DOI: 10.1002/chem.200902309
Red-Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy
Kirill Kolmakov, Vladimir N. Belov,* Jakob Bierwagen, Christian Ringemann,
Veronika Mller, Christian Eggeling, and Stefan W. Hell*
[a]
Introduction
Owing to their high photostabilities, high extinction, high
fluorescent quantum yields, and low degree of triplet forma-
tion, rhodamine dyes are widely used as laser dyes and fluo-
rescent markers for labeling proteins, nucleic acids, lipids,
carbohydrates, toxins, hormones, and other biomolecules.
[1]
Besides their use in biological imaging and single-molecule-
based spectroscopy, rhodamines served well in the practical
implementation of some new physical concepts, for example,
those that helped to overcome the diffraction limit in (far-
field) optical microscopy. “Nanoscopic” techniques
[2]
such as
STED (stimulated emission depletion),
[2a,d,h]
PALM (photo-
activation localization microscopy),
[2i,s]
STORM (stochastic
reconstruction microscopy),
[2j,t–v]
and GSDIM (ground-state
depletion with individual molecular return)
[3]
allowed the
optical resolut ion to be improved from about 200–350 nm to
20–35 nm by switching between the dark and the bright
states of a fluorescent marker. However, the rhodamine
family lacks water-soluble compounds that combine high
photostabilities and fluorescence quant um yields with ab-
sorbance and fluorescence band maxima lying in the far-red
optical region.
Biological applications favor fluorescent dyes that absorb
in the far-red or even near-infrared (IR) optical region. In
contrast to visible and above all ultraviolet, excitation at
wavelengths longer than 600 nm i s mostly noninvasive and
minimizes the unwanted background signal originating from
cellular autofluorescence. The most convenient near-IR
laser lines are between 630 and 650 nm, as supplied by He–
Ne, diode, or krypton ion lasers. A number of fluorescent
dyes have been prepared to match these excitation sour-
ces.
[4]
However, a survey of the patent literature revealed
Abstract: Fluorescent markers emitting
in the red are extremely valuable in
biological microscopy since they mini-
mize cellular autofluores cence and in-
crease flexibility in multicolor experi-
ments. Novel rhodamine dyes excitable
with 630 nm laser light and emitting at
around 660 nm have been developed.
The new rhodamines are very photo-
stable and have high fluorescence
quantum yields of up to 80 %, long ex-
cited state lifetimes of 3.4 ns, and com-
paratively low intersystem-crossing
rates. They perform very well both in
conventional and in subdiffraction-res-
olution microscopy such as STED
(stimulated emission depletion) and
GSDIM (ground-state depletion with
individual molecular return), as well as
in single-molecule-based experiments
such as fluorescence correlation spec-
troscopy (FCS). Syntheses of lipophilic
and hydrophilic derivatives starting
from the same chromophore-containing
scaffold are described. Introduction of
two sulfo groups provides high solubili-
ty in water and a considerable rise in
fluorescence quantum yield. The at-
tachment of amino or thiol reactive
groups allows the dyes to be used as
fluorescent markers in biology. Dyes
deuterated at certain positions have
narrow and symmetrical molecular
mass distribution patterns, and are pro-
posed as new tags in MS or LC-MS for
identification and quantification of var-
ious substance classes (e.g., amines and
thiols) in complex mixtures. High-reso-
lution GSDIM images and live-cell
STED-FCS experiments on labeled mi-
crotubules and lipids prove the versatil-
ity of the novel probes for modern
fluorescence microscopy and nanosco-
py.
Keywords: dyes/pigments · fluores-
cence · fluorescent probes · isotopic
labeling · microscopy
[a] Dr. K. Kolmakov, Dr. V. N. Belov, Dipl.-Chem. J. Bierwagen,
Dr. C. Ringemann, Dipl.-Phys. V. Mller, Dr. C. Eggeling,
Prof. Dr. S. W. Hell
Department of NanoBiophotonics
Max P lanck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
Fax: (
+
49)5512012505
E-mail: vbelov@gwdg.de
shell@gwdg.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002 /chem.200902309.
2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2010, 16, 158 – 166
158
only one commercially available rhodamin e dye, namely,
Alexa 633, which was initially reported as a “sulfonated
rhodamine derivative”.
[5a]
Its exact structure was disclosed in
2007.
[5b]
It can be deduced that the photostability of
Alexa 633 is similar to those of the cyanine dyes.
[4]
Here we present novel rhodamine dyes, excitable at
630 nm and emittin g at around 660 nm. They are highly pho-
tostable, have fluorescence quantum yields of up to 80%,
relatively long fluorescence lifetime (3.4 ns), and low inter-
system-crossing rates. The same chromophore-containing
scaffold allows introductio n of various functional groups and
preparation of fluorescent dyes ranging fr om lipophilic to
highly water soluble (hydrophilic), as well as reactive dyes
for specific biological labeling. High-resolution GSDIM
images of the labeled microtubular network in whole (fixed)
cells and live-cell, single-molecule-based STED-FCS (fluo-
rescence correlation spectroscopy) experiments with lipids
illustrate the potential of the novel markers in modern fluo-
rescence microscopy.
Results and Discussion
Background information on red fluorescent dyes: Fluores-
cence microscopy, nanoscopy, and FCS demand markers
with “red” excitation and emission bands, high fluorescence
quantum yields (F
fl
), high oscillator strengths (absorption
coefficients), high photostability, low rates of format ion of
the “dark” triplet state, relatively long lifetimes of the excit-
ed states (> 3 ns), sufficient solubility in water, and a reac-
tive group with a linker for conjugation with biological ob-
jects or other structures of interest. Moreover, the availabili-
ty of lipophilic and hydrophilic derivatives of the same chro-
mophore is desirable, as long as it provides additional flexi-
bility in labeling substances of various polarities.
Hydrophobicity would be advantageous for labeling polar
substances such as lipid head groups, while lipophilic deriva-
tives are useful for labeling nonpolar domains (e.g., lipid
acyl chains).
Commercially available red-emitting dyes include the pre-
viously mentioned rhodamine Alexa 633, the cyanine dyes
Alexa 647 and Cy 5, oxazines such as Evoblue 30, and the
carbopyronine dyes Atto 647N and Atto 635, designed to
match the preferred characteristics. Carbopyronine dyes are
structurally similar to rhodamines and can serve as their
competitive substitutes in the far-red spectral region.
[6]
In
particular, Atto 647N has gained popularity as a dye for la-
beling in “nanoscopic” studies. Being very photostable,
Atto 647N has quite recently been applied for live-cell
video-rate STED imaging.
[7]
In the course of the study, the
movements of synaptic vesicles inside the axons of cultured
neurons were recorded with a spatial resolution of about
60 nm. However, the low polarity of Atto 647N is often dis-
advantageous.
[4]
The Atto 647N-labeled molecules may
strongly stick to glass, for example, to microscope cover
slides or to the walls of microcapillary injection tubes. As a
consequence, Atto 647N proves difficult for in vitro biologi-
cal assays or for proper cellular injection. In addition, being
lipophilic, Atto 647N produces a considerable background
in our immunostaining experiments largely due to its affinity
for mitochondria. Moreov er, when coupled to an antibody
for immunostaining, Atto 647N sometimes displays a strong
increase in the intensity of an additional absorption peak at
around 605 nm that gives no emission at all. The complex
structure of Atto 647N and the other carbopyronine dyes
precludes any further chemical modifications aimed at in-
creased polarity and better solubility in water. On the other
hand, the cyanine dyes Alexa 647 and Cy 5 and the rhoda-
mine Alexa 633 are less photostable and have lower fluores-
cence quantum yields than Atto 64 7N. The following order
of the fluorescence quantum yields and photoresistance was
observed: Atto 633 Atto 647N> Alexa 647 >Alexa 633>
Cy 5.
[4]
Further, cyanine dyes have rather short fluorescence
lifetimes of about 1 ns, and we can rule them out as poten-
tial lead structures for further improvements. The short life-
times of the excited states, presumably low fluorescence
quantum yields, and moderate photostabilities of oxazine
dyes such as Evoblue 30 (for structures, see ref. [4]) make
them poor candidates for further optimization.
The commercially available dyes mentioned above are
widely used nowadays in numerou s microscopic and nano-
scopic studies. Attempts to devise and improve photostable
red-emitting dyes of other classes are being made in various
research groups. Most recent publications on this topic de-
scribe water-soluble terrylenediimides,
[8a,b]
new hydrophilic
BODIPY derivatives,
[8c]
and dicyanomethylene dihydro-
furans.
[8d]
However, some important data on photophysical
properties of the bioconjugates and microscopic applications
of terrylene diimides and dicyanomethylene dihydrofur-
ans
[8a,b,d]
are lacking. Therefore, here we consider and refer
to commercially available dyes (mentioned above and in the
Supporting Information), whose properties and performance
are well described and are always possib le to explore.
The path to novel red-emitting rhodamines: Having ruled
out cyanines, oxazines, and carbopyronines as lead struc-
tures for the reasons mentioned above, we ultimately chose
rhodamines as development candidates. Although quite a
number of diverse rhodamines with a great variety of sub-
stituents have been explored, certain possibilities to shift the
emission of rhodamine dyes to the red region still remain.
A very large bathochromic shift was reported for Rhoda-
mine 700 with the skeleton of the well-known Rhoda-
mine 101, in which the benzoic acid residue is replaced by a
trifluoromethyl group. All rhodamines with a perfluoroalkyl
group at the 9-position absorb and emit above 600 nm.
[8e]
Unfortunately, the presence of a small and very strongly
electron accepting group at C-9 of the xanthene fragment
(“opposite” to the oxygen atom) makes this position very
vulnerable to nucleophilic attack by water. As a result, such
rhodamines decolorize rapidly in aqueous solution and they
cannot be used as scaffolds.
On the other hand, Rhodamine 101 (Rh 101, for structure
see refs. [1a–c,10]) is one of the most stable and brightest
Chem. Eur. J. 2010, 16, 158 – 166 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
159
FULL PAPER
fluorophores known (F
fl
=
1
[10]
). Due to the high degree
of substitution and the planari-
ty of the molecule, its carboxyl
group is sterically hindered,
and therefore coupling reac-
tions with amines require dras-
tic conditions or powerful acti-
vating agents.
[11]
Importantly,
amidation of Rh 101 with sec-
ondary amines results in a red-
shift of the absorption and
emission bands from 560 and
589 nm to 583 and 604 nm, re-
spectively, with no significant
change in F
fl
.
[12]
There are further tools for
achieving the desired redshift.
First, the excitation and emis-
sion bands are considerably
shifted to the red if tetrafluor-
ophthalic anhydride is used for
rhodamine synthesis instead of
phthalic anhydride.
[13]
Second,
aC=C bond may be intro-
duced into the six-membered
ring, fused with the xanthene
fragment in such a way that it
conjugates with the adjacent
benzene ring. By these means,
the highest reported values of
the adsorption and emission
maxima (630 and 655 nm, re-
spectively) for a “regular”
rhodamine were achieved in
the rigidized xanthene derivative of structure 4 (see
Scheme 1).
[9]
However, 4 is unsuitable for bioconjugation. It
is poorly soluble in water and lacks a convenient binding
site. Moreover, its free carboxyl group may form colorless
and nonfluorescent cyclic esters (lacto nes). This carboxyl
group is sterically hindered, as in all rhodamines, and amida-
tion reactions with primary amines (e.g., in proteins) would
give primary amides that are known to form colorless and
nonfluorescent cyclic spiroamides (due to addit ion of the
NH group across the tetrasubstituted C
9
=C
8a/8b
bond in the
central xanthene ring).
[2m,n,o]
Synthesis of tetrafluororhodamine 4 and its lipophilic and
hydrophilic derivatives: We chose compound 4 as a scaffold
for further derivatization. Synthesis of tetrafluororhodamine
4 (Schem e 1) started with 1, which was prepared from m-
anisidine and aceto ne in the presence of dry ytterbiumACHTUNGTRENNUNG(III)
triflate according to the known general method.
[14]
Alkyla-
tion of 1 with 1-bromo-3-chloropropane was carried out sim-
ilarly to the procedure described for its closest analogue, the
corresponding 10-pivaloyl ester.
[9a]
Demethylation of tricy-
clic ether 2-Me was carried out under conditions reported
for aromatic methyl esters.
[15]
Conversion of the intermedi-
ate 2-H to rhodamine 4 was performed stepwise according
to the known method for the synthesis of asymmetric xan-
thene derivatives.
[9a]
Condensation of 3 and 2-H in the pres-
ence of POCl
3
affords the corresponding acid chloride as an
intermediate. Aqueous workup leads to zwitterionic com-
pound 4, while quenching the reaction with methanol produ-
ces methyl ester 7, easily hydrolyzed to 4 under the proper
conditions. Other esters of rhodamine 4 can be easily ob-
tained in a similar way. In the long run, they may be inter-
esting intermediates, because one of the fluorine atoms in
the aromatic ring can be easily replaced with nucleophilic
(secondary) aliphatic amines.
[2q]
Such transformations may
open a new route to diverse fluorescent dyes. With com-
pound 4 as scaffold, attachm ent of the N-methyl-b-alanine
bridge to the rhodamine core (see Scheme 1 for the synthet-
ic sequence and structures) provided the crucial redshift (>
20 nm in a MeOH solution, see Table 1). Moreover, the ster-
ically unhindered carboxyalkyl or carboxyl groups in com-
pounds 5 are available for further smooth and high-yielding
transformations. Note that in compound 4 the positions of
the absorbance/emission maxima (616 and 641 nm in
Scheme 1. Synthesis of lipophilic (5, 7) and hydrophilic (6) rhodamines. a) BrACHTUNGTRENNUNG(CH
2
)
3
Cl, Na
2
CO
3
, KI, CH
3
CN,
reflux, 25 h; b) 48 % aq. HBr, glacial AcOH, 135–140
8
C, 22 h; c) tetrafluorophthalic anhydride, toluene,
reflux, 5 h; d) POCl
3
, ClCH
2
CH
2
Cl, 63–65
8
C, 3.5 h; e) POCl
3
, ClCH
2
CH
2
Cl, reflux, 2.5 h; f) Et
3
N, CH
3
CN, RT;
g) 10% aq. KOH, THF, 0
8
C; h) 2 m HCl in 1,4-dioxane, RT; i) 96–98 % H
2
SO
4
, RT, 40 h.
www.chemeurj.org 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2010, 16, 158 – 166
160
V. N. Belov, S. W. Hell et al.
MeOH, respectively, see Table 1) are far from suitable. For
attaching the bifunctional b-alanine “bridge” to the carboxyl
group in 4 for providing the lipophilic fluorescent dyes 5-R,
we used our previously developed technique.
[2p]
We estab-
lished that utilization of N-methyl-b-alanine tert-butyl ester
instead of the methyl ester has certain advantages. In con-
trast to n-alkyl esters, tert-butyl esters are easily cleaved
under acidic conditions. Thus, in our synthesis of sulfonated
water-soluble hydrophilic dye 6, the subsequent alkali-assist-
ed saponification step becomes unnecessary. The absence of
this step not only shortens the reaction seque nce, but also
prevents undesired fluorine substitution in the aromatic ring
of dyes 5-R. (Under basic conditions in methanol one of the
four fluorine atoms may be substituted by a strongly nucleo-
philic methoxide or hydroxide anion.) Another crucial step
of the synthesis is introduction of the two sulfo groups. This
is achieved by direct sulfonation, a straightforward proce-
dure which may be considered to be a case of so-called allyl-
ic sulfonation of olefins.
[2r,13]
Introduction of ester 5-tBu or
acid 5-H into a large excess of 96–98% H
2
SO
4
at 0
8
C fol-
lowed by prolonged exposure at room temperature afforded
the desired hydrophilic dye 6. Fortunately, these conditions
(concentrated H
2
SO
4
) left the secondary amido group intact.
Also importantly, the same starting material, tert-butyl ester
5-tBu, can be smoothly converted to the corresponding car-
boxylic acid 5-H by simple treatment with 2m HCl in 1,4-di-
oxane. In fact, the unsulfonated compound 5 -H is the re-
quired lipophilic analogue of rhodamine 6. Later we man-
aged to cleanly saponify methyl ester 5-Me to carboxylic
acid 5-H using an excess of highly dilute aqueous KOH so-
lution at room temperature. Nonetheless, the absence of
methanol in the reaction mixture was important; with meth-
anol, the saponification reaction invariably gave mixtures
containing 5-H and a substance with very similar retention
parameters (both on silica gel and the reversed phase), pre-
sumably the derivative whose aromatic fluorine atom was
replaced by a methoxyl group. The solubility of 6-H in
water is high: up to 40–50 mg in 1 mL of water at room tem-
perature. The dark blue aqueous solutions produce intense
red fluorescence when irradiated with incandescent or halo-
gen lamps. Dissolution of 6 in methanol, aqueous methanol,
or any methanol-containing mixtures is accompanied by no-
ticeable esterification of the b-alanine fragment by metha-
nol. Probably, the close proximity of the sulfonic acid resi-
dues to the carboxyl group assists nucleophilic addition (a
case of an intramolecular acid catalysis). Unlike 6, lipophilic
rhodamine 5 is very poorly soluble in water. On the other
hand, the solubility of dye 5-H in DMF, THF, and even
CHCl
3
is very good. Due to the presence of the asymmetri-
cally substituted secondary amide fragment, the molecules
of compounds 5 and 6 (and their derivatives, see Scheme 1)
have an N
C chirality axis. The rotational barrier in asym-
metrically substituted tertiary amides is high enough (ca.
18 kcal mol
1
) that in the NMR spectrum two signals are ob-
served for the N-methyl group (in ca. 1:2–1:3 ratio). The
presence of the chirality axis makes the “opposite” methyl
groups and the CH atoms attached to the “left” and “right”
sides of the xanthene fragment nonequivalent (diastereotop-
ic), and consequently in the NMR spectra of compounds 5
and 6 two additional sets of equally strong signals can be ob-
served. For example, four signals of the methyl groups at
the C=C bond are clearly seen in
1
H NMR spectra of esters
5-Me and 5-tBu.
[19]
Deuterated analogues: We also prepared the deuterated an-
alogue of fluorescent dye 6 (compound 6D in Scheme 1,
chemically identical to compound 6) for the following rea-
sons. High-frequency C
H stretching vibrations (n
3000 cm
1
) considerably contribute to the nonradiative
S
1
!S
0
transition characterized by the Frank–Condon factor
f
0
, which is associated with the maximum possible rate of
nonemissive decay (f
0
1013 s
1
in most chromophores with
C
H bonds).
[16]
Therefore, isotopic substitution which cre-
ates C
D bonds with considerably lower vibrational energy
(n 2200 cm
1
) may reduce the rate of nonradiative deacti-
vation, improve the fluorescence quantum yield, and pro-
long the excited-states lifetime. These effects are likely to be
observed in rigid molecular systems (e. g., rhodamines) in
which the S
1
and S
0
states are known to have very similar
geometries, so that their potential-energy surfaces do not in-
tersect. Consequently, the overall deactivation rate may be
approximated by the internal conversion, which is deter-
mined by the Frank–Condon factor. Furthermore, deuterat-
ed and non-deuterated fluorophores with the same structure
are expected to have the same (or nearly the same) intersys-
tem-crossing rates (S
1
!T
1
). In view of all these factors, we
hoped that the deuteration would increase the fluorescent
quantum yields and excited-state lifetimes. Compound 6D
was synthesized similarly to compound 6.
Only a very large excess of [D
6
]acetone in the reaction
with m-anisidine catalyzed by anhydrou s ytterbiumACHTUNGTRENNUNG(III) tri-
flate affords compound 1-Me with sufficiently high deuteri-
um content. The structural fragment CACHTUNGTRENNUNG(CD
3
)=CD (the allyl
protons) in compound 1-Me is especially prone to the D/H
Table 1. Spectroscopic properties of the fluorescent dyes prepared in this
study.
[a]
Com-
pound
l
max
ACHTUNGTRENNUNG(abs)
[nm]
l
max
ACHTUNGTRENNUNG(fl.)
[nm]
Solvent e
[10
5
m
1
cm
1
]
F
fl.
[%]
in
H
2
O
[b]
t
fl
[ns]
k
ISC/T
ACHTUNGTRENNUNG[10
6
s
1
]
[c]
4 616 641 MeOH 63
7 632 655.5 MeOH
5-tBu 638 661 MeOH 0.92 61
5-Me 638 661.5 MeOH 0.66 62
5-H 638 661.5 MeOH 0.73 53 3.6
6
[d]
637 660 H
2
O 0.94 80 3.4 2.5/0.3
6D 637 660 H
2
O 0.92 78 3.4 2.5/0.3
[a] l
max
(abs.), l
max
(fl.), e, F
fl
, and t
fl
are absorption maxima, emission
maxima, extinction coefficient, fluorescence quantum yield, and lifetime,
respectively. [b] Atto 633 was used as reference (F
fl
= 64 % in aqueous
buffer). [c] Intersystem crossing rate k
ISC
and rate of T
1
!S
0
transition k
T
measured by FCS under single-molecule conditions in water at 22
8
C.
[17]
[d] The fluorescence quantum yields of bioconjugates were 48% (for dye
6 and sheep anti-mouse antibodies) and 40 % (for dye 6 and goat anti-
rabbit antibodies).
Chem. Eur. J. 2010, 16, 158 – 166 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
161
FULL PAPER
Red-Emitting Rhodamine Dyes
exchange. For this reason, the
subsequent alkylation with Br-
ACHTUNGTRENNUNG(CH
2
)
3
Cl was performed in
[D
3
]acetonitrile in the pres-
ence of Na
2
CO
3
(instead of
NaHCO
3
). Cleavage of deuter-
ated methyl ester 2-Me was
performed with 48% DBr in
D
2
O mixed with AcOD. Other
steps leading to compound 5-
tBu did not involve reagents or
solvents with easily exchangea-
ble protons and were per-
formed as for the undeuterat-
ed precursor. The reaction
with 97% D
2
SO
4
in D
2
O (deu-
terated concentrated sulfuric
acid), due to the fast exchange
of the allyl protons, substan-
tially improved the degree of
deuteration in the fragment C-
ACHTUNGTRENNUNG(CD
3
)=CD. As a result, we
made up for the undesired ex-
change to hydrogen, and target
compound 6D had satisfactory
isotopic purity and a very
narrow molecular mass distri-
bution in its mass spectrum
with two major peaks corre-
sponding to the presence of 17
and 18 D atoms (of 18 possi-
ble).
Reactive derivatives: To dem-
onstrate the potential and per-
formance of the new dyes as
fluorescent markers for bio-
conjugation and microscopy it
was necessary first to prepare
corresponding reactive deriva-
tives (starting from compounds 6/6D and their lipophilic
counterpart 5-H for comparison). Usually, amino- or thio l-
reactive derivatives of fluorescent dyes are used for labeling
of proteins. For conjugation with amines, the free carboxyl
group must be activated by formation of N-hydroxy-
ACHTUNGTRENNUNG(sulfo)succinimidyl (NH(S)S) esters, phenyl esters with elec-
tron-acceptor groups in the benzene ring, or similar com-
pounds with good leaving groups. The corresponding NHS
ester of lipophilic rhodamine 5-H proved to be disappoint-
ingly unstable, and tetrafluorophenyl ester 8 was prepared
instead (Scheme 2). Ester 8 partly decomposed in the course
of chromatographic isolation on silica gel. Therefore, the re-
action mixture containing the active ester 8 was used for la-
beling d-erythro-sphingosine phosphocholine (lyso-SM,
Scheme 2). Low chemical stability of ester 8 and the corre-
sponding NHS derivative may be explained by the presence
of the nucleophilic hydroxide group as counterion. The neg-
atively charged OH
ion is invariably formed in the first
step of any esterification of the carboxyl group in 5-H. If it
could be exchanged quickly and under mild conditions with
another, less nucleophilic anion (e.g., perchlorat e) before
workup of the reaction mixture, then the chances to isolate
the final ester 8 (or the corresponding NHS ester) in a pure
state would have been better. The NHS esters of hydrophilic
rhodamines 6 and 6D, compounds 9 and 9D, turned out to
be comparatively stable. They were isolated by reversed-
phase preparative HPLC with gradient elution with aqueous
acetonitrile containing 0.1 % (v/v) of TFA, lyophilized, and
stored at 20
8
C under argon. Under these conditions, the
content of the corresponding acids 6/6D (the hydrolysis
products) in these dark blue solids increased from 3–5 % up
to 7–10% over two months. The higher stability of the NHS
esters 9 and 11 may be explained by the absence of any nu-
cleophilic counterion (the compounds themselves exist in
Scheme 2. Synthesis of reactive derivatives 8, 9, and 11 of lipophilic (5-H) and hydrophilic (6/6D) rhodamine
dyes. a) 2,3,5,6-Tetrafluorophenol, N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide (EDC), CH
3
CN, RT;
b) N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), Et
3
N, DMF, RT; c) O-(7-aza-
benzotriazol-1-yl)-N, N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), DMF, Et
3
N, RT;
d) 10·CF
3
COOH, DMF, Et
3
N, RT, e) Et
3
N, DMF, MeOH, RT, overnight.
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162
V. N. Belov, S. W. Hell et al.