Highly emissive water-soluble tetraazaperopyrenes as fluorescent markers

Article abstract of DOI:10.1039/c4cc01254b

Water-soluble tetraazaperopyrene (TAPP) derivatives have been synthesized, which show both high photostability and high fluorescence quantum yields (>80%) in water. Furthermore delivery of the dye into cells demonstrated selective staining of the nucleus.

Full text of DOI:10.1039/c4cc01254b

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Highly emissive water-soluble
tetraazaperopyrenes as fluorescent markers†
Lena Hahn,a Simin O¨ z,b Hubert Wadepohla and Lutz H. Gade*a
Cite this: Chem. Commun., 2014,
50, 4941
Received 17th February 2014,
Accepted 10th March 2014
DOI: 10.1039/c4cc01254b
www.rsc.org/chemcomm
Water-soluble tetraazaperopyrene (TAPP) derivatives have been characteristics water-soluble TAPPs were thought to be suitable
synthesized, which show both high photostability and high fluores- candidates for the application as biomarkers. In this work, we
cence quantum yields (480%) in water. Furthermore delivery of the present a first account of the synthesis and photophysical
dye into cells demonstrated selective staining of the nucleus.
properties of such water-soluble systems as well as their appli-
cation as cellular fluorescent markers.
The application of fluorescence microscopy has become a powerful
One strategy to render organic dyes water-soluble is the
tool for the investigation of biological systems as well as in clinical incorporation of charged functional groups into the molecular
diagnosis and therapy.1,2 As a consequence, the use of fluorescent structure.18,24 Based on this concept, the strategy for the syn-
markers for cellular compartments3 or biomolecules such as DNA,4–6 thesis of water-soluble TAPP derivatives was the introduction of
RNA7–9 or proteins10 has expanded rapidly in recent years.11,12 N-heterocycles or amino groups at the 4,7,11 and 14-positions of
However, these markers have to meet certain requirements such the TAPP core and the subsequent quaternization of the nitrogen
as high water solubility, high molecular extinction coefficients atoms. The synthesis of the resulting tetracationic TAPP deriva-
and fluorescence quantum yields in an aqueous medium. This tives 3, 6 and 7 was accomplished in three steps (Scheme 1).
has to be matched by high chemical and photophysical stability,
as well as low cytotoxicity. Even though a large number of water-
soluble dyes are commercially available today, most of them
possess low photochemical stability and/or low fluorescence
quantum yields.1,13 Hence, considerable efforts have been devoted
towards the synthesis of water-soluble chromophores14–21 which
combine the above-mentioned features.22–27 Many of these systems
are functionalized perylenetetracarboxydiimides (PDIs) which bear
charged substituents or polar dendritic sidechains.
In recent years, we have developed a synthesis process for a new
class of polyheterocyclic aromatics, 1,3,8,10-tetraazaperopyrenes
(TAPPs) possessing properties which are complementary to those
of PDIs.28 While the unsubstituted TAPPs are sparingly soluble,
core functionalization of TAPPs demonstrated
a drastic
increase of their solubility in common organic solvents and
resulted in chromophores possessing remarkable absorption
and fluorescence properties.29–31 Owing to these photochemical
a
¨
Anorganisch-Chemisches-Institut, Universitat Heidelberg, Im Neuenheimer Feld 270,
69120 Heidelberg, Germany. E-mail: lutz.gade@uni-hd.de; Fax: (+49) 6221545609
b Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center,
Heidelberg, Germany. E-mail: s.oez@dkfz.de
Scheme 1 (a) 3-Pyridinol, N-methylpyrrolidone (NMP), Cs2CO3, 100 1C,
4 d, 71%; (b/c) 4-dimethylaminophenylboronic acid/pyridine-3-boronic
ester, [PdCl2(dppf)], toluene, K2CO3/H2O (1 M), 105 1C, 3 d, 79% (4), 75% (5);
(d) 1. methyl iodide, methanol, 80 1C, 24 h; 2. silver methanesulfonate,
methanol, rt, 12 h, 90–94%.
† Electronic supplementary information (ESI) available: General experimental
methods; compound characterization and X-ray crystallographic data for com-
pound 5. CCDC 986570 and 986571. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c4cc01254b
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Table 1 Photophysical properties of 3a, 5a and 7a recorded in water
Table 2 Influence of the counter ion on water solubility and FQY
lmax [nm] (log e)
lem [nm] (Stokes-shift)
fem
t [ns]
Counter ion
Water solubility (mol Lꢀ1
)
fem (%)
log e
ꢀ
3
6
7
472 (4.56)
484 (4.31)
474 (4.63)
504 (1344 cmꢀ1
)
)
0.72
0.80
0.80
3.42
2.80
3.03
7
7a
7b
CH3SO3ꢀ
2.0 ꢁ 10ꢀ3
2.1 ꢁ 10ꢀ3
2.2 ꢁ 10ꢀ6
80
82
80
4.63
4.82
4.63
512 (1130 cmꢀ1
CH3SO4
492 (772 cmꢀ1
)
CF3SO3
ꢀ
The first step of the synthesis of 3 was phenoxylation of 2,9-bis-
(7a) and trifluoromethylsulfonate salts (7b), respectively. The
photophysical properties of 7a and 7b remained essentially
unchanged compared to 7 with an absorption maximum at
474 nm and an emission maximum at 492 nm (Table 2).
The FQYs of the three salts are in the range of 80–82%,
demonstrating that the counter ion seems to have no signifi-
cant effect on the FQY. However, the water solubility is strongly
dependent on the counter ion. Whereas compounds 7 and 7a
were found to be highly soluble in water, the solubility of
trifluoromethylsulfonate salt 7b was lower by a factor of 1000.
Crystals of compound 7b, which were suitable for X-ray diffrac-
tion, were grown from methanol and the molecular structure is
displayed in Fig. 2.‡ As observed for most TAPP derivatives,28–31 the
tetraazaperopyrene core of the dye is almost planar (RMSD 0.025 Å)
with the two perfluoroalkyl substituents pointing in opposite
directions above and below the TAPP core. The planes of the
N-methyl pyridinium substituents are at 35.8(1)1 and 46.43(8)1
to the TAPP core.
The water-solubility of the synthesized dyes raised the question
of their applicability as fluorescent markers in biological samples.
In an initial step towards characterizing the effects of the dye in
living cells, a cervical carcinoma cell line (HeLa) was used. We
treated HeLa cells with various concentrations (0 to 100 mM) of 3, 6
and 7a respectively, for 24 h at 37 1C and determined the metabolic
activity of cells by measuring the intracellular ATP levels.32 The
results demonstrated IC50 values in micro molar ranges suggesting
a weak cellular response (Fig. 3). Furthermore, the cytotoxicity of
the methylsulfate salt 7a remained low even at relatively high
concentrations of the dye. Therefore, all subsequent studies
were performed with this compound.
perfluoropropyl-4,7,11,14-tetrabromo-1,3,8,10-tetraazaperopyrene (1),
analogous to the approach chosen by Mu¨llen et al. for PDI deriva-
tives,18 by treatment with 3-pyridinol in 1-methyl-2-pyrrolidone
(NMP) resulting in the formation of 2 in moderate yields. Direct
aryl–aryl coupling employed in the synthesis of 4 and 5 was
accomplished by Suzuki coupling of 1 with either pyridine-3-
boronic ester or 4-dimethylaminophenylboronic acid, respectively.
The next step, the quaternization of the nitrogen atoms, was
achieved by the reaction of 2, 4 and 5 with an excess of methyl
iodide, followed by salt metathesis with silver methanesulfonate to
give the desired water-soluble compounds 3, 6 and 7 in quantita-
tive yields. Each of the TAPP derivatives was structurally characteri-
zed by NMR spectroscopy as well as mass spectrometry. Their
photophysical properties in water were investigated by UV/Vis and
fluorescence spectroscopy. An overview of the optical properties of
compounds 3, 6 and 7 is given in Table 1.
All of the newly synthesized TAPP derivatives are red in the solid
state and display green fluorescence in solution. The UV/Vis absorp-
tion spectra of compounds 3, 6 and 7 recorded in water display
characteristic visible p* ’ p absorption bands with a maximum
between 472 and 484 nm and high extinction coefficients in water
(between log 4.56 and log 4.82). As expected, the emission spectra of
these compounds image the mirror symmetry of absorption spectra
with Stokes shifts in the range of 950–1350 cmꢀ1 (Fig. 1).
Notably, these TAPP salts were found to be characterized by
remarkably high fluorescence quantum yields (FQYs) of up to
80% in water, with compound 3 possessing the lowest FQY of
72%. We attribute the latter to the flexible diarylether linkages
in compound 3 which cause partial fluorescence quenching.
As a next step, the effect of the counter ion on the FQY and the
water solubility of this class of compounds was investigated. Upon
reaction of the neutral precursor 2 with either dimethyl sulfate or
methyl trifluoromethansulfonate we obtained the methylsulfate
Next, we investigated the accumulation of the fluorescent marker
in HeLa cells. We incubated PFA-fixed and permeabilized cells with
Fig. 1 Normalized absorption spectra of 3, 6 and 7 in water (10ꢀ7 M) (left),
normalized absorption and emission spectra (excitation wavelength: 460 nm)
of 7 in water (right).
Fig. 2 Top view (left) and side view (right) of compound 7a, displaying the
structure of the TAPP dye. Thermal ellipsoids are drawn at a 50% prob-
ability level, hydrogen atoms were omitted for clarity.
4942 | Chem. Commun., 2014, 50, 4941--4943
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Financial support from the University of Heidelberg as well
as the doctoral college ‘‘Verknu¨pfung molekularer p-Systeme zu
¨
Funktionsmaterialien’’ funded by the Landesgraduiertenfor-
derung of Baden-Wu¨rttemberg is gratefully acknowledged.
The authors thank Thimon Schwaebel for determining the
fluorescence lifetimes and Nevcin Sentu¨rk for imaging.
Notes and references
‡ Crystal data: C56H32F26N8O12S4,
M = 1631.13, monoclinic, a =
10.5849(6) Å, b = 9.8070(4) Å, c = 29.9716(13) Å, b = 99.183(5)1, V =
3071.3(3) Å3, T = 110(1) K, space group P21/n, Z = 2, Cu Ka X-radiation,
l = 1.5418 Å, 44 255 reflections measured, 5881 unique (Rint = 0.0639),
wR(F2) [all unique data] = 0.1584, R(F) [Fo 4 4s(Fo)] = 0.0609.
Fig. 3 IC50 values of compound 3, 6 and 7a after treatment of HeLa cells
for 24 h.
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investigated by confocal microscopy. The results displayed in Fig. 4
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