Phosphorylated 3-heteroarylcoumarins and their use in fluorescence microscopy and nanoscopy

Article abstract of DOI:10.1002/chem.201202382

Photostable and bright fluorescent dyes with large Stokes shifts are widely used as markers in far-field optical microscopy, but the variety of useful dyes is limited. The present study introduces new 3-heteroaryl coumarins decorated with a primary phosph

Full text of DOI:10.1002/chem.201202382

FULL PAPER
DOI: 10.1002/chem.201202382
Phosphorylated 3-Heteroarylcoumarins and Their Use in Fluorescence
Microscopy and Nanoscopy
Shamil Nizamov, Katrin I. Willig, Maksim V. Sednev, Vladimir N. Belov,* and
[
a]
Stefan W. Hell*
Abstract: Photostable and bright fluo-
rescent dyes with large Stokes shifts
are widely used as markers in far-field
optical microscopy, but the variety of
useful dyes is limited. The present
study introduces new 3-heteroaryl cou-
marins decorated with a primary phos-
of the methyl group at the C=C bond
conjugation with proteins (in aqueous
buffers). In super-resolution light mi-
croscopy with stimulated emission de-
pletion (STED), the new coumarin
dyes provide an optical resolution of
with SeO (to an aldehyde), reduction
2
with NaBH (to an alcohol), and con-
4
version into a primary phosphate. The
4 position in the coumarin system may
be unsubstituted or bear a methyl
group. Phosphorylated coumarins were
found to have high fluorescence quan-
tum yields in the free state and after
40–60 nm with
a
low background
signal. Due to their large Stokes shifts
and high photostability, phosphorylated
coumarins enable to combine multila-
bel imaging (using one detector and
several excitation sources) with diffrac-
tion unlimited optical resolution.
phate group (OP(O)(OH) ) attached
2
to C-4 in 2,2,4-trimethyl-1,2-dihydro-
quinoline fragment fused with the cou-
marin fluorophore. The general syn-
thetic route is based on the Suzuki re-
action of 3-bromocoumarines with het-
Keywords: bioconjugation
·
cou-
marins · dyes/pigments · fluores-
cence · synthetic methods
ACHTUNGTRENNUNGa rylboronic acids followed by oxidation
Introduction
Photostable and bright fluores-
cent dyes with large Stokes
shifts are rare and only a few
are commercially available.
Nearly all these dyes contain
the coumarin fragment as the
fluorophore. For example,
“Mega Stokes” dyes from the
Dyomics company are coumar-
ins absorbing at about 500–
520 nm, and emitting in the
region of 590–670 nm (in etha-
nol). Another practically useful
coumarin dye is AlexaFluor
Figure 1. Examples of the new compounds and commercially available coumarin dyes. For complete data on
the spectral properties of all coumarin dyes of the present study, see Table 1.
430 with absorption and emis-
sion maxima at 434 and 539 nm, respectively (Figure 1).
Many other suppliers (e. g., Invitrogen, BD Biosciences,
Kodak, etc.) offer fluorescent dyes and their conjugates with
large Stokes shifts, but they do not publish their structures.
The lack of essential structural data hinders the proper
choice of the fluorescent dye for a particular biochemical
application (for example, due to the indefinite hydrophilici-
ty, unknown net electrical charge of the molecule, and po-
tential (photo)toxicity). Unavailable structures of the exist-
ing dyes also hamper the progress in basic research aimed at
the creation of new fluorophores. This is particularly true
for the coumarins, whose common features are moderate
photostability and relatively low fluorescence quantum
yields in polar solvents. The lack of brightness and low pho-
toresistance are the main drawbacks that preclude their
[
a] Dr. S. Nizamov, Dr. K. I. Willig, Dipl.-Chem. M. V. Sednev,
Dr. V. N. Belov, Prof. S. W. Hell
Department of NanoBiophotonics
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
Fax : (+49)551-2012505
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.201202382.
[1]
wide use in modern methods of optical “nanoscopy”.
Chem. Eur. J. 2012, 18, 16339 – 16348
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
16339

These new super-resolution methods of the far-field optical
microscopy require fluorescent dyes which demonstrate all
advantages and full scope of the new physical concepts.
Therefore, the synthesis of coumarins emitting in the green
and red spectral regions and the optimization of their photo-
stabilty, quantum yields, solubility in aqueous buffers, and
chemical reactivity with various biologically relevant targets
stituents at the “sensitive” positions susceptible to (photo)
oxidation.
Water is the preferred solvent for operating with the reac-
tive fluorescent dyes, because the conjugation reactions in-
volving biologically relevant macromolecules (e. g. proteins,
nucleic acids, carbohydrates) have to be performed in water
or aqueous buffers. A chemically reactive marker is usually
dissolved in an organic solvent, such as DMF or DMSO, and
then added to the aqueous solution of the substrate. High
concentrations of an organic solvent may cause protein de-
naturation, and should be avoided. On the other hand, a
low coupling efficiency and/or unspecific binding with a
target may be observed if the amount of the organic solvent
is too low, and the dye precipitates from the solution.
Water-soluble fluorescent markers are advantageous in this
regard, because they do not require any organic solvents at
all. Moreover, hydrophilic labels are less prone to aggrega-
tion and to non-specific binding with biological objects, es-
pecially membranes.
[2]
is an important research field.
For example, chemoselective labeling became possible
with “clickable” 3-{2-[N-(3-azidopropyl)-2-sulfopyrid-4-yl]-
[2d,e]
ethenyl}-7-diethylamino coumarins,
presenting an impor-
tant addition to red-emitting fluorescent dyes with large
Stokes shifts possessing the carboxylic acid group as a reac-
tive site (e.g., to DY-480XL in Figure 1). The aldehyde
group at C3 in coumarin enables the formation of hybrids
[2f]
between coumarin and cyanine dyes. These new far-red-
emitting fluorophores with large Stokes shifts are based on
7-hydroxycoumarins. They were decorated with hydrophilic
residues and quenching groups (attached to the phenolic hy-
droxyl) which may be cleaved-off by hydrolytic enzymes,
thus probing the activity of these highly efficient bio-cata-
lysts. A similar approach was used in the course of creating
the fluorimetric sensors for other enzymes or physiologically
active thiols, which trigger the (cascade) reactions and liber-
ate the free hydroxyl group at C7 (responsible for fluores-
The synthesis and properties of hydrophilic coumarins
[5a,b]
decorated with sulfonic acid groups have been disclosed.
An introduction of the sulfonic acid residue into 4-methyl
group of 2,2,4-trimethyl-1,2-dihydroquinolines (precursors to
hydrophilic coumarins with large Stokes shifts) is performed
in concentrated sulfuric acid. Although these harsh condi-
tions provide the required hydrophilic compounds (e.g.,
AlexaFluor 430 in Figure 1), they are incompatible with sev-
eral important functional groups that do not tolerate con-
centrated sulfuric acid (e.g., tert-butyl esters, diazo groups,
etc.) and therefore seriously limit the “synthesis freedom”.
In this respect, the synthesis route to the phosphorylated
coumarins presented here is advantageous, because it pro-
vides the hydrophilic dyes prepared under mild conditions.
Phosphoric acid represents a viable alternative to sulfonic
acid as a source of negatively charged groups preventing the
aggregation of the dye residues, self-quenching of the fluo-
rescence signal and unspecific binding with liphophilic sub-
strates. Indeed, this acid has medium strength, an increased
number of acidic protons (relative to sulfuric acid), and its
primary and secondary alkyl esters are hydrolytically stable
[2g–j]
cence).
Results and Discussion
Motivation of the work and key structures of the new cou-
marins with large Stokes shifts: A very important feature of
a fluorescent dye is the Stokes shift (separation between the
ꢀ
1
absorption and emission maxima measured in nm or cm ).
Fluorescent dyes with large Stokes shifts can be used either
alone, or together with emitters possessing small Stokes
shifts in various imaging techniques. For example, a pair of
dyes emitting approximately at the same wavelength with
well separated absorption bands may be used for labeling,
detection and co-localization of two different (biological)
targets. A great advantage of this approach is that only one
detection channel is used. The “cross-talk” observed in the
course of the excitation with two different light sources
[
5c]
at pH 2–12.
phate residues have been reported.
dyes decorated with phosphonic acid residues were pre-
The BODIPY dyes with two dialkyl phos-
[5d]
Several fluorescent
[5e–i]
(
lasers) has to be low. As mentioned, the chemical structures
A
H
U
G
R
N
N
As ionisable groups, primary phosphates have an
of many commercially available fluorescent dyes with large
Stokes shifts are unknown. For example, the structures of
Pacific Orange (abs. 390 nm, emission 540 nm; Invitrogen)
and BD Horizon V500 (abs. 415 nm, emission 500 nm; BD
Biosciences) have not been disclosed. Despite the very long
history and wide use of coumarin dyes, their structure–prop-
1
2
rived from a stronger acid. For example, pKa and pKa
values for primary alkyl phosphates are 1.5–1.9 and 6.3–6.8,
[5j]
respectively, whereas for primary alkyl phosphonates, the
first and second ionization constants were found to be 2.4–
[5k]
2.8 and 7.8–8.9, respectively.
Therefore, at physiological
[2,3]
erty relationships have not been studied in depth.
Large
pH of 7.3–7.4, fluorescent dyes with primary phosphate
groups will be ionized much better (and their aggregation
will be suppressed more efficiently). If they will be analyzed
by HPLC or gel electrophoresis, in buffer solutions at
pH> 8 they are expected to move as single bands or peaks.
Examples of the commercially available coumarin dyes
and new compounds of the present study are given in
Stokes shifts in coumarins are provided by using intramolec-
ular charge transfer (ICT) and excited state intramolecular
[4]
proton transfer (ESIPT) mechanisms. In the present study,
we relied on the first mechanism. High fluorescence quan-
tum yields and photoresistance were provided by using
planar and rigid molecular frameworks with additional sub-
16340
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 16339 – 16348

Phosphorylated 3-Heteroarylcoumarins
FULL PAPER
Figure 1. They all belong to the family of 7-aminocoumarins,
with variable substituents in positions 3 and 4. The electron-
acceptor group at position 3 with an extended p-system
Synthesis of universal precursors: construction of 3-hetero-
AHCTUNGTRENNUNGa rylcoumarins with hydroxymethyl and primary phosphate
(
e.g., N-alkyl-2-vinylpyridinium salt with a negative induc-
groups: The preparation of the starting building blocks 2–4
for the synthesis of various coumarines dyes (emitting in the
green- and red spectral regions) is given in Scheme 1 and
Scheme 2. Selective protection of the hydroxy-group in m-
aminophenol (1-H) was carried out as described in the liter-
tive (ꢀM) effect in compound DY-480XL) strongly shifts
the absorption and emission bands to the red spectral region
and increases the distance between their maxima. A very
strong acceptor at position 4 (e.g., CF -group in AlexaFluor
4
3
[6]
30) with a ꢀM effect also increases the charge separation
ature and led to compound 1-TBDMS. The Skraup–Doeb-
ner von Miller reaction of 1-TBDMS and acetone was per-
and the “push–pull” character of the chromophore, and pro-
vides similar spectral shifts. Incorporation of the 7-amino
group into the 2,2,4-trimethyl-1,2-dihydroquinoline ring pro-
vides an additional small redshift of 5–10 nm and creates
two variable positions: a methyl group at the double bond
and the disubstituted nitrogen atom. These variable posi-
tions were used to introduce the sulfonic or phosphoric acid
residues and w-carboxyalkyl groups, respectively. The said
transformations do not change the positions of the absorp-
tion and emission bands, but they rather provide more hy-
drophilic compounds with considerably increased fluores-
cence quantum yields (in the free state and in bioconju-
gates) and an additional functional group (COOH) that is
necessary for conjugation reactions. The structures of dyes
[7a]
formed in the presence of ytterbium AHCUTNGRNENUG( III) triflate and pro-
vided precursor 2 with the 1,2-dihydroquinoline moiety in
good yield. At room temperature, this reaction proceeded
smoothly and with good regioselectivity. Alkylation of com-
pound 2 with ethyl or tert-butyl 3-iodobutyrates in the pres-
ence of Hꢁnigꢂs base afforded esters 3. Compound 3-
TBDMS,Et was used for the preparation of the hydrophilic
green-emitting coumarin dye 6-H decorated with sulfonic
acid residue (Scheme 1). Firstly, the hydroxyl group in 3-
TBDMS,Et was deprotected using tetrabutylammonium flu-
oride, and then the obtained phenol 3-H,Et was formylated
according to Vilsmeier–Haack to give salicylic aldehyde
[7b,c]
4-Et in 64% yield.
Next, a coumarin ring was formed by
6
-H and 11-H,H differ in only one respect: compound 6-H
the sequential esterification and Knoevenagel reactions, and
the desired compound 5-Et was obtained in 52% yield.
[7d,e]
bears a sulfonic acid moiety, whereas 11-H,H has a primary
phosphate group. It was interesting to compare the spectral
properties, fluorescence quantum yields (also in bioconju-
gates) and performance of these dyes in optical microscopy.
The betain fragment in the structurally very similar dye 7-H
provides a bathochromic shift of absorption and emission
bands, and further increases the Stokes shift, but the fluores-
cence quantum yield in aqueous solutions decreases drasti-
cally (see Table 1). A variation of the substituent at C4 in
the coumarin system is a more delicate tool that enables a
fine “tuning” of the positions of the absorption bands and
further reduce absorption at 488 nm and, as a result, a cross-
talk between the dyes excited at this wavelength and with
The saponificaton of ethyl ester followed by sulfonation of
4-methyl group in the 2,2,4-trimetyl-1,2-dihydroquinoline
[7f]
fragment provided coumarin 6-H, which readily reacted
with perfluorocyclopentene and afforded red-emitting
[8]
betain 7-H. Overall yields of water-soluble coumarins 6-H
and 7-H were approximately 12 and 6%, respectively.
The phosphorylated analogues of coumarins 5-R were
prepared as depicted in Scheme 2. Although the N-alkyla-
tion of compound 2 with ethyl 4-iodobutanoate proceed
very smoothly, under the same conditions, tert-butyl 4-iodo-
butanoate and amine 2 afforded ester 4-TBDMS,tBu at a
lower yield of 60%. The oxidation of the methyl group at
the C=C-bond in 1,2-dihydroquinolines 3 (to aldehydes 8)
was achieved using selenium dioxide, but even after optimi-
zation, the yields were low (Scheme 2). The low yields of al-
dehydes 8 may be explained by the susceptibility of C(6)-H
4
05 nm laser light.
A detailed discussion and comparison of the spectral
properties of all new coumarin dyes prepared in this study is
given in a separate section below.
Table 1. Properties of the hydrophilic coumarin dyes in PBS buffer at pH 7.4 and room temperature.
ꢀ
5
[a,b]
Dye
Absorption
Emission
eꢃ10
F
fl
Lifetime
t [ns]
Solubility
[mM]
Stability of
NHS-ester
DOL
Ffl [%] in
conjugates
ꢀ
1
ꢀ1
[b]
l
max [nm]
l
max [nm]
A
H
U
G
R
N
U
G
]
[%]
[
c]
[d]
Alexa Fluor 430
434
432, 444
541
506, 514
515
515
512
503
639
631
0.16
0.1, 0.07
0.31
0.23
0.20
0.14
0.40
0.50
55
61, 50
57
–
–
–
3.3
3.2
2.7
–
–
no data
not measured
25
20
good
7
low
no data
–
–
3–7
–
2.7
–
–
[
e,f]
[g]
6
6
1
2
2
7
-H
-H
1-H,H
1c
2c
-H
[
h]
[h]
436
436
432
410
520
504
good
good
good
good
good
–
17
30 (27)
-
27
-
[i]
[i]
[I,b]
[j]
[i]
68
81
44
0.6
2.6 (6.3)
–
2.8
–
[
k]
DY-480XL
–
–
-
[
a] Degree of labeling: average amount of the dye residues attached to one antibody molecule with molecular weight (M
w
)ꢁ150000. [b] Sheep anti-
O. [g] Devi-
Mouse secondary antibodies. [c] Data from the web database: www.fluorophores.org. [d] Recommended DOL-values. [e] In MeOH. [f] In H
2
ations from Beer–Lambert law due to the low solubility; extinction is lower than anticipated due to adsorption of the unpolar substance on quartz sur-
face. [h] Conjugates with Sheep anti-Mouse antibodies have absorption and emission maxima at 437 nm and 512 nm, respectively. [i] Conjugates with an-
tibodies have t=2.9–3.1 ns; their absorption and emission maxima are at 439 and 513 nm, respectively. [j] Goat anti-Rabbit antibodies. [k] Compound
2
2a; 2.2 ns for compound 22d in antibody conjugates.
Chem. Eur. J. 2012, 18, 16339 – 16348
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
16341

V. N. Belov, S. W. Hell et al.
phorylated coumarins 11 and
1
4
2 obtained by this route were
–8%.
The compounds with a free
carboxylic acid group (e.g., 11-
H,H and 12-H,H) can be used
in various (bio)conjugation
procedures. The final activa-
tion step, which involved the
carboxyl group and produced
amino-reactive
N-hydroxy-
AHCTUNGERTNNUNGs uccinimidyl esters 7-NHS
(
Scheme 1), 11-H,NHS, and
1
2-H,NHS (Scheme 2), as well
as 21d and 22d (Scheme 4),
was performed in DMF using
2
1
-(7-aza-1H-benzotriazol-1-yl)-
,1,3,3-tetramethyluronium
hexafluorophosphate (HATU)
in the presence of N-hydroxy-
succinimide and base (Et N).
3
These conditions provided
higher yields of the target
active esters than the use of
other coupling agents (e.g., O-
(
N-succinimidyl)-N,N,N’,N’-tet-
ramethyl uronium tetrafluoro-
borate, which gave yields of
less than 20%). Under these
conditions,
the
carboxylic
Scheme 1. Synthesis of model green-emitting dye 6-H and red-emitting coumarin 7-H decorated with sulfonic
acid residue: a) ClSiMe tBu (TBDMSCl), imidazole, DMF, 0–558C, 2 h; b) acetone, Yb(OTf) , RT, 16 h;
c) ICH CH CH CO Et, DIEA, 1108C, 48 h; d) TBAF·3H O/THF, 08C, 5 min; e) POCl , DMF, 608C, 1.5 h;
f) 4-pyridylacetic acid hydrochloride, NEt , DMAP, CH Cl , RT, 40 h; g) 1m aq. NaOH, THF, MeOH, RT,
6 h; h) H SO , 358C, 2 days; i) perfluorocyclopentene, EtOH, aq. AcOH, 708C, 2 h; j) N-hydroxysuccinimide
HOSu), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), NEt
2
A
H
U
G
R
N
U
G
3
group was activated first,
whereas the phosphoric acid
residue was kept free (in the
absence of the excess of the
coupling reagent and N-hy-
droxysuccinimide). At higher
conversions, partial activation
2
2
2
2
2
3
3
2
2
1
(
2
4
3
,
DMF, RT, 16 h.
to oxidation (followed by destruction); higher yields of alde-
hydes 14-H,R were obtained under more drastic conditions
from coumarines 13-H,R, which are obviously more resist-
ant to oxidative destruction caused by SeO2 (see later;
Scheme 4). The alcohols 9-H were obtained by Luche reduc-
of the phosphate group was also observed, and di(N-succini-
midyl) esters were often detected in the reaction mixtures
by TLC and HPLC. The isolation and purification of the
polar phosphorylated dyes (as carboxylic acids or their N-
hydroxysuccinimidyl esters) was uniformly performed on
silica gel 60 (40–63 m) using acetonitrile-water mixture as an
eluent (e.g., 8:1–4:1), followed by direct lyophylization of
the pooled fractions, dissolution of the solid residues in min-
imal amounts of water or aqueous acetonitrile, filtration
from the silica gel, and lyophilization. In the case of carbox-
ylic acids, triethyl amine may be added to the solvent mix-
ture. Methanol has to be avoided, because it may react with
N-hydroxysuccinimydyl esters, and does not form a solid
during freeze-drying (temperature of the cooler is ꢀ808C).
[
9a]
tion
of aldehydes 8 with NaBH /CeCl . Phosphorylation
4 3
3
of alcohols 9-H,TBDMS,R was performed as described in
the literature.
[9b,c]
3
Formylation of phenols 9-PO ACHTUNGERTNNUNG( OtBu) ,H,R
2
proceeded smoothly and resulted in the target aldehydes 10
with ethyl or tert-butyl ester groups (Scheme 2). According
to the general approach exemplified in Scheme 1 and 2, the
condensation reaction of aromatic aldehydes 4-Et and 10
with the a-substituted acetic acid derivatives directly pro-
[10]
vides coumarins. In particular, the reaction of aldehydes
0 with 4-pyridyl acetic acid was performed under mild con-
2
3
1
If the reaction of alcohols 9-H,R ,R with (iPr) NP-
2
ditions and gave the phosphorylated coumarins 11 with two
protecting groups (Scheme 2). The uniform protection of all
acidic centers as tert-butyl esters saved one synthetic step
later in the synthesis and simplified the isolation of the final
dye 11-H,H (Scheme 2). The overall yields of the phos-
ACHTUNGTRNEN(GU OtBu) and 1H-tetrazole in CH Cl was carried out for a
2 2 2
long time (20 h at 408C), a byproduct (hydrogenphospho-
[11a,b]
nate 9-PHO
ACHTUNGTRENUNNG( OtBu),TBDMS) was isolated (Scheme 3).
Although the phosphonate functionality may be formed
[11b]
using these reagents by various routes,
we consider the
16342
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 16339 – 16348

Phosphorylated 3-Heteroarylcoumarins
FULL PAPER
tions followed by oxidation of
compounds 13-X,R with SeO2,
bromination of coumarin 14-
X,R with a formyl group, con-
version of the corresponding
bromides 14-Br,R into the de-
sired
3-heteroarylcoumarins
1
8–19 by using a Pd catalyst
and postsynthetic modifica-
tions. The second method is
based on the esterification and
Knoevenagel condensation re-
actions using hetaryl acetic
acids followed by conversion
of the methyl group in 1,2-di-
hydroquinoline moiety (com-
pounds 5-Et and 16 and 17)
into the phosphoryl group. The
formation of the coumarin sys-
tems by a well-known Pech-
mann condensation is straight-
forward, albeit low-yielding; it
is short and preferable for the
synthesis of 4-methylcoumarin
derivatives (13-X,Me). The
highest overall yields were ach-
ieved in the following reaction
sequence:
Et/16-R/17-H!18-R/19-H!
0. At the key step, hetaryl
3-H,Et!4-Et!5-
2
acetic acids acylate the phenol-
ic group in compound 4-Et.
The intermediates, 2-formyl-
phenyl acetates, undergo the
ring-closure in Knoevenagel
reaction producing the coumar-
in structure. Unfortunately,
many hetaryl acetic acids are
unstable and lose carbon diox-
Scheme 2. Synthesis of the hydroxylated precursor 9-H,TBDMS of phosphorylated coumarins 11 and 12:
a) ICH
5 min; d) iPr
THF, 08C, 5 min; g) POCl
2
CH
2
CH
2
CO
2
tBu, iPr
, 1H-tetrazole, CH
, DMF, 608C, 1.5 h; h) 4-pyridylacetic acid hydrochloride, NEt
2
NEt, 1108C, 48 h; b) SeO
2
, dioxane, 658C, 0.5 h; c) NaBH
Cl , 08C, 15 min; f) TBAF·3H
2
, DMAP, CH Cl ,
4
, THF, MeOH, 08C,
1
2
NP
A( OtBu)
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
2
Cl , 408C, 1 h; e) mCPBA, CH
2
2
2
2
O,
3
3
2
3
RT, 40 h; i) perfluorocyclopentene/EtOH, aq. AcOH, 708C, 2 h; j) TFA, RT, 16 h; k) HOSu, HATU, NEt /
DMF, RT, 16 h.
[12a]
Ai de.
H
U
G
R
N
N
Therefore, the useful-
acid-catalyzed mono-dealkylation reaction to be the most
plausible one (Scheme 3). If the two reagents (1H-tetrazole
and phosphoroamidite) are used in excess, the first step of
this reaction proceeds quickly. Intercepting the trialkylphos-
phite intermediate 9-X with an oxidizing agent provides the
stable phosphate, whereas the phosphonate is obtained in
ness of this approach is limited only to commercially availa-
ble 2-pyridyl and 4-pyridyl derivatives.
We also prepared and isolated pyrazine acetic acid,
[12b]
which was found to be stable at pH>3 at room tempera-
ture, and was successfully used for the preparation of com-
pound 17-H. Thus, an alternative general approach to 3-het-
eroaryl coumarins was developed. It employs a Suzuki reac-
tion of 3-halo coumarins 13–15 with hetaryl boronic acids
(or a Stille reaction with hetaryl stannanes; see Scheme 4).
A wide range of the commercially available hetaryl boronic
acids makes this route to be an attractive option. However,
if the required aryl- or hetarylacetic acid is stable and can
be readily obtained (e.g., by lithiation of the corresponding
methyl (het)arene followed by carboxylation), then the first
method is superior. The overall yields of the target coumar-
ins obtained by these routes vary in range of 5–35%.
negligible amount. The acidity of 1H-tetrazole (pK =4.9) is
a
similar to that of the acetic acid (pK =4.75), and, probably,
a
this is the reason why one of the two acid sensitive tert-butyl
groups is cleaved in the presence of 1H-tetrazole.
Using the readily available phenolic compound with 1,2-
dihydroquinoline fragment (3-H,Et in Scheme 1) as a start-
ing material, we studied various routes leading to 3-hetero-
aryl coumarins 20 (Scheme 4). They differ according to the
method used for creating the coumarin system. The first ap-
proach includes the formation of the coumarin ring in the
course of the sequential Wittig–Horner and cyclization reac-
Chem. Eur. J. 2012, 18, 16339 – 16348
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16343

V. N. Belov, S. W. Hell et al.
diode laser (405 nm). Therefore, this dye can be efficiently
used in microscopy together with numerous fluorescent
labels with smaller Stokes shifts (e.g., Alexa Fluor 488,
Oregon Green, or Abberior Star 488), which also emit
green light, but absorb strongly at about 480–500 nm and
can be excited with 488 nm light. To compare the spectral
properties of the phosphorylated coumarin dyes with those
of sulfonated ones, we prepared the hydrophilic coumarin
dye 6-H with a sulfonic acid residue attached to the same
position at which compound 11-H,H has the primary phos-
phate group (Figure 1). The emission and absorption spectra
of dye 11-H,H and its analogue 6-H are very similar
(
Table 1). However, after attaching to antibodies, the fluo-
rescence quantum yields of the conjugates obtained from
the phosphorylated coumarin dye 11-H,H were found to be
significantly higher (27–30%) than that of the conjugates
prepared from the sulfonated dye 6-H (17–18%).
The alteration in the fluorescence quantum yield upon
bio ACHUTNGRNEGUNc onjugation (compared to that of a free dye in the same
solvent) is a very important issue. As a rule, the fluores-
cence quantum yield of a (hydrophilic) fluorescent dye de-
[14]
creases upon biocojugation. In addition, the fluorescence
Scheme 3. Monodealkylation of the intermediate trialkylphosphite 9-X
leads to hydrogenphosphonate 9-PHO(OtBu),TBDMS.
quantum yields (F ) in conjugates with antibodies often
A
T
N
T
E
N
N
fl
depend on the degree of labeling (DOL): higher quantum
yields are observed at lower DOL values (see values in last
two columns in Table 1). For dye 11-H,H, in antibody conju-
gates, the fluorescence quantum yields were found to not
Spectral properties and imaging performance of the phos-
phorylated coumarin dyes: Table 1 presents the most impor-
tant photophysical properties of the new coumarin dyes, as
well as compounds Alexa Fluor 430 (Invitrogen Inc) and
DY-480XL (Dyomics GmbH) taken as benchmarks. The flu-
orescence quantum yields were determined by comparison
with reference dyes with known emission efficiencies (see
depend on DOL values: for example, F =30 and 27% at
fl
DOL=2.6 and 6.3, respectively (see data in Table 1).
The 2-pyridyl derivative 21c was found to have nearly the
same properties, as 4-pyridyl analogue 11-H,H (see Table 1).
The absorption and emission maxima of compound 22c
with the methyl group at C4 in the coumarin fluorophore
are shifted to the blue spectral region (D=16 and 12 nm, re-
spectively). This hypsochromic shift may be explained by
the electron-donating effect of the methyl group (+I-effect),
which diminishes the electron-acceptor properties of the 4-
pyridyl substituent and reduces the overall charge separa-
tion in the push–pull 7-amino-4-methyl-3-(4-pyridyl) fluoro-
phore (compared with the 4-unsubstituted compound).
Thus, the broad absorption band of compound 22c (410 nm)
perfectly matches the violet line of the 405 nm laser, and the
absorption at 488 nm is lower than that in the case of dye
11-H,H. Therefore, the cross-talk associated with compound
22c is even less than the cross-talk found for coumarin 11-
H,H, when they are used together with the fluorescent
markers possessing small Stokes shifts and excitable with
Argon laser at 488 nm (e.g., Abberior Star 488).
[13a]
the Supporting Information for details).
As expected, the
polar groups, that is sulfonic acid (in compound 6-H) and
primary phosphate residues (in compounds 11-H,H, 21c and
22c), provide large fluorescence quantum yields and solubil-
ity in aqueous PBS buffer (at pH 7.4, these values varied in
the range of 7–25 mm, or several milligrams in 1 mL). The
intense fluorescence of these hydrophilic dyes is due to the
ionizable groups preventing the aggregation of the dye mol-
ecules and/or the formation of non-emitting dimers in aque-
[13b]
ous solutions.
The solutions of the first five compounds in Table 1 are
green with a very slight blue shade. The color is similar to
that of Fluorescein, Rhodamine 110, Oregon Green 488 and
Alexa Fluor 488 dyes, with the exception of the blue tone.
The color of the emitted light is green. This is true for
Alexa Fluor 430 and the new dyes 6-H, 11-H,H, 21c and
Compound 17-H with a pyrazine substituent (instead of 2-
or 4-pyridyl residues) was found to be a poor emitter: its Ffl
value is only 8%. The decreased fluorescence quantum
yield may be explained by the excited state photoinduced
22c. The last two compounds in Table 1 emit red light, but
nevertheless, they may be excited with the 488 nm line of an
argon laser.
[4]
The Stokes shifts of the green-emitting coumarin dyes 5-
Et, 6-H, 11-H,H, 21c and 22c were found to be approxi-
mately 70–80 nm. For example, the Stokes shift of the phos-
phorylated hydrophilic coumarin dye 11-H,H is 79 nm; its
absorption band matches the excitation line of the violet
electron transfer (PeT) from the lone pair of electrons be-
longing to the nitrogen atom (N4 in pyrazin-2-yl) that is not
directly conjugated with 7-amino substituent in coumarin.
This lone electron-pair is orthogonal to the p-electron
system of the fluorophore and not involved in the charge
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Phosphorylated 3-Heteroarylcoumarins
FULL PAPER
Scheme 4. A general approach to phosphorylated coumarins: a) POCl
3
/DMF, 608C, 1.5 h; b) ethyl acetoacetate or ethyl 2-chloroacetoacetate, ZnCl
/dioxane, heat to reflux, 2 h; f) NaBH , CeCl /THF, MeOH,
, 08C, 15 min; i) Hetaryl-CH COOH, EDC, DMAP/CH Cl , 308C,
, toluene or dioxane, 100–1108C, 2–24 h; k) 4-pyridyl boronic acid, [Pd(PPh ], 2m aq. Na CO /toluene, EtOH,
1108C, 20 h; l) 1m aq. NaOH/MeOH, THF, RT, 16 h; m) TFA, RT, 16 h; n) HOSu, HATU, NEt /DMF, RT, 16 h.
2
/
EtOH, 908C, 40 h; c) Ph
3
P=CH-CO
(OtBu) , 1H-tetrazole/CH
, Pd (dba) , PtBu
2
Et/xylene, 1408C, 3 h; d) Br
2
/AcOH, RT, 15 min; e) SeO
Cl
2
4
3
0
2
8C, 15 min; g) iPr
0 h; j) Hetaryl-SnBu
2
NP
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
2
2
Cl , 408C, 1 h; h) mCPBA/CH
2
2
2
2
2
3
2
A
T
N
T
E
N
N
3
3
A
T
N
T
E
N
N
3
)
4
2
3
+
3
separation. In fact, unlike the lone pair at N1, it was found
to be a nonessential part of the fluorophore. Therefore, if
the molecular orbital associated with this lone pair has
higher energy than the half-occupied HOMO of the excited
state, one of its electrons may easily fill the vacant place in
the HOMO (created in the excited state) and quench the
emission of light. In spite of this plausible explanation given
a posteriori, the spectral properties of compound 17-H are
far from trivial (especially the redshifted absorption and
emission bands). Probably, the more nucleophilic nitrogen
atom (N4 in pyrazin-2-yl) may be selectively alkylated, and
thus a new brightly fluorescent compound with a positively
charged nitrogen atom may be obtained.
The performance of the new coumarins in optical micros-
AHCTUNGTRENNUNG
Compounds 7-H and 12-H,H possess betain structures.
Unfortunately, the fluorescence quantum yield of compound
7
-H is very low (0.6% in the free state), and in this respect
it cannot be favorably compared to the commercial dye
DY-480XL.
Chem. Eur. J. 2012, 18, 16339 – 16348
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16345

V. N. Belov, S. W. Hell et al.
dye (which is considered to be
a benchmark) is that it is not
available as NHS ester or
other reactive derivative, and
its structure is unknown.
In the course of further stud-
ies, we prepared compound
2
2c with an even larger Stokes
shift of 93 nm. This dye has a
very low absorption at 488 nm
and therefore, it was expected
to provide virtually zero cross-
talk when combined with all
standard “green” dyes (e. g.,
Alexa Fluor 488 or Oregon
Green) and excited with the
widely used 488 nm laser line.
Figure 4a illustrates the negli-
gible cross-talk recorded for an
immunolabeled sample stained
with compound 22c and irradi-
ated at 405 nm and at 488 nm.
For comparison, with the same
settings, compound 11-H shows
a cross-talk of 19% when ex-
cited with 488 nm. The emis-
sion maximum of compound
Figure 2. Confocal and STED microscopy images of vimentin (magenta) and microtubule (green), two compo-
nents of the cytoskeleton in a fixed mammalian PtK2 (Potorous tridactylus, kidney) cell. Vimentin was immu-
nolabeled with NHS ester 11-H,NHS (dye 11-H,H, Figure 1 and Table 1); excitation with 405 nm. Tubulin was
immunolabeled with Oregon Green 488; excitation with 488 nm. The excitation of both dyes was alternated
line-by-line to separate the fluorescence in time. Detection at 510–560 nm for both dyes; STED at 590 nm for
both dyes (STED power 84 mW at the back focal plane).
2
2c is at 503 nm and very close
to the emission of these
standard” dyes, and indeed,
both markers (22c and
green dye”) can be detected
“
a
“
simultaneously without signifi-
cant loss. Figure 4 shows that
compound 22c can be used for
immunolabeling and in STED
microscopy, as well; although it
is somewhat “darker” (due to
lower absorption coefficient)
and the STED resolution of
approximately 70 nm is less
than that of compound 11-H.
Figure 3. Confocal and STED microscopy images of single (primary+secondary) antibodies in the background
to determine the resolution of Figure 2. Confocal resolution ꢁ200 nm, STED resolution ꢁ50 nm in both chan-
nels (magenta: 11-H,NHS; green: Oregon Green 488). The boxed area shows a group of antibodies that are
close together; the STED microscopy image resolves separate antibodies that appear as a cloud in the corre-
sponding confocal image.
Conclusion and Outlook
Several synthetic routes to new
3
-heteroaryl coumarins with a
polar group attached to the C4
position in 2,2,4-trimethyl-1,2-
dihydroquinoline
fragment
fused with the coumarin fluo-
rophore were developed, opti-
mized and compared. As a
result, new coumarin dyes 6-H,
Figure 4. Confocal and STED microscopy imaging without noticeable cross-talk. A) Absorption and emission
spectrum of compound 22c with 93 nm Stokes shift. The violet 405 nm laser line is almost in the absorption
maximum whereas the “standard” 488 nm light cannot excite compound 22c. B) Microtubules visualized
through immunolabeling with compound 22c were irradiated with 405 and 488 nm lasers in an alternating
manner with an emission cross-talk less than 1%; images were recorded in confocal and STED modes.
1
1-H,H, 21c and 22c with
Stokes shifts of 80–90 nm suita-
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Phosphorylated 3-Heteroarylcoumarins
FULL PAPER
ble for immunofluorescence assays and two-color imaging
techniques were synthesized. The primary phosphate group
Acta 2008, 620, 120–126; j) for a review on coumarin-derived fluo-
rescent chemosensors, see: H. Li, L. Cai, Z. Chen, Advances in
Chemical Sensors (Ed.: W. Wang), 2012, http://www.intechopen.
com/books/advances-in-chemical-sensors/coumarin-derived-fluores-
cent-chemosensors; k) E. Kim, S. B. Pak, Discovery of New Fluores-
cent Dyes: Targeted Synthesis or Combinatorial Approach. In: Ad-
vanced Fluorescence Reporters in Chemistry and Biology I: Funda-
mentals and Molecular Design (Ed.: A. P. Demchenko), Springer,
Berlin–Heidelberg, 2010.
(
OP(O)(OH) ) as a polar substituent is advantageous, as it
2
provides higher fluorescence quantum yields in bioconju-
[15]
gates, than the sulfonic acid residue.
The confocal and
super-resolution STED images demonstrated good signal-to-
noise ratio and very good optical resolution. Detection and
co-localization of various biological objects can be achieved
by staining with these dyes and a standard “green” marker
with a small Stokes shift (for example, Oregon Green 488),
using one detection channel and two excitation sources (405
and 488 nm lasers, respectively). Under STED conditions,
all new dyes provide an optical resolution below the diffrac-
tion limit (50–75 nm depending on the actual implementa-
tion of the STED microscope and the power of the STED
laser). Brighter dyes were obtained if position 4 in the cou-
marin system was unsubstituted. The presence of two varia-
ble positions (3 and 4) in the coumarin scaffold affords flexi-
ble structural variations in further design of new coumarin
dyes with “tailor-made” properties.
[
3] a) G. A. Reynolds, K.-H. Drexhage, Opt. Commun. 1975, 13, 222–
2
25; b) K.-H. Drexhage, G. R. Erikson, G. H. Hawks, G. A. Rey-
nolds, Opt. Commun. 1975, 15, 399–403; c) G. Jones II, W. R. Jack-
son, A. M. Halpern, Chem. Phys. Lett. 1980, 72, 391–395; d) G. Jo-
nes II, W. R. Jackson, S. Kanoktanaporn, A. M. Halpern, Opt.
Commun. 1980, 33, 315–320; e) A. N. Fletcher, D. E. Bliss, J. M.
Kauffman, Opt. Commun. 1983, 47, 57–61; f) J. E. T. Corrie,
V. R. N. Munasinghe, W. Rettig, J. Heterocycl. Chem. 2000, 37,
1
447–1454; g) C. Uttamapinant, K. A. White, H. Baruah, S. Thomp-
son, M. Fernꢅndez-Suꢅrez, S. Puthenveetil, A. Y. Ting, Proc. Natl.
Acad. Sci. USA 2010, 107, 10914–10919; h) Q. Lin, C. Bao, G. Fan,
S. Cheng, H. Liu, Z. Liu, L. Zhu, J. Mater. Chem. 2012, 22, 6680–
6
688; i) J. Gordo, J. Avꢆ, A. J. Parola, J. C. Lima, A. Pereira, P. S.
Branco, Org. Lett. 2011, 13, 5112–5115; j) C. Murata, T. Masuda, Y.
Kamochi, K. Todoroki, H. Yoshida, H. Nohta, M. Yamaguchi, A.
Takadate, Chem. Pharm. Bull. 2005, 53, 750–758; k) A Guide to Flu-
orescent Probes and Labeling Technologies, Invitrogen, 2010 (11th
ed.), pp. 82–85.
4] F. Vollmer, W. Rettig, E. Birckner, J. Fluoresc. 1994, 4, 65–69.
5] a) P. Czerney, M. Wenzel, B. Schwender, F. Lehmann, (Dyomics
Gmbh) US 7563907 B2, 21.07.2009; b) A. Zilles, J. Arden-Jacob, K.-
H. Drexhage, N. U. Kemnitzer, M. Hamers-Schneider, (Atto-tec
Gmbh) US 2006/0179585A1; WO2005/003086A2; c) F. W. Westheim-
er, Science 1987, 235, 1173–1178; d) T. Bura, R. Ziessel, Org. Lett.
Acknowledgements
[
[
The authors are grateful to Nina Ohm and Jꢁrgen Bienert (MPI BPC
Gçttingen), Reinhard Machinek, Dr. Holm Frauendorf and their co-
workers (Institut fꢁr Organische und Biomolekulare Chemie, Georg-
August-Universitꢄt Gçttingen) for recording spectra. We are indebted to
Bundesministerium fꢁr Bildung und Forschung (BMBF 513) for financial
support in the program Optische Technologien fꢁr Biowissenschaften
und Gesundheit (FKZ 13N11066). We appreciate the assistance of E.
Rothermel with the cell culture and J. Jethwa for proof-reading the
manuscript.
2
011, 13, 3072–3075; e) J. Beechem, K. R. Gee, D. C. Hagen, I. D.
Johnson, H.-C. Kang, C. Pastula, Invitrogen Corp., US7282339,
007; f) E. Lukhatov, EPOCH Biosciences Inc., WO2009/046165;
2
g) F. Bruyneel, L. D’Auria, O. Payen, P. J. Courtoy, J. Marchand-Bry-
naert, ChemBioChem 2010, 11, 1451–1457; h) M. Reddington, Bio-
conjugate Chem. 2007, 18, 2178–2190; i) M. R. Maziꢇres, C. Duprat,
J. Bellan, J. G. Wolf, Dyes Pigm. 2007, 74, 404–409; j) F. Wold, C. E.
Ballou, J. Biol. Chem. 1957, 227, 301–312; k) L. D. Freedman, G. O.
Doak, Chem. Rev. 1957, 57, 479–523.
6] N. Senda, A. Momotake, Y. Nishimura, T. Arai, Bull. Chem. Soc.
Jpn. 2006, 79, 1753–1757.
7] a) M.-E. Theoclitou, L. A. Robinson, Tetrahedron Lett. 2002, 43,
[
1] For reviews on super-resolution optical microscopy, see: a) S. W.
Hell in Single Molecule Spectroscopy in Chemistry, Physics and Biol-
ogy (Eds.: A. Grꢄslund, R. Rigler, J. Widengren), Springer, Berlin,
[
[
2
010, pp. 365–398; b) D. Puliti, D. Warther, C. Orange, A. Specht,
M. Goeldner, Bioorg. Med. Chem. 2011, 19, 1023–1029; c) S. van de
Linde, S. Wolter, M. Sauer, Austr. J. Chem 2011, 503–511; d) T.
Mꢁller, C. Schumann, A. Kraegeloh, ChemPhysChem 2012, 13,
3
907–3910; b) R. Flasik, H. Stankovicova, A. Gaplovsky, J. Donova-
lova, Molecules 2009, 14, 4838–4848; c) H. Takakura, K. Sasakura,
T. Ueno, Y. Urano, T. Terai, K. Hanaoka, T. Tsuboi, T. Nagano,
Chem. Asian J. 2010, 5, 2053–2061; d) W. Groß, W. Benicke, D.
Fuhr, A. Kleen, WO 2009/124800, PCT/EP2009/051801; e) X.
Zhang, Z. Sui, Synthesis 2006, 2568–2572; f) S. M. Pauff, S. C.
Miller, Org. Lett. 2011, 13, 6196–6199.
8] Obtained as a result of the step-wise hydrolytic substitutions of the
four fluorine atoms at the activated positions (in the presence of
water during the work-up and isolation procedures). See: a) S. M.
Polyakova, V. N. Belov, M. L. Bossi, S. W. Hell, Eur. J. Org. Chem.
1
986–2000; for the most recent references, see: e) G. T. Dempsey,
J. C. Vaughan, K. H. Chen, M. Bates, X. Zhuang, Nat. Methods
011, 8, 1027–1040; f) M. Bates, G. T. Dempsey, K. H. Chen, X.
2
Zhuang, ChemPhysChem 2012, 13, 99–107; g) M. Sauer, J. Hofkens,
J. Enderlein, Handbook of Fluorescence Spectroscopy and Imaging,
Wiley-VCH, Weinheim, 2011.
[
[
2] a) For a report on parallel synthesis and screening of fluorescent
coumarins, see: M.-S. Schiedel, C. A. Briehn, P. Bꢄuerle, Angew.
Chem. 2001, 113, 4813–4816; Angew. Chem. Int. Ed. 2001, 40, 4677–
4
680; b) for a basic review on 7-aminocoumarins, see: I. I. Grand-
berg, L. K. Denisov, O. A. Popova, Khim. Geterotsikl. Soedin. 1987,
47–174 (Engl. Transl.: Chem. Het. Comp. 1987, 117–142); c) N. A.
2011, 3301–3312; b) S. E. Ellzey Jr., W. A. Guice, J. Org. Chem.
1966, 31, 1300–1303; c) R. F. Stockel, F. H. Megson, Can. J. Chem.
1967, 45, 1998–2000.
1
Gordeeva, M. A. Kirpichenok, N. S. Patalakha, I. I. Grandberg,
Khim. Get. Soed. 1990, 1600–1609 (Engl. Transl.: Chem. Het. Comp.
[
9] a) J. L. Luche, J. Am. Chem. Soc. 1978, 100, 2226–2227; A. L.
Gemal, J. L. Luche, J. Am. Chem. Soc. 1981, 103, 5454–5459; for
the representative examples of phosphorylation reaction involving
primary alcohols, see: b) G. G. Durgam, T. Virag, M. D. Walker, R.
Tsukahara, S. Yasuda, K. Liliom, L. A. van Meeteren, W. H. Moole-
naar, N. Wilke, W. Siess, G. Tigyi, D. D. Miller, J. Med. Chem. 2005,
48, 4919–4930; c) J. W. Perich, R. B. Johns, Synthesis 1988, 142–144.
1
990, 1329–1337); d) T. Ehrenschwender, B. R. Vagra, P. Kele, H.-
A. Wagenknecht, Chem. Asian J. 2010, 5, 1761–1764; e) K. Nagy, E.
Orbꢅn, S. Bçsze, P. Kele, Chem. Asian J. 2010, 5, 773–777; f) J.-A.
Richard, M. Massonneau, P.-Y. Renard, A. Romieu, Org. Lett. 2008,
1
0, 4175–4178; g) H.-C. Huang, K.-L. Wang, S.-T. Huang, H.-Y. Lin,
C.-M. Lin, Biosens. Bioelectron. 2011, 26, 3511–3516; h) S.-T.
Huang, Y.-X. Peng, K.-L. Wang, Biosens. Bioelectron. 2008, 23,
[10] S. H. Mashraqui, D. Vashi, H. D. Mistry, Synth. Commun. 2004, 34,
1
793–1798; i) S.-T. Huang, K.-N. Ting, K.-L. Wang, Anal. Chim.
3129–3134.
Chem. Eur. J. 2012, 18, 16339 – 16348
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
16347

V. N. Belov, S. W. Hell et al.
[
[
[
11] a) K. Sorensen-Stowell, A. C. Hengge, J. Org. Chem. 2005, 70,
[14] V. P. Boyarskiy, V. N. Belov, R. Medda, B. Hein, M. Bossi, S. W.
Hell, Chem. Eur. J. 2008, 14, 1784–1792.
4
5
805–4809; b) B. Hachꢈ, L. Brett, S. Shakya, Tetrahedron Lett. 2011,
2, 3625–3629; c) E. Lieber, S. H. Patinkin, H. H. Tao, J. Am.
[15] See ref. [5f]: In this study, a diphosphonated oxazine dye with a
large Stokes shift of approximately 100 nm has been prepared, and
its properties compared with those of the sulfonated one. The fluo-
rescence quantum yield of the former dye in PBS buffer was found
to be three times higher than that of the latter one, but in living
cells the diphosphonated dye was found to be non-fluorescent.
Chem. Soc. 1951, 73, 1792–1795.
12] a) For example, upon acidification, pyrazin- and 6-methylpyrimid-4-
yl acetic acids readily lose carbon dioxide. The latter compound
could not be isolated; b) available from Sigma–Aldrich Co.; howev-
er, the preparation of the free acid is not described in the literature.
13] a) http://www.horiba.com/fileadmin/uploads/Scientific/Documents/
Fluorescence/quantumyieldstrad.pdf; b) for a review, see: O. Valdes-
Aguilera, D. C. Neckers, Acc. Chem. Res. 1989, 22, 171–177; howev-
er, fluorescence quantum yield in aqueous solutions does not always
correlate with the polarity and solubility in water.
Received: July 4, 2012
Revised: September 13, 2012
Published online: October 30, 2012
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Chem. Eur. J. 2012, 18, 16339 – 16348