Clickable fluorophores for biological Labeling - With or without copper

Article abstract of DOI:10.1039/b907741c

The synthesis of a set of new clickable fluorophores that virtually cover the whole visible spectrum reaching the near infra-red regime is presented herein. Besides dyes that are capable of participating in classical copper catalyzed 1,3-dipolar cycloaddition reactions with the counterparting function we have also prepared dyes containing a cyclooctyne moiety, an alkyne derivative that enables copper free clicking to azides. The suitability of these dyes for fluorescent labeling of biomolecules is presented by examples on model frameworks representing major biopolymer building blocks. The versatility of these dyes is presented in cell labeling experiments as well as by labeling the azide modified surface glycans of CHO-cells either by copper catalyzed or copper-free click reaction. These dyes are expected to have a large variety of applications in (bio)orthogonal labeling schemes both in vivo and in vitro.

Full text of DOI:10.1039/b907741c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Clickable fluorophores for biological labeling—with or without copper†
Pe´ter Kele,*a,b Xiaohua Li,b,c Martin Link,b Krisztina Nagy,a Andra´s Herner,a Krisztia´n Lo˝rincz,a
Szabolcs Be´nid and Otto S. Wolfbeis*b
Received 17th April 2009, Accepted 12th June 2009
First published as an Advance Article on the web 7th July 2009
DOI: 10.1039/b907741c
The synthesis of a set of new clickable fluorophores that virtually cover the whole visible spectrum
reaching the near infra-red regime is presented herein. Besides dyes that are capable of participating in
classical copper catalyzed 1,3-dipolar cycloaddition reactions with the counterparting function we have
also prepared dyes containing a cyclooctyne moiety, an alkyne derivative that enables copper free
clicking to azides. The suitability of these dyes for fluorescent labeling of biomolecules is presented by
examples on model frameworks representing major biopolymer building blocks. The versatility of these
dyes is presented in cell labeling experiments as well as by labeling the azide modified surface glycans of
CHO-cells either by copper catalyzed or copper-free click reaction. These dyes are expected to have a
large variety of applications in (bio)orthogonal labeling schemes both in vivo and in vitro.
by means of a copper catalyzed azide-alkyne cycloaddition. This
reaction has been shown to be quite versatile in terms of biological
Introduction
applications.3 CuAAC also finds widespread applications in high-
throughput screening of libraries.4 In cases where copper toxicity
can be an obstacle in the ligation process, a strain-promoted
cycloaddition between cyclooctynes and azides can be used as
an alternative as proposed by the Bertozzi and Boons groups.5,6
Very recently, we have presented our results on multiple labeling
of biomolecules using a combination of copper free and copper
mediated click chemistries in a sequential manner.7
Although the extreme sensitivity and the relatively easy detec-
tion modes of fluorescence make these techniques very appealing
there has been little effort to develop clickable dyes for all colors
present in the visible-NIR regime.8 This had initiated our work
presented herein to design clickable fluorophores that meet the
criteria of bioorthogonality.
The in vitro and in vivo labeling of biomolecules by means of
fluorescent tags represents an important tool in order to study
complex biological processes often via imaging. The importance
of fluorescent tags is due to the high sensitivity, the excellent
spatial and temporal resolution and their potential for multi-
channel imaging. The introduction of reporter tags rely on their
selective and efficient reaction under physiological conditions
with functional groups available at the biomolecule of interest.
Amongst the known ligation reactions, so-called bioorthogonal
chemical reporters have drawn much interest lately. The criteria
for bioorthogonal reporters state that these reporter tags should
be “non-native, non-perturbing chemical handles that can be
modified in living systems through highly selective reactions with
exogenously delivered probes”.1a In the bioorthogonal ligation
toolkit the most valuable ones are: (a) the Staudinger ligation1;
(b) the so-called click reaction represented by a Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC)2a,b; (c) photoinduced dipolar
cycloadditions of tetrazoles and alkenes2c,d; and (d) the inverse
electron demand Diels–Alder reaction of tetrazines and strained
alkenes.2e,f A recent review lists all these reactions in more details.2g
Among these reaction types the introduction of fluorophore tags
is synthetically most concise by a click reaction between azides
and alkynes. The extreme rareness of azide and alkyne functions
in biological systems further increases the importance of tagging
Results and discussion
The core part of the fluorescent tags 1–9 (Fig. 1) was selected
on the basis of their emission bands i.e. to have a full coverage
of the visible spectrum with edges at the near- infrared (NIR)
region (Table 1). The incorporation of the clickable azido- or
alkyne-functions was achieved by using appropriate halo-alkyl
or propargyl amine derivatives. In the case of haloalkyl amine
incorporation the halogenes (Br or I) were subsequently converted
into the appropriate azido derivatives.
aInstitute of Chemistry, Eo¨tvo¨s Lora´nd University, H-1117, Pa´zma´ny
Pe´ter se´ta´ny 1a, Budapest, Hungary. E-mail: kelep@elte.hu; Fax: (+36)1
372 2909; Tel: (+36)1 209 0555 ext. 1610
Once the syntheses of these novel dyes were accomplished we
tested their applicability by ligating them to model frameworks.
bInstitute of Analytical Chemistry, Chemo- and Biosensors, Universita¨t
Regensburg, D-93040, Regensburg, Germany. E-mail: otto.wolfbeis@
chemie.uni-r.de; Fax: (+49)941 943 4065
Table 1 Photophysical properties of clickable fluorophoresa
cBeijing National Laboratory for Molecular Sciences, Institute of Chemistry,
Chinese Academy of Sciences, Beijing, 100190, China
1
2
3
4
5
6
7
8
9
dHAS Research Group for Drugs of Abuse, Department of Pharmaceutical
Chemistry, Semmelweis University, Ho˝gyes Endre utca 9, H-1092, Budapest,
Hungary
lmax(abs) 330
lmax(em) 460
450
540
520
535
480
600
4.7
505
630
5.7
625
675
1.8
550
620
7.0
651
673
7.3 10.0
664
718
eb
0.34 0.68 6.3
† Electronic supplementary information (ESI) available: Characterization
data of fluorescent conjugates and details of cell labeling experiments. See
DOI: 10.1039/b907741c
a In methanol b ¥ 104 M-1 cm-1.
3486 | Org. Biomol. Chem., 2009, 7, 3486–3490
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Table 2 Isolated yields of representative ligation experimentsa
Conjugate 1A 1C 2aA 3bD 4A 4C 5C 6aA 6aC 7B 8B 1E 11F
Yield [%] 72 99 75 64 98 83 60 99 42 45 53 80 83
a In each case t.l.c. analysis showed complete consumption of starting
materials.
to develop such (bio)orthogonal labels in this spectral regime.
Nile Blue (6), oxazine (8) and the cyanine dye (9) derivatives are
labels that are compatible with the 635-nm diode laser which is
widely used in fluorescence instrumentation such as cell sorters
and imagers.
Clickable dyes 4 and 5 are noteworthy for their large Stokes-
shifts (120 and 125 nm, respectively). Dyes of this kind are
exceptionally useful in Fo¨rster or fluorescence resonance electron
transfer (FRET) applications as they do not interfere with the
spectral bands of the second fluorophore, a common problem in
FRET technology.
The introduction of strained cyclooctynes in azide-alkyne click
reactions made it possible to overcome the limitations of the
copper catalyzed process (e.g. copper toxicity). To extend our
studies to the copper free version of click tagging we have de-
signed and synthesized compounds that incorporate cyclooctyne
functionality. Due to its relatively long wavelength emission and
synthetic simplicity we have chosen the framework of 4 as a
starting azide. Phosphine reduction of 4 (Staudinger-reduction)9
furnished the appropriate amine which subsequently could easily
be converted into a cyclooctyne derivative, 10, using a cyclooctyne-
carboxylic acid derivative (Scheme 1). Later, the cyclooctyne
derivative (11) of compound 1 was also synthesized in a similar
manner and its versatility was demonstrated by labeling the HIV
drug F.10,11
Fig. 1 Novel clickable fluorophores.
These model scaffolds A–F (Fig. 2) represent examples of major
biological classes including sugars, amino acids, and nucleotides.
Fig. 2 Model frameworks for biological ligation.
Scheme 1 Synthesis of strained alkyne containing dye 10.
Labeling experiments were carried out in an acetonitrile-water
(50 v/v%) mixture in the presence of 10% CuI (10-3 M stock
solution in DMSO and 10% triethylamine. In each case t.l.c.
analyses revealed complete consumption of the starting materials.
Purification of the products was carried out either by simple filtra-
tion of the precipitated products or by column chromatography.
The fluorescently labeled biological building blocks were obtained
in good to excellent yields providing further proof for the efficiency
of CuAAC reaction in ligation processes (Table 2). As expected,
the spectral properties of the dyes did not (or only slightly) changed
upon conjugation.
Azido sugars have frequently been used for click-labeling of
surface glycoproteines, recently.8 We have adapted this system to
demonstrate the ability of our dyes to undergo bioorthogonal
labeling reactions, efficiently. Prior to labeling, Chinese hamster
ovary (CHO) cells were incubated with 9b and 10 to test the
possible cytostatic effects of these dyes. Experimental results have
shown that neither dye is toxic up to 50 mM concentration (see
ESI†). Subsequently, CHO cells were treated with azidoacetyl-
mannosamine (ManNAz) for 3 days.
The resulting cells bearing azido sialic acid on their sur-
face glycoproteins were then fixed and treated with 9b or
10. In case of 9b, CuSO4 with sodium-ascorbate and tris-
(benzyltriazolylmethyl)amine (TBTA)12 were used to facilitate the
reaction, whereas labeling with 10 did not require any further
auxiliary reagents.
Although fluorophores in the spectral region between the far
red to the NIR region are particularly suitable for biological (both
in vitro and in vivo) labeling as they are not interfered with by
biological background luminescence, little effort has been made
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Results have shown that after 20 min reaction, both labels had
efficiently tagged the modified cells, whilst cells not treated with
ManNAz have shown only low intensity fluorescence as a result
of non-specific binding of the dyes (Fig. 3, A–D).
dissolved in MeCN (50 mL). NaN3 (1.6 g, 25 mmol) was added
and the mixture was refluxed overnight. After cooling to r.t. the
solvent was removed and the product was purified on silica using
hexane/EtOAc 1:1 as eluent to afford the product as a greenish-
yellow oil (2.26 g, 71%). 1H (CDCl3): d = 2.89 (6H, s); 3.01–3.07
(2H, m); 3.27 (2H, t, J = 5.8 Hz); 5.08 (1H, t, J = 5.8 Hz); 7.20
(1H, d, J = 7.3 Hz); 7.52 (1H, dd, J = 7.3 Hz, J = 8.8 Hz); 7.59
(1H, dd, J = 7.3 Hz, J = 8.8 Hz); 8.24–8.28 (2H, m); 8.56 (1H,
d, J = 8.8 Hz). 13C (CDCl3) d = 42.2; 45.2; 50.7; 115.2; 118.4; 123.0;
128.5; 129.3; 129.7; 130.5; 144.4; 151.9; 171.1. IR: n(neat) = 3290,
2940, 2832, 2787, 2099, 1572, 1309, 1140 cm-1. HRMS (ESI) [M +
H]+ calcd. for C14H18N5O2S: 320,1181; found: 320,1178.
Synthesis of 2a. A mixture of 4-amino-1,8-naphthalic anhy-
dride (0.91 g, 4 mmol) and ethanolamine (0.543 g, 8 mmol) were
heated at 80 ◦C overnight in 10 mL DMF. After cooling, the
solution was poured into 200 mL of ice-cold water. The precipitate
was filtered, washed with water and acetone and dried in vacuo.
The obtained product was then dissolved in DMF (10 mL), NaN3
(756 mg, 11.4 mmol) was added to it and the reaction mixture was
stirred at 80 ◦C for 24 h. The solvent was then removed in vacuo and
the product was extracted with ethyl acetate, dried over Na2SO4,
and purified by flash chromatography (CHCl3). Yield: 825 mg,
69%. m.p. 214–216 ◦C. 1H (CDCl3) d = 3.57 (2H, t, J = 6.2 Hz);
4.25 (2H, t, J = 6.0 Hz); 6.85 (1H, d, J = 8.4 Hz); 7.64 (1H, t, J =
7.7 Hz); 8.19 (1H, d, J = 8.4 Hz); 8.43 (1H, d, J = 7.4 Hz); 8.62
(1H, d, J = 7.7 Hz). Anal. Calcd. for C14H11N5O2: C: 59.87; H:
4.54; N: 23.85. Found: C: 59.96; H: 4.51; N: 23.74.
Fig. 3 Confocal luminescence images of fixed CHO cells treated with
(A) PBS buffer, (B) 9b/CuSO4/ascorbate/TBTA, (C) ManNAz and 10,
(D) ManNAz and 9b/CuSO4/ascorbate/TBTA and native labeled CHO
cells treated with (E) 10 and (F) ManNAz and 10.
The toxic effects of Cu(I) during the labeling process had been
demonstrated earlier5b and we also observed significant cell death
when we attempted to label live cells with 9b in the presence of a
copper catalyst. On the other hand, as expected, the cyclooctyne
bearing label (10) was found to be quite useful in live-cell tagging
and efficient labeling was observed after 1 hour reaction time
(Fig. 3, E–F).
Synthesis of 4. N-(3-azido-propyl)-4-methyl-pyridinium-
iodide (0.213 g, 0.701 mmol) and 4-(dimethylamino)-
benzaldehyde (0.104 g, 0.701 mmol) were stirred in EtOH
◦
(40 mL) at 60 C for 10 hours. The solvent was evaporated and
the product was purified on silica (MeCN, then 1% NH4PF6 in
MeCN). To the combined fractions was added excess NH4PF6
then the solvent was removed in vacuo. The remainder of the
solid was suspended in water and filtered, washed with several
portions of wate◦r and dried in vacuo. Red solid (0.138 g, 43%).
m.p. = 144–146 C. IR: n(neat) = 1529; 1585; 1645; 2099; 2919;
3096 cm-1. 1H-NMR (DMSO): d = 2.16 (2H, t, J = 6.0 Hz); 3.02
(6H, s); 3.46 (3H, s); 4.49 (2H, t, J = 6.0 Hz); 6.78 (2H, d, J =
7.9 Hz); 7.17 (1H, d, J = 16.3 Hz); 7.60 (2H, d, J = 8.4 Hz); 7.92
(1H, d, J = 16.3 Hz); 8.05 (2H, d, J = 5.5 Hz); 8.75 (2H, d, J =
5.8 Hz). 13C-NMR (DMSO) : d = 29.8; 40.0; 48.0; 57.2; 112.3;
117.4; 122.7; 122.8; 130.6; 142.6; 143.9; 152.3; 154.2. HRMS
Experimental
General
Unless otherwise indicated, all starting materials were obtained
from commercial suppliers (Sigma-Aldrich, Fluka) and used with-
out further purification. Analytical thin-layer chromatography
(TLC) was performed on Polygram SIL G/UV 254 pre-coated
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, Bruker DRX-300 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).
(ESI) [M]+ calcd. for C18H22N5 : 308.1870; found: 308.1866.
+
Synthesis of 5. 7-(Diethylamino)-2-oxo-2H-chromene-3-
carbaldehyde (0.42 g, 1.71 mmol) and N-(3-azido-propyl)-4-
methyl-pyridinium-iodide (0.55 g, 1.71 mmol) were refluxed
for 3 hours in ethanol (20 mL) in the presence of 5 drops of
piperidine. After cooling to room temperature the solvent was
removed and the product was purified on a column (SiO2) using
acetonitrile as eluent. The product was dissolved in acetonitrile,
and an excess of NH4PF6 was added. After solvent removal, the
purple crystalline product was suspended in water and filtered.
The solid was washed several times with water and dried in vacuo
to give the title compound as a purple crystalline sol◦id containing
one molecule of water (0.4 g, 41%). M.p.: 200–201 C. IR (neat)
Syntheses of selected clickable labels‡
Synthesis of 1. Dansyl chloride (2.7 g, 10 mmol) and bro-
moethylamine hydrobromide (2.05 g, 10 mmol) was stirred at r.t.
in 50 mL CH2Cl2 for 3 hours in the presence of Et3N (2.79 mL,
20 mmol). The solvent was evaporated and the residue was
‡ Synthetic procedures for the dyes not listed in the experimental section
can be found in the ESI.
1
n = 1416, 1503, 1570, 1712, 2097, 2978, 3069 cm-1. H NMR
3488 | Org. Biomol. Chem., 2009, 7, 3486–3490
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◦
1
(DMSO-d6)): d = 1.14 (6H, t, J = 6.9 Hz); 2.18 (2H, quint. J =
13.4 Hz); 3.40 - 3.57 (6H, m); 4.52 (2H, t, J = 7.1 Hz); 6.58 (1H,
d, J = 1.9 Hz); 6.78 (1H, dd, J = 1.9 Hz, J = 9.0 Hz); 7.53 (1H,
d, J = 9.0 Hz); 7.66 and 7.82 (2H, AB, J = 16.1 Hz); 8.13 - 8.21
(3H, m); 8.85 (2H, d, J = 6.9 Hz). 13C NMR (DMSO-d6): d =
12.3; 28.4; 44.3; 47.5; 57.2; 96.1; 110.0; 113.6; 122.5; 123.2; 130,7;
137.0; 144.0; 145.4; 151.9; 153.4; 156.3; 159.5. Anal. calcd. for
C23H28F6N5O3P; C: 48.68; H: 4.97; N: 12.34 Found: C: 48,58; H:
4.80; N: 12.45.
93–95 C. IR (neat) n = 3201, 2925, 1666, 1515, 1453 cm-1. H
NMR (DMSO): d = 1.64 (12H, s); 1.69–1.79 (8H, m); 2.50–2.52
(2H, m); 2.74 (3H, s); 3.34 (4H, s); 3.69–3.71 (1H, m); 3.92–4.00
(4H, m); 4.42–4.50 (2H, m); 5.67 (2H, d, J = 12.4 Hz); 7.06 (2H,
t, J = 7.7 Hz); 7.18 (2H, d, J = 8.1 Hz); 7.28 (2H, t, J = 7.7 Hz); 7.45
(2H, d, J = 7.2 Hz); 7.92–7.93 (2H, m). 13C NMR: d = 22.5; 25.3;
25.9; 27.5; 28.1; 35.1; 40.0; 42.3; 47.1; 50.8; 76.9; 80.4; 96.8; 109.2;
121.9; 122.5; 126.0; 128.0; 139.9; 142.7; 163.8; 166.7. HRMS (ESI)
calcd. for C40H48N3O6S2 [M]-: 730.2990, found: 730.2978.
-
Synthesis of 10. Compound 4 (50 mg, 0,11 mmol) and support
bound triphenylphosphine (100 mg, 3 mmol/g) was stirred in
dichloromethane-water (9/1 v/v%) for 24 hours, until all the
starting materials were reduced. The reaction mixture was filtered
through Celite and the filtrate was concentrated in vacuo (43 mg,
91%). This intermediate was used subsequently without further
purification and characterization. The product of the previous
reaction was dissolved in acetonitrile (10 mL) and HBtU (35 mg,
0.0936 mmol), HOBt·H2O (16 mg, 0.103 mmol) and CyOCOOH
(25 mg, 0.103 mmol) were added together with 36 mL of N,N-
diisopropylethylamine. The solution was stirred for 14 hours, and
then concentrated in vacuo. The product was purified on a column
(SiO2, acetonitrile) to give compound 10 as a red solid (35 mg,
70%). 1H NMR (300 MHz, CDCl3): d = 1.38-1.41 (1H, m); 1.53–
1.61 (1H, m); 1.71–1.83 (3H, m); 1.84–2.01 (3H, m); 2.06–2.14 (2H,
m); 2.28 (2H, m); 2.52–2.61 (1H, m); 2.62–2.71 (2H, m); 3.03 (6H,
s); 3.50 (2H, m); 4.38 (2H, m); 6.68 (2H, d, J = 7.9 Hz); 6.76 (1H,
d, J = 15.8 Hz); 7.02 (1H, bs); 7.22 (2H, d, J = 6.6 Hz); 7.43–7.50
(3H, m); 7.64 (2H, d, J = 4.8 Hz); 7.70 (2H, d, J = 6.6 Hz); 8.36
(2H, d, J = 4.8 Hz). 13C NMR: d = 23.5; 31.0; 32.6; 33.3; 37.4;
39.1; 39.2; 42.7; 42.9; 44.4; 60.8; 97.5; 99.1; 114.9; 119.0; 125.2;
129.7; 131.8; 133.3; 133.9; 145.4; 145.6; 147.2; 154.8; 157.0; 170.9;
170.7. HRMS (ESI) calcd. for C34H40N3O+ [M+]: 506.3171; found:
506.3176.
Synthesis of 6a. This was obtained by condensation
of 5-dimethylamino-2-nitrosophenol13 hydrochloride with (3-
azidopropyl)-1-naphthylamine14 in 10 mL acidic ethanol. Specif-
ically, to a cold solution (ice bath) of 5-dimethylamino-2-
nitrosophenol hydrochloride (610 mg, 3 mmol) in ethanol (10 mL),
(3-azidopropyl)-1-naphthylamine (658 mg, 2.91 mmol) and con-
centrated hydrochloride acid (0.25 mL) were added. The mixture
was refluxed for about 9 hours and monitored by TLC (silica:
chloroform and chloroform–methanol, 6:1). The solvent was
removed under reduced pressure and the crude mixture was
purified by dry chromatography (silica: chloroform–methanol,
5.5:0.5). N-[5-(3-Azidopropylamino)-9H-benzo[a]-phenoxazin-9-
ylidene]-N-methyl-methanaminium chloride (6a) was obtained as
a blue solid. Yield: 710 mg, 65%. m.p. 184–186 C. H NMR
(CD3Cl): d = 2.03-2.18 (m, 2H,); 3.20 (s, 6H); 3.54 (t, J = 6.9 Hz,
2H); 3.84 (t, J = 6.9 Hz, 2H); 6.61 (d, J = 2.5 Hz, 1H), 6.72 (s, 1H);
6.97 (dd, J = 2.2 Hz, J = 2.2 Hz, 1H); 7.76 (s, 1H); 7.79 (s, 1H);
7.85 (m, 1H); 8.80 (t, J = 4.6 Hz, 1H); 9.40 (d, J = 8.0 Hz, 1H).
13C NMR (CDCl3): d = 27.1; 30.2; 42.6; 45.8; 99.0; 118.1; 120.6;
120.7; 121.3; 122.2; 122.6; 126.5; 126.9; 127.1; 129.3; 132.9; 147.0;
147.8; 148.6; 164.4. HRMS (PI-LSI) calcd. for C21H21N6O [M+]:
373.1777; found: 373.1770.
◦
1
Synthesis of 8. 5-(Piperidin-1-yl)benzene-1,3-diol15 (0.5 g,
2.6 mmol) was refluxed with propargyl bromide (0.23 mL,
2.6 mmol) in THF for 2 days. 360 mg of K2CO3 was used as
base. After cooling, the reaction was filtered, concentrated on a
rotavapor, and then purified by column to get a colorless oil. Yield:
Synthesis of 11. Compound 1 (0.133 g, 0.41 mmol) was mixed
with support bound triphenylphospine (0.35 g, 3 mmol/g) in
CH2Cl2 (1 mL). After being stirred at r.t. for 2 hours, water
(0.5 mL) was added and the mixture was further stirred at r.t.
for 16 h then filtered through Celite. The solid was washed
thoroughly with CH2Cl2 and the filtrate was concentrated in vacuo
to give 0.111 g of the desired product. The product (0.02 g,
0.068 mmol) was dissolved in acetonitrile (5 mL) and CyOCOOH
(0.018 g, 0.075 mmol), HBTU (0.027 g, 0.682 mmol), HOBt
(0.011 g, 0.075 mmol) and N,N-diisopropylethylamine (24 mL,
0.136 mmol) were added. The solution was stirred at r.t. for 24 h
then concentrated. The product was purified on silica (eluent:
hexane : EtOAc, 1:1 v/v%) to give compound 11 (30 mg, 86%).
1H NMR (300 MHz, CDCl3): d = 1.29–1.41 (2H, m); 1.47–1.59
(1H, m); 1.62–1.94 (5H, m); 2.05–2.11 (2H, m); 2.54–2.67 (3H,
m); 2.78 (6H, s); 3.03 (2H, dd, J = 6.0 Hz, J = 10.4 Hz); 3.38
(2H, dd, J = 6.0 Hz, J = 10.4 Hz); 5.95 (1H, t, J = 6.0 Hz); 6.81
(1H, t, J = 5.5 Hz); 7.04 (1H, d, J = 7.1 Hz); 7.08 (2H, d, J =
8.2 Hz); 7.34–7.43 (2H, m); 7.48 (2H, d, J = 8.4 Hz); 8.14 (1H,
dd, J = 1.1 Hz, J = 7.3 Hz); 8.19 (1H, d, J = 8.7 Hz); 8.43 (1H,
d, J = 8.5 Hz). 13C NMR: d = 22.6; 28.4; 29.9; 34.7; 36.5; 39.7;
40.1; 41.6; 43.0; 45.3; 94.8; 96.3; 115.2; 118.5; 123.1; 127.0; 128.5;
128.9; 129.4; 129.5; 129.8; 130.5, 131.5; 134.3; 144.1, 151.9; 168.0.
HRMS (ESI) calcd. for C30H36N3O3S [M + H]+: 518.2477; found:
518.2471.
1
0.21 g, 35%. H-NMR (DMSO-d6): d = 1.80-1.62 (6H, m); 2.50
(1H, t, J = 2.5 Hz); 3.12-3.04 (4H, m); 4.53 (2H, d, J = 2.5 Hz);
5.23 (1H, t, J = 1.1 Hz); 5.83 (1H, t, J = 1.9 Hz); 5.96 (1H, t, J =
1.9 Hz); 6.01 (1H, t, J = 1.9 Hz). This product (0.2 g, 0.86 mmol)
was further reacted with p-nitroso-N,N-dimethylaniline (0.19 g,
1.3 mmol) and 0.14 mL (1.3 mmol) of perchloric acid. The reaction
was carried out in ethanol at 70 ◦C for 2 hours. The crude product
was recrystallized in ethanol to yield dark blue crystals. Yield:
0.12 g, 30%. 1-H-NMR (acetone-d6) d = 1.78–1-87 (6H, m); 3.35
(1H, t, J = 2.5 Hz); 3.46 (6H, s); 3.97–4.05 (4H, m); 5.19 (2H, d, J =
2.5 Hz); 6.84 (1H, d, J = 2.7 Hz); 6.89 (1H, d, J = 2.5 Hz); 6.89
(1H, d, J = 2.5 Hz); 7.35 (1H, dd, J = 2.7 Hz, J = 9.6 Hz); 7.79 (1H,
d, J = 9.6 Hz). IR (neat) n = 3266, 2921, 2859, 1651, 1540, 1487,
1487 cm-1. HRMS (PI-EI) calcd. for C22H24N3O2+ [M+]: 362.1863;
found: 362.1877.
Synthesis of 9b. IR-806 (Sigma-Aldrich) (0.1 g, 0.136 mmol),
propargylamine (0.009 mg, 0.16 mmol) and triethylamine
(0.016 mg, 0.16 mmol) in DMF (5 mL) were stirred in the dark
for 24 hours. The solution was poured into methyl-t-butyl ether
(MTBE) (50 mL) and the precipitated product was filtered and
washed with several portions of MTBE (0.10 g, 97%). M.p.:
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Preparation of fluorescent adducts
Notes and references
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General procedure for the synthesis of adducts. Azido- or
alkyne bearing fluorophores 1–11 (1 eq.) and modified building
blocks A–F (1.1 eq.) were stirred in an acetonitrile-water (50 v/v%)
mixture at r.t. in the presence of 10% CuI and triethylamine 20%
for 16 hours. Products were purified either by simple filtration or
column chromatography on silica. Characterization data of the
fluorescent ligates can be found in the ESI.†
Fluorescent labeling of CHO cells
Chinese hamster ovary (CHO) cells were incubated for 2 days in a
ManNAz containing medium. The cells bearing azido-sialic acid
on their surface glycans were then fixed or directly treated with
the appropriate labels and auxiliaries where needed. Labeled cells
were then studied under a fluorescent confocal microscope. For a
detailed experimental set up please refer to the ESI.†
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Conclusions
In conclusion, the set of clickable fluorophores presented here is
suitable for fluorescent labeling of biomolecules. These tagging
molecules have moderate to high quantum yields (0.1 to 0.6),
and this is best documented by the brilliant contrast of the
microscopy images. These dyes are expected to have a large variety
of applications in (bio)orthogonal labeling schemes both in vivo
and in vitro. Besides the (cyclo)alkyne function, installation of
additional functional groups e.g. maleimides, amines etc. can lead
to further tools for tagging other desirable reactive groups.
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Acknowledgements
Financial support from the Hungarian Scientific Research Fund
(OTKA-NKTH: H07-B-74291) is greatly acknowledged. P.K.
thanks the Alexander von Humboldt Foundation (Bonn) for
a Humboldt fellowship (3.3-UNG/1126507). M. L. thanks the
DFG (German Research Council) for a stipend within Graduate
College GRK 640. The authors wish to thank Dr Szilvia Bo˝sze and
Ms Erika Orba´n for cytotoxicology and cell labeling experiments
and Prof. Ja´nos Matko´ for fluorescence imaging.
15 Reichspatentamt Patentschrift Nr.: 639125 (1934); “Verfahren zur
Herstellung von Kondensationsprodukten des Phloroglucinols”.
3490 | Org. Biomol. Chem., 2009, 7, 3486–3490
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