Photocontrollable analyte-responsive fluorescent probes: A photocaged copper-responsive fluorescence turn-on probe

Article abstract of DOI:10.1002/chem.201001923

Analyte-responsive fluorescent probes are valuable chemical tools for dissecting complex living systems. However, the major shortcoming of fluorescent probes is that once they enter the cells, control over them is basically lost. It is critical to regulat

Full text of DOI:10.1002/chem.201001923

FULL PAPER
DOI: 10.1002/chem.201001923
Photocontrollable Analyte-Responsive Fluorescent Probes: A Photocaged
Copper-Responsive Fluorescence Turn-On Probe
Lin Yuan, Weiying Lin,* Zengmei Cao, Lingliang Long, and Jizeng Song[a]
Abstract: Analyte-responsive fluores-
cent probes are valuable chemical tools
for dissecting complex living systems.
However, the major shortcoming of
fluorescent probes is that once they
enter the cells, control over them is ba-
sically lost. It is critical to regulate flu-
orescent probes in a spatial and tempo-
ral manner, as functions of biomole-
cules are spatiotemporal. On the other
hand, light can be manipulated in time
and in the application site, so the pho-
tocaging technique allows researchers
to control the biomolecules of interest
ing and photocaging technologies,
which may afford the caging version of
analyte-responsive fluorescent probes,
referred to as photocontrollable ana-
complex biological systems in a spatial
and temporal manner with an innova-
tive chemical tool. In this work, for
proof of concept, we report the rational
design, synthesis, photocontrollable
sensing in solution and in living cells,
and mechanistic studies of a molecular
prototype of PCAFP for copper as the
first paradigm of this new class of
smart fluorescent probes. We believe
that PCAFPs represent a substantial
breakthrough in the sensing and photo-
caging fields, and that the general con-
cept of PCAFPs should be broadly ap-
plicable for a wide variety of biologi-
cally relevant species.
lyte-responsive
fluorescent
probes
(PCAFPs). These “smart” fluorescent
probes apparently have the intrinsic
advantage of spatiotemporal control
when compared to traditional fluores-
cent probes, as the “sensing activity” of
PCAFPs is photocontrollable. This
should enable biologists to interrogate
Keywords: cage compounds · fluo-
rescent probes · photoactivation ·
photochemistry · photolysis
in
a temporal and spatial fashion.
Herein, we propose for the first time
the combination of the merits of sens-
Introduction
sive fluorescent probes for various targets has been devel-
oped. As fluorescent probes exhibit the advantages of high
sensitivity and simplicity for implementation, they are valu-
Biological systems are highly complicated, as they comprise
countless interwoven interactions of biomolecules under
precise temporal and spatial control. To explore the bio-
chemistry of a living system, it is essential to have suitable
chemical tools. One type of useful chemical tool is the fluo-
rescent probe. In this contribution, “fluorescent probes”, in-
cluding fluorescent chemosensors and chemodosimeters, are
defined as synthetic small molecules that react specifically
with analytes to induce a marked change in fluorescence
properties (Scheme 1a).[1] Since the seminal work by Tsienꢀs
group about Ca2+ probes,[2] a wide variety of analyte-respon-
AHCTUNGTREGaNNUN ble in bioimaging applications for dissecting complex living
systems.[3] However, the major shortcoming of analyte-re-
sponsive fluorescent probes is that once they enter cells,
control over them is basically lost.[4] It is critical to regulate
fluorescent probes in a spatial and temporal manner, as
functions of biomolecules are spatiotemporal.
On the other hand, photocaging technology has been
widely employed in studies of molecular and cellular dy-
namics.[4,5] Masking the key functional components of bio-
molecules with photolabile protecting groups may afford
caged compounds, which are essentially biologically inert.
However, upon photolysis, the photolabile groups are depro-
tected to release the native active biomolecules
(Scheme 1b). The pioneering work by Hoffman and co-
workers on caged ATP has stimulated development in the
photocaging field.[6] So far, diverse biologically relevant mol-
ecules including metal ions, neurotransmitters, nucleotides,
peptides, enzymes, nucleic acids, and drugs have been
caged.[4–7] As light can be manipulated in time and in the ap-
[a] L. Yuan, Prof. W. Lin, Z. Cao, L. Long, J. Song
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University, Changsha
Hunan 410082 (P. R. China)
Fax : (+86)731-888-21464
E-mail: weiyinglin@hnu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001923.
Chem. Eur. J. 2011, 17, 689 – 696
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
689

design, synthesis, photocontrollable sensing in solution and
in living cells, and mechanistic studies of a molecular proto-
type of a copper PCAFP.
Results and Discussion
Rational design and synthesis of photocontrollable analyte-
responsive fluorescent probes (PCAFPs): In the develop-
ment of valid PCAFPs, some design criteria should be con-
sidered: 1) the probes should be stable in aqueous solution
at physiological pH; 2) they should be inert to the analyte of
interest in the dark; 3) they should not exhibit a fluores-
cence response after photolysis in the absence of the analyte
of interest; 4) they should display a significant fluorescence
turn-on or ratiometric response after photolysis in the pres-
ence of the analyte; 5) the wavelengths of the uncaging light
should be above 300 nm to minimize UV-induced cell
damage.
To examine the feasibility of the construction of smart
PCAFPs based on the sensing and caging technologies, we
were interested in creating a prototypic PCAFP for Cu2+ as
a step toward the regulation of sensing activity by light.
With the above design criteria in mind, we judiciously de-
signed the probe bisNPE-Fl-Hy as the first paradigm of a
PCAFP for Cu2+ (Scheme 2c), inspired by a rhodamine hy-
drazide based fluorescent copper probe (Scheme 2a) and a
nitrobenzyl-caged fluorescein (Scheme 2b). It is known that
copper may induce the hydrolysis of the non-fluorescent spi-
rolactam form of rhodamine hydrazide into the fluorescent
ring-opened carboxylic form of rhodamine,[10] and that the
non-fluorescent nitrobenzyl-caged fluorescein derivative
could be converted into the fluorescent fluorescein dye
upon photolysis.[9a] Thus, we envisaged that integrating the
sensing platform of the rhodamine hydrazide based copper
fluorescent probe with the caging strategy of the fluorescein
Scheme 1. a) Schematic diagram showing the principle of the traditional
analyte-responsive fluorescent turn-on probes (e.g., chemosensors and
chemodosimeters). b) Schematic diagram illustrating the principle of the
conventional caged compounds. c) Schematic diagram depicting the
working hypothesis of our proposed PCAFPs.
plication site,[4–8] it is then possible to regulate these biologi-
cally relevant molecules with high temporal and spatial reso-
lution using photocaging technology. Thus, the photocaging
method allows researchers to control the biomolecules of in-
terest in a temporal and spatial fashion.
Herein, we propose that combining the advantages of the
analyte-responsive fluorescent probes and the photocaging
technology may afford a novel type of “smart” fluorescent
probes, which we refer to as photocontrollable analyte-re-
sponsive fluorescent probes (PCAFPs). In PCAFPs the sens-
ing activity to the analyte of interest is caged, so PCAFPs
do not exhibit a sensing response to the analyte in the dark
(Scheme 1c). However, upon exposure to light, caged
PCAFPs are uncaged, and are converted to functionally
active analyte-responsive fluorescent probes. Thus, the sens-
ing activity is recovered. When compared to traditional ana-
lyte-responsive fluorescent probes, PCAFPs apparently have
the intrinsic advantage of spatiotemporal control, as the
sensing activity of the PCAFPs is photocontrollable. This
may provide biologists with a novel, powerful, chemical tool
for investigating complex living systems in a spatial and tem-
poral manner. It is important to note that the new concept
of PCAFPs is conceptually distinct from the previous so-
called caged fluorescent probes.[9] Smart PCAFPs are the
caging version of fluorescent chemosensors and chemo-
dosimeters, which, upon photolysis, can specifically interact
with a target analyte to elicit a fluorescence response. In
contrast, the previous so-called caged fluorescent probes are
simply the caging version of dyes, and do not respond to the
analytes of interest after photolysis, as they lack a sensing
unit. The aim of this paper is to introduce the concept of
PCAFPs. For proof of concept, we present the rational
dye could afford
a first-generation prototypic copper
PCAFP (compound bisNPE-Fl-Hy in Scheme 2c). We an-
ticipated that bisNPE-Fl-Hy would be essentially non-fluo-
rescent and resistant to Cu2+-induced hydrolysis in the dark,
as both the hydroxyl groups of the fluorescein dye are alkyl-
AHCTUNGTREGaNNNU ted, which may inhibit the ring-opening reaction (see the
spectral and mass spectrometry studies shown below). How-
ever, upon photolysis, the photolabile nitrobenzyl groups
are removed to uncage the non-fluorescent fluorescein hy-
drazide (Fl-Hy), which may be further hydrolyzed by Cu2+
to provide highly fluorescent fluorescein (Fl).
Notably, bisNPE-Fl-Hy is fundamentally different from
the recently reported “caged copper”[7j] or “light-activated
copper” probes.[7p] In their approach, the probes initially
bind to Cu2+ to form a complex, which is then subjected to
UV light photolysis to release Cu2+[7j] or Cu2+ and fluoro-
phore.[7p] In other words, in this case, the interaction of the
probes with Cu2+ occurs before photolysis, and therefore is
not controllable by light. In sharp contrast, in our PCAFP
strategy, the probes do not interact with Cu2+ before photol-
ysis. Only after photolysis is the caged probe bisNPE-Fl-Hy
690
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Chem. Eur. J. 2011, 17, 689 – 696

Photocontrollable Analyte-Responsive Fluorescent Probes
FULL PAPER
0.8 equivalents 1-(1-bromoethyl)-2-nitrobenzene under basic
conditions. The structures of bisNPE-Fl-Hy and monoNPE-
Fl-Hy were fully characterized by 1H and 13C NMR spectros-
copy, and mass spectrometry.
Fluorescence response of the caged probes to UV light ex-
posure: After acquiring the caged compound bisNPE-Fl-Hy,
we evaluated its fluorescence response to UV light exposure
in the absence or presence of a fixed concentration of Cu2+
.
As the utility of a caged species is dependent on its hydro-
lytic stability at physiological pH in the absence or presence
of Cu2+ in the dark, we initially examined the fluorescence
of the caged compound bisNPE-Fl-Hy in the absence or
presence of Cu2+ before photolysis. As shown in Figure 1,
Figure 1. The fluorescence spectra of compound bisNPE-Fl-Hy (5 mm) in
the absence or presence of Cu2+ in 25 mm HEPES (pH 7.4) containing
20% CH3CN as a co-solvent, kept in the dark for various periods:
Scheme 2. a) Sensing chemistry of a rhodamine hydrazide-based fluores-
cent copper probe. b) Uncaging chemistry of a conventional caged fluo-
rescein. c) The design hypothesis of bisNPE-Fl-Hy, a first-generation pro-
totypic PCAFP for copper.
bisNPE-Fl-Hy (5 mm) in the absence of Cu2+ for 0.5 h ( ); bisNPE-Fl-Hy
~
2+
(5 mm) + Cu2+ (10 equiv) for 0.5 h ( ), bisNPE-Fl-Hy (5 mm) + Cu
&
(10 equiv) for 8 days (N). For comparison, the fluorescence spectra of
bisNPE-Fl-Hy (5 mm) in the absence of Cu2+ after UV light exposure for
2+
!
120 s ( ) and bisNPE-Fl-Hy (5 mm) + Cu (10 equiv) after UV light ex-
*
posure for 120 s ( ) are also shown. Excitation at 492 nm.
released to give the functionally active copper-responsive
probe Fl-Hy, which can then interact with Cu2+ to elicit a
fluorescence turn-on response due to the copper-promoted
hydrolysis. Thus, in the PCAFP strategy described herein,
the sensing of copper can be regulated by light.
The synthesis of compound bisNPE-Fl-Hy is outlined in
Scheme 3. Briefly, condensation of fluorescein with hydra-
zine afforded the key intermediate, fluorescein hydrazide
(Fl-Hy) in high yield.[11] Subsequent alkylation of Fl-Hy with
an excess amount of 1-(1-bromoethyl)-2-nitrobenzene under
basic conditions gave the desired caged compound bisNPE-
Fl-Hy in excellent yield. The compound monoNPE-Fl-Hy
containing only one photolabile nitrobenzyl group was also
synthesized as a reference by reacting Fl-Hy with roughly
bisNPE-Fl-Hy is essentially non-fluorescent (Ff <0.0002, see
Supporting Information) due to the spirolactam structure.
Thus, the caged probe has a very low fluorescence back-
ground. In addition, even in the presence of Cu2+ in the
dark, it is still almost non-fluorescent, indicating that the
caged compound is very stable and does not hydrolyze to Fl
in the presence of Cu2+ in the dark, in good agreement with
the mass spectrometry analysis (Figure S2 in the Supporting
Information). This is critical for the regulation of the sensing
activity of bisNPE-Fl-Hy toward copper by light.
We then proceeded to investigate the emission spectra of
bisNPE-Fl-Hy upon exposure to UV light (350 nm) in the
presence of Cu2+. A large fluo-
rescence
enhancement
at
516 nm (Ff =0.81, see Support-
ing Information) was observed
in the presence of Cu2+ after
exposure
to
UV
light
(Figure 2). It should be noted
that a huge fluorescence en-
hancement (up to 350-fold)
around 516 nm was induced
Scheme 3. Synthesis of caged compound bisNPE-Fl-Hy and the analogue monoNPE-Fl-Hy.
Chem. Eur. J. 2011, 17, 689 – 696
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691

W. Lin et al.
PCAFP for copper according to the above criteria set for
PCAFPs. We decided to focus on the caged probe bisNPE-
Fl-Hy for further studies of sensing sensitivity and selectivity
to copper.
Fluorescence response of bisNPE-Fl-Hy to Cu2+: After
identifying the optimal photolysis time, we further examined
the sensing response of the caged probe bisNPE-Fl-Hy to
various concentrations of Cu2+ at the fixed UV illumination
time (120 s) by fluorescence spectroscopy. The free probe is
essentially non-fluorescent, but upon photolysis, addition of
an increasing concentration of Cu2+ to the solution of
bisNPE-Fl-Hy induced a gradual increase in the emission
around 516 nm (Figure 4). Furthermore, the introduction of
Figure 2. Fluorescence emission spectra of bisNPE-Fl-Hy (5 mm) in the
presence of Cu2+ (10 equiv) before and after UV illumination for various
periods (0—120 s). Excitation at 492 nm.
after photolysis. Such a dramatic fluorescence enhancement
after uncaging is highly favorable for cell imaging applica-
tions. However, no significant fluorescence was observed
after photolysis in the absence of Cu2+ (Figure 1). This evi-
dently indicates that the fluorescence turn-on response was
elicited by Cu2+ after photolysis. As a great fluorescence en-
hancement around 516 nm was achieved after 120 s of pho-
tolysis, this UV light exposure time was selected to examine
further the sensitivity and selectivity of the caged probe
bisNPE-Fl-Hy toward Cu2+
.
The compound monoNPE-Fl-Hy (Scheme 3) is the ana-
logue of bisNPE-Fl-Hy with only a single nitrophenyl group,
and it could also exhibit strong fluorescence in the presence
of Cu2+ upon photolysis (Figure 3). However, monoNPE-Fl-
Figure 4. The emission spectra of bisNPE-Fl-Hy (5 mm) after UV illumi-
nation for 120 s in the presence of various concentrations of Cu2+ (0–
10 equiv). Excitation at 492 nm.
Cu2+ turned the visual emission color of the solution of the
probe bisNPE-Fl-Hy from dark to bright green after photol-
ysis (Scheme 2), further reinforcing the fluorescence turn-on
response. The probe bisNPE-Fl-Hy exhibited a linear fluo-
rescence response toward Cu2+ ranging from 2.5ꢂ10À7 to
1.0ꢂ10À5 m (Figure S5 in the Supporting Information), with
a detection limit of 1.9ꢂ10À7 m under the experimental con-
ditions (Figure S6, in the Supporting Information).[12] More-
over, the studies of the effect of pH on the fluorescence re-
sponse (Figure S7 in the Supporting Information) indicate
that bisNPE-Fl-Hy could be employed to detect Cu2+ upon
photolysis at physiological pH.
Figure 3. The fluorescence spectra of compound monoNPE-Fl-Hy (5 mm)
in the absence or presence of Cu2+ in 25 mm HEPES (pH 7.4) containing
20% CH3CN as a co-solvent, kept in the dark for various periods: mono-
NPE-Fl-Hy (5 mm) in the absence of Cu2+ for 0.5 h ( ); monoNPE-Fl-Hy
&
(5 mm) + Cu2+ (10 equiv) for 0.5 h ( ). For comparison, the fluorescence
*
spectrum of monoNPE-Fl-Hy (5 mm) + Cu2+ (10 equiv) after UV light
Studies of uncaging and sensing chemistry of BisNPE-Fl-
Hy: To gain an insight into the photolysis and sensing pro-
cesses associated with the caged probe bisNPE-Fl-Hy, the
solutions of compound bisNPE-Fl-Hy before and after dif-
ferent durations of UV light exposure in the absence or
presence of Cu2+ were subjected to mass spectrometry and
emission and excitation spectroscopy studies. Prior to re-
lease, in the presence of Cu2+, bisNPE-Fl-Hy only showed a
major peak at m/z 645.0 ([bisNPE-Fl-Hy+H]+); peaks cor-
responding to caged fluorescein or Fl were not present in
the spectrum (Figure S8a in the Supporting Information), in-
dicating that the caged probe bisNPE-Fl-Hy was very stable
~
exposure for 30 s ( ) is also shown. Excitation at 492 nm.
Hy already displayed marked fluorescence in the presence
of Cu2+ even before photolysis (Ff =0.082, see Supporting
Information). This indicates that, unlike bisNPE-Fl-Hy,
monoNPE-Fl-Hy can be hydrolyzed by Cu2+ in the dark to
afford monoNPE-Fl, in accordance with the results of the
mass spectrometry studies for monoNPE-Fl-Hy in the ab-
sence or presence of Cu2+ in the dark or upon UV exposure
(Figures S3 and S4). Thus, monoNPE-Fl-Hy is not ideal as a
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Chem. Eur. J. 2011, 17, 689 – 696

Photocontrollable Analyte-Responsive Fluorescent Probes
FULL PAPER
and was not hydrolyzed to caged fluorescein or Fl by copper
in the dark. This is in accord with the finding of the above
emission spectroscopic studies that bisNPE-Fl-Hy is non-flu-
orescent in the presence of Cu2+ in the dark. However, with
exposure to UV light in the absence of Cu2+, two major
new peaks appeared at m/z 347.0 ([Fl-Hy+H]+) and 496.0
([monoNPE-Fl-Hy+H]+) (Figure S8b–d, in the Supporting
Information). Furthermore, with the increasing photolysis
time, the peak corresponding to Fl-Hy increased, whereas
that corresponding to monoNPE-Fl-Hy decreased, suggest-
ing that the partially photolyzed product monoNPE-Fl-Hy
was eventually converted to the completely photolyzed
product Fl-Hy. However, upon photolysis in the presence of
Cu2+, a major new peak at m/z 315.0 ([Fl+HÀH2O]+) (Fig-
ure S8e in the Supporting Information) was noted. The
same peak was also observed after the photolysis of
bisNPE-Fl-Hy in the absence of Cu2+, followed by addition
of Cu2+ (Figure S8 f in the Supporting Information). In addi-
tion, the emission and excitation spectra of bisNPE-Fl-Hy
after exhaustive photolysis in the presence of Cu2+ were
identical to those of the standard Fl when excited or moni-
tored at the appropriate wavelengths (Figure S9in the Sup-
porting Information).
Figure 5. Fluorescence enhancement factor (FEF) of bisNPE-Fl-Hy
(5 mm) for different competing analytes (200 equiv for Na+, K+, Ca2+
,
,
Mg2+, SO42À, ClÀ, and NO3À, 10 equiv for Zn2+, Cu2+, Co2+, Fe3+, Hg2+
Mn2+, and FÀ) after UV illumination for 120 s: 1) Free; 2) Na+, 3) K+,
4) Ca2+
11) SO4
,
5) Mg2+
,
6) Zn2+
,
7) Co2+
14) PO4
,
8) Fe3+
,
9) Hg2+
16) Cu2+
,
10) Mn2+
+
,
2À
À
3À
,
12) ClÀ, 13) NO3
,
,
15) Cu2+
,
Na+,
17) Cu2+ + K+, 18) Cu2+ + Ca2+, 19) Cu2+ + Mg2+, 20) Cu2+ + Zn2+
,
21) Cu2+ + Co2+, 22) Cu2+ + Hg2+, 23) Cu2+ + Mn2+, 24) Cu2+
+
SO42À, 25) Cu2+ + ClÀ, 26) Cu2+ + NO3À, and 27) Cu2+ + FÀ. Excita-
tion at 492 nm; emission at 516 nm.
the probe has a high selectivity for Cu2+. This is further sup-
ported by the observation that other species have only negli-
gible interference with the fluorescence response. Thus,
bisNPE-Fl-Hy can sense Cu2+ selectively upon photolysis.
Thus, based on the analysis of mass spectrometry and
emission and excitation spectroscopy, the uncaging and sens-
ing chemistry of bisNPE-Fl-Hy is proposed as shown in Fig-
ure S10 in the Supporting Information. The light-mediated
transformation of bisNPE-Fl-Hy to the partially photolyzed
monoNPE-Fl-Hy, and then to the entirely photolyzed Fl-Hy,
followed by the copper-induced hydrolysis, results in the for-
mation of the final product Fl, which exhibits intense fluo-
rescence. This proposal is consistent with the above design
hypothesis of bisNPE-Fl-Hy (Scheme 2). An alternative
pathway from monoNPE-Fl-Hy to the final product Fl may
also be present. The existence of a minor peak at m/z 464.0
([monoNPE-Fl+HÀH2O]+) in the mass spectra of the mix-
ture of bisNPE-Fl-Hy with Cu2+ upon photolysis (Fig-
ure S8e,f in the Supporting Information) implies that the
partially photolyzed product monoNPE-Fl-Hy can also be
hydrolyzed by Cu2+ to afford monoNPE-Fl (Figure S10 in
the Supporting Information), which may be photolyzed fur-
ther to produce Fl, in good agreement with the aforemen-
tioned mass spectrometry analysis and the proposed uncag-
ing and sensing chemistry of monoNPE-Fl-Hy (Figures S3
and S4 in the Supporting Information).
Photoregulated fluorescence sensing of Cu2+ in living cells:
To investigate the photoregulated fluorescence sensing of
Cu2+ in living cells, we treated HeLa cells with bisNPE-Fl-
Hy and Cu2+. The selected cells were irradiated with UV
light through a fluorescence microscope.[9a,d] After photolysis
for 30 s, the irradiated cells became brightly fluorescent
(Figure 6b). However, no marked fluorescence was ob-
Selectivity studies of bisNPE-Fl-Hy: We proceeded to exam-
ine the selectivity of bisNPE-Fl-Hy to Cu2+ over other com-
peting species upon photolysis. 10–200 equiv of various bio-
logically relevant ions were added to solutions of bisNPE-
Fl-Hy, then UV illumination was applied for 120 s. As
shown in Figure 5, introduction of Cu2+ to bisNPE-Fl-Hy
upon photolysis resulted in a significant enhancement of the
fluorescence intensity at 516 nm. By contrast, no visible
changes in the emission were noted upon addition of repre-
Figure 6. Local uncaging and imaging of bisNPE-Fl-Hy (15 mm) + Cu2+
(2 equiv) in living cells. a) DIC image of HeLa cells incubated with probe
bisNPE-Fl-Hy + Cu2+. b) Fluorescence image of the cells in the panel a)
after 30 s illumination for the selected cells with blue light excitation.
c) Overlay of images a) and b). d) DIC image of HeLa cells incubated
with probe bisNPE-Fl-Hy in the absence of Cu2+. e) Fluorescence image
of the cells in panel d) after 30 s illumination as a negative control with
blue light excitation. f) Fluorescence image of the cells only as a negative
control with blue light excitation. g) DIC image of HeLa cells incubated
with Rh-Hy + Cu2+. h) Fluorescence image of the cells in panel g) with
green light excitation. i) Overlay of images g) and h).
sentative species such as Na+, K+, Ca2+, Mg2+, Zn2+, Co2+
,
À
Fe3+, Hg2+, Mn2+, SO42À, ClÀ, NO3 , and FÀ, indicating that
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693

W. Lin et al.
served in the cells that were not photolyzed. In a negative
control experiment, HeLa cells were treated with bisNPE-
Fl-Hy in the absence of Cu2+ upon UV exposure (Fig-
ure 6e), but no significant fluorescence was noted. These
data clearly established that the bisNPE-Fl-Hy probe is cell-
membrane permeable, and that this probe can be employed
for local uncaging and sensing of copper in living cells. It is
important to note that, to our best knowledge, this is the
first proof-of-concept illustration that sensing of an analyte
in living cells can be controlled by light. For comparison,
when the cells were incubated with the traditional copper
probe Rh-Hy and Cu2+, the cells all essentially exhibited
bright red fluorescence (Figure 6h),[13] suggesting no spatial
control fluorescence sensing, which is consistent with the
fact that the fluorescence response of the conventional
probe Rh-Hy to Cu2+ is not amenable to light regulation.
Thus, the newly constructed probe bisNPE-Fl-Hy exhibited
an evident advantage over the traditional copper probe Rh-
Hy in that the copper imaging and sensing in the living cells
is photocontrollable.
probes/reagents reported elsewhere[10,14] is given in Table 1.
Most of the reagents exhibit a high selectivity and sensitivity
to Cu2+. The fluorescent probes exhibited either a fluores-
cence enhancement or quenching response to copper ions.
Furthermore, some of them have been employed for Cu2+
imaging in living cells.[14d–g] However, the fluorescence re-
sponse of the existing fluorescent probes is not photocon-
trollable. By contrast, the sensing ability of the new probe
bisNPE-Fl-Hy can be regulated by light. Thus, the imaging
and sensing of copper can be conducted in a spatiotemporal
manner by using the new probe, as illustrated in the above
studies.
Conclusion
We have described, for the first time, a new type of smart
fluorescent probe, termed photocontrollable analyte-respon-
sive fluorescent probes (PCAFPs). For proof of concept, a
molecular prototype of the caged copper-responsive fluores-
cent probe bisNPE-Fl-Hy was rationally constructed. The
bisNPE-Fl-Hy probe was essentially non-fluorescent and
inert to Cu2+-induced hydrolysis before light exposure.
Comparison with other Cu2+ fluorescent probes/reagents: A
brief comparison of some representative Cu2+ fluorescence
Table 1. Comparison of some Cu2+ fluorescent probes/reagents.
Reagents
Analytical ranges
(detection limit)
Selectivity
Comments
References
phen green FL/
no data available
(near 100 nm)
0.2–0.9 mm
modest
modest
good
MOPS buffer pH 7,
fluorescence quenching
MOPS buffer, pH 6.9,
fluorescence quenching.
HEPES buffer/CH3CN (8:2, v/v),
pH 7, fluorescence enhancement
[14a,b]
[14c]
[10]
phen green FL diacetate
europium tetracycline
hydrogen peroxide complex
Rh-Hy
A
AHCTUNGTRENNUNG
no data available
(0.5 mm)
HEPES buffer/DMSO (9:1, v/v),
pH 4–10, fluorescence quenching, living cell imaging
good
good
[14d]
[14e]
G
8.0ꢂ10À7–1.0ꢂ10À5
m
m
Tris-HCl buffered/C2H5OH (8:2, v/v)
fluorescence enhancement, living cell imaging
(3.0ꢂ10À7 m)
5.0ꢂ10À7–1.0ꢂ10À6
(37 nm)
phosphate buffered saline, pH 6.2–7.8,
fluorescence enhancement, living cell imaging
good
[14f]
HEPES buffer/DMSO (9:1, v/v), pH 7,
fluorescence quenching, living cell imaging
no data available
good
good
[14g]
bisNPE-Fl-Hy
2.5ꢂ10À7–1.0ꢂ10À5
m
HEPES buffer/CH3CN (8:2, v/v), pH 6–10,
fluorescence enhancement, living cell imaging
this work
(1.9ꢂ10À7 m)
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ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 689 – 696

Photocontrollable Analyte-Responsive Fluorescent Probes
FULL PAPER
ure S1 in the Supporting Information, in the absence of Cu2+, compound
Fl-Hy (5 mm) exhibited no observable changes of emission intensities at
516 nm, indicating that Fl-Hy was not converted to Fl when Cu2+ ions
were not present. In contrast, upon addition of Cu2+ at room tempera-
ture, a maximal fluorescence enhancement was noted after approximately
30 min. Thus, the assay time of 30 min was selected for the further exami-
nation of the sensitivity and selectivity of bisNPE-Fl-Hy toward Cu2+
after photolysis. For mass spectrometry analysis, the photolyzed samples
were analyzed using an LCQ Advantage ion trap mass spectrometer.
However, upon photolysis, the photolabile nitrobenzyl
groups were removed to release the active copper-respon-
sive fluorescent probe, which could sense copper in solutions
with a large (350-fold) fluorescence enhancement. In addi-
tion, we have demonstrated that the sensing of Cu2+ in
living cells could also be photoregulated with a spatial reso-
lution. Thus, the new, caged, bisNPE-Fl-Hy probe showed a
clear advantage over the traditional copper probe Rh-Hy in
that the copper sensing in living cells is light-controllable.
Although bisNPE-Fl-Hy, the proof-of-principle paradigm of
a PCAFP for Cu2+ illustrated herein, is regarded as a valu-
Cell culture and fluorescence imaging: HeLa cells were grown in MEM
(modified Eagleꢀs medium) supplemented with 10% FBS (fetal bovine
serum) in an atmosphere of 5% CO2 and 95% air at 378C. The cells
were plated on 6-well plates and allowed to adhere for 24 h. The cells
were washed with HEPES buffer immediately before the experiments,
and then incubated with bisNPE-Fl-Hy (15 mm) and Cu2+ (6 equiv) for
30 min at 378C in HEPES buffer containing 3% CH3CN as a co-solvent.
After washing three times with PBS, the selected cells were illuminated
for 30 s with UV light through a fluorescence microscope.[9a,d] Subse-
quently, the cells were incubated for a further 30 min at 378C, and the
fluorescence images were acquired through a Nikon Eclipse TE2000U in-
verted fluorescence microscope equipped with a cooled CCD camera.
The imaging of copper by Rh-Hy (15 mm) was conducted in a similar way,
but without exposure to UV light.
ACHTUNGTRENNUNGable step toward the ultimate goal to regulate sensing activi-
ty by light, we believe that the concept of PCAFPs repre-
sents a valuable breakthrough in the analyte-responsive flu-
orescent probe and photocaging fields, and that more so-
phisticated PCAFPs will no doubt become powerful chemi-
cal tools for exploring the spatiotemporal information of the
biochemistry of life. Furthermore, because of the great ad-
vance in the sensing chemistry of analyte-responsive fluores-
cent probes and the well-developed uncaging chemistry, the
general strategy of PCAFPs should be broadly applicable
for a wide variety of biologically relevant targets. Our future
efforts will focus on improving the sensitivity of the first-
generation copper PCAFP, and developing various types of
PCAFPs with diverse photolabile groups susceptible to vari-
ous uncaging conditions (i.e., two-photon photolysis) as new
chemical tools for biological exploration in a temporal and
spatial fashion.
Acknowledgements
Funding was partially provided by NSFC (20872032, 20972044), NCET
(08-0175), and the Key Project of the Chinese Ministry of Education
(No. 108167).
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Received: July 8, 2010
Published online: November 9, 2010
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Chem. Eur. J. 2011, 17, 689 – 696