Masked Rhodamine Dyes of Five Principal Colors Revealed by Photolysis of a 2-Diazo-1-Indanone Caging Group: Synthesis, Photophysics, and Light Microscopy Applications

Article abstract of DOI:10.1002/chem.201403316

Caged rhodamine dyes (Rhodamines NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, ei

Full text of DOI:10.1002/chem.201403316

DOI: 10.1002/chem.201403316
Full Paper
&
Fluorescence
Masked Rhodamine Dyes of Five Principal Colors Revealed by
Photolysis of a 2-Diazo-1-Indanone Caging Group: Synthesis,
Photophysics, and Light Microscopy Applications
[
a]
[a]
[b]
[c]
Vladimir N. Belov,* Gyuzel Yu. Mitronova, Mariano L. Bossi,* Vadim P. Boyarskiy,
[a]
[a]
[a]
[a]
[a]
Elke Hebisch, Claudia Geisler, Kirill Kolmakov, Christian A. Wurm, Katrin I. Willig, and
[
a]
Stefan W. Hell*
Dedicated to Armin de Meijere on the occasion of his 75th birthday
Abstract: Caged rhodamine dyes (Rhodamines NN) of five
basic colors were synthesized and used as “hidden” markers
in subdiffractional and conventional light microscopy. These
masked fluorophores with a 2-diazo-1-indanone group can
be irreversibly photoactivated, either by irradiation with UV-
or violet light (one-photon process), or by exposure to in-
tense red light (l~750 nm; two-photon mode). All dyes pos-
sess a very small 2-diazoketone caging group incorporated
into the 2-diazo-1-indanone residue with a quaternary
carbon atom (C-3) and a spiro-9H-xanthene fragment. Initial-
ly they are non-colored (pale yellow), non-fluorescent, and
absorb at l=330–350 nm (molar extinction coefficient (e)
carboxylic and sulfonic acid groups a highly water-soluble
caged red-emitting dye with two sulfonic acid residues was
prepared. Rhodamines NN were decorated with amino-reac-
tive N-hydroxysuccinimidyl ester groups, applied in aqueous
buffers, easily conjugated with proteins, and readily photo-
activated (uncaged) with l=375–420 nm light or intense
red light (l=775 nm). Protein conjugates with optimal de-
grees of labeling (3–6) were prepared and uncaged with l=
405 nm light in aqueous buffer solutions (f=20–38%). The
photochemical cleavage of the masking group generates
only molecular nitrogen. Some 10–40% of the non-fluores-
cent (dark) byproducts are also formed. However, they have
low absorbance and do not quench the fluorescence of the
uncaged dyes. Photoactivation of the individual molecules
of Rhodamines NN (e.g., due to reversible or irreversible
transition to a “dark” non-emitting state or photobleaching)
provides multicolor images with subdiffractional optical res-
olution. The applicability of these novel caged fluorophores
in super-resolution optical microscopy is exemplified.
4
ꢁ1
ꢁ1
ꢀ
10 M cm ) with a band edge that extends to about l=
40 nm. The absorption and emission bands of the uncaged
derivatives are tunable over a wide range (l=511–633 and
25–653 nm, respectively). The unmasked dyes are highly
4
5
4
ꢁ1
ꢁ1
colored and fluorescent (e= 3–8ꢀ10 M cm and fluores-
cence quantum yields (f)=40–85% in the unbound state
and in methanol). By stepwise and orthogonal protection of
Introduction
[
a] Dr. V. N. Belov, Dr. G. Y. Mitronova, E. Hebisch, Dr. C. Geisler,
Dr. K. Kolmakov, Dr. C. A. Wurm, Dr. K. I. Willig, Prof. S. W. Hell
NanoBiophotonics Department
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
Fax: (+49)551-201-2505
Fluorescent dyes that can be converted into a non-fluorescent
state by incorporation of a photosensitive masking group rep-
resent unconventional and indispensable markers applicable in
chemistry, life- and materials sciences, as well as in optical mi-
croscopy. During the course of the masking procedure, the ini-
tial strong color of a fluorescent dye disappears and it be-
comes practically colorless or pale yellow. Irradiation with UV
light removes or transforms the molecular “cage” and creates
the colored, fluorescent form of a dye with the same (or slight-
ly modified) structure. Small photosensitive groups incorporat-
ed into the masked fluorescent dyes are advantageous be-
cause they enable production of compact, cell-permeable la-
beling reagents for biological imaging. In particular, there are
many promising, but yet unexplored, applications of caged flu-
orescent dyes in optical microscopy. They are expected to per-
form extremely well in super-resolution microscopy based on
photoswitching of single molecules, (co)localization of various
E-mail: vbelov@gwdg.de
shell@gwdg.de
[b] Prof. M. L. Bossi
Laboratorio de Nanoscopꢀas Fotonicas
INQUIMAE - DQIAyQF (FCEyN)
Universidad de Buenos Aires & Conicet
Buenos Aires (Argentina)
E-mail: mariano@qi.fcen.uba.ar
[
c] Prof. V. P. Boyarskiy
Chemistry Department
St. Petersburg State University,
Universitetskiy Pr. 26, Petrodvorets
St. Petersburg, 198504 (Russia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201403316.
Chem. Eur. J. 2014, 20, 1 – 13
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[
3a–c,f]
biological objects, tracking the dynamic processes in living
tives.
Other modern photocleavable groups that provide
[
1]
cells, and multicolor experiments. Recently, new types of
caged fluorescent dyes, Rhodamines NN and Carbopyro-
the required absorption in the near-UV region are also bulky,
rather lipophilic, and the procedures for their synthesis and in-
troduction are often difficult. For example, 2-(N,N-dimethylami-
[
2]
nines NN, have been reported. They bear a 2-diazoketone
COCNN) caging group attached to either a spiro-9H-xanthene
(
no)-5-nitrophenol was reported to give photoremovable
[
2a]
[4]
fragment in N,N,N’,N’-tetraalkylrhodamines or a 9,10-dihydro-
0,10-dimethylanthracene residue in carbopyronines with fully
phenyl esters.
7-Diethylamino-4-(hydroxymethyl)-2H-chro-
1
men-2-one is known to form esters which may easily be
[
2b]
[5]
alkylated nitrogen atoms. In both cases, the photosensitive
assembly contains a 2-diazo-1-indanone residue with a quater-
nary carbon atom (C-3) (Scheme 1). In this respect, the struc-
ture of these compounds represents the novel “caging” ap-
proach, which is quite dissimilar to the common strategies
cleaved by irradiation at l=412 nm. Other heterocycles, 8-
[6]
bromo-7-hydroxyquinolines and 6-bromo-7-hydroxycoumar-
[7]
ins, have been proposed as light-sensitive protecting groups.
The irradiation of the caged compounds containing these pho-
tosensitive groups also generates light-absorbing byproducts
and it is not yet clear if these groups may be widely applied
for masking of the fluorescent dyes. In contrast, the reduced
size of the 2-diazoketone caging group in Rhodamines NN and
Carbopyronines NN results in compact structures of the caged
dyes and their photoactivation conveniently generates a mole-
cule of innocuous dinitrogen as a leaving group or byproduct
(Scheme 1).
[
1a,3]
based on the use of bulky masking groups.
Most traditional
caged compounds contain a 2-nitrobenzyl residue as a photo-
cleavable unit (or its derivatives with an alkyl or carboxyl
group attached to the benzylic carbon atom and/or one or
[
1a,3a–f]
two methoxy groups in the aromatic ring).
The synthesis
of the masked rhodamines with substituted 2-nitrobenzyl
caging groups is rather complex, their structures are large and
the photolysis produces highly reactive, toxic, and colored 2-
nitrosobenzaldehyde or 2-nitrosobenzophenone deriva-
All conventional caging groups are directly attached to the
fluorogenic unit as a bulky residue. For example, 2-nitrobenzyl
groups participate in the formation of the two urethane moiet-
ies, which involves both nitrogen atoms in rhodamines, and
provide the presence of the colorless (closed) lactone
[
1a,3a–g]
form.
A similar approach was also used in the masking of
[8]
[9]
coumarins and fluorescein, which were also prepared as
[10]
non-fluorescent derivatives.
Urethane or acetyl caging
groups limit the structural variety of rhodamines, carbopyro-
[11]
nines, and carborhodols that can be masked because they
occupy one position at the nitrogen atom and preclude the
possibility to add an additional group to this atom of the fluo-
rogenic unit (to tune the photophysical properties of the dye).
It is well-known that the alkylation of the nitrogen atom in
xanthene dyes (and coumarins) provides relatively strong bath-
ochromic and bathofluoric shifts (+25 nm) and represents an
[12]
important tool in dye design. However, for the reason men-
tioned above, the former masking strategy was limited to
ꢁ
NH2 and N-monosubstituted rhodamines and carbopyro-
[13]
nines.
In the present report we show that the new approach to
caged rhodamines with a 2-diazoindanone unit enables mask-
ing of lipophilic N,N’-bis-(2,2,2-trifluoroethyl)rhodamines, hy-
drophilic rhodamines with sulfonic acid residues, as well as
red-emitting tetrafluorophenyl-substituted rhodamines. Rho-
damines NN may be decorated with an additional carboxyl
group and hydrophilic residues. The amino-reactive N-hydroxy-
succinimidyl (NHS) esters can be easily synthesized and conju-
gated with proteins. These masked fluorescent dyes can be
readily photoactivated using standard irradiation sources—
lasers or lamps (lꢂ440 nm, or under two-photon condi-
tions)—and form highly fluorescent derivatives of various
colors. They may be applied in aqueous buffers, as well as in
various embedding media used in optical microscopy and
nanoscopy, for obtaining images with subdiffractional optical
Scheme 1. General synthetic route to the masked (colorless) rhodamine de-
rivatives 2a-Y–2e-Y that contain a photosensitive spiro-diazoketone frag-
ment. Irradiation of compounds 2a-Y–2e-Y with UV or violet light affords
brightly fluorescent dyes of various colors. For the mechanism of the forma-
tion of the “bright” and “dark” products see reference [2].
[1b–k,3f]
resolution (e.g., by photoactivation of single molecules).
As examples, caged rhodamine dyes of five basic colors were
synthesized and utilized as fluorescent markers in subdiffrac-
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tional and conventional microscopy in a study on the scope
and limitations of an approach that utilizes 2-diazoketone as
a caging group. The whole spectral area, including the red
region, can be operated over because all principal colors are
available. The latter is especially important in biology and opti-
cal microscopy because red light is less invasive and does not
interfere with autofluorescence of cells and their components.
Importantly, all five dyes bear linkers for conjugation to pro-
teins. The selective and sequential orthogonal protection/de-
protection sequence applicable to carboxylic and sulfonic acid
groups has been developed as a challenging and useful tool in
dye design. As a result, the first example of a caged dye deco-
rated with two sulfonic acid groups is reported. An alternative
way to make a caged dye highly water soluble is to attach
a solubilizing moiety. In an illustrative example a dipeptide
with a sulfocysteine residue was introduced.
[9H]xanthen-3-one fragment (Scheme 1). Without additional
polar groups, their solubility in aqueous buffers is negligible.
All bioconjugates of the fluorescent markers are applied in
aqueous solutions, and the labeling reagent is dissolved in
a minimal amount of organic solvent miscible with water (e.g.,
DMF or DMSO). Rhodamines NN, designed as labeling reagents
for biomolecules, need polar groups to enhance their hydro-
philic properties to be able to achieve the recommended label-
ing efficiencies and minimize the change in the properties of
the formed bioadducts. In this respect, it is advantageous that
upon photoactivation the initial caged dyes turn into more hy-
drophilic zwitterionic structures, which are less prone to aggre-
gation or precipitation from the aqueous buffers. On the other
hand, the polar groups required to provide hydrophilic proper-
ties must be compatible (at least, in their protected form) with
activation agents (e.g., oxalyl chloride or POCl ) and other re-
3
Our synthesis is applicable only to N,N’-bis-(2,2,2-trifluoro-
ethyl)rhodamines or xanthenes with disubstituted nitrogen
atoms. In this respect it is complimentary to the synthetic
strategy mentioned above (based on the attachment of the
caging groups to the fluorogenic nitrogen atoms).
agents required during the course of the synthesis.
[2a]
Compounds 2a-CO Et, 2bc, 2c-CO Et,
2d-CO Et, 2d-
2
2
2
Et,CO Et, and 2e-SCH CO Et (Scheme 1) have an ethyl ester
2
2
2
group, which, after deprotection, liberates the free carboxylic
acid residue and can be used for bioconjugation. Ethyl esters
[2a]
2
a-CO Et and 2c-CO Et were saponified, transformed into
2 2
the corresponding NHS esters (see Scheme 3) and used in the
reaction with secondary antibodies without further structural
modifications (without additional polar groups). The model
compound 2b-H is very lipophilic, and the corresponding NHS
ester derived from it (by using rhodamine 1bb as a starting
material; Scheme 3) was expected to be insoluble in water.
Therefore, we prepared a more hydrophilic version of rho-
damine 1bb, compound 1bc. Conversion of dye 1bc into di-
azoketone 2bc then activation resulted in NHS ester 2bc-
CONHS, which was applicable for conjugation with proteins
under the standard conditions (see Scheme 3). Aside from the
straightforward addition of the methoxy groups, we also used
other ways of increasing the hydrophilic properties of Rho-
damines NN. For example, we synthesized compound 2d-
Results and Discussion
Synthetic strategies: scope and limitations
Before photoactivation, Rhodamines NN (2a-Y–2e-Y, Scheme 1)
are essentially non-fluorescent. Preparation of the chemically
stable non-fluorescent compounds 2a-Y–2e-Y is only possible
if the initial fluorescent dye can form a stable acid chloride
1
(or mixed anhydride), which reacts further with diazome-
[
2]
thane. This requirement poses some limitations on the substi-
tution pattern at the nitrogen atoms in the parent rhodamine,
from which the acid chloride or mixed anhydride has to be
prepared. The primary amino groups (e.g., in Rhodamine 110)
or secondary amino groups (e.g., in Rhodamine 6G) are unac-
ceptable, as they readily react with activating agents used in
the preparation of acyl chlorides 1-Cl, as well as with mixed an-
hydrides or active esters generated from the carboxyl group.
We established that oxalyl chloride, (chloromethylene)dimeth-
Et,CO Et with ethyl sulfonate and ethyl ester groups. Free sul-
2
fonic acid residues are very efficient solubilizing groups, but
they require adequate protection (e.g., as alkyl sulfonates)
before activation of the carboxyl group and its conversion into
the spiro-diazoketone cage (see Scheme 4). This strategy was
successful for the red-emitting dye 2db, but did not work in
the case of tetrafluororhodamine 2e-SCH CO Et (6’ isomer)
yliminium chloride, or POCl (in the case of the more robust
3
compounds) provide acceptable yields of the required spiro di-
azoketones in a subsequent reaction with diazomethane. The
latter should be introduced after complete transformation of
the free carboxyl group into the acid chloride and removal of
the activating agent. All attempts to activate the carboxyl
group by the formation of mixed anhydrides (ClCO R and Et N
2
2
[2b]
decorated with two sulfonic acid residues.
As an alternative, we tried to use the same fluorophore as in
compound 2e-SCH CO Et with two hydroxyl groups as polar
2
2
residues (Scheme 2). The building block 1 f-F,F,H is used in the
synthesis of several bright fluorescent dyes that emit red light
(l=650–750 nm) and is available in quantities of several-hun-
2
3
in THF) resulted in low conversions and low yields of spiro di-
azoketones. Under these conditions, the corresponding esters
[12m–n,14]
(with COOR groups) were found to be the major products. Pre-
dred milligrams.
Hydroxyl groups considerably increase
sumably, they were formed in the course of decarboxylation of
the intermediate mixed anhydrides.
the polarity of organic compounds. In particular, the caged
dye 2 f-H with two hydroxyl groups (Scheme 2) is much more
soluble in aqueous solutions (at pH>7) than the less polar an-
alogue 9-H (see Scheme 5) and the reversed-phase HPLC mobi-
lity of the carboxylic acid 2 f-H is much higher than that of 9-
H. First, tetrafluoride 1 f-F,F,H reacted with ethyl thioglycolate
to give an inseparable mixture of mono- and di-substituted
Another characteristic feature of Rhodamines NN is the in-
herent lipophilic character of their core and, as a result, their
poor solubility in aqueous solutions. These compounds pos-
sess a highly symmetric, rigid, and compact photosensitive
unit based on
a
2-diazo-2,3-dihydro-1H-indene-spiro[1,9’]
Chem. Eur. J. 2014, 20, 1 – 13
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These results forced us to look for another strategy to attach
the hydrophilic groups to spiro diazoketone 2e.
Preparation of Rhodamines NN: Masked dyes with a 2-
diazo-2,3-dihydro-1H-indene-spiro[1,9’][9H]xanthen-3-one
fragment
During the course of caging, rhodamines are transformed into
the corresponding acid chlorides, which further react with di-
[2a]
azomethane or trimethylsilyl diazomethane.
Rhodamines
that emit green or orange light (Rhodamine 110 or Rho-
damine 19) contain primary or secondary amino groups, which
are incompatible with all reagents required for the strong acti-
vation of the carboxylic acid residue (oxalyl or thionyl chlor-
ides, POCl , or (chloromethylene)dimethyliminium chloride).
3
However, if a 2,2,2-trifluoroethyl group replaces one hydrogen
atom in an amino group of any rhodamine dye (e.g., Rho-
damine 110), a derivative with the same absorption and emis-
Scheme 2. Synthesis of the caged red-emitting rhodamines 2 f-R with free
hydroxyl and carboxyl groups and their macrolactonization. a) HSCH CO Et,
MeCN or DMF, Et CO) O, pyridine, 08C–RT, overnight;
N, ꢁ12–188C; b) (CH
c) (COCl) , CH Cl , DMF, 08C–RT, 2–6 h; d) CH
8 h; e) 1m NaOH (aq), THF/H O, 08C– RT; f) N-hydroxysuccinimide, HATU,
N, DMF, RT; g) N-hydroxysuccinimide, CH Cl , N,N’-dicyclohexylcarbodii-
sion spectra is obtained, the carboxylic residue of which can
[12l,16]
2
2
be converted to an ester, or even an acid chloride.
Thus
3
3
2
[
a]
we used rhodamines 1a-H and 1a-CO Et as starting com-
2
2
2
2
2 2 2 3
N , Et O (Et N), 08C–RT, 11–
pounds to prepare masked yellow-green dyes, diazoketones
1
Et
2
3
2
2
2a-H and 2a-CO Et, respectively (Scheme 3). The ethyl ester
2
mide (DCC), RT. [a] The residual acidic impuriries react with diazomethane
and have to be removed completely. If so, the presence of triethyl amine is
not necessary (but it may be added to neutralize the acidic compounds).
group in compound 2a-CO Et can be saponified and used for
further transformations. The orange dyes with green fluores-
2
cence (1b-H and 1bc) were masked by a similar strategy
(
Scheme 3). Dye 1bc is slightly more hydrophilic than rho-
damine 1bb, and its derivatives with N-(2-methoxyethyl) resi-
products in ratio of approximately 2:1. Then, the hydroxyl
dues (2bc, 2bc-CO H, and 2bc-CONHS) were prepared rather
groups
in
compounds
1 f-SCH CO Et,F,H
and
1 f-
2
2
2
than the corresponding N-ethyl counterparts. Though the
SCH CO Et,SCH CO Et,H were protected by acetylation and the
2
2
2
2
mixture
of
dyes
1 f-SCH CO Et,F,Ac,
and
1 f-
2
2
SCH CO Et,SCH CO Et,Ac was subjected to the standard caging
2
2
2
2
procedure (Scheme 2). The free carboxyl group was converted
into an acid chloride and the latter reacted with diazomethane
to give the yellow spiro diazoketones 2 f-SCH CO Et,F and 2 f-
2
2
SCH CO Et,SCH CO Et. This is the decisive step of the whole se-
2
2
2
2
1
2
quence. The relatively non-polar esters 2 f-R ,R are easily sepa-
rated by column chromatography. Deacetylation and saponifi-
cation of 2 f-SCH CO Et,F afforded the required dihydroxyacid
2
2
2
f-H. However, the active NHS ester 2 f-NHS was unstable and
could not be isolated in a pure state. If the reaction of acid 2 f-
H with N-hydroxysucccinimide was performed in the presence
of (7-azabenzotriazol-1-yl)tetramethyluronium hexafluorophos-
phate (HATU) and base, the macrocyclic lactone 2 f was
formed. This interesting result proved unequivocally (and for
the first time) the position of the sulfur atom (C-6’) in all
monosubstituted derivatives 2e (Scheme 1), and 1 f-
SCH CO Et,F,H and 2 f (Scheme 2), as well as in other commer-
2
2
[15]
cial fluorescent dyes with trichloro- or difluoro-phenyl rings.
Scheme 3. Synthesis of the caged green- and orange-fluorescent Rho-
For steric reasons, the macrolactonization is only possible if
the fluorine atom at the opposite position to the carbonyl
group is substituted. If the reaction of N-hydroxysuccinimide
with acid 2 f-H was performed in the absence of base the
active ester 2 f-NHS was formed. Unfortunately, after isolation
by column chromatography or preparative HPLC, 2 f-H decom-
posed upon concentration or lyophilization of the fractions.
damines NN. Conversion of the yellow (1a-H/CO
2
Et) and orange (1b-H and
1
bc) rhodamines into diazoketones 2a-H and 2b-H (model compounds),
ethyl esters 2a-CO
2
Et and 2bc, carboxylic acids 2a-CO
2
H and 2bc-CO
in 1,2-dichloro-
, DMF, 08C–RT, 2–4 h; b) CH in Et
or MeCN, 08C–RT, 1.5–18 h; c) 1m NaOH (aq), EtOH or THF,
RT, 16 h; d) TSTU or, alternatively, N-hydroxysuccinimide with HATU, Et N,
MeCN or CH Cl , RT. [a] See note to Scheme 2.
2
H,
and the corresponding amino-reactive NHS esters. a) POCl
ethane, 708C, 2 h or (COCl) in CH Cl
3
2
2
2
2
N
2
2
O
[
a]
(
3 2 2
Et N), CH Cl
3
2
2
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masked compound 2bc-CONHS does not have highly polar
groups, it was successfully used for labeling of bovine serum
albumin (BSA) and antibodies under standard conditions: 2bc-
CONHS (0.2 mg) in DMF (40 mL) was added to an aqueous solu-
tion of protein (1–2 mg in 1–2 mL of an aqueous buffer with
pH ca. 8.5; see Supporting Information for details). However, it
was very difficult to couple the relatively lipophilic amino-reac-
tive compounds 2bc-CONHS or 2c-CONHS (prepared from the
teresting and promising candidates to select and test the pro-
tecting groups for the sulfonic acid sites. The appropriate pro-
tecting groups should be compatible with the conditions of
the diazoketone synthesis. Moreover, the deprotection of these
acidic centers should not affect the spiro-diazoketone group.
Another challenge was to provide additional protection for at
least one of the two different carboxyl groups in the presence
of the sulfonic acid residue(s) in such a way that these reaction
centers may be addressed independently and in a certain reac-
tion sequence. This issue is of general importance and there is
still no universal solution for this problem for many popular
[
2a]
masked 5’/6’-carboxy tetramethylrhodamines)
with very
polar 5-(3-aminoallyl)uridine-5’-O-triphosphate (AA-UTP). For an
acceptable conversion, this reaction required a great excess of
these markers, which were dissolved in DMF and added to an
aqueous solution of AA-UTP that contained DMF. However,
even under optimized conditions, the yield was low and the
product could only be isolated by preparative HPLC.
[17]
fluorescent dyes, for example, Alexa Fluor 594.
Compounds 7-H,SO H,Y are readily available by sulfonation
3
of the corresponding precursors 7-H,H,Y (Y=H, CO Et), which,
2
[18]
in turn, can be easily synthesized from phenol 5-H,TBDMS
(Scheme 4; TBDMS=tert-butyldimethylsilyl). The condensation
These results indicate that the masked red-emitting rho-
damines 2d-CO Et and 2e-SCH CO Et, which possess extended
[16]
reaction of 5-Me,H with 4-ethoxycarbonyl phthalic anhydride
2
2
2
and bulky fluorophores (and an ethyl ester group; see
Scheme 1), require hydrophilic groups to facilitate their use in
aqueous solution and in various bioconjugation procedures.
These groups may be attached in the course of post-synthetic
modifications of the masked dye core or they may be present
in the initial fluorescent dye before the caging procedure is ap-
plied.
(6-CO Et) was performed in boiling dichlorobenzene and af-
2
forded two diastereomers of rhodamine 7-H,H,CO Et with
2
intact ethyl ester groups. This result is very important because
the protection of one carboxyl group has been provided auto-
matically (by selection of the appropriate starting material).
The synthetic strategy was first elaborated for model com-
pound 7-H,SO H,H (without the second carboxyl group), but
3
In this respect, red-emitting and water-soluble rhodamines
the same approach is applicable to all sulfonated rhodamines
with sulfonic acid residues (7-H,SO H,Y, Scheme 4) represent in-
7-H,SO H,Y (Y=H, CO Et) shown in Scheme 4 and, for example,
3
3
2
Alexa Fluor 594 diastereomers (with 5’- and 6’-carboxyl
[17]
groups).
First, the sterically hindered free carboxyl group
CO R (R=H) in compound 7-R,X,Y was protected as an allyl
2
(
All) ester, then the sulfonic acid residues were alkylated with
Meerwein salt [triethyloxonium tetrafluoroborate (Et OBF )] in
3
4
the presence of a base. Subsequently, the allyl ester was
cleaved with Pd(PPh ) and HCOOH (Scheme 4). The crucial in-
3
4
termediate, 7-H,SO Et,H, was not particularly stable; upon
3
drying (and in the course of further transformations) it partially
rearranged into the corresponding ethyl carboxylate with one
SO Et and one SO H group. However, 7-H,SO Et,H was success-
3
3
3
fully used for the synthesis of 2-diazoketone 2d-Et,H (by the
standard protocol with oxalyl chloride and diazomethane). The
same methodology was used for 7-H,SO Et,CO Et, with an ad-
3
2
ditional ethyl carboxylate group. As we established, the migra-
tion of an alkyl residue from the sulfonic acid to the carboxylic
acid site took place upon concentration of the solutions and
drying. Therefore, to prevent this, we added small amounts of
chlorobenzene to solutions of compounds 7-H,SO Et,H or 7-
3
H,SO Et,CO Et and did not evaporate them to dryness. Rho-
3
2
damine 7-H,SO Et,CO Et was successfully used for the prepara-
3
2
tion of diazoketone 2d-Et,CO Et. The corresponding methyl
2
sulfonates 2d-Me,Y (Y=H, CO Et) were readily obtained when
2
Scheme 4. Synthesis of caged hydrophilic rhodamines 2d decorated with
more reactive Me OBF was used. However, the intermediates
3
4
sulfonic and (activated) carboxylic acid residues. a) MeI, Cs
overnight; b) Bu NF·3H O, THF, 0–48C, overnight; c) o-dichlorobenzene, 170–
908C, 8 h; d) concentrated H SO , RT; e) CH =CHCH OH, DCC, 4-dimethyl-
aminopyridine (DMAP), CH Cl , RT; f) Et OBF , iPr NEt, MeCN, RT; g) Pd(Ph P)
HCOOH, iPr NEt, MeCN, RT; h) (COCl) , CH Cl , 08C–RT, 2–4 h; i) CH , Et
N), 08C–RT, 11–18 h; j) 1m NaOH (aq), THF, 50–608C, 8–16 h; k) 0.17m
NaOH in MeOH/H O, 508C, 7–8 h; l) N-methylimidazole, EtOH, 708C, 16 h;
2 3
CO , DMF, RT,
7
-H,SO Me,Y (not shown in Scheme 4) were found to be even
3
4
2
less stable than the corresponding ethyl sulfonates 7-H,SO Et,Y
1
2
4
2
2
3
2
2
3
4
2
3
4
,
because the methyl groups migrated to the neighboring,
anionic carboxylic acid groups more easily than ethyl groups.
In alkaline solution, methyl sulfonates are more susceptible
to hydrolysis than ethyl sulfonates. However, both are much
more resistant to sodium hydroxide than ethyl carboxylates
2
2
2
2
2
N
2
2
O
[
a]
(Et
3
2
3
m) 0.1m NaOH (aq), 08C–RT, 2 h; n) N-hydroxysuccinimide, HATU, Et N, DMF,
RT. [a] See note to Scheme 2.
Chem. Eur. J. 2014, 20, 1 – 13
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(due to different mechanisms of the cleavage reactions: SN2
for alkyl sulfonates and nucleophilic addition for methyl or
ethyl esters). The alkyl sulfonate groups in compounds 2d-Et,H
and 2d-Me,CO Et may be deprotected by gentle heating in
2
dilute aqueous NaOH solution without affecting the spiro-di-
azoketone group. Ethyl sulfonate 2d-Et,CO Et was the most re-
2
liable and stable intermediate for the successful completion of
the whole reaction sequence. However, it could only be
cleaved without decomposition of the diazoketone residue by
heating with N-ethyl imidazole (a strong nucleophile). Saponifi-
cation of the ethyl ester at the final step afforded the water-
soluble salt 2d-Na,CO Na, which could be converted into the
2
very hydrophilic NHS ester 2d-Na,CONHS (Scheme 4).
Finally, we prepared masked red-emitting rhodamine 2e-F
as a model compound and used its derivative 2e-SCH CO Et
2
2
for the synthesis of hydrophilic analogues (Scheme 5). These
caged rhodamines are non-polar, lipophilic compounds, but
they contain the same fluorophore as the bright, photostable,
hydrophilic fluorescent dye KK114 with two sulfonic acid resi-
dues in allylic positions (in this respect, comparable to com-
Scheme 5. Synthesis of the caged red-emitting rhodamines 2e-F and 2e-
[
12m–o]
pounds 2d in Scheme 4).
Dye KK114 could not be caged
SCH
amino-reactive group. a) HSCH
CH Cl , DMF, 08C–RT, 2–6 h; c) CH
NaOH (aq), THF/H O, 08C–RT; e) N-hydroxysuccinimide, HATU, Et
f) Et N, DMF, RT. [a] See note to Scheme 2.
2
CO
2
Et, and their hydrophilic derivatives (10-R) decorated with an
CO Et, MeCN, Et
N, ꢁ12–188C; b) (COCl)
N), 08C–RT, 11–18 h; d) 1m
N, DMF, RT;
2
2
3
2
,
by conversion into the corresponding spiro diazoketone with-
[
a]
2
2
2 2 2 3
N , Et O (Et
[
2b]
out modification of the structure. Two polar groups are es-
sential to provide the required hydrophilic properties. We in-
troduced the second carboxyl group, replaced the sulfonic
acid residues in KK114 with hydroxyl groups, and obtained the
required diazoketone (2 f-H, Scheme 2), but, at the very end,
we failed to obtain the stable N-hydroxysuccinimidyl ester (2 f-
NHS). As we established, the primary (allylic) hydroxyl groups
are incompatible with the presence of an activated carboxyl
group in close proximity to one of them (Scheme 2). Therefore,
we used another approach based on transformations of ethyl
ester 2e-SCH CO Et (Scheme 5). The conditions for aromatic
2
3
3
Figure S1 in the Supporting Information). Spectra recorded in
solution in methanol or water contain a long-wavelength band
with a maximum or shoulder at about l=330–350 nm (ab-
3
ꢁ1
ꢁ1
sorption coefficient (e)=4–15ꢀ10 m cm ). Although the ab-
sorption intensities in the near-UV region are relatively low, the
band edge extends beyond l=400 nm, up to about l=
450 nm. As a result, uncaging can be performed, not only with
UV light (for example, with a l=375 nm laser), but also with
visible light; in fact, the masked dyes are even sensitive to day-
light. The photolysis of these model masked rhodamines in
methanol is exemplified in Figure 1. Dyes of various colors
(yellow, orange, red, magenta, and blue) with intense fluores-
cence, clear to the naked eye, were readily generated by irradi-
ation of colorless solutions (dye concentrationꢀ10 mm). To
evaluate the uncaging quantum yields (F) of the reactions
2
2
nucleophilic substitution of one fluorine atom in rhodamine 8-
F have been optimized previously (Scheme 2). The selectivity
was slightly better for compound 8-F (Scheme 5) than for rho-
damine 1-F,F,H (Scheme 2), and the product of disubstitution
was formed to a lesser extent. Further transformations were
similar to those shown in Scheme 2. The active ester 9-NHS
(Scheme 5) was obtained and smoothly reacted with the “uni-
versal hydrophylizer” 11, a very hydrophilic dipeptide prepared
[
19]
from sulfocysteine and b-alanine amino acids. Importantly,
the amidation reaction between 9-NHS and 11 proceeded
leading to the fluorescent (F
) and “dark” products
CF!FP
(F ), respectively, we irradiated solutions (c=40–50 mm) of
CF!DP
smoothly in DMF in the presence of Et N, and no protection of
the model caged dyes (2a-H, 2b-H, 2c-CO Et, 2d-CO Et, and
3
2 2
the anionic centers in 11 was required to form acid 10-H,
which represented the caged red-emitting rhodamine decorat-
ed with sulfonic and carboxylic acid residues. The selective ac-
tivation of the carboxylic acid site in the presence of sulfonic
acid residue in compound 10-H was achieved under standard
conditions (see Scheme 5) to afford the amino-reactive caged
dye 10-NHS.
2e-F) in methanol with a l=405 nm monochromatic light
source of ꢀ20 mW (a continious wave (CW) diode laser).
During the course of the irradiation, we recorded the UV/Vis
spectra (Figure 2), calculated the amount of fluorescent prod-
uct (FP) formed and, simultaneously, performed HPLC with UV
detection to evaluate the amount of the photolyzed caged
compound. The amount of FP formed was calculated from the
absorption in the main band in the visible region, neglecting
the contribution from the dark product (DP) because the ab-
sorption coefficients of DPs are at least ten-times lower than
the absorption coefficients of FPs (and the amount of DP
formed is lower). The quantity of DP was calculated from the
difference between the photolyzed Rhodamine NN (HPLC) and
Photophysical properties of Rhodamines NN
In the solid state and in relatively concentrated solutions, all
pure Rhodamines NN are pale yellow. The absorption spectra
of all five dyes in the caged form show similar features (see
&
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2 2
Figure 1. Irradiation of stirred solutions of Rhodamines NN in ethanol (from left to right in each picture: 2a-H, 2b-H, 2c-CO Et, 2d-CO Et, and 2e-F) with UV
light from a middle-pressure mercury lamp (150 W) equipped with Pyrex filter (l>330 nm) provides highly fluorescent dyes of five principal colors. a) Starting
solutions are colorless or light yellow; b) the lamp is ON; c) a daylight picture taken after complete photolysis (several minutes) demonstrates the various
colors of the solutions; d) a picture of the same solutions in c) taken with a torch light demonstrates the various emission colors.
2
Figure 2. a)–c) Irradiation of the model compound 2c-CO Et in methanol and d)–f) conjugate of 2c-CONHS (Abberior Cage 552) with BSA in PBS at pH 7.4.
a),d) Absorption at the maximum of the FP (see Table 1) as a function of time; b),e) a linear fit; c),f) emission spectra. The absorption and emission spectra
were recorded after the irradiation times indicated (1–5 min), with the exception of the spectrum represented by the broken line in d), which was recorded
after complete photolysis.
the quantity of the generated FP, assuming that no other by-
products were produced during the course of the photoly-
vation and minimal photobleaching of the activated dye. The
FP, that is the unmasked fluorescent dyes, demonstrate Stokes
shifts of approximately 20 nm (typical for rhodamines) and
a small redshift (5–10 nm) of the absorption and emission
bands relative to the parent free rhodamine (starting dye), due
to conversion of the free carboxyl group COOH to the homo-
[
2b,20]
sis.
Uncaging quantum yields were calculated at lower
conversions (below 10%), for which a linear regime was ob-
served (Figure 2b and c; see also Figures S3 and S4 in the Sup-
porting Information). The photoisomerization of azobenzene in
solution in methanol was used as a reference actinometric
system to determine the exact irradiation intensity under the
logue CH COOH group or the corresponding ester. The absorp-
2
tion coefficients at the peak of the band of the FP are at least
ten-times higher than those of the DPs at the same wave-
lengths. The grey and non-fluorescent compounds 4a-Y–4e-Y
[
21]
experimental conditions. The main photophysical properties
of Rhodamines NN are listed in Table 1.
ꢁ
1
ꢁ1
All compounds demonstrated moderate uncaging quantum
(Scheme 1) have a low absorption (e<3000m cm ) at wave-
[
22]
yields (F=0.15–1%). In fact, moderate or even low uncag-
ing quantum yields may be advantageous because they allow
the markers and their conjugates to be handled under weak
light rather than in complete darkness. The power of UV lasers
may be easily varied and adjusted to provide quick photoacti-
lengths that correspond to the absorption maxima of the pho-
ꢁ
1
ꢁ1
togenerated fluorescent dyes 3a-Y–4e-Y (e=50000m cm ;
see Table 1 and Figure S2 in the Supporting Information). In
addition, the quantum yield for the photogeneration of the DP
(F=0.05–0.3%) is 3–12 times smaller than the quantum yield
Chem. Eur. J. 2014, 20, 1 – 13
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Table 1. Photophysical properties of the model Rhodamines NN in methanol, including absorption and emission maxima for initial caged dyes (CF), fluo-
rescent products of their photolysis (FP), fluorescence quantum yields of the latter (ffl), uncaging quantum yields of the photoreactions leading to FP
(FCF!FP) and DP (FCF!DP).
[
a]
[a]
Closed form (CF)
Fluorescent product (FP)
Dark product (DP)
3
3
[b]
fl
3
Caged
dye
Abs max
[nm]
e/10
[m cm
Abs max
[nm]
e/10
Em
f
F
CF!FP
Abs max
[nm]
e/10
[m cm
F
CF!DP
F
F
CF!FP
/
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
[f]
ꢁ1
ꢁ1
[f]
]
[m cm
]
max [nm]
ꢀ100
]
ꢀ100
CF!DP
[
[
[
[
[
c]
c]
d]
d]
e]
[g]
[g]
2
2
2
2
2
a-H
b-H
c-CO
d-CO
e-F
230, 299, 340
237, 302, 350
231, 308, 350
261, 296, 329
272, 300, 348
74, 17, 5.1
62, 16, 3.8
74, 18, 4.0
83, 17, 15
61, 16, 9.5
511
533
559
597
633
50
82
43
62
31
525
541
574
609
653
0.85
0.84
0.40
0.87
0.54
0.97
0.77
0.85
0.49
0.15
n.d.
n.d.
0.27
0.06
0.11
0.15
0.12
4
12
8
3
1.3
365, 405, 551
14, 12, 4.5
[g]
[g]
2
Et
Et
n.d.
n.d.
2
315, 476, 609
315, 498, 636
25, 8.1, 5.2
7.3, 2.4, 2.0
[
a] Obtained upon photolysis with l>330 nm in methanol (lifetimes of the excited states vary in the range of 3.3–4 ns). [b] ffl measured in the reaction
[23c] [23d] [12n]
mixtures after photolysis. Refererence values: [c] Atto 532 (f=0.9);
[d] Rhodamine 101 (f=1);
[e] KK114 (f=0.53).
[f] Calculated from the optical
density of the main absorption band and HPLC analysis in irradiation experiments at l=405 nm. [g] The DP was not isolated in sufficient amount.
for the generation of the FP (with the exception of 2e-F, for
which both products are generated with similar efficiency).
Therefore, the color of the dye solutions obtained upon irradia-
tion in methanol or water always looked “clean” (from yellow
to dark blue, depending on the dye). The presence of the DP
in irradiated solutions was observed as a weak band or
a shoulder, appearing at longer wavelengths than the main ab-
sorption band of the FPs (Figure 2b and e; see also Figures S3
and S4 in the Supporting Information). Even though the pho-
tolysis of the caged dyes in solution always leads to a mixture
of FP and DP, the caged fluorescent dyes proved to be valua-
ble markers for biomolecules, such as antibodies in the immu-
nofluorescence method. To investigate the possibility to obtain
conjugates with proteins, evaluate the uncaging parameters,
and the effect of aqueous solutions on the binding and pho-
tolysis of the marker, we first prepared conjugates of NHS
esters 2a-CONHS, 2bc-CONHS, 2c-CONHS (Scheme 3), 2d-Na-
CONHS (Scheme 4), and 10-NHS (Scheme 5) with BSA as
a model protein. The conjugates were prepared by standard la-
405 nm light under conditions similar to those used for the
model caged dyes in methanol (Figure 2). Photolysis of the
BSA conjugates under biologically relevant conditions (in aque-
ous PBS buffer, pH 7.4) also produced clean colors, and the
dark tones were not visible. Upon photoactivation, the fluores-
cence quantum yields of the newly formed dyes were good
(f=0.2–0.4). The uncaging quantum yields for the FPs were
also calculated from absorption measurements (F
=0.1–
CF!FP
0.8; Table 2). Importantly, these values comprise at least 60%
of uncaging quantum yields found for the free model Rho-
damines NN dissolved in methanol. In these cases, the quan-
tum yields for conversion to the DP were not calculated be-
cause HPLC measurements were not possible. Solutions of BSA
conjugates in PBS were also irradiated until complete photoac-
tivation occurred (spectra are shown in Figure 2d and Fig-
ure S4 in the Supporting Information) and the amount of FP
formed was determined (% FP formed, Table 2). These values
may be interpreted as an additional factor to evaluate the ef-
fective DOL (useful labeling efficiencies). A particular case was
observed for the conjugate with compound 2bc (Abberior
Cage 532), for which the amount of FP formed seemed too
small. Considering the absorption of the solution at l=
600 nm (e !F ), we estimate that 11% of the initial cage
[
23a,b]
beling protocols.
Briefly, NHS ester (0.1 mg) was dissolved
in DMF (50 mL) [compound 2d-Na,CONHS was dissolved in
phosphate buffered saline (PBS)], then added to a stirred solu-
tion of BSA (1 mg) in PBS (0.5 mL), which was buffered at pH
FP
DP
ꢀ
8.5. After stirring in the dark at room temperature for 1 h,
compound was converted to the DP, a value in agreement
with that observed for the free dye (see Table 1) if other minor
photolysis products are neglected. This discrepancy may be ex-
plained by photobleaching or aggregation of the uncaged dye
residues attached to BSA. Indeed, the absorption spectra at full
conversion show that the shoulder at short wavelengths prac-
tically disappeared (compare with the spectra at low conver-
sions). This phenomenon indicated the full conversion of the
initial caged compounds, which is in line with the previous
speculation. The amount of DP formed at complete photolysis
for compounds 2d-BSA and 10-BSA is in agreement with the
calculated quantity of the FP, within a reasonable error (S=
100%, within 10% error). The absorption spectrum of 2a-BSA
at full photoactivation also differs from the absorption spectra
obtained at low conversions, despite the fact that the degree
of conversion to the FP is similar to the value observed for the
free dye. Nevertheless, these two compounds displayed higher
labeling efficiencies and in practical applications it may be
the protein fraction was separated from the unreacted marker
by size-exclusion chromatography on a Sephadex G-25 column
(
(
PD-10 desalting column, GE Healthcare). Labeling efficiencies
degrees of labeling (DOL)=average number of markers at-
tached to one molecule of BSA with M=66.5 kDa) were deter-
mined by absorption measurements, assuming e=
ꢁ
1
ꢁ1
43800m cm at l=280 nm for BSA.
The data are given in Table 2. All obtained DOL values are in
the range of 3–6, as expected from the protocol used, which
demonstrates that the amino-reactive derivatives of our caged
fluorescent dyes can be used in the same way as common
commercially available markers. The variation in the values ob-
tained do not seem related to the hydrophilicity of the marker,
and may reflect either the amount of the reacted dye or the in-
accuracy due to overlapping of the BSA and marker bands
(Table 1). Dilute solutions of the conjugates (BSA concentration
ꢀ
3 mm) in PBS at pH 7.4 were subjected to irradiation with l=
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depleted by shelving them to a dark (triplet) state
Table 2. Degrees of labelling (DOL), fluorescence quantum yields (ffl) and uncaging
efficiencies (FCF!FP) of conjugates obtained from NHS esters 2a-CONHS, 2bc-CONHS,
(
GSDIM mode). After that, individual molecules return
2
c-CONHS (Scheme 3), 2d-Na,CONHS (Scheme 4), and 10-NHS (Scheme 5) and BSA in
to the ground state (spontaneously or by triggering
with UV light). These single markers may be localized
by using a charge coupled device (CCD) camera and
collecting the photons emitted by each of them. All
these events occur within micro- or milli-seconds and
can be repeated at different time points during the
course of an experiment until the pool of masked
dyes becomes empty. This algorithm allows the grad-
ual acquisition of a position histogram with a high
spatial resolution (several tens of nm).
PBS solutions. Values for the free dyes were used to calculate ratios ffl/fflfree and
F
CF!FP/FCF!FPfree
.
[
a]
[b]
fl
[c]
Dye–BSA
DOL
f
ffl/fflfree
F
CF!FP ꢀ100
F
F
CF!FP
CF!FPfree
/
FP formed
[%]
[
d]
2
2
2
2
1
a
bc
c
d-Na
0
6.6
4.8
3.4
3.0
2.9
0.24
0.38
0.20
0.36
0.20
0.29
0.45
0.50
0.42
0.37
0.54
0.45
0.77
0.39
0.09
0.56
0.59
0.91
0.81
0.59
60
22 (89)
87
77
65
[
e]
[a] DOL=average number of markers attached to one protein molecule. [b] fluores-
Alternatively, it is possible to employ not only the
transition between singlet and triplet states, but also
to directly use the uncaging reaction to switch be-
tween dark and fluorescent states. Irreversible switch-
ing may be advantageous for the following reason:
in this (ideal) case, each marker contributes to the
final high-resolution image only once and is then
bleached (under the assumption that the population
of long-living “dark” triplet states from which the
marker can come back into the “fluorescence-giving”
singlet state is negligible). Therefore, the number of “signal-
giving” (active) markers is proportional to the number of de-
tected fluorescence bursts. This represents a tool for indirect
estimation of the number of labeled objects or their density in
clusters. Further illustrations to exemplify the applicability of
Rhodamines NN in imaging methods based on single-mole-
cence quantum yields (ffl) measured in the reaction mixtures after photolysis, neglect-
ing the absorption of the DP (for reference compounds see Table 1). [c] calculated
from absorption measurements in irradiation experiments with l=405 nm light, as-
suming that the absorption coefficient of the FP in the conjugate is the same as that
of the free dye in methanol. [d] amount of FP formed in fully irradiated solutions
(
t
IRR!1) calculated from absorption experiments. [e] difference between the amount
of starting dye and the quantity of DP formed, estimated from the absorption at l=
00 nm, at which the FP does not absorb (see Figure S4 in the Supporting Informa-
6
tion).
better to maintain the values between 3 and 4 (and carefully
control the irradiation intensity to reduce bleaching of the un-
caged dyes).
Light microscopy applications of caged dyes as photo-
activatable fluorescent labels
[25]
cule-switching methodologies have been reported.
Photoactivation (switching on) of the single molecules of any
masked fluorescent dye followed by off-switching (e.g., due to
reversible or irreversible transition to a “dark” non-emitting
state or photobleaching) allows acquisition of images with
This approach may be applied to several Rhodamines NN at-
tached to various biological targets and integrated into the
same image. Thus, multicolor optical nanoscopy methods may
be implemented.
[
1]
subdiffractional optical resolution. Caged dyes with a photo-
sensitive 2-diazo-1-indanone group can be readily activated
with UV light (e.g., l=375 or 405 nm lasers) or with very pow-
erful red light (in two-photon mode, e.g., under stimulated
emission depletion (STED) conditions; see below). In the
single-molecule-switching approach, we used antibody conju-
gates prepared from the NHS esters of 2a, 2bc, 2c, 2d-Na,
Additionally, the photoactivation (uncaging) of Rho-
[2b]
damines NN or structurally related caged carbopyronines
may be combined with STED microscopy. After photoactiva-
tion, caged rhodamines 2d-Na and 10, as well as caged carbo-
[2b]
pyronines emit red light and their fluorescent state may be
depleted with standard lasers operating at l=750–780 nm.
Therefore, red-emitting caged dyes are of special importance.
Their emission does not interfere with autofluorescence of
[
2b]
and from 10-CONHS and caged carbopyronines. For exam-
ple, the masked markers of compound 2a were uncaged sto-
chastically in time and space with l=375 nm light in such
a way that only one marker stayed unmasked per camera ex-
posure time in a diffraction-limited volume. The cycle of excita-
tion at l=532 nm, detection of the emitted fluorescence, and
bleaching of the uncaged markers was repeated 20000 times.
Localization of the markers yielded the super-resolved position
histogram with 35 nm FWHM (full width at half maximum)
average localization precision (Figure 3).
The caged dyes (and Rhodamines NN, in particular) offer the
advantage of a choice between two modes of single-marker
switching. They can be imaged in ground state depletion with
[
24]
Figure 3. a) A conventional image of the cellular microtubule cytoskeleton
labeled with compound 2a-CONHS (background-corrected sum of all
frames) and b) its sub-diffraction representation obtained by uncaging of
the single molecules with l=375 nm laser light and subsequent localization.
See text for details.
intramolecular return (GSDIM) mode, that is, a sufficient frac-
tion of all masked molecules is uncaged at first. These may be
used for taking a conventional wide-field reference picture.
Then, the singlet state of the already uncaged dye molecules is
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cells, and their excited state may be depleted with the less-in-
vasive red STED beam. Caged carbopyronines are structurally
similar to Rhodamines NN: the oxygen atom in the xanthene
core is replaced by a tertiary carbon atom (see Figure 4a for
structures and reference [2b] for the preparation and proper-
ties of caged carbopyronines). Here, we describe the results
obtained with caged carbopyronine KK1012 (Figure 4a), the
best-performing dye under standard STED conditions. In partic-
ular, we explored two modes of its uncaging (photoactivation).
The custom-built setup for STED microscopy utilized an exci-
tation wavelength of l=638 nm, STED wavelength of l=
duced by LEICA Microsystems and Abberior Instruments. Both
photoactivation modes lead to images with subdiffractional
optical resolution of about 50 nm. This value is five-times
better than the diffraction-limited resolution of l=250 nm
(Figure 4a and d). The images obtained after an additional un-
caging step at l=375 nm are brighter (by a factor of 1.5) than
the images for which the caged dye was photoactivated by
the l=750 nm depletion laser. This can be explained by in-
complete photoactivation in the latter case.
Conclusion
750 nm, and the possibility of unmasking the dye with
a bright-field UV-illumination of l=375 nm (see the Support-
We have shown that the caging procedure based on incorpo-
ration of a 2-diazoketone fragment can be applied to virtually
ing Information for further details and an additional exam-
[
26]
ple). We showed that unmasking of the dye is possible by
to all N,N,N’,N’-tetraalkylrhodamines (this work) and carbopyro-
either one-photon photoactivation with light of l=375 nm
[2b]
nines.
The compact structure and very small size of the
[
7a]
(
Figure 4a) or a two-photon process that uses the high-in-
caging group facilitate the ability of Rhodamines NN and Car-
bopyronines NN (deprived of the charged groups) to cross the
membranes of living cells (see Figure S5 in the Supporting In-
formation). In combination with the site-specific labeling pro-
tensity infrared (l=750 nm) illumination of the STED laser (Fig-
ure 4b). The second case offers the valuable possibility to skip
the UV-uncaging step. Simultaneous photoactivation and de-
pletion with a STED laser directly provides super-resolved
images. This is especially useful for applications in biological
samples because the absence of a UV laser reduces the photo-
damage to the biological specimen. Moreover, the two-photon
uncaging conditions with a STED beam (l=750–800 nm) are
available for all commercially available STED microscopes pro-
[27]
tocols, realized for the Halo-, SNAP-, and CLIP-tags, the avail-
ability of the novel cell-permeable, caged fluorescent dyes may
extend the set of live-cell labeling strategies based on binding
the genetically encoded protein tags with organic fluoro-
phores. Spatially restricted photoactivation followed by track-
ing of the uncaged molecules may enable measurements of
Figure 4. Conventional confocal and STED microscopy images of the nuclear pore complex protein Nup153 in mammalian cells labeled with the caged carbo-
pyronine KK1012. a) Confocal image of Nup153 before uncaging of the dye. b) Super-resolution image of Nup153 after uncaging the dye by bright-field UV-il-
lumination at l=375 nm and subsequent STED microscopic recording. c) Super-resolution image of Nup153 obtained by simultaneously uncaging and de-
pleting the dye during the STED microscopic recording. d) Confocal image of Nup153 obtained after acquisition of a STED image with the same parameters,
as described for c). b,c) The STED power in the back focal plane was 200 mW. All images show unprocessed (raw) data. The given resolutions denote the
FWHM of exemplarily chosen, Gaussian-fitted object signals (see Supporting Information for details).
&
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ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Full Paper
molecular dynamics (e.g., diffusion parameters or flow veloci-
ties). The combination of Rhodamine NN with nanoscopic tech-
niques (e.g., single-molecule switching followed by localiza-
an acid chloride is the most important step in conversion of rho-
damines and carbopyronines to spiro diazoketones. Before the reaction
with diazomethane, a small sample of an acid chloride must be
quenched by addition of alcohol (after removal of the activating
agent), followed by analysis of the reaction mixture by TLC, HPLC, or
ESI-MS. This is most important in the case of oxalyl chloride, which
often gives incomplete conversions. Oxalyl chloride is a mild activating
agent and is indispensible in some cases. Its reaction may require catal-
ysis with DMF.
[
24]
tion or STED microscopy) is expected to provide additional
[
12e,28]
information on subcellular structure.
Other cationic dyes
are also expected to undergo the caging procedure described
in this work. In particular, caged fluorescence quenchers repre-
sent a very interesting class of photoactivatable dyes. They can
be used as masked acceptors in fluorescence resonance
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ˇ
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5
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1
3
1
9
[
3
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1
2986–12998; n) C. A. Wurm, K. Kolmakov, F. Gçttfert, H. Ta, M. L. Bossi,
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eadmin/user_upload/documents/Downloads/Application_Notes/
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B. Harke, M. Leutenegger, C. Eggeling, S. W. Hell, Eur. J. Org. Chem.
20120316-Labeling_Protocol.pdf;
b) https://www.atto-tec.com/filead-
min/user_upload/Katalog_Flyer_Support/Procedures.pdf. In the case of
the lipophilic NHS esters derived from the caged dyes 2a, 2bc, 2c, and
KK1012, amino-reactive dye (0.2 mg) dissolved in dry DMF (40 mL) was
added slowly to the stirred and buffered (pHꢀ8.5) protein solution
(1 mg of a secondary antibody in 1 mL buffer), followed by incubation
and common isolation procedures (gel-filtration, evaluation of protein
concentrations, etc.). More hydrophilic caged dyes 2d,Na and 10 re-
quire less DMF; c) value provided by the producer (https://www.atto-
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2
010, 3593–3610.
[
13] For phototransformation of photoactivatable azido fluorogens, which
represent a special class of masked fluorescent dyes, to fluorescent
(
“push–pull”) aromatic amines, see: S. J. Lord, H. D. Lee, R. Samuel, R.
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15] Provided that these dyes (e.g., Alexa Fluor 546 or Alexa Fluor 633) were
N
also prepared by the S Ar of one chlorine or fluorine atom in precur-
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ample, see: F. Mao, W.-Y. Leung, R. P. Haugland, US 6130101, 2000.
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[
[
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[
[
[
18] S. Nizamov, K. I. Willig, M. V. Sednev, V. N. Belov, S. W. Hell, Chem. Eur. J.
2012, 18, 16339–16348.
19] Details of preparation, properties, and the use of the “universal hydro-
phlilizer” 11 will be reported later.
20] Methanol was used as a solvent for the model compounds in the prep-
arative photolysis experiments, from which the fluorescent homologues
(
(
3a-Y,Me–3e-Y,Me) of the parent Rhodamines NN and the non-emitting
“dark”) products (4a-Y–4e-Y) were isolated (see Scheme 1 and the
Supporting Information for details).
[
[
21] a) G. Gauglitz, J. Photochem. 1976, 5, 41–47; b) G. Gauglitz, S. Hubig, J.
Photochem. 1985, 30, 121–125; c) H. J. Kuhn, S. E. Braslavsky, R.
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22] Compare with the values for the photactivation (uncaging) quantum
yields reported in references [4a], [5], [6], [8b], [9a], [13], and in the fol-
lowing publications: a) W. Lin, L. Long, W. Tan, B. Chen, L. Yuan, Chem.
Eur. J. 2010, 16, 3914–3917; b) V. Hagen, J. Bendig, S. Frings, T. Eckardt,
S. Helm, D. Reuter, U. B. Kaupp, Angew. Chem. 2001, 113, 1077–1080;
Angew. Chem. Int. Ed. 2001, 40, 1045–1048; c) J. del Mꢄrmol, O. Filevich,
Received: April 29, 2014
Published online on && &&, 0000
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Full Paper
FULL PAPER
Masked fluorophores: New photoacti-
vatable rhodamine dyes (see scheme)
have been designed, decorated with
a reactive group, conjugated with pro-
teins, and used as labels in immuno-
fluorescence and optical microscopy.
These “hidden” markers can be readily
and irreversibly “uncaged” with l=375–
&
Fluorescence
V. N. Belov,* G. Y. Mitronova, M. L. Bossi,*
V. P. Boyarskiy, E. Hebisch, C. Geisler,
K. Kolmakov, C. A. Wurm, K. I. Willig,
S. W. Hell*
&
& – &&
420 nm or intense red light
Masked Rhodamine Dyes of Five
Principal Colors Revealed by
Photolysis of a 2-Diazo-1-Indanone
Caging Group: Synthesis,
Photophysics, and Light Microscopy
Applications
All the beauty is revealed…
when sunlight shines on the
…
statue of Polymnia, one of the nine
Muses in ancient Greek mythology,
masked with snow in winter and
then liberated in all its splendor
from the white cloak by a bright
sunshine. The parallel light-induced
transformation is depicted by the
chemical formulae. Irradiation of
colorless Rhodamines NN, masked
fluorescent dyes, with UV or visible
light irreversibly “releases” the
hidden colors in all their depth and
vibrant fluorescence. For more
details, see the full paper by V. N.
Belov, M. L. Bossi, S. W. Hell et al. on
the page && ff.
Chem. Eur. J. 2014, 20, 1 – 13
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ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ