Ionic perylenetetracarboxdiimides: Highly fluorescent and water-soluble dyes for biolabeling

Article abstract of DOI:10.1002/anie.200353208

Dying to be seen: A series of water-soluble and highly fluorescent perylenetetracarboxdiimides (PDIs) have been synthesized in high yields by introducing charged groups into the bay region of PDI (see picture; C gray, H white, S yellow, O red). They can be used for staining living cells as they are nontoxic.

Full text of DOI:10.1002/anie.200353208

Communications
Water-Soluble Dyes
Up to now, only a few water-soluble PDI derivatives with
functionalities in their imide moieties have been reported.
They exhibit poor water solubility and/or very weak fluores-
cence in water with moderate fluorescence quantum yields of
around 10% because of aggregation of the perylene chromo-
phores.[11,12]
Ionic Perylenetetracarboxdiimides: Highly
Fluorescent and Water-Soluble Dyes for
Biolabeling**
It is well-known that the solubility of PDI derivatives in
organic solvents dramatically increases when substituents are
attached in the bay region, resulting in a twist within the
perylene core,[13] and that water solubility can be achieved by
introducing charged groups on the hydrophobic dyes or
pigments.[14] By successfully combining both structural fea-
tures, we can produce highly fluorescent, water-soluble, and
photostable PDIs for the first time. Furthermore, PDIs that
contain multiple charged groups have been prepared as a
monocarboxylic acid derivative for covalently attached labels
in the field of biology. Initial cell-visualization experiments by
using the novel labels are described below.
Jianqiang Qu, Christopher Kohl, Mark Pottek, and
Klaus Müllen*
The use of water-soluble fluorescent labels for cells, anti-
bodies, and DNA has rapidly expanded in recent years and
has gained additional momentum owing to single-molecule
spectroscopy.[1–3] Although there are a large variety of water-
soluble chromophores commercially available today, most of
them exhibit relatively low fluorescence quantum yields and/
or photochemical stabilities. Cyanine dyes, for example, are
highly instable towards oxygen and light, while xanthene dyes
tend to aggregate in aqueous
medium.[1,4] The ideal dye label
should have the following character-
istics: 1) good water solubility,
2) high fluorescence quantum yield,
3) high chemical and photostability,
4) nontoxicity, 5) good biocompati-
bility, 6) availability of monofunc-
tional derivatives as an extrinsic
covalently bound probe, as well as
7) possible commercial viability and
scalable production. The highly fluo-
rescent
perylene-3,4,9,10-tetracar-
boxdiimide (PDI) chromophore is
widely used as a commercial dye
and pigment due to its outstanding
chemical, thermal, and photochemi-
cal stability.[5] Furthermore, because
of its brilliant color, strong absorp-
tion, and fluorescence, it has been
extensively investigated as an active
compound in reprographic proc-
esses,[6] photovoltaic cells,[7] light-
emitting diodes,[8] other photonic
devices,[9] and light-harvesting com-
plexes.[10] Owing to the unique prop-
erties of PDIs, they should also be
excellent biological probes; however,
the retention of high fluorescence in
aqueous medium is a critical issue.
Scheme 1. a) 3-Pyridinol, N-methylpyrrolidone (NMP), K2CO3, 808C, 15 h, 85%; b) phenol, NMP,
K2CO3, 808C, 15 h, 89%; c) 1) methyl iodide, methanol, reflux, 24 h, 95%; 2) silver methanesulfo-
nate, methanol, room temperature, 5 h, 92%, d) conc. sulfuric acid, room temperature, 15 h, 93%.
[*] J. Qu, C. Kohl, Dr. M. Pottek, Prof. Dr. K. Müllen
Max-Planck-Institut für Polymerforschung
Ackermannweg 10, 55128 Mainz (Germany)
Fax: (+49)6131-379-350
The synthesis of the positively charged PDI 4 involves a
phenoxylation with 3-pyridinol in 1-methyl-2-pyrrolidone
followed by a quaternization, beginning with the tetrachloro-
perylenetetracarboxdiimide 1 (Scheme 1).[15] Because of the
good solubility of PDI 2 in alcohols, the quaternization is
performed with an excess of methyl iodide in methanol
followed by a quantitative exchange of iodide with methane-
sulfonate counterions to give the desired water-soluble PDI 4.
A PDI with sulfonyl substituents 5 is obtained after phenox-
E-mail: muellen@mpip-mainz.mpg.de
[**] Financial support from the Deutsche Forschungsgemeinschaft
(Sonderforschungsbereich 625, Schwerpunktprogramm Orga-
nische Feldeffekt-Transistoren), the Bundesministerium für Bildung
und Forschung (Zentrum für Multifunktionale Werkstoffe und
Miniaturisierte Funktionseinheiten), and BASF AG is gratefully
acknowledged.
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DOI: 10.1002/anie.200353208
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Angewandte
Chemie
ylation of 1 with phenol (by using a similar procedure as for
2), and the subsequent sulfonation with concentrated sulfuric
acid at room temperature.
The asymmetric, chloro substituted PDI 7 is obtained by
imidization by using two different arylamines with and
without the carboxylic function. Isolation of the monoacids
7, 8, and 9 is easily performed by column chromatography, the
eluent being a mixture of dichloromethane/acetone (10:1 v/v).
The monofunctional water-soluble PDIs 10 and 11 are
prepared in high yields by using the same reaction conditions
as for 4 and 5 (Scheme 2). The structures of all compounds are
confirmed by means of field desorption (FD) mass spectra,
matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) spectrometry and/or NMR spectroscopy.[16]
These new chromophores have good thermal stability, with
decomposition temperatures above 3008C, and high water
solubility. The saturation concentration of PDI 4 in water is
above 1.0 ” 10À2 m and 8.0 ” 10À2 m for 5. Monofunctional ionic
PDIs 10 and 11 show similar water solubility in comparison to
the symmetric ionic PDIs. Molecular-modeling studies[17] of
PDIs 4 and 5 reveal a twist of the two naphthalene rings of
about 28o[18] and a polar shell embedding the PDI fluorophore
(not show here).
Figure 1. The normalized absorption spectra and fluorescence spectra
of 4 (dash) and 5 (solid) in water.
Table 1: Absorption (lmax,abs) and fluorescence maxima (lmax,flu), and
fluorescence quantum yields (Ff)[a] in water for 4, 5, 10, 11, and 12.
Compound lmax,abs [nmÀ] (e [mÀ1 cmÀ1])
lmax,flu [nmÀ] Ff
4
5
434 (9766), 516 (24628), 547 (33751) 588 0.66
461 (10708), 541 (21026), 571
(27800)
619
0.58
10
11
432 (9456), 516 (21818), 547 (29974) 592
459 (9540), 541 (19261), 566 (23160) 618
0.58
0.49
The UV/Vis absorption and emission spectra of 4, 5, 10,
and 11 are measured in water (Figure 1) and their fluores-
cence quantum yields are determined. An overview of the
optical properties of all chromophores is given in Table 1.
PDIs 4 and 5 have high extinction coefficients in water in
comparison to tetraphenoxyperylenetetracarboxdiimide 3 in
organic solvents (ꢀ 45000mÀ1 cmÀ1). Furthermore, the
absorption spectra of all ionic PDIs in water in a concen-
[a] Ff was measured at room temperature using Cresyl Violet in
methanol (the standard value is 0.54) as reference.[19]
tration rage of 10 mgmLÀ1–1.0 ” 10À2 mgmLÀ1 exhibited the
same vibrational fine structure of PDI, which suggest that
ionic PDIs still present as monomers in high concentrations.
A PDI that has positive charges, 4, shows a blue shift
Scheme 2. a) 2,6-Diisopropylaniline, 4-(4-aminophenyl)butyric acid, propionic acid, 1408C, 5 h, 30%; b) 8: 3-pyridinol, NMP, K2CO3, 808C, 15 h,
81%; 9: phenol, NMP, K2CO3, 808C, 15 h, 80%; c) 10: 1. methyl iodide, methanol, reflux, 24 h, 94%; 2. silver methanesulfonate, methanol, RT,
5 h, 90%; 11: conc. sulfuric acid, RT 15 h, 92%.
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Communications
(ꢀ 20 nm) in comparison to the negatively charged PDI 5 due
appeared healthy, as judged from their morphology, and were
devoid of vacuoles and any other sign of structural degrada-
tion. In addition, the combination of the PDI treatment with
nuclear staining by the blue fluorescent dye 4,6-diamidino-2-
phenylindole (DAPI)[1] revealed the presence of double
nuclei in some cells (indicated by arrows in Figure 2). Thus,
the cells continued to proliferate in the presence of the PDI
dye, which underlines the nontoxicity of the labels. Photo-
stability and fluorescence intensity remained for a long period
of time after contact with the cell culture, thus indicating that
the dye is not metabolized by the cells. In addition, the
fixation of the cells after staining does not degrade the
fluorescence properties of the water-soluble PDI. These
primary results are encouraging in terms of the applicability
of commercially available dyes/pigments (PDIs) as biological
fluorescent probes.
In summary, novel ionic PDI dyes and monofunctional-
ized ionic PDIs with positively or negatively charged sub-
stituents in the bay regions have been developed with simple
procedures in high yields. This is the first time that water-
soluble PDIs with high fluorescence quantum yields, high
extinction coefficients, as well as significant photostability are
reported. Due to their broad absorption bands, they can be
excited with different excitation sources. The results with
ionic PDI on living cells show that the water-soluble dyes are
nontoxic and maintaining high fluorescence intensity and
photostability. Our future research will concentrate on the
mechanism of water-soluble PDIs entering the cells, the role
of PDI dyes in specific cell organelles, and the study of DNA
and proteins labeled with monofunctional PDIs through
covalent bonds, thereby, also using methods of single mole-
cule spectroscopy.[23,24]
to the stronger electron withdrawing effect of pyridinium. All
synthesized PDIs can be excited with 488 and 514 nm (Ar),
532 nm (Nd:YAG) laser lines, and with a 546 nm mercury arc
line, owing to the presence of a broad and strong absorption
band, to yield intense fluorescence. The dyes have high
fluorescence quantum yields (Ff), which are measured by
using Cresyl Violet in methanol as a reference.[19] PDIs with
positive charges have similar Ff values to those that have
negative charges (0.66 for 4, 0.58 for 5). The monofunctional
dyes 10 and 11 show very similar optical properties to the
analogous symmetric PDIs 4 and 5. However, the fluores-
cence quantum yields of the monofunctional dyes are slightly
lower (0.58 for 10, 0.49 for 11). Clearly the imide structure of
the ionic PDIs influences the fluorescence in water.[20] These
results validate our concept of introducing water solubility
and high fluorescence quantum yield of PDIs in water by
introducing substituents in the bay region and also explain the
failure of approaches found in the early literature that were
based upon functionalization of the imide units.[11,12] The ionic
PDIs in water appear to retain photostability in comparison to
PDIs in organic solvents or in the solid state.[21] The UV/Vis
absorption and the fluorescence spectra of the water solution
of PDIs 4 and 5 remain almost unchanged (their intensities
decrease by about 6% for 4 and 1% for 5) in sun light or after
radiation with under UV light (365 nm) for one week.
The biological applicability of PDI 4 was demonstrated
with a cardiac cell culture.[22] When they were subjected to a
dye concentration of 3.0 mm, these cells became intensely
fluorescent, which was most pronounced in the perinuclear
region (Figure 2). This observation indicates an intracellular
deposition of the dye, although the mechanism for entering
the cells is not yet clear. After they were stained, the cells
Received: October 31, 2003 [Z53208]
Keywords: cell staining · chromophores · dyes/pigments ·
.
fluorescent dyes · water chemistry
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Figure 2. Staining of cardiac fibroblast and muscle cells by PDI 4
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378C, rinsed in nutrient mixture F-10 (Invitrogen Corp.,
Karlsruhe, Germany), and then fixed with 2% paraformalde-
hyde for 15 min at 48C. Subsequently, nuclear staining was
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phenylindole dihydrochloride (DAPI; Sigma, Taufkirchen, Ger-
many) in PBS pH 7.4 for 4 min at room temperature. Cultures
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