Clickable long-wave "mega-stokes" fluorophores for orthogonal chemoselective labeling of cells

Article abstract of DOI:10.1002/asia.200900477

Dyes that can easily be tied: The synthesis of water-soluble far-red/NIR emitting polymethine-based dyes with clickable functionalities (azide, alkyne) and their application in cell-surface labeling are presented.

Full text of DOI:10.1002/asia.200900477

DOI: 10.1002/asia.200900477
Clickable Long-Wave “Mega-Stokes” Fluorophores for Orthogonal
Chemoselective Labeling of Cells
Krisztina Nagy,[a] Erika Orbꢀn,[b] Szilvia Bo˝sze,[b] and Pꢁter Kele*[a]
Dedication to Prof. Dr. Pꢀter Huszthy on the occasion of his 60th birthday
In vitro and in vivo fluorescence imaging of biological
structures that use near-infrared (NIR) fluorophores is be-
coming more and more important for their high sensitivity,
excellent temporal and spatial resolution, and their potential
for multichannel imaging.[1] Fluorophores in the spectral
region between the far-red and NIR region are particularly
suitable for biological (both in vitro and in vivo) labeling as
they are less or not at all interfered by biological back-
ground luminescence. Therefore there is an increasing
demand for water-soluble labels fluorescing in the NIR
regime.[2] It is crucial that the covalent ligation of these
probes to the biomolecule of interest is fast, high-yielding,
and biocompatible and the functional groups do not inter-
fere with other naturally occurring reactive sites. For these
techniques the term “bioorthogonality” was introduced.
Little effort has been made to develop bioorthogonal labels
in this spectral regime.[3] In a bioorthogonal labeling scheme
the introduction of reporter tags relies on their selective and
efficient reaction under physiological conditions with func-
tional groups available at the biomolecules of interest.
nously delivered probes” are particularly suitable for fluo-
rescent labeling of biological species.[4] Among these tagging
reactions, the Staudinger ligation,[5c] the inverse electron
demand Diels–Alder reaction between tetrazines and strain-
ed alkenes,[5d,e] the photoactivable dipolar cycloaddition of
tetrazoles and alkenes,[5f] and the so-called click reaction
represented by the CuI-catalyzed azide–alkyne cycloaddition
(CuAAC)[5g,h] are the most valuable ones.[5] Their superiority
over other methods is due to the inertness of the chemical
reporters, the exogenously delivered probes and their selec-
tive and efficient reaction with each other with the tolerance
of the majority of functional groups. The extreme rareness
of azide and alkyne functions in biological systems as well
as the easy access of azide/alkyne-modified biological build-
ing blocks further increase the importance of tagging by
means of copper-catalyzed azide–alkyne cycloaddition. This
reaction or its copper-free, strain-promoted alternative
(SPAAC) has been shown to be quite versatile in terms of
biological applications.[6,7] CuAAC also finds widespread ap-
plications in high-throughput screening of libraries.[8] The
versatility of the combined use of copper-catalyzed and Cu-
free click chemistries in sequential labeling methods was
also demonstrated recently.[9] Besides the fundamental crite-
ria, for example, chemical stability, water solubility, bright
emission, etc. that a conjugatable dye should meet, a syn-
thetic routine providing fast and good-yielding access to
these dyes is also demanding. Herein we report an efficient
routine to access azide/alkyne-modified, therefore “clicka-
ble”, dyes that emit in the far-red/NIR regime and have
very large Stokes shifts.
ACHTUNGTRENNUNG(Bio)orthogonal chemical reporters that are “non-native,
non-perturbing chemical handles that can be modified in
living systems through highly selective reactions with exoge-
[a] K. Nagy,+ Dr. P. Kele
Institute of Chemistry
Eçtvçs Lorꢀnd University, Faculty of Natural Sciences
H-1117 Pꢀzmꢀny Pꢁter sꢁtꢀny 1a., Budapest (Hungary)
Fax : (+36)1-372-2909
E-mail: kelep@elte.hu
[b] E. Orbꢀn,+ Dr. Sz. Bosze
As part of our program on the development of FRET la-
beled MMP-2 substrates we wished to develop clickable
NIR-dyes with large Stokes shifts.[9] The work of Czerney
et al.[10] on the development of the so-called “mega-Stokes”
dye family has directed our attention to the polymethine
scaffolds shown in Scheme 1. In our recent report we have
shown the applicability of this dye family in the synthesis of
red-emitting clickable dyes.[3b] We expected that installation
˝
Research Group of Peptide Chemistry
Hungarian Academy of Sciences
Eçtvçs Lorꢀnd University
Faculty of Natural Sciences, Institute of Chemistry
H-1117 Pꢀzmꢀny Pꢁter sꢁtꢀny 1a., Budapest (Hungary)
[+] These authors have contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900477.
Chem. Asian J. 2010, 5, 773 – 777
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
773

COMMUNICATION
acceptable quantum yields and excellent photostabilities.
For example, continuous irradiation of 5 and 6 for 1 h re-
sulted in only 4 and 21% loss of the original intensity, re-
spectively. As expected, all photophysical characteristics of
the fluorophores were found to be identical for the a and b
series dyes.
The excitation spectra of the new dyes (Figure 1) show
that each of them is compatible with laser sources (e.g., Ar,
He-Ne, diode lasers) widely used in fluorescence instrumen-
Scheme 1. Synthetic route for preparation of clickable dyes. Reaction
conditions: a) 20% oleum, cat. HgSO4; b) 5-iodo-1-pentyne (2a) or 1,3-
diiodopropane/MeCN, reflux (2b); c) NaN3/MeCN, reflux; d) EtOH, cat.
piperidine, reflux.
of a sulfonyl function onto the picoline ring will increase
water solubility and shift the spectral properties towards the
NIR region of the spectrum relative to derivatives that do
not possess a sulfonyl function.[3b]
The synthetic procedure started with the introduction of
the sulfonyl function onto the picoline ring. Treatment of 4-
picoline with 20% oleum furnished the desired sulfonylpico-
line salt 1 in good yield. Alkylation of 1 was then accom-
plished either by reaction with 5-iodo-1-pentyne or 1,3-diio-
dopropane to obtain the corresponding sulfonylpicolinium
betaines 2. Compound 2b was further subjected to halogen–
azide exchange, which was effected by treatment with NaN3.
Precursors 2 were then condensed with the appropriate al-
dehydes in the presence of a catalytic amount of piperidine.
This synthetic route afforded each dye molecule in accepta-
ble to good yields. The synthetic routine justifies an easy
access to these dyes without the need for special chemicals
or difficult reaction steps.
The fluorescence excitation and emission spectra of the
dyes showed that each possesses a large Stokes shift
(>100 nm). To our delight both excitation and emission
spectra were shifted towards the NIR regime and emission
maxima were positioned at 625, 674, and 734 nm for 4, 5,
and 6, respectively (Table 1). Regardless of the starting alde-
hyde, the photophysical studies revealed that all dyes have
Figure 1. Fluorescence excitation (gray) and emission (black) spectra of
labels in methanol.
tation such as cell sorters and imagers. The large Stokes
shifts make these dyes ideal candidates for fluorescence res-
onance energy transfer (FRET) applications. An often en-
countered problem in FRET assays using common fluoro-
phores is that there is a direct excitation of acceptor emis-
sion by the light used to excite the donor. This problem re-
duces the dynamic range of assays where most of the accept-
or is unbound. Another problem is the overlap between the
emission of the donor and the emission band of the acceptor
dye. The large Stokes shift is likely to reduce these difficul-
ties.
To test the feasibility of clickable dyes in chemoselective
tagging reactions the surface glycoprotein labeling of fixed
cells supplied on azido sugar containing nutrients have fre-
quently been used recently.[3a,7a] We have adapted this
system to demonstrate the ability of our dyes to undergo or-
thogonal labeling reactions efficiently. Prior to labeling, Chi-
nese hamster ovary (CHO) cells were incubated with 4a–6a
to assess the possible cytostatic effects of these dyes. Based
on experimental data, neither dye has a cytostatic effect on
CHO cells (see the Supporting Information). Subsequently,
CHO cells were treated with azidoacetylmannosamine
(ManNAz) for 3 days. The cells, which incorporated the
azido sugar metabolically yielding azido sialic acid residues
in their surface glycoproteins were then fixed with methanol
at À208C for 10 min, and acetone for 1 min. Fixed cells
were then subsequently treated with 4a, 5a, or 6a (20 mm).
Table 1. Spectral characteristics of clickable dyes.
Dye Solvent lmax (exc)
lmax (em)
[nm]
e (ꢃ104)
f[b]
[nm]
[MÀ1 cmÀ1
]
MeOH 519
625
630
674 (695)
675 (697)
735
5.6
4.3
4.8
5.3
4.0
2.8
0.8.
n.d.
15.7
1.0
1.0
n.d.
4
PBS[a]
523
MeOH 538
PBS 544
MeOH 586
PBS 588
5
6
744
[a] PBS: phosphate-buffered saline solution (pH 7.4). [b] Using cresyl
violet as reference standard. n.d.=not determined.
774
www.chemasianj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2010, 5, 773 – 777

Clickable Long-Wave “Mega-Stokes” Fluorophores
The reaction was facilitated with CuSO4-sodium-ascorbate
them to be expected to draw much interest in a large variety
of applications in chemoselective and (bio)orthogonal label-
ing schemes of proteins, sugars, lipids, nucleic acids, or or-
ganelles both in vivo and in vitro.
(100 mm/1 mm)
and
tris(benzyltriazolylmethyl)amine
(100 mm, TBTA[11]) in PBS (pH 7.0) at 378C. The results
have shown that after 25 min reaction all labels had caused
fluorescent tagging of the cells. Variations in the brightness
of the images indicated differences probably in the quantum
yields of the dyes. In the case of control cells that previously
were not supplemented with ManNAz only negligible fluo-
rescence could be observed owing only to unspecific label-
Experimental Section
Unless otherwise indicated, all starting materials were obtained from
commercial suppliers (Sigma–Aldrich, Fluka) and used without further
purification. Analytical thin-layer chromatography (TLC) was performed
on Polygram SIL G/UV 254 precoated plastic TLC plates with 0.25 mm
silica gel from Macherey–Nagel + Co. Silica gel column chromatography
was carried out with flash silica gel (0.040–0.063 mm) from Merck. The
NMR spectra were recorded on a Bruker DRX-250 or Varian Inova
600 MHz spectrometer. Chemical shifts (d) are given in parts per million
(ppm) using solvent signals as the reference. Coupling constants (J) are
reported in Hertz (Hz). Splitting patterns are designated as s (singlet), d
(doublet), t (triplet), quint (quintuplet), m (multiplet), dd (doublet of a
doublet). Cells were imaged using Olympus Fluoview500 software of an
Olympus IX81 confocal laser scanning microscope using the 514 argon-
ion, 543 argon-helium or 633 argon-helium laser lines to excite dyes.
Sodium 4-methylpyridine-3-sulfonate (1)[12]
: A reaction vessel was
charged with mercury-sulfate catalyst (210 mg) and 20% fuming sulphu-
ric acid (53.0 g). To this mixture was added 4-picoline (10.0 g, 0.107 mol)
dropwise at room temperature. After the addition was complete the tem-
perature was raised to 225–2358C and the reaction mixture was vigorous-
ly stirred for 5 h at this temperature. The reaction mixture was neutral-
ized with 50% NaOH to pH 9–10 while it was cooled on an ice-water
bath. The solvent was then evaporated and the product was extracted
with methanol. The methanolic extract was evaporated and the crude
product (12.3 g, 59%) was recrystallized from ethanol to give pure prod-
uct as an off-white solid. 1H NMR ([D6]DMSO): d=2.53 (3H, s), 7.20
(1H, d, J=4.9 Hz), 8.37 (1H, d, J=4.9 Hz), 8.79 ppm (1H, s); 13C NMR
([D6]DMSO): d=19.4, 125.7, 141.4, 144.9, 146.9, 149.7 ppm. M.p.
>2408C. IR (neat): n˜ =3052, 1230, 1213 cmÀ1. HRMS (ESI) calcd for
C6H6NO3SÀ [MÀNa]À: 172.0074; found: 172.0078.
Figure 2. Fluorescent microscopy images of CHO cells. Control experi-
ments with cells not bearing azido sialic acid and treated with 4a, 5a,
and 6a for 25 min (A, B, C); cells modified with ManNAz and labeled
with 4a, 5a, and 6a for 25 min (D, E, F); time dependency: ManNAz-
treated cells labeled with 6a for 5, 25, and 60 min (G, H, I). For color
images, see the Supporting Information.
1-(5-Pentyn-1-yl)-3-sulfonato-4-methylpyridinium betaine (2a): Sodium
4-methylpyridine-3-sulfonate (1.0 g, 5.12 mmol) and 5-iodo-1-pentyne[13]
(2.10 g, 10.8 mmol) were heated at reflux in DMF for 14 h. After cooling
to room temperature the solvent was removed in vacuo and the product
was purified on silica (dichloromethane-methanol-triethylamine 10:1:0.1)
to give 0.86 g (70%) of the desired product as a yellowish solid. 1H
([D6]DMSO): d=2.08 (2H, quint., J=7.1 Hz), 2.21–2.30 (2H, m), 2.79
(3H, s), 2.85 (1H, t, J=2.7 Hz), 4.63 (2H, t, J=7.1 Hz), 8.01 (1H, d, J=
6.2 Hz), 8.92 (1H, dd, J=1.4 Hz, J=6.2 Hz), 9.09 ppm (1H, d, J=
1.4 Hz); 13C ([D6]DMSO): d=14.7, 20.2, 29.2, 59.2, 72.3, 82.4, 129.8,
141.4, 144.0, 145.4, 156.7 ppm. M.p. 175–1798C. IR (neat): n˜ =3278, 3044,
ing of the cells (Figure 2A, B, and C). Contrary to this, cells
bearing azido sialic acid residues in their surface glycans
were labeled efficiently as shown in Figure 2D, E, and F. We
have also studied fluorescent labeling of azido-modified
cells on the time scale. The fixed cells were washed three
times with PBS and incubated with the dyes for 5, 25, and
60 min (Figure 2G, H, and I). Results showed that even
after 5 min reaction time efficient labeling could be ob-
served. For the confocal microscopy images (Figure 2) con-
ventional laser sources as 514 nm Ar, 543 nm He-Ne, and
633 nm He-Ne lasers were used for 4a, 5a, and 6a, respec-
tively, indicating the suitability of the present dyes for mi-
croscopic and/or flow-cytometric applications. Moreover dye
6 was excitable both by green and red lasers.
In conclusion, the set of clickable fluorophores presented
here is suitable for fluorescent labeling of biomolecules. The
synthetic routine provides efficient and easy access to these
dyes. The tagging molecules possess acceptable quantum
yields, which is best documented by the contrast of the mi-
croscopy images. The easy access to these dyes justifies
2935, 1213 cmÀ1
.
HRMS (ESI) calcd for C11H14NO3S+ [M+H]+:
240.0689; found: 240.0685.
1-(3-Iodopropyl)-3-sulfonato-4-methylpyridinium betaine (2b): Sodium
4-methylpyridine-3-sulfonate (1.0 g, 5.12 mmol) and 1,3-diiodopropane
(6.06 g, 20.5 mmol) were heated at reflux in DMF for 4 h. After cooling
to room temperature the solvent was removed in vacuo and the product
was purified on silica (dichloromethane-methanol 10:1 to 5:1 v/v%) to
give 1.43 g (82%) of the desired product as
a
yellowish solid. 1H
([D6]DMSO): d=2.42 (2H, quint., J=7.3 Hz), 2.78 (3H, s), 3.21 (2H, t,
J=7.3 Hz), 4.61 (2H, t, J=7.1 Hz), 8.01 (1H, d, J=6.3 Hz), 8.92 (1H,
dd, J=1.4 Hz, J=6.2 Hz), 9.09 ppm (1H, d, J=1.3 Hz); 13C
([D6]DMSO): d=1.3, 20.2, 34.1, 60.5, 129.8, 141.4, 143.9, 145.4,
156.7 ppm. M.p. 136–1398C. IR (neat): n˜ =3110, 3033, 2952, 1213 cmÀ1
.
HRMS (ESI) calcd for C9H13INO3S+ [M+H]+: 341.9655; found:
341.9649.
1-(3-Azidopropyl)-3-sulfonato-4-methylpyridinium betaine (3b): 1-(3-Io-
dopropyl)-3-sulfonato-4-methylpyridinium betaine (0.85 g, 2.5 mmol) and
NaN3 (0.49 g, 7.5 mmol) were dissolved in an acetonitrile
ACHTUNGTRENUN(NG 40 mL)-DMF
AHCTUNGERTG(NNUN 5 mL) mixture and the resulting solution was heated at reflux for 3 h.
Chem. Asian J. 2010, 5, 773 – 777
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
775

P. Kele et al.
COMMUNICATION
After cooling to room temperature the solution was filtered to remove
salts and the filtrate was brought to dryness. To the remainder solid was
added dichloromethane and the solution was filtered through a plug of
silica. Evaporation of the solvent gave 0.6 g (94%) product as a yellow
crystalline solid. 1H ([D6]DMSO): d=2.15 (2H, quint., J=6.6 Hz), 2.78
(3H, s), 3.45 (2H, t, J=6.6 Hz), 4.64 (2H, t, J=7.1 Hz), 8.03 (1H, d, J=
6.3 Hz), 8.95 (1H, dd, J=1.4 Hz, J=6.3 Hz), 9.10 ppm (1H, d, J=
1.3 Hz); 13C ([D6]DMSO): d=20.1, 29.7, 47.5, 57.6, 129.7, 141.4, 144.0,
1H ([D6]DMSO): d=2.14 (2H, quint, J=6.5 Hz), 3.02 (6H, s), 3.45 (2H,
t, J=6.5 Hz), 4.52 (2H, t, J=6.5 Hz), 6.80 (2H, d, J=8.2 Hz), 7.51 (2H,
d, J=8.5 Hz), 7.93 (2H, AB, J=15.9 Hz), 8.39 (1H, d, J=6.5 Hz), 8.72
(1H, d, J=6.5 Hz), 8.97 ppm (1H, s); 13C ([D6]DMSO): d=29.6, 43.7,
47.6, 56.8, 111.9, 116.2, 121.2, 122.8, 125.6, 130.1, 141.1, 142.1, 142.5,
150.7, 151.9 ppm. M.p. 188–1918C. IR (neat): n˜ =2905, 2095, 1564, 1524,
1158 cmÀ1. HRMS (ESI) calcd for C18H22N5O3S+ [M+H]+: 388.1438;
found: 388.1429.
145.3, 156.6 ppm. M.p. 94–978C. IR (neat): n˜ =3023, 2098, 1214 cmÀ1
.
1-(3-Azidopropyl)-4-([E]-2-(7-diethylamino-2-oxo-2H-chromen-3-yl)-
vinyl]-3-sulfonato pyridinium betaine (5b): 1-(3-Azidopropyl)-3-sulfona-
to-4-methylpyridinium betaine (0.21 g, 0.815 mmol) and 7-diethylamino-
2-oxo-2H-chromene-3-carbaldehyde (0.20 , 0.815 mmol) were stirred at
608C in ethanol (30 mL) in the presence of a catalytic amount of piperi-
dine (six drops) for 14 h. The solvent was removed in vacuo and the
product was purified on silica (acetonitrile-acetonitrile/NH4PF6) to give
0.120 g (36%) product as a dark solid. 1H ([D6]DMSO): d=1.15 (6H, t,
J=7.1 Hz), 2.17 (2H, quint, J=6.9 Hz), 3.45–3.51 (6H, m), 4.58 (2H, t,
J=6.9 Hz), 6.62 (1H, d, J=2.4 Hz), 6.78 (1H, dd, J=2.4 Hz, J=8.9 Hz),
7.61 (1H, d, J=8.9 Hz), 7.76 (1H, d, J=16.3 HZ), 8.14 (1H, s), 8.37 (1H,
d, J=16.3 Hz), 8.39 (1H, d, J=6.9 Hz), 8.82 (1H, dd, J=1.2 Hz, J=
6.9 Hz), 9.09 ppm (1H, d, J=1.2 Hz); 13C ([D6]DMSO): d=12.3, 29.6,
44.2, 47.6, 57.1, 96.2, 108.2, 109.8, 121.9, 122.4, 130.7, 136.1, 142.3, 142.4,
143.2, 143.3, 143.6, 150.8, 151.8, 156.3, 159.5 ppm. M.p.>2408C. IR
(neat): n˜ =3047, 2928, 2086, 1714, 1515 cmÀ1. HRMS (ESI) calcd for
C23H26N5O5S+ [M+H]+: 484.1649; found: 484.1644.
HRMS (ESI) calcd for C9H13N4O3S+ [M+H]+: 257.0703; found:
257.0698.
1-(5-Pentyn-1-yl)-4-([E]-2-(4-dimethylaminophenyl)-vinyl]-3-sulfonato
pyridinium betaine (4a): 1-(5-Pentyn-1-yl)-3-sulfonato-4-methylpyridini-
um betaine (0.25 g, 1.05 mmol) and 4-(dimethylamino)benzaldehyde
(0.156 g, 1.05 mmol) were stirred at 608C in ethanol (30 mL) in the pres-
ence of catalytic amount of piperidine (six drops) for 14 h. The solvent
was removed in vacuo and the product was purified on silica (dichloro-
methane-methanol 9:1 v/v%) to give 0.30 g (77%) product as a red solid.
1H ([D6]DMSO): d=2.07 (2H, quint, J=7.0 Hz), 2.25–2.28 (2H, m), 2.87
(1H, t, J=2.9 Hz), 3.03 (6H, s), 4.51 (2H, t, J=7.0 Hz), 6.81 (2H, d, J=
8.8 Hz), 7.51 (2H, d, J=8.8 Hz), 7.92 (2H, AB, J=15.8 Hz), 8.36 (1H, d,
J=7.0 Hz), 8.70 (1H, d, J=7.0 Hz), 8.97 ppm (1H, d, J=1.8 Hz); 13C
([D6]DMSO): d=14.7, 29.1, 40.0, 58.2, 72.3, 82.5, 111.9, 116.3, 121.1,
122.8, 130.1, 141.2, 141.9, 142.0, 142.4, 150.8, 151.9 ppm. M.p.>2408C. IR
(neat): n˜ =3224, 3072, 2990, 1213 cmÀ1
. HRMS (ESI) calcd for
C20H23N2O3S+ [M+H]+: 371.1424; found: 371.1423.
1-(3-Azidopropyl)-4-([E]-2-(6-diethylamino-benzofuran-2-yl)-vinyl]-3-sul-
fonato pyridinium betaine, (6b): 1-(3-Azidopropyl)-3-sulfonato-4-methyl-
pyridinium betaine (0.21 g, 0.815 mmol) and 6-diethylamino-benzofuran-
2-carbaldehyde (0.20 , 0.815 mmol) were stirred at 608C in ethanol
(30 mL) in the presence of 1.2 equiv piperidine (six drops) for 14 h. The
solvent was removed in vacuo and the product was purified on silica (ace-
tonitrile-acetonitrile/NH4PF6) to give 0.102 g (27%) product as a dark
solid. 1H ([D6]DMSO): d=1.14 (6H, t, J=7.0 Hz), 2.16 (2H, quint, J=
7.0 Hz), 3.41–3.59 (6H, m), 4.55 (2H, t, J=7.0 Hz), 6.76 (1H, dd, J=
2.3 Hz, J=8.8 Hz), 6.88 (1H, s), 7.18 (1H, s), 7.48 (1H, d, J=8.8 Hz),
7.93 (2H, AB, J=15.8 Hz), 8.39 (1H, d, J=7.0 Hz), 8.78 (1H, d, J=
7.0 Hz), 9.05 ppm (1H, d, J=1.8 Hz); 13C ([D6]DMSO): d=12.3, 29.6,
44.2, 47.6, 57.0, 92.2, 110.2, 114.5, 117.2, 118.4, 121.7, 122.7, 127.7, 141.8,
142.3, 142.8, 148.2, 149.7, 150.7, 158.5 ppm. M.p. 163–1668C. IR (neat):
1-(5-Pentyn-1-yl)-4-([E]-2-(7-diethylamino-2-oxo-2H-chromen-3-yl)-
vinyl]-3-sulfonato pyridinium betaine (5a): 1-(5-Pentyn-1-yl)-3-sulfonato-
4-methylpyridinium betaine (0.20 g, 0.836 mmol) and 7-diethylamino-2-
oxo-2H-chromene-3-carbaldehyde[14] (0.21 g, 0.836 mmol) was stirred at
608C in ethanol (30 mL) in the presence of a catalytic amount of piperi-
dine (six drops) for 14 h. The solvent was removed in vacuo and the
product was purified on silica (dichloromethane-methanol 9:1 v/v%) to
give 0.35 g (90%) product as a dark red solid. 1H ([D6]DMSO): d=1.14
(6H, t, J=7.1 Hz), 2.09 (2H, quint, J=6.6 Hz), 2.22–2.32 (2H, m), 2.88
(1H, t, J=2.7 Hz), 3.48 (4H, q, J=6.6 Hz), 4.57 (2H, t, J=7.1 Hz), 6.61
(1H, d, J=2.2 Hz), 6.77 (1H, dd, J=2.4 Hz, J=9.0 Hz), 7.60 (1H, d, J=
9.0 Hz), 7.75 (1H, d, J=16.3 Hz), 8.13 (1H, s), 8.35 (1H, d, J=16.2 Hz),
8.39 (1H, d, J=6.9 Hz), 8.81 (1H, d, J=6.6 Hz), 9.07 ppm (1H, d, J=
1.3 Hz); 13C ([D6]DMSO): d=12.3, 14.7, 29.2, 44.2, 58.6, 72.3, 82.4, 96.2,
108.2, 109.8, 114.3, 121.9, 122.4, 130.7, 136.1, 142.3, 142.4, 143.1, 143.6,
150.8, 151.8, 156.3, 159.5 ppm. M.p.>2408C. IR (neat): n˜ =3272, 3073,
2971, 1610, 1185 cmÀ1. HRMS (ESI) calcd for C25H27N2O5S+ [M+H]+:
467.1635; found: 467.1624.
n˜ =3044, 2100, 1559 cmÀ1
.
HRMS (ESI) calcd for C22H26N5O4S+
[M+H]+: 456.1700; found: 457.1692.
1-(5-Pentyn-1-yl)-4-([E]-2-(6-diethylamino-benzofuran-2-yl)-vinyl]-3-sul-
fonato pyridinium betaine (6a): 1-(5-Pentyn-1-yl)-3-sulfonato-4-methyl-
pyridinium betaine (0.15 g, 0.631 mmol) and 6-diethylamino-benzofuran-
2-carbaldehyde[15] (0.16 g , 0.631 mmol) were stirred at 608C in ethanol
(30 mL) in the presence of a catalytic amount of piperidine (six drops)
for 14 h. The solvent was removed in vacuo and the product was purified
on silica (dichloromethane-methanol 9:1 v/v%) to give 0.056 g (20%)
Acknowledgements
Financial support from the Hungarian Scientific Research Fund and the
National Office for Research and Technology (OTKA-NKTH: H07-B-
74291, K68358, and NKFP-07-1TB INTER-HU) is greatly acknowledged.
We thank Prof. Dr. Jꢀnos Matkꢄ for his help with fluorescent microscopy
imaging, Dr. Szabolcs Bꢁni for NMR data collection, and Ms. Daniela
Achatz for her help in quantum yield determination.
1
product as a dark solid. H ([D6]DMSO): d=1.14 (6H, t, J=7.0 Hz), 2.08
(2H, quint, J=7.0 Hz), 2.21–2.34 (2H, m), 2.89 (1H, t, J=2.4 Hz), 3.44
(4H, q, J=7.0 Hz), 4.53 (2H, t, J=7.0 Hz), 6.76 (1H, dd, J=1.9 Hz, J=
8.8 Hz), 6.88 (1H, s), 7.17 (1H, s), 7.48 (1H, d, J=8.8 Hz), 7.93 (2H, AB,
J=15.9 Hz), 8.38 (1H, d, J=7.0 Hz), 8.76 (1H, d, J=7.0 Hz), 9.03 ppm
(1H, s); 13C ([D6]DMSO): d=12.3, 14.8, 29.1, 44.2, 58.5, 72.3, 82.5, 92.2,
110.2, 114.5, 117.2, 118.4, 121.7, 122.6, 127.7, 141.8, 142.2, 142.8, 148.2,
149.7, 150.7, 158.5 ppm. M.p.>2408C. IR (neat): n˜ =3280, 2968,
2362 cmÀ1. HRMS (ESI) calcd for C24H27N2O4S+ [M+H]+: 439.1686;
found: 439.1682.
Keywords: bioorthogonality · click chemistry · fluorescent
probes · FRET
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Received: September 18, 2009
Published online: February 1, 2010
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www.chemasianj.org
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