Communications
DOI: 10.1002/anie.201000150
Caged Fluorescence
Rhodamines NN: A Novel Class of Caged Fluorescent Dyes**
Vladimir N. Belov,* Christian A. Wurm,* Vadim P. Boyarskiy, Stefan Jakobs, and Stefan W. Hell*
Caged (that is, masked) fluorescent dyes are maintained in
their nonfluorescent state by the incorporation of a photo-
chemical labile group. The photosensitive masking group or
“molecular cage” can be cleaved-off by irradiation with near-
UV light, thereby rendering the dye fluorescent.[1a–c] Caged
fluorescent dyes are of enormous interest for biological
imaging because they may be used, for example, for the
analysis of protein dynamics,[1d,e] multicolor fluorescence
microscopy,[1f] and far-field optical nanoscopy.[1g] o-Nitroben-
zyl groups are often used as masking groups;[1a–c] however, the
use of these dyes is limited because of their rather complex
synthesis and the unwanted by-products liberated by photol-
ysis.
scheme[3] with low cross-talk, despite using three fluorophores
with very similar absorption and emission spectra.[2d,4]
Rhodamines are very photostable and bright fluorescent
dyes which can readily be chemically modified[5] and caged.[6]
Coumarines[7] and fluorescein[8] have also been used as caged
fluorescent dyes.[9] As a photocleavable unit, most of these
caged compounds contain a 2-nitrobenzyl group or a deriv-
ative with an alkyl or a carboxy group in the a position to the
phenyl ring (at the CH2 group) and/or one or two methoxy
groups in the aromatic ring.[10] Compounds with a free
carboxy group are required for bioconjugation. However,
the synthesis of caged rhodamines with a free (“second”)
carboxy group is difficult and their yield is low.[6a]
Herein we report on the synthesis and characterization of
a novel class of caged compounds—rhodamine NN dyes,
which have a 2-diazoketone (COCNN) caging group incor-
porated into a spiro-9H-xanthene fragment (compounds 3
and 9-R in Schemes 1 and 3, respectively). This very simple
and small caging group is the core element of a new class of
masked rhodamines that have remarkable properties. The
rhodamine NN dyes can be easily prepared and conjugated
with biomolecules, they undergo rapid uncaging under
standard irradiation conditions (with wavelengths ꢀ 420 nm)
with formation of highly fluorescent rhodamine derivatives,
and they can be used in aqueous buffers, as well as in various
embedding media utilized in imaging applications.
In microscopy, these novel rhodamines may be used as
labels alone or in combination with conventional fluorescent
dyes and switchable rhodamine spiroamides.[2] In the latter
case, they enable new imaging protocols based on the
stepwise activation and detection of several fluorescent
markers. The combination of the new rhodamine NN deriv-
ative (9-R) with the photochromic spiroamide of rhodami-
ne S[2e] and a normal (uncaged) N,N,N’,N’-tetramethylrhod-
amine resulted in a monochoromatic multilabel imaging
The 2-nitrobenzyl group and its substitutes are bulky and
generate toxic, colored, and highly reactive 2-nitrosobenzal-
dehyde or 2-nitrosobenzophenone derivatives upon photol-
ysis. These compounds or their oligomers are expected to be
poisonous to living cells, and they are also colored and
interfere with optical measurements.
Other modern caging groups with the required absorption
in the near-UV region are also bulky, rather lipophilic, and
the procedures for their synthesis and introduction are often
complex. For example, 2-(N,N-dimethylamino)-5-nitrophenol
was reported to give photocleavable phenyl esters.[11]
7-Diethylamino-4-(hydroxymethyl)-2H-chromen-2-one
is
known to form esters which can be cleaved easily by
irradiation at 412 nm.[12] Derivatives of 8-bromo-7-hydroxy-
quinoline[13] and 6-bromo-7-hydroxycoumarines[14] have also
been proposed as light-sensitive protecting groups. The
photolysis of these caged compounds generates light-absorb-
ing by-products.
We set out to prepare masked fluorescent dyes without
bulky caging groups. A very small 2-diazoketone fragment
would be an ideal caging group, provided that it is still
possible to integrate this group into the colorless form of a
fluorescent dye and then restore the fluorescent state by
photolysis. Rhodamines are ideal for this purpose, because
they contain a carboxy group, which is known to form
colorless and nonfluorescent lactones or lactams with the
spiro-9H-xanthene fragment. Furthermore, this carboxy
group may be transformed into a 2-diazoketone residue.
For the practical realization of this caging strategy, we
used rhodamine B as a model compound and performed the
reaction of diazomethane with its acid chloride 1. The yellow
[*] Dr. V. N. Belov, Dr. C. A. Wurm, Dr. V. P. Boyarskiy, Dr. S. Jakobs,
Prof. Dr. S. W. Hell
Department of NanoBiophotonics
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gꢀttingen (Germany)
Fax: (+49)551-201-2506
E-mail: vbelov@gwdg.de
crystalline diazoketone
3 was obtained in high yield
(Scheme 1).[15] In the course of the facile caging reaction,
the positively charged C9 atom of the xanthene fragment
attacks the negatively charged carbon atom of the diazo-
methane residue in the intermediate 2. The simultaneous
abstraction of a proton furnishes the stable five-membered
ring.
[**] This work was supported by the Leibniz Prize fund of the DFG (to
SWH) and the Max-Planck-Gesellschaft. We are grateful to Donald
Ouw (MPI), Reinhard Machinek, Dr. Holm Frauendorf, and their co-
workers (Institut fꢁr Organische und Biomolekulare Chemie, Georg-
August-Universitꢂt Gꢀttingen) for recording spectra.
Supporting information for this article is available on the WWW
3520
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 3520 –3523