Rhodamine spiroamides for multicolor single-molecule switching fluorescent nanoscopy

Article abstract of DOI:10.1002/chem.200901333

The design, synthesis, and evaluation of new rhodamine spiroamides are described. These molecules have applications in optical nanoscopy based on random switching of the fluorescent single molecules. The new markers may be used in (co)localization studies of various objects and their (mutual) positions and shape can be determined with a precision of a few tens of nanometers. Multicolor staining, good photoactivation, a large number of emitted photons, and selective chemical binding with amino or thiol groups were achieved due to the presence of various functional groups on the rhodamine spiroamides. Rigidized sulfonated xanthene fragment fused with six-membered rings, N,N′-bis(2,2,2-trifluoroethyl) groups, and a combination of additional double bonds and sulfonic acid groups with simple aliphatic spiroamide residue provide multicolor properties and improve performance of the rhodamine spiroamides in photoactivation and bioconjugation reactions. Having both essential parts of the photoswitchable assembly - the switching and the fluorescent (reporter) groups - combined in one chemical entity make this approach attractive for further development. A series of rhodamine spiroamides is presented along with characterizations of their most relevant properties for application as fluorescent probes in single-molecule switching and localization microscopy. Optical images with resolutions on the nanometer scale illustrate the potential of the labels in the colocalization of biological objects and the two-photon activation technique with optical sectioning.

Full text of DOI:10.1002/chem.200901333

DOI: 10.1002/chem.200901333
Rhodamine Spiroamides for Multicolor Single-Molecule Switching
Fluorescent Nanoscopy
Vladimir N. Belov,*[a] Mariano L. Bossi,[a] Jonas Fçlling,[a] Vadim P. Boyarskiy,[a, b] and
Stefan W. Hell*[a]
Dedicated to Professor Armin de Meijere on the occasion of his 70th birthday
Abstract: The design, synthesis, and
evaluation of new rhodamine spiro-
ACHTUNGTRENNUNGamides are described. These molecules
on the rhodamine spiroamides. Rigi-
dized sulfonated xanthene fragment
fused with six-membered rings, N,N’-
bis(2,2,2-trifluoroethyl) groups, and a
combination of additional double
bonds and sulfonic acid groups with
simple aliphatic spiroamide residue
provide multicolor properties and im-
prove performance of the rhodamine
spiroamides in photoactivation and
bioconjugation reactions. Having both
essential parts of the photoswitchable
assembly—the switching and the fluo-
rescent (reporter) groups—combined
in one chemical entity make this ap-
proach attractive for further develop-
ment. A series of rhodamine spiro-
have applications in optical nanoscopy
based on random switching of the fluo-
rescent single molecules. The new
markers may be used in (co)localiza-
tion studies of various objects and their
(mutual) positions and shape can be
determined with a precision of a few
tens of nanometers. Multicolor stain-
AHCTUNGTRENNaGUN mides is presented along with charac-
terizations of their most relevant prop-
erties for application as fluorescent
probes in single-molecule switching
and localization microscopy. Optical
images with resolutions on the nano-
meter scale illustrate the potential of
the labels in the colocalization of bio-
logical objects and the two-photon acti-
ing, good photoactivation,
a large
number of emitted photons, and selec-
tive chemical binding with amino or
thiol groups were achieved due to the
presence of various functional groups
Keywords: cage compounds · dyes/
pigments · fluorescence · molecular
switches · rhodamines
vation
technique
with
optical
sectioning.
Introduction
emitting volume, and thus the resolution, has been squeezed
up to the size of a single (macro)molecule. These techniques
can be subdivided according to the way the observed
volume is reduced. Reversible saturable (switchable) optical
fluorescence transitions (RESOLFT),[2a] the first family of
techniques to overcome the Abbe limit, is based on a local
squeezing of the effective fluorescent volume by means of il-
lumination patterns that have one or more intensity zeros in
the focal plane. This can be achieved using spotlike (focal)
illumination scanned through the sample or by structured il-
lumination. Recently, another family of techniques based on
imaging stochastically switched single emitters has emerged,
in which the position of the observed molecule is a priori
unknown. The main advantage of this single-molecule
switching microscopy (SMS; also known as photoactivation
localization microscopy (PALM),[3] stochastical optical re-
construction microscopy (STORM),[4] or fPALM[5]) over the
RESOLFT techniques is that it is not necessary to force the
marker molecules to undergo many photoswitching cycles.
However, SMS application requires samples with very low
The Abbe principle limits the optical resolution in far-field
microscopy to about half the wavelength of the light used.[1]
Several inventions in the last 10 to 15 years have demon-
strated that the diffraction barrier may be broken by using
reversible or irreversible photoswitching of the fluorescent
probe between a dark and an emitting state.[2] The effective
[a] Dr. V. N. Belov, Dr. M. L. Bossi, Dr. J. Fçlling, Dr. V. P. Boyarskiy,
Prof. Dr. S. W. Hell
Department of NanoBiophotonics
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
Fax : (+49)551-201-2506
E-mail: vbelov@gwdg.de
shell@gwdg.de
[b] Dr. V. P. Boyarskiy
Permanent address: Chair of Physical Organic Chemistry
Chemistry Department, St. Petersburg State University
198504 St. Petersburg (Russia)
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FULL PAPER
background levels and, therefore, a high contrast between
the molecular states of the markers used.
The working principle of the SMS approach consists of
switching on and off a multitude of isolated markers, localiz-
ing them, and then reconstructing the image of the sample
from their positions. The markers are imaged onto the
camera of a wide-field microscope; each marker producing
a diffraction-limited spot. The irradiation conditions are
maintained in such a way that the number of molecules in
the fluorescent state is low enough and their average dis-
tance is larger than the classical resolution. The center of
the spot provides the position of the marker with an accura-
cy that ideally depends only on the number of collected
photons, n, and on the full width at half maximum (FWHM)
of the fluorescence spot, and is approximately given by
Scheme 1. Photochromic (caged) RSAs in the colorless CF and colored
OF forms.
few milliseconds in polar solvents[8] (longer in nonpolar sol-
vents). Because SMS does not require a large number of
cycles per marker to be performed (just one cycle is suffi-
cient), the photobleaching and other secondary processes
shown in Scheme 1 do not limit the use of RSAs in SMS. In
fact, markers in the open form (OF) are bleached by a
strong pulse of the same excitation laser after being imaged,
to reduce imaging times.[9]
The first photochromic RSA used in SMS was aromatic
spiroamide 1d (Figure 1), derived from rhodamine B.[9]
Highly photostable under single-molecule conditions, it al-
lowed the detection of enough photons and provided an
average localization precision of up to 10 nm. The usefulness
of compound 1d was demonstrated in inorganic materials
(silica gel nanobeads) and in biological samples. Photo-
switching between the isomers was realized by a one-photon
activation process (1PA) at l=375 nm and a 2PA process at
l=747 nm. The latter provided optical sectioning, allowing
the measurement of samples several microns thick and a 3D
p
FWHM/ n¯.[6] Thus, provided that enough photons are col-
lected from a single molecule, its position can be determined
with an accuracy below the diffraction limit. After being
imaged and localized, the markers must go back to a dark
state (i.e., become bleached), so other markers can be
switched on, localized, and switched off again. The process
is repeated for a large set of markers and their positions are
mapped to give an image with a resolution far better than
the Abbe limit, ideally equal to the average localization ac-
curacy of the individual markers. Besides the fluorescence
switching, several requirements must be fulfilled for the
markers to perform well in SMS. First, highly photostable
dyes with large fluorescence quantum yields are required. In
the fluorescent “on” state they should yield a large number
of photons. Second, high contrast between the two states
(fluorescent and dark) is necessary for measuring densely
stained samples and allowing the reliable localization of a
large number of markers. This ensures that in the dark state
all markers produce a negligible contribution to the back-
ground when the switched-on molecules are imaged. Finally,
the switching reactions should be easily controlled to keep
the desired fraction of markers in the “on” state during the
acquisition time.
reconstruction by imaging subdiffractionally resolved
z
stacks with ꢀ500 nm spacing. Combined with an asynchro-
nous image acquisition protocol,[10] meaningful images were
recorded in about 2–5 min. Rhodamine amide 1e[11] was
later introduced to improve the two-photon switching effi-
ciency with continuous-wave laser sources, which also sim-
plified the experimental setup.
Initially, SMS was realized by using photoactivable fluo-
rescent proteins (PA-FPs)[3a] and activator–reporters sets of
dyes composed of an Alexa Fluor (activator) and a cyanine
dye (reporter; for example, Cy5).[4a,d] Both of them were
performed in thin samples (cryosectioned) or in TIRF (total
internal reflection fluorescence) geometries without axial
resolution. The introduction of rhodamine spiroamides
(RSAs) as photoactivable markers in SMS addressed the
lack of axial resolution and provided optical sectioning by
using two-photon activation (2PA). The photochromic reac-
tion of RSA, reported in the 1970s by Knauer and Gleiter,[7]
is presented in Scheme 1. The photoinduced ring-opening
reaction generates the rhodamine chromophore, which typi-
cally absorbs in the green region and emits in the red (e.g.,
at lꢀ560–580 nm for rhodamine B derivatives with R1 =
R2 =Et). The closed spiro form is transparent across the
whole visible range, providing a huge contrast between the
signals of the two states. The open isomer returns thermally
to the closed form (CF) with a characteristic lifetime of a
Multicolor extensions of SMS by using PA-FPs,[3b] a com-
bination of different activator–reporter systems,[4b,d] or mix-
tures of a PA-FP and Cy-5[12] have also been reported. In
the last example, an activator was not used. The fluores-
cence of Cy-5 can be temporarily switched off by a strong
laser light (ca. 10 kWcmꢁ2) of the same wavelength used for
its excitation (l=633 nm),[13a] and this process can also be
used without any activator for the acquisition of nanoscopic
images.[13b] However, this switching process is sometimes dif-
ficult to control. The label may spontaneously go into the
“on” state and it is highly sensitive to the (micro)environ-
ment and the presence of oxygen or triplet scavengers. Fluo-
rescent proteins have the advantage of being genetically en-
codable, but they yield a low average number of photons.
Activator–reporter systems are brighter but require special
experimental conditions (e.g., oxygen-scavenging buffers or
a triplet quencher at high concentration). Cyanine dyes
(Cy3, Cy5, and Cy7) are used as reporters, and an adequate
Alexa Fluor dye is typically selected as an activator.[4] Sec-
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V. N. Belov, S. W. Hell et al.
ted photons, selective chemical binding with biomolecules
(1a-NHCH2CH2-maleimide vs. NHS esters obtained with
1b–g), and high photostabilities in both the CF and OF.
New substituents at the nitrogen atoms and in the benzoic
acid residue have been introduced. They predictably change
the properties of the photoactivable assemblies.
Results and Discussion
Design and synthesis: The preparation of lithium salt 1d
and the active esters of the corresponding carboxylic acid
have been previously reported.[9] They provided nanoscopic
images of immunostained (in the tubuline network) whole
fixed cells with the possibility of 2PA and optical sectioning.
Screening of the amines allowed us to obtain a marker with
the desired switching-on efficiency at l=375 nm. At this
and longer wavelengths, conventional lenses have a high
transmittance and inexpensive laser sources are available. It
was found that an aromatic amine was necessary to provide
an adequate activation rate and maintain a sufficient frac-
tion of molecules per frame in the emitting state. An in-
crease in the 2PA rate was achieved with the same 4-amino-
phthalimide derivative simply by changing from rho-
AHCTUNGTRENNUNG
damine B to rhodamine 590 (1e).[11] Though it is widely used
in life science applications, the former is not an ideal fluo-
rescent dye; its fluorescence quantum yield is fairly good
(Ffl =0.65 in basic EtOH) but strongly depends on the tem-
perature and solvent. Amides of rhodamine B absorb at
about l=565 nm and emit at around l=580 nm (in the
OF). Further optimization resulted in compounds 1a, 1c,
and 1g, which have been used for the realization of the mul-
ticolor far-field fluorescence nanoscopy with isolated detec-
tion of distinct molecular species,[15] but the principles of
their design and preparation procedures have not yet been
published. Here we fill this gap and describe the synthesis
and properties of the whole series of RSAs that we have
prepared for different SMS applications.
A spectral shift in the absorption and emission bands of a
rhodamine is an easy and interesting option that may pro-
vide a “brighter” dye, more detectable photons (better local-
ization precision), and another color, valuable for the coloc-
alization studies and other applications in the future. We
screened several amines and rhodamines before we devel-
oped compounds 1a–g for multicolor single-molecule photo-
activation and localization microscopy.[9,11,15] Rhodami-
nes 6G and 110 could in principle afford a blue spectral shift
(compared with rhodamine B), if the reactivity of the mono
(e.g., in rhodamine 6G) or unsubstituted amino groups (e.g.,
in rhodamine 110) did not preclude the implementation of
this plan. Binding with non-nucleophilic aromatic amines re-
quires very strong activation of the sterically hindered car-
boxylic group of the rhodamine.[16] In practice, only acid
chlorides were found to be reactive enough. Unfortunately,
boiling of rhodamine 110 or rhodamine 19 (the free carbox-
ylate of rhodamine 6G) with POCl3 in dichloroethane did
not produce a stable acid chloride but only a tar, probably
Figure 1. RSAs 1a–g for multicolor single-molecule photoactivation ex-
periments; approximate values for the fluorescence maxima of lithium
salts in water or aqueous MeOH: 1a lem ꢀ560 nm, 1b lem ꢀ530 nm, 1c
lem ꢀ560 nm, 1d lem ꢀ590 nm, 1e lem ꢀ620 nm, 1 f lem ꢀ630 nm, 1g lem
ꢀ620 nm. Stokes shifts for all compounds are ꢀ15–20 nm.
ondary antibodies have to be labeled with both the activator
and the reporter in carefully selected proportions to opti-
mize their distance and avoid the presence of more than
one reporter molecule per antibody, which would result in a
drastic reduction in the switching-off efficiency. Therefore,
the photochromic RSA methodology seems to offer a viable
alternative. This procedure is easier to control than the acti-
vator–reporter pair and PA-FP approaches. In fact, multicol-
or SMS based on two or more spectrally distinct photoactiv-
able dyes (rhodamines) attached to different nanoparticles,
antibodies, or “drug-like” molecules that “recognize” vari-
ous bio-objects or receptors have already been proposed
and implemented.[14]
Herein, we describe the design, synthesis, and evaluation
of new fluorophores for SMS with nanoscopic resolution
(Figure 1). The use of some of these new switchable fluores-
cent dyes as labels in the imaging of nanoparticles and (co)-
localization studies of various biological objects with resolu-
tions of up to 10 nm has already been reported.[9,11,15] All of
them may be activated in 1PA or 2PA processes with com-
mercial lasers and provide multicolor staining, improved
performance in aqueous media (1a), a high number of emit-
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Rhodamine Spiroamides
FULL PAPER
due to self-condensation of the amino groups. Rho-
ACHTUNGTRENNUNGdamines B and 590 (14-H) have tertiary amino groups and
clohexane rings turned out to be sufficient for the photoacti-
vation rate required for SMS, even in the absence of any ar-
omatic amine residue. Surprisingly, sodium salt 1a also ab-
sorbs in water with a maximum at l=330 nm (logeꢀ4),
whereas the UV spectrum of reference compound 1d has
only a shoulder at lꢀ314 nm with a similar absorbance.[19]
Photoactivation of colorless sodium salt 1a produces an
orange fluorescent dye (lmax ꢀ530 nm, lem ꢀ550 nm)[18] with
an emission color that can be quite easily distinguished from
that of rhodamine B amides (lem ꢀ570 nm).[9] Alternatively,
chemical “activation” is possible: passing an aqueous solu-
tion of 1a through a cation-exchange resin in the H+ form
produced a highly colored solution of acid 3-OH. Com-
pound 1a, however, could not be transformed into the
stable amino reactive NHS ester under basic conditions be-
cause it acylates its own secondary amino groups, even at
08C. The unprotonated amino groups in 1a and in the corre-
sponding active esters are nucleophilic enough, even if they
are somewhat sterically hindered by the sulfonic acid resi-
dues. N-(2-Aminoethyl)maleimide is much more reactive
than the sterically hindered aromatic amino groups in 1a.
Therefore, we coupled 1a with N-(2-aminoethyl)maleimide
to give thiol reactive derivative 3-NHCH2CH2-maleimide.
The required compound was isolated in the OF as a red
solid by preparative RP-HPLC (with 0.1% v/v of trifluoro-
acetic acid in MeCN/water). It is stable in the absence of
water and in the dark. Column chromatography on SiO2 is
accompanied by partial decomposition by water absorbed
on the silica gel. Compound 3-NHCH2CH2-maleimide may
not only be used for the reaction with cysteine residues in
proteins but also for binding with thiols, which appear after
modification of the terminal amino groups of lysines with
Trautꢁs reagent.[15,20] Under slightly basic conditions
(pH 7.5–8.5) it decolorizes and forms colorless bioconju-
gates.
their amidation is impossible. Therefore, they smoothly give
the corresponding acid chlorides and then amides, even with
relatively unreactive aromatic amines. To prevent an inter-
nal acylation, a new approach was required.
As a viable alternative to the sluggishly reacting aromatic
amines, much more nucleophilic aliphatic amines may be
used for amidation. However, the aromatic 4-amino-N-(me-
thoxycarbonylmethyl)phthalimide (18) has some drawbacks:
its incorporation reduces the water-solubility of the corre-
sponding amides, and the phthalimide cycle is readily de-
stroyed under basic conditions.[17]
We used a much more reactive methyl 3-aminopropio-
nate, which amidated the water-soluble orange fluorescent
dyes with a rigidized xanthene fragment (compound 2 in
Scheme 2).[18] The colorless methyl ester 3-OMe was formed
in the presence of Et3N and isolated from the reaction mix-
ture by chromatography on SiO2 with MeOH/diethyl ether
(1:1) as the eluent. Interestingly, under these conditions
amino sulfonic acid 3-OMe was isolated as an inner salt
(without Et3N). Sodium salt 1a can be efficiently photoacti-
vated by irradiation at l=375 nm even though it has no ex-
tended conjugation in the amide part of the molecule.
Fusion of the 3’,6’-diaminoxanthene fragment with two cy-
A further hypsochromic shift in the absorption and emis-
sion bands may be provided by rhodamine 110, which ab-
sorbs at around l=490 nm and emits at about l=510 nm.
To prevent acylation of the unsubstituted amino groups in
this fluorescent dye, a new protecting group that does not
influence the spectra and allows the formation of the xan-
thene heterocycle from the corresponding aniline is required
for the nitrogen atoms. The inductive effect of the CF3CH2
group is very similar to the inductive effect of the hydrogen
atom.
For
example,
2,2,2-trifluoropropionic
acid
(CF3CH2COOH) is as strong as formic acid (HCOOH).
Therefore, all rhodamine derivatives with 2,2,2-trifluoro-
ethylated nitrogen atoms are expected to have the same ab-
sorption and emission spectra as the parent dyes. Moreover,
the NHCH2CF3 groups in amino acid derivatives are known
to be resistant to acylation (in the presence of “normal” pri-
mary amines).[21] Thus, this residue may also provide suffi-
cient chemical protection for the amino group. The 2,2,2-tri-
fluoroethyl group is easy to introduce (Scheme 3). Trifluoro-
acetylation of m-anisidine (4) and N-ethyl-3-methoxyani-
line (6-Me) provides amides 5-CF3 and 8, which may be re-
duced to 6-CF3 and 9, respectively, by using BH3·THF. The
Scheme 2. Synthesis of primary aliphatic amides of sulfonated rhoda-
mines with a rigidized xanthene fragment. Conditions: i) N,N,N’,N’-tetra-
ꢁ
methyl-O-(N-succinimidyl)uronium·BF4 (TSTU), Et3N, DMF (N,N-di-
methylformamide), RT, overnight; HCl·H2NCH2CH2COOMe, Et3N,
DMF, RT, overnight, 38–66%; ii) aqueous NaOH (0.1–0.15m), RT, 2 h,
100%; iii) Amberlite IR-120 (H+ form); iv) N-(2-amino
ACHTUNGTRENNUNG
trifluoroacetate,
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V. N. Belov, S. W. Hell et al.
Scheme 3. Synthesis of rhodamines 10-R2 with N,N’-bis(2,2,2-trifluoroeth-
yl) groups. Conditions: i) pyridine, CH2Cl2, RT, overnight; ii) BH3·THF
(1m), THF, reflux, overnight; iii) 48% aqueous HBr, AcOH, reflux, 6 h;
iv) 1608C, 3 h, then portion 2 of 7-R2, 85% aqueous H3PO4, 1608C, 3 h.
latter were demethylated, and phenols 7-H and 7-Et were
condensed with phthalic anhydride to afford the required
fluorescent rhodaminic dyes 10-H and 10-Et. These may be
easily isolated from the reaction mixtures in gram quantities
by crystallization, without using column chromatography.
Gratifyingly, the excitation and emission spectra of xanthene
dye 10-H and rhodamine 110 were found to be very similar.
Moreover, compound 10-Et turned out to be a spectral ana-
logue of rhodamine 6G with protected amino groups. Com-
pound 10-H is a red solid that exists in the OF as a zwitter-
ion and is moderately soluble in unpolar solvents (e.g.,
CHCl3). Substance 10-Et is nearly colorless in the solid
state. It readily dissolves in CHCl3 and THF (but not in
water) and its solutions are colorless. In the solid state and
in nonpolar solvents it exists in a CF. The chemical shift of
C9 in the xanthene fragment is diagnostically important; in
the CF the 13C NMR spectroscopic signal of the quaternary
aliphatic spiro carbon atom is found at about d=84 ppm
and in the OF this signal shifts downfield and “disappears”
in the densely populated region between d=110 and
140 ppm. Transformation of the carboxylic groups in 10-H
and 10-Et into the intensely colored acid chlorides can
easily be performed by heating at reflux with POCl3 in di-
chloroethane. Under these conditions, rhodamine 10-H
forms an insoluble intermediate that, however, further
reacts as the corresponding acyl chloride. The amidation re-
actions (Scheme 5) will be discussed below.
Scheme 4. Synthesis of rhodamines 1g (15) and 17, which absorb at l
ꢀ600 nm and emit at lꢀ620 nm. Conditions: i) acetone,
YACHTUNGTRENNUNG(OTf)3
(5 mol%), 24 h, RT; ii) MeI, Cs2CO3, DMF, 908C, 3 h; iii) 48% aqueous
HBr, AcOH, reflux, 6 h; iv) p-TosOH, EtCOOH, 1608C, 24 h; v) 96%
H2SO4, 08C to RT, overnight; vi) H2NCH2CH2OH; HATU, Et3N, DMF,
40–508C, overnight; vii) di(N-succinimidyl)carbonate, Et3N, MeCN,
CH2Cl2, 508C, 2 h.
tended conjugation in compound 1e also resulted in a better
2PA efficiency with respect to 1d. This allowed the use of
continuous-wave light sources in the red region of visible
light for 2PA[11] instead of the more expensive pulsed sour-
ces necessary for 2PA-SMS with markers derived from 1d.
The absorption and emission spectra of compounds 1e and
1g (OF) are also redshifted to an extent of about 40 nm in
comparison with the corresponding spectra of compound 1d
(OF) as a result of the extended conjugation. This allowed
the use of both 1d and 1e in multicolor colocalization stud-
ies.[15] Unfortunately, the low water solubility of lithium salt
1e and the active esters of the corresponding carboxylic
acid preclude their use in biological applications. The pres-
ence of two additional double bonds in the six-membered
rings fused with the xanthene core of compound 14-H is ad-
vantageous. They provide the required absorption in the
near-UV region, even without a heavy and rather nonpolar
4-aminophthalimide fragment as an additional chromo-
phore.
Lithium salt 1e can be photoactivated more easily than
the similar rhodamine B derivative, 1d, due to the presence
of two additional double bonds; the resulting higher absorp-
tion at l=375 nm is assigned to the CF. Improved 1PA with
l=375 nm light has been demonstrated for lithium salt 1e
itself (in a PVA matrix) and silica gel nanobeads stained
with the NHS ester prepared from compound 1e.[11] The ex-
Simple w-amino acids—rather polar and water-soluble
compounds—may be used as linkers instead of the 4-amino-
phthalimide residue. Further hydrophilization of dye 14-H is
provided by sulfonation with conc. H2SO4.[22] However, the
carboxylic group in disulforhodamine 14-SO3H cannot be
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Rhodamine Spiroamides
FULL PAPER
activated by heating with POCl3 because this reagent readily
transforms sulfonic acid residues into the corresponding sul-
fochlorides. Fortunately, selective activation of the carboxyl-
ate with HATU followed by coupling with aliphatic amines
in the presence of sulfonic acid residues is possible. w-
Amino alcohols may be used instead of esters of the w-
amino acids. This modification shortens the reaction se-
quence because it requires commercially available reagents
and omits the saponification of a methyl ester. NHS carbo-
nates are known to be highly amino-reactive and their sta-
bility is sometimes even higher than that of NHS esters.
Starting from 2-aminoethanol and sulforhodamine 14-SO3H,
we synthesized compound 1g (Scheme 4) and isolated it as a
salt with an excess of 2-aminoethanol by using column chro-
matography on SiO2. Before converting 1g into NHS car-
bonate 15-CONHS, the primary amine was removed by ion-
exchange chromatography and the highly colored OF, disul-
fonic acid 15-H-OF, was isolated. Complete conversion of
15-H-OF into 15-CONHS required a large excess of di(N-
succinimidyl)carbonate and was achieved after several hours
at about 508C in the presence of Et3N. The pure final prod-
uct, 15-CONHS, was isolated in the OF as a dark blue solid
by preparative RP-HPLC. It was stable for several weeks
under anhydrous conditions in the dark at about ꢁ208C. It
quickly decolorized in water under neutral or basic condi-
tions. Interestingly, it took several tens of minutes after
acidification for the blue color to reappear. To improve the
photochemical stability of the red marker, we prepared
rhodamine 17, which absorbs and emits at practically the
same wavelengths as 14-R1 (Scheme 4). It is an analogue of
one of the most photostable fluorescent dyes, rhodamine 110
(lem ꢀ589 in MeOH), with a perfluorinated benzoic acid
ring. Electron-acceptor groups at C9 of the xanthene ring
(opposite the oxygen atom) produce a bathochromic shift of
both absorption and emission maxima, so that rhodamine 17
absorbs at about l=595 nm and emits at approximately l=
610 nm. Compound 17 is very nonpolar and lipophilic,
therefore, for better solubility in water and aqueous buffers
in bioconjugation experiments, its spiroamides with a reac-
tive group should contain hydrophilic residues.
Lipophilic rhodamines 10, 14, and 17 were treated with
POCl3 in boiling 1,2-dichloroethane and the corresponding
acid chlorides were amidated with N-(methoxycarbonyl-
methyl)-4-aminophthalimide (18; Scheme 5). The cleavage
of methyl esters and chemical activation of the aliphatic car-
boxy group was carried out as shown in Scheme 5. If a
methyl ester was cleaved with LiI in boiling THF but not in
ethyl acetate, the reaction was slower because of the lower
boiling point of THF. However, ethyl acetate reacted with
LiI and produced lithium acetate, so part of the reagent was
lost. For the conjugation with biomolecules, lithium salts
1b–f can be directly converted into corresponding NHS
esters 20b–f or, if higher solubility in water is desired, into
the N-hydroxy(sulfosuccinimidyl) esters. These transforma-
tions and photoactivation parameters have been already re-
ported for lithium salt 1d,[9] which is also included in the fol-
lowing section for comparison.
Spectral properties: The spectral properties of the new
switchable fluorescent dyes were studied to evaluate their
applicability as molecular markers in SMS. The absorption
and emission spectra of compounds 1a–g were recorded in
water. The effect of pH on the equilibrium between the
open and closed isomers was also analyzed.
The absorption spectra of compounds 1a–g are given in
Figure 2. All the spectra show three main bands (with in-
creasing intensities) at l=310–340 nm, l=270–290 nm, and
l=230–265 nm. The positions of the first band (at longer
wavelengths), which is the most appropriate band for activa-
tion purposes in microscopy, are listed in Table 1. Com-
pounds 1a, 1e, and 1g show a fairly good absorption at
wavelengths of >350 nm. The other compounds displayed
only a marginal absorption above l=350 nm, but it was
enough for the low amount of activation needed for SMS.
As a first approximation, we observed that modifications at
the xanthene chromophore were more efficient at providing
a bathochromic shift in the first band than changing the ni-
trogen substituent of the lactam (X in Figure 1 and
Scheme 6). This conclusion became evident by comparing,
for example, compound 1e with compounds 1d and 1g. For
recording the spectra of the open-ring isomers, irradiation of
Scheme 5. Synthesis of methyl esters 19b–f and the corresponding lithium salts 1b–f. Conditions: i) POCl3, 1,2-dichloroethane, reflux, 4 h, then 18,
MeCN, Et3N, reflux, overnight; ii) LiI, EtOAc or THF, reflux, 2 or 4 d; iii) N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium·BF4ꢁ, Et3N, DMF, RT, over-
night; iv) N-hydroxysulfosuccinimide, HATU, DMF, RT.
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V. N. Belov, S. W. Hell et al.
in nonpolar solvents differ considerably from those in the
aqueous environment normally used in imaging experiments
and as
a mounting media (e.g., buffer solutions and
Mowiol). Nevertheless, nonbuffered saturated aqueous solu-
tions always contain small amounts of the OF due to the
equilibrium presented in Scheme 6.
The emission spectra of the forms (OF-H+) are presented
in Figure 3, and the corresponding absorption maxima are
listed in Table 1. The typical spectral features of the N-sub-
stituted rhodamine amides were observed, with yellow to
red emission colors. This group of compounds comprises a
tool-box from which up to four markers for multicolor ex-
periments can be chosen.
Figure 2. Absorption spectra of compounds 1a–g in water. 1a: c; 1b:
c; 1c: c; 1d: c; 1e: c; 1 f: c; 1g: c.
Table 1. Spectral properties and Ka values of 1a–g with mean numbers
of emitted photons (<n>) after photoactivation of the CF and conver-
sion to the OF.
lmax[a] [nm]
OF
Ka
<n>[c]
CF
OF
(em)
(abs)[b]
(abs)
1a
1b
1c
1d
1e
1 f
1g (15-H)
331
295
301
314
338
307
340
537
510
536
566
596
607
591
555
534
557
589
618
625
620
2.2ꢂ10ꢁ5
1600
1050
2350
1600
1350
1000
850
[d]
–
–
[d]
1.1ꢂ10ꢁ6, 1.2ꢂ10ꢁ4
3.2ꢂ10ꢁ6
[d]
–
2.3ꢂ10ꢁ5
Figure 3. Emission spectra of the OFs (1-OF-H+ in Scheme 6) of com-
pounds 1a–g in aqueous solutions: 1a: c; 1b: c; 1c: c; 1d: c;
1e: c; 1 f: c; 1g: c.
[a] In water. [b] Maxima of the absorption band at the longest wave-
length. [c] In PVA films. [d] Not measured.
The equilibrium presented in Scheme 6 was further stud-
ied in aqueous buffers in at pH values ranging from 2 to 12
(except for compounds 1b, 1c, and 1 f, due to their low solu-
bility). The changes in absorption observed for compound
1g on decreasing the pH value are presented in Figure 4A.
At least three clear isobestic points confirm that only two
species are present in the studied pH range. Similar effects
were observed for compounds 1a and 1e.
Speciation diagrams for these three compounds are pre-
sented in Figure 4B and the calculated equilibrium constants
are listed in Table 1. The proportion of the colored form
(OF-H+) was calculated from the absorption of the red
band (see Table 1). The fluorescence lifetimes measured for
the emitting species were in the range of 3 to 4 ns, and were
similar to those of the unsubstituted parent rhodamines with
free carboxylic groups. Moreover, the lifetimes did not
depend on the pH value (in the studied range).
The acid-catalyzed ring-opening reaction in Scheme 6
competes with the light-induced reaction and, therefore,
should be avoided when using these dyes as probes in
single-molecule localization studies. The useful pH range
can be estimated from the calculated acidity constants given
in Table 1. All the compounds presented here can be used
Scheme 6. Protonation-mediated ring-opening reaction of RSAs in water.
stirred solutions in water was attempted with a mercury
lamp (2–6 mW at l=365 or 313 nm). In all cases, some fluo-
rescence was observed under irradiation. However, thermal
recovery (OF!CF) in water and polar solvents is known to
be very fast (in the millisecond range)[8] and the spectra
were difficult to measure. In less polar solvents (e.g., tolu-
ene), irradiation for about a minute under similar conditions
(ca. 1–3 min) gave colored solutions that persisted for a few
minutes depending on the compound and the solvent (data
not shown). The spectra recorded under these conditions
may be influenced by partial back isomerization to the CF
during the data recording. Moreover, the spectral properties
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Rhodamine Spiroamides
FULL PAPER
PVA was selected because it is commonly used as a mount-
ing media in imaging experiments and, unlike solutions
(e.g., buffer solutions), there is no diffusion of the molecules
during the acquisition of a frame. Because the value may
also depend on the pH, the films for SM experiments were
spin coated from mixtures of the corresponding dye and
PVA in water-buffered solutions at pH 7.4.
Samples containing different dyes were exposed to low
light intensities at l=375 nm in a wide-field microscope to
ensure an appropriate amount of molecules in the OF. In
general, an average of about 15 molecules per frame was
used. A larger number of switching-on events is undesirable
because it could result in two or more molecules being in
the emitting state at a distance closer than the resolution of
the microscope. Switched-on molecules were recorded by
using an EM-CCD camera with a frame rate of 10 ms, and
the number of photons detected per event was calculated.
Figure 5 shows two examples of the histograms obtained
Figure 4. A) Effect of pH on the equilibrium shown in Scheme 6 for com-
pound 1g. The arrows display the direction of the changes as the proton
concentration was increased (the pH was decreased from 12 to 2). B) pH
speciation diagram for the colored forms of compound 1a (&, c), 1d
&
*
*
( , c), 1e ( , c), and 1g ( , c). The symbols correspond to mea-
sured data and the lines are best fits obtained by considering one (com-
pound 1e) or two equilibrium constants (compounds 1a, 1d and 1g).
Figure 5. Histograms of the number of photons per single-molecule event
for compound 1c (black bars) and compound 1 f (red bars). The inset
shows a typical frame from the CCD camera, from which single-molecule
events were localized.
as probes at biologically relevant pH values (6.5–8.0). Com-
pounds 1a and 1g may be also used at pH>5.5. Compound
1d also decolorizes in acidic solutions; probably due to pro-
tonation of the freely rotating N,N-diethyl amino group(s).
In this case, alkyl residues increase the basicity of the nitro-
gen atoms to higher extent than other substituents in com-
pounds 1a, e, and g with a rigidized xanthene fragment.[23]
The data shown in Figure 4 demonstrate another potential
application for the compounds 1a, 1d, 1e, and 1g; they may
serve as fluorescent pH indicators for the pH range of 3 to
7, depending on the dye. Moreover, the color of the indica-
tor and, to some extent, the transition pH range can be in-
dependently tuned by selecting the rhodamine (R1 and R2)
and the substituting amine (X).
Single-molecule behavior was studied in PVA films with
the aim of obtaining the average number of photons that
can be detected per molecule in the OF. This value is very
important because it determines the resolution that can be
achieved when the dyes are used as probes in SMS. The
number of photons depends on the environment, therefore,
from a large number of single-molecule events (>20000), to
ensure the statistic relevance of the data. The threshold to
reject dim events was set to 80 photons; events yielding
over 8000 photons were also neglected because at those
levels the response of the camera is nonlinear at the gain
factors used. The average values extracted from the histo-
grams for all dyes are listed in Table 1. All the compounds
are extremely bright and, calculated from the number of de-
tected photons, theoretical localizations accuracies of single-
molecule events are expected to be below 15 nm for all the
dyes reported in Table 1. It should be noted that these
values are environment dependent, and therefore may differ
slightly in other applications or immersion media used for
the measurements. An example of a CCD camera frame is
presented in Figure 5, inset; the high signal-to-noise ratio
demonstrates the excellent prospect of using RSAs for
single-molecule applications. Based on the observed switch-
ing-on processes and the excellent brightness of all the dyes
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10769

V. N. Belov, S. W. Hell et al.
presented herein, it can be concluded that they are all prom-
ising as probes for SMS. The average number of photons de-
tected per single-molecule event anticipates localization ac-
curacies and, therefore, an achievable spatial resolution in
the order of 10 to 15 nm for all measured compounds.
pounds 14 and 1g (15). They may easily oxidize, and irradia-
tion in the presence of atmospheric oxygen and a dye that
generates singlet oxygen may lead to an oxirane (which fi-
ꢁ
nally gives ring-opening products with a simple C C bond
instead of a double bond). This drawback caused time-de-
pendent channel cross-talk, that is, a false assignment of de-
tected single molecules in multicolor imaging.[15]
Fluorescence nanoscopy at the biomolecular level: Immu-
nostaining is one of the most widespread procedures to de-
liver the required markers to the desired target (an organ-
elle, a biomolecule, etc.) with high specificity and selectivity.
Perhaps cellular biology is the most promising application
field for RSAs as markers in fluorescence nanoscopy. Reac-
tive analogues of the dyes are necessary to specifically stain
the samples to be imaged. The amino-reactive NHS esters
are the simplest choice and were selected whenever possible.
Sometimes thiol reactive analogues were used to label sec-
ondary antibodies according to standard or new protocols.[15]
For compounds 3-NHCH2CH2-maleimide,[15] 15-CONHS,[15]
20c,[15] 20d,[9,15] and 20e,[11] we were successful and high-reso-
lution images were obtained. In case of sodium salt 1a, the
corresponding NHS ester probably reacts with amine groups
of the chromophore to form insoluble oligomeric or poly-
meric condensation products. In this case, a thiol-reactive
derivative, 3-NHCH2CH2-maleimide, was successfully used
for labeling antibodies modified with Trautꢁs reagent (2-imi-
nothiolane). The standard protocol[24] was modified. The
sheep anti-mouse IgG antibody was degassed and incubated
with a ten-fold excess of Trautꢁs reagent under nitrogen. Ter-
Violet or UV light (l=375 nm), which is used for photo-
activation, may cause cell damage and lead to autofluores-
cence, whereas red light minimizes these effects. Irradiation
of RSAs 20b–f with very strong red light also initiates a
ring-opening reaction and produces fluorescent species in
the course of the 2PA process.[9,11] The most important dif-
ference between 1PA and 2PA processes is that 2PA is only
effective in a thin plane,[26] which allows optical sectioning.
To demonstrate the applicability of 2PA with axial section-
ing in cell biology, we imaged lamin proteins in the nucleus
of a human glioma cell specifically labeled with secondary
antibodies stained with N-hydroxysulfosuccinimidyl ester
20d (n=1). Figure 6 shows a 3D reconstruction of nuclear
minal amino groups of lysine residues (H2N
ACHTUGNERTN(NUNG CH2)4) were
transformed into HS(CH2)3C(=NH)NH(CH2)4 groups and
A
R
ACHTUNGTRENNUNG
then thiol groups (and probably 2-iminothiolane) directly re-
acted with 3-NHCH2CH2-maleimide, which was used in an
equal amount with Trautꢁs reagent. Intermediate purifica-
tion of the thiolated antibodies[24] was not performed be-
cause it could have caused oxidation of thiol groups and for-
mation of the corresponding disulfides. Finally, gel filtration
through Sephadex G-25 (with standard phosphate-buffered
saline) gave the labeled protein purified from low-molecu-
lar-weight compounds.
Figure 6. Optical sections of lamin proteins in the nucleus of a U373MG
cell imaged by using 2PA with l=747 nm light; field of view: 12.5ꢂ
12.5 mm; pixel size: 30 nm; distance between z stacks ꢀ600 nm. Nuclear
lamina of whole fixed human glioma cells, U373M, were stained with
antilamin A mouse IgG as a primary antibody and sheep anti-mouse IgG
labeled with N-hydroxysulfosuccinimidyl ester 20d (n=1) as a secondary
antibody.
Lipophilic NHS esters 20b–f (Scheme 5; n=0) are not
soluble in water and under standard reaction conditions
they provide low degrees of labeling. Increasing the amount
of dye per antibody or the content of DMF in the aqueous
buffer (up to 5% v/v) did not improve the degree of label-
ing. However, they were very efficient and gave excellent
images in other applications in which organic solvents can
be used in the labeling process. For example, NHS ester 20c
reacted with 3-aminopropyl triethoxysilane in DMF and was
incorporated into silica nanobeads.[15,25]
After photoactivation of spiroamides 1g (15-CONHS)
with UV light (l=375 nm) followed by excitation by green
light (l=530 nm), fluorescence with lmax ꢀ620 nm was de-
tected. After a large number of activation–excitation cycles,
or prolonged irradiation with UV light (l=375 nm), a blue-
shift of the emission maximum to lꢀ580–590 nm was ob-
served. This behavior may be explained by the (stepwise)
“disappearance” of the two additional double bonds in com-
lamina by combining nine images recorded at different z po-
sitions with a typical separation of 600 nm between slices.
The “tubing” and folding inside the cell nucleus is distinctly
seen in the series of pictures. This kind of tube-like connec-
tions is characteristic of the lamin skeleton of a nucleus. A
good tangential resolution was achieved in this thick sample
(>6 mm), demonstrating the feasibility of imaging in a
whole (fixed) cell with no need for (cryo)sectioning.
Figure 7A shows a two-color high-resolution image of a
PtK2 cell immunostained with compounds 20d and 20e (n=
1) in the tubulin filaments and clatrin vesicles, respectively.
The excellent brightness of the SRA dyes allows the applica-
tion of a marker separation technique through a combina-
tion of single-molecule spectroscopy and microscopy[14,15]
that provides an improved color separation (i.e., reduces
false marker assignment) with respect to the conventional
ensemble techniques normally used (for example, in confo-
cal microscopy). Features beyond the classical resolution are
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Rhodamine Spiroamides
FULL PAPER
intact cell wall (e.g., N,N,N’,N’-tetramethylrhodamine), are
of particular interest. It is better to avoid the bulky aromatic
amines that have been used to shift the absorption band
edge to the near-UV region because they increase molecular
masses and lipophilicity and reduce water solubility. New
chromophores that provide highly efficient photoactivation,
good solubility in water, “atom economy” (absence of un-
necessary structural elements), high fluorescence quantum
yields, and an appropriate reactive group (recognition site)
are going to be developed. To keep the marker molecule
small enough to penetrate through the walls of living cells,
new types of photochemically active groups have to be in-
troduced. In addition, the corresponding NHS ester can
probably be prepared from OF 3-OH, in which the amino
groups are protected by protonation.
Very recently, a simple and yet very powerful super-reso-
lution technique emerged. It is based on ground-state deple-
tion followed by return of the individual molecules
(GSDIM) to the ground state (S0). This method uses com-
mercially available fluorescent dyes and provides an optical
resolution of lꢀ30 nm.[27] However, it requires higher light
intensities (which transfer nearly all dye molecules to a dark
state) and, therefore, 2PA is more difficult. Thus, photoacti-
vation of RSAs is a viable alternative to GSDIM, especially
when imaging with optical sectioning is required.
Figure 7. A) Two-color high-resolution SMS image of PtK2 cells with tu-
bulin and clatrin structures immunostained by using compounds 20d and
20e (n=1), respectively. B) The same conventional wide-field image ob-
tained by adding the individual single-molecule events.
clearly revealed in Figure 7A, reflecting the possibility of re-
solving objects that are blurred in a conventional micro-
scope by employing the probes presented herein. Figure 7B
shows the corresponding conventional wide-field image,
which, apart from lower resolution, also shows worse color
separation. Figure 7 demonstrates the applicability and pro-
spective use of the RSAs for multicolor applications, such as
colocalization of custom-stained objects or structures.
Experimental Section
General remarks: UV/Vis absorption spectra were recorded by using a
Varian Cary 4000 UV/Vis spectrophotometer and fluorescence spectra
were recorded by using a Varian Cary Eclipse fluorescence spectropho-
tometer. Closed quartz cuvettes with a 1 cm path length were used in all
experiments. All reactions were carried out with magnetic stirring in
Schlenk flasks equipped with septa or reflux condensers with bubble-
counters under argon or nitrogen (supplied by using a standard manifold
with vacuum and argon lines). NMR spectra were recorded at 258C by
using Varian MERCURY 300 (300 (1H) and 75.5 MHz (13C, APT)),
Bruker AM 250 (250 (1H) and 62.9 MHz (13C, DEPT), INOVA-500
(125.7 MHz (13C)) and INOVA-600 (600 MHz (1H)) spectrometers. All
spectra are referenced to tetramethylsilane as the internal standard (d=
0 ppm) by using the signals of the residual protons of CHCl3 (7.26 ppm)
in CDCl3, CHD2OD (3.31 ppm) in CD3OD, HOD (4.79 ppm) in D2O,
[D5]acetone (2.04 ppm) in [D6]acetone, or [D5]DMSO (2.50 ppm) in
[D6]DMSO. Multiplicities of signals are described as follows: s=singlet,
br=broad, d=doublet, t=triplet, q=quartet, quint=quintet, m=mul-
tiplet, mc =centrosymmetrical multiplet. Coupling constants (J) are given
in Hz. Multiplicities in the 13C NMR spectra were determined by APT
(attached proton test) measurements. Low-resolution mass spectra (ESI)
were obtained by using an LCQ spectrometer. High-resolution mass
spectra (ESI-HRMS) were obtained by using an APEX IV spectrometer
(Bruker). HPLC system (Knauer): Smartline pump 1000 (2ꢂ), UV detec-
tor 2500, column thermostat 4000 (258C), mixing chamber, injection
valve with 20 and 100 mL loops for the analytical and preparative col-
umns, respectively; 6-port-3-channel switching valve; analytical column:
Eurospher-100 C18, 5 mm, 250ꢂ4 mm, 1 mLminꢁ1; preparative column:
Eurospher-100-5 C18, 5 mm, 250ꢂ8 mm, 4 mLminꢁ1; solvent A: water+
0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA.
Analytical TLC was performed on MERCK ready-to-use plates with
silica gel 60 (F254). Column chromatography: MERCK silica gel 60,
0.04–0.063 mm or Polygoprep 60-50 C18 (Macherey–Nagel). Amberlite
IR-120 (H+ form, VWR) was used as an ion-exchange resin. Elemental
analyses were carried out at Mikroanalytisches Laboratorium des Insti-
Conclusions
Having both essential parts of a dye (the activator (spiro-
ACHTUNGTRENNUNGamide) and the reporter (xanthene) groups) combined in
one chemical entity allows tedious double staining proce-
dures, which have been reported for the related STORM
methodology,[4] to be avoided. New RSAs decorated with a
reactive group may be used for bioconjugation and even
help to overcome some drawbacks of large fluorescent pro-
teins that may interfere with the physiological role of the
studied protein after fusion and are known to be modestly
photostable. In most cases (e.g., for the switchable fluores-
cent protein RSFP rsFastLime),[10] the reported average
number of detected photons per single-molecule burst is
about an order of magnitude lower than the typical values
of 1400–2700 photons reported for RSAs.[15] Moreover,
chemical linking of the photochromic rhodamines with
FlAsH (ReAsH) or benzyl purines (pyrimidines) that recog-
nize very small tetracysteine motifs or SNAP tags, respec-
tively, may help to overcome drawbacks intrinsic to the
PALM imaging of big fused PA-FPs or clusters of antibod-
ies. Other photoactivable (caged) fluorophores, including
those that can penetrate the plasma membrane and the
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10771

V. N. Belov, S. W. Hell et al.
tuts fꢃr Organische und Biomolekulare Chemie der Georg-August-Uni-
versitꢄt, Gçttingen. Organic solutions were dried over MgSO4 or Na2SO4.
Lithium salts 1b–f were converted into the NHS esters 20b–f according
to a previously described procedure.[9]
mode): m/z: 762 (100) [MꢁH]ꢁ, 784 (44) [Mꢁ2H+Na]ꢁ; HRMS: m/z
calcd for C35H33N5O11S2: 656.16907, 656.15102; found: 764.16883 [M+H],
786.15082 [M+Na].
General method for the preparation of N-acyl-m-methoxyanilines 5-Me,
5-CF3, and 8: m-Anisidine 4 or N-ethyl-m-anisidine (6-Me; 0.10 mmol)
and dry pyridine (0.12 mol) were dissolved in dry CH2Cl2 (200 mL) and
cooled to 08C. Acetic or trifluoroacetic anhydride (0.12 mol) in CH2Cl2
(50 mL) was then added dropwise with stirring. The mixture was stirred
overnight at RT and then washed with cold water (3ꢂ100 mL), aqueous
HCl (1m, 100 mL), and saturated aqueous NaHCO3 (100 mL). After
drying, the solvent was evaporated in vacuo and the crystalline residue
was, as a rule, used in the next reduction step without further purification
(yields 85–100%). Compound 9 was purified by using chromatography
on SiO2 with hexane/EtOAc (8:1) to remove the impurity with a lower Rf
(yield 74%).
Compound 3-OMe: Rhodamine 2 (0.29 g, 0.51 mmol) was suspended in
DMF (9.5 mL) and TSTU (0.36 g, 1.2 mmol) was added in one portion,
followed by Et3N (0.5 mL, 3.5 mmol). The solution was stirred at RT for
20 h and then added to a solution of HCl·H2NCH2CH2COOMe (0.56 g,
4.0 mmol) and Et3N (1 mL, 7 mmol) in DMF (10 mL). After stirring for
3 d at RT, the DMF and excess Et3N were removed under vacuum (ca.
1 mbar) and cold MeOH (6 mL) was added to the residue. After stirring
at 08C for 1 h, the precipitate was separated, washed with cold MeOH
(1 mL), and dissolved in MeOH (ca. 100 mL). This solution was diluted
with diethyl ether and immediately filtered through SiO2 (50 g). The title
compound was eluted with MeOH/diethyl ether (1:1) and isolated as a
light violet powder (128 mg, 38%). M.p. >2508C; 1H NMR (600 MHz,
General method for the reduction of amides 5-Me, 5-CF3, and 8; synthe-
sis of amines 6-Me, 6-CF3 and 9: A solution of BH3 in THF (1m;
160 mL) at 08C was added to amide 5-R1 or 8 (80 mmol) in dry THF
(50 mL), and the mixture was heated at reflux overnight before being
cooled to 08C. Excess BH3 was carefully neutralized by adding MeOH
(20 mL), and then aqueous NaOH (1m) was added. After stirring at RT
for 20 min, the mixture was diluted with diethyl ether (300 mL) and the
organic layer was separated. The aqueous layer was extracted with dieth-
yl ether (3ꢂ100 mL), then the combined organic layers were washed
with saturated aqueous NaHCO3 (100 mL) and brine (100 mL), then
dried and evaporated. The residue was directly used in the next demethy-
lation step.
[D6]DMSO): d=1.64 (m, 4H), 2.26 (t, 3J
part of ABXY system, 2H), 2.50 (B part of ABXY system, JAB =12 Hz),
3.24 (m, 6H), 3.50 (s, 2H), 6.01 (s, 2H), 6.98 (d, 3J
(H,H)=7.5 Hz, 2H),
6.98 (d, 3J(H,H)=7.5 Hz, 2H), 7.46 (t, 3J(H,H)=6.9 Hz, 2H), 7.52 (t, 3J-
(H,H)=6.9 Hz, 2H), 8.13 ppm (brs, 2H); ESI-MS (positive mode): m/z:
ACHTUNGERTN(NUNG H,H)=7.6 Hz, 2H), 2.40 (A
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
722 (58) [Mꢁ2H+3Na]+, 732 (100) [MꢁH+2K]+, 747.5 (79); ESI-MS
(negative mode): m/z: 654 (46) [MꢁH]ꢁ, 676 (100) [Mꢁ2H+Na]ꢁ;
HRMS: m/z calcd for C30H29N3O10S2: 656.13671; found: 656.13633
[M+H].
Compound 1a: Methyl ester 3-OMe (115 mg, 0.175 mmol) was suspended
in water (5 mL) at 08C and aqueous NaOH (1m; 0.7 mL) was added
dropwise. After stirring for 2 h at RT, the colorless solution became clear
and HPLC indicated full conversion of 3-OMe (tR =9.1 min) into 1a (tR =
7.1 min; eluent: A/B 80:20–50:50 in 25 min, 254 nm). 1H NMR
(300 MHz, D2O): d=1.78 (m, 4H), 2.11 (m, 2H), 2.40–2.49 (m, 2H),
2.53–2.63 (m, 2H), 3.34 (m, 4H), 3.45 (m, 2H), 6.26 (s, 2H), 7.20 (m,
1H), 7.64 (m, 2H), 7.94 ppm (m, 1H); ESI-MS (positive mode) for
C29H24Na3N3O10S2: m/z: 686 (72) [MꢁNa+2H]+, 708 (100) [M+H]+,
733.5 (29); ESI-MS (negative mode) for C29H24Na3N3O10S2: m/z: 640 (38)
[Mꢁ3Na+2H]ꢁ, 662 (100) [Mꢁ2Na+H]ꢁ.
N-Ethyl-m-methoxyaniline (6-Me): 1H NMR (250 MHz, CDCl3): d=1.26
(t, 3J(H,H)=7 Hz, 3H), 3.16 (q, 3J
ACTHNUGTRENNUGN ACHTUNGTRENNNUG
3.79 (s, 3H), 6.18 (t, 4J
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
99.4 (CH), 104.0 (CH), 106.0 (CH), 130.2 (CH), 147.6 (C), 160.8 ppm
(C).
N-(2,2,2-Trifluoroethyl)-m-methoxyaniline
(250 MHz, CDCl3): d=3.76/3.77 (2ꢂt, 3J
AHCTUNGTRENNUNG
(6-CF3):[29]
(H,F)=7 Hz), 3.78 (s, S 5H),
(H,H)=2.3 Hz, 1H), 6.30
(H,H)=2 Hz, 1H), 6.37 (dd, 3J(H,H)=7.8 Hz, 4J-
(H,H)=2.4 Hz, 1H), 7.12 ppm (t, 3J(H,H)=7.8 Hz, 1H). 13C NMR
(62.9 MHz, CDCl3): d=45.9 (q, 2J
(C,F)=33 Hz), 55.1, 99.4 (CH), 104.0
1H NMR
3.97 (brt, 4J
(H,F)=6.0 Hz, 1H, NH), 6.24 (t, 4J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
(dd, 3J
(H,H)=8 Hz, 4J
U
ACHTUNGTRENNUNG
Compound 3-OH: Ion-exchange resin was washed with methanol, water,
aqueous HCl (1m) and again with water, until the pH value reached 4–5.
Sodium salt 1a (50 mg of freeze-dried solid with ca. 5% (w/w) NaOH)
was dissolved in water (0.5 mL) and passed slowly through the cation ex-
changer (ca. 5 mL). Washing it with water (10 mL) produced a strongly
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
(CH), 106.0 (CH), 125.0 (q, 1J
ACTHNUTRGNEU(GN C,F)=280 Hz), 130.2 (CH), 147.6 (C),
160.8 ppm (C); EI-MS: m/z: 205 (80) [M]+., 136 (100) [MꢁCF3]+.
1
red colored solution that was lyophilized to acid 3-OH (34 mg). H NMR
N-Ethyl-N-(2,2,2-trifluoroethyl)-m-methoxyaniline
(8):
ACHTUNGTRENNUNG
B.p.
(H,H)=7 Hz, 3H),
(H,H)=7 Hz, 2H), 3.80 (s, 3H), 3.83 (q, 3J
(H,F)=8.9 Hz, S
5H), 6.36 (t, 4J(H,H)=1.5 Hz, 1H), 6.38–6.43 (m, 2H), 7.17 ppm (t, 3J-
(H,H)=8.1 Hz, 1H). 13C NMR (62.9 MHz, CDCl3): d=11.4 (Me), 45.6
(CH2), 52.3 (q, 2J
(C,F)=26 Hz), 55.1, 99.9 (CH), 102.5 (C), 105.9 (CH),
125.5 (q, 1J
(C,F)=282 Hz), 130.0 (CH), 148.7 (C), 160.8 ppm (C); ele-
878C
(300 MHz, D2O): d=1.91 (m, 4H), 2.28 (t, 3J
ACHTUNGTRENNUNG
1
3
(2.3 mbar); H NMR (300 MHz, CDCl3): d=1.19 (t, J
(m, 4H), 3.33 (t, 3J
ACHTUNGTRENNUNG
3.47 (q, 3J
A
ACHTUNGTRENNUNG
(m, 1H), 7.76 ppm (m, 3H); 13C NMR (125.7 MHz, D2O, MeOH
49.9 ppm): d=19.8 (2ꢂCH2), 28.3 (2ꢂCH2), 33.9 (CH2), 36.1 (CH2), 43.0
(2ꢂCH2), 111.9 (C), 113.5 (C), 126.6 (C), 128.5 (CH), 130.4 (CH), 131.3
(CH), 131.5 (CH), 131.8 (C), 131.9 (CH), 136.7 (C), 153.0 (C), 155.1 (C),
155.9 (C), 171.3 (CO), 176.1 ppm (CO); ESI-MS (positive mode): m/z:
686 (66) [MꢁH+2Na]+, 708 (100) [Mꢁ2H+3Na]+, 733.6 (25); ESI-MS
(negative mode): m/z: 640 (100) [MꢁH]ꢁ, 662 (98) [Mꢁ2H+Na]ꢁ. In
aqueous solutions at RT, 3-H was found to slowly lose its sulfonic acid
residues. Decomposition was complete in several months.
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
mental analysis calcd. (%) for C11H14F3NO: C 56.65, H 6.32, N 5.74;
found: C 57.03, H 6.32, N 5.74.
General method for the demethylation of amines 6-CF3, 9, and 12; syn-
thesis of amines 7-H, 7-Et, and 13, respectively:[30] The starting amine
(8.6 mmol) was dissolved in glacial AcOH (5 mL), then 48% aqueous
HBr (6 mL) was added and the mixture was heated at reflux for 7 h
under argon (TLC of the neutralized probe indicated full conversion of
the starting amine). After cooling, the solution was carefully neutralized
to about pH 5–6 with aqueous NaOH (30%). The organic phase was sep-
arated and the aqueous phase was extracted with CHCl3 (3ꢂ20 mL). The
combined organic fractions were carefully washed with saturated aqueous
NaHCO3 (50 mL), dried, and evaporated. The residue was dissolved in a
small amount of CHCl3 and filtered through SiO2 (25 g) to remove im-
purities with very high and very low Rf values (eluent CHCl3/MeOH
(50:1) or hexane/EtOAc (2:1); yields 59–80%).
Compound 3-NHCH2CH2-maleimide:
A mixture of sodium salt 1a
(21 mg, 30 mmol) and NaOH (8 mg, 200 mmol) was suspended in DMF
(5 mL) and N-(2-aminoethyl)maleimide trifluoroacetate (39 mg,
153 mmol) was added, followed by HATU (27 mg, 71 mmol) to give a
clear yellow solution with weak fluorescence, which was stirred overnight
at RT. The solvent was evaporated in vacuo (1 mbar) and the glassy resi-
due was separated on SiO2 (25 g) to give the photoactive substance (Rf =
0.8; eluent MeOH/CHCl3 3:1) as a red foam (10 mg). The title compound
(tR =8.8 min) was finally purified from the impurity (tR =10.5 min) by
using HPLC (eluent: A/B 80:20–50:50 in 25 min, 254 nm) and isolated as
red solid after lyophilization (6 mg, 26% OF due to the presence of tri-
fluoroacetic acid in the eluent). 1H NMR (600 MHz, D2O): d=1.93
N-(2,2,2-Trifluoroethyl)-m-hydroxyaniline (7-H):[31] M.p. 388C; 1H NMR
(300 MHz, CDCl3): d=3.67 (t, 3J
ACHTUNGTRENNUNG
(quint, 3J
4H), 3.33 (t, 3J
2H), 6.97 (s, 2H), 7.48 (m, 1H), 7.83 ppm (m, 3H); ESI-MS (negative
A
ACHTUNGERTN(NUNG H,H)=7.0 Hz, 2H), 2.76 (m,
(t, 4J
N
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
10772
www.chemeurj.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 10762 – 10776

Rhodamine Spiroamides
FULL PAPER
147.9 (C), 156.5 ppm (C); 19F NMR (282.4 MHz, [D6]acetone): d=
1508C, was added to a solution of m-anisidine (7.4 g, 60 mmol) in acetone
(300 mL). The mixture was stirred at RT for 4 h, then the acetone was
evaporated in vacuo and the residue was dissolved in EtOAc (500 mL),
washed with water (100 mL) and brine (100 mL), and dried. The residue
was purified on SiO2 (100 g; eluent hexane/EtOAc 8:1) to give an oil that
crystallized into a colorless solid with (8.0 g, 66%). M.p. 688C.
+
ꢁ72.3 ppm (t, 3J
A
C
[MꢁCF3]+; HRMS: m/z calcd for C8H8NOF3: 192.06307; found:
192.06306 [M+H]; elemental analysis calcd (%) for C8H8F3NO: C 50.27,
H 4.22, N 7.33; found: C 49.92, H 3.99, N 7.01.
N-Ethyl-N-(2,2,2-trifluoroethyl)-m-hydroxyaniline
(300 MHz, CDCl3): d=1.18 (t, 3J(H,H)=7 Hz, 3H), 3.45 (q, 3J
7 Hz, 2H), 3.81 (q, 3J(H,F)=9.0 Hz, 2H), 5.39 (brs, 2H), 6.29 (ddd, 3J-
(H,H)=10 Hz, 4J(H,H)=2.3 Hz, 5J
(H,F)=0.7 Hz, 1H), 6.30 (m, 1H),
6.38 (dd, 3J(H,H)=8.3 Hz, 4J(H,H)=2.5 Hz, 1H), 7.17 ppm (t, 3J
(H,H)=
8.1 Hz, 1H); 13C NMR (75.5 MHz, CDCl3): d=11.0 (Me), 45.5 (CH2),
51.9 (q, 2J(C,F)=33 Hz), 100.2 (CH), 104.9 (C), 105.8 (CH), 125.5 (q, 1J-
(7-Et):
1H NMR
A
ACHTUNGTRENNUNG
7-Methoxy-N,2,2,4-tetramethyl-1,2-dihydroquinoline (12): Compound 11
(6.85 g, 33.7 mmol), Cs2CO3 (6.2 g, 19 mmol), and MeI (8 mL, 128 mmol)
were added to DMF (18 mL) and vigorously stirred at 908C for 3 h. The
solvent was evaporated in vacuo (1 mbar), and the residue was taken up
in diethyl ether (200 mL) and water (100 mL). The aqueous layer was
acidified neutralized with 1m HCl and extracted with diethyl ether
(100 mL). The combined organic fractions were washed with brine
(100 mL) and dried. After evaporation of the solvent, the title compound
was isolated by chromatography on SiO2 (200 g; eluent hexane/EtOAc
12:1) to give a colorless oil (5.2 g, 71%).
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG(C,F)=282 Hz), 130.2 (CH), 148.9 (C), 156.2 ppm (C); ESI-MS (negative
mode): m/z: 218 (100) [MꢁH]ꢁ, 435 (79) 2
N
E
1H NMR (300 MHz, CDCl3): d=1.27 (s, 6H), 1.93 (d, 4J
ACHTUNGTRENNUNG
Compound 14-H: A solution of compound 13 (1.82 g, 8.96 mmol) and
phthalic anhydride (1.04 g, 7.03 mmol) in propionic acid (6 mL) was
heated with p-toluene sulfonic acid monohydrate (120 mg) at 1608C for
24 h under Ar. The solvent was evaporated in vacuo and the residue was
dissolved in CH2Cl2 (150 mL). The organic solution was washed with sa-
turated aqueous NaHCO3 (50 mL) and aqueous H2SO4 (0.5m, 50 mL)
and dried. After evaporation of the solvent, the title compound was iso-
lated by chromatography on SiO2 (350 g; eluent CH2Cl2/MeOH 15:1!
0:1) to give a dark blue foam (1.3 g, 56%); HPLC area: 99%, tR =
21.1 min (eluent: A/B 60:40–0:100 in 25 min, 590 nm). 1H NMR
(300 MHz, CDCl3): d=1.27 (s, 6H), 1.30 (s, 6H), 1.62 (s, 6H), 2.83 (s,
3H), 2.73 (s, 3H), 5.14 (q, 4J
1.9 Hz, 1H), 6.09 (dd, 3J
4J
G
E
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
(Meꢂ2), 30.7 (Me), 56.3 (C), 98.5 (CH), 102.5 (CH), 116.9 (C), 124.2
(CHꢂ2), 127.3 (CH), 127.8 (C), 156.4 ppm (C); ESI-MS (positive mode):
m/z: 204 (100) [M+H]+; HRMS: m/z calcd for C13H17NO: 204.13829;
found: 204.13829 [M+H].
Compound 10-H: A mixture of powdered phthalic anhydride (1.23 g,
7.50 mmol) and aniline 7-H (1.08 g, 5.65 mmol) were heated with stirring
under Ar at 160–1708C for 3 h. Then a second portion of 7-H (0.862 g,
4.51 mmol) and H3PO4 (5 mL, 85%) were added and the heating was
continued for another 3 h. After cooling to RT, MeOH (10 mL) and
water (2 mL) were added, the solution was seeded with powdered 10-H,
and the mixture was stirred at RT overnight. The resultant red solid was
collected on a glass filter and recrystallized from MeOH to give the title
compound as a bright red solid (732 mg, 30%); HPLC area: 95%, tR =
9.4 min, impurity with tR =6.9 min (eluent: A/B 60:40–0:100 in 25 min,
254 nm). 1H NMR (300 MHz, [D6]acetone): d=3.97 (brs, 2H, NH), 4.37
6H), 5.17 (s, 2H), 6.29 (s, 2H), 6.30 (s, 2H), 7.14 (dd, 3J
4J(H,H)=1 Hz, 1H), 7.57 (mc, 2H), 8.02 ppm (dd, 3J(H,H)=6.9 Hz, 4J-
(H,H)=1.3 Hz, 1H); 13C NMR (75.5 MHz, CDCl3): d=18.2 (2ꢂMe),
ACHTUNGTREN(NUNG H,H)=6.8 Hz,
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
27.7 (Me), 28.3 (Me), 31.1 (2ꢂMe), 56.9 (C), 96.9 (2ꢂCH), 106.6 (C),
120.0 (C), 122.1 (2ꢂCH), 124.5 (CH), 125.4 (CH), 126.7 (C), 129.2 (CH),
129.3 (2ꢂCH), 134.0 (CH), 147.7 (C), 153.2 (C), 169.8 ppm (CO); ESI-
MS (positive mode): m/z: 519 (100) [M+H]+.
Compound 14-SO3H:[33] Rhodamine 14-H (1.0 g, 1.9 mmol) was dissolved
at 08C in conc. H2SO4 (20 mL, 96%) and the resultant orange–red solu-
tion was stirred at RT overnight. Then the reaction mixture was carefully
diluted with dioxane (50 mL) and diethyl ether (500 mL) and kept at 08C
for 2 h, then the solvent was decanted from the dark blue precipitate.
The solid was dissolved in cold water, and the title compound was isolat-
ed by RP chromatography on Polygoprep 60-50 C18 (100 g; eluent meth-
anol/water 1:1). Lyophilization gave the title compound as a voluminous
dark blue crystalline mass (1.03 g, 76%); HPLC area: 92%, tR =13.7 min
(eluent: A/B 80:20–50:50 in 25 min, 590 nm). 1H NMR (300 MHz,
CD3OD): d=1.49 (s, 6H), 1.53 (s, 6H), 3.14 (s, 6H), 3.53 (A part of AB
system), 3.80 (B part of AB system, JAB =14 Hz, S 4H), 5.87 (s, 2H), 6.73
(q, 3J
[D6]DMSO)), 7.50 (2ꢂdd, 3J
(2ꢂt, 3J(H,H)=7.4 Hz), 7.90 (2ꢂt, 3J
(2ꢂdd, 3J(H,H)=7.7 Hz, 4J(H,H)=1.6 Hz, 1H); 13C NMR (75.5 MHz,
(CD3)2SO): d=43.7 (q, 2J
(C,F)=32 Hz), 97.9 (C4/5), 107.2 (C8a/8b),
109.9 (C1/8), 123.8 (CH), 124.3 (CH), 125.5 (q, 1J
(C,F)=281 Hz), 126.4
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
(C), 128.3 (C2/7), 129.7 (CH), 135.1 (CH), 149.6 (C3/6 or C4a/4b), 152.0
(C4a/4b or C3/6), 152.1 (C2’), 168.5 ppm (CO); 19F NMR (282.4 MHz,
[D6]DMSO): d=ꢁ70.1 ppm (t, 3J
ACHTUNGTRNE(NUNG H,F)=9.0 Hz); HRMS: m/z calcd for
C24H16N2O3F6: 495.11379; found: 495.11374 [M+H].
Compound 10-Et: The title was obtained as described above by using
amine 7-Et (3.4 g, 15.5 mmol) and phthalic anhydride (24 mmol) and
heating at 1608C for 3 h followed by heating with an additional portion
of 7-Et (3.5 g, 16 mmol) and H3PO4 (7.0 mL) at 1608C for 3 h. The warm
reaction mixture was poured into aqueous MeOH (300 mL, 1:1), Et3N
(5 mL) was added to the solution, and the precipitate was filtered off and
dried in vacuo to give the title compound in the CF as a light pink solid
(4.5 g, 51%); HPLC area: 100%, tR =10.1 min (eluent: A/B 50:50–0:100
in 25 min, 254 nm). M.p. 101–1028C;1H NMR (300 MHz, CDCl3): d=1.20
(s, 2H), 7.29 (s, 2H), 7.31 (dd, 3J
7.60 (mc, 2H), 8.06 ppm (dd, 3J(H,H)=7 Hz, 4J
ACHTUNGTRENNUNG
N
ACHTUNGTREN(NUGN H,H)=1.6 Hz, 1H),
U
HRMS: m/z calcd for C34H34N2O9S2: 679.17785; found: 679.17774
[M+H].
Compound 1g: Compound 14-SO3H (110 mg, 0.162 mmol) and 2-aminoe-
thanol (120 mg, 1.97 mmol) were dissolved in dry DMF (1 mL), then
HATU (100 mg, 0.262 mmol) was added, followed by Et3N (0.1 mL,
0.7 mmol). The solution gradually decolorized. The mixture was kept at
408C overnight, then the DMF was removed in vacuo and the residue
was dissolved in methanol/water (1:1, 2 mL) and applied onto a column
with Polygoprep 60-50 C18 (50 g; eluent methanol/water mixture 1:1) to
give 1g·nH2NCH2CH2OH (Rf ~0.5) as a colorless oil (180 mg); HPLC
area: 97%, tR =12.9 min (eluent: A/B 80:20–50:50 in 25 min, 590 or
(t, 3J
(H,F)=8.9 Hz, 4H), 6.44 (dd, 3J
6.59–6.65 (m, 4H), 7.19 (d, 3J
(H,H)=7.6 Hz, 1H), 7.56–7.70 (m, 2H),
8.00 ppm (d, 3J(H,H)=7.2 Hz, 1H); 13C NMR (75.5 MHz, CDCl3): d=
11.3 (Me), 45.8 (CH2), 52.0 (q, 2J
(C,F)=33 Hz), 84.0 (C), 99.5 (CH),
108.4 (C), 108.9 (CH), 124.0 (CH), 124.8 (CH), 125.3 (q, 2J
(C,F)=
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
1
AHCTUNGTRENNUNG
254 nm). H NMR (300 MHz, CD3OD, CF): d=1.29 (s, 6H), 1.36 (s, 6H),
2.83 (s, 6H), 2.95 (m, H2NCH2CH2OH), 3.17 (t, 3J
ACHTUNGTRENNUNG
282 Hz), 127.3 (C), 129.0 (CH), 129.5(CH), 134.7 (CH), 149.0 (C), 152.8
(C), 152.9 (C), 169.6 ppm (CO); 19F NMR (282.4 MHz, CDCl3): d=
ꢁ70.3 ppm (t, 3J
ACHTUNGTRENNUNG(H,F)=8.9 Hz); ESI-MS (positive mode): m/z: 551 (100)
AHCTUNGTRENNUNG
[M+H]+; HRMS: m/z calcd for C30H29N3O10S2: 551.17639; found:
551.17637 [M+H]; elemental analysis calcd (%) for C28H24F6N2O3: C
61.09, H 4.39, N 5.09; found: C 60.80, H 4.53, N 4.93.
(positive
mode):
m/z:
722
(52)
[M+H]+,
844
(89)
[M+2H2NCH2CH2OH+H]+, 1063.8 (88); ESI-MS (negative mode): m/z:
720 (100) [MꢁH]ꢁ, 781 (40) [MꢁH+H2NCH2CH2OH]ꢁ. By using ion-ex-
change resin in the H+ form, 180 mg of the colorless
7-Methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (11):[32,29] Ytterbium
ACHTUNGTNER(NUNG III)
triflate (2.5 g, 5 mmol), which was previously dried in vacuo (1 mbar) at
Chem. Eur. J. 2009, 15, 10762 – 10776
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10773

V. N. Belov, S. W. Hell et al.
1g·nH2NCH2CH2OH were transformed into 99 mg of 15-H-OF (85%
yield), which was isolated as dark blue foam after lyophilisation.
1H NMR (300 MHz, D2O, OF): d=1.50 (s, 6H), 1.60 (s, 6H), 3.13 (s,
Compound 19b: M.p. 1288C (decomp.); 1H NMR (300 MHz, CDCl3):
d=3.71 (s, 3H), 3.73 (m, 4H), 4.15 (t, 3J
ACHTUNGTRENNUNG
2H), 6.30 (dd, 3J
G
(H,H)=8.6 Hz, 4J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
(H,H)=2.4 Hz, 2H), 6.61 (d, 3J
ACHTUNGTRENNUNG
6H), 3.15 (t, 3J(H,H)=6.1 Hz, 2H), 3.27 (t, 3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(m, 1H), 7.53 (mc, 2H), 7.59 (m, 2H), 8.00 (m, 1H); 19F NMR
(282.4 MHz, CDCl3): d=ꢁ72.2 ppm (t, 3J
ACHTUNGERTN(NUNG H,F)=8.7 Hz); HRMS: m/z
AHCTUNGTRENNUNG
calcd for C35H24N4O6F6: 711.16728; found: 711.16743 [M+H]; elemental
analysis calcd (%) for C35H24F6N4O6: C 59.16, H 3.40, N 7.88; found: C
58.88, H 3.53, N 8.02.
7.84 (m, 2H), 7.96 (m, 1H); ESI-MS (positive mode): m/z: 766 (10)
[MꢁH+2Na]+; ESI-MS (negative mode): m/z: 720 (100) [MꢁH]ꢁ.
Compound 15-CONHS: Compound 15-H-OF (11 mg, 43 mmol) was sus-
pended in MeCN (0.5 mL) and CH2Cl2 (0.5 mL), then Et3N was added
(50 mL, 350 mmol), followed by di(N-succinimidyl)carbonate (11 mg,
43 mmol). The clear dark blue–violet solution was stirred at RT overnight
and gradually became colorless. Analysis by using HPLC indicated that
the conversion was only about 15%. Di(N-succinimidyl)carbonate
(40 mg, 156 mmol) was added and the reaction mixture was heated at
508C. After 2 h, HPLC analysis indicated that the reaction mixture con-
tained 77% of a substance with tR =14.3 min, 7% of the starting material
(tR =12.9 min), and 6% of an impurity with tR =9.2 min (eluent: A/B
80:20–50:50 in 25 min, 590 nm). The solvents were evaporated, then the
residue was dissolved in water with trifluoroacetic acid (0.1% (v/v),
0.5 mL) and centrifuged. The title compound was isolated from the su-
pernatant solution by using HPLC. The blue fluorescent eluent, which
Compound 19c: Decomposed with melting on rapid heating up to
ꢀ1708C; 1H NMR (300 MHz, [D6]acetone): d=1.16 (t, 3J
ACHTUNGTRENNUNG
6H), 3.53 (q, 3J(H,H)=7.1 Hz, 4H), 3.68 (s, 3H), 4.12 (q, 3J
ACTHNUGTRENNUGN ACHTUNGTRENNUNG
9.3 Hz, 2H, NH), 4.35 (s, 2H), 6.58 (dd, 3J
2.7 Hz, 2H), 6.62 (d, 4J(H,H)=2.5 Hz, 2H), 6.77 (d, 3J
2H), 7.09 (m, 1H), 7.57 (m, 3H), 7.66 (dd, 3J
(H,H)=8.7 Hz, 4J
ACHUTGTNRENNUG ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
0.6 Hz, 1H), 7.78 (dd, 4J(H,H)=1.9 Hz, 5J
ACTHNUGRTNENUG AHCTUNGTRNEN(UGN H,H)=0.6 Hz, 1H), 7.96 ppm
(m, 1H); 13C NMR (125.7 MHz, [D6]acetone): d=11.5 (Me), 39.3 (CH2),
2
46.4 (CH2N), 51.9 (q, J
N
109.2 (C), 110.6 (CH), 119.9 (CH), 123.3 (CH), 123.4 (CH), 123.6 (CH),
126.9 (q, 1J
AHCTUNGTRENNUNG
(C), 134.9 (CH), 144.4 (C), 149.8 (C), 153.2 (C), 154.6 (C), 167.3 (CO),
167.0 (CO), 168.7 (CO), 168.8 ppm (CO); ESI-MS (positive mode): m/z:
ACTHNUTRGNEUNG
767 (18) [M+H]+, 789 (26) [M+Na]+, 1555 (100) 2[M+Na]+, 1586.8 (56);
contained
a substance with tR =14.3 min, was evaporated in vacuo
ESI-MS (negative mode): m/z: 765 (39) [MꢁH]ꢁ, 1577 (100)
[M+HCOO]ꢁ; elemental analysis calcd (%) for C39H32F6N4O6·0.5CHCl3:
C 57.41, H 3.96, N 6.78; found: C 57.86, H 3.70, N 6.77.
(1 mbar) until the solution froze to remove MeCN. The solid icy residue
was lyophilized and the title compound was isolated as voluminous dark
blue foam. 1H NMR (600 MHz, D2O, OF): d=1.43 (s, 6H), 1.52 (s, 6H),
Compound 19e: M.p. 1728C (decomp.); 1H NMR (300 MHz,
2.91 (s, 4H), 3.05 (s, 6H), 3.39 (t, 3J
ACTHNUTRGNE(NUG H,H)=4.8 Hz, 2H), 3.65 (A part of
[D6]acetone): d=1.24 (s, 6H), 1.29 (s, 6H), 1.62 (d, 4J
6H), 2.83 (s, 6H), 3.68 (s, 3H), 4.35 (s, 2H), 5.23 (q, 4J
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
AB system), 3.83 (B part of AB system, JAB =14.3), 3.99 (t, 3J
5 Hz, 2H), 5.90 (s, 2H), 6.62 (s, 2H), 7.05 (s, 2H), 7.54 (d, 3J
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
2H), 6.24 (s, 2H), 6.44 (s, 2H), 7.12 (m, 1H), 7.57 (mc, 2H), 7.68 (m,
7.3 Hz, 1H), 7.76 (m, 2H), 7.87 ppm (m, 1H); ESI-MS (negative mode):
m/z: 861 (100) [MꢁH]ꢁ; HRMS: m/z calcd for C41H42N4O13S2: 863.22626;
found: 863.22594 [M+H].
2H), 7.89 (dd, 4J(H,H)=1.6 Hz, 5J
N
ACHTUNGTRENNUNG
1H); 13C NMR (125.7 MHz, [D6]acetone): d=18.4 (Me), 27.6 (Me), 28.2
(Me), 31.3 (Me), 39.3 (CH2N), 52.7 (OMe), 57.3 (C), 67.8 (C), 98.3 (2ꢂ
CH), 100.6 (CH), 119.7 (CH), 119.7 (CH), 121.0 (C), 121.9 (2ꢂCH),
124.1 (CH), 124.4 (CH), 124.5 (CH), 127.2 (C), 128.8 (C), 129.5 (CH),
129.7 (C), 130.2 (CH), 130.3 (CH), 133.4 (C), 134.7 (CH), 144.7 (C),
147.6 (C), 153.0 (C), 154.7 (C), 167.2 (CO), 167.4 (CO), 168.6 (CO),
168.7 ppm (CO); ESI-MS (positive mode): m/z: 735 (100) [M+H]+;
HRMS: m/z calcd for C45H42N4O6: 735.31771; found: 735.31788 [M+H].
Compound 17: The title compound was obtained from 8-hydroxyjuloli-
dine 16 (1.18 g, 6.26 mmol) and perfluorophthalic anhydride (1.0 g,
4.54 mmol) in propionic acid (7 mL) with p-toluenesulfonic acid monohy-
drate (80 mg) under the conditions described for rhodamine 14-H. The
solvent was evaporated in vacuo and the residue was separated on Poly-
goprep 60-50 C18 (100 g; eluent methanol/water 1:1!9:1 with 0.1% v/v
TFA) to give
a
shiny dark green solid (0.869 g, 49%). 1H NMR
1
Compound 19 f: Decomp. >2808C; H NMR (600 MHz, CDCl3): d=1.86
(300 MHz, CDCl3): d=1.88 (s, 6H), 1.94 (mc, 3H), 2.05 (s, 3H), 2.64–2.87
(mc, 4H), 1.97 (mc, 4H), 2.53 (mc, 4H), 2.81 (mc, 4H), 3.12 (m, 8H), 3.72
3
(m, 3H), 2.95 (apparent t, J
N
(s, 3H), 4.34 (s, 2H), 6.22 (s, 2H), 7.29 (dd, 3J(H,H)=8.2 Hz, 4J
ACTHNUGTRENNUGN ACHTUNGTRENNUNG
(s, 2H); 19F NMR (282 MHz, CDCl3): d=ꢁ138.4 (ddd, J=24, 13, and
3 Hz), ꢁ140.4 (ddd, J=22, 13, and 2 Hz), ꢁ153.0 (ddd, J=24, 20, and
3 Hz), ꢁ159.3 ppm (dt, J=21 and 2 Hz); ESI-MS (positive mode): m/z:
585 (100) [M+Na]+; HRMS: m/z calcd for C32H26F4N2O3: 653.19523;
found: 563.19540 [M+H].
1.9 Hz, 1H), 7.61 (d, 3J(H,H)=8.2 Hz, 1H), 7.66 ppm (d, 4J
ACTHNUGTRENNUGN ACHTUNGTRENNUNG
1.9 Hz, 1H); 13C NMR (125.7 MHz, CDCl3; due to low intensities, signals
from the fluorinated carbons were not recorded): d=21.0 (CH2), 21.2
(CH2), 21.8 (CH2), 27.2 (CH2), 38.7 (CH2N), 49.3 (CH2N), 49.8 (CH2N),
52.6 (OMe), 66.7 (C), 102.2 (C), 108.0 (C), 117.7 (C), 120.7 (CH), 122.9
(2ꢂCH), 124.0 (CH), 128.8 (C), 130.1 (CH), 132.5 (C), 142.0 (C), 144.3
(C), 147.5 (C), 162.7 (CO), 166.7 (CO), 166.9 (CO), 167.9 ppm (CO);
ESI-MS (positive mode): m/z: 779 (100) [M+H]+; 801 (27) [M+Na]+;
General method for the amidation of rhodamines 10, 10-H, 14-H, and 17
with aromatic amine 18; preparation of the methyl esters 19b–f: Dry
rhodamine 10-H, 10-Et, 14-H, or 17 (2.5 mmol) was suspended in dry 1,2-
dichloroethane (50 mL) and POCl3 (2 mL, 21 mmol) was added, then the
mixture was heated at reflux under Ar for 4 h. All volatile materials
were evaporated in vacuo (1–1.5 mbar) into a trap that was cooled with
dry ice, and the residue was flushed with argon. Dry acetonitrile (50 mL)
was added to the flask, followed by amine 18 (2.7 mmol, 632 mg) and
triethylamine (1 mL, 7 mmol). The reaction mixture was heated at 908C
(bath temp.) for 24 h, then the solvent was evaporated in vacuo. The resi-
due was dissolved in CHCl3 (100 mL) and the organic solution was
washed with water, 1m aqueous HCl, water, and saturated aqueous
NaHCO3 (100 mL each) and dried. After evaporation of the solvent, the
residue was dissolved in the minimum amount of warm dichloromethane
and purified on SiO2 (50 g; eluent hexane/EtOAc 4:1!1:1!1:4 with
0.1% Et3N) to give solutions of methyl esters 19b–f, which moved on the
silica gel as yellowish bands. Colorless spots of 19b–f on TLC (UV detec-
tion at l=254 nm) turned red in several minutes when the dry plates
were exposed to normal laboratory light and air. Evaporation of the sol-
vents gave oils that crystallized on trituration with MeOH or aqueous
MeOH.
[M+H]+; 1557 (19) 2[M+H]+; 1579 (11) 2[M+Na]+; HRMS:
1557 (19) 2ACTHUNGTRENNNGU AHCTUNRTGENNUGN CAHTNUGTRENNGUN
m/z calcd for C43H34F4N4O6: 779.24872; found: 779.24912 [M+H].
General procedure for the cleavage of methyl esters 19b–f to lithium
salts 1b–f: Methyl esters 19b–f (0.5 mmol) were dissolved or suspended
in dry EtOAc (7 mL), then LiI (260 mg, 1.94 mmol) was added and the
mixture was heated in a closed glass thick-wall tube for 48 h (bath temp.
908C). After cooling to RT, the precipitated LiOAc was removed by fil-
tration and washed with EtOAc (2ꢂ1 mL), then dry diethyl ether
(50 mL) was added slowly to the filtrate (concentrated in vacuo to about
half of its volume) with stirring. Precipitated lithium salts 1b–f were col-
lected on a filter, washed with dry diethyl ether, and dried in vacuo. If
THF was used as a solvent, the complete conversion took 4 d (and no
AcOLi formed; it was not necessary to filter the reaction mixture before
precipitating the corresponding lithium salts with diethyl ether).
Compound 1b: The title compound was obtained as a yellow–brown
powder after heating a mixture of 19b (35 mg) and LiI (26 mg) in EtOAc
1
(1 mL) for 20 h (24 mg, 69%). H NMR (300 MHz, CD3OD): d=3.79 (q,
4
3J
C
E
G
10774
www.chemeurj.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 10762 – 10776

Rhodamine Spiroamides
FULL PAPER
2.1 Hz, 2H), 6.41 (d, 4J
2H), 7.11 (m, 1H), 7.18 (dd, 3J
7.42 (dd, 4J(H,H)=1.8 Hz, 5J
(H,H)=0.5 Hz, 2H), 7.59 (m, 3H),
7.98 ppm (ddd, 3J(H,H)=6.1 Hz, 4J(H,H)=1.3 Hz, 5J
(H,H)=0.5 Hz,
1H); 13C NMR (125.7 MHz, CD3OD): d=42.1 (CH2N), 45.8 (q, J
G
G
the course of opening of the phthalimide ring (d=3.85/3.86 (s, NCH2),
6.74 (2ꢂdd, J=8 and 2 Hz), 7.00 (d, J=2 Hz), 7.32 m, 7.40 ppm (d, J=
8 Hz)). The carboxylic acid, which corresponds to lithium salt 1 f, was pu-
rified in the OF by HPLC (injection of a filtered solution of 1 f in
MeCN/water (1:2), followed by gradient elution with the solvents and
0.1% TFA): tR =18.6 min (eluent: A/B 70:30–0:10 in 25 min, 254 nm;
tR =15.3/15.5 min for the impurities).
G
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
G
E
33.5 Hz), 69.5 (C), 100.0 (2ꢂCH), 108.9 (C), 111.3 (2ꢂCH), 121.9 (CH),
124.3 (CH), 124.4 (CH), 125.0 (C), 125.2 (CH), ꢀ127 (q, 1J
ACHTUNGTRNE(NUNG C,F)=
280 Hz), 128.7 (CH), 129.5 (2ꢂCH), 130.0 (CH), 130.8 (C), 131.3 (C),
132.1 (CH), 134.4 (C), 135.3 (CH), 143.5 (C), 150.8 (C), 154.0 (C), 154.7
(C), 167.3 (CO), 168.8 (CO), 168.9 (CO), 169.8 (CO), 174.2 ppm (CO);
19F NMR (282.4 MHz, CDCl3): d=ꢁ73.7 ppm (t, 3J
ACHTUNGTREN(NUGN H,F)=9.0 Hz); ESI-
Acknowledgements
MS (positive mode): m/z: 703 (100) [Mꢁ+H+Li]+; ESI-MS (negative
mode): m/z: 695 (100) [M]ꢁ. The carboxylic acid, which corresponds to
lithium salt 1b, was isolated in the OF by using HPLC (injection of a fil-
tered solution of 1b in MeCN/water (1:2), followed by gradient elution
with the solvents and 0.1% TFA): tR =12.6 min (eluent: A/B 60:40–0:10
in 25 min, 254 nm).
This work was supported by the Max-Planck-Gesellschaft, the Bundesmi-
nisterium fꢃr Bildung und Forschug (BMBF) grant (Biophotonics III pro-
gram), the European Union through a Marie Curie fellowship (to M.B.)
and a project grant (to S.W.H.) within the NEST/Adventure program
(SPOTLITE). The authors are very grateful to R. Machinek, Dr. H.
Frauendorf, and their co-workers for recording numerous NMR and
mass spectra in the Institut fꢃr Organische und Biomolekulare Chemie
(Georg-August-Universitꢄt, Gçttingen). The authors are grateful to Mr.
J. Jethwa for critical reading of the manuscript and to Mr. H. Sebesse for
his help in the preparation of Figure 6.
Compound 1c: The title compound was isolated as a light brown powder
after heating 19c (816 mg) and LiI (584 mg) in THF (5 mL) for 4 d
(702 mg, 87%; isolated as an oil that crystallized after triturating with di-
ethyl ether). 1H NMR (300 MHz, CD3OD): d=1.14 (t, 3J
ACHTUNGTRENNUNG
6H), d=3.47 (dq, 5J(H,F)=1.3 Hz, 3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
4
3
2.5 Hz, 2H), 6.53 (d, JACHTUNGTRENNUNG(H,H)=3 Hz, 2H), 6.65 (d, JACHTUGNTRENNNUG
7.05 (m, 1H), 7.23 (dd, J
4J(H,H)=1.8 Hz, 5J
3
4
ACHUTGTNREN(NUG H,H)=8.1 Hz, JACHTNUGTRENNUNG
[1] E. Abbe, Arch. Mikrosk. Anat. Entwicklungsmech. 1873, 9, 413–420.
[2] For reviews, see: a) S. W. Hell, Science 2007, 317, 1749–1753; b) M.
Heilemann, P. Dedecker, J. Hofkens, M. Sauer, Laser Photonics Rev.
2009, 3, 180–202; c) D. Evanko, Nat. Methods 2009, 6, 19–20;
d) S. W. Hell, Nat. Methods 2009, 6, 24–32; e) J. Lippincott-
Schwartz, S. Manley, Nat. Methods 2009, 6, 21–23; for recent re-
ports, see: f) S. Van de Linde, U. Endesfelder, A. Mukherjee, M.
Schꢃttpelz, G. Wiebusch, S. Wolter, M. Heilemann, M. Sauer, Photo-
chem. Photobiol. Sci. 2009, 8, 465–469; g) V. Westphal, S. O. Rizzoli,
M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, Science 2008, 320,
246–249; h) R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A.
Egner, S. W. Hell, Nat. Methods 2008, 5, 539–544; i) B. Hein, K. I.
Willig, S. W. Hell, Proc. Natl. Acad. Sci. USA 2008, 105, 14271–
14276; j) M. Andresen, A. C. Stiel, J. Fçlling, D. Wenzel, A. Schçnle,
A. Egner, C. Eggeling, S. W. Hell, S. Jakobs, Nat. Biotechnology
2008, 26, 1035–1040; k) S. E. Irvine, T. Staudt, E. Rittweger, J. En-
gelhardt, S. W. Hell, Angew. Chem. 2008, 120, 2725–2728; Angew.
Chem. Int. Ed. 2008, 47, 2685–2688; l) M. C. Lang, T. Staudt, J. En-
gelhardt, S. W. Hell, New J. Phys. 2008, 10, 1–13; m) A. Punge, S. O.
Rizzoli, R. Jahn, J. D. Wildanger, L. Meyer, A. Schçnle, L. Kastrup,
S. W. Hell, Microsc. Res. Tech. 2008, 71, 644–650; n) B. Harke, C.
Ullal, J. Keller, S. W. Hell, Nano Lett. 2008, 8, 1309–1313; o) J. Fçl-
ling, S. Polyakova, V. Belov, A. van Blaaderen, M. L. Bossi, S. W.
Hell, Small 2008, 4, 134–142.
[3] a) E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Ole-
nych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F.
Hess, Science 2006, 313, 1642–1645; b) H. Shroff, C. G. Galbraith,
H. White, J. Gilette, S. Olenych, M. W. Davidson, E. Betzig, Proc.
Natl. Acad. Sci. USA 2007, 104, 20308–20313.
[4] a) M. J. Rust, M. Bates, X. Zhuang, Nat. Methods 2006, 3, 793–796;
b) M. Bates, B. Huang, G. T. Dempsey, X. Zhuang, Science 2007,
317, 1749–1753; c) B. Huang, W. Wang, M. Bates, X. Zhuang, Sci-
ence 2008, 319, 810–813; d) B. Huang, S. A. Jones, B. Brandenburg,
X. Zhuang, Nat. Methods 2008, 5, 1047–1052.
N
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
130.5, 131.1, 131.8, 134.3, 135.3, 143.4, 150.2 (2ꢂ), 153.9 (2ꢂ), 154.5,
168.6 (CO), 168.8 (CO), 169.7 (CO), 174.2 ppm (CO); 19F NMR
(282.4 MHz, CDCl3): d=ꢁ73.7 ppm (t, 3J
ACHTUNGTNER(NUNG H,F)=9.0 Hz); ESI-MS (posi-
tive mode): m/z: 797 (90) [Mꢁ+2Na]+, 1523 (100), 1539 (45); ESI-MS
(negative mode): m/z: 751 (100) [M]ꢁ, 1503 (82), 2
[Mꢁ+Li].
[Mꢁ+H] 1509 (100) 2-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
Compound 1d: The preparation and properties of compound 1d have
been reported in reference [9].
Compound 1e: The title compound was isolated as a green powder after
heating 19e (117 mg) and LiI (56 mg) in EtOAc (1 mL) for 36 h (124 mg;
the product contained ꢀ10% LiI that was very difficult to remove).
1H NMR (300 MHz, CD3OD): d=1.21 (s, 6H), 1.28 (s, 6H), 1.61 (d, 4J-
A
G
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
(Me), 28.2 (Me), 31.4 (Me), 42.1 (NCH2), 57.8 (C), 69.9 (C), 98.6 (2ꢂ
CH), 106.1 (2ꢂC), 121.4 (CH), 121.5 (2ꢂC), 122.2 (2ꢂCH), 124.3 (CH),
124.4 (CH), 125.2 (CH), 127.6 (2ꢂC), 130.0 (C), 130.6 (C), 130.8 (2ꢂ
CH), 131.1 (C), 131.8 (CH), 134.4 (C), 135.3 (CH), 143.6 (C), 148.2 (C),
153.8 (C), 154.8 (C), 168.7 (CO), 168.8 (CO), 169.9 (CO), 174.2 ppm
(CO); ESI-MS (positive mode): m/z: 749 (72) [Mꢁ+Li+Na]+, 765 (100)
[Mꢁ+2Na]+; ESI-MS (negative mode): m/z: 751 (100) [M]ꢁ. The carbox-
ylic acid, which corresponds to lithium salt 1e, was detected in the OF as
a sharp peak in the HPLC (injection of a filtered solution of 1e in
MeCN/water (1:2), followed by gradient elution with the solvents and
0.1% TFA): tR =16.5 min (eluent: A/B 60:40–0:10 in 25 min, 254 nm).
Compound 1 f: The title compound was isolated as a dark blue solid
after heating 19 f (117 mg) of and LiI (48 mg) in boiling EtOAc (2 mL)
for 36 h (95 mg). 1H NMR (300 MHz, CD3OD): d=1.88 (mc, 4H), 1.98
[5] fPALM=fluorescent PALM: a) S. T. Hess, T. P. K. Girirajan, M. D.
Mason, Biophys. J. 2006, 91, 4258–4272; b) M. F. Juette, T. J. Gould,
M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennet, S. T.
Hess, J. Bewersdorf, Nat. Methods 2008, 5, 527–529.
[6] W. E. Moerner, Nat. Methods 2006, 3, 781–782.
[7] K. H. Knauer, R. Gleiter, Angew. Chem. 1977, 89, 116–117; Angew.
Chem. Int. Ed. Engl. 1977, 16, 113–113.
[8] H. Willwohl, J. Wolfrum, R. Gleiter, Laser Chem. 1989, 10, 63–72.
[9] J. Fçlling, V. Belov, R. Kunetsky, R. Medda, A. Schçnle, A. Egner,
C. Eggeling, M. Bossi, S. W. Hell, Angew. Chem. 2007, 119, 6382–
6386; Angew. Chem. Int. Ed. 2007, 46, 6266–6270.
(mc, 4H), 2.58 (mc, 4H), 2.78 (mc, 4H), 3.12 (apparent q, 3J
ACHTUNGTRENNUNG
5.6 Hz, 8H), 4.1 (s, 2H), 6.39 (s, 2H), 7.23 (dd, 3J
(H,H)=1.8 Hz, 1H), 7.39 (d, 4J
ACHTUNGTRENNUNG
N
N
ACHTUNGTRENNUNG
8.1 Hz, 1H); 19F NMR (282.4 MHz, CD3OD): d=ꢁ145.5 (dt, J=19 and
6.7 Hz), ꢁ146.7 (dt, J=18.8 and 3.3 Hz), ꢁ149.8 (dt, J=18.5 and 6.8 Hz),
ꢁ157.0 ppm (dt, J=18 and 3.3 Hz); ESI-MS (positive mode): m/z: 765
(100) [MH+H]+; ESI-MS (negative mode): m/z: 763 (100) [Mꢁ]. The
title lithium salt contained ꢀ9% of both dicarboxylic acids obtained in
Chem. Eur. J. 2009, 15, 10762 – 10776
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10775

V. N. Belov, S. W. Hell et al.
[10] A. Egner, C. Geisler, C. von Middendorf, H. Bock, D. Wenzel, R.
Medda, R. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schçnle,
S. W. Hell, Biophys. J. 2007, 93, 3285–3290.
[11] J. Fçlling, V. Belov, D. Riedel, A. Schçnle, A. Egner, C. Eggeling,
M. Bossi, S. W. Hell, ChemPhysChem 2008, 9, 321–326.
[12] H. Bock, C. Geisler, C. A. Wurm, C. von Middendorf, S. Jakobs, A.
Schçnle, A. Egner, S. W. Hell, C. Eggeling, Appl. Phys. B 2007, 88,
161–165 .
[13] a) M. Heilemann, E. Margeat, R. Kasper, M. Sauer, P. Tinnefeld, J.
Am. Chem. Soc. 2005, 127, 3801–3806; b) J. Vogelsang, R. Kasper,
C. Steinhauer, B. Person, M. Heilemann, M. Sauer, P. Tinnefeld,
Angew. Chem. 2008, 120, 5545–5550; Angew. Chem. Int. Ed. 2008,
47, 5465–5469.
[14] A. Schçnle, S. W. Hell, Nat. Biotechnol. 2007, 25, 1234–1235.
[15] M. Bossi, J. Fçlling, V. N. Belov, V. P. Boyarskiy, R. Medda, A.
Egner, C. Eggeling, A. Schçnle, S. W. Hell, Nano Lett. 2008, 8,
2463–2468.
[20] G. T. Hermanson, Bioconjugate Techniques, Academic Press, San
Diego, 1996, pp. 57–60.
[21] S. Bigotti, A. Volonterio, M. Zanda, Synlett 2008, 958–962.
[22] For a method for the sulfonation of rhodamines, see: F. Mao, W.-Y.
Leung, R. P. Haugland (Molecular Probes), US Patent 6130101,
2000.
[23] The rigidized xanthene fragment in compounds 1a,e, and g improves
the conjugation between the lone electron pair of the nitrogen atom
and the aromatic system and, therefore, decreases the basicity of the
amino group.
[24] See reference [20], pp. 57–59.
[25] A. van Blaaderen, A. Vrij, Langmuir 1992, 8, 2921–2931.
[26] W. Denk, J. H. Strickler, W. W. Webb, Science 1990, 248, 73–76.
[27] J. Fçlling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S.
Jakobs, C. Eggeling, S. W. Hell, Nat. Methods 2008, 5, 943–945.
[28] J. Salazar, S. E. Lopez, O. Rebello, J. Fluorine Chem. 2003, 124,
111–113.
[16] Highly nucleophilic aliphatic amines react sluggishly on heating with
methyl esters of rhodamines in aprotic solvents, and afford the cor-
responding CFs of the primary amides.
[17] Therefore, saponification of the methyl ester group in compounds
19b–f by using aqueous NaOH is accompanied by ring-opening of
the imide and formation of two regioisomeric 2-carboxybenzoylgly-
cines.
[29] For example, see: L. A. Robinson, M. E. Theoclitou, Tetrahedron
Lett. 2002, 43, 3907–3910.
[30] R. Herrmann, H.-P. Josel, K.-H. Drexhage, J. Arden-Jacob (Boeh-
ringer Mannheim GmbH), US Patent 57550409, 1998.
[31] E. R. Bissell, R. W. Swansiger, J. Fluorine Chem. 1978, 12, 293–306.
[32] For example, see: C. S. Yi, S. Y. Yun, J. Am. Chem. Soc. 2005, 127,
17000–17006.
[18] V. P. Boyarskiy, V. N. Belov, R. Medda, B. Hein, M. Bossi, S. W.
Hell, Chem. Eur. J. 2008, 14, 1784–1792.
[33] F. Mao, W.-Y. Leung, R.-P. Haugland, WO9915517, 1999.
[19] Sulfonic acid residues may also result in a slight redshift of the long
wavelength absorption band of 1a (compared with the correspond-
ing unsulfonated heterocycle).
Received: May 19, 2009
Published online: September 16, 2009
10776
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