Synthesis of functional near infrared pyrrolopyrrole cyanine dyes for optical and photoacoustic imaging

Article abstract of DOI:10.1515/HC.2010.006

Fluorescence lifetime and photoacoustic imaging (PAI) provide attractive strategies for in vivo molecular imaging in the near infrared (NIR) region. Pyrrolopyrrole cyanine (PPCy) dyes are promising molecules for these applications. In this study, we synth

Full text of DOI:10.1515/HC.2010.006

Heterocycl. Commun., Vol. 16(4-6), pp. 213–216, 2010 • Copyright © by Walter de Gruyter • Berlin • New York. DOI 10.1515/HC.2010.006
Preliminary Communication
Synthesis of functional near infrared pyrrolopyrrole cyanine
dyes for optical and photoacoustic imaging
Mingfeng Bai and Samuel Achilefu1,2,*
1
1
Unfortunately, conventional NIR cyanine dyes suffer from
some limitations. First, some of these dyes decompose over
time in ambient conditions and tend to aggregate in solutions.
Second, the fluorescence quantum yields of many indocya-
nine dyes are typically <15% in aqueous environment and
are readily photobleached under high laser beams. Finally,
most available NIR cyanine dyes have similar fluorescence
lifetimes (FLTs) around 1 ns, confining the scope of lifetime
imaging applications with these NIR fluorophores to narrow
lifetime range.
Department of Radiology, Washington University School
of Medicine, 4525 Scott Avenue, St. Louis, MO 63110,
USA
Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine, 4525 Scott
Avenue, St. Louis, MO 63110, USA
2
*
Corresponding author
e-mail: achilefus@mir.wustl.edu
Consequently, there is a need to develop novel NIR dyes
to improve signal intensity, sensitivity, photostability, and
diverse FLT ranges for fluorescence imaging. Compared to
conventional fluorescence imaging based on fluorescence in-
tensity measurements, FLT imaging is less affected by dye
concentration, photobleaching, light scattering, excitation in-
tensity, and sample turbidity (O’Leary et al., 1996; Cerussi et
al., 1997; Kuwana and Sevick-Muraca, 2002; Ntziachristos
and Weissleder, 2002; Lichtman and Conchello, 2005).
Development of NIR dyes with long FLTs is challenged by
the energy gap law (Englman and Jortner, 1970; Caspar et al.,
Abstract
Fluorescence lifetime and photoacoustic imaging (PAI) pro-
vide attractive strategies for in vivo molecular imaging in the
near infrared (NIR) region. Pyrrolopyrrole cyanine (PPCy)
dyes are promising molecules for these applications. In this
study, we synthesized a chloro-functionalized non-fluorescent
dye with high absorption coefficient suitable for PAI. Upon
complexation with boron, the dye becomes highly fluorescent
with lifetime of 3.6 ns, which is longer than most cyanine
dyes commonly used for NIR optical imaging. The potential
to use the non-fluorescent or fluorescent analogs of PPCy for
fluorescence or photoacoustic imaging provides a versatile
approach to image molecular processes and physiological
events by these methods.
1
982), causing a decrease in quantum yield and FLT at these
long NIR wavelengths. Another attractive area of research
with NIR dyes is photoacoustic imaging, which provides
strong contrast, high resolution and deep tissue penetration by
combining optical with ultrasound technologies (Wang, 2008).
Imaging capability and accuracy of photoacoustic imaging is
enhanced by using exogenous contrast agents. Because pho-
toacoustic signals originate from heat after light absorption,
contrast agents with high absorption coefficients and low
fluorescence quantum yields are optimal for this application.
Unfortunately, few NIR dyes possess these features.
Keywords: fl uorescence lifetime; molecular probes; photo-
acoustic imaging agent; spectroscopy.
Introduction
Recently, a new class of NIR absorbing and fluorescent
diketopyrrolopyrrole dyes has been developed (Fischer et al.,
2007, 2009). These dyes exhibit high quantum yields (>0.50),
low photobleaching (2% absorption drop after 60 min illu-
mination compared to 93% absorption drop for indocyanine
green) and long FLTs (2.5–3.8 ns) (Berezin et al., 2009). These
properties are suitable for fluorescence imaging, whereas the
non-fluorescent analogs are useful for photoacoustic imag-
ing. However, the application of this class of dye in biomedi-
cal research is greatly limited owing to poor hydrophilicity
and lack of functionality for subsequent conjugation with
biomolecules such as peptides and proteins. The importance
of functionalized pyrrolopyrrole cyanine (PPCy) for conjuga-
tion with peptides and proteins was recently reported (Fischer
et al., 2010).
Owing to relatively deep tissue penetration and low tissue
autofluorescence in the near infrared (NIR) region between
700 and 900 nm, NIR fluorescent dyes are attractive in mod-
ern optical imaging of biological targets in vivo (Akers et
al., 2008). The most common NIR dyes are the polymethine
cyanine dye family, comprising benzoxazole, benzothiazole,
indolyl, 2-quinoline, and 4-quinoline subclasses (Frangioni,
2003). These dyes have adjustable optical properties and high
extinction coefficients, often >150 000 m/cm (Licha, 2002).
In addition, many cyanine dyes can be easily conjugated to
targeting moieties, imparting molecular specificity or selectiv-
ity to targeted cells or tissues (Achilefu, 2004; Bai et al., 2008;
Ye et al., 2008).
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14 M. Bai and S. Achilefu
Si
O
O
O
O
O
O
TBS
O
1
2
. TBS-Cl, imidazole, DMF, overnight
. 4-cyanophenol, Na2CO3, DMF, 1d
O
Br
OH
O
Na, t-amylalcohol, 1d
HN
NH
N
1
O
42%
TBS
2
O
O
POCl3
NH2
N
21%
N
dichlorobenzene
o
1
10 C
0 min
3
2
O
Cl
O
Cl
O
OH
CN
CN
F
B
CN
F
N
B
F
N
N
N
H
DIEA
N
H
N
N
H
N
N
N
F
N
H
N
DCM
NC
NC
o
4
0 C
NC
Cl
O
Cl
O
HO
O
83%
5
4
51%
4
'
Scheme 1 Synthesis of PPCy.
Results and discussion
and FT-IR (data not shown) analysis verified the existence of
chloride, instead of hydroxyl group, on the molecule (com-
pound 4).
After the structure of compound 4 was verified, we stud-
ied the spectroscopic properties of the dye. This PPCy mol-
ecule exhibits major and minor absorption peaks at 730 nm
and 663 nm, respectively (Figure 2), with extinction coef-
ficient of 58 000 m/cm in dimethyl sulfoxide (DMSO). No
significant fluorescence was observed, as the flexible het-
eroaromatic end groups lead to efficient radiationless de-
Here, we report the synthesis of functional PPCy dyes. The
synthetic pathway for the PPCy dyes is shown in Scheme
1
. The hydroxyl group in 3-bromopropanol was first pro-
tected with t-butyldimethylsilyl, and subsequent alkylation
reaction with 4-cyanophenol yielded compound 1 with
4
2% yield. Next, diketopyrrolopyrrole 2 was synthesized
by Stobbe-like condensation of diisopropyl succinate and
in a mixture of sodium t-amyl alcoholate and t-amyl
1
alcohol. Quinoline acetonitrile 3 was prepared as previ-
ously reported (Fischer et al., 2009). Finally, the desired
compound 4′ was obtained by heating a mixture of 2 and
population of the excited S state (Fischer et al., 2009).
1
Therefore, compound 4 is suitable as a contrast agent for
photoacoustic imaging. However, incorporation of BF2
into 4 yields compound 5. This reaction rigidified the
molecule and red-shifted the absorption (Figure 2) with
good absorption coefficient (ε=111 000 m/cm). The resulting
fluorescence quantum yield was high (81% in DMSO) and the
characteristic long FLT of 3.6 ns was retained in compound 5.
These properties are excellent for FLT imaging studies.
In summary, we have reported the synthesis of PPCy and
3
in dichlorobenzene at 110°C in the presence of POCl3.
High reaction yield (52%) was achieved. Compound 2 fi rst
forms a monophosphorylated intermediate with POCl3,
followed by forming condensation product with 3 (Iqbal
et al., 1988; Fischer et al., 2009). The yield of the reaction
was largely affected by the solubility of both condensation
partners. The quinoline acetonitrile 3 is soluble in many
organic solvents, whereas 2 is only partially soluble in lim-
ited solvents. By changing the solvent from toluene to di-
chlorobenzene, the condensation reaction yield improved
from 19% to 52%.
BF ⋅PPCy dyes as functional NIR dyes with attractive opti-
2
cal properties. Compound 4 has strong absorption, and the
boron complexed compound 5 has strong fluorescence in the
NIR region. The use of POCl provided a method for one-pot
3
1
Upon purification, compound 4′ was characterized by H
transformation of the hydroxyl to reactive chloride function.
Derivatization of the chloride to other functional groups, such
as carboxyl and amino, and subsequent bioconjugation of the
PPCy to targeting moieties for biomedical imaging are cur-
rently in process. With a reactive chloride group, good absorp-
tion coefficient, long FLT and/or high fluorescence quantum
yield, these dyes appear to be good candidates for molecular
imaging applications.
NMR. All peaks seemed to correlate with the target structure.
Further derivatization with CDI or chloroformate, however,
failed to produce desired products. Considering that hydroxyl
group and chloride have similar chemical shifts effect for
protons bound to the neighboring carbon, we postulated that
POCl converted the hydroxyl group to chloride. Subsequent
3
1
3
C NMR (Figure 1, peak at 41.7 ppm indicates chloride)
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Functional NIR cyanine dyes 215
ppm
220
200
180
160
140
120
100
80
60
40
20
0
Figure 1 13C NMR spectrum of compound 4.
butyl dimethylsilyl chloride (110 mmol) was added and the result-
ing mixture was allowed to warm to room temperature. After stirring
overnight, the reaction mixture was partitioned between saturated
sodium bicarbonate solution (100 ml) and ethyl ether (200 ml). The
separated ether layer was then washed with 1 m sulfuric acid solution
0
.30
.25
.20
.15
.10
.05
.00
Absorption of 4
Absorption of 5
Fluorescence of 5
2
1
000 000
500 000
0
0
0
1 000 000
(
80 ml), followed by brine (80 ml). Next, the organic solution was
A
dried over sodium sulfate and concentrated by rotary evaporation.
In another flask, a mixture of 4-cyanophenol (80 mmol) and sodium
carbonate (100 mmol) in anhydrous DMF (100 ml) was stirred at 70°C
under argon for 10 min. The freshly synthesized 3-bromopropoxy-t-
butyl-dimethylsilane was added and the mixture was stirred at room
temperature for 24 h. The reaction mixture was quenched with 1 m
sulfuric acid and extracted with ether (3×150 ml). The extracted ether
layers were combined, washed with brine and dried over sodium sul-
fate. After removal of the solvent under reduced pressure, the desired
compound 1 (10 g, 43%) was eluted with 20% ethyl acetate in hexane
0
C
500 000
B
0
0
0
850
550
600
650
700
750
800
Wavelength (nm)
Figure 2 Absorption and fluorescence of PPCy and BF ⋅PPCy in
dichloromethane at room temperature.
A) Absorption of 4 (blue), (B) absorption of 5 (green), and (C) fluo-
rescence of 5, excited at 730 nm.
2
(
1
by flash column chromatography. H NMR 300 MHz (CDCl ): δ 7.57
3
(
(
d, J=9.3 Hz, 2H), 6.94 (d, J=9.3 Hz, 2H), 4.11 (t, J=6.3 Hz, 2H), 3.79
t, J=6.3 Hz, 2H), 1.99 (m, 2H), 0.87 (s, 9H), 0.03 (s, 6H).
Experimental details
Synthesis of 3,6-bis(4-(3-((t-butyldimethylsilyl)oxy)
propoxy)phenyl)pyrrolo[3,4-c]pyrrole-1,4
Compound 3 was synthesized from 4-t-butyl aniline in three steps as
reported previously (Fischer et al., 2009).
(2H,5H)-dione (2)
Synthesis of 4-(3-((t-butyldimethylsilyl)oxy)propoxy)
benzonitrile (1)
Sodium (0.65 mol) in t-amyl alcohol (30 ml) was heated at 100°C
under argon atmosphere overnight. After all the metal sodium had
reacted, compound 1 (30 mmol) was added in one portion. The re-
sulting mixture was stirred at 100°C under argon atmosphere and a
solution of diisopropyl succinate (15 mmol) in t-amyl alcohol (7 ml)
To a solution of 3-bromopropanol (100 mmol) in anhydrous DMF
(
100 ml) was added imidazole (110 mmol) at 0°C. After 5 min, t-
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2
16 M. Bai and S. Achilefu
was added through a syringe pump over 20 h. The reaction mixture
was slowly added to a stirring mixture of 50 ml methanol and 12 ml
acetic acid. The red precipitate was filtered and washed with metha-
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fluorescence lifetime behavior of near-infrared fluorescent con-
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ble ligand for cannabinoid CB2 receptor imaging. Bioconjugate
Chem. 2008, 19, 988–992.
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Zumbusch, A.; Achilefu, S. Long fluorescence lifetime molecu-
lar probes based on near infrared pyrrolopyrrole cyanine fluoro-
phores for in vivo imaging. Biophys. J. 2009, 97, L22–L24.
Caspar, J. V.; Kober, E. M.; Sullivan, B. P.; Meyer, T. J. Application
of the energy-gap law to the decay of charge-transfer excited-
states. J. Am. Chem. Soc. 1982, 104, 630–632.
nol (3×15 ml). After drying, compound 2 was isolated as a red solid
1
(
5 g, 49%). H NMR 300 MHz (CDCl ): δ 11.15 (s, 2H), 8.45 (d,
3
J=8.7 Hz, 4H), 7.12 (d, J=8.7 Hz, 4H), 4.11 (t, J=6.0 Hz, 4H), 3.76
(
(
t, J=6.0 Hz, 4H), 1.92 (m, 4H), 0.86 (s, 16H), 0.03 (s, 12H). MS
MALDI with α-cyano-4-hydroxycinnamic acid as matrix) [M+H]
+
+
calcd. 665.3, found 665.1.
Synthesis of PPCy (4)
POCl (0.8 mmol) was added to a mixture of 2 (0.1 mmol) and 3
3
(0.25 mmol) in anhydrous dichlorobenzene (3 ml) at 110°C. The re-
action mixture was stirred under argon atmosphere for 30 min before
being partitioned between NaHCO3 solution and chloroform. The
combined organic layers (3×30 ml) were combined and dried over
Na SO . The solvent was then removed by rotary evaporation and
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W. W.; Gratton, E. Experimental verification of a theory for the
time-resolved fluorescence spectroscopy of thick tissues. Appl.
Optics 1997, 36, 116–124.
2
4
crude product was purified on silica gel column starting with dichlo-
Englman, R.; Jortner, J. Energy gap law for radiationless transitions
in large molecules. Mol. Phys. 1970, 18, 145–164.
Fischer, G. M.; Ehlers, A. R.; Zumbusch, A.; Daltrozzo, E. Near-
infrared dyes and fluorophores based on diketopyrrolopyrroles.
Angew. Chem. Int. Ed. 2007, 46, 3750–3753.
Fischer, G. M.; Isomaki-Krondahl, M.; Gottker-Schnetmann, I.;
Daltrozzo, E.; Zumbusch, A. Pyrrolopyrrole cyanine dyes: a new
class of near-infrared dyes and fluorophores. Chem. Eur. J. 2009,
romethane to 2% methanol in dichloromethane as eluent. Compound
1
4
(
7
(
was isolated as a green solid (44 mg, 52%). H NMR 300 MHz
CDCl ): δ 7.98 (d, J=8.7 Hz, 2H), 7.81–7.77 (m, 8H), 7.68 (s, 1H),
.65 (s, 3H), 7.18 (d, J=8.7 Hz, 2H), 4.29 (t, J=5.7 Hz, 4H), 3.83
t, J=6.3 Hz, 4H), 2.33 (m, 4H), 1.44 (s, 18H). MS (MALDI with
α-cyano-4-hydroxycinnamic acid as matrix) [M+H] ; calcd. 885.3,
3
+
+
found 885.3.
1
5, 4857–4864.
Fischer, G. M.; Jüngst, C.; Isomäki-Krondahl, M.; Gauss, D.; Möller,
H. M.; Daltrozzo, E.; Zumbusch, A. Asymmetric PPCys: strongly
fluorescing NIR labels. Chem. Commun. 2010, 46, 5289–5291.
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Opin. Chem. Biol. 2003, 7, 626–634.
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O. The synthesis and properties of 1,4-diketo-pyrrolo[3,4-c]pyr-
roles. Bull. Soc. Chim. Belg. 1988, 97, 615–643.
Synthesis of BF ⋅PPCy (5)
2
Amixture of 4 (0.01 mmol) and DIEA(0.2 mmol) was heated to 40°C
in dichloromethane (2 ml) in a sealed vial. After 5 min, BF ⋅Et O
3
2
(
0.4 mmol) was added and the resulting mixture was heated at 40°C
for another 2 h. The reaction mixture was washed with water and
dried over Na SO . After the solvent was removed by rotary evapo-
2
4
ration, the crude product was purified by column chromatography
Kuwana, E.; Sevick-Muraca, E. M. Fluorescence lifetime spectros-
copy in multiply scattering media with dyes exhibiting multi
exponential decay kinetics. Biophys. J. 2002, 83, 1165–1176.
Licha, K. Contrast agents for optical imaging. Top. Curr. Chem.
using chloroform as eluent. BF ⋅PPCy was isolated as a green solid
2
1
(
7.8 mg, 83%). H NMR 300 MHz (CDCl ): δ 8.45 (d, J=9.9 Hz,
3
2H), 8.12 (d, J=9.0 Hz, 2H), 7.78–7.68 (m, 8H), 7.64 (d, J=2.4 Hz,
2H), 7.10 (d, J=8.7 Hz, 4H), 4.26 (t, J=5.7 Hz, 4H), 3.81(t, J=6.3 Hz,
4H), 2.31 (m, 4H), 1.36 (s, 18H).
2
002, 222, 1–29.
Lichtman, J. W.; Conchello, J. A. Fluorescence microscopy. Nat.
Methods 2005, 2, 910–919.
Acknowledgments
Ntziachristos, V.; Weissleder, R. Charge-coupled-device based scan-
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media. Med. Phys. 2002, 29, 803–809.
O’Leary, M. A.; Boas, D. A.; Li, X. D.; Chance, B.; Yodh, A. G.
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This work was supported in part by the US National Institutes of
Health grants NIBIB (R01 EB00811, R01 EB007276) and NCI (R33
CA123537 and U54 CA136398).
2
1, 158–160.
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