding free energy.
1
to simplify the H NMR spectra of the binding assays.
Selective reduction of one of the two nitro groups of
calix[4]arene 7 was necessary for the synthesis of [2]-
rotaxane 1, whereas complete reduction gives calix[4]-
arene 3 and the material needed for the creation of
Syn th esis of th e Hosts. One convenient method for
selective functionalization of calix[4]arenes begins with
selective dialkylation of the lower rim followed by elec-
trophilic substitution of the phenolic units of the dialky-
lated calix[4]arenes.41 The lower rim of calix[4]arene 4
was alkylated with ethyl bromoacetate in a mixture of
THF and DMF (Scheme 1).42 Adding 2 equiv of bromine
(41) van Loon, J .-D.; Arduini, A.; Verboom., W.; van Hummel, G.
J .; Harkema, S.; Reinhoudt, D. N., Tetrahedron Lett. 1989, 30, 2681-
2684.
(42) Arena, G.; Casnati, A.; Mirone, L.; Sciotto, D.; Ungaro, R.
Tetrahedron Lett. 1997, 38, 1999-2002.
(43) van Loon, J .-D.; Arduini, A.; Coppi, L.; Verboom., W.; Pochini,
A.; Ungaro, R.; Harkema, S.; Reinhoudt, D. N. J . Org. Chem. 1990,
55, 5639-5646.
(44) Gutsche, C. D. In Calixarenes; Stoddart, F. J ., Ed.; Monographs
in Supramolecular Chemistry; The Royal Society of Chemistry: Lon-
don, 1989; pp 106-114.
(38) Wong, M. S.; Nicoud, J .-F. Tetrahedron Lett. 1993, 34, 8237-
8240.
(39) Larsen, M.; J orgensen, M. J . Org. Chem. 1997, 62, 4171-4173.
(40) Adruini, A.; McGregor, W. M.; Paganuzzi, D.; Pochini, A.;
Secchi, A.; Ugozzoli, F.; Ungaro, R. J . Chem. Soc., Perkin Trans. 2 1996,
839-846.
(45) Wong, M. S.; Nicoud, J .-F. Tetrahedron Lett. 1993, 34, 8237-
8240.
(46) Negishi, E.; King, A. O.; Okukado, N. J . Org. Chem. 1977, 42,
1821-1823.
J . Org. Chem, Vol. 68, No. 7, 2003 2561

Smukste et al.
SCHEME 1
calix[4]arene 2. We have previously used Sn/HCl to
reduce dinitrophenylcalix[4]arenes, but this method is
unsuitable for monoreduction; the reaction proceeds too
quickly, and partial hydrolysis of the esters gives a
mixture of products. Other conventional reducing meth-
ods (1,4-dicyclohexadiene, Pd/C; H2, Pd/C, HCO2NH4;
2562 J . Org. Chem., Vol. 68, No. 7, 2003

Guest Recognition by Host-[2]Rotaxane
F IGURE 2. (A) Chemical shift dependency of the amide proton of N-Ac-Trp, which was held at a constant concentration of 1.0
mM while the concentration of [2]rotaxane 1 was increased. (B) A J ob plot47 where the concentration of the complex ((∆δ)XG) is
compared against the mole fraction of N-Ac-Trp (XG). The lines are drawn to overlay the data points.
Pd/C, H2 in CHCl3 or in a CHCl3/methanol solution)
produced only a small yield of calix[4]arenes 8 and 3 even
after long reaction times at elevated temperatures. The
best method we found for monoreduction involves the
addition of acetic or formic acid to a solution of dinitrocalix-
[4]arene 7 in a CHCl3/methanol mixture (50/50 (v/v)) in
the presence of Pd/C under a H2 atmosphere. The
reaction was monitored by TLC and terminated when
∼70-80% of calix[4]arene 7 was consumed, usually
within 12-18 h. The crude reaction mixture was sepa-
rated by column chromatography to give calix[4]arenes
7, 8, and 3 in yields of 1, 2.7, and 4.3 ratio, respectively.
To create calix[4]arenes with arginines displayed on
their upper rims, e.g., calix[4]arene 2, synthetic meth-
odology was needed to attach derivatized arginines to
diaminophenylcalix[4]arenes. Initial attempts to con-
struct calix[4]arene 10 through the coupling reaction of
calix[4]arene 3 with N-Boc-Arg-OH with CDI, EDC, or
DCC/HOBT as activating agents in DMFsneeded to
solubilize N-Boc-Arg-OHswere unsuccessful. Fully Boc-
protected arginine (Boc)3-Arg-OH was attached using
EDC/HOBT activation in CHCl3 to provide (Boc)3-Arg-
calix[4]arene 9a in an 84% yield. Complete removal of
Boc protecting groups required exposure to a 50-70%
TFA solution in CH2Cl2 and extended reaction times,
which resulted in a moderate yield of calix[4]arene 10.
N-Ac-(Boc)2-Arg-OH was attached to calix[4]arene 3 also
through EDC/HOBT coupling. Deprotection of the result-
ing calix[4]arene 9b required only exposure to a 30% TFA
solution in CHCl3 for 1 h for complete removal of the Boc
groups to give calix[4]arene 2 in a good yield.
Host-[2]Rota xa n e F or m a tion . Synthesis of [2]ro-
taxane 1 was based on the general method of coupling
DCC-[2]rotaxanes to primary amines to give [2]rotax-
anes.1 The addition of a primary amine to calix[4]arene
8, which is necessary for coupling, was easily accom-
plished by attaching Boc-â-alanine using EEDQ in re-
fluxing pyridine or through CDI coupling in refluxing
CHCl3. Subsequent deprotection of Boc-â-alanylcalix[4]-
arene 11 with 20% TFA in CH2Cl2 gave â-alanylcalix[4]-
arene 12. DCC-rotaxane 13,18 which contains a Boc-
protected ring, was used to make [2]rotaxane 14.
Deprotection with exposure to TFA proceeded smoothly
to give the diamino[2]rotaxane 15. N-Ac-Arg was at-
tached to the ring of diamino[2]rotaxane 15 instead of
Boc-protected arginines. Experience with the synthesis
of rotaxanes in our laboratory showed that the removal
of multiple Boc groups requires a large percentage of TFA
in CH2Cl2 or DMF and long reaction times, which tends
to cleave some of the tert-butyl groups of the di-tert-
butylphenyl blocking group. The poor solubility of N-Ac-
Arg in most organic solvents is problematic. Attempts to
couple diamino[2]rotaxane 15 with the sodium salt of
N-Ac-Arg with activating reagents commonly used for
amidation (CDI, DCC/HOBT, or PyBroP, as a suspension
in DMF or DMSO) were unsuccessful. [2]Rotaxane 1 was
obtained in good yields only when the hydrochloride salt
of N-Ac-Arg was activated with BOP in the presence of
DIEA.
1H and 13C NMR spectra of the final [2]rotaxanes were
too complex for accurate determination of their preferred
conformation in solution. However, the precursor calix-
[4]arenes were clearly locked in a cone conformation37
(see the Supporting Information), which strongly sug-
gests that the [2]rotaxanes are also locked in a cone
conformation.
Mon itor in g Associa tion th r ou gh 1H NMR Sp ec-
tr oscop y. A series of guests were chosen to test the
ability of the hosts to recognize charged, polar, or
aromatic groups and to determine whether the arginine
group of host 1 or 2 and the aromatic pocket of all three
hosts interact with a guest. N-Ac-Trp was chosen because
it contains a variety of functional groups and the tryp-
tophan side chain is a key recognition amino acid in
protein binding domains.23
[2]Rotaxane 1 strongly interacts with N-Ac-Trp in
DMSO-d6 and in DMSO-d6/buffer (1 mM phosphate, pH
7.0) mixtures. Association was too favorable to derive an
accurate KA from 1H NMR binding assays. Plots of the
changes in the chemical shifts of the aromatic N-H
proton of N-Ac-Trp versus the change in concentration
of [2]rotaxane 1 did show that a 1/1 complex is most likely
formed (Figure 2A). To verify this result, a J ob plot47 was
constructed by plotting the concentration of the complex
versus the mole fraction of N-Ac-Trp (Figure 2B). A
maximum in the curve is reached at a mole fraction of
ca. 0.5, which is consistent with a 1/1 complex. The
formation of a 1/1 complex with a single arginine group,
according to molecular modeling results, suggests that
the syn and anti constitutional isomers of the host-[2]-
rotaxane bind with the same association constant.
The proton resonance of the aromatic N-H shifted
upfield with an increase in the concentration of [2]-
(47) (a) Blanda, M. T.; Horner, J . H.; Newcomb, M. J . Org. Chem.
1989, 54, 4626-4636. (b) J ob, P. Ann. Chim. 1928, 9, 113-134.
J . Org. Chem, Vol. 68, No. 7, 2003 2563

Smukste et al.
TABLE 1. Associa tion Con sta n ts for Va r iou s Host-Gu est Com p lexes Mea su r ed a t 25 °Ca
KA × 10-4
b
c
host
guest
solution
(M-1
)
KA(1)/KA(2)
KA(1)/KA(3)
1
N-Ac-L-Trp
DMSO
10 ( 0.5
10 ( 0.2
111
420
>60
>190
11
14
2
DMSO/H2O (80/20)
DMSO/H2O (50/50)
DMSO/H2O (25/75)
DMSO/H2O (2/98)
DMSO
DMSO/H2O (80/20)
DMSO/H2O (50/50)
DMSO/H2O (25/75)
DMSO-d6
DMSO-d6/H2O (80/20)
DMSO
DMSO/H2O (80/20)
DMSO
DMSO/H2O (2/98)
DMSO/H2O (2/98)
DMSO/H2O (2/98)
DMSO-d6
DMSO-d6/H2O (80/20)
DMSO-d6/H2O (50/50)
DMSO-d6/H2O (25/75)
DMSO-d6
DMSO-d6/H2O (80/20)
DMSO-d6
DMSO-d6/H2O (80/20)
DMSO-d6
1.2 ( 0.1
3.75 ( 0.08
1.9 ( 0.3
4.2 ( 0.4
1.8 ( 0.1
0.9 ( 0.1
1.1 ( 0.1
0.16 ( 0.03
<0.01
>19
indole
>400
>180
>45
>55
4
10
1
1
1
>16
N-Ac-Gly
L-Trp
1.0 ( 0.4
1.5 ( 0.4
0.9 ( 0.1
5.5 ( 0.5
470 ( 80
3.2 ( 0.3
0.09 ( 0.01
0.024 ( 0.006
<0.02
20
> 150
D-Trp
1,5-DNS
fluorescein
pyrene
4
320
3
10
480
3
2
N-Ac-L-Trp
<0.02
L-Trp
0.05 ( 0.01
NAd
N-Ac-Gly
indole
0.041 ( 0.005
NA
NA
DMSO-d6/H2O (80/20)
DMSO-d6/H2O (50/50)
DMSO-d6/H2O (25/75)
DMSO/H2O (10/90)
DMSO/H2O (10/90)
DMSO/H2O (10/90)
DMSO
DMSO-d6/H2O (80/20)
DMSO-d6/H2O (50/50)
DMSO-d6/H2O (25/75)
DMSO-d6
<0.01
<0.02
<0.02
1,5-DNS
fluorescein
pyrene
1.50 ( 0.04
1.45 ( 0.05
1.3 ( 0.1
0.91 ( 0.05
0.7 ( 0.2
0.5 ( 0.1
<0.2
3
N-Ac-L-Trp
N-Ac-Gly
indole
<0.01
DMSO-d6
0.42 ( 0.05
1.2 ( 0.1
0.8 ( 0.04
0.8 ( 0.1
0.58 ( 0.06
0.98 ( 0.06
1.22 ( 0.08
DMSO/H2O (80/20)
DMSO/H2O (50/50)
DMSO/H2O (25/75)
DMSO/H2O (10/90)
DMSO/H2O (10/90)
DMSO/H2O (2/98)
1,5-DNS
fluorescein
pyrene
a
KA values determined from analysis of fluorescence spectra except for experiments performed in a deuterated solvent, which were
determined from analysis of 1H NMR spectra. Water solutions were buffered with 1 mM phosphate buffer, pH 7.0. Values with less
b
than signs are estimated. c A comparison of KA values, with the numerical subscript indicating a host. NA indicates no association was
d
detected.
rotaxane 1 in DMSO-d6, suggesting that the indole ring
of N-Ac-Trp becomes embedded in the electron-rich
environment of the aromatic pocket of the host. The
signal for some aromatic protons of the indole ring shifted
downfield. A large downfield shift (∆δ ) 0.3 ppm) was
observed for the aromatic protons of N-Ac-Trp in the
presence of 1 equiv of [2]rotaxane 1 as compared to the
guest alone in a 50/50 DMSO/buffer solution. A downfield
shift indicates that these parts of the indole ring are held
in a face-to-face manner with the phenyl rings of the
calix[4]arene pocket, and not in an edge-to-face align-
ment.6 This geometrical arrangement is observed in the
molecular modeling results vide infra. Only small changes
in chemical shifts were observed with the other hosts,
which suggests that the aromatic ring of DB24C8 par-
tially encloses the pocket of the rotaxane.
indole and N-Ac-Gly bound to [2]rotaxane 1 (Table 1).
The KA for the complex between indole and [2]rotaxane
1 was too large for accurate determination; however, the
KA for the N-Ac-Gly could be determined. A downfield
shift in the resonance of the acetyl protons of N-Ac-Gly,
caused by the increase in concentration of [2]rotaxane
1, provided for an easily traceable signal. The signifi-
cantly sized association constant (KA ) 1.6 × 103 M-1) in
DMSO-d6 suggests that a salt bridge formed between the
carboxylate of N-Ac-Gly and the host. Consistent with a
salt bridge, adding water to the solution reduced the
association constant for the N-Ac-Gly-[2]rotaxane 1
complex.
Calix[4]arenes 2 and 3 bound N-Ac-Trp weaker than
[2]rotaxane 1 in DMSO-d6. A downfield shift of the N-H
proton of the indole ring was observed with an increase
in the concentration of either host. In contrast to the [2]-
rotaxane 1 complex, the indole ring is not placed in an
electron-rich environment of calix[4]arenes 2 and 3. The
An estimation of the contribution to the binding free
energy provided by the functional groups of N-Ac-Trp was
obtained by comparing the association constants for
2564 J . Org. Chem., Vol. 68, No. 7, 2003

Guest Recognition by Host-[2]Rotaxane
TABLE 2. F lu or escen ce P r op er ties of th e Hostsa
solution, calix[4]arene 3 has only a small drop in fluo-
rescence. On the other hand, the fluorescence intensity
of [2]rotaxane 1 was approximately nonexistent, and the
intensity was greatly reduced for calix[4]arene 2 with the
addition of water to a DMSO solution. The change in
fluorescence caused by the addition of water may be due
to a greater water solvation of hosts 1 and 2 or a change
in their structure.
b
host
solvent
λex,max
λem,max
Fhost/Findole
1
DMSO
286
290
287
290
280
272
388
360
343
350
390
393
0.05
<0.005
0.45
DMSO/H2Oc (25/75)
DMSO
2
3
DMSO/H2O (25/75)
DMSO
DMSO/H2O (25/75)
0.01
0.36
0.29
a
b
Fluorescence measured at 25.0 °C. Ratio of a host’s fluores-
cence at its maximum excitation and emission wavelengths versus
indole’s fluorescence at λex,max 280 nm and λem,max 333 nm. c Water
solutions are buffered with 1.0 mM phosphate, pH 7.0.
The reduction in fluorescnece intensity observed with
the addition of water was not caused by a precipitation
of hosts. For the least soluble host, calix[4]arene 3, host
concentration was verified by removing the fluorescence
solution, spinning the sample to condense any precipitate,
and comparing the absorbance, and thus concentration,
of the solution to a predetermined calibration curve.
Significant quenching of the fluorescence of N-Ac-Trp
occurs in the presence of [2]rotaxane 1, allowing an
association constant to be determined for the complex.
The derived large KA for association in DMSO (Table 1)
is consistent with the fact that an accurate association
N-H proton is either held near an electron-poor edge of
a host’s aromatic ring or involved in a H-bond complex.
This result suggests that N-Ac-Trp is held in a different
binding geometry for [2]rotaxane 1.
With the addition of water to the solutions (80/20 (v/v)
DMSO-d6/buffer), no shifts could be detected in the signal
of the N-H proton of N-Ac-Trp’s indole ring in the
presence of calix[4]arene 2. There was a small downfield
shift for an aromatic proton of N-Ac-Trp, and this signal
was used to derive the association constant. Apparently,
water disrupts a salt bridge, and association occurs via
pocket penetration. The similar KA values observed for
N-Ac-Trp and N-Ac-Gly bound to calix[4]arene 2 in
DMSO-d6 also suggest that a salt bridge provides a
significant proportion of the binding free energy for the
association of N-Ac-Trp. The very weak association
between N-Ac-Gly and calix[4]arene 3 further demon-
strates the importance of the arginine groups for the
complexes formed between calix[4]arene 2 and N-Ac-Gly.
The addition of water to a millimolar solution of calix-
[4]arene 3 precipitated the host, which prevented further
NMR analysis of the calix[4]arene 3-N-Ac-Trp complex.
The chemical shifts of indole or calix[4]arene 2 did not
change when they were mixed in the millimolar concen-
tration range in DMSO-d6, indicating no association
occurs in this solvent. With the addition of 20% buffer (1
mM phosphate, pH 7.0) to the solutions, a weak complex
formed. The N-H proton of indole shifted upfield with
an increase in the concentration of calix[4]arene 2. The
aromatic ring enters a shielding environment, which
suggests that the ring becomes buried within the aro-
matic pocket in a manner similar to that of the complex
formed between [2]rotaxane 1 and indole. Because the
binding plot was linear with the host and guest combined
in the low millimolar range, a KA < 100 M-1 was
assigned.
F lu or escen ce Titr a tion s. Fluorescence binding as-
says were performed to determine the larger association
constants and to discover whether complexes form in
solutions that contain a large percentage of water.
Besides tryptophan derivatives and indole, other fluoro-
phores, having a variety of functional groups, were
chosen as guests. The host’s were fluorescent as well,
albeit weaker than the fluorophores (Table 2). In DMSO,
calix[4]arene 2 is the strongest fluorophore, followed
closely by calix[4]arene 3, and [2]rotaxane 1 only fluo-
resces weakly. The ring or arginine derivative may
quench the fluorescence of the biaryl rings of [2]rotaxane
1 in DMSO. As the percentage of water is increased in
the solutions, the fluorescence of the hosts drops. In a
25/75 (v/v) DMSO/buffer (1 mM phosphate buffer, pH 7.0)
1
constant could not be obtained from the H NMR experi-
ments. With the addition of a small amount of water to
the DMSO solution (20% (v/v)), the KA for this complex
does not change within experimental error. At higher
concentrations of water, KA drops only by a small amount.
A 5-fold reduction occurs in an essentially pure buffer
solution (98/2 (v/v) buffer/DMSO).
Indole also interacts strongly with [2]rotaxane 1. The
magnitude of its KA becomes more similar to the values
derived for the N-Ac-Trp-[2]rotaxane 1 complex for
solutions having higher percentages of water. This result
indicates that the major driving force for the interaction
of N-Ac-Trp with [2]rotaxane 1 is the placement of its
indole ring into the aromatic pocket. However, the larger
KA observed for N-Ac-Trp as compared to indole suggests
that either a salt bridge or H-bonds exist between the
rotaxane’s and guest’s functional groups, even in the
presence of a high percentage of water.
The advantage of converging functional groups for
recognition is clearly evident upon comparing the as-
sociation constants for [2]rotaxane 1 and calix[4]arene 2
binding the various guests. A 10-fold smaller KA is
observed for the calix[4]arene 2-N-Ac-Trp complex in
DMSO, as compared to [2]rotaxane 1. As the percentage
of water is increased in the solutions containing N-Ac-
Trp and calix[4]arene 2, the association constant drops
by at least a factor of 5, maintaining a 2 orders of
magnitude reduction in KA compared to those of com-
plexes formed with [2]rotaxane 1. Association constants
for complexes of calix[4]arene 2 in solutions containing
50% and 75% water are approximations. A change in
fluorescence was observed only with the addition of a
large amount of the host. The KA < 200 M-1 limiting
value was chosen because of the results obtained for the
assay performed in the 80/20 (v/v) DMSO/buffer mixture.
In this solvent system, the plot of the small change in
fluorescence versus host concentration was linear and
could not be fitted using a nonlinear least-squares fitting
procedure. The association constant in this solvent
1
mixture was thus determined by H NMR experiments
to give a value of KA ) 240 M-1. Binding assays that
produced linear plots at a high concentration of hosts
(10-5 to 10-4 M) were assigned KA values less than 200
M-1
.
J . Org. Chem, Vol. 68, No. 7, 2003 2565

Smukste et al.
Calix[4]arene 2 binds indole with KA values similar to
those observed for N-Ac-Trp. Because of the relatively
weak association, most of the values are estimates. We
cannot determine which functional group drives associa-
tion. The fluorescence titrations were complicated by the
fact that calix[4]arene 2 and indole have almost identical
excitation and emission spectra. Fortunately at higher
concentrations of water, the fluorescence intensity of
calix[4]arene 2 is greatly diminished. In the 25/75 (v/v)
DMSO/buffer solution, the host’s fluorescence is very
weak (Table 2), and a high concentration of host (10-4
M) was needed to produce a change in the fluorescence
intensity of indole. Thus, the limiting value of KA ) 200
M-1 was assigned. The same KA was assigned to the
complex formed in a 50/50 (v/v) DMSO/buffer solution for
the same reasons.
Fluorescence assays of calix[4]arene 3 were compli-
cated by an apparent self-association of the host, espe-
cially in solutions containing a high percentage of water.
Dimerization would be consistent with the synthetic
capsules developed by Rebek.33 Association constants for
guest complexes were estimated by using the Benesi-
Hildebrand method48 for the data obtained with low
concentrations of components, which should have had an
insignificant amount of the dimer in the formation in the
solution. These values were consistent with KA values
derived using all the data through a nonlinear least-
squares fitting of the data to a standard binding equation
for a 1/1 complex.49 Thus, if dimerization occurs, only a
small percentage of the hosts interact at the concentra-
tions used in the experiment.
Small changes in the fluorescence of N-Ac-Trp and
indole were observed with the addition of calix[4]arene
3 to a solution containing either guest. For the N-Ac-Trp
complex, the binding strength diminishes with the ad-
dition of water to the solution. A small association
constant (KA ) 5 × 103 M-1) was derived for complexation
in a 50/50 (v/v) DMSO/water solution. In the 25/75 (v/v)
DMSO/water solution, the fluorescence plot was linear
and could not be fitted. We arbitrarily assigned a KA
value that is half as large as the one derived in the
50/50 (v/v) DMSO/water solution, which is a conservative
estimate. The KA for the indole complex is actually
weakest in DMSO and increases with the addition of
water to the solution. Overlapping fluorescence spectra
did not occur for these experiments. Calix[4]arene 3
fluoresces at 408 nm, leaving the indole and N-Ac-Trp
window at 334 nm open.
1-(Dimethylamino)-5-naphthalenesulfonate (1,5-DNS),
fluorescein, and pyrene form tight complexes with all
three hosts, with [2]rotaxane 1 having the strongest
interactions. The largest association constant is observed
for the complex of [2]rotaxane 1 and fluorescein; its KA
is over 2 orders of magnitude greater than the association
constants observed for the other dye complexes. For the
weaker complexes, i.e., KA values of 1 × 104 M-1 or
smaller, a 10/90 (v/v) DMSO/buffer solution was neces-
sary to solubilize the higher concentrations of hosts that
were necessary to obtain curvature in the binding plots.
For example, small changes in the fluorescence of fluo-
rescein was observed with the addition of calix[4]arene
2 in the same concentration range as used for [2]rotaxane
1 (10-7 to 10-6 M), which resulted in a linear binding plot.
A higher concentration of calix[4]arene 2 (10-6 to 10-5
M) did result in significant curvature in the plot. A 10/
90 (v/v) DMSO/buffer solution was also used in the calix-
[4]arene 3 and pyrene study. Calix[4]arene 3 has a slight
preference for pyrene as compared to its complexes with
the other fluorophores.
The long-wavelength emissions of these dyes do not
overlap with the fluorescence of the hosts except for the
case of pyrene and calix[4]arene 3. The fluorescence of
calix[4]arene 3 is substantially weaker than that of
pyrene (<10%), and thus, the addition of host should not
have produced a large error in the fluorescence measure-
ments. This appears to be the case since the shape of
pyrene’s fluorescence bands did not change with the
addition of calix[4]arene 3 and the derived KA has a small
standard deviation.
Discu ssion
The arginine-derivatized ring of [2]rotaxane 1 contrib-
utes favorable binding free energy for most of the
complexes investigated in this study. A one order of
magnitude enhancement in KA is observed for the binding
of N-Ac-Trp by [2]rotaxane 1 as compared to calix[4]arene
3 in DMSO. The ca. 1.5 kcal/mol difference in binding
free energy can easily be accounted for if a salt bridge or
H-bonds form between the guanidinium moiety of [2]-
rotaxane 1 and the amino acid backbone of N-Ac-Trp. As
the percentage of water is increased, the difference in
KA increases except for the solution containing 50%
buffer. The reduction in KA observed for the calix[4]arene
3-N-Ac-Trp complex with the addition of water is
reasonable considering that the polar groups of the guest
become strongly solvated with water, which results in a
greater desolvation penalty for guest inclusion into the
hydrophobic pocket. Apparently, the guanidinium moiety
of [2]rotaxane 1 can accommodate the polar groups of the
guest and lessen the desolvation penalty.
The importance of desolvation is evident by the 10-
fold drop in the KA for L-Trp association with [2]rotaxane
1, as compared to the N-Ac-Trp complex. The additional
charge of the ammonium ion of L-Trp interacts favorably
with solvent molecules, which may need to be removed
for complexation. Since D-Trp associates with [2]rotaxane
1 as favorably as L-Trp, chiral recognition does not occur
for this guest in DMSO.
The importance of the hydrophobic effect is exemplified
by the strength of the indole interaction with the hosts.
A 10-fold larger KA is observed for the binding of indole
by [2]rotaxane 1 as compared to calix[4]arene 3 in DMSO.
A possible H-bond may exist between the arginine of the
ring and the N-H of indole. With the addition of water
to DMSO, the complex between indole and [2]rotaxane
1 is weakened, which could be explained by the breaking
of this H-bond. On the other hand, the presence of water
enhances the association between calix[4]arene 3 and
indole, which is consistent with the hydrophobic effect.
In DMSO/water mixtures, hosts 1 and 3 bind indole with
approximately the same large association constant. Fur-
thermore, the N-H proton of indole shifts upfield in the
presence of either host. These results suggest that these
(48) Benesi, H. A.; Hildebrand, J . H. J . Am. Chem. Soc. 1949, 71,
2703.
(49) Conners, K. A. Binding Constants, The Measurement of Molec-
ular Complex Stability; Wiley: New York, 1987.
2566 J . Org. Chem., Vol. 68, No. 7, 2003

Guest Recognition by Host-[2]Rotaxane
hosts have similar hydrophobic cavitiessthe ring of [2]-
rotaxane 1 does not significantly distort its pocketsand
the hydrophobic effect is an important component for
complex formation. The fact that N-Ac-Gly associates
very weakly with [2]rotaxane 1 in DMSO/water mixtures
suggests that the indole ring is necessary for N-Ac-Trp
to strongly interact with [2]rotaxane 1.
Examination of the association constants derived for
complexes formed with calix[4]arene 2 shows that it is a
poorly designed host. The arginine side chains appear
not to operate jointly with the hydrophobic pocket to
obtain a highly effective host. For most guests, the KA
values for complexes formed with calix[4]arene 2 are
actually smaller than the ones derived for calix[4]arene
3 with the same guests. This result suggests that the
pocket of calix[4]arene 2 is closed, possibly through
intramolecular interactions between the amino acids on
the upper rim. Accordingly, indole does not bind in a
DMSO solution, because this solvent does not break
interactions between the amino acids. Adding water to
the solution weakens the intramolecular interactions and
promotes indole binding through the hydrophobic effect.
On the other hand for charged guests, the hydrophobic
effect does not appear to contribute to the binding free
energy. The binding free energy derived for the complex
of calix[4]arene 2 and N-Ac-Trp (-4 kcal/mol) is consis-
tent with a salt bridge.50 A similar free energy for
association was derived for L-Trp and N-Ac-Gly, which
suggests that only a salt bridge forms between calix[4]-
arene 2 and small, polar guests in DMSO. Consistent
with a salt bridge, adding water to the solutions reduces
the association constants.
Guests with large aromatic surfaces, however, do
strongly bind calix[4]arene 2. In fact all fluorophores
associate with calix[4]arene 2 to the same extent, which
suggests that contact between aromatic surfaces provides
the favorable binding energy. The other possibility is that
the interaction energies between the functional groups
of fluorescein and 1,5-DNS just happen to be equal in
magnitude to the hydrophobic interaction energies of
pyrene. This situation is unlikely since interactions
between charged groups of a guest and calix[4]arene 2
are weak even in a high percentage of DMSO. The fact
that calix[4]arene 2 interacts strongly with pyrene shows
that its pocket can interact with a large aromatic ring
better than a small ring such as indole. Besides the
greater hydrophobic effect, the pocket of calix[4]arene 2
could also be more accommodating of the fluorophores
because intramolecular interactions between the arginine
residues should be very weak in solutions containing a
high percentage of water.
[2]Rotaxane 1 forms the strongest complexes with the
fluorophores. According to the KA values, [2]rotaxane 1
prefers fluorescein by two orders of magnitude over the
other fluorophores. Fluorescein has a large aromatic
surface area and a carboxylate moiety; both components
were targeted in the design of the host. A 3-fold larger
KA is observed for the association of pyrene by [2]rotaxane
1, as compared to calix[4]arene 3, whereas the smaller
indole ring binds both hosts with the same association
constant within experimental error. Therefore, the ro-
F IGURE 3. Fluorescence titrations of 1,5-DNS and [2]-
rotaxane 1, calix[4]arene 2, and calix[4]arene 3 in DMSO/
buffer (1 mM phosphate, pH 7) mixtures at 25 °C. An
enhancement in the fluorescence intensity with the addition
of host 1 or 3 suggests that 1,5-DNS enters the hydrophobic
pocket of the hosts.
taxane can contribute a greater amount of binding free
energy to a complex with a large aromatic guest, most
likely, through favorable interactions with the crown
ether’s aromatic ring or an enhancement in the hydro-
phobic effect. As also seen with small charged guests, [2]-
rotaxane 1 can accommodate the charged fluorophores
better than calix[4]arene 3. For the latter host, the
highest affinity is observed for pyrene followed by
fluorescein and then 1,5-DNS. This trend is consistent
with the number of charged groups; the greater the
desolvation penalty, the weaker the complex.
One major difference observed in these studies is that
the fluorescence of 1,5-DNS is increased with exposure
to [2]rotaxane 1 or calix[4]arene 3, whereas in the
(50) Thompson, S. E.; Smithrud, D. B. J . Am. Chem. Soc. 2002, 124,
442-449.
J . Org. Chem, Vol. 68, No. 7, 2003 2567

Smukste et al.
were observed in the chemical shifts of either the host or guest,
we assigned a KA value of zero. One caveat with 1H NMR
experiments is that a change in environment must occur upon
complexation to produce a change in the chemical shift of a
proton. Literature precedence6 has shown that in most cases
the resonances of a guest’s aromatic protons will change upon
complexation. In these studies, the N-H of the indole ring
should be a likely indicator of association. It is highly solvated
in DMSO-d6 or DMSO-d6/buffer solutions and should experi-
ence a dramatic change in environment once bound to a host.
presence of calix[4]arene 2 it is decreased (Figure 3).
Enhanced fluorescence suggests that 1,5-DNS is embed-
ded within a hydrophobic environment of hosts 1 and
3.51-53 Even in a high concentration of water, it appears
that the aromatic pocket of calix[4]arene 2 with divergent
functional groups does not associate strongly through the
hydrophobic effect.
Con clu sion
Fluorescence binding assays were performed on hosts and
fluorescent guests, including ones that did not have an
observable association in the 1H NMR experiments. In the
latter cases, we wanted to verify that association does not
occur. 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 were
added. The change in volume caused by the addition of host
was less than 10%. For assays containing a mixture of DMSO
and buffer (1 mM phosphate, pH 7.0), the host stock solution
contained the same ratio of solvents except for the calix[4]-
arene 3-pyrene experiment. In this study, the stock and assay
solutions contained 10/90 and 2/98 (v/v) DMSO/buffer, respec-
tively. Several assays were repeated with different concentra-
tions of host and guest to obtain a curvature in the binding
plots. The concentrations of the hosts and guests are given in
the Supporting Information. Complexes with large association
constants (KA > 2 × 104 M-1) had an observed maximum
change in fluorescence equal to 50-75% of the calculated value
(∆Fmax). Because of solubility problems, weaker complexes had
an observed maximum change in fluorescence equal to 30-
The convergent arrangement of the functional groups
of the host-[2]rotaxane is advantageous for binding
guests that have an aromatic ring and a negative charge.
For these guests, [2]rotaxane 1 has stronger interactions
than a host with divergent functional groups or one that
just has an aromatic pocket. This result suggests that
the pocket and arginine derivative of [2]rotaxane 1
interact with an aromatic ring and anion, respectively.
The aromatic ring of a guest provides the majority of
binding free energy in DMSO and DMSO/water mixtures.
In the case of indole, the ring of the rotaxane does not
appear to help or hinder association. But for larger
aromatic guests, the ring does contribute and provide
favorable binding free energy. The arginine derivative
on the rotaxane’s ring makes a significant contribution
to the binding free energy for charged guests. Although
the strengths of these complexes are diminished in the
presence of water, a greater binding free energy is
observed for [2]rotaxane 1 binding N-Ac-Trp than for its
association with indole, which essentially binds through
the hydrophobic effect alone. Thus, [2]rotaxane 1 still
binds charged guests strongly in solutions with a high
percentage of water by forming favorable interactions
with its convergent functional group. The divergent
groups of calix[4]arene 2 are actually counterproductive
for guest binding; the recognition elements and aromatic
pocket do not cooperate in complex formation.
50% of ∆Fmax
.
Maximum excitation and emission wavelengths were used
for the guests except in the cases of a high concentration of
the components or an overlap of the emission bands. At high
concentration of a fluorophore, a shoulder of the excitation or
emission band was used or monitored, respectively. If overlap
occurred, λem was chosen such that the total emission of the
host was less than 10% of the emission of the guest. In the
case of calix[4]arene 2 mixed with N-Ac-Trp and indole in
DMSO and DMSO/buffer (80/20 (v/v)), significant overlap in
the emission spectra occurred. No quenching occurred in these
assays, but there was a slight increase in fluorescence intensity
consistent with the amount of host added. At higher concen-
trations of water, the fluorescence of calix[4]arene 2 is
negligible. A small amount of quenching was only observed
with a high concentration of host, which is consistent with the
degree of association observed by 1H NMR spectroscopic
experiments.
Exp er im en ta l Section
Gen er a l In for m a tion . All air-sensitive reactions were
carried out under positive argon pressure, and solvents were
freshly distilled under vacuum over a suitable drying agent.
All reagents were used as purchased. The melting points of
the compounds are given uncorrected. NMR spectra used for
product identification and conformational analysis were mea-
sured in CDCl3 or DMSO-d6 using a spectrometer operating
at 400.14 MHz for proton nuclei and 100.23 MHz for carbon
nuclei or one operating at 250 MHz for proton nuclei and 62.9
MHz for carbon nuclei. Chemical shifts are in parts per million
and are referenced using an internal TMS standard.
Molecu la r Mod elin g. Energy-minimized conformers of [2]-
rotaxane 1 bound to N-Ac-Trp and fluorescein were determined
using a conjugate gradient method (Polak-Ribiere) to an rms
gradient of 0.01 kcal/(mol‚Å) (semiempirical calculations, AM1
force field to assign charges, and then molecular mechanics
calculations, MM+ force field as presented by HyperChem,
Hypercube, Inc., Gainesville, FL). A guest was combined with
the host in several different orientations, and the complexes
were minimized (Polak-Ribiere) to an rms gradient of 0.1 kcal/
(mol‚Å). The conformation-energy space of the lowest energy
complex was further explored through Monte Carlo simula-
tions. A complex was heated from 0 to 300 K over 10 steps,
and the simulations were run for 1000 steps at 300 K. Low-
energy complexes were chosen out of the 1000 structures, and
a final round of energy minimization was performed using a
conjugate gradient method (Polak-Ribiere) to an rms gradient
of 0.1 kcal/(mol‚Å) (molecular mechanics calculations, MM+
force field). The lowest energy complexes for host-[2]rotaxane
bound to N-Ac-Trp and fluorescein are presented.
1
Mon itor in g Associa tion . For H NMR assays, six separate
solutions were made that contained a guest at a constant
concentration of 1.0 mM. Five of the solutions contained a
different amount of a host from 0.2 to 3.0 mM. At this
concentration range, KA values in the range of 103 M-1 can
usually be accurately derived. Weaker complexes will generally
provide linear binding plots. Assays that had linear plots were
repeated with the guest held at a constant concentration of
5.0 mM and the host varied from 1.0 to 10 mM. If no changes
(51) Yamana, K.; Ohashi, Y.; Nunota, K.; Nakano, H. Tetrahedron
1997, 53, 4265-4270.
(52) Li, Y.-H.; Chan, L.-M.; Tyer, L.; Moody, R. T.; Himel, C. M.;
Hercules, D. M. J . Am. Chem. Soc. 1975, 97, 3118-3126.
(53) Chen, R. F.; Kernohan, J . K. J . Biol. Chem. 1967, 242, 5813-
5823.
Com p ou n d Syn th esis. The mixture of syn and anti
isomers of the crown ether derivatives or the sliding of the
rotaxane ring complicate most 1H and 13C NMR spectra.
2568 J . Org. Chem., Vol. 68, No. 7, 2003

Guest Recognition by Host-[2]Rotaxane
Changing the temperature did not significantly reduce the
number of conformers.
Hz, 1/2H), 7.75 (s, 1/2H), 7.70-7.50 (m, 11/2H), 7.32 (s, 2H), 7.25
(s, 2H), 7.17-7.15 (m, 1H), 7.06-6.76 (m, 8H), 6.60-6.50
(m, 1H), 4.79-4.73 (m, 4H), 4.57-4.45 (m, 4H), 3.90 (s, 3H),
3.88 (s, 3H), 3.51-3.42 (m, 4H); 13C NMR (63 MHz, CDCl3) δ
169.25, 152.29, 148.68, 146.29, 133.16, 132.74, 132.53, 132.40,
130.83, 130.21, 129.49, 129.23, 128.94, 128.51, 128.21, 127.25,
125.77, 117.50, 113.45, 110.62, 72.47, 72.25, 52.27, 31.65,
31.45; TOF MS m/z calcd for C46H41N2O10Na+ 803.2581, found
803.2604.
Da ta for 5,17-bis(3-a m in op h en yl)-25,27-bis(m eth oxy-
ca r bon ylm eth oxy)ca lix[4]a r en e-26,28-d iol (3): mp 156 °C
dec; 1H NMR (250 MHz, CDCl3) 7.68 (s, 2H), 7.25 (s, 4H), 7.16
(t, J ) 7.6 Hz, 2H), 6.90-6.98 (m, 6H), 6.83 (s, 2H), 6.76 (t, J
) 7.4 Hz, 2H), 6.59 (d, J ) 7.6 Hz, 2H), 4.76 (s, 4H), 4.50 (d,
J ) 13.0 Hz, 4H), 3.87 (6H, s), 3.40-3.80 (br s, 2H), 3.45 (d, J
) 13.0 Hz, 4H); 13C NMR (63 MHz, CDCl3) δ 169.23, 152.65,
152.35, 146.58, 142.57, 133.03, 132.52, 129.44, 129.32, 128.26,
127.88, 127.25, 125.67, 117.34, 113.58, 113.24, 77.21, 72.32,
52.19, 31.65; TOF MS ES+ m/z calcd for C46H43N2O8 (MH+)
751.3019, found 751.3069.
5,17-Bis((Boc)3a r gin yla m in op h en yl)-25,27-b is(m et h -
oxyca r bon ylm eth oxy)ca lix[4]a r en e-26,28-d iol (9a ). A 140
mg (0.185 mmol) sample of calix[4]arene 3, 266 mg (0.560
mmol) of (Boc)3-Arg-OH, and 76 mg (0.560 mmol) of HOBT
were dissolved in 10 mL of CHCl3, and the mixture was cooled
to 0 °C. After 118 mg (0.616 mmol) of EDC was added, the
reaction mixture was warmed to rt and stirred under Ar for 1
h. The reaction mixture was refluxed for 12 h to facilitate
coupling. The crude reaction mixture was diluted with ca. 30
mL of CHCl3 and extracted with 5% HCl and then water. The
organic layers were collected and dried over NaSO4, and the
product was purified by column chromatography (SiO2, CHCl3,
C2H5OH) to give 0.26 g (84%) of calix[4]arene 9a as a yellow
oil: 1H NMR (250 MHz, CDCl3) δ 9.60-9.15 (m, 6H), 9.08 (s,
2H), 7.64 (s, 2H), 7.53-7.45 (m, 4H), 7.37-7.27 (m, 8H+solvent),
6.99-6.95 (m, 4H), 6.77 (t, J ) 7.5 Hz, 2H), 5.94 (d, J ) 8.1
Hz, 2H), 5.36 (d, J ) 7.8 Hz, 1H), 4.76 (s, 4H), 4.50 (d, J )
13.3 Hz, 4H), 4.60-4.40 (m, 2H), 3.89 (s, 6H), 4.00-3.80 (m,
2H), 3.80-3.60 (m, 2H), 3.47 (d, J ) 13.3 Hz, 4H), 1.93-1.67
(m, 8H), 1.52-1.34 (m, 54H); 13C NMR (100 MHz, CDCl3) δ
170.67, 169.20, 163.46, 161.15, 155.50, 154.83, 152.96, 152.28,
141.81, 137.86, 132.88, 131.53, 129.35, 129.02, 128.39, 127.75,
127.22, 125.68, 124.57, 124.28, 122.92, 119.22, 118.18, 111.38,
105.07, 84.25, 83.80, 79.69, 72.33, 61.19, 52.21, 44.16, 44.03,
31.56, 31.21, 29.49, 28.43-27.91 (m), 27.02, 24.75, 24.63, 14.18;
TOF MS ES+ m/z calcd for C88H114N10O22Na+ 1685.8007, found
1685.8002.
5,17-Bis(N-a cetyl(Boc)2a r gin yla m in op h en yl)-25,27-bis-
(m eth oxyca r bon ylm eth oxy)ca lix[4]a r en e-26,28-d iol (9b).
A 200 mg (0.266 mmol) sample of calix[4]arene 3, 334 mg
(0.800 mmol) of Ac-(Boc)2-Arg-OH, and 108 mg (0.800 mmol)
of HOBT were dissolved in 15 mL of CHCl3, and the mixture
was cooled to 0 °C. A 170 mg (0.880 mmol) sample of EDC
was added to the reaction mixture, which was warmed to rt
and refluxed under argon for 1 h (TLC analysis indicated
complete conversion). The crude reaction mixture was diluted
with ca. 30 mL of CHCl3 and extracted with 1% HCl and water.
The product was purified by column chromatography (SiO2,
CHCl3, CH3OH) to give 0.29 g (70%) of calix[4]arene 9b as a
transparent oil: 1H NMR (250 MHz, CDCl3) 9.70-9.40 (br m,
4H), 9.12 (s, 1H), 7.53 (s, 2H), 7.46-7.42 (m, 4H), 7.35-7.27
(m, 8H), 6.70-6.98 (m, 4H), 6.79-6.77 (m, 2H), 6.37 (m, 2H),
5.00-4.51 (m, 2H), 4.76 (s, 4H), 4.50 (d, J ) 13.2 Hz, 4H),
4.20-4.00 (m, 2H), 3.90 (6H, s), 3.83-3.80 (m, 1H), 3.70-3.34
(m, 3H), 3.47 (d, J ) 13.2 Hz, 4H), 2.60-2.45 (m, 1H), 2.17-
2.03 (m, 6H), 1.92-1.67 (m, 8H), 1.51-1.31 (m, 36H); 13C NMR
(63 MHz, CDCl3) δ 170.29, 170.20, 169.15, 163.04, 154.72,
152.90, 152.22, 141.76, 138.01, 137.79, 132.86, 132.36, 131.46,
129.32, 128.98, 128.35, 127.16, 125.90, 125.65, 122.90, 119.07,
118.51, 115.05, 84.34, 79.74, 72.27, 53.16, 52.19, 44.05, 31.52,
5,17-Dibr om o-25,27-bis(eth oxyca r bon ylm eth oxy)ca lix-
[4]a r en e-26,28-d iol (5). To a solution of 6.00 g (10.0 mmol)
of calix[4]arene diethyl ester42 in 40 mL of CHCl3 was slowly
added 3.10 g (20.0 mmol) of Br2 in 20 mL of CHCl3. The
reaction mixture was stirred at rt for 3 h, followed by the
removal of CHCl3 and residual Br2 in vacuo. Recrystallization
from CHCl3 gave 7.54 g (99%) of calix[4]arene 5 as a white
powder: mp 122 °C dec; 1H NMR (250 MHz, DMSO-d6) δ 7.97
(s, 2H), 7.37 (s, 4H), 7.07 (d, J ) 7.6 Hz, 4H), 6.82 (t, J ) 7.6
Hz, 2H), 4.75 (s, 4H), 4.34-4.21 (m, 6H), 3.46 (d, J ) 13.2 Hz,
4H), 1.27 (t, J ) 7.4 Hz, 6H); 13C NMR (63 MHz, DMSO-d6) δ
168.61, 151.86, 132.77, 130.59, 130.17, 129.24, 125.43, 109.88,
72.23, 60.89, 30.09, 13.89; TOF MS m/z calcd for C36H35O8-
+
Br2 753.0699, found 753.0740.
5,17-Bis(3-n it r op h en yl)-25,27-b is(h yd r oxyca r b on yl-
m eth oxy)ca lix[4]a r en e-26,28-d iol (6). A 1.13 g (1.50 mmol)
sample of dibromocalix[4]arene 5 and 1.00 g (5.99 mmol) of
3-nitrophenylboronic acid were dissolved in a 55 mL of toluene/
methanol (9/1) solution. A 1.11 g (10.5 mmol) sample of Na2-
CO3 was dissolved in 10 mL of water, and this mixture was
added to the reaction mixture. The flask was flushed twice
with Ar, and charged with 0.15 g (0.15 mol) of Pd(PPh3)4. After
the reaction mixture was stirred at 70 °C for 40 h, the organic
phase was separated, diluted with 100 mL of CH2Cl2, and
extracted with 5% HCl. The organic phases were collected and
dried with Na2SO4. After the solvents were removed in vacuo,
the crude product was purified via column chromatography
(SiO2, CHCl3, CH3OH) to give 0.91 g (78%) of calix[4]arene 6
as a yellow solid: mp 270 °C dec; 1H NMR (250 MHz, DMSO-
d6) δ 8.40 (s, 2H), 8.09 (d, J ) 7.9 Hz, 4H), 7.66 (s, 4H), 7.71-
7.64 (m, 2H), 7.17 (d, J ) 7.6 Hz, 4H), 6.79 (t, J ) 7.6 Hz,
2H), 4.75 (s, 4H), 4.43 (d, J ) 12.9 Hz, 4H), 3.59 (d, J ) 12.9
Hz, 4H); 13C NMR (63 MHz, DMSO-d6) δ 170.29, 153.29,
152.78, 148.43, 141.82, 133.12, 132.57, 130.09, 129.23, 128.56,
128.44, 127.13, 125.27, 120.88, 120.13, 72.33, 30.55; TOF MS
+
m/z calcd for C44H35N2O12 783.219, found 783.2256.
5,17-Bis(3-n it r op h en yl)-25,27-b is(m et h oxyca r b on yl-
m eth oxy)ca lix[4]a r en e-26,28-d iol (7). A 3.40 g (4.34 mmol)
sample of calix[4]arene 6 was refluxed in a solution of CHCl3/
CH3OH (2/1 (v/v)), containing concd H2SO4 (2-3%) for 12 h.
Upon solvent removal, 3.50 g (99%) of the calix[4]arene 7 was
obtained as an off-white solid, which was pure by 1H NMR
analysis: mp 151-155 °C; 1H NMR (250 MHz, CDCl3) δ 8.37
(s, 2H), 8.11 (d, J ) 8.1 Hz, 2H), 8.00 (s, 2H), 7.84 (d, J ) 7.8
Hz, 2H), 7.54 (t, J ) 8.3 Hz, 2H), 7.33 (s, 4H), 7.02 (d, J ) 7.4
Hz, 4H), 6.83 (t, J ) 7.4 Hz, 2H), 4.80 (s, 4H), 4.55 (d, J )
13.2 Hz, 4H), 3.92 (s, 6H), 3.52 (d, J ) 12.5 Hz, 4H); 13C NMR
(63 MHz, CDCl3) δ 169.23, 153.79, 152.36, 148.69, 142.86,
132.87, 132.48, 130.16, 129.66, 129.44, 128.90, 127.26, 125.86,
121.22, 121.00, 77.21, 72.28, 52.31, 31.64; MS HiResESI m/z
calcd for C46H38N2O12Na+ 833.2322, found 833.2300.
P a r tia l Red u ction of 5,17-Bis(3-n itr op h en yl)-25,27-bis-
(m eth oxycar bon ylm eth oxy)calix[4]ar en e-26,28-diol. A 2.00
g (2.47 mmol) sample of calix[4]arene 7 was dissolved in 130
mL of a CHCl3/CH3OH/CH3CO2H (10/5/1) solution. The flask
was flushed with Ar and charged with 100 mg of Pd/C, H2 (50
psi), and the reaction mixture was stirred at rt for 21 h. The
progress of reduction was monitored by TLC, and the reaction
was ended when ca. 80% of dinitrophenylcalix[4]arene 7 was
consumed. The reaction mixture was filtered through a plug
of Celite, the solvents were removed in vacuo, and the products
were separated by column chromatography (SiO2, CHCl3/CH3-
OH) to give 0.23 g (0.28 mmol), 0.60 g (0.77 mmol, 35%), and
0.91 g (1.2 mmol, 56%) of calix[4]arene 7, 8, and 3, respectively
(yields are calculated on the basis of the amount of consumed
calix[4]arene 7).
Da ta for 5-(3-a m in op h en yl)-17-(3-n itr op h en yl)-25,27-
b is(m et h oxyca r b on ylm et h oxy)ca lix[4]a r en e-26,28-d iol
(8): mp 141-145 °C; 1H NMR (400 MHz, CDCl3) δ 8.38 (s,
28.14, 27.93, 24.90, 23.29, 23.12; TOF MS m/z calcd for
1
1
+
1/2H), 8.10 (d, J ) 8.0 Hz, /2H), 8.01 (s, /2H), 7.83 (d, J ) 7.5
C
82H103N10O20 1547.7350, found 1547.7360.
J . Org. Chem, Vol. 68, No. 7, 2003 2569

Smukste et al.
5,17-Bis(3-(2-a m in o-5-gu a n id in op e n t a n oyla m in o)-
p h e n yl)-25,27-b is(m e t h oxyca r b on ylm e t h oxy)ca lix[4]-
a r en e-26,28-d iol (2). A 254 mg (0.16 mg) sample of calix[4]-
arene 9b was dissolved in 5 mL of CHCl3, and 1.5 mL of TFA
was added to the reaction mixture. Deprotection was complete
within 6 h, as determined by TLC analysis. The solvent and
TFA were removed in vacuo, and the crude product was
purified via column chromatography (SiO2, CHCl3, CH3OH)
to give 0.15 g (82%) of calix[4]arene 2 as a yellow oil: 1H NMR
(250 MHz, CDCl3) 8.00-7.80 (m, 2H), 7.63 (s, 2H), 7.50 (d, J
) 7.6 Hz, 2H), 7.36-7.26 (m, 8H), 6.99 (d, J ) 7.4 Hz, 4H),
6.77 (t, J ) 7.4 Hz, 2H), 4.76 (s, 4H), 4.49-4.45 (m, 2H), 4.43
(d, J ) 13.1 Hz, 4H), 3.89 (s, 6H), 3.45 (d, J ) 13.1 Hz, 4H),
3.40-3.11 (m, 4H), 2.04 (s, 6H), 2.00-1.40 (m, 8H); 13C NMR
(63 MHz, CDCl3) δ 170.02, 169.88, 169.12, 157.73, 153.06,
152.47, 152.19, 150.25, 140.42, 139.22, 132.82, 132.31, 131.46,
130.71, 128.85, 128.28, 128.05, 126.74, 124.91, 120.68, 119.83,
116.34, 78.52, 52.58, 51.69, 47.55, 31.52, 28.14, 24.90, 21.60;
13C NMR (63 MHz, DMSO-d6) δ 169.35, 168.92, 154.20, 153.56,
153.13, 152.48, 151.52, 148.40, 141.96, 140.77, 139.48, 133.18,
132.62, 131.21, 130.14, 129.30, 129.14, 128.98, 128.64, 127.20,
126.83, 125.45, 121.38, 120.92, 120.20, 117.39, 117.00, 79.33,
79.17, 72.24, 51.95, 36.40, 35.78, 31.43, 31.49, 30.81, 30.62;
TOF MS m/z calcd for C49H46N3O11+ 852.3132, found 852.3105.
Ca lix[4]a r en e Di[(ter t-bu toxyca r bon yla m in o)ben zo]-
[24]cr ow n -8 Host [2]Rota xa n e (14). A 400 mg (0.589 mmol)
sample of di-Boc-crown ether and 275 mg (0.589 mmol) of
5-(3,5-di-tert-butylbenzylamino)pentanoic acid hexafluorophos-
phate were dissolved in 3 mL of CHCl3. The solution was cooled
to -10 °C (ice/salt bath) for 10 min, and then 146 mg (0.707
mmol) of DCC in 0.5 mL of CHCl3 was added.18 After the
reaction mixture was stirred for 1.5 h, 153 mg (0.442 mmol)
of calix[4]arene 12, dissolved in 0.3 mL of CHCl3, was added.
The reaction was stirred at rt overnight. The solvent was
removed in vacuo, the oily residue was dissolved in CH3CN,
and DCU was removed by filtration. CH3CN was removed by
evaporation, and the crude reaction mixture was triturated
with ethyl ether to remove cyclic tether. The crude product
was purified using rotary chromatography (SiO2, CH2Cl2, CH3-
OH) to give 546 mg (67%) of [2]rotaxane 14. Using this
procedure, [2]rotaxane 14 was obtained in 30-70% yield; the
success of the reaction is very sensitive to the purity of di-
Boc-crown ether: 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H),
8.34 (s, 1H), 8.06 (d, J ) 8.0 Hz, 1H), 8.01 (s, 1H), 7.84-7.83
(m, 2H), 7.81 (s, 1H), 7.72 (d, J ) 6.8 Hz, 1H), 7.50 (dt, J )
8.0, 1.6 Hz, 1H), 7.36-6.74 (m, 25H), 6.40 (q, J ) 6.4 Hz, 1H),
4.77-4.76 (m, 4H), 4.55-4.45 (m, 6H), 4.20-4.00 (m, 6H),
4.00-3.93 (m, 4H), 3.89 (s, 6H), 3.60-3.42 (m, 18H), 3.20-
3.05 (m, 4H), 3.90 (m or br s, 2H), 2.63 (m, 2H), 1.85 (m, 2H),
1.49 (s, 18H), 1.31-1.22 (m, 4H), 1.23 (s, 18H); 13C NMR (63
MHz, CDCl3) δ 172.65, 170.25, 169.23, 153.83, 153.16, 152.86,
152.19, 151.17, 148.56, 147.37, 143.00, 141.30, 138.94, 133.15,
132.56, 131.75, 131.30, 129.64, 129.44, 129.11, 128.94, 128.35,
127.97, 127.17, 126.96, 125.81, 124.31, 123.07, 121.61, 121.04,
120.85, 117.68, 113.09, 111.59, 104.92, 80.21, 72.26, 70.34,
69.94, 69.67, 68.41, 68.15, 52.63, 52.20, 48.72, 37.12, 35.71,
35.12, 34.77, 31.55, 31.35, 30.59, 29.62, 28.30, 25.74, 22.24,
+
TOF MS ES+ m/z calcd for C62H71N10O12 1147.5253, found
1147.5204.
5,17-Bis(N-a cetyla r gin yla m in op h en yl)-25,27-bis(m eth -
oxyca r bon ylm eth oxy)ca lix[4]a r en e-26,28-d iol (10). A 120
mg (0.071 mg) sample of of calix[4]arene 9a was dissolved in
5 mL of CH2Cl2, and 3.0 mL of TFA was added to the reaction
mixture. Complete deprotection required 24 h. The solvent and
TFA were removed in vacuo, and the crude product was
purified via column chromatography (SiO2, CHCl3, CH3OH)
to give 0.030 g (41%) of calix[4]arene 10 as a yellow oil: 1H
NMR (250 MHz, CDCl3) δ 7.15-7.33 (8H, m), 6.93-7.00 (6H,
m), 6.88 (2H, s), 6.78 (2H, t, J ) 7.5 Hz), 6.65 (2H, d, J ) 8.3
Hz), 4.78 (4H, s), 4.50 (4H, d, J ) 13.1 Hz), 4.20-4.40 (2H,
m), 3.90 (6H, s), 3.47 (4H, d, J ) 13.1 Hz), 1.10-1.90 (6H, m),
0.86-0.89 (4H, m); 13C NMR (63 MHz, CDCl3) δ 171.5, 168.3,
167.2, 153.8, 153.4, 143.2, 139.3, 134.3, 133.2, 130.5, 130.3,
130.0, 128.3, 126.7, 124.0, 119.3, 73.3, 55.1, 52.9, 41.8, 32.1,
29.8, 25.5; TOF MS ES+ m/z calcd for C58H67N10O10+ 1063.5041,
found 1063.5146.
5-[3-(3-ter t-Bu t oxyca r b on yla m in op r op ion yla m in o)-
p h en yl]-17-(3-a m in op h en yl)-25,27-bis(m eth oxyca r bon yl-
m eth oxy)ca lix[4]a r en e-26,28-d iol (11). To a solution of 436
mg (2.30 mmol) of Boc-â-alanine in 20 mL of CHCl3 was added
374 mg (2.30 mmol) of CDI. The reaction was stirred at rt for
1 h, and then 600 mg (0.768 mmol) of calix[4]arene 8 and 52
mg (0.77 mmol) of imidazole were added to the solution. The
reaction mixture was stirred at rt for 30 min and then refluxed
for 4 h. The solvents were removed in vacuo. Flash chroma-
tography (SiO2, CHCl3, C2H5OH) afforded 703 mg (96%) of
+
20.95, 14.14, 13.63; TOF MS m/z calcd for C103H127N6O24
1831.8902, found 1831.8905.
Ca lix[4]a r en e Di(a m in oben zo)[24]cr ow n -8 Host [2]-
Rota xa n e (15). A 480 mg (0.243 mmol) sample of calix[4]-
arene rotaxane 14 was dissolved in 10 mL of CH2Cl2, and 3
mL of TFA was added. After the reaction was stirred at rt for
3 h, volatile materials were removed under high vacuum. To
remove traces of TFA, the crude product was dissolved in 10
mL of a CH2Cl2/CH3OH (2/1) solution, which was subsequently
removed under high vacuum. This procedure was repeated
several times. Crude [2]rotaxane 15 was dissolved in CH2Cl2/
CH3OH (9/1) and extracted with 0.1 N NHCO3. The organic
phase was collected and dried over Na2SO4, and the solvent
was removed in vacuo to give 378 mg (89%) of the rotaxane 9,
which was used without further purification: 1H NMR (400
MHz, CDCl3) δ 8.03-7.50 (m, 6H), 7.40-6.84 (m, 17H), 6.70-
6.60 (m, 6H), 6.56-6.52 (m, 2H), 6.30-6.20 (m, 2H), 4.71 (s,
4H), 4.53-4.39 (m, 6H), 4.12-3.20 (m, 6H (-OCH3), 24H
(DB24C8), 4H (ArCH2Ar), 4H (-NH2)), 3.09-2.80 (m, 6H),
2.75-2.70 (m, 4H), 1.75-1.79 (m, 2H), 1.27 (s, 18H), 1.27-
1.14 (4H); 13C NMR (63 MHz, CDCl3) δ 171.12, 169.53, 169.44,
152.69, 152.57, 151.81, 151.06, 147.82, 141.76, 140.87, 139.74,
132.95, 132.79, 132.41, 131.93, 131.58, 130.29, 129.06, 128.52,
128.14, 126.87, 125.66, 124.54, 123.00, 121.00, 119.27, 117.42,
114.00, 109.94, 103.60, 77.17, 72.16, 70.10, 69.03, 68.65, 68.02,
60.25, 52.27, 48.60, 45.53, 36.10, 34.76, 31.33, 25.77, 22.11,
20.93; TOF MS m/z calcd for C93H111N6O20+ 1631.7853, found
1631.7805.
1
calix[4]arene 11 as a yellow solid: mp 145-150 °C; H NMR
(250 MHz, CDCl3) δ 7.70-7.67 (m, 2H), 7.54-7.26 (m, 8H),
7.05 (d, J ) 7.4 Hz, 1H), 6.97-6.91 (m, 4H), 6.90-6.76 (m,
2H), 6.65 (t, J ) 7.5 Hz, 1H), 4.75 (s, 4H), 4.53-4.44 (m, 4H),
3.88 (s, 6H), 3.53-3.38 (m, 6H), 2.63 (t, J ) 5.5 Hz), 1.44 (s,
9H); 13C NMR (63 MHz, CDCl3) δ 169.78, 169.11, 156.22,
153.66, 152.77, 152.18, 148.47, 142.69, 141.89, 138.22, 132.93,
132.82, 132.68, 132.52, 132.40, 132.18, 131.68, 130.67, 130.08,
129.33, 129.08, 128.81, 128.38, 128.22, 128.01, 127.11, 125.60,
125.48, 122.50, 121.04, 120.83, 119.11, 117.93, 117.77, 79.37,
77.17, 72.14, 52.09, 37.35, 36.46, 31.43, 31.27, 28.27; TOF MS
+
m/z calcd for C54H54N3O13 952.3657, found 952.3703.
5-[3-(3-Am in op r op ion yla m in o)p h en yl]-17-(3-a m in o-
p h e n yl)-25,27-b is(m e t h oxyca r b on ylm e t h oxy)ca lix[4]-
a r en e-26,28-d iol (12). A 667 mg (0.700 mmol) sample of calix-
[4]arene 11 was dissolved in 10 mL of CHCl3, and 3 mL of
TFA was added. After the reaction mixture was stirred at rt
for 1 h, the solvent and TFA were removed in vacuo, and the
product was purified by column chromatography (SiO2, CHCl3,
CH3OH) to give 504 mg (84%) of calix[4]arene 12: mp 284 °C
dec; 1H NMR (400 MHz, CDCl3) δ 9.99 (br s, 1H), 7.72 (s, 1H),
7.47-7.46 (m, 2H), 7.33-7.22 (m, 6H), 7.05 (d, J ) 4.5 Hz,
1H), 6.98-6.91 (m, 4H), 6.77-6.73 (m, 2H), 6.66 (t, J ) 4.5
Hz, 1H), 4.75 (s, 4H), 4.56-4.44 (m, 4H), 3.88 (s, 6H), 3.51-
3.36 (m, 4H), 3.09 (t, J ) 3.3 Hz, 2H), 2.49 (t, J ) 3.3 Hz, 2H);
Ca lix[4]a r en e Di[(N-a cetyla r gin yla m in o)ben zo][24]-
cr ow n -8 Host-[2]Rota xa n e (1). A 370 mg (0.227 mmol)
sample of amino[2]rotaxane 15, 148 mg (0.681 mmol) of Ac-
Arg-OH‚HCl, and 302 mg (0.681 mmol) of BOP were dissolved
in 3 mL of DMF. A 316 µL sample of DIEA was added to the
2570 J . Org. Chem., Vol. 68, No. 7, 2003

Guest Recognition by Host-[2]Rotaxane
solution. The reaction mixture was stirred at rt for 12 h. TLC
analysis indicated incomplete conversion; therefore, 50 mg
(0.23 mmol) of BOP and Ac-Arg-OH‚HCl were added. The
reaction was continued for another 12 h, and then the solvent
and DIEA were removed under high vacuum. Triturating the
crude material with an acetone/ether (1/1) solution removed
DIEA‚HCl, HMPA, and HOBt, which are soluble. The precipi-
tate was dissolved in 20 mL of CH2Cl2/CH3OH (8/2) and
extracted with water. The organic phase was separated, the
solvents were removed, and the product was purified by
column chromatography to give 297 mg (66%) of [2]rotaxane
1 as a yellow oil: 1H NMR (400 MHz, CDCl3) δ 10.00 (s, 2H),
8.42 (s, 1H), 8.19 (d, J ) 8 Hz, 1H), 8.11 (s, 2H), 7.98 (s, 1H),
7.80-7.75 (m, 3H), 7.69 (s, 2H), 7.59-6.97 (m, 28 H), 6.77 (t,
J ) 7.6 Hz, 4H), 6.73 (d, J ) 7.6 Hz, 2H), 6.21 (s, 1H), 6.06 (d,
J ) 7.6 Hz, 1H), 4.86 (s, 4H), 4.83 (s), 4.56 (br s, 2H), 4.39 (d,
J ) 12.8 Hz, 4 H), 4.13-4.08 (m, 8H), 3.93-3.91 (m, 2H), 3.82
(s, 6H), 3.81-3.52 (m, 24 H), 3.37-3.10 (m, 10H), 1.87 (s, 6H),
1.75-1.21 (m, 14 H), 1.17 (s, 18 H); 13C NMR (101 MHz, CDCl3)
δ 171.39, 170.21, 170.09, 169.49, 169.35, 158.30, 157.98,
156.70, 153.36, 152.73, 152.41, 150.49, 148.40, 140.71, 139.44,
133.11, 132.52, 131.49, 130.94, 130.05, 129.16, 128.96, 128.86,
128.53, 128.12, 127.11, 126.69, 125.17, 123.81, 122.61, 121.17,
120.83, 120.07, 113.74, 111.63, 79.09, 72.35, 69.97, 69.71-
67.63 (m), 53.52, 52.93, 51.92, 48.13, 47.60 (several signals are
overlaid by the CD3OD signal), 36.31, 34.98, 34.29, 31.08,
30.94, 30.74, 30.52, 29.19, 25.31, 25.14, 22.35, 21.90; TOF MS
+
m/z calcd for C109H139N14O24 2028.0087, found 2027.9873.
Ack n ow led gm en t is made to the donors of the
Petroleum Research Fund, administered by the Ameri-
can Chemical Society, for partial support of this re-
search. Thanks are also due to the University of
Cincinnati for partial funding. I.S. was supported by the
University of Cincinnati Research Council Fellowship
and the Stecker Fellowship.
Su p p or tin g In for m a tion Ava ila ble: NMR properties of
the calix[4]arenes, a representative binding plot, and concen-
trations of the components in the fluorescence assays. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O026522+
J . Org. Chem, Vol. 68, No. 7, 2003 2571