Self-assembly of fluorescent inclusion complexes in competitive media including the interior of living cells

Article abstract of DOI:10.1021/ja075567v

Anthracene-containing tetralactam macrocycles are prepared and found to have an extremely high affinity for squaraine dyes in chloroform (log K _a = 5.2). Simply mixing the two components produces highly fluorescent, near-infrared inclusion comp

Full text of DOI:10.1021/ja075567v

Published on Web 11/10/2007
Self-Assembly of Fluorescent Inclusion Complexes in
Competitive Media Including the Interior of Living Cells
Jeremiah J. Gassensmith, Easwaran Arunkumar, Lorna Barr, Jeffrey M. Baumes,
Kristy M. DiVittorio, James R. Johnson, Bruce C. Noll, and Bradley D. Smith*
Contribution from the Department of Chemistry and Biochemistry and the Walther Cancer
Research Center, UniVersity of Notre Dame, Notre Dame, Indiana 46556
Received July 25, 2007; E-mail: smith.115@nd.edu
Abstract: Anthracene-containing tetralactam macrocycles are prepared and found to have an extremely
high affinity for squaraine dyes in chloroform (log Ka ) 5.2). Simply mixing the two components produces
highly fluorescent, near-infrared inclusion complexes in quantitative yield. An X-ray crystal structure shows
the expected hydrogen bonding between the squaraine oxygens and the macrocycle amide NH residues,
and a high degree of cofacial aromatic stacking. The kinetics and thermodynamics of the assembly process
are very sensitive to small structural changes in the binding partners. For example, a macrocycle containing
two isophthalamide units associates with the squaraine dye in chloroform 400 000 times faster than an
analogous macrocycle containing two 2,6-dicarboxamidopyridine units. Squaraine encapsulation also occurs
in highly competitive media such as mixed aqueous/organic solutions, vesicle membranes, and the
organelles within living cells. The highly fluorescent inclusion complexes possess emergent properties;
that is, as compared to the building blocks, the complexes have improved chemical stabilities, red-shifted
absorption/emission maxima, and different cell localization propensities. These are useful properties for
new classes of near-infrared fluorescent imaging probes.
with limited water solubility,3 which means that in biological
samples they will be confined to hydrophobic environments such
Introduction
The selective and reversible self-assembly of biomolecules
is a central requirement for cell function as demonstrated by
the recognition properties of DNA, bilayer membranes, enzyme/
substrates, and protein receptor/ligand complexes. Indeed, a
major goal of the pharmaceutical industry is to discover
“biologically active” drug molecules that selectively inhibit or
activate these recognition processes. In addition, there is an
articulated need to develop synthetic host:guest systems that
can operate in biological media.1 A notable success has been
the invention of synthetic chemosensors for cell imaging, that
is, small fluorescent host molecules that can recognize and sense
the presence of cellular analytes such as metal cations, anions,
and biomolecules.2 In contrast to this success, there are
comparatively few examples of synthetic host:guest partners that
can selectively self-assemble in complicated biological environ-
ments and form discrete complexes with well-defined structures.
The focus of this Article is on uncharged organic molecules
as protein surfaces,4 micelles,5 and the interior of bilayer
membranes.6 Programmed host-guest assembly under these
competitive conditions is particularly challenging because the
association must be driven by the relatively weak noncovalent
interactions of hydrogen bonding, aromatic stacking, and dis-
persion forces.7 Furthermore, it is difficult to prove unambigu-
ously that assembly has occurred as designed because common
spectroscopic methods (e.g., NMR, UV absorption, etc.) are
generally not applicable in these complex matrices.8 Therefore,
it is particularly helpful if the host:guest complex can be
(3) For supramolecular assembly of synthetic molecules in water, see:
Oshovsky, G. V.; Reinhoudt, D. N.; Verboom, W. Angew. Chem., Int. Ed.
2007, 46, 2366-2393.
(4) Nishijima, M.; Pace, T. C. S.; Nakamura, A.; Mori, T.; Wada, T.; Bohne,
C.; Inoue, Y. J. Org. Chem. 2007, 72, 2707-2715.
(5) Baglioni, P.; Berti, D. Curr. Opin. Colloid Interface Sci. 2003, 8, 55-61
and references therein.
(6) (a) Sisson, A. L.; Shah, M. R.; Bhosal, S.; Matile, S. Chem. Soc. ReV.
2006, 35, 1269-1286 and references therein. (b) McNally, B.; Leevy, W.
M. Supramol. Chem. 2007, 19, 29-37 and references therein. (c) Percec,
V.; Dulcey, A. E.; Peterca, M.; Adelman, P.; Samant, R.; Balagurusamy,
V. S. K.; Venkatachalapathy, S. K.; Heiney, P. A. J. Am. Chem. Soc. 2007,
129, 5992-6002. (d) Percec, V.; Smidrkal, J.; Peterca, M.; Mitchell, C.
M.; Nummelin, S.; Dulcey, A. E.; Sienkowska, M. J.; Heiney, P. A. Chem.-
Eur. J. 2007, 13, 3989-4007. (e) Percec, V.; Dulcey, A. E.; Peterca, M.;
Ilies, M.; Nummelin, S.; Sienkowska, M. J.; Heiney, P. A. Proc. Natl. Acad.
Sci. U.S.A. 2006, 103, 2518-2523. (f) Yan, X. H.; He, Q.; Wang, K. W.;
Duan, L.; Cui, Y.; Li, J. B. Angew. Chem., Int. Ed. 2007, 46, 2431-2434.
(7) There are, of course, several water-soluble macrocyclic host molecules,
such as cyclodextrins and cucurbiturils, which can form inclusion complexes
with organic guests in biological samples. In these cases, the hydrophobic
effect is a major driving force for guest inclusion. For recent examples,
see: (a) Hwang, L.; Baek, K.; Jung, M.; Kim, Y.; Park, K. M.; Lee, D.-
W.; Selvapalam, N.; Kim, K. J. Am. Chem. Soc. 2007, 129, 4170-4171.
(b) Liu, Y.; Chen, Y. Acc. Chem. Res. 2006, 39, 681-691.
(1) (a) Sessler, J. L.; Gale, P. A.; Cho, W.-S. Anion Receptor Chemistry; RSC
Publishing: Cambridge, 2006. (b) Macrocyclic Chemistry: Current Trends
and Future PerspectiVes; Gloe, K., Ed.; Springer: Dordrecht, 2005. (c)
James, T. D.; Phillips, M. D.; Shinkai, S. Boronic Acids in Saccharide
Recognition; RSC Publishing: Cambridge, 2006. (d) Ariga, K.; Kunitake,
T. Supramolecular Chemistry-Fundamentals and Applications; Springer:
Berlin Heidelberg, 2006.
(2) For recent reviews on fluorescent chemosensors that operate in biological
samples, see: (a) Creative Chemical Sensor Systems. Topics in Current
Chemistry 277; Schrader, T., Ed.; Springer: New York, 2007 and all nine
chapters therein. (b) Carol, P.; Sreejith, S.; Ajayaghosh, A. Chem. Asian J.
2007, 2, 338-348. (c) Johnsson, N.; Johnsson, K. ACS Chem. Biol. 2007,
2, 31-38. (d) Umezawa, Y. Chem. Asian J. 2006, 1, 304-312. (e)
Katerinopoulos, H. E. Curr. Pharma Des. 2004, 10, 3835-3852. (f) Bell,
T. W.; Hext, N. M. Chem. Soc. ReV. 2004, 33, 589-598. (g) Prodi, L.
New J. Chem. 2005, 29, 20-31.
9
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J. AM. CHEM. SOC. 2007, 129, 15054-15059
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Self-Assembly of Fluorescent Inclusion Complexes
A R T I C L E S
Chart 1
identified in situ by a unique and sensitive spectroscopic
signature.9
have shown that squaraine-rotaxanes have tremendous promise
as extremely stable molecular probes for fluorescence bioim-
aging.14 These first generation squaraine-rotaxanes were syn-
thesized in yields of 10-30% using a Leigh-type clipping
reaction (a templated macrocyclization around the squaraine
dye).15 Preparation by a direct self-assembly of the two
components (macrocycle and squaraine) does not work with the
phenylene tetralactam macrocycle (i.e., the macrocycle in
rotaxane structure 1) because it is quite insoluble.16 To
circumvent this problem, we designed the novel anthrylene
tetralactam macrocycles 2a and 2b.17 We find that these
macrocycles are soluble in many different solvents, and they
can associate strongly with squaraine dyes in weakly polar
Here, we describe a novel, photoactive host:guest system
whose self-assembly can be monitored directly by the com-
plexation-induced changes in its photophysical properties.10 The
guest molecules are squaraines, intensely fluorescent near-
infrared dyes that have been investigated for potential applica-
tions in many photonic-based technologies.11,12 Recently, we
described the synthesis of permanently interlocked squaraine-
rotaxanes, 1 (Chart 1), and demonstrated that encapsulation of
the squaraine fluorophore within a phenylene tetralactam
macrocycle provides substantial steric protection of the squaraine
chromophore and inhibits aggregation induced self-quenching,
as well as attack by biological nucleophiles.13 In addition, we
(12) Recent papers using squaraine dyes include: (a) Ros-Lis, J. V.; Marcos,
M. D.; Mart´ınez-Ma´n˜ez, R.; Rurack, K.; Soto, J. Angew. Chem., Int. Ed.
2005, 44, 4405-4407. (b) Basheer, M. C.; Santhosh, U.; Alex, S.; Thomas,
K. G.; Suresh, C. H.; Das, S. Tetrahedron 2007, 63, 1617-1623. (c)
Ajayaghosh, A.; Chithra, P.; Varghese, R. Angew. Chem., Int. Ed. 2007,
46, 230-233. (d) Snee, P. T.; Somers, R. C.; Nair, G.; Zimmer, J. P.;
Bawendi, M. G.; Nocera, D. G. J. Am. Chem. Soc. 2006, 128, 13320-
13321. (e) Tatarets, A. L.; Fedyunyayeva, I. A.; Dyubko, T. S.; Povrozin,
Y. A.; Doroshenko, A. O.; Terpetschnig, E. A.; Patsenker, L. D. Anal.
Chim. Acta 2006, 570, 214-223. (f) Avirah, R. R.; Jyothish, K.; Ramaiah,
D. Org. Lett. 2007, 9, 121-124. (g) Hsueh, S.-Y.; Lai, C.-C.; Liu, Y.-J.;
Peng, S.-M.; Chiu, S.-H. Angew. Chem., Int. Ed. 2007, 46, 2013-2017.
(13) (a) Arunkumar, E.; Forbes, C. C.; Noll, B. C.; Smith, B. D. J. Am. Chem.
Soc. 2005, 127, 3288-3289. (b) Arunkumar, E.; Fu, N.; Smith, B. D.
Chem.-Eur. J. 2006, 12, 4684-4690. (c) Arunkumar, E.; Sudeep, P. K.;
Kamat, P. V.; Noll, B. C.; Smith, B. D. New J. Chem. 2007, 31, 677-683.
(d) For a general review on dye encapsulation, see: Arunkumar, E.; Forbes,
C. C.; Smith, B. D. Eur. J. Org. Chem. 2005, 4051-4059.
(8) Most literature reports of self-assembly in bilayer membranes describe
membrane transport systems, and proof of the assembled structure is
typically inferred from the observation of enhanced transport activity,
perhaps with some structural characterization in the solid state or a
homogenous organic solution that mimics the membrane polarity. For a
thoughtful discussion on the practicality of structural studies of pore forming
systems, see: Baudry, Y.; Bollot, G.; Gorteau, V.; et al. AdV. Funct. Mater.
2006, 16, 169-179.
(9) (a) Bhosal, S.; Matile, S. Chirality 2006, 18, 849-856. (b) Bhosale, S.;
Sisson, A. L.; Talukdar, P.; Furstenberg, A.; Banerji, N.; Vauthey, E.; Bollot,
G.; Mareda, J.; Roger, C.; Wurthner, F.; Sakai, N.; Matile, S. Science 2006,
313, 84-86. (c) Burazerovic, S.; Gradinaru, J.; Pierron, J.; Ward, T. R.
Angew. Chem., Int. Ed. 2007, 46, 5510-5514. (d) Milov, A. D.; Samoilova,
R. I.; Tsvetkov, Y. D.; Formaggio, F.; Toniolo, C.; Raap, J. J. Am. Chem.
Soc. 2007, 129, 9260-9261.
(10) For reviews of photoactive self-assembly systems, see: Supramolecular
Dye Chemistry. Topics in Current Chemistry 258; Wu¨rthner, F., Ed.;
Springer: New York, 2005 and all seven chapters therein.
(14) Johnson, J. R.; Fu, N.; Arunkumar, E.; Leevy, W. M.; Gammon, S. T.;
Piwnica-Worms, D.; Smith, B. D. Angew. Chem., Int. Ed. 2007, 46, 5528-
5531.
(11) Reviews of squaraine dyes: (a) Das, S.; Thomas, K. G.; George, M. V. In
Organic Photochemistry; Ramamurthy, V., Schanze, K. S., Eds.; Marcel
Dekker: New York, 1997; pp 467-517. (b) Law, K.-Y. In Organic
Photochemistry; Ramamurthy, V., Schanze, K. S., Eds.; Marcel Dekker:
New York, 1997; pp 519-584. (c) Ajayaghosh, A. Acc. Chem. Res. 2005,
38, 449-459.
(15) Gatti, F. G.; Leigh, D. A.; Nepogodiev, S. A.; Slawin, A. M. Z.; Teat, S.
J.; Wong, J. K. Y. J. Am. Chem. Soc. 2001, 123, 5983-5989.
(16) (a) Johnston, A. G.; Leigh, D. A.; Murphy, A.; Smart, J. P.; Deegan, M.
D. J. Am. Chem. Soc. 1996, 118, 10662-10663. (b) Inoue, Y.; Kanbara,
T.; Yamamoto, T. Tetrahedron Lett. 2003, 44, 5167-5169.
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A R T I C L E S
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Table 1. Absorption and Emission Maxima for Selected Squaraine
Derivativesa
λabs (nm)
log
ꢀ
λem (nm)
Φfb
3b
630
648
658
642
5.65
5.45
5.44
5.70
657
699
695
667
0.60
0.45
0.45
0.60
2a⊃3b
2b⊃3b
4
a Chloroform solvent. b Solutions were excited at 580 nm and emission
monitored in the region 600-800 nm for estimating Φf. Fluorescence
quantum yields were determined using 4,4-[bis-(N,N-dimethylamino)phe-
nyl]squaraine dye as the standard (Φf ) 0.7 in CHCl3), error limit ( 5%.
Figure 1. Two views of the X-ray crystal structure of 2a⊃3a.
organic solvents and aqueous/organic mixtures. Furthermore,
the assembly process occurs in highly competitive biological
media such as bilayer membranes and the organelles within a
living cell. The assembly process produces host:guest inclusion
complexes that possess emergent properties such as improved
chemical stability, red-shifted absorption/emission maxima, and
altered intracellular targeting. Because one of the practical goals
of this research is to produce new classes of near-infrared
fluorescent imaging probes, these properties are highly desired.18
Results
Structure of Inclusion Complexes. The novel 2+2 macro-
cycle 2a, with 2,6-dicarboxamidopyridine and 9,10-anthrylene
components, was prepared in 30% yield by a straightforward,
pseudo-dilution macrocyclization reaction. Simply stirring a
mixture of macrocycle 2a (9 mM) and an excess of poorly
soluble squaraine 3a in CDCl3 produces the corresponding
inclusion complex 2a⊃3a in quantitative yield as judged by 1H
NMR spectroscopy. The solution-state 1H NMR spectrum
exhibits a number of upfield chemical shifts that are highly
characteristic of dye encapsulation inside the macrocycle (see
Supporting Information). Additional structural characterization
was gained from single-crystal X-ray diffraction analysis of
2a⊃3a (Figure 1). Notable features of the solid-state structure
include the expected hydrogen bonding between the squaraine
oxygens and the macrocycle amide NH residues, and the high
degree of cofacial aromatic stacking.19 An unexpected feature
is the macrocyclic conformation, which is almost perfectly flat
and not the usual chair conformer that is seen with the phenylene
macrocycle in previously published squaraine-rotaxane struc-
tures (the macrocyclic conformation is discussed further be-
low).13,20
Figure 2. (A) Absorption (black full line) and fluorescence emission (red
dashed line, ex: 580 nm) spectra for rotaxane 2a⊃3b in chloroform. (B)
Fluorescence emission spectra (ex: 350 nm) for an equimolar mixture of
2a and 3b (both 5 µM) (black full line) and a solution of 2a⊃3b (red dashed
line) in chloroform.
alkyne squaraine 3b, which has relatively larger and more
flexible end-groups. As expected,21 a 1:1 mixture of 2a and 3b
in chloroform forms 2a⊃3b in quantitative yield, but with a
very slow rate constant (second-order rate constant is 2 × 10-4
M-1 s-1 at 25 °C; the half-life is 8.5 days). However, the
assembly process can be pushed to completion within a few
days by heating the sample at 120 °C.22 A room-temperature
solution of rotaxane 2a⊃3b does not dissociate upon extreme
dilution in organic solvents, which allows its photophysical
properties to be measured. A listing of the relevant absorption
and emission maxima is provided in Table 1. It is apparent that
encapsulation of the squaraine dye inside the anthrylene macro-
cycle induces a much larger red-shift effect than the phenylene
macrocycle. Shown in Figure 2A are the absorption spectrum
for anthrylene rotaxane 2a⊃3b and the fluorescence emission
Self-Assembly in Weakly Polar Organic Solvent. Inclusion
complex 2a⊃3a is classified as a pseudo-rotaxane because there
is partial dissociation when a chloroform solution is diluted to
1 µM. To inhibit dissociation, we prepared the symmetrical bis-
(17) Recent examples of host molecules with anthracene units include: (a)
Gunnlaugsson, T.; Davis, A. P.; O’Brien, J. E.; Glynn, M. Org. Biomol.
Chem. 2005, 3, 48-56. (b) Neelakandan, P. P.; Hariharan, M.; Ramaiah,
D. Org. Lett. 2005, 7, 5765-5768.
(21) Affeld, A.; Hu¨bner, G. M.; Seel, C.; Schalley, C. A. Eur. J. Org. Chem.
2001, 2877-2890.
(18) Rao, J. H.; Dragulescu-Andrasi, A.; Yao, H. Q.; Yao, H. Q. Curr. Opin.
Biotechnol. 2007, 18, 17-25.
(22) For examples of rotaxane synthesis by heat-induced slippage, see: (a)
Raymo, F. M.; Stoddart, J. F. Pure Appl. Chem. 1997, 69, 1987-1997. (b)
Ha¨ndel, M.; Plevoets, M.; Gestermann, S.; Vo¨gtle, F. Angew. Chem., Int.
Ed. Engl. 1997, 36, 1199-1201. For rotaxane synthesis by pressure-induced
slippage, see: Tokunaga, Y.; Wakamatsu, N.; Ohbayashi, A.; Akasaka,
K.; Saeki, S.; Hisada, K.; Goda, T.; Shimomura, Y. Tetrahedron Lett. 2006,
47, 2679-2682.
(19) Distances (Å) and angles (deg) for the two unequal N‚‚‚O hydrogen bonds
in 2a⊃3a: 2.942, 167; 3.005, 161. The centroid to centroid distance between
the two parallel anthrylene units is 6.8 Å.
(20) It appears that the energy differences between the macrocycle chair and
flat conformations are quite small and that the flat conformation may be
induced by solid-state packing.
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Self-Assembly of Fluorescent Inclusion Complexes
A R T I C L E S
that is observed upon excitation at 580 nm (squaraine chro-
mophore). Shown in Figure 2B are the fluorescence emission
upon excitation at 350 nm (anthrylene chromophore), overlaid
with the emission spectrum for an equimolar mixture of
unassembled anthrylene macrocycle 2a and squaraine 3b. While
the free macrocycle is highly fluorescent, there is no similar
anthrylene emission band when it is part of the rotaxane.23,24
While the quantitative formation of stable rotaxane 2a⊃3b
from a mixture of 2a and 3b is very useful, the slow kinetics of
the self-assembly process is a potential limitation for many
eventual applications. We suspected that a contributing factor
was the known propensity of the 2,6-dicarboxamidopyridine unit
in 2a to form a locked and less-flexible macrocyclic conforma-
tion due to internal hydrogen bonding between the pyridyl
nitrogen and the amide NH residues.21 Therefore, we prepared
the 2+2 isophthalamide macrocycle 2b with the expectation
that it would be more flexible and exhibit faster association
kinetics. We were gratified to find that simply dissolving a 1:1
mixture of macrocycle 2b and squaraine 3b in CDCl3 (6 mM
each) at room temperature leads to extremely rapid and
quantitative formation of 2b⊃3b. Like 2a⊃3b, the absorption
and emission maxima for 2b⊃3b are significantly red-shifted
when compared to the parent squaraine 3b (Table 1); however,
unlike the stable 2a⊃3b, there is measurable dissociation upon
dilution of a chloroform solution (i.e., 2b⊃3b is a pseudo-
rotaxane). The encapsulation-induced change in absorption
maxima allowed direct measurement of association kinetics and
thermodynamics. The association constant for a mixture of 2b
and 3b to form 2b⊃3b is (1.8 ( 0.4) × 105 M-1 in chloroform,
and the initial second-order rate constant is 80 M-1 s-1 at 25 °C.
In other words, association to form the pseudo-rotaxane 2b⊃3b
is essentially quantitative when the concentrations of 2b and
3b are above 100 µM. Furthermore, the rate of association of
squaraine 3b with isophthalamide macrocycle 2b is 400 000
times faster than the association with pyridyl macrocycle 2a.25
Self-Assembly in Polar Solvents. There was no spectrometric
evidence for squaraine encapsulation by macrocycle 3b in the
polar aprotic solvents acetonitrile and DMSO, which agrees with
previous studies of uncharged inclusion processes that are driven
primarily by hydrogen bonding and aromatic stacking.26
We next examined the self-assembly process in water/
acetonitrile mixtures, although only semiquantitatively, because
the water slowly attacks and bleaches the unprotected mono-
disperse squaraine dye. In 1:1 water:acetonitrile, there was no
evidence for encapsulation of symmetric squaraine 3b by
macrocycle 2b; however, the admixture of asymmetric squaraine
Figure 3. Time-dependent changes in fluorescence emission for association
of unsymmetrical squaraine 3c (10 µM) and macrocycle 2b (10 µM) to
form 2b⊃3c in 1:1 water/acetonitrile at 25 °C.
Figure 4. The relatively high kinetic and thermodynamic stability of
pseudo-rotaxane 2b⊃3c is attributed to aromatic stacking interactions that
are absent in 2b⊃3b.
3c and macrocycle 2b produced substantial amounts of 2b⊃3c,
as judged by the time-dependent red-shift in squaraine absorp-
tion and fluorescence maxima. The spectra in Figure 3 indicate
that the binding equilibrium was reached after approximately
17 min and that the association constant was >105 M-1. The
enhanced stability of asymmetric inclusion complex 2b⊃3c as
compared to symmetric 2b⊃3b is attributed to favorable
stacking interactions in 2b⊃3c between the bridging isophthal-
imide units on the macrocycle and the terminal N,N′-dibenzyl
rings on the squaraine (Figure 4). This structural model is
supported by unpublished X-ray diffraction evidence, from three
different squaraine-rotaxane crystal structures, that the encap-
sulating macrocycle can easily adopt a macrocyclic boat
conformation.27
Self-Assembly in Vesicle Membranes. The uncharged
squaraine dye 3b is essentially nonfluorescent in water (due to
aggregation and self-quenching), but it fluoresces strongly upon
insertion into vesicle membranes. Figure 5 shows time-depend-
ent absorption and fluorescence emission spectra after addition
of 3b to vesicles composed of 1-palmitoyl-2-oleoylphosphati-
dylcholine (POPC). The dye undergoes slow chemical bleaching,
due to hydrolysis, over a period of several hours. In Figure 6
are the absorption and fluorescence emissions obtained after
3b is added to vesicles that contain the lipophilic macrocycle
2b. In this case, the absorption and fluorescence maxima
undergo time-dependent red-shifts of 17 and 35 nm, respectively,
reflecting the bimolecular formation of 2b⊃3b. The spectra for
this inclusion complex hardly change with time, indicating that
squaraine 3b is strongly protected inside the macrocycle 2b. A
graphic illustration of the enhanced stability is the photograph
in Figure 7, which shows the vesicles from Figures 5 and 6
after they have been allowed to sit for 24 h. At this point in
(23) Future studies of rotaxane 2a⊃3b will determine the degree of energy
transfer from the surrounding anthrylene macrocycle to the encapsulated
squaraine thread. The supramolecular photochemistry of the free anthrylene
macrocycles, 2, will also be reported in due course.
(24) Another significant photophysical property produced by squaraine encap-
sulation inside a protecting macrocycle is insulation from the effects of an
external quencher. It is known that the squaraine quantum yield is
significantly decreased in alcohol solvents, due to hydrogen bonding
between the solvent hydroxyl residue and the oxygens on the squaraine
dye (see ref 11a). Thus, as expected, the quantum yield for squaraine dye
3b drops by almost a factor of 10 as the solvent is changed incrementally
from pure chloroform to pure methanol (see Figure S9 in the Supporting
Information). With phenylene-containing rotaxane 4, the quantum yield
decreases by about a factor of 3, whereas it is only lowered by about 20%
in the case of anthrylene-containing rotaxane 2a⊃3b. Thus, the anthrylene
tetralactam macrocycle provides superior steric protection of the encapsu-
lated dye.
(25) This large difference in association kinetics agrees with a previous
observation using a similar hydrogen-bonded pseudo-rotaxane. See ref 21.
(26) Wu, A.; Mukhopadhyay, P.; Chakraborty, A.; Fettinger, J. D.; Isaacs, L. J.
Am. Chem. Soc. 2004, 126, 10035-10043 and references therein.
(27) Arunkumar, E.; Noll, B. C.; Smith, B. D., unpublished results.
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Figure 7. Vesicles containing squaraine 3b (left) or a mixture of 3b and
macrocycle 2b (right) after 24 h. In both cases, the vesicles have settled to
the bottom of the tubes. The vesicles containing only 3b have completely
lost color due to hydrolytic bleaching (left), whereas the vesicles containing
the highly stable 2b⊃3b retain the blue color (right).
Figure 5. Time-dependent changes in absorbance (A) and fluorescence
(B) maxima for squaraine 3b (1.9 µM) after addition to vesicles (200 nm
diameter) composed of POPC. The two fluorescence bands centered at 740
nm are artifacts due to the excitation at 370 nm.
Figure 8. Fluorescence microscopy images of live CHO cells treated with
separate aliquots of 3c (10 µM) and 2b (10 µM). Panel A: Phase contrast
image. Panel B: Blue emission of uncomplexed 2b. Panel C: Far-red
emission of 2b⊃3c. Panel D: Overlay of panels B and D.
be observed using a far-red filter set where there is no
interference from the assembly components (macrocycle or
squaraine). The anthrylene chromophore does not fluoresce after
it forms an inclusion complex, so only the uncomplexed
macrocycle (2) appears in the blue channel. In contrast, the
uncomplexed squaraine dye (3) cannot be observed selectively
because of bleed-through emission from the inclusion complex.
Typical cell microscopy results are provided in Figure 8, which
shows micrographs of live CHO (Chinese Hamster Ovary) cells
24 h after treatment with separate aliquots of macrocycle 2b
and asymmetric squaraine 3c. The blue-emitting, uncomplexed
anthrylene macrocycle is seen in panel B, the far-red emitting
2b⊃3c complex appears in panel C, and an overlay of the two
is in panel D. It is apparent that both the uncomplexed
macrocycle and the inclusion complex are inside the cells but
they are not colocalized. The far-red emission of lipophilic
2b⊃3c is confined to small punctate compartments, whereas
the blue emission of the uncomplexed macrocycle 2b is
distributed fairly evenly throughout the cell. The far-red staining
due to 2b⊃3c is quite photostable and allows the acquisition
of real time fluorescence movies (see Supporting Information).
Figure 6. Time-dependent changes in absorbance (A) and fluorescence
(B) maxima for squaraine 3b (1.9 µM) after addition to vesicles (200 nm
diameter) composed of 99.5:0.5 POPC:2b. The two fluorescence bands
centered at 740 nm are artifacts due to the excitation at 370 nm.
time, the vesicles have settled to the bottom of the tubes. The
vesicles containing only squaraine 3b have completely lost color
due to hydrolytic bleaching, whereas the vesicles containing
the highly stable inclusion complex 2b⊃3b retain their blue
color.
Self-Assembly Inside Living Cells. The complexation-
induced red-shift in absorbance and emission allows the self-
assembly process to be monitored by multichannel epifluores-
cence microscopy. The appearance of an inclusion complex can
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15058 J. AM. CHEM. SOC. VOL. 129, NO. 48, 2007

Self-Assembly of Fluorescent Inclusion Complexes
A R T I C L E S
The encapsulation of squaraine dyes (3) by macrocycles (2)
produces inclusion complexes with emergent properties;30 that
is, the complexes have properties (i.e., improved chemical
stabilities, red-shifted absorption/emission maxima, different cell
localization propensities) that are not the linear sum of an
uncomplexed mixture of binding partners.31 Squaraines 3b and
3c have terminal acetylene residues, and in a future publication
we will describe how click chemistry (copper catalyzed Huisgen
cycloaddition) can be used to cap these end groups with azide-
containing stopper groups and produce permanently interlocked
squaraine-rotaxanes. Our eventual goal is to convert these highly
stable dyes with bright, near-infrared fluorescence into new
families of molecular probes for cell and whole animal imaging.
The first generation squaraine-rotaxanes, 1, with a phenylene
tetralactam macrocycle have absorption/emission profiles (Table
1) that closely match those of the highly popular cyanine dye,
Cy-5, whereas squaraine-rotaxanes with an anthrylene macro-
cycle have a red-shifted absorption/emission that matches that
of the homologous cyanine, Cy-5.5.32 The different emission
colors of these two squaraine-rotaxane systems can easily be
resolved by commercial fluorescence microscopes, flow cy-
tometry instruments, and in vivo animal imaging stations using
common filter sets. Furthermore, it may be possible to employ
these two families of squaraine-rotaxane dyes as a donor/
acceptor pair for photonic technologies that employ FRET
(fluorescence resonance energy transfer), potentially inside living
organisms.33 Finally, the self-assembly process described here
can be employed to make various interlocked fluorescent
switches and shuttles, with applications in materials science and
nanotechnology.34
Figure 9. Flow cytometry of CHO cells at time zero (gray) and 24 h (red)
after treatment with squaraine 3c and macrocycle 2b. The filter set only
detects cells that emit far-red fluorescence due to time-dependent formation
of 2b⊃3c.
They indicate that the punctate compartments are vesicle-like
structures, trafficking slowly inside the living cells.28 Flow
cytometry was used to show that the far-red fluorescence of
2b⊃3c appears over time, as expected for a bimolecular
association process. Shortly after the cells were treated with
separate aliquots of macrocycle 2b and squaraine 3c, the fraction
of cells with far-red emission due to 2b⊃3c was negligible,
but it increased substantially after 24 h (Figure 9). These live
cell staining experiments were repeated with macrocycle 2b and
symmetric squaraine dye 3b with a similar outcome (see
Supporting Information).
Discussion
The X-ray structure of 2a⊃3a shows that squaraine dyes 3
and anthrylene macrocycles 2 are a highly complementary host:
guest pair (Figure 1). The very strong association in chloroform
solvent (Ka ) 1.8 × 105 M-1) is driven by the formation of
four hydrogen bonds and extensive overlap of aromatic surfaces.
Association cannot be detected in polar aprotic solvents like
acetonitrile and DMSO because these solvents have a combina-
tion of high polarity that disrupts hydrogen bonds and moderate
polarizability that disrupts aromatic stacking.26 However, as-
sociation can be very strong (Ka > 105 M-1) if the solvent
includes significant amounts of water, which provides a strong
hydrophobic driving force for aromatic stacking.29 Inclusion of
the squaraine inside the macrocycle is a “snug fit” as reflected
by the large changes in association kinetics that are induced by
small structural variations in the bridging unit of the macrocycle
(2a vs 2b) or the terminal groups on the squaraine (3a vs 3b).
Although the macrocyclic hosts (2) have not yet been fully
evaluated for guest selectivity, it is notable that the two assembly
partners (e.g., macrocycle 2b and squaraine 3c) can find each
other within the extreme molecular complexity of a living cell.
In other words, macrocycle/squaraine association does not
appear to be greatly inhibited by the presence of biomolecules
that contain aromatic rings or functional groups that can act as
hydrogen-bond acceptors.
Acknowledgment. This work was supported by the NIH,
Walther Cancer Research Center, and the University of Notre
Dame.
Supporting Information Available: Synthesis and spectra,
X-ray crystal structures, analytical measurements, vesicle stud-
ies, cell studies, and crystallographic information (CIF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA075567V
(30) The reviewers of this Article feel that it is inappropriate to apply the term
“emergent properties” because the self-assembly system is not sufficiently
complex. However, to the best of our knowledge, the concept of emergence
has not been accurately defined at a molecular level. Recently, De Haan
has provided a broad treatment that can be applied to various scientific
fields (De Haan. J. Ecol. Complex 2006, 3, 293-301) and includes the
following definition of weak, type-I emergence: “Appearance of a pattern,
property, or phenomenon on the higher level that cannot be explained by
the objects on the lower level. The emergence is not trivial and is only
explainable by simulation.” It seems to us that the debate hinges on whether
the “emergent properties” that are observed in this study are considered
complex or trivial.
(31) Using this definition, supramolecular systems that exhibit emergent
properties are described in the following recent articles: (b) Shiki, Y. J.
Photochem. Photobiol., C 2006, 7, 164-182. (c) Schmittel, M.; Kalsani,
V. Top. Curr. Chem. 2005, 245, 1-53. (d) Ruben, M.; Ziener, U.; Lehn,
J. M.; Ksenofontov, V.; Gutlich, P.; Vaughan, G. B. M. Chem.-Eur. J.
2005, 11, 94-100.
(32) A search of Scifinder Scholar using the term “Cy5”, refined by year (2000-)
and document type (Journal, Review, Letter), produces over 1200 separate
publications covering various topics in biomedicine, materials science, and
nanotechnology.
(28) Cell morphology appears to be another biological property that is altered
by in situ squaraine encapsulation. For example, treatment of CHO cells
with squaraine alone (3b or 3c) induced about 30% of the cells to become
rounded after 24 h, whereas there was much less morphology change when
they were treated with a binary mixture of squaraine dye and macrocycle
2b. MTT cell viability assays showed no difference in toxicity, which was
essentially negligible when the compound concentrations were in the low
micromolar range. The reasons for this subtle cell morphology effect require
further study.
(33) Sapsford, K. E.; Berti, L.; Medintz, I. L. Angew. Chem., Int. Ed. 2006, 45,
4562-4588.
(34) Recent reviews of functional interlocked nanostructures include: (a) Tian,
H.; Wang, Q.-C. Chem. Soc. ReV. 2006, 35, 361-374. (b) Saha, S.; Stoddart,
J. F. Chem. Soc. ReV. 2007, 36, 77-92. (c) Balzani, V.; Credi, A.; Silvi,
S.; Venturi, M. Chem. Soc. ReV. 2006, 35, 1135-1149.
(29) Meyer, E. A.; Castellano, R. K.; Diederich, F. Angew. Chem., Int. Ed. 2003,
42, 1210-1250.
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