Fast fluorescence switching within hydrophilic supramolecular assemblies

Article abstract of DOI:10.1002/chem.201201184

We designed a supramolecular strategy to modulate fluorescence in water under optical control. It is based on the entrapment of fluorophore-photochrome dyads within the hydrophobic interior of an amphiphilic polymer. The polymeric envelope around the dyad

Full text of DOI:10.1002/chem.201201184

FULL PAPER
DOI: 10.1002/chem.201201184
Fast Fluorescence Switching within Hydrophilic Supramolecular Assemblies
Janet Cusido,[a] Mutlu Battal,[a] Erhan Deniz,[a] Ibrahim Yildiz,[a]
Salvatore Sortino,*[b] and FranÅisco M. Raymo*[a]
Abstract: We designed a supramolec-
ular strategy to modulate fluorescence
in water under optical control. It is
based on the entrapment of fluoro-
phore–photochrome dyads within the
hydrophobic interior of an amphiphilic
polymer. The polymeric envelope
around the dyads protects them from
the aqueous environment, while impos-
ing hydrophilic character on the overall
supramolecular construct. In the result-
ing assemblies, the photochromic com-
ponent can be operated reversibly on
a microsecond timescale under the in-
fluence of ultraviolet stimulations. In
turn, the reversible transformations
control the emission intensity of the
adjacent fluorophore. As a result, the
fluorescence of such nanostructured
constructs can be photomodulated for
hundreds of cycles in water with micro-
second switching speeds. Thus, our pro-
tocol for fast fluorescence switching in
aqueous solutions can eventually lead
to the realization of functional probes
for the investigation of biological sam-
ples.
Keywords: fluorescence · heterocy-
cles · micelles · photochromism ·
polymers
Introduction
able components for the assembly of photoswitchable fluo-
rescent constructs. In fact, their photoinduced and reversible
transformations can be engineered to regulate the emission
of fluorescent partners.[25–28] Specifically, the integration of
fluorescent and photochromic components within the same
molecular or supramolecular assembly can be exploited to
modulate the emission of the former by operating the latter
with optical inputs. Generally, electron or energy transfer
processes are invoked to establish communication between
the functional components in the excited state and manipu-
late the excitation dynamics of the emissive species.[29–37] In
particular, the excited fluorophore can be designed to ex-
change an electron with or transfer energy to only one of
the two interconvertible states of the photochromic compo-
nent. Under these conditions, the photochromic transforma-
tion activates or suppresses fluorescence reversibly. Indeed,
numerous examples of fluorophore–photochrome constructs
have already been developed successfully on the basis of
these mechanisms.[38]
Photochromic spiropyrans undergo ring-opening reac-
tions, followed by cis!trans isomerizations, upon ultraviolet
irradiation to produce merocyanine chromophores.[39–50] The
photogenerated isomers revert back to the original species
either thermally, over the course of hundreds of seconds, or
photochemically, upon visible illumination. Such reversible
structural transformations are accompanied by pronounced
absorbance changes in the visible region as well as by signif-
icant shifts in redox potentials. In the presence of compati-
ble fluorescent partners, the photoinduced modification of
these spectroscopic and electrochemical parameters can acti-
vate energy- and electron-transfer pathways, respectively,
and quench fluorescence.[38] On the basis of these operating
principles, we have been able to modulate the emission of
a diversity of fluorophores in liquid solutions[51] and within
The ability to switch fluorescence under optical control can
offer the opportunity to monitor the translocation of labeled
targets within a biological sample and provide valuable in-
formation on the fundamental factors governing the diffu-
sion of biorelevant species.[1–3] Similarly, fluorescence photo-
switching can permit the separation in time of spatially in-
distinguishable probes and allow the reconstruction of
images with subdiffraction resolution.[2c,e,4–13] As a result,
photoswitchable fluorophores are becoming invaluable ana-
lytical tools in the biomedical laboratory for the investiga-
tion of cellular dynamics and the visualization of cellular
substructures. Indeed, the significant implications that such
molecular switches can have in biomedical research, togeth-
er with the need to advance our basic understanding on the
excitation dynamics of organic chromophores, are encourag-
ing the identification of practical mechanisms to activate
fluorescence under the influence of optical stimulations.[14–19]
In this context, photochromic compounds[20–25] can be valu-
[a] J. Cusido, M. Battal, Dr. E. Deniz, Dr. I. Yildiz, Prof. F. M. Raymo
Laboratory for Molecular Photonics
Department of Chemistry, University of Miami
1301 Memorial Drive, Coral Gables, Florida, 33146-0431 (USA)
Fax : (+1)305-284-4571
E-mail: fraymo@miami.edu
[b] Prof. S. Sortino
Laboratory of Photochemistry
Department of Drug Sciences, University of Catania
Viale Andrea Doria 6, I-95125 Catania (Italy)
Fax : (+39)095-580138
E-mail: ssortino@unict.it
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201184.
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rigid matrices.[52] In search of strategies to impose biocom-
patibility on these photoswitchable systems, we then devised
a supramolecular protocol to transport mixtures of boron-
dipyrromethene (BODIPY) fluorophores and spiropyran
photochromes within the intracellular environment and op-
erate them successfully within live cells.[53] Our method is
based on the encapsulation of separate fluorescent and pho-
tochromic components within the hydrophobic interior of
appropriate polymer micelles. Nonetheless, the slow switch-
ing speeds and modest fatigue resistances of the photochro-
mic components limit significantly the performance of these
photoswitchable supramolecular assemblies. Specifically,
a single switching cycle requires more than 104 s and signifi-
cant photodegradation occurs within a few cycles.
In parallel to our investigations on fluorescence photo-
switching with spiropyrans,[51–53] we developed a new family
of photochromic compounds with fast switching speeds and
excellent fatigue resistance.[54] They are based on the photo-
induced opening and thermal closing of an oxazine ring.
These photochromic switches can complete a single cycle on
a submicrosecond timescale and tolerate hundreds of cycles
with no sign of degradation even in air. In fact, we have
been able to modulate the emission of BODIPY and cou-
marin fluorophores with microsecond switching speeds and
for hundreds of cycles on the basis of such photochromic
transformations.[54k–m,r] As a result, we envisaged the possibil-
ity of introducing appropriate members of this family of
photochromic compounds within our biocompatible supra-
molecular constructs, in place of their spiropyran compo-
nents, with the ultimate goal of improving their perfor-
mance. In this article, we report the design and synthesis of
two hydrophobic photochromic oxazines, their introduction
within biocompatible polymer micelles as well as the photo-
chemical and photophysical properties of the resulting
supramolecular assemblies and appropriate model com-
pounds.
Figure 1. Amphiphilic co-polymer 1 and hydrophobic fluorophore 2.
Figure 2. Photoinduced and reversible transformation of the oxazines
3a–7a into the corresponding zwitterionic isomers 3b–7b.
Results and Discussion
Design and synthesis: The co-polymer 1 (Figure 1) has mul-
tiple hydrophobic and hydrophilic side chains along
a common poly(methacrylate) backbone. In aqueous envi-
ronments, this particular amphiphilic macromolecule forms
micellar assemblies with a hydrodynamic diameter of ap-
proximately 40 nm that are capable of trapping organic dyes
in their hydrophobic interior.[53] Specifically, the BODIPY
fluorophore 2 can be embedded within these supramolecular
constructs together with a spiropyran photochrome. The
photoinduced transformation of the spiropyran into the cor-
responding merocyanine activates electron- and energy-
transfer pathways from the excited fluorophore to the pho-
tochrome that encourage the nonradiative deactivation of
the former. In principle, the spiropyran component can be
replaced with the photochromic oxazine 3a (Figure 2) to en-
hance the switching speeds and fatigue resistance of the
overall fluorescent construct. Indeed, the oxazine ring of
this particular compound opens to produce the zwitterionic
isomer 3b in less than 6 ns upon ultraviolet illumination in
acetonitrile.[54j] The photogenerated isomer reverts sponta-
neously to the original one in less than 10 ms, under these
experimental conditions, and the photochromic system can
be switched back and forth between its two states for hun-
dreds of cycles with no sign of degradation. The photoin-
duced transformation of 3a into 3b brings the 4-dimethyl-
aminostyryl appendage in conjugation with the resulting
3H-indolium cation. This extended chromophoric fragment
absorbs[54j] in the same range of wavelengths at which 2
emits[53] and, therefore, can accept the excitation energy of
this fluorophore. Furthermore, the redox potentials of simi-
lar compounds[54k] indicate that the transfer of an electron
from the excited fluorophore to the 3H-indolium chromo-
phore is exergonic with a free energy change of ꢀ0.5 eV.
Thus, both electron- and energy-transfer pathways can be
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Fast Fluorescence Switching
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activated with the photoinduced transformation of 3a into
3b to quench the emission of 2, if fluorescent and photo-
chromic components are both trapped within the same
supramolecular assembly. On the basis of these considera-
tions, we envisaged the possibility of appending an oligo-
(methylene) tail to this photochromic oxazine, in the form
of compound 4a (Figure 2), to encourage the entrapment of
the resulting molecule within micelles of the amphiphilic co-
polymer 1. In particular, we prepared 4a in two synthetic
steps (Figure S1 in the Supporting Information), starting
from commercial and known precursors.
The integration of 2 and 4a within the same micellar con-
struct can offer the opportunity to suppress the emission of
the former with the photoinduced ring opening of the latter.
A similar photochemical transformation, however, can also
be exploited to activate, rather than deactivate, fluorescence.
Specifically, the photoinduced opening of the oxazine ring
of 5a (Figure 2) brings its coumarin appendage in conjuga-
tion with the 3H-indolium cation of 5b and shifts the main
absorption band of this fluorescent appendage from 410 to
570 nm in acetonitrile.[54m,r] As a consequence, illumination
at 532 nm results in significant fluorescence only after the
photoinduced opening of the oxazine ring. Thus, the intro-
duction of such a fluorophore–photochrome dyad within mi-
cellar assemblies of the amphiphilic co-polymer 1 can trans-
late into the realization of photoactivatable and biocompat-
ible fluorescent constructs. Therefore, we designed an ana-
logue of 5a with a pendant oligo(methylene) tail, in the
form of compound 6a (Figure 2), to facilitate the encapsula-
tion of the resulting dyad within micelles of 1. In particular,
we prepared 6a in four synthetic steps (Figure S2 in the
Supporting Information), starting from commercial and
known precursors. Following a similar synthetic procedure
(Figure S3 in the Supporting Information), we also prepared
the hydrophilic analogue 7a (Figure 2). The pendant oligo-
(ethylene glycol) tail of 7a is designed to impose aqueous
solubility on the fluorophore–photochrome dyad and, there-
fore, permit the investigation of its photochemical and pho-
tophysical properties in aqueous solutions lacking any micel-
lar host.
Figure 3. Absorption spectra of solutions (MeCN, 258C) of 3a (a, 5 mm),
4a (b, 5 mm), and the hexafluorophosphate salt of 8 (c, 1 mm). Absorption
spectra of dispersions (PBS, pH 7.0, 258C) of 1 (50 mgmLꢀ1) and either
3a (d, 5 mgmLꢀ1) or 4a (e, 5 mgmLꢀ1). Emission spectrum (f) of a disper-
sion (PBS, pH 7.0, 258C, lEx =490 nm) of
1 ) and 2
(50 mgmLꢀ1
(5 mgmLꢀ1).
Steady-state spectroscopy: The absorption spectra (a and
b in Figure 3) of acetonitrile solutions of 3a and 4a are very
similar, indicating that the hydrophobic tail of the latter has
a negligible influence on the photophysical properties of the
heterocyclic core. In both instances, the spectrum shows
bands at 305 and 555 nm that correspond to the 4-nitrophen-
oxy fragment of the ring-closed isomer and the 3H-indolium
cation of the ring-open species respectively.[54j] Indeed, the
absorption in the visible region resembles that of the model
compound 8 (c in Figure 3 and Figure 4), which has essen-
tially the same chromophoric fragment of the ring-open iso-
mers 3b and 4b. Thus, the two isomers of each system co-
exist under these experimental conditions with a ratio of
90:10 in favor of the ring-closed species.[55]
Figure 4. Model 3H-indolium chromophores 8–10.
tions. However, they readily dissolve in neutral phosphate
buffer saline (PBS) in the presence of the amphiphilic co-
polymer 1. The absorption spectra of the resulting disper-
sions (d and e in Figure 3) predominantly show bands at 390
and 560 nm for the 4-nitrophenolate anion[56] and 3H-indol-
The two oxazines 3a and 4a are relatively hydrophobic
and, as a result, they are sparingly soluble in aqueous solu-
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S. Sortino, F. M. Raymo et al.
ium cation, respectively, of the zwitterionic species 3b and
4b. These observations suggest that both molecular switches
are partially exposed to the aqueous medium, despite their
interactions with the amphiphilic co-polymer 1. Indeed, the
ring-open isomers are significantly more polar than their
ring-closed counterparts and, as a result, the transition from
organic to aqueous environments tends to encourage their
population. Specifically, the absorbance of the 3H-indolium
chromophore in the visible region indicates that the ratio
between 3a and 3b is 30:70, whereas that between 4a and
4b is 85:15.[55,57] The different ratios suggest that the two
molecular switches reside in different domains within their
polymeric host. Presumably, the hydrophobic tail of one
system limits the exposure of the heterocyclic core to the
polar aqueous environment and, therefore, decreases the
fractional concentration of ring-open isomer relative to that
associated with the other system.
In agreement with our design logic, the absorption band
of the 3H-indolium chromophore of the ring-open isomers
3b and 4b is, indeed, positioned in the same range of wave-
lengths at which 2 emits (f in Figure 3).[53] This pronounced
spectral overlap together with the redox potentials of model
compounds[54k] suggests that the concomitance of energy-
and electron-transfer pathways can quench the fluorescence
of 2, if this component and either one of the two molecular
switches are trapped within the same micellar assembly.
Nonetheless, the fraction of ring-open isomer is relatively
large for both systems, under these experimental conditions.
This unexpected complication prevents the implementation
of our operating principles for photoinduced fluorescence
suppression, which instead require a significant population
of the quenching species only after ultraviolet illumination.
The absorption spectra (a and b in Figure 5) of acetoni-
trile solutions of 5a and 6a show a band at 410 nm for the
coumarin appendage of the ring-closed isomer,[54m,r] but do
not reveal any significant absorbance for the 3H-indolium
cation of the ring-open species. Indeed, the spectrum (c in
Figure 5) of the model compound 9 indicates that this par-
ticular cationic chromophore absorbs at 570 nm. Analogous
to 3a and 4a, compounds 5a and 6a are also relatively hy-
drophobic and sparingly soluble in water, but can readily be
dispersed in aqueous solution together with the amphiphilic
co-polymer 1. In contrast to 3a and 5a, however, the spectra
(d and e in Figure 5) of the resulting aqueous dispersions
are very similar to those of the acetonitrile solutions. Once
again, only the band for the coumarin appendage of the
ring-closed isomers 5a and 6a can be observed at 400 nm.
Similarly, the absorption spectrum (a in Figure S5 in the
Supporting Information) of an acetonitrile solution of their
hydrophilic analogue shows exclusively a band at 405 nm for
the ring-closed isomer 7a. However, an additional band is
clearly present at 610 nm in the spectrum (b in Figure S5 in
the Supporting Information) of the very same species dis-
solved in PBS in the absence of 1. This band resembles that
(c in Figure S5 in the Supporting Information) of the model
3H-indolium 10 (Figure 4) and indicates the co-existence of
7a and 7b in a ratio of 90:10 under these conditions.[57,58]
Figure 5. Absorption spectra of solutions (MeCN, 258C) of 5a (a, 5 mm),
6a (b, 5 mm), and the hexafluorophosphate salt of 9 (c, 1 mm). Absorption
spectra of dispersions (PBS, pH 7.0, 258C) of 1 (50 mgmLꢀ1) and either
5a (d, 5 mgmLꢀ1) or 6a (e, 5 mgmLꢀ1). Emission spectrum (f) of a solution
(MeCN, 258C, lEx =530 nm) of the hexafluorophosphate salt of
(10 mm).
9
These observations demonstrate that the micellar host effec-
tively protects 5a and 6a from the aqueous environment
and discourages the population of their ring-open isomers
5b and 6b. Thus, the supramolecular encapsulation of these
fluorophore–photochrome dyads within the hydrophobic in-
terior of the amphiphilic co-polymer offers the opportunity
to implement our operating principles for fluorescence pho-
toactivation, which demand the population of the emissive
isomer only after ultraviolet stimulations. Indeed, the photo-
induced opening of the oxazine ring of 5a and 6a within the
micellar assemblies can extend the conjugation of the cou-
marin fluorophore within the photogenerated isomers 5b
and 6b to produce a 3H-indolium chromophore able to emit
at 645 nm, according to the emission spectrum of the model
compound 9 (f in Figure 5).[59]
Time-resolved spectroscopy: The illumination of an aqueous
dispersion of 1 and either 5a or 6a at 355 nm with a pulsed
laser opens the oxazine ring of the molecular switch, entrap-
ped within the polymeric construct, to generate the zwitter-
ionic isomer 5b or 6b within the laser pulse (6 ns). Consis-
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tently, the absorption spectrum (a in Figure 6 and Figure S6
in the Supporting Information) of the dispersion, recorded
0.1 ms after illumination, shows the appearance of band at
620 nm. This transient absorption resembles the steady-state
band (c in Figure 5) observed for the model compound 8
and corresponds to a ground-state absorption of the 3H-
indolium chromophore of the ring-open isomer 5b or 6b.
Essentially the same transient absorption is observed also in
the spectrum (a in Figure S7 in the Supporting Information)
of an aqueous solution of the hydrophilic analogue 7a in the
absence of the polymeric host, under otherwise identical
conditions. However, the dependence of the absorbance of
this band on the laser intensity indicates the quantum yield
for the photochromic transformation to be approximately
0.02 for 5a and 6a within the micellar assemblies and only
0.006 for 7a in water. These values suggest that the poly-
meric envelope around the photochromic components limits
their exposure to the aqueous environment, in agreement
with the indications that emerged from the analysis of the
steady-state spectra. Furthermore, the quantum yield for the
photochromic transformation of 5a and 6a in the micellar
assemblies is identical to that measured for 5a in acetoni-
trile,[54m,r] suggesting that the polymeric host provides an en-
vironment similar to this particular organic solvent.
The isomers 5b and 6b, photogenerated within the poly-
mer micelles, as well as the ring-open species 7b, produced
in the absence of the polymeric host, all revert spontaneous-
ly back to the ring-closed species with first-order kinetics.
As a result, the absorbance for their 3H-indolium chromo-
phore decays monoexponentially (d in Figure 6 and Fig-
ures S6 and S7 in the Supporting Information). Curve fitting
of the temporal absorbance profiles indicates the lifetime of
these species to be approximately 0.2 ms in all instances. This
value is essentially identical to that of 5b measured in aceto-
nitrile.[54m,r] Furthermore, all three photochromic systems
can be switched back and forth between their two states for
hundreds of cycles with no sign of degradation. Thus, the en-
vironment around the photochromic components has a negli-
gible influence on their reisomerization kinetics and fatigue
resistances.
The illumination of 5a and 6a within the polymer micelles
and of 7a in the absence of the polymeric host at 532 nm
does not result in any significant fluorescence (b in Figure 6
and Figures S6 and S7 in the Supporting Information).
Indeed, the coumarin fluorophore of 5a–7a does not absorb
at this particular wavelength and, therefore, it cannot emit.
After the photoinduced ring-opening process, however, the
coumarin absorption shifts from 400 to 620 nm (a in
Figure 6 and Figures S6 and S7 in the Supporting Informa-
tion). Therefore, the fluorescent appendage within the ring-
open isomers 5b–7b can absorb the exciting radiations at
532 nm and emit as a result. In fact, the simultaneous illumi-
nation of the sample at 355 nm, to open the oxazine ring of
5a–7a, and at 532 nm, to excite the coumarin fluorophore of
5b–7b, is accompanied by the appearance of an intense
band at 650 nm in the corresponding emission spectrum (c
in Figure 6 and Figures S6 and S7 in the Supporting Infor-
mation). This band disappears after the spontaneous re-
isomerization of 5b–7b back to 5a–7a. As a consequence,
the emission intensity at 650 nm can be switched on and off
for multiple cycles (e in Figure 6) simply by turning on and
Figure 6. Absorption spectrum (a) of
a dispersion (H2O, 25 8C) of
1 (2 mgmLꢀ1) and 6a (9 mgmLꢀ1), recorded 0.1 ms after pulsed illumina-
tion (355 nm, 6 ns, 15 mJ). Emission spectra of the same dispersion, re-
corded upon pulsed irradiation at 532 nm (6 ns, 30 mJ) without (b) and
with (c) simultaneous pulsed illumination at 355 nm. Absorbance evolu-
tion at 620 nm of the same dispersion upon pulsed irradiation at 355 nm
with the corresponding monoexponential fitting (d). Relative emission in-
tensity at 650 nm of the same dispersion, recorded upon pulsed irradia-
tion at 532 nm without and with simultaneous pulsed illumination at
355 nm (e).
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S. Sortino, F. M. Raymo et al.
tography (SiO2: Hexane/EtOAc (2:1, v/v)) to give 4a (350 mg, 50%) as
a red oil. 1H NMR (CDCl3): d=0.91 (t, J=7 Hz, 3H), 1.28 (s, 26H),
1.59–1.61 (m, 2H), 2.28 (t, J=7 Hz, 2H), 3.02 (s, 3H), 3.62 (t, J=6 Hz,
2H), 4.25 (t, J=6 Hz, 2H), 7.51 (d, J=8 Hz, 1H), 4.61 (s, 2H), 6.15 (d,
J=16 Hz, 1H), 6.64–6.76 (m, 4H), 6.89 (t, J=8 Hz, 2H), 7.11–7.17 (m,
2H), 7.32 (d, J=9 Hz, 2H), 7.96–8.01 ppm (m, 2H); 13C NMR (CDCl3):
d=13.9, 22.8, 24.2, 25.1, 29.3, 29.6, 29.9, 32.1, 34.1, 38.2, 44.3, 50.7, 61.3,
109.3, 111.2, 111.6, 112.3, 116.4, 117.9, 122.7, 123.1, 123.2, 124.3, 124.6,
125.3, 128.2, 131.7, 133.5, 136.1, 140.7, 141.9, 162.5, 173.2 ppm; HRMS
(ESI): m/z calcd for C45H62N3O5: 724.4689 [M+H]+; found: 724.4711.
off an excitation source at 355 nm, while illuminating the
sample at 532 nm.
Conclusion
Hydrophobic fluorophore–photochrome dyads can be dis-
solved in aqueous environments with the assistance of an
amphiphilic polymer. In the resulting supramolecular assem-
blies, the photochromic component retains its photochemi-
cal properties essentially unaltered. Specifically, its oxazine
ring opens upon ultraviolet illumination to bring the adja-
cent coumarin fluorophore in conjugation with a 3H-indol-
ium cation. This structural transformation shifts the main
absorption of the fluorophore to the visible region. As
a result, the visible illumination of aqueous dispersions of
such nanostructured constructs results in significant fluores-
cence only under concomitant ultraviolet irradiation. Fur-
thermore, the photochromic component reverts to the origi-
nal state on a microsecond timescale after the spontaneous
closing of its oxazine ring. In fact, the fluorescence of these
supramolecular assemblies can be modulated for hundreds
of cycles with microsecond switching speeds simply by turn-
ing on and off an ultraviolet source under visible illumina-
tion. Thus, our operating principles for fluorescence modula-
tion under optical control can translate into the realization
of switchable probes compatible with aqueous environments
and, ultimately, lead to valuable analytical tools for the in-
vestigation of biological samples.
Synthesis of 6a: A solution of 15a (71 mg, 0.3 mmol), 16 (120 mg,
0.2 mmol), and TFA (0.5 mL, 6.5 mmol) in EtOH (10 mL) was heated for
4 h under reflux. After cooling down to ambient temperature, the solvent
was distilled off under reduced pressure and the residue was dissolved in
CH2Cl2 (20 mL). The organic phase was dried over Na2SO4 and the sol-
vent was distilled off under reduced pressure. The residue was purified
by column chromatography (SiO2: Hexane/EtOAc (3:2, v/v)) to give 6a
(24 mg, 14%) as a green solid. 1H NMR (CDCl3): d=0.88 (t, J=7 Hz,
3H), 1.20–1.40 (m, 26H), 1.55–1.60 (m, 2H), 3.42 (q, J=7 Hz, 6H), 4.63
(d, J=9 Hz, 3H), 6.03 (t, J=5 Hz, 1H), 6.47 (d, J=3 Hz, 1H), 6.56–6.63
(m, 3H), 6.87–6.95 (m, 2H), 7.23 (d, J=2 Hz, 1H), 7.51–7.56 (m, 2H),
7.61 (d, J=2 Hz, 1H), 7.99–8.05 ppm (m, 2H); HRMS (ESI): m/z calcd
for C43H53N4O6: 721.3965 [M+H]+; found: 721.3959.
Synthesis of 7a:
A solution of 18a (48 mg, 0.1 mmol), 16 (21 mg,
0.1 mmol), and trifluoroacetic acid (20 mg, 0.2 mmol) in EtOH (20 mL)
was heated under reflux for 18 h. After cooling down to ambient temper-
ature, the solvent was distilled off under reduced pressure and the resi-
due was dissolved in CH2Cl2 (20 mL). The resulting solution was washed
with H2O (2ꢁ20 mL), dried over Na2SO4 and then the solvent was dis-
tilled off under reduced pressure to yield 7a (55 mg, 80%) as a blue
solid. 1H NMR (CDCl3): d=1.14 (t, J=6 Hz, 6H), 1.25 (brs, 3H), 1.37
(brs, 3H), 3.23 (brs, 3H), 3.39–3.57 (m, 22H), 4.60 (d, J=6 Hz, 1H), 4.70
(d, J=6 Hz, 1H), 6.44 (s, 1H), 6.62 (d, J=9 Hz, 1H), 6.64–6.77 (m, 2H),
6.90 (d, J=9 Hz, 1H), 7.10–7.20 (m, 1H), 7.28 (d, J=9 Hz, 1H), 7.60 (d,
J=6 Hz, 1H), 7.76 (s, 1H), 7.94 (dd, J=3, 9 Hz, 1H), 8.06 ppm (d, J=
3 Hz, 1H); HRMS (ESI): m/z calcd for C42H51N4O10: 771.3600 [M+H]+;
found: 771.3596.
Synthesis of the hexafluorophosphate salt of 10:
A solution of 19
Experimental Section
(317 mg, 0.6 mmol) and 16 (146 mg, 0.6 mmol) in EtOH (10 mL) was
heated under reflux for 18 h. After cooling to ambient temperature, the
solvent was distilled off under reduced pressure and the residue was dis-
solved in CH2Cl2 (3 mL). The addition of Et2O (20 mL) caused the for-
mation of a precipitate, which was filtered off to give the hexafluorophos-
phate salt of 10 (40 mg, 94%) as a blue solid. 1H NMR (CD3CN): d=
1.24 (t, J=8 Hz, 6H), 1.80 (s, 6H), 3.23 (s, 3H), 3.51–3.65 (m, 20H), 3.90
(s, 3H), 6.60 (s, 1H), 6.85 (d, J=8 Hz, 1H), 7.51 (d, J=8 Hz, 1H), 7.65
(d, J=8 Hz, 1H), 7.85 (d, J=16 Hz, 1H,), 8.02 (d, J=8 Hz, 1H), 8.13 (s,
1H), 8.19 (d, J=16 Hz, 1H), 8.42 ppm (s, 1H); 13C NMR (CD3CN): d=
12.3, 26.2, 34.0, 40.1, 44.7, 45.7, 52.1, 58.3, 69.5, 70.4, 70.5, 70.6, 72.0, 97.1,
110.0, 110.5, 112.0, 113.0, 114.2, 122.1, 129.0, 133.1, 135.3, 143.5, 144.5,
151.2, 151.9, 155.3, 158.8, 160.0, 166.2, 183.0 ppm; HRMS (ESI): m/z
calcd for C36H48N3O7: 634.3487 [MꢀPF6]+; found: 634.3506.
Materials and methods: Chemicals were purchased from commercial
sources and used as received with the exception of MeCN, which was dis-
tilled over CaH2. Compounds 1, 2, 3a, 5a, 8, 9, 12a, 13, and 16 were pre-
pared according to literature procedures.[53,54a,j,m,60–62] All reactions were
monitored by thin-layer chromatography, using aluminum sheets coated
with silica (60, F254). Electrospray ionization mass spectra (ESIMS) were
recorded with a Bruker micrOTO-Q II spectrometer. Nuclear magnetic
resonance (NMR) spectra were recorded with a Bruker Avance 400 spec-
trometer. Steady-state absorption spectra were recorded with a Varian
Cary 100 Bio spectrometer, using quartz cells with a path length of
1.0 cm. Steady-state emission spectra were recorded with a Varian Cary
Eclipse spectrometer in aerated solutions. Time-resolved absorption and
emission spectra were recorded with a Luzchem Research mLFP-111
spectrometer in aerated solutions by illuminating orthogonally the
sample with a Continuum Surelite II-10 Nd:YAG pulsed laser. For ab-
sorption measurements, the laser was operated at 355 nm (10 mJ) and
the transmittance was measured in the 350–700 nm spectral range. For
fluorescence measurements, the laser was operated simultaneously at 355
(10 mJ) and 532 nm (30 mJ) and the emission intensity was measured in
the 400–800 nm spectral range. The quantum yields for the photochromic
transformation were determined with a benzophenone standard, follow-
ing a literature protocol.[54m]
Synthesis of 11:
A solution of N,N-dicyclohexylcarbodiimide (DCC,
205 mg, 1.7 mmol) in CH2Cl2 (5 mL) was added dropwise over the course
of 10 min to a solution of N-methyl-N-(2-hydroxyethyl)-4-aminobenzal-
dehyde (300 mg, 1.7 mmol), heptadecanoic acid (453 mg, 1.7 mmol) and
4-(dimethylamino)pyridine (DMAP, 205 mg, 1.7 mmol) in CH2Cl2
(20 mL) maintained at 08C under Ar. The reaction mixture was allowed
to warm to ambient temperature and stirred for 24 h under these condi-
tions. The solvent was distilled off under reduced pressure and the resi-
due was purified by column chromatography (SiO2, Hexane/EtOAc (1:1,
v/v)) to afford 11 (670 mg, 93%) as a yellow solid.; 1H NMR (CDCl3):
d=0.89 (t, J=6 Hz, 3H), 1.27 (s, 26H), 1.56–1.59 (m, 2H), 2.25 (t, J=
7 Hz, 2H), 3.10 (s, 3H), 3.71 (t, J=6 Hz, 2H), 4.30 (t, J=6 Hz, 2H), 6.77
(d, J=9 Hz, 2H), 7.75 (d, J=9 Hz, 2H), 9.76 ppm (s, 1H); 13C NMR
(CDCl3): d=13.9, 22.9, 25.0, 29.3, 29.6, 29.9, 32.2, 34.0, 38.5, 50.7, 61.3,
111.6, 126.1, 131.8, 154.0, 173.1, 189.5 ppm; HRMS (ESI): m/z calcd for
C27H46NO3: 432.3478 [M+H]+; found: 432.3471.
Synthesis of 4a: A solution of 11 (417 mg, 1.0 mmol), 12a (300 mg,
1.0 mmol), and trifluoroacetic acid (TFA, 72 mL, 0.967 mmol) in EtOH
(10 mL) was heated under reflux for 24 h. After cooling down to ambient
temperature, the solvent was distilled off under reduced pressure and the
residue was dissolved in CH2Cl2 (15 mL) and washed with H2O (20 mL).
The organic phase was dried over Na2SO4 and the solvent was distilled
off under reduced pressure. The residue was purified by column chroma-
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Fast Fluorescence Switching
FULL PAPER
Synthesis of 14: A solution of DCC (764 mg, 3.7 mmol) in CH2Cl2 (5 mL)
was added dropwise over the course of 10 min to a solution of 13
(500 mg, 2.5 mmol), N-hydroxysuccinimide (NHS, 425 mg, 3.7 mmol),
and DMAP (30 mg, 0.2 mmol) in CH2Cl2 (80 mL) maintained at 08C
under Ar. The reaction mixture was allowed to warm to ambient temper-
ature and was stirred for 15 h under these conditions. The resulting pre-
cipitate was filtered off and n-decylamine (580 mg, 3.7 mmol) was added
dropwise to the filtrate over the course of 10 min. The solution was
stirred for 12 h at ambient temperature. The resulting precipitate was fil-
tered off and the solvent was distilled off under reduced pressure. The
residue was purified by column chromatography (SiO2, Hexane/EtOAc
tion of NH4PF6 (5 mL), the mixture was concentrated under reduced
pressure to half of its original volume and the resulting suspension was
extracted with CH2Cl2. The organic phase was dried over Na2SO4 and the
solvent was distilled off under reduced pressure to give the hexafluoro-
phosphate salt of 22 (317 mg, 74%) as dark red oil. 1H NMR (CDCl3):
d=1.56 (s, 6H), 2.75 (s, 3H), 3.27 (s, 3H), 3.45–3.47 (m, 2H), 3.52–3.73
(m, 14H), 3.98 (s, 3H), 7.80 (d, J=8 Hz, 1H), 8.05 (d, J=8 Hz, 1H),
8.13 ppm (s, 1H); 13C NMR (CDCl3): d=14.9, 22.3, 35, 40.8, 55.7, 5.89,
70.0, 70.2, 70.9, 71.1, 72.5, 72.6, 116.1, 123.2, 129.6, 137.1, 142.9, 145.1,
167.0, 167.1, 199.5 ppm; ESIMS: m/z=407 [MꢀPF6]+.
Polymer micelles: A solution of 1 (2.5 mgmLꢀ1, 200 mL) in CHCl3 was
added to a solution of 2, 3a, 4a, 5a, or 6a (0.1 mgmLꢀ1, 50 mL) in
CHCl3. Alternatively, a solution of 1 (2.5 mgmLꢀ1, 200 mL) in CHCl3 was
mixed with solutions of 2 (0.1 mgmLꢀ1, 20 mL) and 3a, 4a, 5a, or 6a
(0.1 mgmLꢀ1, 100 mL) in CHCl3. Each mixture was heated at 408C in an
open vial. After the evaporation of the solvent, the residue was purged
with air and dispersed in PBS (1 mL, pH 7.0). After vigorous shaking, the
dispersion was filtered and the filtrate was used for the spectroscopic and
imaging experiments without further purification.
1
(1:2, v/v)) to afford 14 (542 mg, 43%) as a white solid. H NMR (CDCl3):
d=0.89 (t, J=6 Hz, 3H), 1.28–1.36 (m, 20H), 1.62–1.73 (m, 2H), 2.32 (s,
3H), 3.47 (q, J=6 Hz, 2H), 6.18 (brs, 1H), 7.54 (d, J=8 Hz, 1H), 7.66
(dd, J=2, 8 Hz, 1H), 7.81 ppm (d, J=2 Hz, 1H); 13C NMR (CDCl3): d=
14.0, 15.5, 22.6, 22.8, 27.0, 29.2, 29.3, 29.5, 29.7, 31.8, 40.2, 53.8, 119.4,
120.8, 126.4, 131.8, 146.0, 156.2, 157.2, 167.7 ppm; HRMS (ESI): m/z
calcd for C22H35N2O: 343.2749 [M+H]+; found: 343.2734.
Synthesis of 15a: A solution of 14 (342 mg, 1.2 mmol) and 4-nitro-2-
chloromethyl-phenol (225 mg, 1.2 mmol) in MeCN (20 mL) was heated
for 24 h under reflux and Ar. After cooling to ambient temperature, the
solvent was distilled off under reduced pressure and the residue was dis-
solved in CH2Cl2 (15 mL) and washed with H2O (20 mL). The organic
phase was dried over Na2SO4 and the solvent was distilled off under re-
duced pressure and the residue was purified by column chromatography
(SiO2, Hexane/EtOAc (2:1, v/v)) to afford 15a (180 mg, 36%) as a white
solid. 1H NMR (CDCl3): d=0.87 (t, J=7 Hz, 3H), 1.18–1.30 (m, 20H),
1.54–1.66 (m, 5H), 3.39 (q, J=6 Hz, 2H), 4.65 (s, 2H), 6.21 (t, J=6 Hz,
1H), 6.55 (d, J=8 Hz, 1H), 6.72 (d, J=9 Hz, 1H), 7.50 (dd, J=2, 8 Hz,
1H), 7.62 (d, J=2 Hz, 1H), 7.95 (dd, J=3, 9 Hz, 1H), 8.07 ppm (d, J=
3 Hz, 1H); 13C NMR (CDCl3): d=13.9, 16.2, 18.5, 22.8, 27.3, 29.6, 29.8,
30.1, 32.1, 34.0, 39.9, 48.1, 103.7, 108.3, 118.2, 121.9, 124.6, 124.7, 127.9,
128.5, 138.6, 141.1, 150.1, 159.2, 167.0 ppm; HRMS (ESI): m/z calcd for
C29H40N3O4: 494.3019 [M+H]+; found: 494.2987.
Acknowledgements
F.M.R. thanks the National Science Foundation (CAREER Award CHE-
0237578, CHE-0749840, and CHE-1049860) for supporting his research
program and for providing funds to purchase a mass spectrometer (CHE-
0946858). S.S. thanks the MIUR (PRIN 2008) for financial support and
the Royal Society of Chemistry for a travel grant.
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Synthesis of 17: DCC (405 mg, 2.0 mmol) was slowly added to a solution
of 13 (200 mg, 1.0 mmol), NHS (227 mg, 2.0 mmol), and DMAP (25 mg,
0.2 mmol) in CH2Cl2 (30 mL) maintained at 08C under Ar. The mixture
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solution was stirred for 12 h at ambient temperature. The solvent was dis-
tilled off under reduced pressure to give 17 (300 mg, 90%) as a yellow
oil. 1H NMR (CDCl3): d=1.19 (s, 6H), 2.56 (s, 3H), 3.18 (t, J=4 Hz,
2H), 3.23 (t, J=4 Hz, 2H), 3.40–3.54 (m, 15H), 7.43 (d, J=8 Hz, 1H),
7.65 (t, J=6 Hz, 1H), 7.75 ppm (d, J=8 Hz, 1H); ESIMS: m/z=393
[M+H]+.
Synthesis of 18a: A solution of 17 (100 mg, 0.3 mmol) and 2-chlorometh-
yl-4-nitrophenol (48 mg, 0.3 mmol) in MeCN (10 mL) was heated for
24 h under reflux. After cooling to ambient temperature, the solvent was
distilled off under reduced pressure and the residue was dissolved in
CH2Cl2 (20 mL). The resulting solution was washed with H2O (20 mL),
dried over Na2SO4 and the solvent was distilled off under reduced pres-
sure. The residue was purified by column chromatography (SiO2: CHCl3/
MeOH (99:1!95:5, v/v) to afford 18a (80 mg, 59%) as a yellow oil.
1H NMR (CD3CN): d=1.54 (s, 3H), 1.59 (s, 3H), 2.40 (s, 3H), 3.28 (s,
3H), 3.34–3.52 (m, 16H), 4.59 (d, J=16 Hz, 1H), 4.73 (d, J=6 Hz, 1H),
6.69 (d, J=8 Hz, 1H), 6.73 (d, J=8 Hz, 1H), 7.12 (brs, 1H), 7.58 (d, J=
8 Hz, 1H), 7.63 (s, 1H), 7.93 (d, J=8 Hz, 1H), 8.14 ppm (s, 1H);
13C NMR (CD3CN): d=16.8, 18.9, 26.1, 40.4, 40.5, 48.7, 58.8, 70.2, 70.9,
71.1, 72.5, 104.1, 108.9, 118.6, 120.5, 22.5, 124.6, 124.9, 127.8, 128.5, 139.4,
141.5, 150.8, 159.7, 167.8 ppm; ESIMS: m/z=544 [M+H]+.
Synthesis of the hexafluorophosphate salt of 19:
A solution of 17
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Fast Fluorescence Switching
FULL PAPER
j) E. Deniz, M. Tomasulo, S. Sortino, F. M. Raymo, J. Phys. Chem. C
2009, 113, 8491–8497; k) E. Deniz, S. Ray, M. Tomasulo, S. Impelliz-
zeri, S. Sortino, F. M. Raymo, J. Phys. Chem. A 2010, 114, 11567–
11575; l) E. Deniz, S. Sortino, F. M. Raymo, J. Phys. Chem. Lett.
2010, 1, 1690–1693; m) E. Deniz, S. Sortino, F. M. Raymo, J. Phys.
Chem. Lett. 2010, 1, 3506–3509; n) M. Tomasulo, E. Deniz, S. Sorti-
no, F. M. Raymo, Photochem. Photobiol. Sci. 2010, 9, 136–140; o) E.
Deniz, S. Impellizzeri, S. Sortino, F. M. Raymo, Can. J. Chem. 2011,
89, 110–116; p) E. Deniz, M. Tomasulo, J. Cusido, S. Sortino, F. M.
Raymo, Langmuir 2011, 27, 11773–11783; q) E. Deniz, J. Cusido, S.
Swaminathan, M. Battal, S. Impellizzeri, S. Sortino, F. M. Raymo, J.
Photochem. Photobiol. A 2012, 229, 20–28; r) E. Deniz, M. Tomasu-
lo, J. Cusido, I. Yildiz, M. Petriella, M. L. Bossi, S. Sortino, F. M.
Raymo, J. Phys. Chem. C 2012, 116, 6058–6068.
ring-open isomer has the 4-nitrophenolate anion in a protonated
form.
[58] The relative concentrations of the two isomers at equilibrium were
estimated from the absorbance of the 3H-indolium chromophore of
7b in the visible region, using the molar extinction coefficient of the
model compound 10.
[59] The fluorescence quantum yield of the hexafluorophosphate salt of
9 is 0.09 in acetonitrile[54m,r] and 0.04 in PBS (pH 7.0). Thus, the tran-
sition from organic to aqueous solutions tends to discourage the ra-
diative deactivation of this particular fluorophore. In the presence
of the amphiphilic co-polymer 1, however, the quantum yield in
PBS increases to 0.13. These observations suggest that the polymeric
host limits the exposure of the fluorescent guest to the aqueous en-
vironment.
[55] The relative concentrations of the two isomers of each system at
equilibrium were estimated from the absorbance of the 3H-indolium
chromophore of 3b and 4b in the visible region, using the molar ex-
tinction coefficient of the model compound 8.
[56] The pH dependence of the absorption spectrum (a and b in Fig-
ure S4 in the Supporting Information) of 4-nitrophenol confirms the
assignment of the band at 390 nm to the anionic chromophore of
the ring-open isomers 3b and 4b. Indeed, this band resembles the
absorption observed for the 4-nitrophenolate anion at a pH of 8.0 (b
in Figure S4 in the Supporting Information).
[60] The synthesis of 12a was originally reported in: a) A. A. Shachkus,
J. Degutis, A. Jezerskaite in Chemistry of Heterocyclic Compounds
(Eds.: J. Kovac, P. Zalupsky), Elsevier, Amsterdam, 1987, vol. 35
pp. 518–520; b) A. A. Shachkus, J. Degutis, A. G. Urbonavichyus,
Khim. Geterotsikl. Soedin. 1989, 5, 672–676; A. A. Shachkus, Yu. A.
Degutis, A. G. Urbonavichyus, Chem. Heterocycl. Compd. 1989, 25,
562–565.
[61] M. Tomasulo, S. L. Kaanumal, S. Sortino, F. M. Raymo, J. Org.
Chem. 2007, 72, 595–605.
[62] J. Wu, W. Liu, X. Zhuang, F. Wang, P. Wang, S. Tao, X. Zhang, S.
Wu, S. T. Lee, Org. Lett. 2007, 9, 33–36.
[57] The pKa of 4-nitrophenol is 7.15 (CRC Handbook of Chemistry and
Physics (Ed.: W. M. Haynes), CRC Press/Taylor and Francis, Boca
Raton, 2012). This value suggests that a significant fraction of the
Received: April 6, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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S. Sortino, F. M. Raymo et al.
Supramolecular Chemistry
J. Cusido, M. Battal, E. Deniz,
I. Yildiz, S. Sortino,*
F. M. Raymo* ................. &&&& — &&&&
Fast Fluorescence Switching within
Hydrophilic Supramolecular Assem-
blies
Photoswitchable fluorophores can be
entrapped within the hydrophobic inte-
rior of an amphiphilic polymer. The
fluorescence of the resulting supra-
molecular assemblies can be switched
for hundreds of cycles on a microsec-
ond timescale in aqueous environ-
ments (see scheme).
&
10
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These are not the final page numbers!