Water-soluble pyrrolopyrrole cyanine (PPCy) NIR fluorophores

Article abstract of DOI:10.1039/c4cc01014k

Water-soluble derivatives of pyrrolopyrrole cyanines (PPCys) have been synthesized by a post-synthetic modification route. In highly polar media, these dyes are excellent NIR fluorophores. Labeling experiments show how these novel dyes are internalized into mammalian cells. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01014k

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Water-soluble pyrrolopyrrole cyanine (PPCy)
NIR fluorophores†
Cite this: Chem. Commun., 2014,
50, 4755
Simon Wiktorowski, Christelle Rosazza, Martin J. Winterhalder, Ewald Daltrozzo
and Andreas Zumbusch*
Received 7th February 2014,
Accepted 21st March 2014
DOI: 10.1039/c4cc01014k
www.rsc.org/chemcomm
Water-soluble derivatives of pyrrolopyrrole cyanines (PPCys) have been dyes, BODIPYs or others.7 Notable are also advances in other fields,
synthesized by a post-synthetic modification route. In highly polar like the engineering of GFP-related fluorescing proteins or quantum
media, these dyes are excellent NIR fluorophores. Labeling experiments dots, which have resulted in the synthesis of novel systems with NIR
show how these novel dyes are internalized into mammalian cells.
emission.8 To date, however, only a few water-soluble dyes with strong
NIR absorptions and emissions have been known. Apart from the
Near-infrared (NIR) light absorbing and emitting compounds have general scarcity of NIR absorbing molecules, the main reason for this
attracted a lot of interest since the 1990’s.1 Initially, this was motivated is that NIR absorption is commonly observed in extended p-systems
by their use in optical data storage or as laser dyes. Recently, however, which most often are hydrophobic. The incorporation of hydrophilic
new applications of NIR dyes have emerged, which has led to a surge of functionalities into hydrophobic dye structures, however, poses experi-
interest in the synthesis of such compounds. Examples for new mental difficulties. Additionally, even after the successful establish-
applications are NIR chromophores as emitters in light-emitting diodes ment of synthetic routes, one has to face problems with hydrolytic
or as sensitizers in light-conversion materials, relevant in renewable decomposition or dye aggregation, the latter often being accompanied
energy related fields.2 In biomedical applications, NIR dyes are used as by significant fluorescence quenching.7d,9 The currently most widely
labels for fluorescence microscopy imaging of biomolecules and used water-soluble NIR dyes are heptamethine dyes like indocyanine
cellular structures.3 In addition, NIR fluorophores serve as contrast green (ICG, cardio green), Cy7 or more stable and long-wavelength
agents in cancer-targeted imaging, angiography and as sensitizers in shifted derivatives thereof, which have been synthesized in recent
photodynamic therapy.4 Here, NIR dyes offer several advantages over years.3f,10 ICG is the only NIR fluorophore which has been approved
dyes absorbing and emitting in the UV/visible region: NIR light by the U.S. Food and Drug Administration for medical use.3e
penetrates deeper into tissues due to lower scattering and lower
Pyrrolopyrrole cyanines (PPCys) are a class of NIR dyes which has
absorption, natural autofluorescence of cellular components is lower been introduced recently.11 PPCy chromophores exhibit narrow-band
and generally significantly reduced photodamage is observed in this absorption and strong emission in the NIR region up to almost 1 mm.
spectral region.5 The use of NIR absorbing dyes is also facilitated by the In general they are very photostable.12 The optical features and the
ready availability of diode lasers as cheap excitation sources and the fact structure–property relationships of PPCy dyes have been investigated
that Si-based detectors such as photodiodes and CCD cameras have in detail.13 They have proven to be attractive candidates for labeling
their sensitivity maximum at around 800 nm.6
applications, fluorescence lifetime imaging in vivo or as photoacoustic
In all of the aforementioned applications, NIR dyes with strong probes.14 While very recently, PPCy derivatives with highly polar
absorptions and emissions as well as with high photostability are structures have been reported,15 to date, however, no water-soluble
desired. In order to be applicable in biological environments, the dyes derivatives of PPCys have been synthesized. In this contribution, we
need to be water-soluble. Therefore, many research efforts are present a synthetic strategy to the development of water-soluble PPCys
currently dedicated to the development of new water-soluble NIR and discuss their optical properties. Live-cell imaging experiments are
dyes or to the respective derivatization of NIR dyes from existing presented which demonstrate great potential of these compounds in
classes, like (naphthalo)cyanines, rylenes, squaraines, croconium imaging applications.
The preparation of water-soluble PPCys faces the problem that the
¨
¨
Fachbereich Chemie, Universitat Konstanz, Universitatsstrasse 10, 78457 Konstanz,
Germany. E-mail: andreas.zumbusch@uni-konstanz.de; Fax: +49 7531-88-3139
† Electronic supplementary information (ESI) available: Synthesis and characterisation
of intermediates and final compounds. Additional spectroscopic data. Time-dependent
changes in absorption spectra of final compounds in water. Data of photostability tests
and intracellular emission spectra of 600 . See DOI: 10.1039/c4cc01014k
hydrophobic core of the dyes needs to be functionalized with polar
groups which mediate the solubility in polar solvents. Functionalities
of this type, e.g. sulfonates, can generally be introduced either during
the synthesis of the core chromophore structure or as a post-synthetic
modification. The established routes for PPCy synthesis11–14 lead to
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specific problems which have to be solved. Since the use of compounds are converted to the water-soluble derivatives 60
e.g. alcohols as polar solvents for polar reactants is precluded by the and 600 by NHS activation of the four carboxylic acid functions
use of phosphoryl chloride as an activator, we decided to introduce and subsequent fourfold amidation through the sodium salt of
the polar groups via the modification of a suitably functionalized 2-aminoethanesulfonic acid.
PPCy. However, also the introduction of suitable functionalities to
Fig. 1 (top) shows the normalized absorption spectra of benzo-
which water-soluble groups can be attached after PPCy synthesis thiazole substituted BPh2-PPCy 60 and quinoline substituted BPh2-PPCy
00
poses problems because PPCys are synthesized in a strongly acidic 6 in water with absorption maxima at around 800 nm. The solubility
reaction from diketopyrrolopyrroles (DPPs), which themselves are of both compounds in pure water is in the mM range. When dissolving
prepared under strongly basic reaction conditions. Possible protective these compounds in water the absorption spectra shift very slightly over
group strategies are hampered by the pigment character of DPPs, a period of hours (cf. Fig. S1 in ESI†). These changes in absorbance
00
i.e. their low solubility in common organic solvents. Moreover, we for the pure aqueous milieu are more pronounced in the case of 6
found that the electrophilic character of phosphoryl chloride prevents compared to 60. We assign them to conformational rearrangements of
conversion to PPCys if the reactants contain nucleophilic functional the long alkyl chains and solvation effects. As is the case for other
groups such as amines. A post-synthetic Pd-catalyzed alkyne substitu- PPCys dissolved in nonpolar solvents, the half widths of the absorption
tion of aryl bromide functions in PPCys (cf. compounds 3b, 4b from and emission bands of these compounds are also very small in polar
ref. 13a) also fails, most likely due to the insufficient solubility of these solvents (Dn~A1/2 = 650 (575) cmꢀ1 for 60 and 859 (539) cmꢀ1 for 600 in
compounds. We therefore chose to synthesize a new DPP with water (methanol)). We do not observe any bands which could be
terminal double bonds which can be converted to a PPCy before attributed to dye aggregation. Therefore we conclude that if any
sulfonate groups are attached via a linker.
aggregation occurs in these systems, the respective chromophore
The synthetic scheme for the generation of water-soluble PPCys interactions must be very weak. A possible explanation for the absence
is shown in Scheme 1 and follows the general established route for of aggregation is the steric demand of both the BPh2 groups and the
PPCys described by our group up to BPh2-PPCys 4 (cf. experimental long alkyl chains, which hinder p–p-interactions. For this reason the
section in the ESI†).12a DPP 1 is allowed to react with two equivalents complexing agent BPh2Cl was preferably chosen over BF3ꢁOEt2, which
of 2-(6-tert-butylbenzothiazol-2-yl)-acetonitrile 20 or 2-(6-tert-butyl- was also frequently used in order to obtain strongly fluorescent PPCys.
quinoline-2-yl)acetonitrile 200, respectively, by activation through The fluorescence quantum yields for 60 and 600 range between 1 and
phosphoryl chloride to yield the hydrogen chelates 30 or 300, respec- 34% in water, methanol and DMSO, respectively (cf. Table 1; for
tively. These H-PPCys 3 are converted to the benzothiazole supplemented spectroscopic data see Table S1, ESI†). Since there is
substituted BPh2-PPCy 40 or quinoline substituted BPh2-PPCy no evidence for dye–dye aggregates from the absorption spectra, we
400, respectively. Subsequent radical addition of mercaptoacetic attribute the reduction of fluorescence quantum yields in water to
acid to the four terminal double bonds yields the carboxylated solvation effects rather than to dye aggregation. Reduced quantum
BPh2-PPCys 50 and 500. In the final step, the carboxylated yields in water are often found for different dyes, as exemplarily shown
Scheme 1 Reagents and conditions (in each step ‘‘R’’ corresponds to the same alkyl substituent as in the para position to the DPP core; A = aromatic
heterocycle): (a) anhydrous toluene–POCl3, reflux; (b) CH2Cl2–diisopropylethylamine, chlorodiphenylborane, reflux; (c) anhydrous 1,4-dioxane–mercaptoacetic
acid, AIBN, 90 1C or reflux. (d) (1) CH2Cl2–DCC–NHS, room temperature; (2) DMF, 2-aminoethanesulfonic acid sodium salt, room temperature.
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Table 1 Spectroscopic data for BPh2-PPCys 60 and 600 in different solvents
and in CHO cells compared to ICG. lAmax: wavelength of the absorption
maximum, lFmax: wavelength of the fluorescence maximum, emax: molar
decadic absorption coefficient of the absorption maximum, FF: fluores-
cence quantum yield, emax FF: optical brightness
lAmax
lFmax
emax
emax FF
a
[nm] [nm] [Mꢀ1 cmꢀ1
]
FF
[Mꢀ1 cmꢀ1
]
60
DMSO
MeOH
H2O
DMSO
MeOH
H2O
794
782
793
825
810
805b
805
800
800
840
825
825
840
807
820
171 000
166 000
125 000
195 000
183 000
110 000
—
204 000
155 000
0.27
0.20
0.01
0.34
0.25
0.01
0.34
0.08
0.003
46 000
33 000
1000
600
66 000
46 000
1000
c
c
c
CHO cell
—
—
ICG MeOH16b 785
16 000
500
H2O17
775
a
Values corrected for ten and three molecules of water per formula unit
for 60 and 600, respectively (cf. general remarks in the experimental
section, ESI). Can range from 805 to 820 nm (conformation/solvation
effect; see text). Not determined.
b
c
aqueous solution. For the microscopy experiments, we employed a
scanning stage confocal microscope which allowed excitation with
690 nm light as the longest available wavelength. Despite the fact that
this wavelength is far off the absorption maximum of 6 such that it
has only a low extinction at this wavelength (see Fig. 1) and the fact that
Fig. 1 (top) Normalized absorption spectra of benzothiazole substituted
BPh2-PPCy 60 (black) and quinoline substituted BPh2-PPCy 600 (red) in
water. (bottom) Absorption spectra (left) and normalized fluorescence
spectra (right) of benzothiazole substituted BPh2-PPCy 60 in methanol
(black) and DMSO (red) and quinoline substituted BPh2-PPCy 600 in methanol
(green) and DMSO (blue).
00
low excitation powers were used (B5 mW), a strong fluorescence signal
00
was readily obtained. Fig. 2 (left) shows a high concentration of dye 6
in vesicular structures in the cytoplasm and low concentrations in the
00
for ICG (Table 1). In all solvents, the emission maxima are at or slightly cell plasma membrane. The data show that BPh2-PPCy 6 enters the
above 800 nm (cf. Fig. 1 (bottom) and Table 1). Two important cells without application of any permeability-enhancing techniques like
parameters for imaging applications are the optical brightness, defined e.g. electroporation. This is surprising because the molecular weight of
in different publications as the product of the extinction coefficient of the dye is comparably high (B2.3 kDa). Together with its overall
the absorption maximum emax and the fluorescence quantum yield negative charge, this should make direct diffusion through the plasma
16
FF and the photostability of the dyes. As shown in Table 1, the membrane less probable. In order to learn more about the entry
00
brightness of 60 and 6 is high in polar organic solvents with values of process, especially whether the dye enters by endocytosis or by
33 000 to 66 000 Mꢀ1 cmꢀ1 compared to ICG in methanol with a value transmembrane diffusion, and about the compartments it ends up
of 16 000 Mꢀ1 cmꢀ1
.
in the cells, incubation experiments were performed at different
Concerning the photostability, we have shown earlier that temperatures. Since endocytotic processes are strongly suppressed at
PPCys exhibit very little photobleaching in organic solvents like 4 1C, one expects to find a very low dye concentration in the cytoplasm
chloroform.12a In order to test the photostability in water, a cuvette upon incubation at this temperature if endocytosis is the main entering
containing an aqueous solution of 600 was exposed to daylight for pathway. Several incubations of CHO cells were performed at 4 1C
00
several hours. Solutions of ICG in water and of compound 10e from along with control experiments at 37 1C using a 100 mM solution of 6
ref. 12a in chloroform were exposed to the same illumination condi- in cell culture medium for 30 minutes. Confocal fluorescence images of
00
tions. These experiments show that while BPh2-PPCy 6 is less photo- fixed cells were taken in a z-scanning mode (see maximum projections
stable in water than 10e in chloroform, it is much more photostable in Fig. 2 (middle) for incubation at 4 1C and Fig. 2 (right) for incubation
than ICG in water (cf. Fig. S2 in ESI†). The main advantage of ICG is its at 37 1C; excitation powerso1 mW). Cells incubated at 4 1C indeed show
low toxicity. While no data on the toxicity of PPCys are available, one only very weak, diffuse fluorescence emission and do not feature the
can note that several days after injection of PPCy solutions, no adverse vesicular dye structures observed in cells incubated at 37 1C, even
effects on mice were observed.14b Moreover, DPPs, the core structure of though a very high dye concentration of 100 mM was used. This result
PPCys, have been shown to be non-toxic by either oral or dermal uptake points at endocytosis rather than diffusion through the plasma
and show no mutagenic effects in vitro and in vivo.18
As pointed out before, water-soluble NIR fluorophores have great means that the observed structures are most likely endosomes.
potential in biomedical applications. In order to evaluate the suitability
Because emission of 600 in CHO cells seemed to be fairly strong,
membrane being the main pathway for cellular uptake of 600 . It also
of the new water-soluble BPh2-PPCys as fluorescence probes, live-cell we were interested in determining the intracellular quantum yield of
00
images of CHO cells incubated with 6 were taken using a confocal this dye. For this purpose, first emission spectra of the vesicular
fluorescence 00microscope. Incubation was performed with a 1 mM structures of a series of CHO cells incubated with 600 were recorded
solution of 6 in cell culture medium for 30 minutes. No additional (cf. Fig. S3 in ESI†). It was found that the fluorescence spectrum
00
solvents or surfactants had to be used since 6 is well soluble in matches that of 600 in a DMSO solution, indicating that emission of
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Fig. 2 Confocal fluorescence images of CHO cells incubated with 600 taken
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In conclusion, we report the synthesis of two novel pyrrolopyrrole
cyanine dyes as the first water-soluble representatives of this class of
NIR fluorophores. The synthesis followed a post-synthetic functionali-
zation route. The new PPCy dyes thus obtained exhibit good solubility
in pure water (mM range). The half widths of the absorption spectra in
water are very low and no distinct spectral bands attributable to dye–dye
aggregates were found. Quantum yields and extinction coefficients are
high in polar organic solvents like DMSO and methanol resulting in a
high optical brightness of up to 66 000 Mꢀ1 cmꢀ1. The photostability of
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00
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fluorescence lifetime imaging data. All of these results in summary
show that the new water-soluble PPCy dyes hold great promise for
imaging applications both on a cellular and on a tissue level.
Financial support by the DFG, SFB 767, TP B3 is acknowledged.
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