Coumarin-caged rosamine probes based on a unique intramolecular carbon-carbon Spirocyclization

Article abstract of DOI:10.1002/chem.201000015

(Figure Presented) Caged fluorophore: We report the first caging strategy for rosamine fluorophores based on a unique intramolecular carbon-carbon spirocyclization. The new class of carbon-carbon spirocyclic caged probes exhibit several sig-nificant advantages over the traditional nitrobenzyl-caged xanthene probes and can be employed for spatially controlled fluorescence imaging of living cells (see graphic).

Full text of DOI:10.1002/chem.201000015

DOI: 10.1002/chem.201000015
Coumarin-Caged Rosamine Probes Based on a Unique Intramolecular
Carbon–Carbon Spirocyclization
Weiying Lin,* Lingliang Long, Wen Tan, Bingbing Chen, and Lin Yuan[a]
To investigate the biochemistry of life, it is essential to
have suitable chemical tools that are able to provide spatio-
temporal information because the interactions of biomole-
cules in living systems are under precise temporal and spa-
tial control. Masking the key functional groups of biomole-
cules with photolabile protecting groups may afford caged
compounds, which are essentially biologically inert.[1] How-
ever, upon photolysis, the photolabile groups are deprotect-
ed to release the native, active biomolecules. Since light is
able to access/activate the desired excitation site at any
time, it is then possible to regulate these biomolecules with
high temporal and spatial resolution by using the photocag-
ing technology. Caged fluorophore probes are essentially
nonfluorescent and can be converted into highly fluorescent
species after photolysis.[2] This light-mediated fluorescence-
enhancement method, with the intrinsic advantage of high
spatiotemporal resolution, renders caged fluorophore probes
very useful for studying cell lineage,[3] cell–cell communica-
tion,[4] cellular protein fluorescence labeling,[2a] hydrodynam-
ic properties of the cytoplasmic matrix, and lateral diffusion
in membranes.[5] Thus, the development of new caged fluo-
rophore probes is very important for studies of molecular
and cellular dynamics.
the nitrobenzyl-caged xanthenes have several drawbacks.
First, the photolytic efficiency (the efficiency of photolysis is
defined by the product of the uncaging quantum yield and
the extinction coefficient)[9] of these nitrobenzyl-caged xan-
thenes are very low (typically less than 100).[10] This is highly
unfavorable for bio-applications because the intense light
that is employed to photolyze caged probes with low photo-
lytic efficiency may be detrimental to living species.[11]
Second, the photolabile nitrobenzyl groups have to be re-
moved by UV light, which can cause photodamage to cells,
such as changes in morphology and phenotypes, or can even
kill cells. Third, the parent dyes of caged fluoresceins suffer
from photobleaching.[2a,b] Thereby, these shortcomings have
apparently constrained the potential bio-applications of
these caged probes.
Rosamines are another key class of xanthenes with favor-
able photophysical properties. Structurally, in comparison to
rhodamines, rosamines lack a carboxylic acid functional
group.[12] Thus, the traditional rhodamine caging method is
not applicable for rosamines, since they can not lactonize to
turn off the fluorescence due to the lack of a carboxylic acid
group. To the best of our knowledge, rosamines have not
been successfully caged yet. Herein, we introduce the first
photocaging strategy for rosamines based on new photocag-
ing chemistry, that is, a unique intramolecular carbon–
carbon spirocyclization (Scheme 1c). For proof of concept,
rosamine (3) was caged with the photolabile coumarin-4-yl-
methyl moieties as the representative examples of the novel
class of photocaged rosamine probes. Compound 3 is highly
fluorescent with red emission at 577 nm. Our hypothesis was
that the hydroxyl chemical handle of 3 could allow the in-
corporation of the photolabile coumarins by alkylation, and
the 4-ylmethyl group of the coumarin intermediate could be
deprotonated under basic conditions and the resulting nucle-
ophilic carbon (C2’) could attack the electrophilic carbon
(C9) of the xanthene core to undergo an intramolecular
carbon–carbon spirocyclization. This should render caged
probes 1 and 2 nonfluorescent because the conjugation of
the xanthene ring is destroyed. Since coumarin-4-ylmethyl
derivatives are known to be efficiently photolyzed under
Xanthene fluorophores, such as fluoresceins, rhodamines,
and TokyoGreens have been caged. Typically, caged fluores-
cein (Scheme 1a) or caged rhodamine probes (Scheme 1b)
are prepared by incorporating nitrobenzyl groups on the hy-
droxyl or amino groups of the xanthene core to form decon-
jugated and nonfluorescent cyclic lactone structures.[2a,6,7]
Recently, in an elegant piece of work, Naganoꢀs group re-
ported nitrobenzyl-caged TokyoGreens by exploiting the
fluorescence quenching effect of nitro groups.[8] However,
[a] Prof. W. Lin, L. Long, W. Tan, B. Chen, L. Yuan
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering, Hunan University
Changsha, Hunan 410082 (P.R. China)
Fax : (+86)731-8882-1464
E-mail: weiyinglin@hnu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000015.
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Chem. Eur. J. 2010, 16, 3914 – 3917

COMMUNICATION
ring to the deconjugated form.
Although the fluorescence of
xanthenes can be switched off
by xanthene deconjugation
through the formation of the
deconjugated cyclic lactone or
lactam structure[2a,6,7,14] or the
zinc
coordinated
m-oxygen
structure,[15] these methods are
apparently not applicable for
photocaging rosamines.
In the absence of light, caged
probe 1 displayed only the char-
acteristic absorption of 7-N,N-
diethylaminocoumarin around
400 nm with a molar extinction
coefficient of 21300mÀ1 cmÀ1
under the neutral aqueous con-
ditions (25 mm sodium phos-
phate buffer, pH 7.0, containing
10% DMF as a cosolvent) (Fig-
ure S7 in the Supporting Infor-
mation). However, the typical
absorption of conjugated rosa-
mine dyes around 550 nm was
not observed. This is in good
agreement with the carbon–
carbon spirocyclic structure,
which obviously breaks the con-
jugation of the xanthene moiety
in the caged species. A similar
absorption profile was also no-
ticed for caged probe 2 (Fig-
ure S8 in the Supporting Infor-
mation). However, caged probe
2 has much lower absorption in
Scheme 1. a) Caging chemistry of fluoresceins; b) Caging chemistry of rhodamines (HMPA=hexamethylphos-
phoramide); c) New caging chemistry of rosamine: mechanistic design and synthesis of coumarin-caged rosa-
mine probes 1 and 2.
visible light,[13] we envisioned that the caged probes may be
uncaged to release the fluorescent conjugated xanthene ring
upon exposure to visible light.
The synthetic routes to the new coumarin-caged rosamine
derivatives 1 and 2 are outlined in Scheme 1c and Figure S1
in the Supporting Information. Alkylation of 3 with coumar-
ins 5 or 6 provided coumarin-caged rosamines 1 or 2, respec-
tively, in 48 or 55% yield. Reference coumarin aldehydes 7
and 8 (Figure S1 in the Supporting Information) were also
prepared by the standard chemistry.
the visible region than caged probe 1. Consequently, we de-
cided to focus on caged probe 1 for further spectroscopic
and photolysis studies. Upon visible light photolysis (the in-
expensive and simple-to-use 275 W tungsten lamp with a
visible light bandpass filter (390–600 nm) was chosen as the
visible light source[16]), the typical conjugated xanthene ab-
sorption at around 554 nm increased gradually in a light-ex-
posure and time-dependent manner indicating the formation
of a conjugated rosamine species.
Probe 1 showed no emission in the rosamine emission
region, consistent with the formation of the deconjugated
carbon–carbon spirocyclic structure. In contrast, upon expo-
sure to visible light, a dramatic fluorescence enhancement at
577 nm was observed (Figure 1), indicating the formation of
a conjugated rosamine species. Notably, visible light expo-
sure on caged probe 1 induced a large fluorescence en-
hancement (360-fold), which is much larger than that of a
nitrobenzyl caged rhodamine (<50-fold).[7b] Such a magni-
tude of fluorescence jump after a brief photolysis is desira-
ble for cellular fluorescence imaging applications.[8] In addi-
tion, the uncaging also resulted in light-exposure and time-
The structures of the novel coumarin-caged rosamine
1
probes 1 and 2 were fully characterized by H and 13C NMR
spectroscopy (Figures S2–S5 in the Supporting Information),
and HRMS. Furthermore, the X-ray crystallographic analy-
sis authenticated that, indeed there is a carbon–carbon spi-
rocyclic structure in both the caged probes 1 and 2 (Fig-
ure S6 in the Supporting Information). Thus, the novel
carbon–carbon spirocyclic structure in the caged probes 1
and 2 indicates that they represent a new class of xanthene-
based spirocyclic derivatives. Notably, the formation of the
carbon–carbon spirocyclic ring clearly confines the xanthene
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W. Lin et al.
hydrorosamine,[18] a probable mechanism of photorelease
was proposed (Figure S15 in the Supporting Information).
The uncaging quantum yield of probe 1 was calculated to
be 0.26 by means of HPLC analysis (see the Supporting In-
formation), and was comparable to other 4-methyl-7-N,N-di-
ethylaminocoumarin caged species.[13b] The photolytic effi-
ciency of caged probe 1 is 5538. In contrast, the photolytic
efficiencies of nitrobenzyl-caged fluoresceins are only
around 60.[10] Thus, the photolytic efficiency of caged probe
1 is about a hundred times larger than those of caged fluo-
resceins. We concluded that caged probe 1 is superior to
caged fluoresceins in terms of photolytic efficiency.
The fluorescence quantum yields of 3 and 7 were deter-
mined as 0.20 and 0.10, respectively (see the Supporting In-
formation). In addition, we also evaluated the photostability
of the photolyzed product 3, under visible light illumination.
The fluorescence spectra of 3 are practically the same after
15 min of illumination under the tungsten lamp (Figure S16
in the Supporting Information), suggesting that the photo-
lyzed product is highly resistant to photobleaching. This is in
contrast with the parent dyes of caged fluoresceins, which
are prone to photobleaching.[2a–b]
Figure 1. The emission spectra of probe 1 (0.6 mm in 25 mm sodium phos-
phate buffer, pH 7.0, containing 10% DMF) prior to exposure to visible
light, and after increasing duration of illumination with visible light.
A) Emission changes in the rosamine emission region with lex =554 nm
(ex=excitation); B) Emission changes in the coumarin emission region
with lex =396 nm. The duration of light exposure is in seconds.
Since the utility of caged dyes depends on their hydrolytic
stability at physiological pH in the absence of light, we ex-
amined the rosamine emission of caged probe 1 in phos-
phate-buffered saline (PBS) in the dark. After three weeks,
essentially no rosamine fluorescence emission was observed
(Figure S17 in the Supporting Information), indicating that
probe 1 was very stable in the absence of light. Thus, caged
probe 1 has a low fluorescence background in the rosamine
emission region due to the high stability of the carbon–
carbon spirocyclic structure at physiological pH in the dark.
To examine the photoactivation of caged probe 1 in cell-
based systems, Hela cells were incubated with caged probe 1
(1 mm) and the selected cells were photolyzed by visible light
through a fluorescence microscope.[2a,8] After photolysis for
10 s, the illuminated cells showed intense red emission (Fig-
ure 2B, for a color version, see Figure S19 in the Supporting
Information) and green emission (Figure 2C) in the rosa-
mine and coumarin emission regions, respectively. In con-
trast, no fluorescence was observed in the cells that were
not photolyzed. These data established that probe 1 is cell-
membrane permeable and can be employed for spatial con-
trol imaging in living cells.[19] The cells exhibited no appar-
ent cytotoxicity after photo-uncaging. For comparison, when
the cells were incubated with 3 (the parent dye of caged
probe 1), all the cells exhibited bright red fluorescence (Fig-
ure 2J), indicating no spatial-restricted imaging, since the
fluorescence of 3 is not amenable to light regulation. Thus,
probe 1 displayed an advantage over 3 in that the imaging
of probe 1 is light controllable.
dependent changes in the coumarin emission spectra: a
large fluorescence increase accompanied by a redshift from
450 to 490 nm. Interestingly, an isoemission point at 454 nm
was noted implying that only one new coumarin species was
formed upon visible light illumination.
To investigate the identity of the photolysis products, the
solutions of probe 1 before and after different durations of
visible light exposure were subjected to HPLC analysis (Fig-
ure S9 in the Supporting Information), which indicates that
caged probe 1 generated rosamine 3 and coumarin 7 after
visible light exposure. The character of the photolysis prod-
ucts was further substantiated by mass spectrometric analy-
sis. The expected molecular peaks at 415.2 [3]+ and 278.0
[7+CH3OH+H]+ were displayed in the mass spectrum of a
partially photolyzed solution of probe 1 (Figure S10 in the
Supporting Information). Furthermore, the emission and ex-
citation spectra of probe 1 after exhaustive photolysis are
identical to those of standard 3 and 7 when excited or moni-
tored at the appropriate wavelengths (Figures S11 and S12
in the Supporting Information). Thus, the HPLC, mass spec-
trometry, and emission and excitation spectroscopy studies
confirmed that the uncaging of caged probe 1 afforded 3
and 7 (Figure S13 in the Supporting Information). Interest-
ingly, upon exposure of caged probe 1 to visible light in the
presence of hydroquinone (a free-radical scavenger), almost
no fluorescence enhancement in both the rosamine and cou-
marin channels was observed (Figure S14 in the Supporting
Information). The preliminary finding that a free-radical
scavenger could completely inhibit the photolysis of caged 1
suggests that a radical mechanism is likely to be involved in
the photolysis. Based on this preliminary study and the pho-
tochemistry of coumarin-4-ylmethyl derivatives[1a,17] and di-
In conclusion, we have reported the first photocaging
strategy for rosamines based on novel photocaging chemis-
try, that is, an intramolecular carbon–carbon spirocycliza-
tion. The formation of the carbon–carbon spirocyclic struc-
ture in the caged probes 1 and 2 has been unambiguously
1
characterized by H and 13C NMR spectroscopy, HRMS, X-
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Coumarin-Caged Rosamine Probes
COMMUNICATION
Acknowledgements
Funding was partially provided by NSFC (20872032 and 20972044),
NCET (08-0175), and the Key Project of the Chinese Ministry of Educa-
tion (no.: 108167).
Keywords: cage compounds · dyes/pigments · fluorescent
probes · photoactivation · photolysis
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Supporting Information.
ray crystallographic analysis, and absorption and emission
spectra. We also demonstrated that upon local photoactiva-
tion by visible light, caged probe 1 could release 3 and 7 for
living cell fluorescence imaging in a light-dependent fashion.
In comparison with the nitrobenzyl caged xanthenes, our
newly constructed carbon–carbon spirocyclic probes clearly
exhibited several significant advantages: high photolytic effi-
ciency, photoactivation by visible light instead of UV light,
and high photostability of the parent dyes. Thus, these
highly favorable photochemical properties should render the
novel caged probes powerful chemical tools for studies of
the spatiotemporal dynamics of a wide variety of biological
processes. In addition, the general and novel rosamine pho-
tocaging strategy could be widely applicable for caging
other classes of the xanthene family that also lack a carbox-
ylic acid group with the various photolabile groups suscepti-
ble to diverse uncaging conditions.
Received: January 5, 2010
Published online: March 10, 2010
Chem. Eur. J. 2010, 16, 3914 – 3917
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