Asymmetric PPCys: Strongly fluorescing NIR labels

Article abstract of DOI:10.1039/c0cc00359j

By a stepwise synthesis strategy biofunctionalized Pyrrolopyrrole Cyanines (PPCy) with an asymmetric substitution pattern were obtained. These exhibit extremely strong and narrowband NIR absorption and fluorescence. Internalization of a peptide bound PPCy is demonstrated using live cell microscopy.

Full text of DOI:10.1039/c0cc00359j

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Asymmetric PPCys: Strongly fluorescing NIR labelsw
Georg Michael Fischer, Christian Jungst, Magnus Isomaki-Krondahl, Dominik Gauss,
¨
¨
¨
Heiko Michael Moller, Ewald Daltrozzo and Andreas Zumbusch*
Received 12th March 2010, Accepted 24th May 2010
First published as an Advance Article on the web 11th June 2010
DOI: 10.1039/c0cc00359j
By a stepwise synthesis strategy biofunctionalized Pyrrolopyrrole
Cyanines (PPCy) with an asymmetric substitution pattern were
obtained. These exhibit extremely strong and narrowband NIR
absorption and fluorescence. Internalization of a peptide bound
PPCy is demonstrated using live cell microscopy.
PPCys show high chemical stability and photostability in
nonpolar solvents such as chloroform or toluene. In the PPCys
reported so far, the alkyl chains which are necessary for their
synthesis complicate their use in polar or aqueous environment.
Nevertheless, first in vivo experiments have been carried out by
presolving the dyes in unpolar solvents and subsequently
diluting them in buffer. This allowed fluorescence and
fluorescence lifetime imaging in live mice, where PPCys
exhibited good stability, bright fluorescence, very long
fluorescence lifetimes, and no adverse effects in mice.7 While
these results are promising, labeling applications with a more
general scope require the functionalization and selective
binding of PPCys to biological target molecules. Here, we
report the implementation of a synthetic strategy improving
the solubility in polar solvents and leading to functionalized
PPCys. The resulting chromophores feature very strong
absorptions above 730 nm and extraordinarily high fluorescence
quantum yields in the NIR region.
Fluorescence microscopy is one of the key technologies in
modern biology.1 It relies on the availability of strongly
fluorescing and highly photostable chromophores, which can
be functionalized for binding to biological target structures.2
The desire to investigate more complex biological samples,
especially thick tissues, however, necessitates new methodological
developments. One is the development of new fluorophores
absorbing and emitting in the near infrared (NIR) spectral
region.3 NIR excitation and detection offers many important
advantages: (i) great sample penetration depths due to weak
absorption and strongly reduced scattering; (ii) sensitive
detection because of negligible NIR autofluorescence back-
ground; and (iii) less photodamage compared to visible
excitation.4 These are reasons which motivated the recent
development of a broad variety of new NIR-fluorophores.5
An important aspect here is the increase of the typically very
low fluorescence quantum yields in the NIR.
The scheme for the synthesis of PPCys as it has been
followed earlier (cf. Scheme 1 in ref. 6a) leads to symmetric
1 : 2 condensation products. The high symmetry of these
would lead to bifunctionalization, which is undesirable for
labeling applications. Monofunctionalization requires
a
different strategy. Our rationale to reach this aim is to
assemble the chromophores in a stepwise manner. For this
purpose, the monosubstituted 1 : 1 condensation products of
a DPP (1) with a HAA (3) have to be isolated (Scheme 1).
Previously, this has only been achieved for the PPCy resulting
as the 4-(N-methyl-N-octyl amino)phenyl DPP 1 : 1 condensation
product.6 All other PPCys were obtained as 1 : 2 condensation
products. Mono-phosphorylated intermediates (2) from the
reaction of a DPP with POCl3 have however been reported.8
These can be isolated in good yields by removing excess POCl3
in vacuum. The intermediates thus obtained can be dissolved
in abs. THF and reacted with one equivalent HAA (3) to
the monosubstituted product (4). The longest wavelength
absorption (S0 - S1) of 4 is broad with rather intense vibronic
bands. As is the case for symmetrically substituted PPCys,
solutions of 4 do not show fluorescence at room temperature.
Complexation with BF2 leads to products (5) with S0 - S1
transitions whose intensity ratio of the purely electronic
00-transition vs. the vibronic 01, 02, . . . transitions, as reflected
by the Franck–Condon factors, are shifted in favour of the
00-transition. The BF2-complexes 5 exhibit strong fluorescence
with Stokes shifts well above 1100 cmÀ1 (cf. Table 1 and ESIw).
The monosubstituted PPCys (4) serve as the starting
material for the synthesis of asymmetrically bisubstituted
PPCys. For this purpose, 4 is reacted with a second equivalent
of a HAA (3) leading to asymmetric H-PPCys (6) in yields
In earlier work, we have described the synthesis and spectro-
scopic characterisation of pyrrolopyrrole cyanine dyes (PPCy),
a new class of NIR dyes and fluorophores.6 They are obtained
by heating a diketopyrrolopyrrole (DPP 1) and a hetero-
aromatic acetonitrile (HAA 3) in the presence of an excess
of phosphoroxychloride. The resulting condensation products
feature strong and narrow band absorption in the NIR.
Solutions of these compounds do not yet fluoresce. Extra-
ordinarily strong fluorescence at NIR wavelengths can however
be induced by stiffening the PPCys through complexation with
either BF2 or BPh2. Interestingly and in contrast to many
other NIR dyes, PPCys show hardly any absorption in the
visible spectral range which makes them attractive candidates
for applications necessitating selective NIR absorption. By
variation of the heterocyclic endgroups and the complexing
agent, the absorption maximum can be shifted over a broad
spectral range.
Fachbereich Chemie and Center of Applied Photonics, Universitat
¨
Konstanz, 78457 Konstanz, Germany.
E-mail: andreas.zumbusch@uni-konstanz.de; Fax: +49 7531-883870;
Tel: +49 7531-2357
w Electronic supplementary information (ESI) available: Synthesis
and characterisation of the intermediates and the final compounds.
Absorption spectra of the dyes and details of the live cell imaging. See
DOI: 10.1039/c0cc00359j
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Fig.
1
Absorption of 7a: A1
=
4,6-dimethyl-pyrimidine (3a)
A2 = 6-tert-butylquinoline (3e) (solid line) and the corresponding
symmetric PPCys6: A1 = A2 = 3a (dotted line) and 7e: A1 = A2 = 3e
(dashed line).
Scheme 1 Reagents and conditions: (a) POCl3, reflux; (b) absolute
THF, reflux; (c) absolute toluene, POCl3, reflux; and (d) methylene
chloride, di-iso-propylethylamine, BF3ÁOEt2, reflux; R = p-octyl-
oxyphenyl; 3: (4,6-dimethyl-pyrimidine-2-yl)-acetonitrile (3a), (5-tert-
butyl-benzooxazol-2-yl)-acetonitrile (3b), (6-methyl-pyridin-2-yl)-acetonitrile
(3c), 2-(6-tert-butylbenzothiazol-2-yl)-acetonitrile (3d), 2-(6-tert-butyl-
quinoline-2-yl)-acetonitrile (3e), 2-(6-bromoquinoline-2-yl)-acetonitrile (3f),
quinoxalin-2-yl-acetonitrile (3g), tert-butyl 5-(2-(cyanomethyl)quinolin-
6-yl)pent-4-ynoate (3h). A: aromatic ring.
the electron density distribution, which is relevant for the
Franck–Condon factors of the first electronic transition.
Thus the spectral properties (absorption maxima, extinction
coefficients and oscillator strengths) of the A1A2 types lie
exactly between the corresponding values of the symmetrical
A1A1 and the A2A2 types (see Fig. 1) supporting the classification
of PPCys as typical cyanine dyes. The heteroaromatic
substituents however shift the energy of the observed electronic
transitions in a fashion which is also known from other
cyanine chromophores.9 Table 2 shows that by suitable
combinations of 3, the absorption maxima can be shifted at
will in a broad spectral range.
Table 1 Yield and spectroscopic data of the first electronic transition
(S0 2 S1) of 4 and their BF2-complexes 5a
Yield l00A/nm
e
00/MÀ1 cmÀ1 l00F/nm
D
n~A–F/cmÀ1 FF
While the one-pot synthesis described earlier leads to
symmetric PPCys (A1 = A2), the stepwise synthesis of the
chromophores now permits the introduction of a single
functional group. As an example, we introduced a carboxylic
acid function by a Sonogashira reaction of 3f with tert-butyl-4-
pentyonate which yields 3h. 3h can be converted with 4e to the
asymmetric H-PPCy 6h, which is deprotected to the free acid
6i, and subsequently complexed to 7i. All these dyes bear
unfavorable octyl chains which are helpful in increasing the
solubility during the syntheses and purifications, but hamper
their use in polar solvents and especially in water. They can be
removed by cleaving the octyl ethers with BBr3 which leads to
derivative 8 (Scheme 2) which is insoluble in unpolar solvents
such as methylene chloride, but sufficiently soluble for
coupling reactions in polar solvents like NMP. This allows
its coupling to biological target molecules, e.g. peptides.10
As a demonstration for the suitability of this approach
for applications of asymmetric PPCys as labels in live cell
fluorescence imaging, we linked 8 to the N-terminus of an Arg9
peptide that is well known as a cell penetrating peptide.11 This
construct was then used to incubate HeLa cells which were
grown in microscope dishes for 30 min (cf. ESIw).12 After
washing, live cell images were taken with a standard confocal
microscope. It can clearly be seen that the Arg9-8 constructs
were internalized into the cells in the form of small aggregates
4d 40
618
611
618
630
33 000
37 000
43 000
58 000
—
—
672
678
—
—
1320
1130
—
—
0.50
0.40
4e 84
5d 44
5e 72
a
In chloroform at room temperature, absorption/emission
wavelength l00A/l00F, Stokes shift Dn~A–F, molar decadic absorption
coefficient e00, fluorescence quantum yield FF.
between 58 and 87%. Complexation with BF3ÁOEt2 yields
asymmetric BF2-PPCys (7) between 60 and 86% (Scheme 1).
Obviously, as in the case of the classical cyanine cations9a and
both the cationic and anionic cyanines,9b the asymmetric
structure, with two different terminal groups A1 and A2, does
not produce a significant deviation from the C2 symmetry of
Scheme 2 Reagents and conditions: (a) TFA, CH2Cl2, reflux; (b)
CH2Cl2, DIPEA, BF3ÁOEt2, reflux; and (c) CH2Cl2, BBr3, room
temperature.
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Table 2 Spectroscopic data of the first electronic transition (S0 2 S1) of the H-PPCys 6 and the BF2-PPCys 7: A1 = 3ea
A2
6
l00A/nm
e
00/MÀ1 cmÀ1
f
7
l00A/nm
e
00/MÀ1 cmÀ1
f
l00F/nm
Dn~A–F/cmÀ1
FF
3a
3b
3c
3d
3e
3f
6a
6b
6c
6d
6e
6f
6g
6h
6i
708
720
718
734
731
734
739
739
739
100 000
108 000
97 000
116 000
118 000
122 000
132 000
134 000
134 000
0.62
0.69
0.61
0.72
0.71
0.70
0.73
0.76
0.76
7a
7b
7c
7d
7e
7f
7g
—
7i
719
718
730
742
754
758
770
—
178 000
162 000
179 000
191 000
205 000
236 000
213 000
—
0.76
0.77
0.76
0.79
0.83
0.86
0.83
—
740
739
751
759
773
774
786
—
400
350
350
350
300
300
300
—
0.66
0.58
0.58
0.60
0.59
0.59
0.41
—
3g
3h
763
244 000
0.91
782
300
0.56
a
In chloroform at room temperature, absorption/emission wavelength l00A/l00F, molar decadic absorption coefficient e00, oscillator strength f,
Stokes shift Dn~A–F and fluorescence quantum yield FF.
the derivatization of this new class of compounds and their use
in ultrasensitive microscopy in live cells tissues.
Support by the Bioimaging Center of the University of
Konstanz and the SFB 767, as well as by C. Strasser for help
with the cellular samples, is gratefully acknowledged.
Notes and references
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2 M. S. T. Goncalves, Chem. Rev., 2009, 109, 190.
3 (a) R. Weissleder, Nat. Biotechnol., 2001, 19, 316; (b) Near-Infrared
Dyes for High Technology Applications, NATO Series 3, ed.
S. Dahne, U. Resch-Genger, O. S. Wolfbeis, Kluwer, Dordrecht,
1998, vol. 52.
4 (a) A. Becker, C. Hessenius, K. Licha, B. Ebert, U. Sukowski,
W. Semmler, B. Wiedenmann and C. Grotzinger, Nat. Biotechnol.,
2001, 19, 327; (b) R. Weissleder, C. H. Tung, U. Mahmood and
A. Bogdanov, Nat. Biotechnol., 1999, 17, 375; (c) Y. Ye, S. Bloch,
B. Xu and S. Achilefu, J. Med. Chem., 2006, 49, 2268.
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Opin. Chem. Biol., 2010, 14, 64; (b) J. J. Gassensmith,
J. M. Baumesm and B. D. Smith, Chem. Commun., 2009, 6329;
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A. D. Bhise, H. L. Kee, D. F. Bocian, D. Holten and J. S. Lindsey,
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Angew. Chem., Int. Ed., 2007, 46, 3750; (b) G. M. Fischer,
M. Isomaki-Krondahl, I. Gottker-Schnetmann, E. Daltrozzo and
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7 M. Y. Berezin, W. J. Akers, K. Guo, G. M. Fischer, E. Daltrozzo,
A. Zumbusch and S. Achilefu, Biophys. J., 2009, 97, L22.
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O. Wallquist, Bull. Soc. Chim. Belg., 1988, 97, 615;
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Recording Materials, EP0217245B1, Int. Cl. C09B 23/10, G11B7/
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Fig. 2 Images of live HeLa cells labeled with Arg9-8. Upper left:
transmission bright-field; upper right: Hoechst 33342 staining of the
cell nucleus; bottom left: NIR fluorescence intracellular Arg9-8
(excitation wavelength: 633 nm, detection wavelengths: 4650 nm,
see ESIw); bottom right: overlay of Hoechst and Arg9-8 channels
showing the localization of Arg9-8 close to but outside the nucleus,
5 h after incubation; scale bar: 20 mm.
(Fig. 2). Additional Hoechst staining shows that these aggregates
concentrate around the nucleus, but do not enter inside.
In summary, a synthetic strategy for BF2-PPCys with
asymmetric substitution pattern has been described. The
compounds show strong NIR absorption and very bright
fluorescence. The spectral properties of different derivatives
show typical cyanine dye behavior. The asymmetric substitution
allows the introduction of one functional group which can be
used for labeling applications with NIR excitation and
detection. First in vivo experiments show that the resulting
dye is brightly fluorescing in live cells and has a very good
chemical stability. We did not observe harmful effects on the
cells. The findings reported here open exciting possibilities for
10 (a) G. T. Hermanson, Bioconjugate Techniques, Academic Press,
1996; (b) A. El-Faham, R. S. Funosas, R. Prohens and
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11 P. Lundberg and U. Langel, J. Mol. Recognit., 2003, 16, 227.
12 T. Holm, H. Johansson, P. Lundberg, M. Pooga, M. Lindgren and
U. Langel, Nat. Protocols, 2006, 1, 1001.
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Chem. Commun., 2010, 46, 5289–5291 | 5291