Phenanthrene-fused boron-dipyrromethenes as bright long-wavelength fluorophores

Article abstract of DOI:10.1021/ol800271e

A new class of boron-dipyrromethene (BDP or BODIPY) dyes was obtained by phenanthrene fusion to the ss-pyrrole positions, absorbing in the wavelength range of important laser sources. Despite a 'propeller-like' distorted structure in the crystalline state, the chromophore absorbs (log ε ≥ 5) and fluoresces (φ_f ≥0.8) strongly and can be easily turned into a fluorescence light-up probe. Incorporation into latex beads produces bright and photostable single-dye and Foerster Resonance Energy Transfer (FRET) particles for microscopy applications.

Full text of DOI:10.1021/ol800271e

ORGANIC
LETTERS
2008
Vol. 10, No. 8
1581-1584
Phenanthrene-Fused
Boron Dipyrromethenes as Bright
−
Long-Wavelength Fluorophores
Ana B. Descalzo,† Hai-Jun Xu,‡ Zhao-Li Xue,‡ Katrin Hoffmann,† Zhen Shen,*,‡
Michael G. Weller,† Xiao-Zeng You,*,‡ and Knut Rurack*,†
BAMsFederal Institute for Materials Research and Testing, DiVision I.5,
Richard-Willsta¨tter-Strasse 11, D-12489 Berlin, Germany, and State Key Laboratory of
Coordination Chemistry, Nanjing UniVersity, Nanjing, 210093, PR China
knut.rurack@bam.de; zshen@nju.edu.cn; youxz@nju.edu.cn
Received February 6, 2008
ABSTRACT
A new class of boron−dipyrromethene (BDP or BODIPY) dyes was obtained by phenanthrene fusion to the
â
-pyrrole positions, absorbing in
the wavelength range of important laser sources. Despite a ‘propeller-like’ distorted structure in the crystalline state, the chromophore absorbs
(log E g 5) and fluoresces (Φf g 0.8) strongly and can be easily turned into a fluorescence light-up probe. Incorporation into latex beads
produces bright and photostable single-dye and Fo1rster Resonance Energy Transfer (FRET) particles for microscopy applications.
Rapid advances in optical technologies have recently led to
significant progress in medical diagnostics, drug discovery,
and (bio)chemical analysis, in particular for methods that
use fluorescence detection in microscopy, for microarrays
or single-molecule tracking.1 These developments have
fueled the search for novel fluorophores (stains, labels, and
indicators)2 with high brightness that can be excited and that
emit well within the visible or near-infrared (NIR) region
of the spectrum.3 Research in this field has basically two
aims: (i) to create dyes with ever red-shifted spectra4 and
(ii) to develop functional dyes that match well the emission
lines or excitation/emission filter ranges of commercial light
sources and equipment.5 In the latter case, besides the
advancement of well-known dyes such as cyanines or
rhodamines,5 the design of long-wavelength boron-dipyr-
romethene (BDP) dyes6 has especially attracted attention in
the past decade. Dyes with the parent BDP chromophore
(2, Scheme 1) usually carry alkyl substituents in the 1,7-,
3,5-, and/or 2,6-positions of the core and show absorption
and emission maxima in the 495-545 nm range.7,8 Red-shifts
of the optical spectra can be realized by (a)symmetric
substitution with aryl, vinyl, styryl, or ethynylphenyl groups
at the core.9-11 Alternatively, exchange of Cmeso for an N
(4) Wu, D.; Descalzo, A. B.; Weik, F.; Emmerling, F.; Shen, Z.; You,
X.-Z.; Rurack, K. Angew. Chem., Int. Ed. 2008, 47, 193-197. Fischer, G.
M.; Ehlers, A. P.; Zumbusch, A.; Daltrozzo E. Angew. Chem., Int. Ed. 2007,
46, 3750-3753. Tian, M.; Tatsuura, S.; Furuki, M.; Iwasa, I.; Pu, L. S. J.
Am. Chem. Soc. 2003, 125, 348-349.
(5) Hilderbrand, S. A.; Weissleder, R. Chem. Commun. 2007, 2747-
2749. Liu, J.; Diwu, Z.; Leung, W.-Y.; Lu, Y.; Patch, B.; Haugland, R. P.
Tetrahedron Lett. 2003, 44, 4355-4359.
† BAMsFederal Institute for Materials Research and Testing.
‡ Nanjing University.
(1) Cornish, P. V.; Ha, T. ACS Chem. Biol. 2007, 2, 53-61. Fricker,
M.; Runions, J.; Moore, I. Annu. ReV. Plant Biol. 2006, 57, 79-107. Nagl,
S.; Scha¨ferling, M.; Wolfbeis, O. S. Microchim. Acta 2005, 151, 1-21.
(2) Johnsson, N.; Johnsson, K. ACS Chem. Biol. 2007, 2, 31-38. Carol,
P.; Sreejith, S.; Ajayaghosh, A. Chem. Asian J. 2007, 2, 338-348. Wang,
F.; Tan, W. B.; Zhang, Y.; Fan, X.; Wang, M. Nanotechnology 2006, 17,
R1-R13.
(6) Ziessel, R.; Ulrich, G.; Harriman, A. New J. Chem. 2007, 31, 496-
501. Loudet, A.; Burgess, K. Chem. ReV. 2007, 107, 4891-4932.
(7) Kollmannsberger, M.; Rurack, K.; Resch-Genger, U.; Daub, J. J. Phys.
Chem. A 1998, 102, 10211-10220.
(3) Go´mez-Hens, A.; Aguilar-Caballos, M. P. Trends Anal. Chem. 2004,
23, 127-136. Achilefu, S. Technol. Cancer Res. Treat. 2004, 3, 393-409.
(8) Beer, G.; Rurack, K.; Daub, J. Chem. Commun. 2001, 1138-1139.
10.1021/ol800271e CCC: $40.75
© 2008 American Chemical Society
Published on Web 03/21/2008

NMR measurements revealed 10 signals between 6.42 and
8.55 ppm (e.g., for 1d). The two doublets at 8.55 and 8.43
ppm arise from Hortho and Hmeta of the phenyl ring, since they
are directed toward the BDP core and are thus deshielded
by the indacene and phenanthrene planes. The phenanthrene
protons lying over the phenyl ring are shielded and give
upfield-shifted signals at 6.42, 6.72, 7.01, and 7.19 ppm
compared with those of II. These data indicate that 1d
possesses a distorted structure yet maintains its aromatic
Scheme 1. (A) Chemical Structures of BDPs from the
Literature: 2,7 3,10 and 4;14 Data in MeCN. (B) Preparation
Scheme of 1a-e.
1
character. The H NMR spectra of 1a-c,e also confirmed
the proposed structures.
Single-crystal X-ray structures were obtained for 1a and
1d (Figures 1,S1). As compared to the structures of alky-
atom or fusion of aromatic rings yields more deeply colored
dyes.12-15 In particular, ring fusion is a promising way to
long-wavelength BDPs while maintaining all possibilities of
functionalization. Up to now, BDP analogues with isoin-
dole,11,14,15 and 3H-benzo[e]indole13 have been realized. On
the basis of our previous attempts in this regard11,14 and the
motivation to develop a potent dye platform for widespread
commercial equipment, we report here on the use of the 2H-
dibenzo[e,g]isoindole (or â-phenanthropyrrole) unit to con-
struct highly annelated BDP derivatives that match the output
range (594-635 nm) of the main HeNe or diode laser
excitation sources.
Figure 1. Crystal structure of 1a viewed from the top (A) and
along the B(1)-C(8) axis (B).
lpyrrole or -isoindole BDPs that possess planar indacene
planes and virtually orthogonal meso-phenyl rings,14,17 1a
and 1d display distorted, ‘propeller-like’ conformations. The
indacene plane deviates from planarity with an average
dihedral angle θav ) 12.2° between the central six-membered
ring B and the two adjacent pyrrole rings C,D.18 The
phenanthrene rings19 are also distorted from the mean plane
of indacene (rings B-D) with θav ) 21.4°. The torsion angle
between the two phenanthrene rings19 is 31.7° for 1a and
47.3° for 1d. The highly distorted structure can be ascribed
to the steric hindrance between the large phenanthrene units
and the C8-appended phenyl group, forcing the molecules
into the ‘propeller-like’ conformation. The latter determines
the angle between the planes of indacene ring B and phenyl
ring A, θAB amounting to only 57.9°/49.7° for 1a/1d. In
contrast, θAB ) 90.0° for 4.14 The more distorted conforma-
tion of 1d compared to 1a is most likely due to packing
effects because of the alkylamino group.
1a-e were synthesized from ethylphenanthro[9,10-c]-
pyrrole-1-carboxylate (I)16 via 1-methylphenanthro[9,10-c]-
pyrrole (II) and subsequent condensation with various
aromatic aldehydes according to a published procedure
(Scheme 1).14 Oxidation with DDQ, treatment with triethy-
lamine and BF3‚Et2O and purification by column chroma-
1
tography and recrystallization completed the procedure. H
(9) Thivierge, C.; Bandichhor, R.; Burgess, K. Org. Lett. 2007, 9, 2135-
2138. Dost, Z.; Atilgan, S.; Akkaya, E. U. Tetrahedron 2006, 62, 8484-
8488. Rohand, T.; Qin, W.; Boens, N.; Dehaen, W. Eur. J. Org. Chem.
2006, 4658-4663. Mei, Y.; Bentley, P. A.; Wang, W. Tetrahedron Lett.
2006, 47, 2447-2449. Rurack, K.; Kollmannsberger, M.; Daub, J. Angew.
Chem., Int. Ed. 2001, 40, 385-387.
(10) Rurack, K.; Kollmannsberger, M.; Daub, J. New J. Chem. 2001,
25, 289-292.
(11) Yu, Y.-H.; Descalzo, A. B.; Shen, Z.; Ro¨hr, H.; Liu, Q.; Wang,
Y.-W.; Spieles, M.; Li, Y.-Z.; Rurack, K.; You, X.-Z. Chem. Asian J. 2006,
1, 176-187.
Selected spectroscopic data and optical spectra of 1a-e
are presented in Figure 2 and Table 1. It is evident that the
(12) McDonnell, S. O.; O’Shea, D. F. Org. Lett. 2006, 8, 3493-3496.
Zhao, W.; Carreira, E. M. Angew. Chem., Int. Ed. 2005, 44, 1677-1679.
(13) Zhao, W. L.; Carreira, E. M. Chem. Eur. J. 2006, 12, 7254-7263.
(14) Shen, Z.; Ro¨hr, H.; Rurack, K.; Uno, H.; Spieles, M.; Schulz, B.;
Reck, G.; Ono, N. Chem. Eur. J. 2004, 10, 4853-4871.
(15) Ulrich, G.; Goeb, S.; De Nicola, A.; Retailleau, P.; Ziessel, R. Synlett
2007, 1517-1520.
(17) Yu, Y. H.; Shen, Z.; Xu, H. Y.; Wang, Y. W.; Okujima, T.; Ono,
N.; Li, Y. Z.; You, X. Z. J. Mol. Struct. 2007, 827, 130-136.
(18) All θ between the adjacent ring planes are listed in Table S1.
(19) As defined by the mean planes of rings G, E, F and rings I, H, J.
The mean planes of the phenanthrene units were taken as a reference here
because the single phenanthrene rings are not co-planar, Table S1.
(16) Novak, B. H.; Lash, T. D. J. Org. Chem. 1998, 63, 3998-4010.
1582
Org. Lett., Vol. 10, No. 8, 2008

fusion of the larger phenanthrene π-electron systems induces
the ‘propeller-like’ distortion of the title chromophore, yet
also accounts for the favorably intense bands of log ꢀ g 5.
The second important observation relates to the correlation
between Hammett constant of the 4-substituents of the meso-
phenyl ring and the absorption maximum (Figure 2, inset).
An increase of the substituent’s electron accepting nature
results in a bathochromic shift of the absorption and emission
bands, reflecting the charge redistribution that takes place
in indacene chromophores upon excitation: the electron
density increases at the meso position.7,14 Electron-accepting
substituents thus stabilize these features: 1c with the
strongest acceptor (CN) shows the longest wavelength and
1d with the strongest donor [N(CH3)2] the shortest wave-
length absorption. In contrast, for BDPs such as 2 or 4 with
a dihedral angle between the indacene and meso ring plane
θAB ≈ 90°, identical absorption maxima are found for
different meso-(4-X)-phenyl groups.7,14 These results stress
the influence of the crowded structure on the electronic
properties and enable a fine-tuning of the spectral bands.21
First experiments on the suitability of the phenanthro-
BDPs as efficient analyte-responsive fluorescent switches and
labels also yielded promising results. For instance, Figure 3
demonstrates the potential of 1d as a pH light-up probe.22
In the polar protic solvent mixture, 1d is quenched. Upon
Figure 2. Normalized absorption (left) and emission (right) spectra
of 1a (black), 1b (red), 1c (green), 1d (orange), and 1e (blue) in
hexane at 298 K. Inset: correlation of absorption maximum (in
cm-1) and Hammett constant σP, r ) -0.982.
spectra of 1a-e possess similar shapes as those of the classic
BDP dyes. Depending on the substituent in the meso position,
Table 1. Spectroscopic Data of 1a-e in Dibutyl Ether at
298 Ka
λabs /nm
λem /nm
Φf ((0.03)
τf /ns
1a
1b
1c
1d
1e
630
633
642
621
626
648
654
668
636
642
1.01
0.92
0.95
0.96
1.02
5.75
5.65
5.92
5.54
5.58
a cdye ) 2 × 10-6 M; λexc ) 580 nm.
the absorption maxima are found between ca. 620 nm for
1d and 640 nm for 1c (Table 1). Like for pyrrole- or
isoindole-BDPs, solvatochromic shifts are not observed.
Molar absorption coefficients are favorably high and have
1a
been determined exemplarily in acetonitrile to log ꢀ625
)
1b
1c
1d
5.07, log ꢀ628 ) 4.99, log ꢀ637 ) 4.98, log ꢀ617 ) 5.06,
and log ꢀ622 ) 5.02. Excitation of the phenanthro-BDPs
1e
yields fluorescence spectra of mirror image shape with
maxima in the 630-670 nm region (Figure 2). The emission
maxima lack a particular solvatochromic trend. Except for
1d in polar solvents (see below), the fluorescence quantum
yields of all dyes in solvents of any polarity between hexane
and 1:1 v/v mixtures of water/methanol are favorably high
with Φf g 0.8.20 The quenched fluorescence of 1d (e.g., Φf
) 0.02 in MeCN) is due to a fast charge-transfer process
from the strong donor in the meso position to the BDP core,
in agreement with previous reports on meso-dialkylanilino-
BDPs.7,10,14 A more detailed account of the solvent-modulated
photophysics of 1a-e will be published separately.
The spectroscopic data reveal two important findings. First,
the molar absorption coefficients of the phenanthro-BPDs
and the boron-diindomethene (BDI) dyes (4, Scheme 1) are
equally high despite the fact that the absorption and emission
spectra of the BDI dyes are narrower. This suggests that
Figure 3. Absorption and emission spectra of 1d (black) and 1d-
H+ (red) in ethanol/water 1:1. Blue emission spectra correspond
to intermediate steps of a pH titration with HClO4. Inset: repre-
sentative titration curve (9) and fit (s) for 1d; the emission of
1a-c,e is pH independent (1a, O; 1c, ×; 1b,e not shown).
addition of acid, the typical BDP fluorescence is recovered
with an enhancement factor of 150 in the 650 nm region.
The spectral red-shifts reflect the conversion of 1d into 1d-
H+, i.e., toward 1c (cf. Table 1). Analysis of several titration
curves yields a pKa of 1.92 ( 0.05, indicating that the
dimethylamino group is rather acidic.23
(21) Because θAB ≈ 50-60°, the molecular orbitals involved in the S1
r S0 transition are presumably also localized in part on the meso unit.
Current theoretical studies address these aspects in detail.
(22) All the dyes 1a-e are readily soluble in 1:1 (v/v) H2O/alcohol or
H2O/MeCN mixtures. For 1a-c,e, the absorption and emission spectra are
similar as in organic solvents, independent of pH and Φf are equally high
as in MeOH. The ‘propeller-like’ distortion of the chromophore’s structure
thus prevents aggregation of the dyes in polar aqueous media.
(20) A heavy atom substituent such as the iodo atom in 1b has obviously
no influence on the fluorescence of such chromophores when attached
through the 4-position of the meso group.
Org. Lett., Vol. 10, No. 8, 2008
1583

To test the performance of phenanthro-BDPs in bead-based
applications, 1a was incorporated into commercially available
polystyrene nano- and microparticles (mean particle sizes
of 300 nm and 1.2 µm). This allows one to use the dyes in
aqueous media and would also permit the delivery of such
dyes into cells.24 The dye was loaded into the beads simply
by swelling in an organic solvent (cf. Supporting Informa-
tion). Both the resulting 1a-loaded nano- and microparticles
(red particles, RP) show virtually identical spectroscopic
properties in aqueous suspensions as the free dye in organic
solvents, i.e., the typical spectra (cf. Figures 2 and S2), Φf
of ca. 0.98, and τf ≈ 4.3 ns. To demonstrate the versatility
and complementarity of ring-fused BDPs for fluorescent
beads applications, we prepared FRET-active nano- and
microparticles (FP) with 4 as the FRET donor and 1a as the
FRET acceptor. On one hand, such FRET particles are very
interesting for single wavelength excitation multicolor FRET
applications.25 On the other hand, FRET-based sensing
systems can easily be obtained by adequate substitution of
dyes such as 1 or 4. Figure 4 shows the efficiency of the
energy transfer as recorded by confocal laser scanning
microscopy (CLSM). Excitation of the control particles with
4 (yellow particles, YP) and RP with the common 543 nm
line yields a bright fluorescence image only for YP in a 555-
625 nm detection window with the other three images
remaining dim (Figure 4A,B); YP show only residual
emission of the long-wavelength tail in the red channel
(detection at >650 nm) and RP absorb only negligibly at
543 nm. However, employing FP, brightly fluorescing beads
are only observed in the red channel (Figure 4C). Measure-
ments on a steady-state fluorometer revealed similar spec-
troscopic and FRET features for nano- and micro-FP,
suggesting that size-tailored particles can easily be prepared.
Analysis of the spectra of YP and RP according to the FRET
formalism26 yields an overlap integral J ) 7.5 × 1015 M-1
cm-1 nm4 that is more than 10 times higher than that of a
common FRET pair such as FITC-TRITC27 with 4.9 × 1014
M-1 cm-1 nm4.27 That the emission of the title dyes is not
quenched by oxygen and is pH-independent and that the
particles do not photobleach (data not shown) stress their
potential.
Figure 4. Confocal laser scanning microscope (CLSM) images
of control [YP (A); RP (B)] and FRET polystyrene microparticles
FP (C). λexc ) 543 nm; yellow (filter combination for 555-625
nm detection) and red channels (filter for >650 nm detection) shown
in the left and right panels, respectively; identical parameters (laser
power, PMT voltage, gain) used for all acquisitions.
fluorescence imaging and flow cytometry. The results of 1d
as a pH indicator for the acidic range suggest that phenan-
thro-BDPs are a promising platform for designing more
elaborate red/NIR labels and probes. The versatility of the
chromophore is obvious when considering that it offers more
flexibility in functionalization than azaBDPs, especially
through the meso position, and that it allows to obtain novel
NIR dyes; in accordance with known BDP tuning procedures,
disubstitution with dimethylamino-styryl groups at the C3,-
C5-positions should for instance lead to spectral red-shifts
of 200 nm.11 Attempts to improve the dyes’ water solubility
are currently also under way.
In conclusion, a novel series of highly annelated phenan-
thro-BDPs is presented that show intense absorption and
fluorescence bands in the long-wavelength region with Φf
close to 1. The distorted structure facilitates a stronger
electronic influence of the meso group while retaining the
advantages of the virtually decoupled architecture of meso-
substituted BDPs. The particle experiments revealed that the
title dyes are a suitable alternative for red beads in
Acknowledgment. We thank the European Commission’s
Human Resources & Mobility Programme (MC-EIF Fel-
lowship), the National Basic Research Program of China (No.
2006CB806104, 2007CB925103), the National Natural Sci-
ence Foundation of China (No. 20401009 and 20721002),
and the Fok Ying Tung Education Foundation (No. 104013)
for financial support.
(23) Similar features have been found for related dyes: Maus, M.;
Rurack, K. New. J. Chem. 2000, 24, 677-686. Shizuka, H.; Ogiwara, T.;
Kimura, E. J. Phys. Chem. 1985, 89, 4302-4306.
(24) Sanchez-Martin, R. M.; Muzerelle, M.; Chitkul, N.; Eng, How, S.;
Mittoo, S.; Bradley, M. ChemBioChem 2005, 6, 1341-1345.
(25) Wang, L.; Tan, W. Nano Lett. 2006, 6, 84-88.
Supporting Information Available: Experimental de-
tails, additional X-ray data, and bead spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
(26) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;
Springer: Berlin, 2006; p 445ff.
(27) FITC ) fluorescein isothiocyanate, TRITC ) tetramethylrhodamine
isothiocyanate. Heidecker, M.; Yanmarriott, Y.; Marriott, G. Biochemistry
1995, 34, 11017-11025.
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